The FASEB Journal article fj.14-266023. Published online February 12, 2015.

The FASEB Journal Research Communication

Armet is an effector protein mediating

aphidplant interactions
Wei Wang,*, Huaien Dai, Yi Zhang, Raman Chandrasekar, Lan Luo,* Yasuaki Hiromasa,
Changzhong Sheng, Gongxin Peng, Shaoliang Chen, John M. Tomich, John Reese,{
Owain Edwards,k Le Kang,* Gerald Reeck,,1 and Feng Cui*,,1
*State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology,
Chinese Academy of Sciences, Beijing, China; College of Biological Sciences and Technology, Beijing
Forestry University, Beijing, China; Department of Biochemistry and Molecular Biophysics, Kansas State
University, Manhattan, Kansas, USA; Department of Mathematics, Hebei University of Science and
Technology/Hebei Laboratory of Pharmaceutic Molecular Chemistry, Shijiazhuang, Hebei, China;
{
Department of Entomology, Kansas State University, Manhattan, Kansas, USA; and kCommonwealth
Scientic and Industrial Research Organisation Ecosystem Sciences, Centre for Environment and Life
Sciences, Floreat, Australia
Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding.
Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet,
namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armets physical and chemical
properties and its intracellular role are comparable to
those reported for mammalian Armets. Uniquely, we
detected Armet in aphid watery saliva and in the phloem
sap of fava beans fed on by aphids. Armets transcript
level is several times higher in the salivary gland when
aphids feed on bean plants than when they feed on an
articial diet. Knockdown of the Armet transcript by
RNA interference disturbs aphid feeding behavior on
fava beans measured by the electrical penetration graph
technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves
induced a transcriptional response that included pathogenresponsive genes. The data suggest that Armet is an effector protein mediating aphidplant interactions.Wang,
W., Dai, Zhang, Y., Chandrasekar, R., Luo, L., Hiromasa,
Y., Sheng, C., Peng, G., Chen, S., Tomich, J. M., Reese, J.,
Edwards, O., Kang, L., Reeck, G., Cui, F. Armet is an effector protein mediating aphidplant interactions. FASEB J.
29, 000000 (2015). www.fasebj.org

ABSTRACT

Key Words: saliva protein salivary gland feeding behavior

MANF calcium binding

NUMEROUS APHID SPECIES are major crop pests through direct

damage or as vectors of plant viruses (1). Aphids feed on
phloem sap from sieve elements while keeping these cells
Abbreviations: CDNF, conserved dopamine neurotrophic
factor; dsRNA, double-stranded RNA; EPG, electrical
penetration graph; ER, endoplasmic reticulum; ERSE,
endoplasmic reticulum stress response element; GFP,
green uorescent protein; MALDI-TOF-MS, matrix-assisted
laser desorption-ionization time-of-ight mass spectrometry;
(continued on next page)

0892-6638/15/0029-0001 FASEB

alive and their sieve-plate pores open (2). Continuous

production of watery saliva occurs as aphids probe sieve
elements and ingest phloem sap from individual sieve
elements for hours or even days (3). Saliva is predicted to
contain numerous proteins that overcome plant defenses
(46) and is thought to deliver effector proteins in analogy
to plant pathogens (68). However, the functions of individual protein effectors in aphid saliva remains poorly
understood.
The identication of individual proteins of aphid saliva
has progressed considerably in recent years. Nine proteins
secreted in the saliva of the pea aphid, Acyrthosiphon pisum,
5 enzymes in the saliva of the green peach aphid Myzus
persicae, 12 and 7 proteins in the saliva of 2 cereal aphid
species, Sitobion avenae and Metopolophium dirhodum, respectively, have been identied using proteomics (911). A
catalog of candidate effector proteins from the salivary
glands of the pea aphid has been generated using a combined proteomics and transcriptomics approach, 42 of
whose transcripts were enriched in salivary glands (6).
When fed different diets, the Russian wheat aphid, Diuraphis
noxia, secretes saliva with qualitative and quantitative differences in soluble proteins (12).
Several studies on the effects of individual proteins of
saliva have appeared. Protein C002 (aphidbase: ACYPI008617;
National Center for Biotechnology Information [NCBI]
reference sequence [RefSeq]: XP_001948358) was detected in pea aphidinfested host plants, and knockdown of its transcript affected the foraging and feeding
behavior of aphids (13). The role of protein C002 as an
1
Correspondence: Feng Cui, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of
Zoology, Chinese Academy of Sciences, Beijing 100101,
China. E-mail: cuif@ioz.ac.cn. Gerald Reeck, Department of
Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA. E-mail: reeck@ksu.edu
doi: 10.1096/fj.14-266023
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.

effector has been supported by overexpression of green

peach aphid C002 (RefSeq: EC389283) in Nicotiana
benthamiana or Arabidopsis thaliana, which enhanced
green peach aphid colonization (14, 15), and by feeding
aphids on transformed plants producing double-stranded
RNA (dsRNA) of protein C002 that induced a contrary
effect (16). Two candidate effectors, Me10 (RefSeq:
GAAF01000080) and Me23 (RefSeq: GAAF01000028)
from the potato aphid, Macrosiphum euphorbiae, increase
aphid fecundity with their overexpression in N. benthamiana (17). A candidate effector, Mp10 (RefSeq:
ES225905), from the green peach aphid, was found to
induce chlorosis and local cell death in planta (14). An
unknown salivary gland protein (ACYPI39568; RefSeq:
NP_001232999) of A. pisum, belonging to an aphidspecic cysteine-rich protein family, was characterized
for its physical and chemical properties and possible roles
in the aphidplant interactions (18).
Here we report work on a protein, Armet, not previously
studied in aphids. Armet is bifunctional in mammals, both
intracellularly, as a component of the unfolded protein
response in the lumen of the endoplasmic reticulum (ER)
(19, 20), and extracellularly, as a neurotrophic factor. In
the latter role, the protein is typically referred to as mesencephalic astrocytederived neurotrophic factor (MANF)
(21, 22). Mammalian Armet is widely distributed in many
organs and tissues; it is most highly expressed in tissues or
organs that are characterized by especially active protein
secretion (23, 24). In invertebrates, published work on
Armet (MANF) has been limited to Drosophila (RefSeq:
AAF55303), documenting both the neurotrophic role of
the protein (22) and its function as a unfolded protein
response component (25) in that species. We note that
mammals have a MANF homolog called conserved dopamine neurotrophic factor (CDNF), which, in contrast to
MANF, is not increased by ER stress (20).
Our attention was drawn to Armet for 2 reasons. One is
that the pea aphid salivary gland has a much higher level of
the Armet transcript than does the rest of the organism (6).
The other is that pea aphid Armet was predicted to have
calcium-binding ability in an early round of genome annotation. Calcium-binding proteins in aphid saliva are
thought to undermine the calcium-triggered sieve-tube
occlusion mechanism by conversion of proteinaceous
forisomes in sieve elements of legume plants to a contracted state, thus preventing the forisomes from occluding
the sieve tubes (26). Suppression of sieve-tube occlusion by
aphid saliva is a general phenomenon, independent of
aphid species, host plant species, and host family (27, 28).
In this study, we explored both the intracellular and
extracellular roles of Armet (ACYPI008001, RefSeq:
XP_001949541) in the pea aphid, with an emphasis on the
extracellular role of Armet as a component of saliva. Our
results suggest that Armet is an effector protein that mediates the compatible interaction between aphid and plant.

