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Running Title: Actin-depolymerizing factor in plant defense 1
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Author for Correspondence: Jyoti Shah 4
Department of Biological Sciences and BioDiscovery Institute, University of North Texas, 5
Denton, TX 76203, USA 6
Phone: (940) 565-3535; Fax: (940) 565-4136; E-mail: [email protected] 7
8
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Research Area: Signaling and Response 11
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Plant Physiology Preview. Published on November 13, 2017, as DOI:10.1104/pp.17.01438
Copyright 2017 by the American Society of Plant Biologists
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Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 is required for controlling aphid 16
feeding from the phloem 17
18
Hossain A. Mondal1,3, Joe Louis1,4, Lani Archer1,2, Monika Patel1,2, Vamsi J. Nalam1,5, Sujon 19
Sarowar1, Vishala Sivapalan1, Douglas D. Root1, and Jyoti Shah1,2 20
21 1Department of Biological Sciences and 2BioDiscovery Institute, University of North Texas, 22
Denton, TX 76203, USA 23 3Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar, India 24
4Department of Entomology and Department of Biochemistry, University of Nebraska, Lincoln, 25
NE 68583, USA 26 5Department of Biology, Indiana University-Purdue University, Fort Wayne, IN 46805, USA 27
28
Present address for Sujon Sarowar: Botanical Genetics, Buffalo, NY 14203, USA 29
30
One sentence summary: Green peach aphid feeding from the sieve elements is shown to be 31
restricted by ACTIN-DEPOLYMERIZING FACTOR3, thus implicating actin-dependent process 32
in controlling insect feeding from the phloem. 33
34
Author contributions: HAM, JL, LA, MP, DDR and JS conceived and designed the study. 35
HAM, JL, LA, MP, VJM, SS and VS conducted the experiments, and analyzed and interpreted 36
the data. DDR contributed to methods development and data analysis. The manuscript was 37
written by HAM, JL and JS with input from all authors. 38
39
Funding information: This work was partially supported by a grant from the National Science 40
Foundation (IOS-0919192) to JS. MP was supported by graduate assistantship from the 41
University of North Texas. 42
43
The author responsible for distribution of materials integral to the findings presented in this 44
article in accordance with the Journal policy described in the Instructions for Authors 45
(http://www.plantphysiol.org) is Jyoti Shah ([email protected] ). 46
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ABSTRACT 47
The actin cytoskeleton network has an important role in plant cell growth, division and stress 48
response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that 49
contribute to reorganization of the actin network. Here we show that the Arabidopsis thaliana 50
ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly 51
known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of 52
more than fifty plant families. In agreement with a role for the actin-depolymerizing function of 53
ADF3 in defense against the GPA, we show that resistance in adf3 was restored by 54
overexpression of the related ADF4, and the actin cytoskeleton destabilizers, cytochalasin D and 55
latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets 56
found sieve elements faster when feeding on the adf3 mutant compared to the wild-type (WT) 57
plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period 58
from sieve elements of adf3 compared to the WT plant. The longer feeding period correlated 59
with an increase in fecundity and population size of the GPA and a parallel reduction in callose 60
deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by 61
expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus 62
confirming the importance of ADF3 function in the phloem. We further demonstrate that the 63
ADF3-dependent defense mechanism is linked to the transcriptional upregulation of 64
PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA. 65
66
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INTRODUCTION 67
In eukaryotes, the actin cytoskeleton, which is composed of filamentous (F)-actin, has a 68
central role in multiple cellular processes, including cell growth, division and differentiation, 69
regulation of polarity, and facilitating cytoplasmic streaming, organelle movement and response 70
to the environment (Staiger, 2000; Hussey et al., 2006; Staiger and Blanchoin, 2006; Pollard and 71
Cooper, 2009; Szymanski and Cosgrove, 2009; Day et al., 2011; Henty-Ridilla et al., 2013). 72
This microfilament network is dynamic and requires continuous reorganization. Remodeling of 73
the actin network involves severing, depolymerization and polymerization of F-actin, which 74
needs to be spatially and temporally regulated. A variety of actin-binding proteins are involved 75
in remodeling of the actin cytoskeleton (Hussey et al., 2006). These include the actin-nucleating 76
and filament stabilizing proteins like the formins and fimbrins, respectively, and the actin-77
depolymerizing factor (ADF) family of proteins (Vidali et al., 2009; Ye et al., 2009; Wu et al., 78
2010). As a result of their ability to sever and depolymerize F-actin and yield products with ends 79
that can serve as sites for new filament initiation, the ADFs, which are small proteins 80
(approximately 17 kDa), increase the dynamics of the actin cytoskeleton and the balance of F-81
actin versus the free globular (G)-actin (Andrianantoandro and Pollard, 2006; Pavlov et al., 82
2007). ADF’s are also involved in shuttling actin into the nucleus (Nebl et al., 1996; Jiang et al., 83
1997), where actin is a component of chromatin remodeling activities that control gene 84
expression (Farrants, 2008; Jockusch et al., 2006). In addition to the cytosol, some ADF’s also 85
localize to the nucleus (Ruzicka et al., 2007). 86
The ADF family in Arabidopsis thaliana consists of eleven expressed genes, which based 87
on their relatedness to each other have been grouped into four subclasses (Feng et al., 2006). 88
These ADF subclasses exhibit novel and differential tissue-specific and developmental 89
expression patterns (Ruzicka et al., 2007). For example, the subclass I genes, which include 90
ADF1, ADF2, ADF3 and ADF4, are constitutively expressed in a variety of vegetative and 91
reproductive tissues, including roots, leaves, flowers, and young seedlings. Amongst the 92
subclass I genes, ADF3 was the most strongly expressed. The subclass II genes can be 93
subdivided into clade IIa (ADF7 and ADF10) and clade IIb (ADF8 and ADF11). ADF7 and 94
ADF10 exhibit high levels of expression in the reproductive tissues, with strongest expression in 95
mature pollen grain. In comparison, expression of the clade IIa genes is relatively poor in roots 96
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and leaves. By contrast, the clade IIb ADF8 and ADF11 genes, show highest expression in 97
epidermal cells that develop into root hairs. ADF5 and ADF9, which belong to subclass III, 98
exhibit strongest expression in the fast growing tissues, including the root meristem and 99
emerging leaves. ADF6, which is the lone member of subclass IV, is expressed in all tissues 100
including pollen. Biochemical characterization of Arabidopsis ADFs indicated that all Class I, II 101
and IV ADFs possess F-actin-severing/depolymerizing activity, with Class I ADFs possessing 102
the strongest activities (Nan et al., 2017). In comparison, the class III ADFs lacked F-actin-103
severing/depolymerizing activity, and instead possessed F-actin bundling activity (Nan et al., 104
2017). 105
ADFs and actin cytoskeleton dynamics are involved in plant response to pathogens. In 106
Arabidopsis, the actin polymerization inhibitor cytochalasin E attenuated non-host resistance 107
