Figure 2.

A diagram showing the procedure of identifying the 5′ termini of begomoviral mRNAs using the cap snatching of rice stripe tenuivirus (RSV). In a co-infected host cell, RSV snatches capped RNA leaders from host mRNAs (black) as well as those of the co-infecting begomovirus (red). RSV mRNAs (blue) with heterogeneous 5′ terminal sequences were extracted from the co-infected host plant (step 1); the RNA was treated with alkaline phosphatase to remove the 5′-phosphate groups of some RNA species (step 2) and RppH (NEB) to de-cap mRNAs and leave a monophosphate at their 5′ ends (step 3); a RNA oligo was added to the 5′ monophosphate-bearing mRNAs (step 4) and the oligo-tagged mRNAs were reverse transcribed and PCR amplified (Step 5); The PCR products were used for library construction and high-throughput sequencing. The primer sequences obtained were mapped to the genome of a begomovirus (Step 6).