PVP 40

Tissues were collected from five or six year-old ramets (genetically identical trees) of a single female poplar hybrid clone (P. trichocarpa × P. deltoidies) over a seven-month period in 2001 (Table 1). The trees had been growing in commercial plantations in the Columbia River basin northwest of Portland, Oregon USA. Bud scales were removed and tissues were frozen in liquid N₂ and stored at -80°C until RNA extraction. Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) with modifications. Tissues (0.2 g) were ground to a fine powder with mortar and pestle in liquid N₂. The powder was added to a tube containing 1 ml of RNeasy RLT buffer and 0.01 g soluble polyvinylpyrrolidone (PVP-40; Sigma, St. Louis, MO, USA), and homogenized using a polytron for approximately 30 sec. Four volumes of 5 M Potassium acetate, pH 6.5 was added to the homogenate, the mixture was incubated on ice for 15 min, and the precipitate removed by a 15 min centrifugation (12,000 rpm) at 4°C. Supernatant was transferred to two 1.5 ml microcentrifuge tubes and 0.5 volume of 100% EtOH was added. Samples were transferred to RNeasy mini columns and the remaining steps were as directed by the manufacturer's instructions for plant RNA isolation (steps 6–11). RNA was quantified using spectrophotometric OD₂₆₀ measurements and quality was assesed by OD₂₆₀/ OD ₂₈₀ ratios and by electrophoresis on 1% formaldehyde agarose gels followed by ethidium bromide staining. RNAs were stored at -80°C.

Fresh plant material (1g) was homogenized in 100 mM Tris-HCl (pH 7.5) in presence of DTT (Dithiothreitol, 5 mM), MgCl₂ 10 mM, Ethylenediaminetetraacetic acid (EDTA, 1 mM), magnesium acetate 5 mM, Polyvinylpyrolidone (PVP-40 1.5%), phenylmethanesulfonyl fluoride (PMSF 1 mM) and aproptinin 1 μgmL^-1. After the filtration, the homogenate was centrifuged at 10,000 rpm for 15 min. The supernatant collected after centrifugation served as enzyme source. For the analysis of APX activity, tissues were separately homogenized with 2 mM AsA. All experiments were performed at 4°C.
Activity of SOD was estimated according to Kono (1978) (link) following the photo reduction of nitroblue tetrazolium (NBT). The absorbance was recorded spectrophotometerically (Beckman 640 D, USA) at 540 nm. SOD unit is the quantity of enzyme that hamper 50% photoreduction of NBT and is expressed as EU mg^-1 protein.
The activity of POD was estimated according to the method proposed by Putter and Becker (1974) . The rate of production of oxidized guaiacol was estimated spectrophotometerically (Beckman 640 D, USA) at 436 nm. The activity of POD was expressed as EU mg^-1 protein.
Catalase activity was estimated by the method of Aebi (1984) (link). The OD was taken spectrophotometerically (Beckman 640 D, USA) at 240 nm and the activity was expressed as EU mg^-1 protein.
For the determination of APX activity, the procedure of Nakano and Asada (1981) was used. The OD was recorded at 265 nm by spectrophotometer (Beckman 640 D, USA) and the activity was expressed as EU mg^-l protein.

During the fall of 2017, a total of 2,415 plant samples (S1 Table) were collected from grapevine populations with a historically high incidence of GLRaV-3 or with observable GLD symptoms such as interveinal reddening and downward rolling of leaf margins in red wine cultivars, and interveinal chlorosis and downward rolling of leaf margins in white cultivars [1 ]. These grapevine populations included: 1) the USDA National Clonal Germplasm Repository (NCGR) in Winters, CA, in which a previous study [18 (link)] identified plants infected with GLRaV-3 and originating from 12 different countries (1,206 samples); 2) the Davis Virus Collection (DVC) at University of California-Davis [21 ], which primarily consists of domestic GLRaV-3 isolates and is also the source of an isolate which is 99% identical to the GH24 isolate (109 samples); 3) the FPS pipeline of foreign and domestic introductions (417 samples); and 4) 89 vineyards in the main grape-growing areas of California including 77 samples from Napa Valley, 14 samples from Sonoma, 44 samples from San Luis Obispo, 39 samples from Monterey, 156 samples from Central Coast, 10 samples from Coachella Valley, 70 samples from the North Coast, 164 samples from the San Joaquin Valley and 109 samples from the Central Sierra region. Additionally, 45 samples from different GLRaV-3 collections outside of the USA were included in this study. These included 23 South African grapevines from eight different selections that represent the different GLRaV-3 variant groups present in that country; six samples originated from New Zealand, where several new GLRaV-3 isolates have been reported recently [8 (link)]; seven GLRaV-3 positive plants originated from Australia that showed mild leaf roll symptoms [22 ]; an asymptomatic GLRaV-3 infected ‘Pinot noir’ vine originating from Spain; and lastly, eight grapevine samples determined positive for GLRaV-3 by end-point RT-PCR and HTS during a study validating this technology for virus detection in Canada, including a sample (ON936) from a ‘Vidal Blanc’ vine infected with a putative new GLRaV-3 variant based on preliminary sequence analysis. Positive controls (described above) were included during the testing process, as well as a grapevine that tested negative by HTS for viruses and virus-like pathogens.
All the above-mentioned samples (leaf petioles or bark scrapings) were subjected to TNA extraction: 0.2 g plant tissue was homogenized in 2 ml of guanidine isothiocyanate lysis buffer (4 M guanidine isothiocyanate; 0.2 M sodium acetate, pH 5.0; 2 mM EDTA; 2.5% (w/v) PVP-40) and TNA extracts were prepared using a MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, USA) as per the manufacturer’s protocol. Subsequently, the integrity of RNA was verified using an 18S rRNA assay [18 (link)].