against Blumeria graminis f. sp. tritici (Yun et al., 2003). Cytochalasin E also interfered with the 108
targeting of the resistance protein RPW8.2 to the extrahaustorial membrane in Arabidopsis 109
inoculated with the powdery mildew Golovinomyces spp fungi (Wang et al., 2009). Similarly, 110
RPW8.2 localization was also affected in plants overexpressing ADF6, thus suggesting that the 111
specific targeting of RPW8.2 to the extrahaustorial membrane is dependent on actin cytoskeleton 112
dynamics (Wang et al., 2009). Arabidopsis ADF4 is required for resisting infection by the 113
bacterial pathogen Pseudomonas syringae pv tomato DC3000 expressing the AvrPphB effector 114
protein (Tian et al., 2009). ADF4 function in defense against this pathogen was linked to the 115
transcriptional regulation of the Arabidopsis RPS5 gene, which encodes a resistance protein that 116
facilitates the recognition of the AvrPphB effector protein and the activation of downstream 117
defense signaling (Porter et al., 2012). Thus, it was suggested that ADF4 links actin cytoskeleton 118
dynamics to pathogen perception and defense activation (Porter et al., 2012). ADF4 was also 119
shown to regulate actin dynamics and callose deposition in response to elf26, a microbial elicitor 120
of immunity (Henty-Ridilla et al., 2014). In contrast, knockdown of ADF4 resulted in enhanced 121
resistance against an Arabidopsis adapted strain of the powdery mildew fungus (Inada et al., 122
2016), while knockdown of ADF2 resulted in enhanced resistance to the root-knot nematode 123
Meloidogyne incognita (Clément et al., 2009). Hence, there is a wider involvement of ADFs in 124
plant defense as well as susceptibility to pests. 125
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Aphids (Hemiptera: Aphididae) encompass a large group of insects that consume phloem 126
sap. Nearly 250 species of aphids are considered as pests of plants that limit plant growth and 127
productivity due to removal of nutrients from sieve elements and their ability to alter source-sink 128
patterns, and thus the flow of nutrients to growing parts of the infested plant (Pollard, 1973; 129
Blackman and Eastop, 2000; Goggin, 2007). Furthermore, some aphids also vector viral 130
diseases (Kennedy et al., 1962; Matthews, 1991; Guerrieri and Digilio, 2008). Aphids utilize 131
their mouthparts, which are modified into slender stylets, to feed from the sieve elements. Prior 132
to inserting stylets into the plant tissue, aphids utilize chemosensory hairs on their antennae to 133
assess the plant surface, potentially for gustatory cues (Powell, 2006). Subsequently the stylets 134
briefly penetrate non-vascular cells to sample cell contents for additional gustatory cues. The 135
puncturing of non-vascular cells along the path of the stylet penetration likely results in the 136
activation of host defenses that could potentially interfere with the ability of the stylet to reach a 137
sieve element. Aphids produce two distinct salivary secretions that facilitate infestation (Miles, 138
1999). The proteinaceous gelling saliva, which is released by the stylets on their way to the 139
sieve elements, forms a sheath that facilitates stylet movement through the plant tissue and 140
simultaneously minimizes wound-responses in the plant by quickly sealing off wounds. In 141
comparison, the watery saliva, which is released when the stylets penetrate the sieve elements, 142
contains factors suggested to enable the insect to prevent and maybe reverse phloem occlusion 143
and thus facilitate feeding from the sieve elements (Miles, 1999; Will et al., 2007, 2009). 144
The plant surface provides the first line of defense (e.g. trichomes and glandular 145
secretions) that could deter aphid settling on a plant (Walling, 2008). In addition, during the 146
different stages of stylet penetration into the plant tissue, the insect encounters defenses that 147
deter feeding and adversely impact insect fecundity (Walling, 2008; Louis and Shah, 2013). 148
These include factors that contribute to sieve element occlusion (e.g. callose deposition and 149
phloem protein aggregation) as well as insecticidal factors present in the phloem sap (Pedigo, 150
1999; Smith, 2005; Powell et al., 2006; Goggin, 2007; Walling, 2008). The interaction between 151
Arabidopsis and the green peach aphid (GPA; Myzus persicae Sülzer) has been utilized as a 152
model system to characterize plant genes and mechanism that contribute to defense (Louis et al., 153
2012; Louis and Shah, 2013). GPA is a polyphagous insect that is an important pest of more 154
than 400 plant species belonging to over 50 plant families, and the vector of more than 100 viral 155
diseases (Kennedy et al., 1962; Matthews, 1991; Blackman and Eastop, 2000). The 156
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PHYTOALEXIN-DEFICIENT 4 (PAD4) gene is an important regulator of Arabidopsis defenses 157
that deter GPA feeding and adversely impact GPA fecundity (Pegadaraju et al., 2005, 2007; 158
Louis et al., 2010a). Genetic studies have indicated that the upregulation of PAD4 expression in 159
response to GPA infestation correlated with PAD4’s involvement in deterring GPA feeding from 160
sieve elements. This feeding deterrence was intensified in plants overexpressing PAD4 from the 161
Cauliflower mosaic virus 35S gene promoter (Pegadaraju et al., 2007). In contrast, basal 162
expression of PAD4 was sufficient for the accumulation of an antibiotic activity that adversely 163
impacts insect fecundity (Louis et al., 2010a, 2012). 164
ADF proteins, and profilin, an actin-binding protein that influences actin polymerization, 165
have been identified in phloem exudates from a variety of plants (Schobert et al., 1998, 2000; 166
Kulikova and Puryaseva, 2002; Lin et al., 2009; Rodriguez-Medina, 2011; Fröhlich et al., 2012). 167
Furthermore, microfilament meshwork have been revealed in sieve elements of fava bean (Vicia 168
faba) injected with the actin-binding fluorescent phalloidin (Hafke et al., 2013). Immunolabeling 169
with anti-actin antibodies further confirmed the presence of actin cytoskeleton in the sieve 170
elements (Hafke et al., 2013). The identification of an actin-binding protein in aphid saliva has 171
led to the suggestion that an actin-dependent process contributes to plant defense in the phloem 172
and aphids attempt to curtail these defenses by targeting actin dynamics (Nicholson et al., 2012). 173
We therefore investigated the contribution of ADF genes in the interaction of Arabidopsis with 174
the GPA. We show that an ADF3-dependent mechanism is required for controlling GPA feeding 175
from the sieve elements. We further demonstrate that the PAD4 gene is a critical downstream 176
component of this ADF3-dependent defense mechanism. 177
178
RESULTS 179
ADF3 is Required for Limiting GPA Infestation on Arabidopsis 180
To determine if the ADF genes influence infestation of Arabidopsis by the GPA, 181
Arabidopsis lines that were previously shown to lack or accumulate reduced levels of the ADF1 182
(At3g46010), ADF2 (At3g46000), ADF3 (At5g59880), ADF4 (At5g59890), ADF5 (At2g16700) 183
and ADF9 (At4g34970) transcripts (Clément et al., 2009; Tian et al., 2009) were utilized in no-184
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choice bioassays with the GPA. For the no-choice bioassay, twenty adult apterous asexually 185
reproducing insects were released on each plant and the insect population size (adults + nymphs) 186
determined two days post infestation (dpi). As shown in Figure 1A and 1B, only plants lacking 187
ADF3 function repeatedly showed GPA numbers that were significantly higher than GPA 188
numbers on the wild-type (WT) plant. Compared to the WT plant, the GPA population was 189
significantly larger (P < 0.05) on the adf3-1 (Salk_139265) and adf3-2 (SAIL_501_F01) 190
mutants. adf3-1 and adf3-2 contain T-DNA insertions within the first intron and last exon of 191
