Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Abstract

P osttranscriptional gene silencing (PTGS) refers to the transinactivation of homologous genes caused by increased RNA degradation (1, 2). This is a stable, reversible, epigenetic modification triggered by sequence-specific signals that, in some cases, can spread systemically. First described for transgenic plants, it is now recognized that very similar phenomena occur in a wide variety of organisms. Examples include quelling in Neurospora crassa (3) and RNA interference (RNAi) mediated by double-stranded RNA (dsRNA) in Caenorhabditis elegans (4), Drosophila melanogaster (5), and mouse (6). The finding that RNAi can spread systemically; the identification of small, ca. 25-nt sense-and antisense-RNAs (smRNAs) associated with PTGS in plants and RNAi in Drosophila (7, 8); and the strong genetic links between quelling, RNAi in C. elegans, and PTGS in Arabidopsis provide compelling evidence for a common, highly conserved molecular mechanism (9-12). Although the function of PTGS in plants is still unclear, the findings that PTGS can be suppressed by virus-encoded proteins that also suppress antiviral defense in the host (13-16) and that PTGS mutants also affect virus infection (10, 11) strongly support a role in virus resistance. PTGS-deficient mutants (12) and plants overexpressing viral suppressor proteins (17) can show striking abnormalities, suggesting that PTGS may also be important in normal development.Expression of endogenous plant genes can also be posttranscriptionally silenced by antisense transformation. Although little is known about the mechanism for antisense-mediated gene silencing (ASGS) (18)(19)(20), the involvement of dsRNAs and smRNAs in PTGS (1, 2, 7, 21) supports the early hypothesis proposed by Grierson et al. (22) and Mol et al. (23) that ASGS and PTGS are mechanistically linked and that antisense RNAs (asRNA) have a role in sequence-specific degradation of target RNAs in both processes (24).We have obtained direct evidence linking ASGS and PTGS by comparing the silencing of genes encoding class I chitinases (CHN) and ␤-1,3-glucanases (GLU) in tobacco plants transformed with the homologous cDNAs in sense and antisense orientation. Earlier we showed that all members of the CHN and GLU gene families can be posttranscriptionally silenced in sense Nicotiana sylvestris transformants (25-27). Here we report that posttranscriptional sense-and antisense-silencing of these genes in tobacco is suppressed by plant viruses known to suppress PTGS and that smRNAs diagnostic for PTGS (7) are associated with both forms of silencing. These findings suggest that PTGS and ASGS share, at least in part, a common mechanism. (29) and a bacterial hptII gene conferring hygromycin resistance, and shows stochastic silencing starting at the two-leaf stage of seedling development. Antisense-chitinase line TAC11.7 (TAC) carries a tobacco chitinase CHN50 cDNA (29) in inverted orientation and a bacterial nptII gene conferring kanamycin resistance. Antisense-␤-1,3-glucanase line TAG4.4 (TAG) carries a tobacco GLU cDNA in in...

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“…In the initiation stage, the invading RNA triggers a pathway that results in its being degraded into a small RNA species of discrete size (21 to 25 nucleotides [nt]) called small interfering RNAs (siRNAs) that function as a guide for further degradation in the maintenance stage (22,71). The most potent initiator of PTGS is thought to be double-stranded RNA (dsRNA) (8,18,30,68), although single-stranded RNA (ssRNA), both sense and antisense orientations, or even DNA trigger RNA silencing (15,65,66). ssRNA is most likely converted to a double-stranded form with the help of a host RNAdependent RNA polymerase (RdRP) in order to be effective (9,11,42,52,57).…”

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