(continued from previous page)

MANF, mesencephalic astrocyte-derived neurotrophic factor;
MS, mass spectrometry; NCBI, National Center for Biotechnology Information; ORF, open reading frame; qPCR,
quantitative PCR; RefSeq, reference sequence; TCEP, Tris (2carboxyethyl) phosphine

Vol. 29

May 2015

MATERIALS AND METHODS

Aphids
Pea aphids were collected from peas (Pisum sativum) in 2010 at
Yuxi, Yunnan Province, China. Offspring of several female adults
were reared on fava beans (Vicia fabae) in incubators at 21 6 1C,
60 6 5% relative humidity, and 16 hour light/8 hour dark photoperiod to form a stable clone named YYC. The experiments
conducted at Kansas State University used aphids maintained on
fava beans, as described elsewhere (29), with a clone of aphids
derived from the LSR1 clone maintained by D. Stern at Princeton
and used in the pea aphid genome project. The YYC clone was
used in the experiments of injection of ER stress inducer, analysis
of aphid survival and feeding behavior after dsRNA injection. The
LSR1 clone was used in the rest of experiments. When necessary,
the developmental stage of aphids was synchronized by collecting
rst instar aphids, then rearing these aphids at 23C for 3 days to
reach the third instar and 7 days to reach adult stage.

Cloning of aphid Armet transcript, protein sequence

analysis, homology modeling, and molecular
evolution analysis
Total RNA was extracted from 16 pairs of salivary glands of adult
aphids using Trizol (Invitrogen, Carlsbad, CA, USA) and then
reverse transcribed into cDNA with SuperScript III rst-strand
synthesis system for RT-PCR (Invitrogen). A pair of primers,
namely, Armet-ORF-F and Armet-ORF-R (Supplemental Data 1),
was designed for amplifying Armet open reading frame (ORF)
from the salivary gland cDNA library. The predicted protein
sequence was analyzed with SignalP (http://www.cbs.dtu.dk/
services/SignalP) and PSORT (http://psort.ims.u-tokyo.ac.jp)
servers for identifying signal peptide. Prediction of possible
O- and N-glycosylation sites was performed online at http://
www.cbs.dtu.dk/services/NetOGlyc and http://www.cbs.dtu.dk/
services/NetNGlyc, respectively. A phylogenetic tree containing
homologous proteins from 31 organisms with complete genomes
was constructed by the maximum likelihood method. Molecular
evolution analysis was carried out using Datamonkey (http://www.
datamonkey.org). Positive selection sites were identied with single
likelihood ancestor counting and xed effects likelihood methods.
Protein expression, purication, and antibody preparation
The nucleotide sequences encoding Armet lacking the N-terminal
secretion signal or its 2 domains individually (N-terminal and Cterminal domains) were constructed in the pET28a vector, with
a hexa-histidine-tag added at the 39 terminus for purication
convenience. Recombinant plasmids were used to transform
Escherichia coli BL21 (DE3) cells for expression after sequence
verication. The supernatant of the sonicated cells was used for
purication. Also, full-length Armet, including the signal peptide,
was inserted in vector pFastBac1 and expressed in the Bac-to-Bac
baculovirus system (Invitrogen) with or without a hexa histidinetag at the 39 terminus. The resulting plasmids, after sequence
verication, plus the blank vector, were used to generate
recombinant baculoviruses. Spodoptera frugiperda Sf9 cells were
infested with the recombinant baculovirus. The cell-free medium
and the supernatant of sonicated cells were kept for purication.
The primers for our constructions are presented in Supplemental
Data 1. The expressed recombinant proteins were puried sequentially with Ni-NTA agarose column (Qiagen, Germantown,
MD, USA) and Q-Sepharose Fast Flow column (Sigma-Aldrich,
St. Louis, MO, USA), and then treated with Chelex-100 resin
(Sigma-Aldrich) to remove the remaining calcium ion. Puried

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WANG ET AL.

protein was served as antigen to produce rabbit anti-Armet polyclonal antibody in Cocalico Biologicals Inc. (Reamstown, PA, USA).
Molecular weight measurement and determination of
disulde bonding pairs by mass spectrometry
Molecular weights of recombinantly expressed proteins were
measured by matrix-assisted laser desorption-ionization timeof-ight mass spectrometry (MALDI-TOF-MS) with a Bruker
Ultraex II (Bruker Daltonics, Fremont, CA, USA) using Bruker
protein molecular weight standard I for molecular weight calibration. Armet was subjected to SDS-PAGE in the absence of
a reducing agent. After subjected to alkylation of free cysteine
residues using 100 mM iodoacetamide, the gel sections were
treated with 100 ng of sequencing-grade trypsin or chymotrypsin.
To measure the digested peptide prole for each sample, 1.0 ml of
the peptide solution was dispensed on 1.0 ml of 30 mg/ml 2,5dihydroxylbenzonic acid (Sigma-Aldrich) onto a stainless steel
target plate. MALDI-TOF spectra were acquired with a Bruker
Ultraex II and selected precursor ions were subjected to TOF/
TOF (MS/MS) analysis with laser-induced fragmentation. The
spectra were processed by FlexAnalysis 3.0 and Biotools software
3.0. The generated peak lists were transferred to ProteinScape 1.3
software for protein identication. Tris (2-carboxyethyl) phosphine (TCEP) was applied to do on-target reduction of disulde
bonded cysteines, which were conrmed by the +2 Da shift of
peptide molecular mass in the spectra.
Measurement of circular dichroism spectra
Circular dichroism spectra were recorded from 190 nm to 260 nm
at 25C on a J-815 spectropolarimeter (Jasco, Tokyo, Japan). The
spectra were an average of 5 consecutive scans at a scan rate of
50 nm/min. All spectra were corrected by subtracting the signal
from the protein-free solutions recorded under the same conditions. Data were recorded as the mean residue ellipticity in
degrees cm2 dmol21.

