Wheat germ extract kit

In order to perform in vitro translation, both the Wheat Germ Extract kit (Promega, Madison, WI, USA) and the FluoroTect™ GreenLys Labeling System (Promega, Madison, WI, USA) were used according to manufacturer’s instructions with the following modifications. Briefly, the reaction was performed in 25 µL containing 5 µg viroid RNA (specifically, (+) dimeric, (−) dimeric, (+) monomeric and (−) monomeric) and 2 µL of FluoroTect™. The reactions were carried out at 25 °C for 60 min, followed by an incubation at 30 °C for 60 min. The reactions were then terminated by the addition of RNase A (Promega, Madison, WI, USA). For PSTVd-derived translational analysis, 5 µL of the in vitro translation reactions were separated on a 12% SDS-PAGE gel and were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, CA, USA). Anti- BODIPY™ FL rabbit IgG (ThermoFischer Scientific Inc, Waltham, MA, USA) at a dilution of 1:500 dilution was used to detect the translation according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), followed by a subsequent incubation with a 1:10,000 dilution of the IRDye 800CW donkey anti-rabbit-IgG polyclonal antibody (LI-COR). The proteins were subsequently visualized using an LiCOR scanner (LI-COR, Lincoln, NE, USA) at 700 nm.