ADF3, respectively (Supplemental Fig. S1A), which is associated with reduced accumulation of 192
the ADF3 transcript in these mutant lines compared to the WT (Supplemental Fig. S1B). The 193
increase in GPA population on the adf3-1 mutant correlated with the significantly higher 194
fecundity of GPA on the mutant compared to the WT (Fig. 1C). Compared to an average of 1.2 195
nymphs/day produced by a GPA on the WT plant, 1.9 nymphs/day were produced on the adf3-1 196
mutant. 197
To confirm that loss of ADF3 function is indeed responsible for the better performance of 198
the GPA on the adf3 plants compared to the WT plants, a genomic clone of ADF3 was 199
transformed into the adf3-1 mutant. As shown in Figure 1D, ADF3 expression was restored in 200
two independently-derived gADF3 transgenic lines. In comparison to the adf3-1 mutant, the 201
GPA population size was significantly lower on these gADF3 transgenic lines and comparable to 202
that on the WT plants (Fig. 1D). Similarly, resistance was restored in adf3-1 plants expressing 203
ADF3 from the heterologous Cauliflower mosaic virus 35S gene promoter. The GPA population 204
size on two independently-derived 35Spro:ADF3 (in adf3-1 background) lines was significantly 205
smaller than that on the adf3-1 mutant, and comparable to that on the WT plant (Fig. 1E and 206
Supplemental Fig. S2). Taken together, these results confirm that ADF3 has a critical role in 207
limiting GPA infestation on Arabidopsis. 208
209
Actin Cytoskeleton Destabilizers Restore Resistance to the adf3 Mutant 210
ADF3 is an actin-binding protein that was recently shown to possess F-actin-211
severing/depolymerizing activity (Nan et al., 2017). F-actin depolymerization assays conducted 212
by us confirm the ability of ADF3 to depolymerize F-actin (Supplementary Fig. S3). To 213
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determine if ADF3-dependent actin reorganization is critical for controlling GPA infestation, the 214
ability of the actin cytoskeleton destabilizers cytochalasin D and latrunculin B to compensate for 215
ADF3 deficiency in the adf3-1 mutant was evaluated. Two adult aphids were caged onto 216
cytochalasin D- and latrunculin B-treated, and as control on dimethyl sulfoxide (DMSO)-treated 217
leaves of adf3-1 and WT plants, and the number of nymphs born per adult aphid monitored two 218
days later. As expected, significantly larger number of nymphs were born on the DMSO-treated 219
adf3-1 compared to the WT plant (Fig. 2A). However, compared to DMSO treatment, 220
significantly fewer nymphs were born on adf3-1 leaves that were treated with cytochalasin D and 221
latrunculin B. The number of nymphs born on the cytochalasin D- and latrunculin B-treated 222
adf3-1 leaves was comparable to that observed on the cytochalasin D- and latrunculin B-treated 223
leaves of WT plants, thus indicating that these actin destabilizers compensate for the lack of 224
ADF3 function in the adf3-1 mutant. Taken together, these results confirm the importance of 225
ADF3’s actin-depolymerizing function in Arabidopsis defense against the GPA. 226
To further test the involvement of ADF3’s actin-depolymerizing function in controlling 227
GPA infestation, we tested if the adf3-1 defect could be compensated by overexpression of 228
another actin depolymerizing factor, ADF4 (Henty et al., 2011; Henty-Ridilla et al., 2014; Nan et 229
al., 2017), which exhibits 83% identity and 91% similarity to ADF3 (Supplemental Fig. S4). A 230
previously described 35Spro:ADF4 construct (Henty-Ridilla et al., 2014), in which the ADF4 231
protein coding sequence is expressed from the 35S promoter, was transformed into the adf3-1 232
mutant. No choice assays were conducted on three independently-derived adf3-1 35Spro:ADF4 233
lines, and as a control on the WT and adf3-1 mutant. As shown in Figure 2B, GPA population 234
size was comparable on the WT and the adf3-1 35Spro:ADF4 plants, and significantly lower than 235
that on the adf3-1 mutant, thus confirming the ability of ADF4 overexpression to overcome the 236
adf3-1 defect in limiting GPA infestation. Collectively, the above results in conjunction with the 237
studies of Nan et al. (2017) lead us to conclude that the impact of ADF3 on controlling GPA 238
population size is linked to ADF3’s function in actin cytoskeleton reorganization. 239
240
ADF3 Expression in the Phloem is Required for Controlling GPA Infestation 241
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ADF3 expression was reported to be the strongest amongst the ADF family of genes in 242
Arabidopsis (Ruzicka et al., 2007). Gene expression data available on the Arabidopsis eFP 243
Browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi) revealed that ADF3 is expressed 244
throughout the development of Arabidopsis in most organs, except pollen. Furthermore, biotic 245
stress, including GPA infestation did not have a pronounced effect on ADF3 expression 246
(http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Real-time RT-PCR analysis confirmed 247
that ADF3 expression was not significantly different between uninfested and GPA-infested 248
plants (Fig. 3A). Histochemical analysis for GUS activity in leaves of transgenic ADF3pro:UidA 249
plants in which the bacterial UidA-encoded GUS reporter was expressed from the ADF3 250
promoter, further confirmed that ADF3 promoter activity was comparable between uninfested 251
and GPA-infested leaves (Supplemental Fig. S5). ADF3 promoter activity was strong in the 252
lateral and minor veins compared to the non-vascular tissues (Fig. 3B and Supplemental Fig. S5). 253
Within the vasculature, GUS activity was found to be strong in the phloem (Fig. 3C). 254
To test if ADF3 expression in phloem is important for ADF3’s role in controlling GPA 255
infestation, we tested if the ADF3 coding sequence expressed from the phloem-specific SUC2 256
promoter (Gottwald et al., 2000) was sufficient to restore resistance against the GPA in the adf3-257
1 mutant background. Transgenic adf3-1 plants expressing the ADF3 coding sequence from the 258
35S promoter provided the positive controls for this experiment. As shown in Figure 3D, the 259
SUC2pro:ADF3 chimera complemented the adf3-1 defect. GPA population size was significantly 260
lower on three independently-derived adf3-1 SUC2pro:ADF3 lines compared to the adf3-1 261
mutant. The level of resistance observed in the adf3-1 SUC2pro:ADF3 lines was comparable to 262
that observed in adf3-1 35Spro:ADF3 line. There results confirm an important role for ADF3 in 263
the phloem in controlling GPA infestation on Arabidopsis. 264
265
ADF3 is Required for Limiting GPA Feeding From Sieve Elements 266
The adverse effect of ADF3 on insect fecundity could result from its effect on the 267
accumulation of antibiosis activity, or alternatively due to its impact on the insects feeding 268
behavior. Previous studies have indicated the presence of an antibiotic activity, which is 269
detrimental to GPA fecundity, in leaf petiole exudates that are enriched in phloem sap (Louis et 270
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al., 2010a; Nalam et al., 2012). To determine if ADF3 controls the accumulation of this antibiotic 271
activity, petiole exudates collected from leaves of WT, and the adf3-1 and adf3-2 mutants were 272
added to a synthetic diet and GPAs were reared on the supplemented diet for five days. As 273
shown in Figure 4, in comparison to the GPA population on the synthetic diet lacking petiole 274
exudates, insect population size was significantly smaller (P < 0.05) on diet supplemented with 275
petiole exudates from WT plants. The GPA population size was similarly lower on diet 276
supplemented with petiole exudates collected from the adf3-1 and adf3-2 mutant plants, thus 277
confirming that ADF3 does not significantly influence the accumulation of this antibiotic activity 278
