Insect Molecular Biology

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Insect Molecular Biology (2014) 23(1), 67–77

doi: 10.1111/imb.12061

Comparative analyses of salivary proteins from three aphid species

S. Vandermoten*, N. Harmel*, G. Mazzucchelli†, E. De Pauw†, E. Haubruge* and F. Francis* *Gembloux Agro-Bio Tech, Department of Functional and Evolutionary Entomology, University of Liege, Gembloux, Belgium; and †Mass Spectrometry Laboratory, Department of Chemistry, Chemistry Institute, University of Liege, Liege, Belgium Abstract Saliva is a critical biochemical interface between aphids and their host plants; however, the biochemical nature and physiological functions of aphid saliva proteins are not fully elucidated. In this study we used a multidisciplinary proteomics approach combining liquid chromatography-electrospray ionization tandem mass spectrometry and two-dimensional differential in-gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry to compare the salivary proteins from three aphid species including Acyrthosiphon pisum, Megoura viciae and Myzus persicae. Comparative analyses revealed variability among aphid salivary proteomes. Among the proteins that varied, 22% were related to DNA-binding, 19% were related to GTP-binding, and 19% had oxidoreductase activity. In addition, we identified a peroxiredoxin enzyme and an ATP-binding protein that may be involved in the modulation of plant defences. Knowledge of salivary components and how they vary among aphid species may reveal how aphids target plant processes and how the aphid and host plant interact. Keywords: aphids, salivary proteome, saliva, plant– aphid interactions, Myzus persicae, Acyrthosiphon pisum, Megoura viciae. First published online 1 November 2013. Correspondence: Sophie Vandermoten, Gembloux Agro-Bio Tech, Functional and Evolutionary Entomology, University of Liege, Passage des Déportés 2, B-5030 Gembloux, Belgium. Tel.: +32(0)81 622 287; Fax: +32(0)81 622 312; e-mail: [email protected]; [email protected]

© 2013 The Royal Entomological Society

Introduction The components of insect saliva play crucial roles in the early stages of food processing. The saliva of plant-feeding insects is predicted to contain bioactive compounds capable of modulating, suppressing or counteracting plant defences (Hogenhout & Bos, 2011). Moreover, it is assumed that the saliva of plant-feeding insects may determine the severity of damage to host plants. The knowledge of the biochemical nature and physiological functions of herbivorous insect saliva may be a key factor for the development of new pest control strategies. The family Aphididae comprises more than 4300 species, each of which are specialized to feed on phloem sap (Blackman & Eastop, 1985). In addition to transmitting plant viruses, aphids can damage host plants by manipulating host plant physiology (Goggin, 2007). As a result, aphids are considered to be a major agricultural and forest pest. Aphids produce two types of saliva during feeding. Gelling saliva is produced before sustained feeding, and it forms a continuous protective sheath around an aphid’s mouthparts (stylets). Aphids also produce a watery saliva that is secreted into plant cells and the plant phloem during sap ingestion (Miles, 1999; Tjallingii, 2006). Gelling saliva is composed of proteins including phenoloxidases, peroxidases, pectinases and β glucosidases, phospholipids, and conjugated carbohydrates; therefore, watery saliva is a complex mixture of enzymes and other components capable of eliciting plant defence signals (Baumann & Baumann, 1995; Urbanska et al., 1998; Miles, 1999; Cherqui & Tjallingii, 2000; Ni et al., 2000; Tjallingii, 2006; Harmel et al., 2008; Carolan et al., 2009; Will et al., 2009; Ma et al., 2010; Nicholson et al., 2012). Nevertheless, no elicitors, suppressors or modulators of plant physiology have been identified in aphid salivary secretions, and the effects of aphid saliva on plants are only partially understood. Recent advances in genomics, transcriptomics and proteomics provide new opportunities for identifying proteins that may mediate aphid–host plant interactions 67

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(Hogenhout & Bos, 2011). The essential roles of the salivary gland gene C002 from the pea aphid Acyrthosiphon pisum in feeding behaviour and the survival of its host Vicia faba have been demonstrated using RNA interference (Mutti et al., 2006, 2008). Although the function of the C002 protein is still unknown, its role in aphid feeding was confirmed by overexpressing the Myzus persicae C002 gene in Nicotiana benthamiana, which resulted in enhanced aphid performance (Bos et al., 2010). The enzyme glucose oxidase, a plant defence suppressor was first identified in the chewing insect Helicoverpa zea (Eichenseer et al., 1999; Musser et al., 2005). It was also found in M. persicae saliva, which suggests that the suppression of plant defences by this enzyme may be a common strategy for chewing and sucking insects (Harmel et al., 2008). In addition, a functional genomics approach enabled the identification of two candidate elicitors, Mp10 and Mp42, from the aphid species M. persicae. The overexpression of both in N. benthamiana triggered defence responses and resulted in decreased aphid fecundity (Bos et al., 2010). Although salivary gland extracts of diverse aphid species have been investigated, a limited number of studies have investigated the proteomes of aphid saliva (Harmel et al., 2008; Carolan et al., 2009; Nicholson et al., 2012). Because these studies were performed with different sampling procedures and different proteomic technologies, comparing the saliva proteomes between aphid species is difficult. An early report directly compared the salivary proteomes of different aphid species, but protein quantities were low and identification of salivary proteins was missing (Cooper et al., 2011). More recently, Rao et al. (2013) compared the salivary profiles of two cereal aphid species and A. pisum. In the present study, we used an optimized sampling procedure and combined two-dimensional differential in-gel electrophoresis (2D-DIGE)/matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/mass spectrometry (MS) and liquid chromatography (LC)electrospray ionization (ESI)-tandem MS (MS/MS) proteomic technologies, with the aim of investigating the salivary proteomes of A. pisum, M. persicae and Megoura viciae. These species were chosen because the genome of A. pisum was recently sequenced (International Aphid Genomics Consortium, 2010), and transcriptomic data for M. persicae was available (Ramsey et al., 2007). The vetch aphid M. viciae was included in our analyses because its feeding behaviour is similar to that of A. pisum. M. viciae and A. pisum are oligophagous species that feed on leguminous plants of the Fabaceae family, and M. persicae is a polyphagous aphid that feeds on hundreds of species in >40 plant families (Blackman & Eastop, 1985). The salivary proteomes were investigated in order to identify salivary

