Journal of Experimental Botany, Vol. 66, No. 2 pp. 559–570, 2015
doi:10.1093/jxb/eru472 Advance Access publication 11 December, 2014
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Research Paper
The ethylene response factor Pti5 contributes to potato
aphid resistance in tomato independent of ethylene signalling
Chengjun Wu1,*,†, Carlos A. Avila1,2,† and Fiona L. Goggin1,‡
1 2 Department of Entomology, University of Arkansas, Fayetteville, AR 72701, USA
Department of Horticultural Sciences, Texas A&M AgriLife Research, Weslaco, TX 78596, USA
* Present address: Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA.
These authors contributed equally to this work.
‡ To whom correspondence should be addressed. E-mail: [email protected]
† Received 28 August 2014; Revised 23 October 2014; Accepted 28 October 2014
Abstract
Ethylene response factors (ERFs) comprise a large family of transcription factors that regulate numerous biological
processes including growth, development, and response to environmental stresses. Here, we report that Pti5, an
ERF in tomato [Solanum lycopersicum (Linnaeus)] was transcriptionally upregulated in response to the potato aphid
Macrosiphum euphorbiae (Thomas), and contributed to plant defences that limited the population growth of this
phloem-feeding insect. Virus-induced gene silencing of Pti5 enhanced aphid population growth on tomato, both on an
aphid-susceptible cultivar and on a near-isogenic genotype that carried the Mi-1.2 resistance (R) gene. These results
indicate that Pti5 contributes to basal resistance in susceptible plants and also can synergize with other R gene-mediated defences to limit aphid survival and reproduction. Although Pti5 contains the ERF motif, induction of this gene
by aphids was independent of ethylene, since the ACC deaminase (ACD) transgene, which inhibits ethylene synthesis,
did not diminish the responsiveness of Pti5 to aphid infestation. Furthermore, experiments with inhibitors of ethylene
synthesis revealed that Pti5 and ethylene have distinctly different roles in plant responses to aphids. Whereas Pti5
contributed to antibiotic plant defences that limited aphid survival and reproduction on both resistant (Mi-1.2+) and
susceptible (Mi-1.2–) genotypes, ethylene signalling promoted aphid infestation on susceptible plants but contributed
to antixenotic defences that deterred the early stages of aphid host selection on resistant plants. These findings suggest that the antixenotic defences that inhibit aphid settling and the antibiotic defences that depress fecundity and
promote mortality are regulated through different signalling pathways.
Key words: Basal resistance, ERF, EREBP, insect resistance, Macrosiphum euphorbiae, Mi-1.2.
Introduction
Aphids are a large and economically damaging group of
insects that are specialized to feed on phloem sap and that
can reduce crop yields by withdrawing photoassimilates,
manipulating plant growth and resource allocation, and
transmitting phytopathogenic viruses (Goggin, 2007). Plants
limit losses to aphids through a variety of defences, including sensory cues that deter aphids from settling on the plant
(i.e. antixenosis), and toxins or other resistance factors that
suppress aphid fecundity and promote mortality (i.e. antibiosis) (Smith and Chuang, 2014). In some cases, host plant
resistance depends upon single dominant resistance genes
(so-called R genes) that act against specific pest biotypes,
resulting in an incompatible interaction. For example, the
Mi-1.2 gene in tomato [Solanum lycopersicum (Linnaeus)],
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACD, ACC deaminase; ANOVA, analysis of variance; AVG, aminoethoxyvinylglycine; DAI, days after
infestation; EREBP, ethylene-responsive element binding protein; ERF, ethylene response factor; HAI, h after infestation; JA, jasmonic acid; PR, pathogenesisrelated; R, resistance; RT-qPCR, real-time quantitative PCR; SA, salicylic acid; TRV, tobacco rattle virus; VIGS, virus-induced gene silencing; WT, wild type.
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
560 | Wu et al.
blocks infestation by some but not all populations of the
potato aphid, Macrosiphum euphorbiae (Thomas) (Rossi
et al., 1998; Goggin et al., 2001). However, even in compatible
interactions in which relevant R genes are absent and aphids
can colonize the host, plants deploy defences that reduce the
magnitude of infestations. These defences, present in compatible or ‘susceptible’ hosts, are described as basal resistance and provide a foundation upon which R gene-mediated
resistance builds. Basal resistance encompasses not only
direct defences with toxic or repellent effects on the pest but
also any plant traits that intercept the chain of events leading
to susceptibility (Dangl and Jones, 2001; de Wit, 2007; Niks
and Marcel, 2009; Anderson et al., 2014). Both basal and R
gene-mediated resistance involve induced responses including
hormone signalling and transcriptional reprogramming, and
there is often extensive overlap in the signalling networks and
transcription factors that contribute to these two forms of
defence (Navarro et al., 2004).
This study explored the role of the Pti5 transcription factor
in basal and Mi-1.2-mediated aphid resistance tomato, as well
as the potential interactions of this ethylene response factor (ERF) with the hormone ethylene. Ethylene is a gaseous
hormone that regulates plant responses to numerous biotic
and abiotic stimuli including mechanical wounding, chewing insects, and pathogen infection (O’Donnell et al., 1996;
Knoester et al., 1998; Solano and Ecker, 1998; von Dahl and
Baldwin, 2007; Mantelin et al., 2009). Ethylene signalling
activates the transcription of numerous pathogenesis-related
(PR) proteins and other genes that contribute to defence,
and many of these ethylene-inducible genes contain a conserved 11 bp promoter sequence (TAAGAGCCGCC) called
the GCC box (Eyal et al., 1993). ERFs, formerly known as
ethylene-responsive element binding proteins (EREBPs), are
transcription factors that specifically bind to genes having
the GCC box. The ERF family has important roles in regulating biological processes related to plant growth, development, metabolism, and response to biotic and abiotic stresses
(Ohme-Takagi and Shinshi, 1995; Stockinger et al., 1997;
Liu et al., 1998; Yamamoto et al., 1999; Gu et al., 2000; van
der Fits and Memelink, 2000; Berrocal-Lobo et al., 2002;
Nakano et al., 2006). Ethylene may regulate PR gene expression through ERF transcription factors (Eyal et al., 1993;
Ohme-Takagi and Shinshi, 1995; Solano et al., 1998), and
ERFs can act as either transcriptional activators or repressors of GCC box-mediated gene expression (Fujimoto et al.,
2000; Yang et al., 2005).