in the phloem sap-enriched petiole exudates. 279
To test if ADF3 adversely influences GPA feeding, GPA feeding behavior was monitored 280
on leaves of the WT and adf3-1 mutant plants with the Electrical Penetration Graph (EPG) 281
technique in which the plant and the insect, with a wire glued to its dorsum, are part of a low-282
voltage circuit (van Helden and Tjallingii, 2000). The different waveform patterns in an EPG 283
are characteristic of the different feeding behavioral activities of the insect. EPG provides 284
information on the time the insect takes to first probe (FP) the plant with its stylets, the time 285
spent by the insect to find and tap into a sieve element for the first time (f-SEP; first sieve 286
element phase), and the sum of time spent during the recording period by the insect in all the 287
SEPs (s-SEP), in the xylem phase (XP) when the stylet is in the xylem and the insect is 288
consuming xylem sap, the pathway phase (PP) when the stylet is inserted into the leaf tissue but 289
is outside the sieve elements and likely sampling other cells, and the non-probing (NP) phase 290
when the stylet is not inserted into the plant tissue. As shown in Figure 5A, comparison of these 291
behavioral activities of the GPA on the WT and the adf3-1 mutant revealed that the insect spent 292
significantly less time (P < 0.05) attaining the f-SEP on the adf3-1 mutant. In addition, the s-293
SEP was significantly longer (P < 0.05) when the insects were on the adf3-1 mutant compared to 294
the WT, thus indicating that the insects spent significantly more time feeding from the sieve 295
elements of the adf3-1 mutant than the WT plant. A corresponding reduction in the time spent 296
by GPA in PP was observed on the adf3-1 mutant compared to the WT plant, thus confirming 297
that the insect encounters fewer obstacles that allow it to find sieve elements faster and feed for 298
longer periods on the adf3-1 mutant. In contrast to f-SEP, s-SEP and PP, the insect took 299
comparable amount of time to reach the FP, and spent comparable time in the NP and XP on the 300
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WT and adf3-1 mutant. These results suggest that ADF3 and/or an ADF3-dependent 301
mechanism(s) interferes with the ability of the GPA to find and feed from the sieve elements. 302
During the SEP, the watery saliva injected by the insect into the sieve elements may 303
inhibit phloem-sealing mechanisms (Powell et al., 2006; Tjallingii, 2006; Will and van Bel, 304
2006). This watery saliva injection phase, the E1 phase, is followed by the E2 phloem sap-305
ingestion phase. A prolonged E1 and/or reduced E2 is suggestive of a lowered ability of the 306
insect to suppress rapid phloem-sealing mechanisms (Garzo et al., 2002). To determine if insects 307
feeding from sieve elements of the adf3 mutant encounter less resistance that results in a shorter 308
duration of watery saliva release into the sieve elements, the length of the E1 waveform in the 309
first SEP was compared between insects released on the WT and the adf3-1 mutant. As shown in 310
Figure 5B, the length of the E1 phase was comparable between insects placed on the WT and 311
adf3-1 mutant, thus indicating that ADF3 does not influence the length of the period of watery 312
saliva release by GPA into Arabidopsis sieve elements. However, the E2 phase was significantly 313
shorter (P < 0.05) on the WT compared to the adf3-1, thus confirming that ADF3 and/or an 314
ADF3-dependent mechanism in the WT plant limits ingestion of phloem sap by the GPA. 315
316
ADF3 Controls Callose Deposition in GPA-infested Plants 317
Callose deposition is one of the processes involved in phloem occlusion (Will and van 318
Bel, 2006; Kempema et al., 2007; Hao et al., 2008). Similarly, callose deposited outside cells that 319
are in the path of the stylets trying to reach the sieve element, could interfere with the ability of 320
the stylets to reach sieve elements. Considering that ADF3 is required for limiting GPA feeding, 321
and callose synthesis is subject to the participation of the actin cytoskeleton (Cai et al., 2010), we 322
tested the impact of ADF3 on callose deposition in GPA-infested plants. As shown in Figure 6, 323
in response to GPA infestation a significant increase in the number of callose spots was observed 324
in WT plants. In comparison to the WT, the number of callose deposits were lower in the GPA-325
infested leaves of adf3-1 mutant. The number of callose spots in the GPA-infested adf3-1 mutant 326
was comparable to that observed in the GPA-infested pad4-1 mutant (Fig. 6), which like adf3-1, 327
is also defective in controlling GPA feeding from the sieve elements (Pegadaraju et al., 2007; 328
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Louis et al., 2012), thus further relating callose deposition with the ability of the plant to control 329
GPA feeding. 330
331
An ADF3-dependent Mechanism Controls PAD4 Expression in GPA-infested Plants 332
In the nucleus, actin is a component of the chromatin remodeling complexes that function 333
to regulate gene expression (Jockusch et al., 2006; Farrants, 2008). Furthermore, some ADFs are 334
involved in shuttling actin into the nucleus and thus in regulating gene expression (Nebl et al., 335
1996; Jiang et al., 1997). We noted that the upregulation of PAD4 expression, which is observed 336
in the GPA-infested leaves of the WT, was attenuated in the adf3-1 mutant. As shown in Figure 337
7A, in comparison to the significant (P<0.05) increase in PAD4 expression observed in GPA-338
infested compared to uninfested WT plants, in the adf3-1 mutant, PAD4 transcript levels did not 339
exhibit a significant increase in response to GPA infestation. These results suggest that an 340
ADF3-dependent mechanism regulates PAD4 transcript accumulation in GPA-infested WT 341
plants. 342
To further define the relationship between ADF3 and PAD4 in Arabidopsis defense 343
against the GPA, adf3-1 35Spro:PAD4 and pad4-1 35Spro:ADF3 plants in which PAD4 and 344
ADF3, respectively, are expressed from the 35S promoter were generated. adf3-1 35Spro:ADF3 345
plants provided the controls. As expected, ADF3 expression from the 35S promoter restored 346
basal resistance against GPA in the adf3-1 35Spro:ADF3 plants (Fig. 7B and Supplemental Fig. 347
S6). By comparison, in two independently-derived transgenic lines, ADF3 expression from the 348
35S promoter was unable to restore resistance in the absence of a functional PAD4 gene in the 349
pad4-1 35Spro ADF3 plants (Fig. 7B and Supplemental Fig. S6), thus suggesting a critical role 350
for PAD4 in ADF3-dependent defense against the GPA. Moreover, in no-choice assays 351
conducted on plants of two independent adf3-1 35Spro PAD4 lines, GPA population size was 352
comparable to that on the WT plants, but smaller than on the adf3-1 mutant (Fig. 7B and 353
Supplemental Fig. S6), thus indicating that overexpression of PAD4 from the heterologous 35S 354
promoter is sufficient to restore significant level of resistance in the absence of ADF3 function. 355
Collectively, these results reaffirm the importance of ADF3-dependent regulation of PAD4 356
expression in Arabidopsis defense against the GPA. 357
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358
DISCUSSION 359
The actin cytoskeleton and actin-binding proteins are present in the phloem (Schobert et 360
al., 1998, 2000; Kulikova and Puryaseva, 2002; Lin et al., 2009; Rodriguez-Medina, 2011; 361
Fröhlich et al., 2012; Hafke et al., 2013). However, their biological function in the phloem is 362
poorly understood. We provide multiple lines of evidence indicating an important role for ADF3 363
and actin reorganization in Arabidopsis defense against the GPA, particularly in controlling GPA 364
feeding from the sieve elements. (i) Compared to the WT plant, GPA population was larger on 365
