Tissuelyzer bead mill

Ventricular myocardium (30 mg) was collected in Qiazol (Qiagen) and homogenized using the TissueLyzer bead mill (Qiagen) for 4 min at a frequency of 50/s. For RNA isolation and subsequent cDNA synthesis the miRNAeasy kit including DNAse1 digestion (Qiagen) was used according to the manufacturer’s instructions. RNA (1 µg) was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit (BioRad) following the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) was performed using the CFX96 C1000 TouchTM thermal cycler Real-Time System (BioRad) in combination with the SsoAdvancedTM Universal SYBR® Green Supermix (BioRad) according to the manufacturer’s instructions. Samples were amplified in duplicates and gene expression was quantified using the CFX manager 3.1 software (BioRad) applying the 2^−ΔΔCt method^{48 (link)}. Relative gene expression was calculated to the housekeeping gene Hprt1. Specific primer sequences are provided in Supplementary Table 1.

Liquid nitrogen-frozen plant tissue was homogenized in a TissueLyzer bead mill (Qiagen). Total proteins were extracted, separated by glycine-SDS-PAGE and electroblotted onto a nitrocellulose membrane, as reported
[45 (link)]. Proteins were detected using rabbit anti-PPV CP and -PPV HCPro sera, and mouse anti-GFP monoclonal antibody (clones 7.1 and 13.1, Roche) as primary antibodies; horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson) or sheep anti-mouse IgG (GE Healthcare) were used as secondary antibody. For signal quantification, chemiluminescence was acquired in a ChemiDoc XRS imager (BioRad) and analyzed with ImageJ.

Plant tissue was ground in a mortar in liquid nitrogen or homogenized in a TissueLyzer bead mill (Qiagen). Total proteins were extracted in 150 mM Tris-HCl pH 7.5, 6 M urea, 2% (w/v) SDS, 5% (v/v) glycerol and 5% (v/v) β-mercaptoethanol; heat denatured (96°C, 5 min) and centrifuged (14000 rpm, 10 min) to remove cell debris. Protein samples were separated by glycine-SDS-PAGE and electroblotted onto a nitrocellulose membrane. Ponceau red staining was used to control protein loading equivalence. Proteins were detected using anti-PPV CP and anti-PPV HCPro rabbit sera, and anti-GFP monoclonal antibody (clones 7.1 and 13.1, Roche) as primary antibodies. Antibodies raised against tobacco class II PR-2 [44] (link) and class II PR-3 proteins [47] (link) were kindly provided by Dr. T. Heitz (IBMP-CNRS, Strasbourg, France). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson) or sheep anti-mouse IgG (GE Healthcare) were used as secondary antibody. Immunostained proteins were visualized by enhanced chemiluminescence detection with a LiteABlot kit (Euroclone). For signal quantification, chemioluminescence was acquired in a ChemiDoc XRS imager (BioRad) and analyzed with ImageJ software [110] (link).