ERFs have been characterized in several model plants
including Arabidopsis thaliana (Büttner and Singh, 1997;
Solano et al., 1998; Fujimoto et al., 2000; Nakano et al.,
2006), tomato (Zhou et al., 1997), Oryza sativa (Nakano
et al., 2006), and Nicotiana tobaccum (Ohme-Takagi and
Shinshi, 1995; Suzuki et al., 1998). In tomato, the Pti5 transcription factor containing the ERF binding domain was isolated based on its physical interaction with a serine–threonine
kinase encoded by the Pto gene (Pti=Pto interacting) (Zhou
et al., 1997). Pto acts in conjunction with the Prf R gene to
confer resistance against certain races of the bacterial pathogen Pseudomonas syringae pv. tomato (Salmeron et al., 1996;
Pedley and Martin, 2003). Pti5 acts downstream of Pto and
Prf to upregulate expression of defence genes including the
GCC box-containing PR genes encoding β-1,3-glucanase
and osmotin (He et al., 2001). Furthermore, overexpression
of Pti5 in a susceptible tomato background enhances basal
resistance to P. syringae pv. tomato, indicating that Pti5 contributes to pathogen resistance (He et al., 2001). Analysis of
transcriptional responses in melon (Cucumis melo) to infestation by the cotton-melon aphid (Aphis gossypii) also indicates that aphid feeding upregulates ERFs, and that the Vat
gene for aphid resistance may enhance this response (Anstead
et al., 2010). Thus, ERFs could potentially play a role in R
gene-mediated aphid resistance.
While the impact of ERFs on aphid resistance has not
been tested previously, several earlier studies have examined
the role of ethylene in plant–aphid interactions, and aphids
have been shown to induce an ethylene burst in several different plant species (Dillwith et al., 1991; Anderson and Peters,
1994; Miller et al., 1994; Argandona et al., 2001; Mantelin
et al., 2009). In barley, this ethylene burst was reported to
be greater in an aphid-resistant genotype than in a susceptible cultivar, suggesting that ethylene may be associated with
resistance (Argandona et al., 2001). However, the opposite
trend was observed in alfalfa plants challenged with the spotted alfalfa aphid (Therioaphis maculata) and wheat plants
challenged with the Russian wheat aphid (Diuraphis noxia);
in these cases, ethylene generation in response to aphids was
greater in susceptible genotypes than in resistant or tolerant
lines (Dillwith et al., 1991; Miller et al., 1994). In tomato,
artificially exposing plants to ethylene also rendered them
more susceptible to the green peach aphid, Myzus persicae
(Boughton et al., 2006). Furthermore, Mantelin et al. (2009)
found that inhibiting ethylene synthesis in tomato reduced
host suitability for the potato aphid on a susceptible genotype
but had no effect on a resistant tomato genotype that carries
the Mi-1.2 R gene. These results indicate that ethylene signalling in response to aphids can actually facilitate the infestation process in some cases, but that the effects of ethylene
vary among genotypes.
In this study, we investigated the role of the ERF Pti5 in
plant–aphid interactions in resistant (Mi-1.2+) and susceptible (Mi-1.2–) tomato genotypes, and compared its effects
with those of ethylene. We demonstrated that the potato
aphid causes transcriptional upregulation of Pti5, and that
this gene contributes to antibiotic defences against aphids
in both susceptible and resistant plants. Ethylene was not
required for upregulation of Pti5 by aphids and did not
contribute to antibiosis in either genotype. Therefore, we
concluded that Pti5 contributes to potato aphid resistance
in tomato independent of ethylene signalling. Surprisingly,
we also discovered that, even though ethylene promoted
infestations on susceptible tomato plants, it contributed
to the early stages of antixenotic defences in plants that
carried the Mi-1.2 gene for aphid resistance. These results
indicate that antibiosis and antixenosis depend upon different signalling pathways and further our understanding
of the molecular basis of basal and R gene-mediated aphid
resistance.
Pti5 and ethylene in plant–aphid interactions | 561
Materials and methods
Plant and insect materials
The following tomato (S. lycopersicum L.) genotypes were used
in this study: three aphid-susceptible cultivars that lack the Mi1.2 resistance gene (‘MoneyMaker’, ‘UC82B’, and ‘Castlemart’);
a transgenic line (143-25) that was generated by transforming ‘MoneyMaker’ with Mi-1.2 (Milligan et al., 1998); a cultivar,
‘Motelle’, that is near-isogenic to ‘MoneyMaker’ but that carries
a 650 kb introgressed region containing Mi-1.2 (Kaloshian et al.,
1998); a transgenic line, ACD, that was generated by transforming
the cultivar ‘UC82B’ with a gene from Pseudomonas chloroaphis that
encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase
(ACD; Klee et al., 1991); two mutant tomato lines, acyl-CoA oxidase
1A (acx1) and suppressor of prosystemin-mediated responses2 (spr2),
that were generated in the ‘Castlemart’ genetic background (Li et al.,
2003); and a transgenic line that was generated in a ‘MoneyMaker’
background and that expresses the naphthalene/salicylate hydroxylase (NahG) transgene from Pseudomonas putida (Gaffney et al.,
1993). The aphid-resistant (Mi-1.2+) transgenic line 143-25 was used
in initial gene expression experiments because it provides an isogenic
control to the susceptible (Mi-1.2–) line ‘MoneyMaker’. However,
for subsequent bioassays, ‘Motelle’ (Mi-1.2+) was utilized instead
of 143-25 because levels of Mi-1.2-mediated resistance appear to
decline as the Mi-1.2 transgene is passed through more than one
generation (Goggin et al., 2004).
Tomato plants were grown in 0.2-l pots using LC1 Sunshine potting mix (Sungro Horticulture, MI, USA) supplemented with 15-9-12
Osmocote Plus slow-release fertilizer (Scotts-MiracleGro Company,
OH, USA). Seedlings were grown under uniform greenhouse conditions (24–27 °C and a 16:8 h light:dark photoperiod) until imposition of experimental treatments. Plants were watered with a dilute
nutrient solution containing 1000 ppm CaNO3 (Hydro Agri North
America, FL, USA), 500 ppm MgSO4 (Giles Chemical Corp, NC,
USA), and 500 ppm 4-18-38 Gromore fertilizer (Gromore, CA,
USA). The potato aphid (M. euphorbiae, isolate WU11) was reared
on a combination of tomato (cv. ‘UC82’), potato (Solanum tuberosum L.), and jimson weed (Datura stramonium L.) plants at 23 °C
with a 16 h light photoperiod.