adf3 mutant plants. (ii) The adf3 deficiency in controlling GPA infestation was compensated by 366
the actin cytoskeleton destabilizers cytochalasin D and latrunculin B, and by overexpression of 367
actin depolymerizing factor ADF4. (iii) ADF3 was expressed in the phloem and expression of 368
ADF3 from the phloem-specific SUC2 promoter was sufficient to restore resistance against the 369
GPA in the adf3-1 mutant. (iv) In agreement with it functioning in the phloem, ADF3 was 370
required for limiting GPA feeding from the sieve elements. Our results further demonstrate that 371
ADF3’s involvement in controlling GPA infestation is in part mediated via its influence on 372
PAD4 expression and hence defense signaling. 373
EPG analysis indicated that besides limiting GPA feeding from sieve elements, an ADF3-374
dependent mechanism interferes with the ability of the insect stylets to reach the sieve elements. 375
Thus, we propose that an ADF3-dependent process also impacts events occurring prior to the 376
first penetration of a sieve element by the aphid stylets. On their way to finding a sieve element, 377
the stylets penetrate and sample contents of non-vascular cells (Pollard, 1973; Powell et al., 378
2006). These punctures of plant cells by the stylets could stimulate plant defenses. Salivary 379
components that are released into the plant tissue are also known to stimulate host defenses 380
(DeVos and Jander, 2009; Bos et al., 2010). For example, callose deposition increases in 381
response to GPA infestation (Elzinga et al., 2014) as well as application of GPA-derived elicitors 382
(Prince et al., 2014). Callose has been implicated in the control of infestation by insects that feed 383
from the phloem (Will and van Bel, 2006; Kempema et al., 2007; Hao et al., 2008). Callose 384
deposited in the sieve elements interferes with the ability to the aphid to feed from the sieve 385
elements. In addition, callose deposited outside the sieve elements could interfere with the ability 386
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of the stylet to reach sieve elements to further limit insect feeding. The GPA infestation-387
associated increase in callose deposition was attenuated the adf3-1 mutant, suggesting an 388
important role for ADF3 in this process. The actin cytoskeleton is involved in the localization of 389
callose synthases to the cell membrane, presumably due to the involvement of the actin 390
cytoskeleton in vesicular trafficking (Cai et al., 2010). Whether ADF3 is similarly involved in 391
the localization of callose synthase is not known. However, since the GPA infestation-associated 392
increase in callose deposition was also attenuated in the pad4 mutant, we suggest that the impact 393
of ADF3 on callose deposition in response to GPA infestation is likely exerted via ADF3’s 394
engagement of the PAD4 defense signaling pathway. The contribution of PAD4 in promoting 395
callose deposition is further supported by a recent study, which showed that the endophytic 396
Bacillus velezensis-induced resistance against the GPA was accompanied by an increase in 397
callose deposition, which was dependent on PAD4 (Rashid et al., 2017). 398
Actin is a component of chromatin remodeling activities that control gene expression in 399
the nucleus (Jockusch et al., 2006; Farrants, 2008), and ADF’s are involved in shuttling actin 400
into the nucleus (Nebl et al., 1996; Jiang et al., 1997) and in regulating gene expression (Burgos-401
Rivera et al., 2008; Porter et al., 2012). Amongst the subclass I ADFs in Arabidopsis, ADF4’s 402
involvement in effector-triggered immunity is linked to downstream activation of gene 403
expression (Porter et al., 2012). Similarly, our results indicate that ADF3’s involvement in 404
defense against the GPA is linked to the upregulation of PAD4. However, although PAD4 is 405
required for ADF3-conferrred resistance to the GPA, our results also indicate that ADF3 and 406
PAD4 have additional functions in Arabidopsis defense against the GPA that are independent of 407
each other. For example, unlike PAD4, an ADF3-dependent mechanism hinders with the ability 408
of the insect to find sieve elements. In contrast, unlike ADF3, a PAD4-dependent mechanism is 409
required for the accumulation of an antibiosis activity in the vascular sap (Pegadaraju et al., 410
2005, 2007; Louis et al., 2010a). Previous studies have shown that while basal expression of 411
PAD4 contributes to the antibiosis activity, the upregulation of PAD4 expression is associated 412
with the feeding deterrence function of the PAD4-dependent pathway (Pegadaraju et al., 2007; 413
Louis et al., 2010a, 2010b). These conclusions are in agreement with the observations described 414
here that ADF3, which is required for the upregulation of PAD4 expression in response to GPA 415
infestation, but not for the basal expression of PAD4, is required for controlling GPA feeding, 416
but not for the accumulation of the antibiotic activity. It is likely that different cell types are 417
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involved in exerting these effects. For example, the contribution of ADF3 and PAD4 to feeding 418
deterrence is exerted at the level of the sieve elements, while ADF3’s involvement in hindering 419
with the ability of the GPA to find sieve elements is exerted outside the sieve elements. 420
Similarly, PAD4’s contribution to antibiosis is likely exerted in tissues where these antibiotic 421
factors are produced. 422
ADF3 (At5g59880) and ADF4 (At5g59890), which are located in tandem on 423
chromosome 5, are both subclass I ADFs that share high level of sequence identity. Both possess 424
actin-severing/depolymerizing activity (Henty-Ridilla et al., 2014; Nan et al., 2017). However, 425
while ADF3, not ADF4, is essential for defense against the GPA, ADF4, but not ADF3, is 426
required for AvrPphB -triggered immunity against the bacterial pathogen P. syringae (Tian et al., 427
2009). The ability of ADF4 expressed from the strong and ubiquitously expressed 35S promoter 428
to limit GPA population in the adf3-1 mutant background indicates that ADF3 and ADF4 have 429
overlapping biochemical functions. We therefore conclude that the unique biological functions of 430
ADF3 in defense against the GPA is likely determined by differences in the spatial expression 431
pattern and/or overall level of expression of ADF3, compared to ADF4, and likely other subclass 432
I ADF genes. 433
434
CONCLUSION 435
Our results demonstrate that an ADF3-dependent mechanism hinders with the ability of 436
the GPA to find and feed from sieve elements. Considering that ADF3 is required for 437
upregulating PAD4 expression and callose deposition in GPA-infested leaves, we postulate that 438
an ADF3-dependent mechanism is involved in signaling associated with Arabidopsis defense 439
against the GPA. 440
441
MATERIAL AND METHODS 442
Plant and Insect Materials 443
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All plants were cultivated at 22°C under a 14 h light (100 μE m2 s-1) and10 h dark regime 444
on a peat-based soil mix (Fafard #2, fafard.com). The Arabidopsis adf1 (Salk_144459) (Tian et 445
al., 2009), adf2 (RNAi line; Clément et al., 2009), adf3-1 (Salk_139265C) (Tian et al., 2009), 446
adf3-2 (SAIL_501_F01), adf4 (Garlic_823_A11.b.1b.Lb3Fa) (Tian et al., 2009), adf5 447
(Salk_030145C), and adf9 (Salk_056064) (Tian et al., 2009) lines were used in this study. 448
Silencing of ADF2 in the ADF2 RNAi line was induced by treating plants with 0.5% ethanol 449
solution (Clément et al., 2009). 450
A GPA (Kansas State University, Museum of Entomological and Prairie Arthropod 451
Research, voucher specimen 194) colony was reared at 22°C under a 14 h light (100 μE m2 s-1) 452
and10 h dark regime on a 1:1 mixture of commercially available radish (Raphanus sativus) 453
