JOURNAL OF VIROLOGY, Jan. 2008, p. 291–299
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 1
Coupling Genetics and Proteomics To Identify Aphid Proteins
Associated with Vector-Specific Transmission of
Xiaolong Yang,1,2,3T. W. Thannhauser,3Mary Burrows,4Diana Cox-Foster,2
Fred E. Gildow,1and Stewart M. Gray4*
Departments of Plant Pathology1and Entomology,2The Pennsylvania State University, University Park, Pennsylvania 16802;
Plant Protection Research Unit, USDA, ARS, and Department of Plant Pathology, Cornell University, Ithaca,
New York 148534; and Functional and Comparative Proteomics Center, USDA-ARS Plant, Soil,
and Nutrition Laboratory, Ithaca, New York 148533
Received 9 August 2007/Accepted 15 October 2007
Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and
Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from
the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow
virus to transverse the gut and salivary tissues. Studies of F2progeny from crosses of vector and nonvector
genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple
genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically
linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and
nonvector parental and F2genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated
with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with
purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry
identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular
transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic
system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically
inherited and conserved in different generations of vector genotypes and suggests that they play a major role
in regulating polerovirus transmission.
Viruses in the family Luteoviridae, including Barley yellow
dwarf virus (BYDV), Cereal yellow dwarf virus (CYDV), Potato
leafroll virus, and Beet western yellows virus, are collectively
referred to in this paper as luteovirids. They are transmitted in
a circulative persistent nonpropagative manner only by aphids
(Aphididae: Hemiptera) (20). Ultrastructural studies indicate
that all luteovirids follow a similar pathway through their aphid
vectors (12). Aphids acquire the viruses from infected phloem
cells while feeding with their piercing-sucking stylets. Virions
are drawn up the food canal of the stylets and into the gut
lumen within the aphid. Subsequently, virions traverse the lin-
ing of the midgut, hindgut, or both (7, 8, 25) and are released
into the body cavity (hemocoel) to circulate in the hemolymph.
Virions suspended in hemolymph that contact the paired ac-
cessory salivary glands (ASG) are actively transported by en-
docytosis into the ASG cells and then transported into the
salivary duct to be transmitted into potential host plants. In-
terestingly, each luteovirid species is transmitted most effi-
ciently only by a specific set of aphid species or populations
within an aphid species, thus demonstrating a high level of
vector specificity. The cellular mechanisms responsible for vec-
tor specificity are regulated by distinct interactions between the
two virus structural proteins and unknown proteins in the
The discovery of Gildow and Rochow (9) that competition
occurred between serologically related luteovirids for transmis-
sion by a common vector supported the hypothesis that recep-
tor-mediated endocytosis is the mechanism that regulates the
vector-specific transmission of each luteovirid species. Subse-
quently, three cellular sites in aphids that are associated with
differential virus passage that may determine vector transmis-
sion ability have been identified (reviewed in reference 12).
Potential barriers to transmission occur at the gut and two
distinct sites at the ASG, the basal lamina surrounding the
ASG, and the basal plasmalemma of each of the four cells that
comprise each gland. The collective information thus far sug-
gests that aphids possess specific protein receptors to facilitate
binding and endocytosis on the surfaces of the gut endothelial
cells, on the ASG basal lamina, and on the ASG cell plasma-
lemma. Additional aphid proteins likely regulate the transport
of virions across the cell cytoplasm and their subsequent re-
lease into the hemolymph or salivary duct.
Five proteins from the aphid vector Myzus persicae were
identified that were capable of binding potato leafroll virus
(32), one of which was subsequently identified as symbionin, an
Escherichia coli GroEL homologue produced by the aphid
endosymbiont Buchnera aphidicola. Symbionin may protect the
virus from recognition by the aphid immune system (5, 33).
Using similar methods, Li et al. (18) isolated a number of
proteins from the head of the English grain aphid (Sitobion
* Corresponding author. Mailing address: Department of Plant Pa-
thology, Cornell University, Ithaca, NY 14853. Phone: (607) 255-7844.
Fax: (607) 255-2459. E-mail: firstname.lastname@example.org.
?Published ahead of print on 24 October 2007.
avenae) that were able to bind to BYDV-MAV, a virus that is
transmitted specifically by this aphid. Two proteins SaM35
(molecular mass of 35 kDa, pI 4.35) and SaM50 (molecular
mass of 50 kDa, pI 4.51) bound in vitro with high affinity to
BYDV-MAV virions and also to an anti-idiotypic antibody
that mimics an epitope on the virion surface and competes with
virions in antibody-binding competition assays (15). These two
proteins were not detected in the corn leaf aphid, Rhopalosi-
phum maidis, which is not a vector of BYDV-MAV. Symbionin
was among the proteins detected, but interestingly, although it
bound to the anti-idiotypic antibody, it did not bind virions. A
protein with a size similar to that of SaM50 was detected in the
aphids Schizaphis graminum and S. avenae that bound to
BYDV-GAV virions, a virus transmitted specifically by these
two aphid species (34). In situ labeling localized this protein to
the aphid ASG. Furthermore, aphids fed an antibody made
against this protein prior to virus acquisition were less efficient
at transmitting the virus. With the exception of symbionin,
none of the other virus-binding aphid proteins have been iden-
tified. A more recent study (29) identified a number of M.
persicae proteins that bound beet western yellows virus, includ-
ing symbionin. Other proteins were identified by mass spec-
trometry (MS) as homologues of an aphid cuticle protein,
various actins, the Drosophila melanogaster receptor for acti-
vated C kinase (protein kinase C) (Rack-1) and the D.