proteins that may be universal among aphid species, have general roles in the feeding process, or that may be related to specific feeding behaviours.

Results and discussion Comparison of protein patterns on gel electrophoresis A preliminary investigation of the salivary protein profiles using one-dimensional gel electrophoresis (1-DE) was performed for each aphid species. The 1-DE analyses revealed variability in protein banding patterns between the aphid species (Fig. 1). While A. pisum and M. viciae, both oligophagous species, shared similar protein expression patterns, with the most intense protein bands located below 20 kDa, the polyphagous species M. persicae expressed a greater number of salivary proteins. The protein variability detected by 1-DE analyses was then confirmed by 2D-DIGE. Protein spots on the 2D-DIGE gels displayed isoelectric points (pI) ranging from pH 3 to pH 10, and the spots were concentrated between pH 3 and pH 7 (Fig. 2). The complexity of the two-dimensional gel electrophoresis (2-DE) gels was consistent with previous reports for other aphid species (Harmel et al., 2008; Carolan et al., 2009, 2011; Nicholson et al., 2012).

MW

Mp

Ap

Mv

97

66

45

30 20

Figure 1. One-dimensional gel electrophoresis comparing salivary proteins (20 μg total protein/sample) from Myzus persicae (Mp), Acyrthosiphon pisum (Ap) and Megoura viciae (Mv). Proteins were electrophoresed on 12.5% resolving gels with a 4% stacking gel.

© 2013 The Royal Entomological Society, 23, 67–77

Salivary proteomes from three aphid species

Acyrthosiphon pisum

Megoura viciae

Myzus persicae

Figure 2. Two-dimensional gel electrophoresis comparing salivary proteins from Myzus persicae, Acyrthosiphon pisum and Megoura viciae. CyDye labeled proteins (20 μg) were separated by isoelectric focusing at pH 3–10, followed by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Comparison of salivary proteomes The salivary proteome of each aphid species was investigated using LC-ESI-MS/MS on unseparated salivary concentrates (in-solution samples), and MALDI-TOF/MS was used on tryptic peptides obtained from salivary proteins separated by 2D-DIGE (in-gel samples). More than 300 peptides were generated from the LC-ESI-MS/MS while pairwise comparisons of the protein profiles generated by 2D-DIGE allowed 132 protein spots to be selected © 2013 The Royal Entomological Society, 23, 67–77

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for identification. The number of spots analysed for each saliva sample did not change in a significant way. Database mining was performed using stringent MASCOT database searches and MS BLAST sequence-similarity searches with deduced sequence candidates. These analyses allowed the identification of 14 and 61 individual proteins for LC-ESI-MS/MS and MALDI-TOF/MS analyses, respectively. Proteins identified by MS were categorized based on their molecular functions. Analysis of in-solution samples using LC-ESI-MS/MS revealed that proteins with predicted oxidoreductase activity accounted for 50 and 43% of the total proteins identified in M. persicae and A. pisum saliva, respectively while proteins with peptidase activities made up the majority of salivary proteins from M. viciae (40% of the total proteins identified). Comparisons of salivary proteomes by 2D-DIGE-MALDI-TOF/MS demonstrated that the protein constituents of aphid saliva varied among aphid species (Fig. 3). Although the number of spots analysed and the number of proteins identified in each saliva sample did not change significantly, we observed more protein function diversity in M. persicae. This may be because M. persicae is a polyphagous species that is exposed to a greater diversity of plant defences, which may require a larger complement of salivary proteins. This was observed for detoxification enzymes (Ramsey et al., 2007); however, our analyses did not allow the assignment of specific molecular functions to a specific aphid species, nor to a specific feeding habit. Most of the variability among aphid salivary proteomes was associated with DNA-binding (22%), GTP-binding (19%) and oxidoreductase activity (19%) (Fig. 3). Although transcripts for DNA- and GTP-binding proteins were found in the salivary glands of A. pisum (Carolan et al., 2011), the involvement of these proteins in host plant–aphid interactions is speculative at best. We hypothesize that GTP-binding proteins may affect intracellular signalling by interfering with G-protein GTPases and that DNA-binding proteins may affect transcriptional regulation of host plant genes. Indeed, among the phytopathogenic bacteria of the genus Xanthomonas, DNA-binding proteins in the transcription activator-like effector family were found to be key virulence factors that mimicked eukaryotic transcription factors to modulate host cell functions and enable invasion (Sugio et al., 2007; Boch et al., 2009). In the following section, with the aim of uncovering host plant–aphid interactions, the proteins identified by MS were grouped based on their molecular functions and discussed in terms of their potential roles as mediators of plant–aphid interactions. Oxidoreductases. Proteins with assumed oxidoreductase activity were identified in all aphid species in the present study. This was consistent with previous substrate-specific

