Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequen ces encoding beta -glucuronidase (GUS) and the satellite RNA (satRNA) of ce real yellow dwarf luteovirus. When transgenic plants were infected with pot ato leafroil luteovirus (PLRV), which replicated the transgene-derived satR NA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the up per strand and 73 of the 75 cytosines in the lower strand were either parti ally or fully methylated, in contrast, very low levels of RNA methylation w ere detected in the satellite sequence of the transgene in uninfected plant s and in the flanking non-satellite sequences in both infected and uninfect ed plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleoti des within the first 60 bp of the satellite sequence. Whereas this RNA trun cation was associated with high levels of satRNA replication, it appeared t o be independent of the levels of DNA methylation in the satellite sequence , suggesting that it is not caused by methylation. Ail the sequenced GUS:Sa t DNA molecules were hypermethylated in plants with replicating satRNA desp ite the phloem restriction of the helper PLRV. Also, small, sense and antis ense similar to 22 nt RNAs, derived from the satRNA, were associated with t he replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred a nd that this was mediated by the spread of unamplified satRNA and/or its as sociated 22 nt RNA molecules.