(Early Scarlet Globe) and mustard (Brassica juncea) (Florida Broadleaf). 454
455
No-choice Tests 456
No-choice assays were conducted as previously described (Pegadaraju et al., 2005; Louis 457
et al., 2010a). Unless stated otherwise, 20 adult asexually reproducing apterous (wingless) 458
aphids were placed on each 4-week-old plant. Two days later, the number of insects on each 459
plant was counted. To monitor aphid fecundity, one young nymph (3-4 day old) was placed on 460
each plant and the total number of progeny born over a 10-day period was recorded. 461
462
Monitoring Aphid Feeding Behavior 463
The electrical penetration graph (EPG) technique (van Helden and Tjallingii, 2000; 464
Walker, 2000) was used to simultaneously monitor the feeding behavior of GPA on 4-week-old 465
plants of two different genotypes, as previously described (Pegadaraju et al., 2007). An eight-466
channel GIGA-8 direct current amplifier (http://www.epgsystems.eu/systems.htm) was used for 467
simultaneous recordings of eight individual aphids. The waveform recordings were analyzed 468
using the EPG analysis software PROBE 3.4 (provided by W.F. Tjallingii, Wageningen 469
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University, The Netherlands). The analysis of the first sieve element phase was divided into the 470
length of the first salivation E1 and the time of active ingestion phase E2. 471
472
Petiole Exudate Collection and Artificial Diet Feeding Assays 473
Phloem sap-enriched petiole exudates were collected from Arabidopsis leaves as 474
previously described (Chaturvedi et al., 2008). After concentration, equal volumes of petiole 475
exudates were added to a synthetic diet (Mittler et al., 1965) contained in a feeding chamber 476
(Louis et al., 2010b). Three adult aphids were released on each feeding chamber and the total 477
number of GPA in each feeding chamber was determined 5 days later. 478
479
Actin Cytoskeleton Destabilizer Treatment 480
Two leaves of each 4-week-old plant were infiltrated with 2 μM of Cytochalasin D or 481
Latruculin B solubilized in 0.5% dimethylsulfoxide (DMSO), followed by release of two adult 482
GPA on each leaf. Each leaf was caged in a perforated 2 ml microfuge tube to prevent escape of 483
insects. Total number of nymphs on each leaf were determined two days later. Plants treated 484
with 0.5% DMSO provided the negative control. A minimum of twelve leaves of each genotype 485
were used for each treatment. 486
487
PCR Analysis for Mutant Screening 488
All primers used in this study to confirm the knock-down of individual ADF genes in the 489
adf mutants and the ADF2 RNAi knock-down lines are listed in Supplemental Table S1. PCR 490
with gene-specific primers was performed under the following conditions: 94°C for 5 min, 491
followed by 36 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, with a final 492
extension of 72°C for 5 min. PCR for genotyping the adf mutants utilized the T-DNA left border 493
primer and a gene-specific primer. The PCR conditions included a 5 min denaturation at 94°C, 494
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followed by 36 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, with a final 495
extension of 72°C for 5 min. 496
497
Gene expression 498
RNA extraction from leaves was performed as previously described (Pegadaraju et al., 499
2005). Each sample included a minimum of two leaves. A minimum of three samples were 500
analyzed for each treatment. For Real-time RT-PCR, gene expression levels were normalized to 501
that of EF1α, while for RT-PCR ACT8 was used as a control. Gene-specific primers used for 502
Real-time RT-PCR and RT-PCR are listed in Supplemental Table S1. 503
504
Expression and Purification of Recombinant ADF3 Proteins 505
The primers 5’-CGCCCCATATGGCTAATGCAGCATCAGGAATGGCAGTCC-3’ and 506
5’- AAGCTTCTCGAGTCAATTGGCTCGGCTTTTGAAAAC -3’ were used to amplify the 507
ADF3 coding sequence, using cDNA prepared from mRNA harvested from Arabidopsis Col-0 as 508
the template. The resultant product was digested with NdeI and XhoI, which cut within the two 509
primer regions. The digested product was ligated between the NdeI and XhoI sites of pET28a 510
vector. The resultant pET28a-ADF3 plasmid in which a 6X-His tag was incorporated at the N-511
terminal end of ADF3 was transformed into E. coli strain BL21-DE3. Expression of the 512
recombinant ADF3 protein was induced by the addition of Isopropyl-β-D-thiogalactopyranoside 513
(IPTG) prior to bacterial cell harvest. The recombinant, 6X His-tagged protein was purified over 514
an affinity Ni-NTA column (QIAexpress, Qiagen, http://www.qiagen.com). The purified ADF3 515
protein was centrifuged at 20,000g for 30 min at 4°C before use in the F-actin co-sedimentation 516
assays. The GST-ADF3 and GST-ADF4 clones used in the actin depolymerization assays 517
encode recombinant proteins that contain GST fused to the N-terminal end of ADF3 and ADF4. 518
These were a gift of Brad Day. The recombinant proteins were purified over a GST-affinity 519
column before use in actin depolymerization assays. 520
521
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Transgenic ADF3 and ADF4 Expressing Plants 522
The ADF3 genomic fragment (gADF3) was amplified using the primers 5’-523
TAAATGAATTTTTTTTACGGGA-3’ and 5’-TCAATTGGCTCGGCTTTTGA-3’, and the 524
resultant product cloned into the pCR8/GW/TOPO® vector (Life technologies; 525
www.lifetechnologies.com). Gateway LR clonase® (Life technologies; 526
www.lifetechnologies.com) was used to mobilize the cloned fragment into the binary vector 527
pMDC107 (Curtis and Grossniklaus, 2003) to yield pMDC107-gADF3. To generate a construct 528
for in planta expression of the ADF3 coding sequence from the Cauliflower mosaic virus 35S 529
gene promoter, the pET28a-ADF3 plasmid (see above) was used as a template with the primer 530
5’-ATGGGCAGCAGCCATCATC-3’, which is derived from the pET28a vector and primer 5’-531
TCAATTGGCTCGGCTTTTGA-3’ to amplify the ADF3 coding region. The resultant PCR 532
product was cloned into pCR8/GW/TOPO® from where it was mobilized into the destination 533
vector pMDC32 (Curtis and Grossniklaus, 2003) to yield the pMDC32-35Spro:ADF3 plasmid. 534
The pMDC107-gADF3 and pMDC32-35Spro:ADF3 plasmids were individually 535
electroporated into Agrobacterium tumefaciens strain GV3101, which was subsequently used for 536
transforming the adf3-1 plants by the floral-dip method (Zhang et al., 2006) to generate 537
transgenic plants in which the adf3-1 mutant phenotype was complemented by expression of the 538
gADF3 and 35Spro:ADF3. Transgenic plants were selected on ½ strength MS agar plates 539
containing hygromycin (25 μg ml-1). The 35Spro:PAD4 construct has been previously described 540
(Xing and Chen, 2006). 541
The 35Spro:ADF4 construct (Henty-Ridilla et al., 2014), in which the ADF4 coding 542
sequence containing a N-terminal T7 epitope tag is expressed from the 35S promoter was used for 543
generating the adf3-1 35Spro:ADF4 plants by transforming adf3-1 plants with the recombinant 544
construct. Transformed plants were screened based on their resistance to kanamycin (50 μg ml-1) 545
and the presence of the recombinant construct and its expression confirmed by PCR and RT-PCR, 546
respectively. To verify the construct in the adf3-1 35S:ADF4 transgenic lines, a forward primer 547
5’-GGTGGTCAACAAATGGGT- 3’ that is specific to the region containing the T7 tag and a 548
reverse primer 5’-TTAGTTGACGCGGCTTTTC- 3’, which is specific to the ADF4 coding 549
sequence were utilized. The same primers were also utilized to monitor expression of the 550
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21
recombinant constructed by RT-PCR. The PCR conditions were as follows: 94°C for 5 min, 551
followed by 30 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, followed by a 552
