To rule out co-sedimentation of plant proteins with purified virus based on density alone, we used LC-MS/MS to thoroughly characterize sucrose gradient fractions of healthy plant homogenates (Table S1 ). Healthy plants were grown in the greenhouse alongside infected plants and the tissue was used in the virus purification protocol. Fractions from the gradient that would normally contain virus particles when infected plant tissue is used in the purification were subjected to trypsin digestion and LC-MS/MS to identify any plant proteins that could co-sediment with virus particles based on density and not a physical association. Four proteins ( Table 4 ) that are likely not associated with virions, Figure 2 . RT-PCR of total RNA extracted from R. padi aphids that fed on different virus preparations was performed to detect RPV using RPV coat protein primers, amplifying a 614 bp product. Aphids were collected after initially feeding for a 24 h AAP (A) and after 3 d IAP (B). Three independent replicates using RNA collected from small pools of aphids are shown for each treatment. doi:10.1371/journal.pone.0048177.g002 Table 2 . Summary of R. padi transmission and virion detection following membrane feeding of different cereal yellow dwarf virus-RPV preparations used for RT-PCR and TEM evaluations. including homologues of three ribosomal proteins and fructokinase, were also identified in the analysis of the gradient fractions from healthy plants. Other abundant proteins detected in the sucrose gradients from healthy tissue included Rubisco, pyrophosphate-dependent phosphofructo-1-kinase, 6-phosphofructokinase, ribosomal proteins, and histone proteins (Table S1 ). Although many of these proteins have been detected in phloem sap in other plant species [26] and in the gradients of purified virus, the presence of these proteins is probably unrelated to the transmissibility of virions since they were detected in the fractions from healthy tissue. Importantly, no peptides were identified as belonging to CYDV-RPV indicating that the plants used for the healthy controls were not infected. Analysis of purified sodium sulfite and EDTA-treated and nontreated CYDV-RPV using 1-D gel electrophoresis often showed distinct protein profiles (Fig. S1A ). These differences were initially thought to be due to degradation products of the RTP; since the C-terminal half of the RTP is truncated during purification [30, 31] . For both transmissible and non-transmissible virus, we analyzed gel bands corresponding to viral and host proteins. MS analysis of peptides recovered from an in-gel trypsin digestion of band containing the truncated RTP from purified virions produced nearly identical tryptic fragments representing both the CP and RTP from both treated (Fig. S1B ) and untreated virions (Fig. S1C ). These data indicate minimal or no differences in the virus protein composition of the virion particles. Consistent with previous reports of C-terminal truncation of the RTP during purification, no peptides were discovered in the C-terminal portion of the RTP from either type of purified virus. The highly abundant proteins (Table S1 ) were also found in analysis of the gel bands from Fig. S1 (data not shown) and enabled us to attribute some of the variability in the gel bands to differences in plant protein complexes that co-sediment based on their density alone.