Genetic crosses and genotype selection
The cultivar ‘Motelle’ (MT) and the transgenic line ACD were
crossed to produce MT×ACD plants carrying both the Mi-1.2
gene and the ACD gene. The (MT×ACD) F1 generation was PCR
screened for the presence of both genes using the following sets of
primers: ACD forward, 5ʹ-CGATACGCTGGTTTCCATC-3ʹ, and
reverse, 5ʹ-CGTCCTCTTCCGTAATCTCG-3ʹ; and Mi-1.2 forward, 5ʹ-CTAGAAAGTCTGTTTGTGTCTAACAAAGG-3ʹ, and
reverse, 5ʹ-CTAAGAGGAATCTCATCACAGG-3ʹ. Similar to Mi1.2, the ACD transgene behaves as a single dominant gene inherited
in a Mendelian fashion when transgenic tomato plants are crossed
with other tomato lines (Reed et al., 1995); therefore, heterozygous
F1 plants were used for subsequent assays.
Gene expression analysis
In order to assess the effects of aphid feeding on gene expression in
plants with and without the Mi-1.2 resistance gene, ‘MoneyMaker’
and 143-25 were mock infested with empty nylon sleeve cages that
each covered a single leaflet (control), or infested with potato aphids
(25 aphids per sleeve cage; two sleeve cages per plant; three plants per
treatment group). At 6 and 24 h after infestation (HAI), the aphids
were gently removed with a paintbrush, and the leaf tissue was
flash frozen with liquid nitrogen and stored at –80 °C. Total RNA
was extracted from each sample using TRI Reagent (Molecular
Research Center, OH, USA), and then was DNase treated with
TURBO DNA-free (Ambion, Austin, TX, USA) followed by
reverse transcription of 0.5 µg of RNA using Superscript II reverse
transcriptase (Invitrogen, Carlsbad, CA, USA) in a 20 µl reaction
volume. The following set of primers were then used for quantitative PCR (qPCR): Pti5 (GenBank accession no. U89256) forward,
5ʹ-GCGAGGTGCTAAGGCACTAC-3ʹ, and reverse, 5ʹ-TGCCA
AGAAATTCTCCATGC-3ʹ; E4 (GenBank accession no. S44989.1)
forward, 5ʹ-TGATGCTCAGGCTCAACTGG-3ʹ, and reverse,
5ʹ-ACTGCTTACAACCTCTGCCC-3ʹ; RPL2 (GenBank accession
no. X64562) forward, 5ʹ-GAGGGCGTACTGAGAAACCA-3ʹ, and
reverse, 5ʹ-CTTTTGTCCAGGAGGTGCAT-3ʹ. Real-time (RT)qPCR was performed with a QuantiTect SYBR Green PCR kit
(Qiagen, CA, USA) in a 20 μl reaction. The StepOnePlusTM RealTime PCR system (Applied Biosystems) was used for fluorescence
detection. Two technical replicates per biological sample were run.
The PCR conditions were as follows: 15 min of activation at 95 °C,
40 amplification cycles (94 °C for 15 s, 55 °C for 30 s, and 72 °C
for 30 s, with data acquisition at the end of each cycle), and a final
data acquisition step to generate melting curves from 65 to 95 °C
every 0.3 °C. Melting curves were performed for all samples to
verify single amplification product. For each primer set, the amplification efficiency was calculated using the formula E=10(–1/Ct slope)
from data generated from serial dilutions of a set of bulked cDNA
standards having equal aliquots from all the samples (Rasmussen,
2000). Relative gene expression was calculated using Pfaffl methodology (Pfaffl, 2001). Data were normalized to the expression levels
of the endogenous control ribosomal protein L2 gene (RPL2), and
gene expression for each treatment group was calculated relative to
the wild-type (WT) control group in each experiment. For statistical analysis, the relative expression values for each treatment group
were log2 transformed to stabilize variances. Data were analysed by
two-way analysis of variance (ANOVA) and means for significant
effects at α=0.05 were separated using Student’s t-test with JMP®
Pro 9.0 (SAS Institute, NC, USA).
Aphid bioassays
No-choice tests Aphids were restricted to single leaflets using lightweight mesh clip cages (~3.5 cm diameter). Six-week old tomato
plants were infested with four to five apterous young adult potato
aphids per cage, with at least two cages per plant, and maintained
at 20 °C and with a 16:8 h light:dark photoperiod. At 5–7 days
after infestation (DAI), the total number of live aphids (adults and
nymphs) was recorded in each cage. Each plant was treated as a biological replication, and separate cages were treated as subsamples.
The average number of aphids per cage per plant was analysed by
one- or two-way ANOVA, depending upon whether there were one
or two independent variables in the experiment. If the ANOVAs
revealed statistically significant main effects or significant interactions between independent variables, mean separations were performed using Student’s t-test to identify differences among treatment
groups (α=0.05). All statistics were performed with JMP® Pro 9.0.
Choice tests Eight-week-old ‘MoneyMaker’ and ‘Motelle’ plants
were sprayed with 0.01% Tween 20 with or without 1 mM aminoethoxyvinylglycine (AVG; Valent USA Corporation, IL, USA). Six
hours after treatment, when the foliage was fully dry, the plants were
arranged in pairs on laboratory benches. For each possible pairing
of plants, there were six replicate pairs per experiment. A choice
arena consisting of a pedestal with a styrofoam platform approximately 10 cm in diameter was placed half way between the plant
pairs as described by Avila et al. (2012). Each arena was in contact
with one terminal leaflet of a mature leaf on each of the two plants
in the pair. The leaf positions 6–10 (first true leaf=1) were used for
this assay. Each leaflet used for the choice test was isolated from the
rest of the plant using a circular plastic shield (11 cm in diameter)
with Tanglefoot Pest Barrier (The Tanglefoot Company, MI, USA)
around the edge. Twenty adult apterous potato aphids were released
onto the centre of each platform at the start of the experiment. The
number of aphids and offspring were counted on each leaflet at
four time points after release (1, 6, 24, and 48 HAI). The data for
each comparison were analysed by a matched-pairs one-sided t-test
within each time point in JMP® Pro 9.0.
