Replication data for: A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

Abstract

The accumulation of GFP mRNA and GFP mRNA-derived siRNA was evaluated by northern blot analysis in leaf tissues agroinfiltrated for coexpression of GFP and P1, P1N-PISPO, SPFMV HCpro, SPLV P1, PVA HCpro or GUS. Total RNA was extracted with TRIzol LS Reagent (Invitrogen) according to the manufacturer’s recommendations alow-molecular-weight (LMW) and high-molecular-weight (HMW) RNA fractions were separated as described in Cuellar et al. (2009). LMW RNA (15 lg/lane) and HMW RNA (10 lg/lane) were separated by electrophoresis in a denaturing 15% polyacrylamide gel and 1.2% (w/v) agarose gel, respectively, both containing formaldehyde. The two gels were transferred to a nylon membrane (GE Healthcare) and crosslinked chemically with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (Pall and Hamilton, 2008) or by UV light (Sambrook and Russell, 2001), respectively. A GFP mRNA-specific radioactive RNA probe was synthesized, fractionated with carbonate hydrolysis and used to probe the membranes for GFP mRNA and GFP mRNA-derived siRNA, as described previously (Cuellar et al., 2009). Signals were detected by autoradiography on an X-ray film (Kodak)