melanogaster glyceraldehyde-3-phosphate dehydrogenase 3
(GAPDH3). The last three proteins can function in various
aspects of membrane and cytosolic transport and may be in-
volved in some aspect of luteovirid movement into, across, or
out of gut or salivary tissues. GADPH3 also functions in gly-
colysis, and recent evidence suggests that glycosylation of lu-
teovirid structural proteins is required for aphid transmission
(28). Although the aforementioned studies have identified lu-
teovirid-binding proteins in aphids, they have not directly
linked these proteins to the virus transmission phenotype of
Recently we have established multiple parthenogenetic col-
onies of S. graminum that have a common genetic background
but differ in their ability to transmit two viruses that cause
barley yellow dwarf disease. Two naturally occurring S. grami-
num genotypes, Sg-F and Sg-SC, differ in the ability to transmit
CYDV and BYDV; Sg-F is an efficient vector, while Sg-SC is
a poor vector (11, 13). Sexual crosses with aphids with these
genotypes coupled with transmission efficiency studies on F1
and F2progeny indicated that the hybrids segregated not only
for the ability to transmit each virus species but also for which
cellular barrier (hindgut or ASG) blocked virus movement and
transmission (3). Subsequent genetic analysis indicated that
genetic inheritance of vector competence was a multigenic trait
involving only a few major genes and several minor genes that
function in an additive manner (4). Evidence that multigenetic
factors in aphids regulated luteovirus transmission is consistent
with other work describing transmission barriers to luteovirus
transmission occurring at different sites in aphid tissues, such
as the gut and salivary gland (12). Based on this information,
multiple gene products (proteins) would be expected to be
involved in luteovirus movement through aphid gut and sali-
vary gland tissues, and it should be possible to identify proteins
unique to or overexpressed in vector genotypes. In this study
we compared the proteomes of S. graminum vector (Sg-F) and
nonvector (Sg-SC) genotypes to identify aphid proteins specif-
ically expressed by the vector phenotype. F2genotypes derived
from Sg-SC ? Sg-F crosses were then used to confirm that the
proteins that were either specifically expressed or significantly
upregulated in Sg-F were correlated with expression levels in
all vector competent F2genotypes. Furthermore, aphid pro-
teins involved in luteovirus transmission should bind to virus
particles. To test this hypothesis, whole-aphid protein extracts
were incubated in vitro with purified CYDV-RPV and then
immunoprecipitated using anti-CYDV immunoglobulin G
MATERIALS AND METHODS
Aphids and viruses. Parthenogenetic colonies of several genotypes of the
aphid S. graminum were maintained on caged barley seedlings (Hordeum vulgare)
at 20 to 22°C with an 18- or 24-h photoperiod. The Sg-F genotype is an efficient
vector of BYDV-SGV, BYDV-PAV, and CYDV-RPV; the Sg-SC genotype is an
inefficient vector of these viruses (13). The F1and F2matings and subsequent
testing and analyses of virus transmission efficiency were described previously
(3). Six of the F2hybrid genotypes differing in their ability to transmit CYDV
were used in this study, along with the Sg-F and Sg-SC parental genotypes.
CYDV-RPV was purified from oat plants (?Coast black’) inoculated 4 to 5
weeks previously using nonviruliferous aphids (Sg-F or Rhopalosiphum padi) that
were allowed a 48-h acquisition feeding on detached leaves from RPV-infected
oat source plants and then allowed a 5-day inoculation feeding on the 7-day-old
Coast black oat seedlings. Infection was determined by obvious yellowing and
dwarfing symptoms and then verified as CYDV-RPV by double antibody sand-
wich enzyme-linked immunosorbent assay (13). CYDV-RPV was purified from
freshly harvested oat tissues or oat tissues stored at ?80°C by the pectinase-
cellulase enzyme-assisted protocol described by Hammond et al. (14). After
purification through sucrose density gradients and high-speed pelleting, the virus
was resuspended in 0.01 M phosphate buffer (pH 7) and stored at ?80°C.
Extraction of aphid proteins. For each aphid genotype (Sg-F, Sg-SC, A3, G8,
G11, K2, K3, and C2), soluble proteins were extracted from aphid whole-body
homogenates. Aphids previously stored frozen at ?80°C were placed in a cold
mortar and immediately covered with liquid nitrogen. The aphids were homog-
enized to a dry frozen powder with a chilled pestle. An extraction buffer, 0.1 M
phosphate buffer (pH 6.7) containing 2.1% of the EDTA-free Halt protease
inhibitor cocktail (Pierce, Rockford, IL), was added to the aphid powder at a
ratio of 1 gram of aphids per 1 ml of buffer. The aphid extract was homogenized
a second time in the extraction buffer at 4°C on ice with the pestle. The homog-
enates were then transferred into plastic tubes, sonicated for 4 min in an ice-
water bath to disrupt aphid cells, frozen at ?80°C overnight, and thawed at 4°C
the next day. A total of four freeze-thaw cycles were performed to further disrupt
aphid cell membranes, and vigorous vortexing (1 min) was carried out after each
cycle. The homogenates were then centrifuged at 16,000 ? g for 5 h at 4°C, and
the supernatants were collected and frozen at ?80°C in aliquots for proteomics
analyses. The protein concentrations were determined with a Bio-Rad DC pro-
tein assay kit using bovine serum albumin as a standard.