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Figure 3. Functional annotation of the differentially expressed proteins identified by 2D-DIGE-MALDITOF/MS in all the three aphid species addressed. Proteins were classified on the basis of data available in the literature and using the information available in the Swiss-Prot/TrEMBL and Gene Ontology databases.

assays demonstrating oxidoreductase activity in the saliva of several aphid species (Madhusudhan & Miles, 1998; Ma et al., 2010). Oxidoreductase enzymes are capable of converting molecular oxygen to free radicals including superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. In aphids, oxidoreductases are involved in the detoxification of plant defence compounds (Miles & Oertli, 1993). Among oxidoreductase enzymes, cytochrome P450 constitutes the largest class of insect detoxification enzymes (Li et al., 2006; Ramsey et al., 2007). We identified cytochrome P450 in saliva from M. viciae using 2D-DIGE/ MALDI-TOF/MS. Previously, cytochrome P450 was identified in the saliva of Diuraphis noxia (Nicholson et al., 2012), and transcripts were identified in the salivary glands of A. pisum (Carolan et al., 2011). This was not confirmed by our proteomics approach. Together, these results suggest that cytochrome P450 may be a common constituent of aphid saliva and have general roles in aphid feeding. Peroxiredoxin, an oxidoreductase belonging to the family of peroxidases, was identified in the oligophagous species A. pisum and M. viciae. In response to pathogen attacks, plant cells produce cytotoxic oxygen radicals such as hydrogen peroxide, which is a signalling molecule for pathogen defence responses in plants. Hydrogen per-

oxide can also kill pathogens directly and strengthen plant cell walls by oxidative cross-linking of cell wall structural proteins. In many pathogens and insects, peroxiredoxins are peroxide-detoxifying enzymes that play important roles in protection against oxidative stress (Piacenza et al., 2008; Dubreuil et al., 2011; Shi et al., 2012). Although a peroxidase has been identified in the saliva of the grain aphid Sitobion avenae (Rao et al., 2013), and peroxidase activity has been measured in the salivary secretions of several aphid species (Cherqui & Tjallingii, 2000), the present study is the first to report the identification of peroxiredoxins in aphid saliva. In addition to the general detoxification activities of oxidoreductase enzymes, insect glucose-methanolcholine oxidoreductases have been implicated in modifying plant defences. For example, the salivary glucose oxidase of H. zea reduces the production of nicotine in tobacco plants and suppresses jasmonic acid-related plant defence pathways (Musser et al., 2002, 2005, 2006). Although a glucose oxidase was previously identified in the saliva proteome of M. persicae (Harmel et al., 2008), we did not detect one in the present study; however, we identified a glucose dehydrogenase in both oligo- and polyphagous aphid species. This was consistent with other studies conducted on aphid saliva and salivary glands (Carolan et al., 2009, 2011; Nicholson et al., 2012). © 2013 The Royal Entomological Society, 23, 67–77

Salivary proteomes from three aphid species Peptidases. Pest- and pathogen-associated peptidases capable of digesting host defence proteins and peptides are essential for pathogenesis. These enzymes are common constituents of aphid salivary proteomes (Cherqui & Tjallingii, 2000; Carolan et al., 2009; Nicholson et al., 2012). We detected aminopeptidase enzymes in the saliva from all three aphid species using LC-ESI-MS/MS. An aminopeptidase was previously characterized in A. pisum with the authors suggesting an involvement in the detoxification of entomotoxic plant-expressed lectins (Cristofoletti et al., 2006). A previous proteomic analysis of secreted saliva from D. noxia biotypes also identified aminopeptidases (Nicholson et al., 2012); therefore, it is likely that aminopeptidases are common protein constituents of aphid saliva. We identified metalloproteases, another class of peptidases, in both the oligo- and polyphagous species, which are known to be components of insect (Macours & Hens, 2004) and aphid saliva (Carolan et al., 2009, 2011). Although the roles in saliva and potential involvement in plant–insect interactions are not known, it is likely that metalloproteases may detoxify plant defence proteins. Lipid-binding proteins. Apolipophorins were identified in the salivary proteomes of M. persicae and A. pisum. Apolipophorins belong to a family of proteins that play critical roles in lipid transport and lipoprotein metabolism. Lipids and free fatty acids function as plant defence and signalling compounds; therefore, secreted apolipophorins may interfere with signalling defence responses in the plant. In addition, a lipocalin was identified in M. persicae saliva. Although, its roles in aphid saliva remain to be determined, data suggest it may interfere with plant defence by altering lipid transport. ATP-binding proteins. Proteins with ATP-binding activity were observed in the saliva of all three aphid species. In mammals, extracellular ATP mediates diverse responses including platelet aggregation, inflammatory responses, neurotransmission and apoptosis (Ralevic & Burnstock, 1998). Hematophagous arthropods such as mosquitoes and ticks can suppress platelet aggregation by degrading extracellular ADP with apyrases (Ribeiro & Garcia, 1980; Ribeiro et al., 1984; Valenzuela et al., 2001; Faudry et al., 2004). Recent reports suggest that extracellular ATP may also serve as a signalling agent in plant cells (Demidchik et al., 2003). For example, physical injury to plants occurring as a result of herbivory or environmental stresses enhances the expression of a multitude of genes that function in the repair of damaged tissues and in defence mechanisms to prevent further damage (Reymond et al., 2000). Among responses to wounding, the accumulation of extracellular ATP increases the cytosolic calcium concentration and subsequently elicits plant defence © 2013 The Royal Entomological Society, 23, 67–77

