Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

Wilkinson TL (2013) Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins. PLoS ONE 8(2): e57413. doi:10.1371/journal.pone.0057413 Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins Sohail A. K. Rao. 0 1 James C. Carolan. 0 1 Tom L. Wilkinson 0 1 Guy Smagghe, Ghent University, Belgium 0 a Current address: Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture , Faisalabad , Ireland b Current address: Department of Biology, National University of Ireland Maynooth , Maynooth , Ireland 1 School of Biology and Environmental Science, University College Dublin , Dublin , Ireland The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. - Funding: Financial support was provided through Department of Agriculture, Food and the Marine Research Stimulus Grant 07/553, and in part by Science Foundation Ireland Principal Investigator Grant 03/IN3/B381 and UCD Seed Funding SF268. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Insects that feed on living prey rely on bioactive compounds present in their saliva to negate the defences of their host. The saliva of animal and plant feeding insects alike comprises a diverse suite of proteins that suppress, circumvent, modulate and even induce host defence and immune responses. Whilst the saliva of blood feeding insects has been extensively studied, due in part to their role as vectors of mammalian disease e.g. [13], it is becoming increasingly clear that the salivary repertoires of plant feeding insects are equally complex. Aphids (Hemiptera: Aphidoidea) are phytophagous insects that feed on the phloem sap of plants. They are unusual herbivores because their feeding site is a single phloem cell in the sieve element buried deep within plant tissues, yet they represent one of the most important insect pests of temperate agriculture [4,5]. The aphid mouthparts are modified into fine, needle-like stylets that can penetrate between plant cells and puncture individual cells, including the sieve element. Aphid feeding reduces plant fitness by removing photoassimilates, transmitting plant viruses and, in some cases, altering plant growth and development [6]. The intimate relationship between the aphid and plant is mediated by the secretion of copious amounts of saliva during all stages of feeding, including probing and ingestion [7]. The saliva is secreted as a liquid but during probing it hardens around the stylets to form a sheath that remains in the plant after the aphid has withdrawn the stylets. When the stylets are located within cells the saliva remains in liquid form (so-called watery saliva). The secretion of saliva is divided into four phases: (i) intercellular sheath secretion; (ii) intracellular salivation into cells along the stylet path; (iii) phloem salivation into sieve elements; and (iv) phloem feeding salivation i.e. feeding interspersed with sporadic periods of salivation [8,9]. The composition of aphid saliva and the specific functions of salivary proteins have become clear only recently, driven primarily by the increase in available genomic resources for aphids, particularly the sequenced genome of the pea aphid Acyrthosiphon pisum (Harris, 1776) [10]. Salivary gland expressed sequence tag (EST) libraries for A. pisum and the green peach aphid Myzus persicae (Sulzer, 1776) have been exploited to provide candidate salivary proteins [1113]. Subsequent characterisation of a number of these proteins has indicated their importance in facilitating the plant-aphid interaction [12,14]. Mass spectrometry (MS)-based proteomics has also been used to identify proteins directly from saliva secreted into chemically-defined diets by M. persicae [15] and A. pisum [16]. These studies have identified metalloproteases, glucose oxidases, regucalcin, NADH dehydrogenase and several novel proteins lacking homologues outside aphids, including a putative constituent of the salivary sheath. In this paper, we identify salivary proteins secreted into chemically-defined diets by Sitobion avenae (Fabricius, 1775) and Metopolophium dihrodum (Walker, 1849), two aphid species that are important agricultural pests of cereals in Europe [17,18]. The results represent one of the first MS-based characterisations of the saliva of aphids that are restricted to feeding on plants from within the monocot family Poaceae, and are a test of the utility of using genomic information derived from A. pisum (see www.aphidbase. com) to identify proteins in other aphid species from peptide mass spectrometry. The identification and functional characterisation of aphid salivary proteins has ecological and applied implications since variation in salivary protein composition could be an important driver in plant acceptance, perhaps mediating aphid host plant range and the ability of specific genotypes to exploit different crop varieties. Salivary profiles from S. avenae and M. dirhodum are compared to those obtained for A. pisum [16] to identify common proteins secreted by the different aphids. cDNA sequencing of the common salivary genes informed the design o (...truncated)