final extension step of 72°C for 5 min. 553
To generate a SUC2pro:ADF3 construct, the ADF3 coding sequence with a 6X-His tag at 554
the N-terminus was amplified from plants containing the gADF3 construct which contains a His 555
tag on the 5' end. The primers 5’- ATGGGCAGCAGCCATCATC -3’ and 5’-TCAATTG 556
GCTCGGCTTTTGA-3’ were used to amplify the ADF3 coding sequence and the resultant PCR 557
product was cloned into pCR8/GW/TOPO®. The pCR8 product was then mobilized into a 558
pMDC85 destination vector (Curtis and Grossniklaus, 2003) which, had been modified by the 559
removal of the 2x 35S promoter and replaced by the SUC2 promoter (Elzinga et al., 2014). The 560
resultant construct, pMDC85-SUC2pro:ADF3 was transformed into adf3-1 using Agrobacterium 561
tumefaciens via the floral-dip method (Zhang et al., 2006). Transformed plants were screened 562
based on resistance to hygromycin (20 μg ml-1) and presence of the recombinant construct and its 563
expression confirmed by PCR and RT-PCR, respectively. A forward primer 5’-564
AGCCATCATCATCATCATCAC-3’ designed to the 6X-HIS tag at the 5’ end of the ADF3 565
coding sequence and a reverse primer 5’-TCAATTG GCTCGGCTTTTG-3’ that is specific to 566
the ADF3 coding sequence were utilized in PCR reactions to confirm the presence of the 567
SUC2pro:ADF3 construct. These same primers were also utilized to monitor expression of the 568
recombinant construct by RT-PCR. The PCR conditions were as follows: 94°C for 5 min, 569
followed by 30 cycles of 94°C for 30 sec, 57°C for 30 sec and 72°C for 45 sec, followed by a 570
final extension step of 72°C for 5 min. 571
572
GUS Reporter Constructs and Histochemical Analysis 573
A 2035 bp DNA fragment upstream of the transcriptional start site of ADF3 was 574
amplified with Platinum® Taq polymerase (Invitrogen) using Arabidopsis Col-0 genomic DNA 575
as template and the primers 5’-TAAATGAATTTTTTTTACGGGA-3’ and 5’- 576
GGTTGAATCAAAGCTAGTCTCA-3’. The PCR product was cloned into pCR8/GW/TOPO 577
from where it was mobilized into the pMDC132 vector (Curtis and Grossniklaus, 2003), which 578
contains the coding sequence of the bacterial UidA gene, which encodes the GUS protein 579
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(Jefferson et al., 1987). Transgenic plants containing the ADF3pro:UidA construct were 580
generated in the accession Col-0 background. Histochemical analysis of GUS activity was 581
performed using X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucopyranosiduronic acid) as the 582
substrate. 583
584
Callose Staining 585
Callose staining and quantification of callose deposits was conducted using a modification 610
of a previously described protocol (Ton and Mauch-Mani, 2004). Briefly, leaf samples were 611
cleared overnight in 96% ethanol, followed by a 30 min incubation in sodium phosphate buffer 612
(0.07 M, pH 9). Leaves were then incubated for 60 min in a solution containing 0.005% aniline 613
blue in sodium phosphate buffer (0.07 M, pH 9), followed by the addition of calcofluor to a final 614
concentration of 0.005%. The tissues were immediately observed under a Nikon e600 615
epifluorescence microscope equipped with a X-cite 120 Fluor System, UV (DAPI) filter and a 616
digital SPOT color camera. Digital images were used to quantify callose spots, which were 617
expressed as number of callose spots per mm2 of leaf tissue. 618
619
Actin Depolymerization Assays 620
Rabbit skeletal muscle G-actin was prepared by the method of Spudich and Watt (1971) 621
and flash frozen in liquid nitrogen in 0.5 mL aliquots for storage at -70°C until use. After rapid 622
thawing, the G-actin was chromatographed on a superdex 75 gel filtration column immediately 623
prior to measurements. Concentrations of G-actin were determined using an extinction 624
coefficient of 0.63 mg/ml/cm at 290 nm. A portion of the actin was labeled with pyrene 625
iodoacetamide by the method of Cooper et al. (1983) and stored by the same method as the 626
unlabeled G-actin. 627
Measurements of pyrene-labeled actin were performed with an Aminco-Bowman II 628
luminescence spectrometer using methods similar to those previously described (Xu and Root 629
2000). Briefly, 15% pyrene-labeled actin was polymerized by the addition of 0.1 M KCl and 2 630
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23
mM MgCl2 yielding a 20-fold enhancement of pyrene fluorescence intensity. Actin 631
concentrations were tested in the ranges of 3 to 7.1 μM for separate experiments. The 632
polymerized actin was titrated at 25°C with a range of concentrations of recombinant GST-633
tagged ADF3 and ADF4 or buffer as a control, and changes in fluorescence intensity measured. 634
Control experiments indicated a reproducibility of ±10% error or better. 635
636
Statistical Analysis 637
When comparing two treatments or genotypes, 2-tail t-test was used to determine if the 638
mean values were significantly different from each other (P<0.05). When simultaneously 639
comparing multiple genotypes and/or treatments to each other, analysis of variance (ANOVA) 640
performed following the General Linear Model GLM was used followed by Tukey’s multiple 641
comparison test to identify mean values that were significantly different from each other 642
(P<0.05) (Minitab v15; www.minitab.com). When comparing multiple experimental groups to a 643
single control group, Dunnett’s multiple comparison test was used to compare means to identify 644
values that were significantly different (P<0.05) from the control group. All data conform to the 645
assumptions of ANOVA and no transformations were necessary. For the EPG analysis, the 646
nonparametric Kruskal-Wallis test (Minitab v15; www.minitab.com) was used to analyze the 647
mean time spent by aphids on various activities. 648
649
Arabidopsis Gene Accession Numbers 650
ADF1 (At3g46010); ADF2 (At3g46000); ADF3 (At5g59880); ADF4 (At5g59890); ADF5 651
(At2g16700); ADF9 (At4g34970); ACT8 (At1g49240); EF1α (At5g60390); PAD4 (At3g52430). 652
653
ACKNOWLEDGEMENTS 654
We thank the Arabidopsis Biological Resource Center for Arabidopsis seeds, Brad Day 655
for adf1, adf3-1, adf4, aadf5 and adf9 mutant lines, the GST-ADF3, GST-ADF4 and 35S:ADF4 656
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24
clones, Janice de Almeida Engler for the ADF2-RNAi line, Zhixiang Chen for the 35Spro:PAD4 657
construct, and Jane Parker and Bart Feys for discussion and making available unpublished 658
results. 659
660
661
SUPPLEMENTAL DATA 662 The following materials are available in the online version of this article. 663
Supplemental Table S1. Primers used for monitoring gene expression. 664
Supplemental Figure S1. Arabidopsis lines carrying T-DNA insertions at the ADF3 locus. 665
Supplemental Figure S2. ADF3 expression from the 35S promoter restores resistance against 666
the GPA in the adf3-1 mutant. 667
Supplemental Figure S3. ADF3 exhibits actin depolymerizing activity. 668
Supplemental Figure S4. Alignment of ADF3 and ADF4 669
Supplemental Figure S5. Histochemical staining for GUS activity in uninfested (-GPA) and 670
GPA-infested (+GPA) leaves of a ADF3pro:UidA plant. 671
Supplemental Figure S6. Expression of PAD4 from the constitutively expressed 35S promoter 672
restores resistance against GPA in plants lacking ADF3 function. 673
674
675
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FIGURE LEGENDS 676
677
Figure 1. Arabidopsis ADF3 gene is required for limiting GPA infestation. A, GPA population 678
size on Arabidopsis wild-type (WT) accession Col-0 and adf1 (a1), adf2 (a2), adf3-1 (a3-1), 679
adf4 (a4), adf5 (a5) and adf9 (a9) mutants (n=10) two days post infestation. B, GPA population 680
size on WT accession Col-0 and adf3-1 (a3-1) and adf3-2 (a3-2) mutant plants (n=10) two days 681
post infestation. C, One nymph was released on each WT and a3-1 plant and the number of 682