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responses (Demidchik et al., 2003). It would therefore be beneficial for herbivorous insects to harbour proteins that reduce extracellular ATP and prevent plant responses during feeding. This hypothesis is supported by the identification of proteins with ATPase activity in the saliva of the caterpillar H. zea (Wu et al., 2012) and the whitefly Bemisia tabaci (Su et al., 2012). Although ATP scavenger enzymes have not been identified in aphid saliva to date, the demonstration of these proteins in the saliva of each species included in the present study suggests that the regulation of plant extracellular ATP may be an important process in establishing successful feeding by aphids.

Conclusions We investigated the salivary proteomes of aphid species A. pisum, M. viciae and M. persicae in an attempt to (1) understand the components of the saliva produced by these economic pests and to (2) test the hypothesis that polyphagous and oligophagous aphid species differ in term of abundance and functional diversity of salivary proteins. Comparative analyses revealed variability among aphid salivary proteomes. We observed a higher functional diversity of proteins in the generalist species M. persicae. These results may reflect the different host ranges of the three aphid species, namely members of the Fabaceae for A. pisum and M. viciae and 40 different plant families for M. persicae. This is consistent with the hypothesis that M. persicae would require a larger complement of salivary proteins to combat a greater diversity of plant defences; however, the present results did not allow the assignment of specific molecular functions to aphid species nor to specific feeding habits. Most of the variability observed in salivary proteomes was related to DNA-binding (22%), GTP-binding (19%) and oxidoreductase activity (19%). While oxidoreductase enzymes are ubiquitious enzymes in aphid saliva, with a well understood role, the involvement of salivary DNA- and GTP-binding proteins in aphid feeding and host interactions is still uncertain. Finally, in addition to known aphid salivary proteins, we identified peroxiredoxin enzymes and ATP-binding proteins that may play roles in aphid feeding. The present study contributes to the characterization of aphid saliva and may help reveal the functions of salivary proteins in aphid– plant interactions.

Experimental procedures Insects Acyrthosiphon pisum, M. viciae and M. persicae clones were collected from beans in a crop field in Gembloux (Belgium) in 1997. The insects were reared under standard conditions at

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20 ± 2 °C in a 16h light:8 h dark cycle. They were grown in separate growth chambers on broad beans (V. faba) in 12-cm pots.

Saliva collection and protein preparation Aphids were removed from plants by gentle shaking. Aphids were collected on white paper, cleaned of debris, and fed a diet of 15% sucrose (weight/volume). The diet was prepared under aseptic conditions with Milli-Q water, filtered through 0.45 μm filters (Millipore, Billerica, MA, USA), and sealed between two layers of stretched Parafilm (SERVA Electrophoresis GmbH, Heidelberg, Germany) on the bottoms of 27-mm diameter cylinders (PVC tube). For all the experiments aphids were delicately transferred to the feeding chambers. After 1 h, aphids that were not on the top of the cylinder were removed to ensure that the surface of the Parafilm layer was fully covered with aphids. The aphids were then allowed to feed for 48 h. The diet after feeding (containing saliva) was collected under aseptic conditions by gently peeling back a small portion of the Parafilm and slowly pipetting the liquid. The parafilm was washed with 200 μl of 15% sucrose diet and was pooled with the saliva-containing diet. The samples were concentrated using Vivaspin 20 concentrators with 3000 Da cut-offs (Sartorius Stedim, Sartorius stedim Biotech, Goettingen, Germany). The extracts were treated with the two-dimensional clean-up kit according to the manufacturer’s instructions (GE Healthcare, Little Chalfont, UK) and resuspended in the corresponding buffer for 1-DE, 2-DE, or in-solution digestion. Proteins were quantified using the RCDC quantification kit (Bio-Rad, Hercules, CA, USA).

One-dimensional gel electrophoresis Salivary protein pellets were dissolved in Laemmli buffer (BioRad). The 1-DE gels were run using 20 μg of total protein per sample. Proteins were electrophoresced on 12.5% resolving gels coupled to 4% stacking gels, run using the Bio-Rad Mini Protean System, and visualized with silver nitrate stain. Each collection was repeated in duplicate.