from the genomic DNA using primers Armet-pro-F and Armetpro-R (Supplemental Data 1). The ERSE region was then deleted
or replaced with a 19 base random sequence CAGCAGTAATACATTTGAA to generate mutant promoters, which were sequenced for verication. The intact or mutant promoters were
cloned into the psiCHECKTM-2 vector (Promega, Madison, WI,
USA), allowing simultaneous expression of Renilla and rey
luciferases, and then the vector was transferred to Drosophila S2
cells. The Armet promoter replaced the SV40 early promoter of
the vector to regulate the expression of Renilla luciferase. The
rey luciferase served as control. ER stress inducer tunicamycin
(2 and 5 mg/ml) or DTT (2 and 3 mM) was added to the cell
cultures. Cells were collected after 24 hours of treatment, and the
activities of rey and Renilla luciferases were measured using the
dual-luciferase reporter assay system (Promega). Light emission
was determined by a modulus microplate multimode reader
(Turner Biosystems, Sunnyvale, CA, USA). Fifteen replicates were
prepared and assayed for each treatment. Differences were evaluated statistically by SPSS 17.0 (IBM, Armonk, NY), either by t test
to compare 2 means, or by 1-way ANOVA followed by a Tukeys
test for multiple comparisons.
Injection of ER stress inducer into pea aphids
The ER stress inducer tunicamycin was injected in the third instar
stage of the aphids. Exactly 23 nl of tunicamycin (about 0.5 mg/ml
in water) was injected in each aphid, which caused 30% to 40%
mortality over 3 days of treatment. The mortality of control
aphids that were injected with 23 nl of water was approximately
20% over 3 days. Ten surviving aphids as one biologic replicate
were collected on the third day; 5 replicates were conducted.
Armet transcript levels in the treated aphids were compared with
those in the aphids injected with water using quantitative PCR
(qPCR). Ribosomal protein L27 transcript (RefSeq: CN584974) was
amplied as internal control (Supplemental Data 1). qPCR was performed on a Roche LightCycler 480 (Roche, Mannheim, Germany).
Differences were evaluated by t test statistically by SPSS 17.0.
Treating insect cells with ER stress inducers

Analytical ultracentrifugation
Sedimentation velocity was conducted at 20C using an Optima XL-I
ultracentrifuge (Beckman Coulter, Fullerton, CA) with an An-60 Ti
rotor. Recombinant Armet at 0.4 mg/ml was buffered in 20 mM
Tris-HCl and 120 mM NaCl at pH 8.0. Sedimentation was monitored
at A280 for 120 minutes at 5 minute intervals. Data were analyzed
using DCDT1 software 1.16 (http://www.jphilo.mailway.com).
Calcium binding assay of pea aphid Armet and its 2 domains
The binding of Ca2+ to Armet or its domains was examined by
isothermal titration calorimetry using the MCS-ITC system
(MicroCal, Northampton, MA) at 30C. Recombinant Armet, its Nand C- terminal domains, and CaCl2 were buffered in 20 mM TrisHCl and 120 mM NaCl at pH 8.0. Armet (48 mM) was titrated with
3 mM CaCl2. The N-terminal domain (50 mM) was titrated with
3.5 mM CaCl2, and the C-terminal domain (100 mM) was titrated
with 15 mM CaCl2. Data were tted to possible binding models, and
thermodynamic parameters were determined from nonlinear leastsquares tting using Origin ITC software supplied by MicroCal.
Dual luciferase assay
A 427 base region containing an endoplasmic reticulum stress
response element (ERSE) motif CCAATN9CCAAG was cloned

ARMET MEDIATES APHIDPLANT INTERACTIONS

S2 and Sf9 cells were treated with 2 mM or 5 mM DTT (dissolved

in water) and 5 mg/ml or 10 mg/ml tunicamycin (dissolved in
DMSO) for 12, 24, or 48 hours. The transcript levels of the Drosophila melanogaster Armet and S. frugiperda Armet (RefSeq:
DY898745) in S2 and Sf9 cells, respectively, were quantied using
qPCR. The ribosomal protein L27 transcripts of D. melanogaster
(RefSeq: NM_143160) and S. frugiperda (RefSeq: AF400191), as
internal controls, were quantied (Supplemental Data 1). Differences were statistically evaluated by t test by SPSS 17.0. Three
biologic replicates of the cells were collected for assay.
Protein preparation from phloem sap of plants
and aphid saliva
Fava bean plants (13 days old) were infested with aphids for 2 days,
then washed 3 times in water. Ten leaves with petioles were cut off
and each immediately placed in 2.5 ml of 20 mM EDTA, pH 8.0, in
one well of a 12-well tissue culture plate in the dark. After 4 hours,
the buffer-containing phloem sap was collected and passed
through a 0.2 mm lter. Phloem sap from plants not infested by
aphids was collected in the same way, as the control. Cooled trichloroacetic acid was added to the collected phloem sap to attain
15% nal concentration and kept at 220C for at least
30 minutes, and then at 4C overnight. After centrifugation for
10 minutes at 12,000 3 g, the precipitate was washed 3 times with
cold acetone and then dissolved in a 1:1 (v/v) mixture of solution

guts using qPCR. The change in protein level of Armet in heads

(containing salivary glands) at the third day after injection of
dsRNA was examined by Western blot analysis.

