The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Abstract

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE-LC-MS/MS and LC-MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin-converting enzyme (an M2 metalloprotease), an M1 zinc-dependant metalloprotease, a glucose-methanol-choline (GMC)-oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium-binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium-mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.

Predicted Effector Molecules in the Salivary Secretome of the Pea Aphid (Acyrthosiphon pisum): A Dual Transcriptomic/Proteomic Approach

“…Seven nonsynonymous substitution sites were identified as evolving significantly faster than synonymous substitutions in the same genes, an indication that they are potentially under positive selection. Interestingly, the two glucose dehydrogenases identified in this study (ACYPI-000986 and ACYPI000288) also evolved at a faster rate relative to homologues in other insects, but in contrast the glucose dehydrogenase (ACYIPI000113; GMC oxidoreductase) identified in pea aphid saliva 28 has evolved at a significantly lower rate relative to other insect homologues. We found no evidence for positive selection among the predicted salivary glucose dehydrogenase genes (ACYPI000986 and ACYPI000288).…”

“…The validity of this approach is supported by the fact that our resulting data set includes all but one (an unknown protein ACYPI008138) of the proteins identified by direct MS analysis of the saliva. 28 Further validation of our approach can be inferred from the high proportion of annotatable salivary-gland enriched transcript supported proteins that possess a secretion signal. This includes some of the proteases, oxidoreductases, and apolipophorin as well as some hypothetical proteins with homologues in other insect species.…”

“…28 The bestsupported of the predicted secreted salivary proteins were subjected to PhylomeDB analysis (http://phylomedb.org) to identify related genes arising by speciation (orthologs) or duplication (paralogs). Putative paralogs were cross-referenced to the EST and proteome data sets (salivary gland and saliva) to determine whether there was any evidence that the paralogs could also be secreted salivary proteins.…”

“…Using antibody-based approaches, Mutti et al 33 confirmed that a protein called C002, initially identified by prospecting salivary gland ESTs and genome analysis, was detected in the plants upon which aphids had fed, confirming that it is a component of the secreted saliva. More recently, mass spectrometry (LC-MS/MS) resulted in the identification of nine secreted salivary proteins, four of which exhibited orthology to proteins in existing databases: 28 a GMC oxidoreductase, an SMP-30-like protein (regucalcin), an angiotensin converting enzyme (Ance)-like protein (carboxyl dipeptidase) and an M1 metalloprotease.…”

“…Aphids initially secrete a gelling saliva that hardens and forms a protective sheath around the mouthparts, followed by watery saliva which is secreted into the phloem immediately before and during feeding but also into cells as the tip of the mouthparts progress toward the phloem. 21,22 A number of strategies have been employed in an attempt to catalogue and characterize the salivary proteins of various aphid species and include mass spectrometry of collected saliva, 27,28 RNA interference, 29 salivary gland EST analysis, 30 in planta analysis of phloem occlusion mechanisms, 23 plant differential gene expression studies 31 in response to aphid salivary concentrates and in planta expression of candidate salivary effector proteins. 32 The availability of a genome sequence for the pea aphid, Acyrthosiphon pisum, has made this species a particularly good candidate for saliva studies.…”