Coimmunoprecipitation of aphid proteins and virus. Aphid proteins were
allowed to bind to RPV virions by mixing 3 ml of whole-body aphid protein
extracts from 2 g of aphids (?8,000 adult aphids) with 200 ?g of purified RPV
and incubating at 4°C for 4 h. Extracts from aphids with Sg-F and Sg-SC geno-
types were incubated in parallel experiments. Following the aphid protein-RPV
incubations, anti-RPV IgG antibody (20 ?g in 10 ?l) was added and incubated
overnight with agitation at 4°C to allow binding to the RPV component of the
complex. Protein A agarose (Sigma, St. Louis, MO) was then added at a 1:25
ratio (vol/vol), and this mixture was incubated for an additional 24 h with
agitation at 4°C to allow binding of the protein A to the IgG component of the
aphid protein-RPV-IgG complex. Finally, the mixture was centrifuged at 1,500 ?
g at 4°C for 5 min and the agarose bead-protein complex pellet was washed six
times in 3 ml of 0.025 M phosphate-buffered saline containing 0.15 M NaCl (pH
7) to remove unattached or loosely bound proteins. Elution of the aphid proteins
bound to the protein A-antibody complex was achieved by adding 125 ?l of
elution buffer (pH 2.8 containing 0.5 M NaCl) (ProFound pull-down kit; Pierce,
Rockford, IL). This buffer separates aphid proteins from the RPV antibody-
protein A-agarose complex. Two more elutions were performed, and the result-
ing solutions were combined. The eluted solutions were concentrated with Mi-
crocon YM-3 centrifugal filters with a 3-kDa molecular mass cutoff (Millipore,
Billerica, MA). Controls included aphid proteins precipitated by the antibody-
292YANG ET AL.J. VIROL.
protein A complex when no purified virus was added. Thus, aphid proteins
binding specifically to RPV were differentiated from proteins binding nonspe-
cifically to anti-RPV antibody–protein A–agarose complexes.
Two-dimensional difference gel electrophoresis (2D DIGE) protein analysis.
Whole-body protein extracts from vector and nonvector genotypes or proteins
from coimmunoprecipitation were compared in individual 2D gels to identify
proteins unique to or significantly upregulated in either the vector or nonvector.
The parent vector (Sg-F) and nonvector (Sg-SC) proteins were compared in the
initial experiments, and then proteins of six F2genotypes (three that are vectors,
A3, G8, and G11, and three that are nonvectors, C2, K2, and K3) were analyzed
to establish whether the presence or absence of proteins in Sg-F or Sg-SC was
correlated with the vector phenotype of each aphid genotype. Proteins were
labeled with CyDye DIGE Fluors (Cy2, Cy3, and Cy5) (GE Healthcare Bio-
Sciences Corp., Piscataway, NJ) according to the manufacturer’s recommenda-
tions. Proteins from a pair of vector and nonvector genotypes (50 ?g total
proteins/fluor/gel) were labeled with Cy3 or Cy5 fluor (4 pmol/?g proteins) and
corun on the same gels. A dye swap was used to normalize for any bias intro-
duced by the dyes, since different CyDye DIGE Fluors may have different
efficiencies in labeling different proteins. An internal standard was prepared; this
standard contained a mixture of equal amounts of proteins from each extract and
from all genotypes included in the experiment. This standard was labeled with
Cy2 fluor (4 pmol/?g proteins) and was included in each gel for intergel com-
parisons used by gel image analysis software. For the coimmunoprecipitated
proteins, the nonspecific control was labeled with Cy3 and the treatment was
labeled with Cy5. The protein samples were loaded onto immobilized pH gra-
dient (IPG) strips (pH 3 to 10 nonlinear, 24 cm) during an overnight passive
rehydration of the strips according to the manufacturer’s specifications. The first
dimension was run on the IPGphore II (GE Healthcare Bio-Sciences Corp.,
Piscataway, NJ) at 20°C with the following settings: step 1, 500 V, 1 h; step 2,
1,000 V, 1 h; and step3, 8,000 V, 9.3 h. Before the second dimension was run, the
strips were reduced for 15 min with 64.8 mM of dithiothreitol in sodium dodecyl
sulfate (SDS) equilibration buffer (50 mM Tris-HCl [pH 8.8], 6 M urea, 30%
glycerol, 2% SDS, 0.002% bromophenol blue) and then alkylated for 15 min with
135.2 mM of iodoacetamide in the same equilibration buffer. The second dimen-
sion was carried out in the Ettan DALT Six system (GE Healthcare Bio-Sciences
Corp., Piscataway, NJ) at 25°C in an electrode buffer (25 mM Tris, 192 mM
glycine, and 0.1% [wt/vol] SDS) with the following settings: step 1, 2.5 W/gel, 25
min; step 2, 17 W/gel, 4 h. The gels used in the second dimension were 12.5%
homogenous acrylamide gels cast in the laboratory. Immediately after electro-
phoresis, the gels were scanned with a Typhoon 9400 variable-mode imager (GE
Healthcare Bio-Sciences Corp., Piscataway, NJ). Four replicate 2D gels for each
pair of vector-nonvector genotypes were used, and one 2D gel of the coimmu-
noprecipitated proteins for each genotype was run. The gel images were initially
analyzed with the Decyder 2D V 6.0 software (GE Healthcare Bio-Sciences
Corp., Piscataway, NJ) and then further confirmed by Progenesis SameSpots,
V2.0 (Nonlinear USA Inc., Durham, NC). To identify the conserved proteins
associated with CYDV-RPV transmission with the SameSpots image analysis
software, the gel images were clustered into vector and nonvector groups (i.e.,
parent vector and nonvector groups and F2vector and nonvector groups). To
analyze the numbers of upregulated and specific protein spots in the parent and
F2aphids, upregulated or specific spots were determined by spot-by-spot and
gel-by-gel manual confirmation on all the 2D and 3D images for the group. The
spots reported all had at least 1.3-fold intensity (normalized volume) difference,
and all were statistically significant, which was measured with the built-in statis-
tical tool in the SameSpots software.
