To test if a DEAD-box helicase from plants might have similar stimulatory function on the activity of tombusvirus replicase, we have cloned and purified RH20 cytosolic DEAD-box helicase from Arabidosis thaliana, which shows high degree of similarity to yeast Ded1p ( Figure S7 ). Addition of the recombinant AtRH20 to the CFE prepared from Ded1p-depleted yeast strain increased TBSV repRNA replication by ,2-fold ( Figure 6A, lane 3) . Also, adding the recombinant RH20 to the replicase assay led to almost Yeast with depleted Ded1p co-expressing p33 and p92 pol replication proteins and DI-72 (+)repRNA were used to affinitypurify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (2)repRNA, and they also contained purified recombinant Ded1p, mutants or MBP in addition to ATP/CTP/GTP and 32 P-UTP. (B) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 0.5 mg or 1.0 mg of purified recombinant Ded1p or its mutants is shown. The level of complementary RNA synthesis producing ''repRNA'' (marked as ''FL'', the full-length product, made via de novo initiation from the 39-terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein (lane 1). Note that this replicase preparation also synthesizes de novo internal initiation products (''ii'') and 39-terminal extension products (''39TEX''). Each experiment was repeated three times. (C) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p or its mutants is shown. Note that the FHV replicase can use TBSV (2)repRNA as a template in vitro. Each experiment was repeated three times. (D) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p or its mutants is shown. Note that the FHV DI-634 (2)repRNA was used as a template in the FHV replicase assay. doi:10.1371/journal.ppat.1002537.g004