Title: The identification of protein biomarkers distinguishing virus transmission competent and refractive insect populations by coupling genetics with quantitative intact proteomics Authors
Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Proceedings
Publication Acceptance Date: May 27, 2010
Publication Date: May 27, 2010
Citation: Cilia, M., Tamborindeguy, C., Thannhauser, T.W., Gray, S.M. 2010. The identification of protein biomarkers distinguishing virus transmission competent and refractive insect populations by coupling genetics with quantitative intact proteomics. Journal of American Society for Mass Spectrometry. 58:42. Technical Abstract: Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are vectored by aphids. The identification of vector proteins mediating virus transmission is critical to develop agriculturally-sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Previously, we generated F2 populations by crossing transmission competent and refractive aphids. The refractive F2 genotypes differed in their transmission barrier: the gut or the salivary gland, and in their ability to transmit distinct species of yellow dwarf virus. Thus, a partially overlapping set of alleles act at the gut and salivary gland to control the circulative movement of each barley yellow dwarf virus species in the aphid. Aphids are perfectly suited for coupling genetics with quantitative intact proteomics (G-QIP) because genotypes can be clonally propagated indefinitely, or mated sexually. Competent and refractive F2 genotypes were compared using DIGE-LC-MS/MS. Multivariate statistics were applied to identify proteins that are coordinately regulated and differentially expressed between the F2 clones. Principle components analysis was applied to the data to identify the sources of variation, and serendipitously indicated incorrectly categorized genotypes with respect to virus transmission phenotype. Ten proteins had pI isoforms specific to competent and refractive aphid clones. We tested the robustness of these ten proteins as biomarkers to distinguish between transmission competent or refractive aphid populations using DIGE and virus transmission assays with aphids of uncharacterized vector competency. Thirty-five aphid proteins involved in energy metabolism, membrane trafficking and the cytoskeleton were identified as differentially expressed between competent and refractive aphids; among them a receptor with homology to the human Epstein Barr virus receptor, PITP, GAPDH3, and tubulin. Surprisingly, proteins from the aphid’s obligate and maternally-inherited bacterial endosymbiont Buchnera were also identified, suggesting its involvement in virus transmission. G-QIP made it possible to identify where the proteins are expressed in refractive aphids with different barriers to transmission. For example, to identify proteins that promote virus translocation through the gut, refractive aphids with a pure salivary gland barrier (refractive type-2, or RT-2) were grouped with competent aphids because both permit virus movement through the gut. Refractive type-1 (RT-1) aphids have a strong gut barrier. Identification of proteins differentially expressed between RT-1 and the group containing competent aphids and RT2 could identify candidates predicted to act at the gut for virus movement. In this class, cuticle proteins were identified. Portions of the aphid gut are known to be lined with cuticle, therefore our analysis suggests a novel involvement for cuticle proteins in virus movement across the gut epithelium at these sites. Ten of the 35 proteins had pI isoforms specific to competent and refractive aphids. We categorized 10 aphid populations with unknown vectoring ability using the DIGE profile of an aphid population at these ten protein biomarkers and their respective isoforms. Based on the biomarker profiles, we predicted 3 aphid populations to be competent and 7 populations to be refractory. We then conducted virus transmission assays. Expression of the at least 9 of the biomarker isoforms found in the competent F2 genotypes distinguished between competent and refractory aphids in different genetic backgrounds with 100% accuracy. G-QIP pinpointed the functional location of candidate proteins and delivered a robust set of protein biomarkers for virus transmission by insects.