For MS analyses, a new set of 2D gels was run for each genotype of the parent
aphids with the same settings and procedure, except that the gels were run at a
protein concentration of 500 ?g/gel and without labeling the proteins with CyDye
DIGE Fluors. These “picking gels” were stained by using a colloidal blue stain kit
(Invitrogen, Carlsbad, CA) and scanned with a Typhoon 9400 variable-mode
imager (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). By manually com-
paring the colloidal blue images with the 2D DIGE images, we were able to
identify a set of spots that were unique to or obviously upregulated in vector or
nonvector genotypes. These spots were then excised from the picking gels and
digested with trypsin, and the resulting peptides were extracted for analysis by
MS (19). Special attention was paid to unique protein spots associated with
vector or nonvector phenotypes that were conserved in both parent aphids and
F2offspring. The digestions were carried out with sequencing-grade modified
porcine trypsin (Promega, Madison, WI), and the peptide extracts were desalted
and concentrated with C18 ZipTips (Millipore, Billerica, MA) according to the
Analysis by MS. All mass spectra were obtained using a model 4700 proteom-
ics analyzer with tandem time of flight optics using 4000 Explorer software
(version 3.0) (Applied Biosystems, Foster City, CA). The sample was reconsti-
tuted in 3 ?l of 0.1% trifluoroacetic acid in 50% CH3CN prior to MS analysis.
Samples of 1 ?l each were applied to a target plate and mixed with 0.5 ?l of
matrix (10 mg/ml ?-cyano-4-hydroxycinnamic acid in 50% CH3CN–0.1% triflu-
oroacetic acid–1 mM ammonium phosphate) using the dried droplet method
(16). Prior to analysis, the mass spectrometer was calibrated, externally, using a
six-peptide calibration standard available from Applied Biosystems (4700 Cal
mix). Most samples were calibrated internally, using the common trypsin autol-
ysis products (at m/z values of 842.51, 1045.5642, and 2211.1046 Da) as mass
calibrants. The external calibration was used as the default if the trypsin autolysis
products were not observed in the spectra of the samples. MS spectra were
acquired across the mass range of 800 to 4,000 Da using the 1 kV positive ions
and the reflector mode with a laser power of 4100. The signal from 1,000 laser
shots was averaged to produce the final MS spectra. For tandem MS (MS-MS)
experiments, the instrument was operated at a laser power of 4,900 with the
collision-induced dissociation and metastable ion suppressor off. Calibration was
external, using the known fragments of Glu-fibrinopeptide B as calibrants. The
10 most abundant ions not appearing on the exclusion list with a minimum
signal/noise ratio of 25 were selected automatically as precursor ions for MS-MS
analysis. The signal from 4,000 laser shots was averaged to produce each MS-MS
spectra. All m/z values reported in this study are monoisotopic.
Protein identification. The MS and MS-MS data collected were submitted as
a combined search to Mascot (23) using the GPS Explorer software, V3.5
(Applied Biosystems, Foster City, CA). Preliminary protein identifications
were obtained by comparing the experimental data to the NCBI nonredun-
dant (nr) and Acyrthosiphon pisum expressed sequence tag (EST) databases.
Multiple searches of the current NCBI databases (April 2007) were per-
formed using following taxonomies: nr, Drosophila, Aphididae, Insecta, and
viruses. The search criteria used were as follows: carbamidomenthyl-cysteine
and methionine oxidation were selected as variable modifications, and one
missed tryptic cleavage was allowed. The searches were done with a mass
tolerance of 75 ppm for the MS mode and 0.4 Da in the MS-MS mode. The
preliminary protein identifications obtained automatically from the software
were inspected manually for conformation prior to acceptance. Homology to
known proteins was determined by searching against protein databases in
NCBI with protein BLAST programs (1).
RESULTS AND DISCUSSION
Vector competency of S. graminum genotypes. S. graminum
genotypes differ in their ability to transmit several luteovirids
(13). Aphids collected in the field with the Sg-F and Sg-SC
genotypes represent two extremes in their ability to act as a
vector for CYDV-RPV. Similar to previously reports (11, 13),
Sg-F aphids transmitted CYDV-RPV efficiently, while Sg-SC
aphids rarely transmitted the virus. As previously described
(3), there are multiple genetically controlled barriers to
CYDV-RPV movement in aphids with the Sg-SC genotype,
and these barriers can segregate independently in the sexual
progeny. Sg-SC aphids are not a vector of CYDV-RPV due to
an efficient barrier to virus movement through the ASG and an
incomplete barrier to movement at the hindgut (3). In addi-
tion, six F2progeny from a random mating of 13 F1progeny of
the Sg-F and Sg-SC mating were used in this proteomic anal-
ysis. Aphids with genotypes Sg-A3, Sg-G8, and Sg-G11 are
efficient vectors of CYDV-RPV, and aphids with genotypes
Sg-C2, Sg-K2, and Sg-K3 are inefficient vectors or nonvectors
(Table 1). Aphids with the Sg-C2 genotype possess an efficient
barrier to virus movement at the hindgut, since introduction of
virus directly into the hemocoel by microinjection resulted in a
transmission efficiency (55%) similar to that of the Sg-F pa-
rental vector. Introduction of virus into the hemocoel of aphids
with Sg-K2 and Sg-K3 genotypes resulted in transmission effi-
ciencies of 21 to 30% compared to 0% by feeding acquisition,
suggesting that there were incomplete barriers to virus move-
ment functioning at the ASG but a complete barrier at the
VOL. 82, 2008APHID PROTEINS REGULATING CYDV TRANSMISSION IN VECTORS 293
hindgut of these two aphids (Table 1). This provides a unique
genetic system to study the proteomes of vector and nonvector
aphids in an attempt to identify proteins that may define the
vector phenotype of an aphid genotype. Although a wide vari-
ation in luterovirus transmission efficiency exists in natural
populations of S. graminum (13), the F2progeny and the
segregating transmission barriers provide a method to cor-
relate the presence or absence of protein(s) in vector and
nonvector genotypes with the same genetic background.