Indirect ELISA was conducted to detect TSWV in dsRNA-fed insect samples. The protein was extracted from insect samples at a 1/20 dilution with a coating buffer (0.05 M sodium carbonate, pH 9.6, containing 0.01% sodium azide). The mouse monoclonal TSWV antibody (Agdia) was diluted with conjugate buffer (PBST containing 2% PVP-40 and 0.2% BSA) and was used at a 1/8000 dilution. The alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma-Aldrich Korea) was used at a 1/5000 dilution as the secondary antibody. AP substrate tablets (p-nitrophenyl phosphate disodium salt hexahydrate) (BCIP/NBT, Sigma-Aldrich Korea) dissolved in substrate buffer (9.7% diethanolamine and 0.02% NaN₃, pH 9.8) were used for color development. The absorbance at 405 nm was determined after a 10 to 40 min incubation.

Characterization of assembled VLPs was performed by SDS-PAGE, Western blot, ELISAs, and transmission electron microscopy (TEM, ICTS-CNME, Madrid, Spain).
Proteins from the crude extracts were separated in 12% SDS-PAGE (with a stacking gel of 4%) and transferred to a PVDF membrane (Amersham^TM Hybond^TM P 0.45 PVDF, Cytiva, Cornellá de Llobregat, Spain). The membrane was blocked afterwards with 2% skimmed milk in PBS. CP detection was carried out through incubation with the primary monoclonal antibody anti-POTY (Agdia, Elkhart, IN, USA) diluted 1:200 and the subsequent treatment with the secondary antibody anti-mouse (Agdia) conjugated to alkaline phosphatase diluted 1:500. The presence of GFP was detected by incubating the membrane with the primary antibody anti-GFP (Clontech, Mountain View, CA, USA) diluted 1:1000, followed by the binding of the secondary antibody anti-mouse (Agdia) conjugated to alkaline phosphatase diluted 1:500. The detection of VIP was performed by incubating the membrane with the primary antibody anti-VIP (Invitrogen, Waltham, MA, USA) diluted 1:1000, followed by a treatment with the secondary antibody anti-rabbit (Invitrogen) conjugated to alkaline phosphatase diluted 1:2000.
To detect the presence of CP and VIP in the corresponding VLPs, indirect enzyme-linked immunosorbent assays (ELISAs) were carried out. High binding plates (Fisher Scientific, Waltham, MA, USA) were coated with 5 µL of purified VLPs diluted with 195 µL of 50 mM sodium carbonate buffer, pH 9.6, and incubated overnight at 4 °C. Plates were incubated for 1 h at 37 °C with the corresponding primary antibody in PBS, 0.05% Tween 20, 2% PVP-40, 2 mg/mL BSA (anti-POTY diluted 1:200; anti-VIP diluted 1:2000). Then, an incubation with the corresponding alkaline phosphatase-conjugated secondary antibody was performed for 1 h at 37 °C. Alkaline phosphatase activity was detected using nitrophenylphosphate. The optical density of samples was measured at 405 nm (SPECTROstar Nano^®; BMG Labtech, Ortenberg, Germany).
To check VLPs’ assembly, TEM was applied. Electron microscopy grids (400 mesh copper-carbon-coated) were floated at room temperature with a 10 μL drop of VLPs (1:25 dilution of the crude extracts in 50 mM borate buffer, pH 8.1). After 10 min, the grids were washed with five drops of distilled H₂O for 5 min in each drop and incubated with 10 µL of anti-GFP or anti-VIP (both antibodies diluted 1:50 in 50 mM borate buffer, pH 8.1) for 10 min for decoration. No blocking step was carried out. Only in the case of VLPs-VIP, the grids were washed again in distilled H₂O and then incubated for 10 min with 10 µL of a 5 nm gold-labelled anti-rabbit antibody for immunogold electron microscopy.
Finally, all the grids were rinsed five times with five drops of distilled H₂O and stained with 2% uranyl acetate for 2 min. Samples were eventually examined on a transmission electron microscope (JEM JEOL 1400, Tokyo, Japan).