Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase

Abstract

RNA silencing is a sequence-specific RNA degradation process that follows the recognition of double-stranded RNA. Here, we show that virus vectors carrying parts of a green fluorescent protein (GFP) transgene targeted RNA silencing in Nicotiana benthamiana and Arabidopsis against the entire GFP RNA. These results indicate that there was spreading of RNA targeting from the initiator region into the adjacent 5' and 3' regions of the target gene. Spreading was accompanied by methylation of the corresponding GFP DNA. It also was dependent on transcription of the transgene and on the putative RNA-dependent RNA polymerase, SDE1/SGS2. These findings indicate that SDE1/SGS2 produces double-stranded RNA using the target RNA as a template. RNA silencing of ribulose-1,5-bisphosphate carboxylase/oxygenase and phytoene desaturase was not associated with the spreading of RNA targeting or DNA methylation, indicating that these endogenous RNAs are not templates for SDE1/SGS2.

Figure 1.

Spreading of siRNA Production. (A) N. benthamiana 35S:GFP:NOS transgenic plants (line 16c) infected with TRV:00, TRV:35S, TRV:GF, TRV:P, or TRV:NOS and photographed 21 days later under UV light. Silencing of GFP is evident as red fluorescence, which is caused by the chlorophyll. Infection with TRV:35S induced transcriptional gene silencing of the transgene, and infection with TRV:GF, TRV:P, or TRV:NOS induced post-transcriptional gene silencing. Infection with the TRV vector carrying no sequences of the transgene (TRV:00) did not trigger any GFP silencing. (B) PVX RNA levels in TRV-infected plants challenge-inoculated with PVX:P. RNA samples were extracted from upper leaves of PVX:P-challenged plants at 10 DAI, and a probe specific for PVX was used in RNA gel blot analysis. At this time, TRV:00-infected plants were not yet showing GFP silencing by PVX:P. Ethidium bromide (EtBr)–stained rRNAs are shown at bottom. (C) RNA gel blot analysis of siRNAs (21 to 25 nucleotides in length [21/25]) from TRV-inoculated plants at 21 DAI. Sense RNA probes were specific for antisense RNAs corresponding to the P region of GFP (top) or TRV (bottom).

Figure 2.

Spreading and Endogenous Genes. (A) N. benthamiana nontransgenic plants inoculated with TRV:00, TRV:PD, or TRV:S and photographed at 21 DAI. Only infection with TRV vectors carrying fragments of the PDS cDNA (TRV:PD or TRV:S) induces PDS silencing, which was manifested as photobleaching of the infected tissue. (B) PVX RNA levels in TRV:00-, TRV:PD-, or TRV:S-infected plants challenge-inoculated with PVX:PD or PVX:S. RNA samples were extracted from PVX-inoculated leaves at 4 DAI, and a probe specific for PVX was used in RNA gel blot analysis. At this time, TRV:00-infected plants were not yet showing PDS silencing by PVX:PD or PVX:S. Ethidium bromide (EtBr)–stained rRNAs are shown at bottom. (C) RNA gel blot analysis of siRNAs (21 to 25 nucleotides in length [21/25]) from TRV:PD-, TRV:S-, TRV:RU-, or TRV:BISCO-inoculated plants at 21 DAI. Sense RNA probes were specific for antisense RNAs corresponding to the PD, S, RU, and BISCO regions of the PDS and Rubisco genes.

Figure 3.

Spreading Requires Transgene Transcription. (A) RNA gel blot analysis of siRNAs (21 to 25 nucleotides in length [21/25]) from nontranscriptionally silenced (TRV:00-infected) or transcriptionally silenced (TRV:35S-infected) 35S:GFP:NOS N. benthamiana plants (line 16c) infected with PVX:GF or PVX:P. RNA samples were extracted from upper leaves at 28 days after PVX infection. At this time, TRV:00-infected control plants were showing full GFP silencing by PVX:GF or PVX:P. Sense RNA probes were specific for antisense RNAs corresponding to the GF or P region of GFP (top and bottom, respectively). (B) Structure of the GFP transgene, including the 35S promoter (35S), the GF and P regions, the NOS terminator, and Sau96I sites (S). Sau96I sites analyzed by TaqMan quantitative PCR are indicated with arrowheads. (C) and (D) Analysis of DNA methylation within the GF and P regions by Sau96I digestion from TaqMan quantitative PCR. DNA samples were prepared from TRV:00 (C) or TRV:35S (D) plants infected with PVX:00, PVX:GF, or PVX:P at 28 DAI. There is a linear relationship between the amplification values and the amount of amplifiable starting material. Because cytosine methylation inhibits Sau96I digestion, the greater the degree of DNA methylation, the higher the amplification value. Amplification values are the average of three PCRs of three independent DNA samples. Error bars indicate ±sd.

Figure 4.

Spreading Requires SDE1/SGS2. (A) Detection of GFP and TRV:GF RNAs in mock-inoculated or TRV:GF-inoculated wild-type (wt) or sde1/sgs2 mutant (sde1) 35S:GFP:NOS Arabidopsis plants (top). RNA preparations were made from pools of 10 plants at 7 to 10 DAI, and the probe used was specific to the GF region of the GFP RNA. Ethidium bromide (EtBr)–stained rRNAs are shown at bottom. (B) RNA gel blot analysis of siRNAs (21 to 25 nucleotides in length [21/25]) in wild-type (wt) and sde1/sgs2 (sde1) TRV:GF-inoculated plants at 7 to 10 DAI. Sense RNA probes were specific for antisense RNAs corresponding to the GF or P region of GFP (bottom and top, respectively). (C) Scheme of the 35S:GFP:NOS transgene in Arabidopsis. Expected sizes (kb) for total and relevant partial Sau96I restriction enzyme digestions and the P probe used for DNA gel blot analysis are indicated. (D) DNA gel blot analysis of Sau96I-digested DNA extracted from pooled plants as described in (A). Sizes (kb) of relevant DNA fragments are indicated. Fragments marked with asterisks are attributable to a low level of methylation at the Sau96I site within the 35S promoter.