progeny nymphs produced by each insect over a 10 day period determined (n=6). D, GPA 683
population size on WT accession Col-0, a3-1 mutant, and two independently-derived gADF3 684
plants in which a genomic clone of ADF3 was transformed into the a3-1 mutant (n= 6). Top 685
panel shows RT-PCR analysis of ADF3 and as a control ACT8 expression in uninfested plants of 686
indicated genotypes. E, GPA population size on WT, adf3-1 and a transgenic 35Spro:ADF3 line 687
#1 in which the ADF3 coding sequence was expressed from the heterologous 35S promoter 688
(n=6). See Supplemental Fig. S2 for results from an independently derived 35Spro:ADF3 line. 689
In A, B, D and E, twenty adult apterous aphids were released on each plant and the population 690
size (adults + nymphs) on each plant determined two days later. All values are mean + SE for 691
each genotype. In A and B, asterisks above bars denote values that are significantly different 692
from the WT (P < 0.05; Dunnett test). In C, an asterisk denotes significant difference from the 693
WT (P < 0.05; t-test). In D and E, different letters above bars denote values that are significantly 694
different from each other (P < 0.05; Tukey test). 695
696
697
Figure 2. Actin cytoskeleton destabilizers restore resistance to GPA in the adf3 mutant. 698
A, Effect of the actin destabilizers cytochalasin D (CytD, left panel) and latrunculin B (LatB, 699
right panel) on insect numbers. Number of nymphs on leaves of WT and adf3-1 (a3-1) mutant 700
treated with 2 μM CytD or LatB, and as control with DMSO (0.5%), which was used as a solvent 701
for CytD and LatB. The number of nymphs per adult was determined two days after release of 702
two adult aphids on each leaf (n=12). B, Complementation of adf3-1-conferred susceptibility to 703
GPA by constitutive expression of ADF4 from the 35S promoter. Upper panel: RT-PCR analysis 704
of ADF4 expression from the 35S promoter in three independent a3-1 35SPpro:ADF4 lines, and 705
as control in WT and a3-1 plants. EF1α expression provided the control for RT-PCR. Lower 706
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26
panel: No choice assay comparing aphid numbers two days post release of 20 adult aphids per 707
leaf on the WT, a3-1 and three independently-derived a3-1 35Spro:ADF4 transgenic lines. 708
Values are mean + SE of a minimum of six replicates for each genotype. Different letters above 709
bars denote values that are significantly different from each other (P < 0.05; Tukey test). 710
711
Figure 3. ADF3 function in the phloem is required for controlling GPA infestation. A, Real-712
time RT-PCR analysis of ADF3 expression relative to that of EF1α in uninfested (-GPA) and 713
GPA-infested (+GPA) leaves of the Arabidopsis accession Col-0 at the indicated hours post 714
infestation (hpi). Values are mean + SE (n = 3). B, Histochemical staining for GUS activity 715
(blue color) in a GPA-infested leaf of a ADF3pro:UidA plant in which UidA expression is driven 716
from the ADF3 promoter. Right panel shows a close-up showing strong GUS activity in the 717
veins. C, Histochemical staining in a section through a vein showing strong GUS activity in the 718
phloem (P). X, xylem. D, ADF3 expression in the phloem is sufficient for controlling GPA 719
infestation. Upper panel: RT-PCR analysis of ADF3 expression from the SUC2 promoter in 720
three independent a3-1 SUC2Pro:ADF3 lines, and as control in wild-type (WT) accession Col-0, 721
a3-1 and a3-1 35Spro:ADF3 plants. Primers specific for the SUC2 promoter-driven ADF3 722
construct were used. EF1α expression provided the control for RT-PCR. Lower panel: GPA 723
population size on Arabidopsis plants of indicated genotypes. Twenty adult apterous aphids 724
were released on each plant and the population size (adults + nymphs) on each plant determined 725
two days later. Values are mean + SE of a minimum of six replicates for each genotype. 726
Different letters above bars denote values that are significantly different from each other (P < 727
0.05; Tukey test). 728
729
730
Figure 4. ADF3 is not required for accumulation of antibiosis activity in petiole exudates. 731
Artificial diet assay showing a comparison of GPA numbers on diet containing petiole exudates 732
(Pet-ex) collected from leaves of wild-type (WT), adf3-1 (a3-1) and adf3-2 (a3-2) plants. 733
Control was an artificial diet supplemented with the buffer used to collect petiole exudates. 734
Values are mean + SE of a minimum of seven replicates for each treatment. Different letters 735
above bars denote values that are significantly different from each other (P < 0.05; Tukey test). 736
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27
Figure 5. ADF3 is required for controlling GPA feeding on Arabidopsis leaves. A, Electrical 737
penetration graph (EPG) comparison of time spent by GPA in various activities on the WT and 738
adf3-1 (a3-1) mutant plants in an 8 h period. FP: time taken for first probe; f-SEP: time taken to 739
reach first sieve element phase; PP: time spent in pathway phase during the 8 h period; NP: total 740
time spent non-probing during the 8 h period; s-SEP: total time spent in sieve element phase 741
during the 8 h period; XP: total time spent in the xylem phase during the 8 h period. B, EPG 742
comparison of time spent by GPA in the E1 (salivation) and E2 (ingestion) phases during the 743
first SEP. In A and B, values are the mean + SE (n=10). Asterisks indicate significant 744
differences (P < 0.05; Kruskal-Wallis test) in an individual feeding parameter between insect 745
feeding on the a3-1 compared to the WT plant. 746
747
Figure 6. ADF3 is required for promoting callose deposition in response to GPA infestation. 748
Upper panel: Callose deposits (light blue spots) in leaves of wild-type (WT) plants of the 749
Arabidopsis accession Col-0 and the adf3-1 (a3-1) mutant, 24 hpi with the GPA (+GPA) or as 750
control in uninfested (-GPA) plants. Bar = 200 μM. Lower panel: Mean number of callose spots 751
+ SE per mm2 of leaf tissue in GPA-infested and uninfested leaves (n=5). Different letters above 752
bars denote values that are significantly different from each other (P < 0.05; Tukey test). 753
754
Figure 7. ADF3 is required for promoting PAD4 expression in response to GPA infestation. A, 755
qRT-PCR analysis of PAD4 expression relative to EF1a expression at 24 hpi in GPA-infested 756
and uninfested WT and adf3-1 (a3-1) mutant plants. Values are mean + SE (n=3). Asterisks 757
indicate values that are significantly different from the corresponding mock-treatment (P < 0.05; 758
t-test). B, GPA population size on WT, a3-1, a3-1 35Spro:ADF3 line #1, a3-1 35Spro:PAD4 line 759
#1, pad4-1, and pad4-1 3Spro:ADF3 line #1. See Supplemental Fig. S6 for data for additional 760
independently-derived transgenic lines. The no-choice assay was conducted by releasing twenty 761
adult apterous aphids on each plant and the population size (adults + nymphs) on each plant 762
determined two days later. Values are mean + SE (n=6) for each genotype. Different letters 763
above bars denote values that are significantly different from each other (P < 0.05; Tukey test). 764
765
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Mondal et al. Fig. 1
A
a3-1 a4a1 a2WT a9a5
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ymph
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Mondal et al. Fig. 2
A
02468
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CytD
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Mondal et al. Fig. 3
B
C
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X
A
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0.2
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Mondal et al. Fig. 4
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Mondal et al. Fig. 5
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Mondal et al. Fig. 7
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