Proteome comparison between S. graminum parental vector
and nonvector genotypes. The soluble proteomes extracted
from whole Sg-F (vector) and Sg-SC (nonvector) aphid bodies
were directly compared by 2D DIGE (31). Four replicate gels
were run with samples that included in each gel 50 ?g of total
protein extracts from Sg-F aphids and 50 ?g of total protein
extracts from Sg-SC aphids labeled with Cy3 or Cy5, respec-
tively. Internal standards consisted of Cy2-labeled combined
protein samples (25 ?g from Sg-F and 25 ?g from Sg-SC). This
allowed for quantitative measurement of the degree of fluctu-
ation in the spot volume ratios of image pairs from identical
protein samples. Additionally, a dye swap between Sg-F and
Sg-SC samples was done as a replicate analysis that controlled
for any bias introduced by differences in the reactivities of the
dye lots. Figure 1 shows an overlay image of Sg-F (red) and
Sg-SC (green). The red spots represent the Sg-F-specific or
upregulated protein spots, and the green spots represent the
Sg-SC-specific or upregulated protein spots. The yellow spots
indicate proteins that are expressed similarly or equally in both
genotypes. A different set of 2D gels (picking gels) was used to
separate proteins (500 ?g/gel/aphid) extracted from the vector
and nonvector aphids. These were stained with colloidal blue
to provide sufficient material for isolation of individual protein
FIG. 1. Proteomic profiling of vector and nonvector genotypes as revealed by 2D DIGE analysis. Proteins extracted from whole-body protein
extracts of the vector genotype Sg-F (red) and the nonvector genotype Sg-SC (green) were labeled with Cy5 and Cy3, respectively. An internal
standard comprising proteins from a combination of both aphid genotypes, labeled with the Cy2 dye, was included in all gels. IPG strips (pH 3 to
10) were used for isoelectric focusing prior to standard SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide) for the second dimension.
Spots marked with a number indicate proteins whose abundance was significantly different for the vector and nonvector extracts, within a 95%
confidence level. The yellow-colored spots are proteins expressed equally in both genotypes. Molecular mass (MM) (in kilodaltons) is shown on
the y axis, and pI is shown on the x axis.
TABLE 1. Transmission efficiency of CYDV-RPV by parental and
selected F2progeny genotypes of Schizaphis graminum aphidsa
No. of plants infected/no. of plants infested by
aphids (%) following virus acquisition by:
aTransmission efficiency of CYDV-RPV by parental (Sg-F and Sg-SC) and
selected F2progeny genotypes of S. graminum aphids following feeding on
infected plants or by injecting virus directly into the aphid hemocoel.
bNT, not tested.
294 YANG ET AL.J. VIROL.
spots from the gel and subsequent protein identification by
The 2D DIGE images were clustered into vector and non-
vector groups in two ways: parent vector versus parent non-
vector and F2vector versus F2nonvector. The gel images were
initially analyzed with DeCyder 2D software and then further
analyzed and confirmed with SameSpots software. A total of
1,945 protein spots were detected by the SameSpots software
program in the parental aphid whole-body protein extracts
(Fig. 1). A comparison of the parental genotypes (Sg-F and
Sg-SC) identified 120 differentially expressed protein spots.
The molecular masses ranged from 15.0 to 77.3 kDa, and the
pIs ranged from 4.40 to 9.25. The vector with Sg-F genotype
had 68 upregulated/specific protein spots; 13 were Sg-F specific
and 55 were expressed at significantly higher levels than in
Sg-SC (by analysis of variance [ANOVA], 7.00 ? 10?9? P ?
0.012) (Table 2). The nonvector Sg-SC aphids had 52 upregu-
lated/specific protein spots; 11 were detected only in Sg-SC and
41 were significantly upregulated (by ANOVA, 2.46 ? 10?8?
P ? 0.003).
Detection of proteins specifically linked to CYDV-RPV
transmission. Aphid proteins that may be associated with virus
recognition, endocytotic and exocytotic pathways, and trans-
cellular transport through aphid tissues resulting in circulative
virus transmission were expected to be conserved in both the
parental (Sg-F) and F2(Sg-A3, Sg-G8, and Sg-G11) vector
genotypes. Similarly, proteins that may be specifically associ-
ated with preventing CYDV-RPV transmission by blocking
pathways and preventing transmission would be expected to be
shared by both the parental (Sg-SC) and F2(Sg-C2, Sg-K2, and
Sg-K3) nonvector genotypes. We recognize that vector and
nonvector genotypes may not be distinguished by the presence
or absence of proteins. The difference may be more subtle such
as a slightly altered amino acid sequence or posttranslational
modification that could alter protein function. Some subtle
changes would not be discovered using the methods described
here, but many changes or alterations can be identified by
slight alterations in protein spot migration in 2D gels.