A (phenol, pH8.0) and B (0.1 M Tris-HCl, 2% SDS, 30% sucrose,

5% b-mercaptoethanol). The supernatant (phenol phase) was
retained after centrifugation, mixed with 5 volumes of 0.1 M
ammonium acetate in methanol, and incubated for 30 min at
220C and then at 4C overnight. The precipitate was collected
by centrifugation, washed 3 times with cold acetone, and solubilized in SDS-PAGE loading buffer.
To collect aphid saliva, about 1000 aphids were reared on articial diet (30) for 2 days at room temperature under a 16 hour
photoperiod. The diet was sealed between 2 layers of Paralm in
a petri plate (150315 mm). Saliva-containing diet from 8000 aphids
(from 8 petri plates) was collected after feeding, passed through
0.2 mm lter, and dialyzed against 20 mM Tris-HCl, pH 8.0, overnight. Protein was precipitated with 20% trichloroacetic acid. After
washing 3 times with cold acetone, the precipitate was dissolved in
SDS-PAGE loading buffer. Diet that had not been fed on was processed in the same way as control. The gelling saliva was collected
and proteins in the gelling saliva were viewed in a silver-stained SDSPAGE gel following the method presented by Will et al. (31).

The electrical penetration graph (EPG) technique was used to

analyze the feeding behavior of individual aphids on fava bean
plants according to a procedure previously described (13). The
feeding behavior of dsGFP-RNA-injected aphids and dsArmetRNA-injected aphids was monitored continuously for 8 h on the
third day after injection. Valid data sets from 23 and 29 aphids
were collected for the dsGFP-RNA and dsArmet-RNA groups,
respectively. The EPG waveforms were recorded by a Giga amplier series, GIGA-8 model (EPG-Systems, Wageningen, The
Netherlands) and analyzed with the software Stylet+ according to
the manufacturers manual and the information from Tjallingii
(5). Differences between the 2 groups were analyzed statistically
by independent-sample t test using SPSS 17.0.

Western blot analysis and immunohistochemistry

Armet protein injection from leaf petiole

In vitro detection of Armet protein in the phloem sap, aphid saliva,

and aphid heads (containing salivary glands) was performed with
Western blot analysis using puried anti-Armet polyclonal antibody and X-ray exposure method. Immunohistochemistry was
carried out to localize in vivo the expression of Armet in aphid
salivary glands. Detailed experimental steps have been described
in a previous study (13).
Comparison of Armet or C002 transcript in diet-fed
and plant-fed pea aphids
Adult pea aphids were placed on a healthy fava bean leaf inserted
into sterilized 2% agar [supplemented with 0.1% miracle growth
fertilizer (Miracle-Gro, Marysville, OH, USA) and 0.03% methyl
4-hydroxybenzoate] in a petri plate for feeding for 24 hours.
Another group of adult aphids were transferred to an articial
diet (30) in a sachet for feeding for 24 hours. The transcript levels
of Armet or C002 in the heads (containing salivary glands) of dietfed and plant-fed pea aphids were compared through qPCR.
Actin transcript (RefSeq: NM_001142636) was amplied as internal control (Supplemental Data 1). Forty-ve to 50 aphid
heads were used for a measurement, and 3 replicates were performed. Differences were evaluated by t test using SPSS 17.0.
dsRNA synthesis and delivery
A 286 and 439 bp dsRNA of Armet and green uorescent protein
(GFP), respectively, were generated using T7 RiboMAX Express
RNAi System (Promega) and puried using Wizard SV Gel and
PCR Clean-Up System (Promega) following the manufacturers
protocol. Injection of 23 nl of dsRNAs at 6 mg/ml was performed
on the third instar stage of aphids. The dsRNAs were delivered
into hemolymph from dorsal abdomens by microinjection
through a glass needle at slow speed using Nanoliter 2000 (World
Precision Instruments, Sarasota, Florida, USA). Six groups of
aphids and 15 individuals in each group were injected and then
reared on fava bean plants or an articial diet for 9 days (31).
Survival rates were recorded every day and reported as means 6
SE. The survival curves of interference and control groups were
compared for statistical differences using the log-rank (MantelCox) test (SPSS 14.0). Four groups of aphids and 5 aphids in each
group were collected on the rst and third days after injection of
dsRNA for testing inhibition efciency of Armet transcription in
aphid whole bodies or in heads (containing salivary glands) and

Vol. 29

May 2015

Analysis of aphid feeding behavior

Approximately 2.5 mg of puried pea aphid Armet, recombinantly expressed in E. coli, in 50 ml buffer (20 mM Tris-HCl, 120 mM
NaCl, pH 8.0) was injected through leaf petiole in the abaxial
side of the leaves of 1 mo old tobacco N. benthamiana using a 1 ml
sterile syringe and a 0.4 3 13 RWLB needle (Shanghai Misawa
Medical Industry, Shanghai, China). An equal volume of puried
product from pET28a empty vector was injected as control. The
leaves from the treatment group and the control group were
collected for RNA extraction after 60 hours.
Transcriptomic analysis of N. benthamiana
One-month-old tobacco N. benthamiana was used for inltration
of puried pea aphid Armet protein that has been expressed
recombinantly in E. coli. Five micrograms of pea aphid Armet in
100 ml of buffer (20 mM Tris-HCl, 120 mM NaCl, pH 8.0) was
inltrated in 1 leaf, and 2 leaves were treated in each plant.
Control leaves were inltrated with 100 ml of buffer. Eight leaves
from the treatment group and the control group were collected
for RNA extraction after 60 h. Total RNA was sent to BGI
Shenzhen, China, for RNA-Seq analysis using the Illumina HiSeq
2000 sequencer. At least 10 million 49 bp clean reads were
obtained for each sample. High-quality reads were mapped to
N. benthamiana gene set using SOAP2. The reads per kilobase of
transcript per million reads mapped values are used for comparing the difference in transcript levels between samples. A false
discovery rate of #0.001 and the absolute value of log2 ratio $1
were used as a threshold to judge the signicance of the gene
expression difference. Unigenes were assigned to Kyoto Encyclopedia of Genes and Genomes pathways. Twenty up-regulated genes
(with initials of Nb in the N. benthamiana gene set) of the plant
pathogen interaction pathway were veried with qPCR in the
petiole injection groups of Armet or the pET28a empty vector
control (Supplemental Data 1). The elongation factor 1a
(NbS00007372g0013) was quantied as an internal control. Six
to 8 replicates and 8 leaves in each replicate were prepared for
qPCR verication.