562 | Wu et al.
Silencing of Pti5
Construction of TRV-Pti5 Virus-induced gene silencing (VIGS)
was performed to suppress expression of Pti5 in tomato using
the tobacco rattle virus (TRV) system. VIGS was performed as
described previously (Wu et al., 2011; Avila et al., 2012, 2013). The
TRV-Pti5 construct consisted of a 302 bp fragment corresponding to nt 379–680 of the tomato Pti5 gene (GenBank accession no.
U89256), which was amplified using the following primers: forward,
5ʹ- CGGTCTAGAAGTGAACGCCTCTGTTTCAG-3ʹ and reverse
5ʹ-CGGGGATCCGTCGCTGCAAACAATTCCAT-3ʹ. The Pti5
fragment was cloned into the pYL156 vector to generate the TRVPti5 silencing construct. The pYL156-Pti5 vector was transformed
into Escherichia coli strain DH10B, and the insert was confirmed
by sequencing and then introduced into Agrobacterium tumefaciens strain GV3101. For the empty control vector (TRV-CV), we
used pYL156 carrying a 396 bp insert from the β-glucuronidase
reporter gene (GenBank accession no. S69414.1) to achieve a control vector that would have similar movement and proliferation in
the plant compared with the experimental vector (Wu et al., 2011).
Additionally, a construct that silences the phytoene desaturase gene
(PDS, GenBank accession no. M88683.1) (Wu et al., 2011) was used
in a separate set of plants as a visual reporter to monitor the onset
of VIGS.
Agro-infiltration and bioassays The TRV vectors were infiltrated into
tomato as described previously (Wu et al., 2011). Briefly, 2-weekold tomato plants were vacuum infiltrated with TRV-Pti5, TRV-CV,
or TRV-PDS and kept at 20 °C in a growth chamber with a 16:8h
light:dark photoperiod until ready for bioassay. No-choice aphid
bioassays were performed as described above when monitor plants
infiltrated with the TRV-PDS vector showed a widespread uniform
bleaching phenotype (~4 weeks after infiltration; inoculum level of
five aphids per cage, four cages per plant; the number of live aphids
per cage scored at 4 and 7 DAI).
Results
Aphid infestation induces Pti5 expression in tomato
Gene expression of Pti5 was monitored by RT-qPCR at
6 and 24 h HAI in the aphid-susceptible tomato cultivar
‘MoneyMaker’ and the aphid-resistant transgenic line 14325, which carries the Mi-1.2 transgene in a ‘Moneymaker’
genetic background (Fig. 1). No change in gene expression
was observed at 6 HAI, but at 24 HAI, Pti5 transcripts were 3to 4-fold higher in infested plants than in uninfested controls
for both genotypes (P<0.05). No difference in Pti5 transcript
abundance was observed between ‘MoneyMaker’ and 143-25
(P=0.7697), which suggested that Mi-1.2 does not influence
constitutive or aphid-responsive levels of Pti5 expression.
The Pti5 gene contributes to antibiotic defences
against aphids in both resistant and susceptible
tomato genotypes
To investigate if Pti5 influenced plant defences against
aphids, VIGS was performed to suppress Pti5 expression in
the aphid-susceptible tomato ‘Moneymaker’ and the nearisogenic resistant line ‘Motelle’ carrying the Mi-1.2 gene.
Plants were infiltrated either with a construct designed to
silence Pti5 (TRV-Pti5) or with a control vector of comparable size (TRV-CV). Compared with plants that received
the vector control, plants infiltrated with TRV-Pti5 had significantly lower abundance of Pti5 transcripts in the foliage
Fig. 1. Tomato Pti5 gene expression in response to aphid infestation. The
aphid-susceptible tomato cv. ‘MoneyMaker’ (MM) and a transgenic line
transformed with the Mi-1.2 aphid resistance gene (143-25) were mock
infested with empty cages (control) or infested with aphids (25 aphids
per sleeve cage; two sleeve cages per plant). Relative gene expression
was measured by RT-qPCR at 6 and 24 HAI. Expression was normalized
relative to the endogenous control RPL2 gene. Asterisks (*) denote
statistically significant differences at α=0.05 [n=3; error bars represent
standard error of the mean (SEM)].
(Fig. 2A; P=0.0013), and the efficacy of silencing was similar in the two genotypes. To measure antibiosis, plants were
inoculated with caged adult aphids in a no-choice test, and
insect survivorship and reproduction was monitored at 4 and
7 DAI. The aphid-susceptible cultivar ‘MoneyMaker’ had
higher numbers of live adults (Fig. 2B; P=0.0098) and offspring (Fig. 2C; P<0.0001) than the aphid-resistant genotype
‘Motelle’ at 4 DAI. Regardless of the genotype tested, plants
treated with TRV-Pti5 also supported higher numbers of surviving adults (Fig. 2B; P=0.0033) and live offspring (Fig. 2C;
P=0.0063) than the TRV-CV-treated plants. The percentage
increase in surviving adults (35% on ‘MoneyMaker’ and 35%
on ‘Motelle’) and offspring (37% on ‘MoneyMaker’ and 40%
on ‘Motelle’) that resulted from silencing of Pti5 was similar
on the two genotypes, and there was no statistically significant interaction between VIGS treatment and plant genotype
(P>0.1). This suggested that the effects of silencing by TRVPti5 was similar in the two genetic backgrounds. The same
patterns of aphid population growth observed at 4- DAI persisted at 7 DAI (Fig. 2D, E), indicating that the effect of Pti5
silencing on aphid population growth remained stable for the
time period tested.
In contrast to Pti5, ethylene signalling depresses
basal resistance in susceptible tomato and does not
contribute to antibiosis in resistant plants
Because Pti5 is classified as an ERF, we also investigated the
influence of ethylene on defences that affect herbivore survival
and reproduction (i.e. antibiosis) using the ACD transgene.