Two-dimensional differential in-gel electrophoresis Sample preparation and CyeDye labeling. Before electrophoresis, protein extracts (20 μg samples, three replicates/sample) were labelled with dyes (GE Healthcare) according to the standard 2D-DIGE protocol. Salivary protein samples were subjected to pairwise comparison. Six gels were run in total. Two salivary protein samples from two different species, labelled with Cy3 or Cy5, were mixed with an internal reference. The internal reference was made from pooled aliquots from all experimental samples labelled with Cy2. A conventional dye swap for 2D-DIGE was performed. First-dimensional isoelectric focusing for 2D-DIGE. The mixture of labelled proteins was adjusted to a volume of 450 μl and was used to rehydrate 24 cm immobilized pH gradient (IPG) strips (pH 3–10 NL) (GE Healthcare) for 12 h at 20 °C and a constant voltage of 50 V. Isoelectric focusing (IEF) was carried out at 200 V for 200 Vh, 500 V for 500 Vh, 1000 V for 1000 Vh, and 8000 V for 60000 Vh at 20 °C. A maximum current setting

of 50 μA/strip was used in an isoelectric focusing unit (GE Healthcare). Equilibration of IPG strips and 2-DE. After IEF, the IPG strips were equilibrated for 15 min in 375 mM Tris (pH 8.8) containing 6 M urea, 20% (v/v) glycerol, 2% (w/v) sodium dodecyl sulphate (SDS), and 130 mM 1,4-Dithio-DL-threitol (DTT). Then IPG strips were incubated for 15 min in the same buffer without DTT containing 135 mM iodoacetamide. The IPG strips were then sealed with 0.5% agarose in SDS running buffer at the top of slab gels [240 mm × 200 mm × 1 mm, 12% (w/v) acrylamide and 0.1% N,N′-methylenebisacrylamide]. Electrophoresis was performed at 20 °C in an Ettan Dalt-six electrophoresis unit (GE Healthcare) at 25 W/gel for 5 h. Gel scanning, image analyses, and protein digestion. Gels were scanned with a Typhoon fluorescence imager (Amersham, Little Chalfont, UK) at wavelengths corresponding to each dye. Images were analysed with PROGENESIS SAMESPOTS software (Nonlinear Dynamics Ltd, Newcastle, UK) according to the manufacturer’s instructions. Protein spots were excised (based on their presence/absence among the three aphid species) from the gel using an Ettan spot picker robot (GE Healthcare). Selected gel pieces were collected in 96-well plates designed for the Proteineer dp automated Digester (Bruker, Bremen, Germany). Briefly, gel pieces were washed with three incubations in 100% of 50 mM ammonium bicarbonate, and a mix of 50% acetonitrile 50% of 50 mM ammonium bicarbonate. Two additional washes were performed with 100% acetonitrile to dehydrate the gel. Freshly activated trypsin (Roche, porcine, proteomics grade) was used to rehydrate the gel pieces at 8 °C for 30 min. Trypsin digestions were performed for 3 h at 30 °C. Peptide extractions were performed with 10 μl of 1% formic acid for 30 min at 20 °C. Protein identification by MALDI-TOF/MS. Protein digests (3 μl) were adsorbed for 3 min on pre-spotted Anchorchips (R) using the Proteineer dp automaton. Spots were washed on-target using 10 mM ammonium dihydrogen phosphate in 0.1% TFA and MilliQ water (Millipore) to remove salts. High throughput spectra were acquired using an Ultraflex II MALDI mass spectrometer (Bruker) in positive reflectron mode with close calibration enabled. The Smartbeam laser focus was set to medium and the laser fluency setting was 65–72% of the maximum. Delayed extractions were set to 30 ns. Spectra in the range of 860–3800 Da were acquired at a 200 Hz laser shot frequency with automated evaluation of intensity, resolution and mass range. Six hundred successful spectra per sample were summed, treated, and de-isotoped in line with an automated SNAP algorithm using Flex Analysis 2.4 software (Bruker), and subsequently submitted in batch mode to the Biotools 3.0 software suite (Bruker) with an in-house hosted MASCOT search engine (www.MatrixScience.com). Two databases were used: (1) the public National Center for Biotechnology Information (NCBI) non-redundant database with parameters set for Arthropoda (released on 1January 2013 and including 1 766 009 sequences) and (2) a homemade database containing all available aphid 347 and aphid symbiont sequences (Table 1). A mass tolerance of 80 ppm with close calibration and one missing cleavage site were allowed. Partial oxidation of methionine residues and complete carbamylation of cystein residues were © 2013 The Royal Entomological Society, 23, 67–77

© 2013 The Royal Entomological Society, 23, 67–77

amidase ras-related protein Rab-26 zinc finger protein ADP-ribosylation factor-like protein multiprotein bridging factor 1 reverse transcriptase-like protein peroxiredoxin‡ ATP-binding cassette sub-family B metalloprotease*,§ cytochrome P450 reductase*,† ribosomal protein S28e-like salivary lipocalin salivary protein of the 35 kDa family neuroglobin-like ADP-ribosylation factor-like protein 3 GTPase 1 golgin subfamily A member 7 ATP-dependent RNA helicase PI10 DNA-binding protein rfxank macrophage migration inhibitory factor dynamin-1-like protein GTP-binding protein 10 endothelial differentiation-related factor 1 fatty acid/phospholipid synthesis protein multiprotein bridging factor 1 peroxiredoxin-6‡ neuroglobin-like 39S ribosomal protein L19 zinc finger MYM-type protein phospholipase DDHD1 zinc finger-containing protein 1 unc-112-related protein metalloprotease*,§ oxidoreductase*,† homer protein homologue 2 epoxide reductase ras-related protein Rap-1A DNA mismatch repair protein dihydrolipoamide dehydrogenase guanosine monophosphate reductase methyltransferase-like protein 6 dynamin-1-like protein