RESULTS
Armet gene, transcript, and predicted protein in
pea aphid
The Armet gene is located in scaffold 17420 of the pea
aphid genome. By aligning with Armet expressed

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WANG ET AL.

sequence tags, the gene is found to have 4 exons (of 94,

122, 142, and 167 bases) partitioned by 3 introns (of 114,
156, and 79 bases). A 58-base intron is observed in the 59
UTR, separating this region into 130- and 44-base sections
(Fig. 1A). A putative cis-acting ERSE, CCAATN9CCAAG,
occurs 76 bases upstream of the transcription start site (Fig.
1A), and differs only at one nucleotide (the underlined A)
from the canonical ERSE sequence in mammals (32). The
ORF of the Armet transcript contains 522 bases and encodes a protein of 174 amino acid residues, 8 of which are
cysteines (Fig. 1B). SignalP and PSORT predict an Nterminal signal secretion peptide with cleavage predicted
either between Ala20 and Gln21 (SignalP) or between Ser22
and Arg23 (PSORT). The proteins sequence includes
KEEL at its C-terminus, a putative ER retention signal, and
no predicted O- or N-glycosylation sites.
Molecular evolution of Armet
Using BLAST analysis, transcripts homologous to pea
aphid Armet were identied in many animal species, from

invertebrates to mammals. A related protein, CDNF,

occurs in vertebrates, whereas only Armet exists in invertebrates (Fig. 2). Common features of Armet across
invertebrates and vertebrates include a secretory signal
peptide at the N-terminus, a KDEL-like ER retention motif
at the C-terminus, and 8 cysteines (Supplemental Data 2).
For Armet, no positive selection sites with P , 0.05 were
identied, but we found positive selection in the codon
encoding Leu62 in CDNF (Supplemental Data 2), in which
the dN-dS value was 2.36 (P = 0.08) based on the single
likelihood ancestor counting method and 2.24 (P = 0.02)
based on the xed effects likelihood method.
Characterization of recombinantly expressed pea
aphid Armet
Armet (excluding the signal peptide) was expressed in
E. coli and puried, as were the proteins N- and C-terminal
domains individually (Supplemental Data 3A, B). The circular dichroism spectrum of pea aphid Armet exhibited
minima at 208 and 222 nm (characteristic of high helix

Figure 1. Nucleotide sequence of pea aphid

Armet gene region. A) 59-Upstream region. The
transcription start site is marked with an arrow;
ERSE is enclosed in a box. The 58 base intron is
underlined. B) The 522-base ORF with the
encoded amino acid sequence. The amino acid
residues of the signal secretion peptide are in
red. The KDEL-like ER retention motif is
underlined by double strands. The linker between domains is enclosed in brackets. Eight conserved cysteines are marked in green.

ARMET MEDIATES APHIDPLANT INTERACTIONS

Figure 2. Phylogenetic tree for Armet and CDNF from invertebrates and vertebrates. The tree was constructed using the
maximum likelihood method based on the alignment of amino acid sequences. The bar indicates branch scale and genetic
distance. The RefSeq numbers in NCBI are indicated.

content) and was unaffected by the presence of calcium

(Fig. 3A). Analysis of sedimentation velocity revealed that
pea aphid Armet exists as a monomer with a sedimentation
coefcient of 1.70 S (Fig. 3B). The frictional coefcient
ratio (f/f0 = 1.45) ts a somewhat extended globular
structure (Fig. 3C).
Thermodynamic properties of calcium binding to pea
aphid Armet were determined by isothermal titration calorimetry. The titration data t best to a 1-site modelthat
is, to binding at a 1:1 molar ratio (Fig. 3D). The dissociation
constant Kd was estimated to be 240 6 12 mM. Armets
binding of Ca2+ was driven mostly by a change in enthalpy
(DH = 25.9 6 0.2 kcal/mol) rather than entropy (TDS =
0.9 kcal/mol). When either the N- or C-terminal domain or
the 2 domains, mixed, were titrated with Ca2+ under the
same conditions, no binding was observed.
No suggestion of intermolecular disulde bonding between Armet molecules was observed from the SDS-PAGE
performed in the absence of mercaptoethanol, and no free
cysteine residue was detected by MS analysis. Three disulde bonds, namely Cys82Cys11, Cys822Cys93, and
Cys1272Cys130, were identied after digestion of the
protein with trypsin, and a fourth disulde bond, Cys392
Cys51, was identied in chymotrypsin digests (Fig. 3EH).
6

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May 2015

Response of Armet from 3 insect species to ER stress

Transcription driven by the promoter of the pea aphid
Armet gene was examined in Drosophila S2 cells using a dual
luciferase assay. Transcription was signicantly increased
by the treatment of cells with either DTT (a reducing agent)
or tunicamycin (an N-glycosylation inhibitor), whereas no
signicant increase in transcriptional activity was observed
after deletion of ERSE or its replacement with a random
sequence (Fig. 4A).
An aphid cell line is not available, so we used insect cell
lines S2 and Sf9 to examine their Armet transcript levels
with and without ER stress. Normalized levels of Drosophila
Armet transcript in S2 cells ranged from about 5-fold to
over 15-fold higher in the presence of DTT (Fig. 4B), and
from about 2-fold to 3-fold higher in the presence of
tunicamycin (Fig. 4C). In Sf9 cells, the increase in normalized Spodoptera Armet transcript levels was somewhat
less than 4-fold in the presence of DTT (Fig. 4D) and up to
10-fold in the presence of tunicamycin (Fig. 4E).
To impose ER stress throughout aphids, tunicamycin
was injected into pea aphids at a level that caused
30% to 40% mortality over 3 days of treatment. After 3
days, the normalized transcript level of the pea aphid

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WANG ET AL.

