Targeted protein sequences were imported into Skyline [32] and converted into trypsin fragments. Refinement was performed as described [91] . Briefly, to optimize collection of SRM data, we focused initial analysis on peptides from host proteins that could be detected in the matrix of total protein homogenates extracted from R. padi fed on CYDV-RPV infected tissues. From these samples, MS/MS data were collected for Skyline-predicted tryptic peptide ions from host proteins of interest. These data were imported back into Skyline for refinement of the SRM method. During refinement, we selected proteotypic peptides that ionized well (3-4 abundant y-ions for each peptide) and showed reproducible chromatographic retention properties and made a new SRM method. The new, data-driven, refined SRM method was exported to the mass spectrometer. Three biological replicates were analyzed using the refined SRM method, and a Student's Ttest was used to compare total peak areas. A normalization factor of 0.92 was calculated by monitoring for peptides derived from two different proteins that were not differentially expressed between treatments and applied to the peptides derived from healthy samples. Both raw and normalized peak areas for each transition are reported (Table S2 ). Figure S1 (A) 1-D PAGE of proteins from purified virus showing bands that were excised and digested with trypsin. Lane 1, Broad range molecular weight standards (Biorad) in kDa; Lane 2, transmissible virus; Lane 3; non-transmissible virus. The band containing the RTP is encircled in red. Other bands subjected to LC-MS/MS analysis are indicated with a red *. Multiple proteins were found in each lane. These proteins were abundant contaminants (reported in the text and Table S1) also found in sucrose gradients separating healthy tissue. Tryptic peptides matched to the full-length RTP in (B) non-transmissible virus purified with sodium sulfite and EDTA and (C) transmissible virus are highlighted in red. Virions were purified from the same infected source tissue (oats), the only difference was addition of sodium sulfite and EDTA in homogenization buffer. Peptides were identified from the same region of the protein in both treated and untreated virions indicating no cleavage of the RTP in the nontransmissible virion preparations. (TIF) Figure S2 Clustal W alignment showing CYDV-PRV cysteine residues in the RTP in the context of a multiple alignment of twelve luteovirid species. C136 and C373 are highly conserved among luteovirids whereas C112 is unique to CYDV-RPV. In CYDV-RPV, all three cysteine residues within the RTP are flanked by at least one basic amino acid, making them especially reactive and likely to be involved in disulfide bonding. (TIF) Figure S3 SRM transitions of the peptides from host plant proteins that were detected in tryptic digests of pooled, whole-body R. padi protein samples (from data shown in Table 5 ). One replicate per peptide is shown. Two peptides from cellulose synthase show no statistically significant fold-change in aphids reared on CYDV-RPV infected or healthy plants (A) SQTGDFDHNR detected aphids fed on CYDV-RPV infected plants or (B) healthy plants, (C) IPMFAYVSR detected in aphids fed on CYDV-RPV infected plants or (D) healthy plants. The peptide FGGDTYCCR from thaumatin-like protein 5 detected in aphids fed on CYDV-RPV infected plants in (E) or healthy plants in (F). The peptide VLYSSCYVR from 33-kD secretory protein detected in aphids fed on CYDV-RPV infected plants (G) or healthy plants (H). The peptide VLYSSCYVR was at the lower limit of detection in the samples derived from aphids reared on healthy plants. The peptide SDSIITAYR from pyruvate dehydrogenase E1 derived from samples of aphids collected from CYDV-RPV infected plants (I) or healthy plants (J). One peptide was detected from pyruvate dehydrogenase E2: GLGMIAEEVK was only detected in samples of aphids reared on CYDV-RPV infected tissues (K), and not in aphids reared on healthy tissue (L). The next step to confirm these differences in aphids fed on healthy or infected plants is validation and absolute quantification of these peptides via the use of stable isotope labeled peptides. (TIF)