Plants that express ACD are impaired in ethylene synthesis
because this enzyme degrades ACC, an essential precursor
for ethylene synthesis (Klee et al., 1991). We performed nochoice tests to compare the survival and offspring production
of adult aphids on the transgenic line ACD (which carries
the ACD gene in an aphid-susceptible ‘UC82B’ background),
hybrids that carry both the ACD transgene and the Mi-1.2
Pti5 and ethylene in plant–aphid interactions | 563
no significant impact on offspring production (P=0.4326) or
adult aphid survival (P=0.3226) in a genetic background that
carried the Mi-1.2 resistance gene (Fig. 3B). A similar trend
was observed when we used a pharmacological approach to
disrupt ethylene synthesis in ‘MoneyMaker’ and ‘Motelle’ by
applying AVG to block the activity of 1-aminocyclopropane1-carboxylate synthase, which generates a necessary precursor
for ethylene synthesis (Yu and Yang, 1979; Huai et al., 2001).
AVG treatment lowered offspring production on the susceptible genotype ‘MoneyMaker’ but not on the resistant isoline
‘Motelle’, and as denoted by the statistically significant interaction between AVG treatment and plant genotype (Fig. 3C;
two-way ANOVA, main effect of AVG treatment: P=0.4889;
main effect of genotype: P<0.0001; and the interaction effect
between AVG treatment and genotype: P=0.0401). AVG did
not impact adult survival on either genotype (P>0.10). These
results indicated that ethylene signalling promotes aphid
infestation on susceptible tomato genotypes but not on resistant genotypes that carry the Mi-1.2 resistance gene.
Ethylene signalling promotes aphid settling on
susceptible plants but may contribute to antixenosis in
plants that carry Mi-1.2
Fig. 2. Aphid population growth on Pti5-silenced tomato plants. The
aphid-susceptible tomato cultivar ‘MoneyMaker’ (MM) and the nearisogenic line ‘Motelle’ (MT) carrying the Mi-1.2 resistance gene were
treated with a TRV vector modified to suppress expression of Pti5 (TRVPti5) or with a control vector of comparable size that does not silence any
endogenous genes in tomato (TRV-CV). The silencing efficiency for TRVPti5 was corroborated by RT-qPCR normalized relative to the endogenous
RPL2 gene (A). Plants were infested with aphids confined to clip cages
(five young adult potato aphids per cage; five cages per plant; eight plants
per treatment group), and the number of live adults (B, D) and offspring
(C, E) were recorded at 4 and 7 DAI. Values for Pti5 expression (A) and
aphid survival and reproduction (B–E) were analysed by two-way ANOVA
and means separated by Student’s t-test. Asterisks (*) denote statistically
significant differences at α=0.05, and error bars represent SEM (n=3 for A,
n=8 for B–E).
resistance gene (MT×ACD), and resistant (‘Motelle’) and
susceptible (‘UC82B’, ‘Moneymaker’) cultivars with WT
ethylene signalling. At 7 DAI, the average number of aphid
offspring (i.e. nymphs) was ~40% lower on the ACD transgenic line than on the WT background ‘UC82B’ (Fig. 3A,
P=0.0450). Since ACD did not affect adult survival (Fig. 3A,
P=0.5000), the decrease in reproduction on ACD was probably due to a decrease in adult fecundity. In contrast, ACD had
We also tested the effect of ethylene on the plant’s ability to deter aphid settling during the host selection process
(i.e. antixenosis or non-preference). To avoid the potential
effects of multiple loci that are segregating in the ‘MT×ACD’
hybrids, we chose instead to manipulate ethylene signalling by
applying AVG or a blank carrier solution to the near-isolines
‘MoneyMaker’ and ‘Motelle’. Aphid settling was tested in
choice tests in which groups of 20 adult aphids were placed
on platforms (choice arenas) between paired plants with and
without AVG treatment. Aphids typically moved off the arena
onto the foliage within minutes of introduction, and were free
to move back and forth between the two plants by crossing
the arena. At 1, 6, 24, and 48 HAI, we recorded the position
of adults, as well their offspring production, which is a wellestablished marker of host plant acceptance (Powell et al.,
2006). On the susceptible cultivar ‘MoneyMaker’, aphids
had a significant preference to settle on plants treated with
the control solution compared with plants treated with AVG
(Fig. 4A, P<0.05 at all time points), and by 24 HAI, reproduction was also higher on the controls (Fig. 4B; matchedpairs test, 6 HAI: P=0.1146; 24 HAI: P=0.0304; and 48 HAI:
P=0.0350. No nymphs were observed at 1 HAI). In contrast, on the resistant line ‘Motelle’, aphids preferred to settle (Fig. 4C) and reproduce (Fig. 4D) on plants treated with
AVG than on plants treated with the blank carrier solution
(adult settling: matched-pairs test, 1 HAI: P=0.0343; 6 HAI:
P=0.1054; 24 HAI: P=0.0189; and 48 HAI: P=0.0272; offspring production: matched-pairs test, 6 HAI: P=0.0421; 24
HAI: P=0.0654; and 48 HAI: P=0.0351). These results indicated that ethylene signalling promotes aphid host acceptance
on susceptible plants, but contributes to antixenosis (i.e. deterrent defences) in plants that carry the Mi1.2 resistance gene.
We also performed additional choice tests to investigate
whether ethylene influenced aphid host preferences between
564 | Wu et al.
Fig. 3. Aphid population growth on tomato plants with compromised ethylene signalling. Aphid survival and reproduction was compared in no-choice
tests on an ACD transgenic line deficient in ethylene synthesis and the WT background for this line, ‘UC82B’ (A); and on hybrids between ACD and
the resistant cultivar ‘Motelle’ (MT×ACD) (B). Additionally, aphid survival and reproduction was measured on resistant ‘Motelle’ (MT) and susceptible
‘MoneyMaker’ (MM) plants treated with AVG or a blank carrier solution (control). The numbers of adult aphids and their offspring (nymphs) were analysed
by two-way ANOVA and means separated by Student’s t-test. Asterisks (*) denote statistically significant differences at α=0.05, and error bars represent
SEM (n=14 for A and n=6 for B and C).