Figure 3. Biophysical and biochemical characterizations of pea aphid Armet. A) Far-ultraviolet circular dichroism spectra of pea
aphid Armet in 20 mM Tris-HCl, 120 mM NaCl (pH 8.0), and EDTA and CaCl2 as indicated. B) Sedimentation velocity proles of
analytical ultracentrifugation of pea aphid Armet in 20 mM Tris-HCl, 120 mM NaCl (pH 8.0). Scans at A280 across the cell at
5 min intervals are shown. Direction of sedimentation is to the right. These are the primary data from the measurement. C) Timederivative analysis of the sedimentation velocity proles using DCDT1 software (see Methods). The y-axis is the time derivative of
the protein concentration, in units of ABS/s, as a function of sedimentation coefcient, s*, in units of svedbergs. The solid curve
is the result of a least-squares t to the data for a single, homogenous species with a sedimentation coefcient of 1.70 S. The
excellent t to the time-derivative data indicates that the protein is monodisperse, without tendency to aggregate. D) Isothermal
titration calorimetry of pea aphid Armet with Ca2+. At top is heat release with incremental addition of CaCl2 to recombinant pea
aphid Armet; at bottom are data tted with best-t single-site binding model. E) MS prole of TFTEEDCPVCVLTIDK peptide by
tryptic digestion. F) MS prole of CLSTKIDKEKRLCY peptide by chymotryptic digestion. G) MS prole of ICERLKKMDAQVCDIK
(continued on next page)

ARMET MEDIATES APHIDPLANT INTERACTIONS

Figure 4. Response of Armet from 3 insects to ER stress. A) Dual luciferase reporter gene for the analysis of transcription driven
by pea aphid Armet promoter with intact, deleted, or replaced ERSE region in S2 cells. The activity of the Renilla luciferase is
divided by the activity of the rey luciferase. This ratio of luciferase activities is obtained at different DTT and tunicamycin
concentrations as indicated, then divided by the same ratio in the absence of DTT and tunicamycain (i.e., the control ratio of
luciferase activities). This value, compared with the control value, is reported as mean 6 SE. *P , 0.05 for the difference between
the treatment and the control. Letters above bars indicate results of multiple comparisons among the 3 types of promoters. BE)
Transcript level ratios of Drosophila Armet and Spodoptera Armet in S2 cells or Sf9 cells after the treatment of cells from 12 to 48 h
with DTT or tunicamycin relative to control (in DTT-free cells or in DMSO-treated cells). Drosophila and Spodoptera Armet
transcript levels were measured by real-time PCR and normalized with the transcript level of respective ribosomal protein L27.
Ratios are reported as mean 6 SE. *P , 0.05, **P , 0.01 for the difference between the treatment and the control.

Armet had increased from 0.040 6 0.003 (SE) to 0.077 6

0.008 (SE) (P , 0.05).
Pea aphid Armet is secreted
When full-length Armet, including the signal secretion
peptide, was expressed in Sf9 cells, with or without a His tag
at the C-terminus, Armet was detected in both cell lysate and
the culture medium, indicating that some Armet protein is
secreted (Fig. 5A). The molecular mass of the secreted
Armet (carrying a His tag at the C-terminus) in the medium
of Sf9 cells was found to be 18,556 Da (Supplemental Data
3C, D), which is consistent with cleavage of the signal peptide
between Ser22 and Arg23. MALDI-TOF-MS analysis of tryptic
peptides revealed the peptide RTFTEEDCPVCVLTIDK,
thus conrming that cleavage site (Fig. 5B).
Immunohistochemistry using antipea aphid Armet
polyclonal antibody revealed that Armet occurs preferentially in a symmetrically disposed pair of large secretory
cells in middle of the 2 lobes of principal salivary glands

(Fig. 5C). Knockdown of Armet transcription by dsRNA

greatly reduced the immune signal in those middle large
secretory cells (Fig. 5C). Using immunoblotting, a 17.7 kDa
band, the size of secreted Armet, appeared in watery saliva
(800 aphids), head extract (10 aphids), and phloem sap of
fava beans fed on by aphids (800 aphids), but not in
phloem sap from fava beans not exposed to aphids or
gelling saliva (800 aphids) (Fig. 5D).
We conclude that pea aphid Armet is a component of
aphid watery saliva and is secreted into fava beans during
the feeding process.
Armet promotes pea aphid feeding on plants
The transcript levels of Armet and of protein C002,
a protein that is required for the feeding of pea aphid on
fava bean plants (13), were measured in RNA isolated
from aphid heads (containing salivary glands). The normalized level of the Armet transcript was about 3.5-fold
higher in plant-feeding aphids compared with the level

peptide by tryptic digestion. H) MS prole of ILDNWGEICDGCLEK peptide by tryptic digestion. The proteolytic peptide of
Armet was subjected to MALDI-TOF-MS analysis before (top) and after (bottom) reduction with TCEP. The shift of 2 amu mass
after reduction with TCEP indicates addition of 2-H atoms. Cysteines are underlined.

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May 2015

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WANG ET AL.

Figure 5. Pea aphid Armet is secreted. A) Western blot assay of pea aphid Armet expressed in insect Sf9 cells using antipea aphid
Armet polyclonal antibody. B) MALDI-TOF-MS spectrum of Sf9-expressed pea aphid Armet in cell medium after trypsin
digestion. The peptides in red are conrmed further with MALDI-TOF-TOF. C) Immunohistochemical localization of Armet in
pea aphid salivary glands (YYC clone) using puried antipea aphid Armet antibody before or after dsArmet-RNA injection on
the third day. Red is the positive signal. Nuclei are shown in blue. PG, principal gland; AG, accessory gland. Negative control is
depleting the anti-Armet antibody by pretreatment with recombinant Armet for the immunohistochemistry. D) Western blot
analysis of Armet in aphid saliva (LSR1 and YYC clones) and plant phloem sap fed by aphids using antipea aphid Armet
polyclonal antibody. 1, aphids Armet expressed in the medium of insect Sf9 cells; 2, aphids gelling saliva collected from articial
diet fed by aphids; 3, aphids watery saliva collected from articial diet fed by aphids; 4, aphids heads; 5, phloem sap of fava bean
fed by aphids; 6, phloem sap of fava bean without aphids infestation; 7, articial diet without aphids infestation.

