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

Abstract

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding b-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ;22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

Extensive 3′ modification of plant small RNAs is modulated by helper component-proteinase expression

“…1. smRNAs to their primary targets. In support of this, viral satellite siRNA-directed DNA methylation and RNA cleavage are highly restricted to the viral satellite sequence in a fusion transgene and its transcript (30). Also, siRNA-directed heterochromatin silencing in Arabidopsis is restricted to transposons and repetitive sequences from which the siRNAs originate and does not spread to the neighboring gene-rich areas (31).…”

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites

“…To make the ␤-glucuronidase (GUS) fusion constructs, GUS:PSTVd[ϩ] and GUS:PSTVd[Ϫ], the full-length sequence (with minor mutations; see Fig. 1) of the RG1 strain (15) of PSTVd, was assembled by PCR with overlapping oligonucleotides and then cloned into a 35S-GUS-Ocs cassette (11) in either sense (for GUS:PSTVd[ϩ]) or antisense (for GUS:PSTVd[Ϫ]) orientation. For Agrobacterium-mediated transformation, the three constructs were all cloned into the binary vector pWBVec2a (16).…”

“…GUS activity in transgenic tobacco was measured at 37°C by the fluorometric 4-methylumbelliferryl-␤-glucuronide assay (20) by using 5 g of leaf protein extract. For Northern blot hybridization analysis, total RNA was prepared by using the TRIzol reagent (Invitrogen), separated in formaldehyde-agarose gels (for normal Northern analysis) or in 15% polyacrylamide gels (for siRNA detection), blotted to Hybond-N filter, and hybridized with T7 or SP6 polymerasesynthesized, ␣-32 P-labeled riboprobes (11).…”

“…Extracts of the CMV͞Y-Sat-infected tobacco leaves were then used for subsequent inoculation. Infection by potato leaf roll virus, which was required to support RPVSat replication, was performed as described (11).…”

“…40. Both viroids and satellites induce heavy de novo cytosine methylation of homologous nuclear DNA (11,41), which could lead to transcriptional silencing of cognate endogenous genes. However, this model would not account for the prevention of Y-Sat symptom development by Hc-Pro, which does not block RNAdirected methylation (22,25).…”