Fig. 4. Aphid host preference between plants with and without an inhibitor of ethylene synthesis. The aphid-susceptible cultivar ‘MoneyMaker’ (MM) and
the near-isogenic resistant line ‘Motelle’ (MT) were treated with an inhibitor of ethylene synthesis (AVG) or mock treated with solvent (control). To determine
if aphids had a preference between AVG-treated or mock-treated plants, adult aphids were placed on choice arenas between paired plants and allowed
to move back and forth between the plants (20 aphid per /area; six arenas per comparison). The percentage of adults that were located on treated plants
versus controls (A, C) was recorded at 1, 6, 24, and 48 HAI, along with the number of offspring produced on each treatment group (B, D). Comparisons
labelled with an asterisk (*) were statistically different at α=0.05 according to one-sided matched-pair comparisons. Error bars in (B) and (C) represent SEM.
genotypes. When aphids were offered a choice between
‘MoneyMaker’ plants treated with AVG or ‘Motelle’ plants
treated with AVG, they consistently preferred ‘MoneyMaker’
over ‘Motelle’ (Fig. 5B, P<0.05 at all time points tested),
indicating that inhibiting ethylene synthesis did not eliminate
antixenosis in the resistant line. However, when aphids were
offered a choice between AVG-treated ‘Motelle’ and mocktreated ‘MoneyMaker,’ AVG appeared to delay the aphids’
ability to discriminate between the resistant and susceptible
genotypes (Fig. 5C). Whereas aphid numbers were significantly
higher on mock-treated ‘MoneyMaker’ compared with
mock-treated ‘Motelle’ as early as 1 HAI (Fig. 5A, P<0.05
at all time points), aphids presented with ‘MoneyMaker’
controls and AVG-treated ‘Motelle’ plants did not show a
statistically significant preference for ‘MoneyMaker’ until
24 HAI (Fig. 5C; 1 HAI, P=0.6222; 6 HAI, P=0.0744; 24
HAI P=0.0014; and 48 HAI, P=0.0356). In contrast, applying AVG to ‘MoneyMaker’ plants only (MM-AVG) did not
inhibit aphids from selecting ‘MoneyMaker’ over mocktreated ‘Motelle’ plants (Fig. 5D; P< 0.01 at all time points).
Pti5 and ethylene in plant–aphid interactions | 565
Fig. 5. Aphid host preference between plants with and without Mi-1.2, in the presence or absence of an inhibitor of ethylene synthesis. The aphidsusceptible cultivar ‘MoneyMaker’ (MM) and the near-isogenic resistant line ‘Motelle’ (MT) were treated with AVG or mock treated with solvent (control),
and choice assays were performed to determine whether AVG influenced the aphids’ ability to discriminate between the two genotypes (20 aphids per
area; six arenas per comparison). The percentage of adults that were located on treated plants versus controls was recorded at 1, 6, 24, and 48 HAI.
Comparisons labelled with an asterisk (*) were statistically different at α=0.05 according to one-sided matched-pair comparisons.
Together, these results suggested that, in resistant plants, ethylene signalling may contribute to antixenotic defences that
act early (<24 h) in the plant–aphid interaction.
Induction of Pti5 expression by aphids is independent
of ethylene, jasmonic acid (JA), or salicylic acid (SA)
accumulation
Although several studies on ERF-like genes encoding GCC
box-binding factors have focused on their role in ethylene
signalling (Solano and Ecker, 1998; Suzuki et al., 1998), our
results indicated that Pti5 and ethylene signalling have different roles in plant–aphid interactions. To further corroborate
that induction of Pti5 acted independent of ethylene signalling, we monitored Pti5 gene expression at 48 HAI in the
ethylene-deficient ACD transgenic line. Pti5 gene expression
was induced by aphids to a similar extent in both ACD and
the WT control ‘UC82B’, indicating that aphid induction was
independent of ethylene levels. However, basal levels of the
Pti5 transcript in uninfested plants were higher in ACD than
in ‘UC82B’, suggesting that constitutive expression of Pti5
may be inhibited by ethylene (Fig. 6A, two-way ANOVA,
main effect of genotype: P=0.0312; main effect of aphid
infestation: P=0.168; effect of the interaction between genotype and aphid infestation: P=0.2140). The ethylene-induced
marker gene E4 was downregulated in ACD plants as compared with ‘UC82B’ (Supplementary Fig. S1 at JXB online),
confirming that ethylene signalling was impaired in the ACD
transgenic plants.
Since induction of Pti5 by aphids did not require ethylene accumulation, we investigated the possible role of the
alternative plant defensive hormones SA and JA. Transcript
abundance of Pti5 in response to aphid infestation was measured in the NahG tomato line, which expresses salicylate
hydroxylase, an enzyme that diminishes SA accumulation by
degrading SA to catechol (Gaffney et al., 1993). Expression
of Pti5 was upregulated at 48 HAI in both the NahG and
WT background tomato cultivar ‘MoneyMaker’ in response
to aphid infestation (Fig. 6B; P=0.0277). No significant interaction between genotype and aphid treatment was observed
(P=0.7489), indicating that induction of Pti5 by aphids is
also SA independent. However, constitutive transcript levels of Pti5 were ~40 times higher in NahG as compared with
‘MoneyMaker’ (P<0.0001). We hypothesized that high constitutive Pti5 expression in NahG plants resulted from the
development of necrotic lesions in this genotype and not
from a deficiency in SA. Several lines of evidence support
our hypothesis: (i) constitutive expression of Pti5 is higher in
older than younger leaves, suggesting that expression of Pti5
is regulated upon cell senescence (Gu et al., 2000); (ii) NahG
plants develop necrotic lesions through the vegetative growth
stage (Brading et al., 2000; Li et al., 2002); (iii) expression of
senescence-related genes in NahG plants has been related to
the appearance of necrotic lesions (Avila et al., 2013); and (iv)
soil drenches of the SA analogue benzothiadiazole induce
Pti5 transcripts in tomato, which argues against the idea that
SA is a negative regulator of this gene (Harel et al., 2014).
We also measured transcript abundance of Pti5 in the
JA-deficient mutant tomato lines acx1 and spr2. The acx1
mutation in tomato blocks the first β-oxidation step of
JA synthesis reducing JA content by 95% (Li et al., 2005).
The spr2 line carries a loss-of-function mutation in FATTY
ACID DESATURASE 7 (FAD7) resulting in a 90% reduction in linolenic acid, a necessary precursor for JA synthesis
(Li et al., 2003). Both mutations almost completely eliminate
expression of the JA-responsive marker gene PROTEINASE
INHIBITOR II (PI-II) (Howe and Ryan, 1999; Li et al., 2003;
Li et al., 2005; Avila et al., 2012). Transcript abundance of
566 | Wu et al.