The comparison of F2genotypes identified 14 protein spots
that were specific or upregulated in the F2vector genotypes
(Table 2). Two of them, S8 and S29, were F2vector-specific
spots, and they were among the 13 parent vector-specific spots
(Fig. 2). The other 12 spots were significantly upregulated (by
ANOVA, 8.2 ? 10?5? P ? 0.033), two of which (S4 and S405)
were also identified in the parent vector Sg-F (Fig. 2). There-
fore, four protein spots (S4, S8, S29, and S405) are conserved
between the parental and F2vectors that efficiently transmit
CYDV-RPV (Table 3), indicating that these four proteins,
especially the two vector-specific proteins S8 and S29, are
linked to CYDV-RPV transmission. Although S4 was signifi-
cantly downregulated at the group level in the F2nonvectors,
one exception was that S4 was highly expressed in the nonvec-
tor genotype Sg-C2. However, Sg-C2 is an efficient vector of
BYDV-SGV (3). Thus, S4 expression in Sg-C2 may be related
to BYDV-SGV transmission. The other two nonvector F2ge-
notypes do not transmit BYDV-SGV. With the exception of
S4, the other three proteins were all significantly downregu-
lated in Sg-C2 (Table 3). S4, S8, and S405 have similar molec-
ular masses (68.4 to 70.2 kDa) and pIs (6.2 to 6.6), suggesting
these may be related proteins; S29 is a smaller basic protein
(26.6 kDa; pI 9.0).
The F2nonvector group had only one upregulated protein
spot (by ANOVA, P ? 0.022). This spot is not among the 52
upregulated/specific spots in the nonvector parent, Sg-SC, i.e.,
none of these 52 proteins were conserved in F2nonvector
genotypes. Therefore, no strong association could be made
with the occurrence of a specific protein and the nonvector
phenotype. This suggests that the inability of vectoring CYDV-
RPV in nonvectors is not due to the presence of proteins
preventing transmission of CYDV-RPV in the nonvectors but
more likely due to a lack of or inactivity of specific CYDV-
RPV-binding proteins that facilitate transmission.
Eliminating false-positive results and the power of using
genetic screens to identify target proteins. There were 68 and
52 specific/upregulated protein spots detected in the vector
and nonvector parents, respectively (Table 2). Since these par-
ents represent two distinct genetic populations, it is difficult to
conclude which proteins are actually related to virus transmis-
sion with this information alone. It can be presumed that
proteins associated with CYDV-RPV transmission by the pa-
rental vector Sg-F would be represented within these 68 pro-
teins. Naturally, one would assume that the parental vector-
specific or upregulated protein spots are related to virus
transmission, but this may not be true. For example, S6 is
upregulated and S7 is vector specific in the parent generation;
however, their levels of expression are not significantly differ-
ent between F2vectors and nonvectors (Fig. 2). In addition,
any of the 52 proteins associated specifically with the Sg-SC
nonvector phenotype could be involved in preventing CYDV-
RPV transmission due to mechanisms like irreversible binding
of the virus to certain Sg-SC proteins. The numbers of specific/
upregulated protein spots consistently identified in all F2vec-
tor and nonvector genotypes were reduced to 14 and 1, respec-
tively, probably due to a more similar (homologous) genetic
background among the F2hybrids than between the parental
types. Through the proteome comparison among parent vector
and F2vectors, only four protein spots (S4, S8, S29, and S405)
were found to be conserved between generations (Fig. 2 and
TABLE 2. Summary of upregulated or specific protein spots in
comparisons between parent vector and nonvector genotypes and
between F2vector and nonvector genotypes
No. of protein spots
Total no. of
Vector135568 7.00 ? 10?9?
P ? 0.012
2.46 ? 10?8?
P ? 0.003
148.2 ? 10?5?
P ? 0.033
P ? 0.022
aThe statistical significance of the intensity of the protein spots of vector and
nonvector aphids was calculated by ANOVA.
bOnly 2 of these 12 spots are upregulated in the parent vector.
cThis protein was not upregulated in the parent nonvector.
VOL. 82, 2008APHID PROTEINS REGULATING CYDV TRANSMISSION IN VECTORS 295
Tables 2 and 3). The number of protein spots of interest was
reduced from 68 to 4, enabling one to focus efforts on signif-
icantly fewer proteins more likely to be involved in virus trans-
mission. Similar comparisons between parent nonvector and F2
nonvectors eliminated the need to look further into the non-
vectors for virus-binding proteins, since no protein was found
to be conserved in all nonvector genotypes. Thus, coupling the
proteomics data with the genetic analysis greatly reduced the
number of false-positive results and thereby the number of
protein spots of interest requiring further analysis. Our data
demonstrated the power and usefulness of using a genetic
system in proteomics research, something that may have a
Coimmunoprecipitation of CYDV-RPV-binding aphid pro-
teins. We expect that many aphid proteins involved in virus
transport into and through gut or salivary tissues will bind
virions due to the well-described vector-specific transmission
of each virus species (12). Other aphid proteins involved in
general macromolecular transport are undoubtedly involved in
virus transmission, but not the specificity of virus species trans-
mission; these proteins may not bind directly to the virus. The
population of virus-binding proteins would also be expected to
overlap with proteins identified in the 2D DIGE experiments if
the proteins are involved in regulating virus transmission. Sol-
uble protein extracts from whole aphid bodies were incubated
with purified, transmissible CYDV-RPV. Virus-aphid protein
complexes were subsequently immunoprecipitated with anti-
bodies to whole virus. The same aphid protein extracts were
also immunoprecipitated with antibodies without the previous
addition of virus. Following dissociation of the immunoprecipi-
tated protein complexes and labeling with Cy3 or Cy5 dye,
proteins were separated and compared by 2D DIGE. This
technique provides an advantage over far-Western blots used
previously to identify virus-binding proteins (18, 29, 32, 34) in
that the aphid proteins are in their native conformation, and
the DIGE format provides a direct comparison of virus-pre-
cipitated proteins with proteins precipitated nonspecifically.