859 297 324 34 51 677 25 405 217 11 799 159 204 638 171 26 29 475 376 75 19 467 120 303 343 134 18 165 324 102 615 100 34 52 363 689 85 100 175 299 155 24

Aedes aegypti Apis mellifera Culex quinquefasciatus Acyrthosiphon pisum A. mellifera Ixodes scapularis Gryllotalpa orientalis A. mellifera C. quinquefasciatus Drosophila melanogaster A. pisum Triatoma brasiliensis Ae. aegypti A. pisum A. pisum A. pisum A. pisum A. pisum C. quinquefasciatus A. pisum A. pisum A. pisum A. pisum Serratia symbiotica A. mellifera A. pisum A. pisum A. pisum A. pisum Bombus terrestris Acyrthosiphon pisum A. pisum C. quinquefasciatus Buchnera aphidicola A. pisum A. mellifera A. pisum A. pisum Bombyx mori Acyrthosiphon pisum B. terrestris A. pisum

Organism gi|157104752 gi|66505876 gi|170037796 gi|193580240 gi|66512104 gi|56267945 gi|60300018 gi|328781281 gi|170072559 gi|17137192 gi|187175305 gi|116267203 gi|94468400 gi|242247087 gi|193580240 gi|229577161 gi|193706985 gi|328706401 gi|170029252 gi|193605965 gi|328709535| gi|328718050 gi|240849174 gi|320538805 gi|66512104 gi|240848687 gi|242247087 gi|240848691 gi|328717580 gi|340715461 gi|328716888 gi|328697257 gi|170072559 gi|311086271 gi|328703533 gi|110761015 gi|326320031| gi|193636599 gi|295852987 gi|237858775 gi|340718634 gi|328709533

NCBI Accession 58269 20149 59793 21149 16265 57849 25245 75485 36433 77040 7454 18150 8942 16452 21149 70409 13686 77601 23613 14682 79113 45686 16068 38079 16265 25245 16452 35597 66820 84115 210975 79202 36433 43904 41631 19864 20975 77118 52481 37952 32055 79113

Theoretical MW

NCBI, National Center for Biotechnology Information; MW, molecular weight; pI, isoelectic point. Previously reported by *Carolan et al. 2011; †Nicholson et al. 2012; ‡Rao et al. 2013; §Carolan et al. 2009.

Protein identification

Spot Nb

Table 1. Proteins identified by 2D-DIGE-MALDI-TOF/MS

7.66 5.54 8.20 8.77 10.20 10.13 6.16 9.22 9.38 5.62 10.38 9.40 5.81 5.92 8.77 8.58 8.35 5.96 5.33 4.89 5.74 9.21 9.89 9.59 10.20 6.16 5.92 9.33 9.15 5.69 8.51 6.74 9.38 9.30 8.90 8.45 5.65 6.22 7.18 6.67 7.58 5.74

Theoretical pI 58 57 58 52 52 60 71 69 56 55 56 47 49 64 52 45 59 58 48 48 45 47 56 48 52 71 64 47 54 56 63 58 62 48 49 48 56 72 48 71 45 46

Score 14 29 12 33 40 18 41 16 22 14 55 34 19 62 33 10 39 17 19 35 11 12 37 11 32 41 62 13 14 12 12 18 19 10 21 25 29 18 15 24 14 14

Coverage 9/38 5/12 6/17 4/16 6/37 11/45 9/47 10/40 5/16 6/19 7/35 4/19 5/29 9/75 4/16 7/38 5/31 11/51 4/25 4/35 6/32 6/27 6/42 5/26 5/27 9/47 9/45 4/38 9/45 6/15 23/75 12/50 4/14 5/30 7/48 7/39 4/53 12/53 6/40 7/40 4/32 8/34

Peptide Nb

x

x

x

x x

x x

x

x x x x

x

x

x x x

x x

x

x x x x

x x

x x x x

x

x x x

x

x

x x x x

Acyrthosiphon pisum

x

x

x

x

Myzus persicae

x x

x x x x

x

x

x x

x x x

x

x x

x

Megoura viciae

Salivary proteomes from three aphid species 73

search

5

2

6

4

gi|193659536

gi|193610809

gi|193669489 gi|193627355

gi|193610805

gi|193636568

gi|193607327

gi|193676365

gi|209571509

gi|33520007

gi|193659536

A. pisum

A. pisum A. pisum

A. pisum

A. pisum

A. pisum

A. pisum

A. pisum Megoura viciae Buchnera aphidicola A. pisum

1

2

gi|193676365

gi|27904343

gi|193678823

gi|193671609

gi|38491462 gi|193575603

gi|193620211

gi|10039177

A. pisum

Buchnera aphidicola A. pisum

A. pisum

B. aphidicola A. pisum

A. pisum

B. aphidicola

26569

27989

57475 106723

69222

112714

79598

128504

65760

80591

43732

75113

128504

78337

56037

88858

26994 65760

102225

80591

69222

Theoretical MW

10.40

5.67

4.84 6.67

5.80

5.81

6.89

8.28

5.61

5.05

5.71

6.80

8.28

4.99

5.06

5.41

5.29 5.61

5.25

5.05

5.80

Theoretical pI

K.NEFVATDFNLVPIDK.N R.HKLAVHVR.S K.LAVHVR.S K.EVIICAGPIK.S K.SPQLLMLSGIGPK.E R.EMLPVLEAVAK.A K.KVMDTWTRQAGFPLVSAIR.N R.DALTASK.E K.HFKEAENILATALFQAKQK.L K.EAENILATALFQAKQK.L K.HPVSSEDLIK.Y K.GIKVEVSGR.L

