Grapevine red blotch is a recently identified viral disease that was first recognized in the Napa Valley of California. Infected plants showed foliar symptoms similar to leafroll, another grapevine viral disease, on vines testing negative for known grapevine leafroll-associated virus. Later, the Grapevine red blotch virus (GRBV) was independently discovered in the US states of California and New York and was demonstrated to be the causal agent of red blotch disease. Due to its wide occurrence in the US, vector transmission and impacts on grape industry, this virus has the potential to cause serious economic losses. Despite numerous attempts, it was not possible to isolate or visualize viral particles from GRBV infected plants. Consequently, this has hampered the development of a serological assay that would facilitate GRBV detection in grapevine. We therefore decided to explore mass spectrometry approaches in order to quantify GRBV in infected plants and to identify potential biomarkers for viral infection. We present for the first time the physical detection on the protein level of the two GRBV genes V1 (coat protein) and V2 in grapevine tissue lysates. The GRBV coat protein load in leaf petioles was determined to be in the range of 100 to 900 million copies per milligram wet weight by using three heavy isotope labeled reference peptides as internal standards. The V1 copy number per unit wet tissue weight in leaves appeared to be about six times lower, and about 200-times lower in terms of protein concentration in the extractable protein mass than in petioles. We found a consistent upregulation of several enzymes involved in flavonoid biosynthesis in leaf and petiole extracts of GRBV-infected plants by label-free shotgun proteomics, indicating the activation of a defense mechanism against GRBV, a plant response already described for grapevine leafroll associated virus infection on the transcriptome level. Last but not least, we identified some other microorganisms belonging to the grapevine leaf microbiota, two bacterial species (Novosphingobium sp. Rr 2-17 and Methylobacterium) and one virus, Grapevine rupestris stem pitting associated virus.

For the targeted, absolute quantification of GRBV coat protein, we used the three proteotypic stable-isotope labelled peptides NDVSGGGRNDVER[13C6,15N4], IYLSAASASGHTFK[13C6,15N2], and AAFNIFQR[13C6,15N4] (Bachem AG, Bubendorf, Switzerland) with a PRM approach on a QExactive HF mass spectrometer coupled with an Easy-nLC 1000 (Thermo Fischer, Bremen; Germany). Protein digests were loaded onto a pre-column (PepMap C18, 5µm, 300A, 300µm x 5mm) at a pressure of 500 bars with loading solvent (0.05% TFA in water/acetonitrile 98:2). After loading, peptides were eluted in back flush mode onto the analytical nano-column (C18, 3µm, 155Å, 0.075 mm i.d. x 150mm length, Nikkyo Technos, Tokyo, Japan) using an acetonitrile gradient of 5% to 40% solvent B (0.1% FA in water/acetonitrile 4,9:95) in 60min at a flow rate of 400nL/min. PRM acquisition was done with a scheduled inclusion list between 8 to 15minutes for NDVSGGGRNDVER (3+ ion, m/z= 458.8834, and m/z= 462.21950 for the light and heavy form, respectively), between 25 to 33 minutes for IYLSAASASGHTFK (3+ ion, m/z= 484.9209, and m/z= 487.5923), and between 34 to 41 minutes for AAFNIFQR (2+ ion, m/z= 483.76143, and m/z= 488.76560). Resolution was set to 30’000 (at m/z=400), AGC target to 2e5, mIT to 130ms, and a relative HCD energy of 28%, respectively.

For 50 mg of leaf powder, 0.2 mL of 50 mM Tris/HCl pH 8.0 / 4% (w/w) SDS / 50 mM DTT / protease inhibitor cocktail (Roche, Rotkreuz, Switzerland) was added. The mixture was vortexed well with subsequent agitation for 30 min. at 4°C. The resulting suspension was centrifuged for 15 min. at 16’000×g and 4°C. The supernatant was either used directly for SDS-PAGE or transferred to another reaction vial, heated for 5 min. at 95°C, cooled to room temperature before addition of ¼ volume of 1M iodoacetamide in 100 mM Tris/HCl pH 8.0 and incubation for 30 min. at 37°C. Proteins were precipitated by addition of 10% (w/w) TCA, 0.07% DTT (w/w) in acetone by overnight incubation at -20°C. Proteins were spun down for 15 min. at 16’000×g and 4°C. The supernatant was discarded and the pellet washed four times with ice-cold 0.07% (w/w) DTT in acetone. The final pellet was dried in ambient air and resuspended in 8 M urea / 50 mM Tris/HCl pH 8.0.