Fig. 6. Role of plant defensive hormones in aphid-induced Pti5 gene expression. Relative expression of Pti5 was measured by RT-qPCR 48 h after
potato aphid infestation in the following tomato hormone-deficient plants: ethylene-deficient ACD and untransformed control ‘UC82B’ (A); SA-deficient
NahG and untransformed control ‘MoneyMaker’ (MM) (B); and JA-deficient acx1 (C) and spr2 (D) with their WT background ‘Castlemart’ (CM). Gene
expression was normalized relative to the RPL2 gene. Asterisks (*) denote statistically significant differences at α=0.05, and error bars represent SEM
(n=6 for A, n=4 for B and C, and n=3 for D).
Pti5 was induced in both JA mutants in response to aphids
at 48 HAI when compared with the WT background tomato
cultivar ‘Castlemart’ (Fig. 6C, D; P<0.05). Similarly, there
was no significant interaction between genotype and aphid
treatment in either mutant (P>0.10), indicating that accumulation of JA is not required for aphid induction of Pti5
transcription. However, JA mutants showed a small decrease
in constitutive Pti5 transcription levels compared with WT
plants, although statistical significance was only reached in
the acx1 mutant (acx1 genotype main effect P=0.0390; spr2
genotype main effect P=0.6736). Thus, JA may potentially
promote basal levels of Pti5 expression.
In summary, our results clearly demonstrated that ethylene,
SA, and JA accumulation are not required for transcriptional
upregulation of Pti5 by aphid infestation on tomato. While
ethylene, SA, and JA are not the primary signals that trigger
Pti5 expression in response to aphids, they may potentially
fine-tune constitutive or inducible Pti5 expression levels;
moreover, these phytohormone pathways may interact with
Pti5 signalling downstream of Pti5 transcription. In fact, Mimediated aphid resistance is known to require SA (Martinez
de Ilarduya et al., 2003); therefore, SA-dependent defences
and Pti5-dependent defences may act synergistically.
Discussion
VIGS of Pti5 resulted in increased aphid infestations on
tomato, indicating that this ERF contributes to plant defences
against piercing/sucking insects. These results illustrated
the overlap between plant responses to aphids and plant
responses to microbes, since Pti5 and other ERFs are known
for their roles in plant–pathogen interactions (Licausi et al.,
2013). As mentioned previously, Pti5 contributes to host plant
resistance against the bacterial pathogen Pseudomonas syringae pv. syringae (Thara et al., 1999; He et al., 2001). Moreover,
it is also transcriptionally upregulated by Trichoderma harzianum, a beneficial soil fungus that promotes induced systemic
resistance against the grey mould fungus Botrytis cinerea and
other pathogens (Harel et al., 2014). The Pti5 gene product
may contribute to aphid resistance by upregulating expression of PR genes, as is seen in the defence response against
P. syringae (Gu et al., 2000). In addition, ERFs are known
to play other diverse roles in plant stress responses, including
transcriptional regulation of secondary metabolite synthesis
(Shoji et al., 2010), cell death (Ogata et al., 2013), reactive oxygen species signalling (Sewelam et al., 2013), adjustments in
primary metabolism (Vogel et al., 2014), and crosstalk among
hormone signalling pathways (Van der Does et al., 2013). In
other words, the suppressive effects of Pti5 on aphids may be
due to activation of direct defences and/or enhancement of
defensive signalling by this transcription factor.
Silencing of Pti5 resulted in increased aphid infestations
on a genotype that lacks the Mi-1.2 aphid resistance gene,
which indicates that this gene contributes to basal levels of
aphid resistance in susceptible hosts (Fig. 7). Suppression of
Pti5 expression also increased aphid population growth on
a resistant (Mi-1.2+) cultivar, suggesting that this transcription factor might also contribute to R gene-mediated aphid
resistance (Fig. 7). Previous studies have shown that transcriptional upregulation of Pti5 in response to Pseudomonas
syringae is enhanced when the pathogen is recognized by the
R gene Prf (Thara et al., 1999), and that Pti5 contributes to
Prf/Pto-mediated resistance against P. syringae through a
direct physical interaction with the Pto kinase (Zhou et al.,
1997). However, transcriptional upregulation of Pti5 by aphid
infestation was similar in magnitude in genotypes with and
Pti5 and ethylene in plant–aphid interactions | 567
Fig. 7. Pti5, Mi-1.2, and ethylene signalling in response to potato aphid
feeding on tomato. Tomato plants recognize potato aphid secretions,
which contain effectors that promote infestation, as well as elicitors that
turn on defences. In the susceptible cultivar, ethylene signalling promotes
aphid infestation by increasing aphid fecundity. In the resistant cultivar,
Mi-1.2 signalling reduces fecundity and increases mortality (antibiosis),
and ethylene contributes to early stages of antixenotic defences. In both
the susceptible and resistant cultivars, Pti5 contributes to antibiosis.
The shaded bar indicates the overlap between basal and R genemediated resistance. Arrows indicate steps that contribute to defence or
susceptibility. Dashed arrows indicate unknown steps/mechanisms. ET,
ethylene; SA, salicylic acid.
without Mi-1.2, so this R gene does not appear to enhance
transcription of Pti5. Upregulation of Pti5 by aphids was
also relatively slow and was not evident at 6 h, whereas upregulation by Pseudomonas syringae is detectable as early as 1
HAI (Thara et al., 1999). These differences suggest that Pti5
might not play a direct role in Mi-1.2-dependent defences
against aphids; instead, the effects of silencing Pti5 on aphid
numbers on the resistant (Mi-1.2+) cultivar might be due to
additive effects between Pti5-dependent basal defences and
R gene-mediated resistance. Alternatively, Pti5 may participate in R gene-mediated aphid resistance, but enhancement
of Pti5 activity by Mi-1.2 may rely on processes other than
transcriptional activation. The activity of some ERFs is
regulated by alternative splicing of the mRNA, post-translational modifications of the protein, and interactions with
other transcription factors and structural proteins (Licausi
et al., 2013). Phosphorylation of the Pti5 protein by the Pto
kinase enhances the DNA-binding activity of this transcription factor (Zhou et al., 1997), and phosphorylation of ERF6
in Arabidopsis promotes its role in B. cinerea resistance (Meng
et al., 2013). Moreover, the susceptibility of ERF proteins to
proteolysis and the affinity and specificity with which they
bind DNA can be influenced by interacting proteins. For
example, ORF1, an ERF in tobacco, interacts synergistically with basic helix–loop–helix transcription factors to
promote expression of genes for nicotine synthesis (De Boer
et al., 2011). Thus, it is possible that Mi-1.2 could impact
­post-transcriptional regulation of Pti5 or the expression of
interacting partners that modify Pti5 activity.