in diet-fed aphids (Fig. 6A). The normalized level of

the protein C002 transcript was 6-fold higher in plantfeeding aphids compared with that in diet-fed aphids (Fig.
6A). Both values suggest a major increase in synthesis of
proteins of saliva when aphids are moved from liquid diet
to plants.
When dsArmet-RNA was injected into pea aphids, the
Armet transcript level in the aphid whole bodies was reduced by 39 6 10% and 34 6 6% at 1 and 3 days after
injection, respectively (Fig. 6B). The knockdown was as
high as 48% in aphid heads (containing salivary glands),
while no change was observed in aphid guts 3 d after injection (Fig. 6C). A decrease in Armet protein was observed
in the heads (containing salivary glands) of the knockdown
insects (Fig. 6D). Injection of dsArmet-RNA signicantly
ARMET MEDIATES APHIDPLANT INTERACTIONS

reduced the life span of aphids feeding on host plants (P ,

0.001) (Fig. 6E) but did not inuence that of aphids
feeding on articial diet (P = 0.08) (Fig. 6F).
Knockdown of the Armet transcript led to several statistically signicant changes in the feeding behavior of pea
aphids on fava bean plants as monitored by the EPG
technique (Fig. 6G). Most notably, the total (average)
length of E2 (passive ingestion from sieve elements) was
reduced to 50 min from 190 min in control insects (Supplemental Data 4). Furthermore, the dsArmet-injected
aphids showed a higher frequency of E1 (watery salivation)
resurgence and E2/E1 transition than the dsGFP-injected
aphids, as illustrated in Fig. 6G, where 4 E2/E1 transitions
were observed in the dsArmet-injected aphid. This suggests
that aphids have to secrete more saliva for phloem feeding
9

Figure 6. Armet promotes aphid feeding on plants. A) Armet and protein C002 transcript levels in articial diet-fed and fava
bean-fed pea aphids (LSR1 clone) that were normalized with actin transcript. Values are reported as mean 6 SE. ***P , 0.001. B,
C) The Armet transcript levels in aphid whole bodies, in heads that contain salivary glands, or in guts at the third day after dsRNA
injection (YYC clone). *P , 0.05. Interference ratios are indicated above the bars. D) Western blot analysis of Armet in aphid
heads (YYC clone) that contain salivary glands at the third d after dsRNA injection using antipea aphid Armet polyclonal
antibody. E, F) Survival graphs of aphids (YYC clone) on fava beans or on articial liquid diet after injection of dsArmet-RNA or
dsGFP-RNA. G) Representative EPG waveforms of dsArmet-injected or dsGFP-injected (as control) aphids (YYC clone) on fava
beans. np, nonprobing; pd, potential drop; C, pathway phase; E1, watery salivation; E2, passive ingestion.

with decreased levels of Armet in saliva. Thus, Armet is

needed to sustain phloem feeding to normal duration.
Correspondingly (and necessarily, given the time-limited
design of the experiments), the combined lengths of the
nonprobing and pathway components were increased in
the knockdown insects (Supplemental Data 4).
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Vol. 29 May 2015

Pea aphid Armet induces antipathogen reaction in

tobacco plants
We inltrated E. coli recombinantly expressed and puried
pea aphid Armet into leaves of tobacco, N. benthamiana,
a nonhost plant but the species that is best established for

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WANG ET AL.

inltration experiments (33). The transcriptome of tobacco was compared between Armet-inltrated and bufferinltrated leaves. Compared with the latter, 144 genes in
the plantpathogen interaction pathway were up-regulated
in the Armet-inltrated plants. These genes were mainly
categorized as kinases (45 genes), WRKY transcription
factors (28 genes), calcium-binding proteins (11 genes),
and disease resistance proteins (6 genes), along with a large
group of uncharacterized proteins (31 genes). qPCR was
performed to verify the expression levels of 20 genes in the
plantpathogen interaction pathway when the puried
product from pET28a empty vector was used as negative
control. Among the 20 genes, 18 were conrmed to be upregulated in the Armet-delivered group relative to the
pET28a empty vector group (Fig. 7).
DISCUSSION
Aphids deliver proteins in their saliva to their host plants
during feeding. Although numerous proteins of aphid saliva have now been identied (6, 9, 10), we are still in the
early stages of studying individual proteins to unravel their
modes of action. Many such studies on individual proteins
will be needed in order to develop meaningful insight into
the molecular basis of aphidplant interactions.
Here we report work on the intracellular and extracellular roles of the protein Armet, which occurs in saliva of
the pea aphid. In a broad sense, pea aphid Armet appears
similar to mammalian Armet in that the protein serves both
intracellular and extracellular roles in the aphid and in
mammals. We present a physicochemical characterization
of the aphid protein and demonstrate that its promoter is
responsive to ER stress. Our greater emphasis, however, is
on a novel extracellular role of the protein as a secreted
effector protein that facilitates successful aphid feeding on
host plants. Interference with Armet expression in the
aphid undermined the compatible interaction between
aphid and plant.
Our focus here on the role of Armet in saliva is not
intended to imply that the extracellular, neurotrophic role
of Armet is absent in aphids. It is clear from gene knockout
studies that the protein (under its alternative name,
MANF) acts on the nervous system in another insect,
namely D. melanogaster (22). Although we have no direct
evidence for a neurotrophic role of Armet in the pea
aphid, it can be assumed that such a role exists. Extrapolations from the results on Drosophila to the pea aphid
system would be hazardous at best, not only because of the
evolutionary distance between ies and aphids, but also
because of the very different methods used (gene knockout in Drosophila, transcript knockdown in pea aphid) and
different developmental stages studied (embryogenesis in
Drosophila, adult aphids in our work). We believe the most
likely interpretation of our results is that the effects of
Armet transcript knockdown are due to decreased production of Armet as a component of saliva, based partly on
comparable results (in altered feeding and reduced life
span) obtained by transcript knockdown for another effector protein in pea aphid, protein C002 (13, 29).
Protein C002 is perhaps the most clearly identied
effector-protein in aphids (1316, 29), and it is therefore
interesting to compare some of the results reported here
ARMET MEDIATES APHIDPLANT INTERACTIONS