Numerous CYDV-RPV-binding proteins were identified from
the vector genotype Sg-F, including proteins with molecular
masses and pIs corresponding to those of proteins S4, S8, S29,
and S405 (Fig. 3). The identities of these spots were confirmed
by aligning the gel images of the immunoprecipitated proteins
to the gel images of the whole-body protein extracts from
parent vector Sg-F using the SameSpots software. Similar pro-
teins were not observed in controls lacking CYDV-RPV or in
the nonvector Sg-SC. It should be noted that the amount of
immunoprecipitated proteins was increased due to the concen-
trating effect of the virus antibody (Fig. 3). Our results indi-
cated that these four proteins recognize and bind to CYDV-
RPV, thus fulfilling a functional requirement expected of
proteins involved in virus-specific uptake and transport
through aphid cells. Additionally, numerous protein spots were
observed on the immunoprecipitated gels that were not
present on the gels with whole-aphid extracts (data not shown).
These may represent proteins that are involved in virus recog-
nition and transport and that are either minor components of
the total proteome or proteins not resolved on 2D gels loaded
with the total protein extracts. Isolation of CYDV-RPV-bind-
ing aphid proteins by virus coimmunoprecipitation further sub-
stantiates the hypothesis that these proteins are linked to
FIG. 2. 2D and 3D images showing comparisons of key protein
spots between vector and nonvector genotypes of the parent aphids or
the F2progeny. 2D DIGE analysis identified significantly increased
expression of proteins S4, S8, S29, and S405 in all vector genotypes
(parent Sg-F and F2hybrids) compared to the nonvector genotypes
(parent Sg-SC and F2hybrids). Densitometric volume is shown in a 3D
view generated by the SameSpots image analysis software package.
Computational analysis of the images with the SameSpots software
allowed for the detection of significant abundance changes (95% con-
fidence level) based on the variance of the mean change within the
cohort. The amount of the protein is proportional to the volume of
the protein peak. A spot circled with a red or blue line represents the
protein identified by the label. S6 and S7 are examples of noncon-
served proteins. In the parent vector, the expression of S6 was signif-
icantly increased relative to the nonvector parent, and S7 was specific
to the vector parent; however, no significant difference in S6 or S7
expression was found between F2vector and nonvector groups. The
size and pI of S28 are similar to those of S29, and both proteins were
identified by MS analysis as cyclophilin-like proteins.
296YANG ET AL.J. VIROL.
CYDV-RPV recognition and binding associated with cellular
MS analysis and protein identification. To further charac-
terize and identify the four protein spots identified in both the
analysis of whole-aphid protein extracts and coimmunoprecipi-
tated proteins (i.e., S4, S8, S29, and S405), protein spots were
cut from 2D picking gels and digested with trypsin, and the
resulting peptides were analyzed by MS using a model 4700
proteomics analyzer with 4000 series Explorer software.
MS-MS data from the aphid proteins were analyzed by Mascot
using GPS Explorer software, and the results were compared
for protein homology in various protein databases. Both pep-
tide mass fingerprint and peptide fragmentation (MS-MS) data
were used to identify proteins. The Mascot protein score
judged the significance of the peptide mass fingerprint data,
and the Mascot ion score judged the significance of the MS-MS
data. The protein score confidence interval percentages and
total ion confidence interval percentages were all 100%. For
S29, the average protein score was 214, and average mass
accuracy was ?6.9 ppm within the range of 1 to 16 ppm; for S4,
the protein score was 122, and average mass accuracy was ?5.3
ppm within the range of 0 to 16 ppm. MS data for each of the
four proteins matched ESTs from available aphid databases,
but only two proteins had homology to known proteins by
BLAST search. S4 was identified as a luciferase-like protein,
and S29 was identified as a cyclophilin-like protein (Table 4).
In both cases, the E values were ?10?27and amino acid
identity was ?68%. These types of proteins are associated with
endoplasmic reticulum (cyclophilin) (22) or peroxisomes de-
rived from endoplasmic reticulum (luciferase) (21).
Luciferase contains a microbody targeting signal (10) that
directs the protein to subcellular organelles that are bound by
a single membrane (17). Luteovirids are transported across the
gut and ASG cell cytoplasm in single-membrane-bound vesi-
cles, and the transport pathway is often observed to include
endosomes, lysosomes, and possibly other vesicles (2, 12). A
search of the protein databases at NCBI reveals that homo-
logues of luciferase are found in all sequenced genomes of
insects. Firefly luciferase is a member of a superfamily of
homologous enzymes, which includes acyl-coenzyme A (acyl-
CoA) ligases and peptide synthetases. Recently, luciferase has
been found to have additional activities in long-chain fatty
acyl-CoA synthesis (21). Its functional roles in insects outside
of the luminescence reaction in fireflies are not well charac-
terized, and additional roles of these proteins are likely to be
discovered, some of which may be applicable to luteovirus
transport in aphids.
The cyclophilin present in the aphid is most closely related
to cyclophilin A-like and cyclophilin B-like proteins that are
localized in endoplasmic reticulum via an N-terminal signal
peptide. Cyclophilin B proteins are reported to function in the
secretory pathway, possibly by chaperoning of membrane pro-
teins or having a role in receptor signaling pathways (22).