R.AFDQIDNAPEEK.A R.HYAHVDCPGHADYVK.N K.KSTTYKK.K K.ALSINTLEMCQK.Y K.YGYNVEGLYVVPEFLR.L K.FLTEQEDNLFK.G K.NLATSIFHSVGTNK.M K.IQPDSTTGFGIGGNMK.I K.IVCSGFEHIGCK.K K.AYLSPIFGR.E K.HLSEMEVPVVK.D K.IATDPFVGNLTFFR.V

K.LMSVLGTDALPEDK.L

R.GVDFLFPDSK.Y K.IADIYEYYQR.D R.DYSAGTVTLSQER.Y K.IADIYEYYQR.Q K.LEDIDLDGCAK.Y

K.LGSSKPWFDAI ELLTGQR.E K.NRPIEDIVAESQK.L K.NLATSIFHSVGTNK.M K.VIGIDKLR.V K.GLGYAQYINSDLEDVIIK.I

K.ALSINTLEMCQK.Y K.WSWEDVLK.Y K.EVILCAGPIGSPK.L K.MIGISLLRPK.N R.FFADTSSAHLNAFLQSLCR.T

K.SPQLLMLSGIGPK.E K.NITSLGLSK.L

Peptides

17.39

17.39

36

33

17.39 17.39

62 38

17.39

17.39

91

74

17.39

17.39

113

97

17.39

17.39

117

116

17.39

2.94

2.94

2.94

2.94

2.94

2.94 2.94

172

48

51

55

64

83

118 108

2.94

2.94

147

120

50.00

FDR†

60

score

MASCOT

*Cover: expressed in % of total sequence. NCBI, National Center for Biotechnology Information; MW, molecular weight; pI, isoelectic point. †FDR, false discovery rate calculated according to peptide matches above identity threshold under MASCOT software. Previously reported by ‡Carolan et al. 2011; §Nicholson et al. 2012; **Carolan et al. 2009; ††Cherqui and Tjallingii 2000.

3

11

2 2

3

4

gi|193627355

A. pisum

4

1

3

4

2

7 5

2

3

Cover*

gi|193671609

NCBI Accession

Myzus persicae Acyrthosiphon pisum A.pisum A. pisum

Organism

MASCOT

Table 2. Proteins identified by LC-ESI-MS/MS using the homemade database

30S ribosomal protein S3

mediator of RNA polymerase II transcription

chaperonin GroEL glutamyl aminopeptidase§,**,††

glucose dehydrogenase‡,§,**

metalloprotease‡,**

elongation factor G

glucose dehydrogenase‡,§,**

glucose dehydrogenase‡,§,**

glucose dehydrogenase‡,§,**

elongation factor Tu

angiotensin-converting enzyme

aminopeptidase N-like isoform 3§,**,†† glucose dehydrogenase‡,§,**

aminopeptidase§,**,††

lipophorin

angiotensin-converting enzyme glucose dehydrogenase‡,§,**

apolipophorins

glucose dehydrogenase‡,§,**

glucose dehydrogenase‡,§,**

Protein identification

BLAST search

B. aphidicola

A. pisum

B. aphidicola A. pisum

A. pisum

Culex quinquefasciatus

B. aphidicola

D. noxia

A. pisum

A. pisum

B. aphidicola

Diuraphis noxia A. pisum

A. pisum

Nilaparvata lugens A. pisum

Pediculus humanus corporis A. pisum A. pisum

A. pisum

A. pisum

Organism

3,00E-162

3,00E-154

0 0

0

0

0

1,00E-170

0

0

0

0

1,00E-170

0

1,00E-126

5,00E-88

gi|15617112

gi|193620211

gi|15616648 gi|193575603

gi|328710729

gi|170039557

gi|15617121

gi|347546083

gi|328709186

gi|193659536

gi|28952057

gi|328705709

gi|347546083

gi|328702668

gi|328702664

gi|209571429

gi|328705709 gi|328709186

gi|242008029

2,00E-60

2,00E-167 0

gi|193659536

gi|328710729

NCBI Accession

0

0

Score (E value)

74 S. Vandermoten et al.

© 2013 The Royal Entomological Society, 23, 67–77

© 2013 The Royal Entomological Society, 23, 67–77

glutamyl aminopeptidase§,**,†† 16.67

60S acidic ribosomal protein P2 angiotensin converting enzyme glyceraldehyde 3 phosphate dehydrogenase 2 3.70 3.70 3.70

glucose dehydrogenase‡,§,†† 3.70

translation elongation factor chaperonin groEL glutamyl aminopeptidase §,**,†† m1 zinc metalloprotease‡,†† 0.00 0.00 0.00 0.00

0.00

FDR†

75

2 gi|167872260

NCBI, National Center for Biotechnology Information; MW, molecular weight; pI, isoelectic point. *Cover: expressed in % of total sequence. †FDR: false discovery rate calculated according to peptide matches above identity threshold under MASCOT software. Previously reported by ‡Carolan et al. 2011; §Nicholson et al. 2012; **Carolan et al. 2009; ††Cherqui and Tjallingii 2000.

