In vitro TBSV replication assay in cell-free yeast extract CFEs from BY4741 or TET::DED1 strains capable of supporting TBSV replication in vitro were prepared as described earlier [23, 40] . Briefly, the in vitro TBSV replication assays were performed in 20-ml total volume containing 2 ml of CFE, 0. [23, 40] . Host proteins were added in different amounts as indicated in the Figure legends. The reaction was performed as described [23, 40] . The newly synthesized 32 P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.56 Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85uC for 5 min in the presence of 50% formamide [27] .