on Armet with earlier results on protein C002. Both proteins occur in subsets of secretory cells of the principal
salivary glands, but those subsets are different for protein
C002 and Armet. Armet occurs in a pair of large secretory
cells in middle of the 2 lobes of principal salivary glands,
while protein C002 is present in several other secretory
cells (13). We suggest that a preferential expression of
a protein in a subset of secretory cells of the salivary gland
may be a hallmark of saliva proteins. As regards the effects
of transcript knockdown, in each case, the life span of
knockdown aphids feeding on fava beans is reduced
compared to control insects, but the effect is less marked in
the case of Armet. Correspondingly, there are differences
in the effects of transcript knockdown on feeding behavior,
as shown by EPG. Both the total duration of passive ingestion and the total duration of pathway phase are affected in the Armet-knockdown insects, whereas only the
total duration of passive ingestion is affected in protein
C002knockdown insects, leading essentially, in that case,
to a great reduction in sustained phloem feeding in the
knockdown insects. Thus, the role of Armet as effector
appears to be signicantly different from the role of protein C002 as effector.
An important tool in this work is knockdown of the
Armet transcript in the pea aphid salivary gland by injection of dsArmet RNA. We have previously obtained efcient knockdown of the transcripts encoding protein
C002 and the cysteine-rich protein ACYPI39568 by the
same method (13, 18, 29). It appears that dsRNA injection
into the hemolymph of the pea aphid is quite effective in
transcript knockdown in the salivary gland and considerably less effective in other organsfor instance the gut,
which also has relatively high Armet transcript levels (Fig.
6C) but did not incur a statistically signicant knockdown.
Our interpretation of the phenotype resulting from
knockdown of the Armet transcript (i.e., altered EPG patterns, reduced life span) is that these are most likely due to
transcript knockdown in the salivary glands.
As do other plant pathogens or parasites, aphids must
secrete bioactive effector proteins into host plants to
modulate the plant defense response and allow successful
feeding (14). These effectors are almost certainly introduced into the plant as a part of the aphid saliva, and
plant responses typical of pathogen effectors (e.g., chlorosis, necrosis) have been associated with some proteins of
aphid saliva (14, 17). Plant defense responses to aphid
feeding have been characterized in several systems and
often involve induction of elements of the salicylic acid,
jasmonic acid, and ethylene defense response pathways
(3436). Jasmonic acid appears to be most important in
regulating effective defense responses in incompatible
aphidplant interactions (37, 38). It has been demonstrated that some components of aphid saliva elicit local
defenses even in compatible interactions (39), and hence
other components of aphid saliva must act to effectively
neutralize these plant defenses (40). Inltration of pea
aphid Armet into tobacco, a nonhost, induced a transcriptional response that included many genes associated with
pathogen infection, suggesting that Armets function may
induce rather than suppress defenses. One possible way of
interpreting these results is to imagine Armet (in a host
plant) as being a double-edged swordon the one hand,
an agent that is useful or required to sustain feeding from
11

Figure 7. Real-time qPCR verication of the transcript levels of 20 plantpathogen interaction genes of Nicotiana benthamiana
when Armet was delivered by petiole injection. The puried product from pET28a empty vector was used as negative control. *P ,
0.05; **P , 0.01.

sieve elements, but on the other hand, an agent that also

stimulates plant defense mechanisms. However, it is also
possible that Armet might elicit plant defenses in a nonhost even if its function in a compatible plant is to suppress
12

Vol. 29 May 2015

host defenses. Additional functional work is required to

clarify Armets effector function.
One of the important phloem-related mechanisms of
plants that aphids have to overcome is an occlusion

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WANG ET AL.

response to injury in the sieve tubes. It has been shown that

calcium binding by components of aphid saliva may undermine a calcium-triggered occlusion mechanism (26).
We have shown here that pea aphid Armet binds calcium,
despite the absence of any recognized calcium-binding
motif in its sequence. Thus, we should consider the possibility that Armet might conceivably take part in counteracting the calcium-triggered occlusion mechanism in
plants. However, any physiologically signicant effect of
calcium binding by Armet (or, for that matter, by any other
calcium-binding protein in phloem sap) would have to be
highly localized because the molar concentrations of individual aphid proteins in phloem sap are undoubtedly
very low, and thus the effect of calcium binding by such
a protein on global calcium concentration in phloem sap
would be negligible. Such localization would result from
specic interactions between a calcium-binding protein and
other components (presumably proteins) in sieve elements.
A potentially important aspect of our studies on pea
aphid Armet is the pairing of cysteine residues in the proteins disulde bonds. Actually, this is an issue that is already represented, but has never been discussed, in the
literature on the structure of mammalian Armet. Studies of
the 3-dimensional structures of human (RefSeq: NP_006001)
and mammalian Armets (4143) report disulde bonds in
the following pairings: Cys8-Cys93, Cys11-Cys82, Cys39Cys51, and Cys127-Cys130. (For convenience, we use the
numbering for the pea aphid Armet sequence, with Arg of
the sequence RTFTEED taken as residue 1 of the mature
protein.) It is interesting to note, however, that a MS-based
approach on mouse Armet (RefSeq: NP_083379) (19)
revealed 2 differences from the pairings listed above
namely, the existence of Cys8-Cys11 and Cys82-Cys93.
These 2 pairs are in fact what we have found here for
pea aphid Armet, also using an MS approach. Both
approachesthe elucidation of Armets 3-dimensional
structure and MS of Armet peptidesare valid; neither
supplants or invalidates the other as regards the disulde
bonding pattern. We therefore hypothesize that Armet,
whether mammalian or insect in origin, has alternative
disulde arrangements in a portion of the N-terminal
domain, both of which exist simultaneously in a population of Armet molecules. Cys8, Cys11, Cys82, and Cys93
are all clustered in the 3-dimensional structure of Armet,
and both disulde pairings that have been reported are
entirely feasible spatially. It seems that the different
methods of identifying the disulde pairings in this region
preferentially detect one of the 2 forms. The structure
determinations would emphasize the major pairings, and
the MS approach could detect the minor pairings preferentially. The hypothesis of alternative disulde pairings
clustered in the N-terminal domain, if validated, could be
important in understanding the function of Armet intracellularly, extracellularly, or both.
This work was supported by grants from the Strategic
Priority Research Program of the Chinese Academy of
Sciences (Grant XDB11040200), the Major State Basic
Research Development Program of China (973 Program;
Grant 2012CB114102), the Cooperative Research Centre for
National Plant Biosecurity (Australia), the Grains Research
and Development Corporation (Australia), and the Kansas
Agricultural Experiment Station (publication 15-035-J).

ARMET MEDIATES APHIDPLANT INTERACTIONS

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The FASEB Journal x www.fasebj.org

Received for publication October 14, 2014.

Accepted for publication December 23, 2014.

WANG ET AL.