Cyclophilin A is required for the attachment of human immu-
nodeficiency virus (HIV) to host cells by binding a proline-rich
loop on the surface of the HIV type 1 capsid (27, 30) and is
involved in additional postentry events associated with HIV
infection (26). This function of cyclophilin A may have rele-
vance to CYDV-RPV movement in vector aphids. Also, cyclo-
philin A associates with the dynein/dynactin motor protein
complex and has been suggested to perform a general function
related to molecular binding for movement along microtubules
(6). Cyclophilin B is targeted to the endoplasmic reticulum and
FIG. 3. Comparison of protein spots following 2D DIGE analysis
of whole-aphid-body protein extracts (top) from vector (red) and non-
vector (green) aphids and CYDV-RPV-binding proteins immunopre-
cipitated from whole-aphid-body protein extracts (bottom) (virus
treatment [red]; control treatment [green]). The four proteins, S4, S8,
S405 and S29, were specific to or significantly upregulated in vector
genotypes, and all were confirmed to have CYDV-RPV binding prop-
erties by coimmunoprecipitation (Co-IP). S28 was a cyclophilin iden-
tified in all genotypes, S29 was a cyclophilin identified specifically in
vector genotypes. Only S29 was able to bind virus and was identified in
the coimmunoprecipitation experiments.
TABLE 3. Four aphid proteins are conserved between parent and F2vectors efficiently transmitting CYDV-RPV
Expression of protein ina:
Sg-FSg-A3Sg-G8 Sg-G11Sg-SCSg-K2Sg-K3 Sg-C2
?3.40 ? 10?4
?6.78 ? 10?4
aThe expression of proteins S4, S8, S29, and S405 in vector or nonvector aphids was examined. Symbols: ?, specific or significantly upregulated; ?, absent or
bStatistical significance was determined by ANOVA done on Sg-F versus Sg-SC and F2vectors (A3, G8, and G11) versus F2nonvectors (K2, K3, and C2) for each
spot. The greater of the two P values was reported.
VOL. 82, 2008 APHID PROTEINS REGULATING CYDV TRANSMISSION IN VECTORS 297
functions within secretory pathways and in complexes on the
plasma membrane (24). They chaperone plasma membrane
proteins and function in receptor signaling pathways (22). Lu-
teovirids are dependent upon receptor-mediated endocytosis
pathways to enter both the gut and ASG cells and appear to be
directed to specific transcytosis pathways (2, 12).
Intriguingly, cyclophilin B in Drosophila melanogaster has
undergone a gene duplication event and the duplicated gene is
expressed in a tissue-specific manner (22). Such a gene dupli-
cation event may have occurred in aphids also, since we detect
another cyclophilin B isoform in the proteomes of both vector
and nonvector aphids (S28 [Fig. 2]). Only the cyclophilin pro-
tein identified in the vectors (S29) was detected in the coim-
munoprecipitation reaction. S28, the cyclophilin isoform iden-
tified in both vector and nonvector genotypes, was not detected
in the coimmunoprecipitation reaction, indicating it does not
bind to CYDV-RPV (Fig. 3). This result also shows the utility
of this assay to determine relative expression of proteins in
different aphid genotypes.
Although these analyses have not identified the same virus-
binding proteins as reported by others, the common denomi-
nator is the identification of proteins involved in receptor bind-
ing or targeting and transport of macromolecules (i.e., viruses)
through cell cytoplasm. We have not identified symbionin as an
aphid protein involved in RPV transmission in the S. graminum
experimental system, but our biological data suggest that sym-
bionin does not play a role in preventing transmission in the
nonvector genotypes (3).
The proteomics approach utilizing 2D DIGE and mass spec-
tral data is an effective way to identify aphid proteins unique to
specific aphid genotypes that differed in their ability to transmit
viruses. These data alone identified a relatively large number
of proteins that be involved in virus transmission; however, the
different proteins may represent only genetic differences
among aphid populations that could not be associated with
virus transmission. Coupling these approaches with the aphid
genetic system allowed us to directly correlate the presence or
upregulation of specific proteins in multiple aphid genotypes
with the same genetic background and the same vector com-
petency phenotypes. This allowed the identification of four
proteins out of an initial pool of 120 candidates. Furthermore,
these data show that specific aphid proteins associated with
CYDV-RPV vector competence are heritable. Coimmunopre-
cipitation was a method other than 2D DIGE to identify a
different set of aphid proteins that were able to bind to virus
particles, but again this provides only indirect evidence that the
proteins are linked to virus transmission, and not all proteins
involved in virus transmission may be involved in direct inter-
actions with the virus. In our studies the four proteins that were
conserved in different generations of only the vector compe-
tent genotypes of S. graminum were also immunoprecipitated
with CYDV-RPV, indicting that these proteins specifically
bind to CYDV-RPV. This strengthens the evidence that these
proteins are involved in circulative virus transport, although
their specific function in luteovirid transmission remains to be
determined. Previous research does support a potential role of
cyclophilins and luciferase in viral transmission. Our experi-
mental system now allows us to investigate virus-aphid inter-
actions from both the perspective of the aphid and the per-
spective of the virus. The results of these studies may provide
a means to screen aphid populations in the future to determine
their capacity to serve as efficient virus vectors. We anticipate
that our methods and results will have relevance to the mech-
anisms underlying the transmission of both plant and animal
viruses by other arthropod vectors.
We thank William Sackett in the Department of Plant Pathology at
the Pennsylvania State University for assistance in the greenhouse and
growth chambers for preparing and maintaining the aphid colonies,
plants, and virus stocks needed to complete this research. We appre-
ciate Yong Yang, Kevin Howe, and Tara Fish in the Functional and
Comparative Proteomics Center, USDA-ARS Plant, Soil, and Nutri-
tion Laboratory at Cornell University for assistance in the laboratory.
This project was supported in part by the National Research Initia-
tive of the USDA Cooperative State Research, Education and Exten-
sion Service (NRI award 03-01647).
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Protein(s) matched by
aTotal number of sequence matches identified for each aphid protein sequence compared to NCBI nonredundant and Acyrthosiphon pisum EST databases using
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bBLAST E values were between 10?99and 10?27.
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