Our results also indicated that ethylene is not required for
transcriptional upregulation of Pti5 by aphids, despite this
gene’s classification as an ERF. Aphid infestation induces
Pti5 expression even in genotypes that express ACD, a
transgene that inhibits ethylene synthesis and suppresses
expression of E4 and other ethylene-responsive genes. This
is consistent with previous reports that Pti5 is not induced by
exogenous ethylene (Thara et al., 1999; Gu et al., 2000), and
that Pseudomonas syringae is able to upregulate this gene in
a mutant tomato line with impaired ethylene perception, the
Never-ripe (Nr) mutant (Thara et al., 1999). These findings
highlight the fact that transcription factors are classified as
ERFs based not on empirical evidence of ethylene responsiveness but on the presence of a conserved domain that can
bind the GCC box, which is in turn common among genes
that are upregulated by ethylene. In contrast to this classification scheme, a growing body of evidence has revealed that the
ERF family does much more than mediate ethylene-responsive induction of GCC box-containing genes. For example,
chromatin immunoprecipitation assays have shown that
another ERF in tomato, Pti4, can bind to promoters that do
not contain a GCC box, and microarray analysis has revealed
that the majority of genes that were upregulated as a result
of overexpression of Pti4 lacked this motif (Chakravarthy
et al., 2003). The specificity of ERF binding can also vary
in response to different stimuli; ERF1 in Arabidopsis binds
to the GCC box in response to biotic stresses but interacts
with the DRE/CRT motif when activated by abiotic stimuli
(Cheng et al., 2013). Furthermore, ERFs can mediate plant
responses to hormones other than ethylene, including jasmonates, abscisic acid, and cytokinins (Licausi et al., 2013).
While upstream ethylene signalling is not required for Pti5
induction, it has been shown previously that ERFs can act as
a point of convergence between ethylene and other signalling
pathways (Lorenzo et al., 2003). However, our results suggest that ethylene and Pti5 play divergent rather than convergent roles in plant–aphid interactions (Fig. 7). In susceptible
plants that lack Mi-1.2, the Pti5 gene contributes to basal
resistance, whereas ethylene signalling promotes aphid infestation. In the absence of Mi-1.2, we found that suppressing
ethylene synthesis with the ACD transgene depressed aphid
population growth; likewise, Mantelin et al. (2009) showed
that inhibiting ethylene synthesis by silencing ACC synthases
decreased aphid life spans. Furthermore, we observed higher
levels of constitutive Pti5 expression in the ethylene-deficient
ACD plants than in WT controls, suggesting that ethylene
suppresses basal Pti5 expression. Similarly, application of
the ethylene precursor ACC inhibited induction of the jasmonate-responsive ERFs at the NIC2 locus in tobacco (Shoji
et al., 2010). Thus, it is possible for ethylene and ERFs to act
antagonistically, and this appears to be the case for ethylene
and Pti5 in basal aphid resistance.
Pti5 and ethylene also play distinctly different roles in
tomato genotypes that carry the Mi-1.2 aphid resistance gene.
Pti5 is induced sometime after 6 h and contributes to antibiotic defences that limit aphid survival or reproduction on the
host. In contrast, ethylene does not help or hinder Mi-1.2mediated antibiosis, which is unaffected by the ACD transgene
or AVG treatment (our results), the Nr mutation, or the
pharmacological inhibitor 1-methylcyclopropene (Mantelin
et al., 2009). Instead, we found that ethylene contributes to
568 | Wu et al.
Mi-1.2-mediated antixenotic defences that limit aphid settling
on the host and that act early in the interaction (6 h and earlier). These results illustrate that ethylene can play different
roles in different genetic backgrounds, promoting infestation
on compatible hosts but contributing to resistance in backgrounds that carry a relevant R gene. This is consistent with
the fact that ethylene-associated transcripts are responsive to
aphid feeding on both resistant (Mi-1.2+) and susceptible
(Mi-1.2–) cultivars (Anstead et al., 2010). In addition, our
findings suggest that the antibioitic and antixenotic components of Mi-1.2-mediated aphid resistance are due to separate
defence mechanisms regulated through different signalling
pathways. Louis et al. (2012) also reported a divergence in
molecular signalling between antibiotic and antixenotic components of basal aphid resistance in Arabidopsis. We have
shown previously that, in addition to factors that inhibit
ingestion from the phloem, Mi-1.2-mediated resistance also
involves earlier-acting factors that deter sampling in the mesophyll or epidermis (Pallipparambil et al., 2010). Therefore,
it is possible that these resistance factors localized outside the
vascular tissue are involved in ethylene-dependent antixenosis. These results advance our understanding of the mechanisms of R gene-mediated aphid resistance and also expand
the potential applications of ERFs, overexpression of which
has been shown previously to enhance pathogen resistance
and abiotic stress tolerance in multiple plant species (Xu
et al., 2011).
Supplementary data
Supplementary data are available at JXB online.
Supplementary Fig. S1. Expression of E4 ethylene-responsive gene in the ACD transgenic line.
Acknowledgements
We thank Dr Stephanie Defibaugh-Chávez, Lingling Jia, Dr L. Milenka
Arevalo-Soliz, Dr Carmen S. Padilla, and Dr Godshen Palliparambil for technical assistance and/or materials. We also thank Dr Harry Klee (University of
Florida, FL, USA) for providing the ACD transgenic line, Valent USA Corp.
for AVG, and Dr Dinesh-Kumar (University of California, Davis, USA) and
Dr Kyle Willis (USDA-ARS, Madison, WI, USA) for VIGS vectors. This
research was supported by the National Research Initiative of the USDA
Cooperative State Research, Education and Extension Service (CSREES),
grant no. 2006-35607-330 16612, the National Science Foundation IOS grant
no. 0951287, and the Arkansas Agricultural Experiment Station.
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