67 R.QAFPCFDEPQLK.A R.NQKSVADWLK.T 5.86

5.11 6.80 7.05 14125 74533 26820 12 2 5

Culex quinquefasciatus A. pisum Drosophila affinis Megoura viciae Culex quinquefasciatus

gi|167869979 gi|209571509 gi|60099994

116883

54 48 38

208

K.ALSINTLEMCQK.Y K.WSWEDVLK.Y K.EVILCAGPIGSPK.L K.MIGISLLRPK.N K.VVNEMKGKSVVELIATR.R LMSVLGTDALPEDK VPTPNVSVADLTVR 5.05 5 gi|193659536

80591

111 62 62 51 5.59 4.92 5.86 4.77 43296 57121 116883 109893 3 2 1 1 gi|215501888 gi|7443844 gi|167872260 gi|157120775

Ixodes scapularis Sitophilus oryzae Culex quinquefasciatus Aedes aegypti A. pisum A. pisum

gi|193659536 Myzus persicae Acyrthosiphon pisum

3

80591

5.05

K.ALSINTLEMCQK.Y K.YGYNVEGLYVVPEFLR.L R.HYAHVDCPGHADYVK.N R.EMLPVLEAVAK.A R.QAFPCFDEPQLK.A R.TAFPCFDEPALK.A

112

score MASCOT

Peptides Theoritical pI Theoritical MW Cover* NCBI Accession Organism

Table 3. Proteins identified by LC-ESI-MS/MS using the NCBI database with parameters set for Arthropoda

Protein digestion and LC-ESI-MS/MS analyses. Salivary protein pellets were re-suspended in 100 μl of 50 mM ammonium bicarbonate pH 8. In-solution digestions were performed using 20 μg total protein/sample. Five μl of reduction solution (200 mM DTT, 100 mM ammonium bicarbonate pH 8) was added to the samples and they were incubated at 100 °C for 10 min. The alkylation solution (4 μl) (1 M iodoacetamide, 100 mM ammonium bicarbonate pH 8) was added and the samples were incubated at room temperature (22 °C) for 60 min in the dark. The alkylation reaction was stopped by adding 20 μl of reduction solution. Trypsin digestions were performed overnight at 37 °C. Digested samples were dried in a vacuum concentrator. The experiments was repeated in duplicate. Peptide separations were performed on an Ultimate LC system (LC Packings) with a Famos autosampler and a Switchos II microcolumn switching device for sample clean-up and preconcentration. Each sample (30 μl) was loaded in duplicate at a flow rate of 200 nl/min on a micro-precolumn cartridge (300 μm i.d. x 5 mm, packed with 5 μm C18 100 Å PepMap). After 5 min, the pre-column was connected with the separating nano-column (75 μm i.d. x 15 cm, packed with C18 PepMap100, 3 μm, 100 Å) and the gradient was initiated. The elution gradient varied from 0 to 30% buffer B for 30 min. Buffer A consisted of 0.1% formic acid in acetonitrile:water 2:98 (vol/vol) and buffer B consisted of 0.1% formic acid in acetonitrile:water 20:80 (vol/vol). The outlet of the LC system was directly connected to the nano electrospray source of an Esquire HCT ion trap mass spectrometer (Bruker). Mass data were acquired from 50 to 1700 m/z using the standardenhanced mode (8100 m/z per s). For each mass scan, a datadependent scheme picked the four most intensely charged ions to be isolated and fragmented in the trap, and the resulting fragments were mass analysed using the Ultra Scan mode (50– 3000 m/z at 26 000 m/z per s). The MS/MS spectra were searched against against two databases: (1) the public NCBI non-redundant database with parameters set for Arthropoda (released on 2 August 2013 and including 1 766 449 sequences) and (2) a homemade database containing all available aphid and aphid symbiont sequences. For protein matches without a known function, BLAST searches were performed; BLAST E-scores are provided in Tables 2 and 3. The significance threshold used was P < 0.05; P was the probability that the observed match was a random event. All proteins with peptide scored above 50 were accepted. A false discovery rate filter was applied to the peptides and proteins identified using a decoy approach. False-positive identifications were removed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral .proteomexchange.org/cgi/GetDataset) via the PRIDE partner repository with the dataset identifier PXD000476 and DOI10.6019/PXD000476.

Protein identification

considered. The probability score calculated by the software was used as one criterion for correct identification. In order to confirm the identifications, experimental molecular weights (MW) and pI were compared to the predicted values resulting from the MASCOT analysis (data not shown). Significant interpretation was also correlated to the identified organism (mainly A. pisum) and protein nature and function in the studied biological matrix. Proteins were classified based on the literature and information available in the Swiss-Prot/TrEMBL and Gene Ontology databases.

glucose dehydrogenase‡,§,**

Salivary proteomes from three aphid species

76

S. Vandermoten et al.

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