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DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS, Bowman LH, Vance V - PLoS ONE (2008)

Bottom Line: Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs.Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis.We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

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P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.(A) Gel blot analysis of HMW and LMW RNA is shown for progeny of crosses between the ΔGUS-SUG hairpin line 306-1 and lines expressing the indicated viral suppressors of silencing. All procedures and designations are as described in the legend to Figure 2. Grouped lanes are all from the same gel, blot, and exposure. (B) RNA gel blot analysis is shown for lines that express the indicated viral suppressors and are transgenic for both the ΔGUS-SUG hairpin and 6b4 GUS expressing locus. Controls that carry 6b4 GUS alone (lane 10) or ΔGUS-SUG alone (lanes 1, 2, 9) are also included. All procedures and designations are as described in the legends to Figures 2 and 3. A longer exposure of the loop band in lanes 7–9 is also shown. Accumulation of viral suppressor mRNA in lanes 1–6 is shown for a duplicate gel that was blotted to one membrane, cut into sections corresponding to the indicated viral suppressors, and hybridized with an RNA probe specific for that suppressor; otherwise, grouped lanes are all from the same gel, blot, and exposure. (C) RNA gel blot analysis is shown for progeny of crosses between the silenced L1 GUS line and lines expressing the indicated viral suppressor. All procedures and designations are as described in the legend to Figure 1 except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.
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pone-0001755-g004: P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.(A) Gel blot analysis of HMW and LMW RNA is shown for progeny of crosses between the ΔGUS-SUG hairpin line 306-1 and lines expressing the indicated viral suppressors of silencing. All procedures and designations are as described in the legend to Figure 2. Grouped lanes are all from the same gel, blot, and exposure. (B) RNA gel blot analysis is shown for lines that express the indicated viral suppressors and are transgenic for both the ΔGUS-SUG hairpin and 6b4 GUS expressing locus. Controls that carry 6b4 GUS alone (lane 10) or ΔGUS-SUG alone (lanes 1, 2, 9) are also included. All procedures and designations are as described in the legends to Figures 2 and 3. A longer exposure of the loop band in lanes 7–9 is also shown. Accumulation of viral suppressor mRNA in lanes 1–6 is shown for a duplicate gel that was blotted to one membrane, cut into sections corresponding to the indicated viral suppressors, and hybridized with an RNA probe specific for that suppressor; otherwise, grouped lanes are all from the same gel, blot, and exposure. (C) RNA gel blot analysis is shown for progeny of crosses between the silenced L1 GUS line and lines expressing the indicated viral suppressor. All procedures and designations are as described in the legend to Figure 1 except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.

Mentions: Both of these suppressors restored accumulation of GUS mRNA and eliminated accumulation of GUS siRNAs in progeny of crosses with the sense transgene silenced line L1 (Figure 4C), similar to the effect of TEV P1/HC-Pro on sense transgene-induced silencing in tobacco. Neither suppressor, however, enabled accumulation of high levels of the full-length ΔGUS-SUG transcript in progeny of crosses with the hairpin-transgenic line 306-1, although accumulation of the full-length transcript was increased (Figure 4A, lanes 1–5). In contrast to the full-length transcript, high levels of loop mRNA accumulated in both the P1/HC-Pro and P38 lines (Figure 4A, lanes 1–4). Thus, P1/HC-Pro and P38 have little or no inhibitory effect on processing the dsRNA stem of the hairpin, but they suppress the secondary siRNA-dependent pathway responsible for degradation of the loop. Consistent with this interpretation, P1/HC-Pro and P38 reduced accumulation of secondary (loop) siRNAs (Figure 4A, lanes 1–5). Interestingly, the suppressors also increased accumulation of primary (stem) siRNAs (Figure 4A, lanes 1–5). The relatively minor accumulation of full-length transcript in the P1/HC-Pro and P38 lines suggests that although most ΔGUS-SUG transcripts are degraded via processing of the dsRNA stem into primary siRNAs, some are degraded as a result of being targeted by primary siRNAs and that P1/HC-Pro and P38 block this latter pathway. The increase in primary siRNA accumulation in the viral suppressor lines compared to wild type plants is consistent with the idea that some full-length hairpin transcripts are degraded by transitive silencing in wild type plants, thereby reducing production of primary siRNAs. Alternatively, the suppressors might increase primary siRNA stability, perhaps by binding siRNA duplexes [41], [42].


DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS, Bowman LH, Vance V - PLoS ONE (2008)

P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.(A) Gel blot analysis of HMW and LMW RNA is shown for progeny of crosses between the ΔGUS-SUG hairpin line 306-1 and lines expressing the indicated viral suppressors of silencing. All procedures and designations are as described in the legend to Figure 2. Grouped lanes are all from the same gel, blot, and exposure. (B) RNA gel blot analysis is shown for lines that express the indicated viral suppressors and are transgenic for both the ΔGUS-SUG hairpin and 6b4 GUS expressing locus. Controls that carry 6b4 GUS alone (lane 10) or ΔGUS-SUG alone (lanes 1, 2, 9) are also included. All procedures and designations are as described in the legends to Figures 2 and 3. A longer exposure of the loop band in lanes 7–9 is also shown. Accumulation of viral suppressor mRNA in lanes 1–6 is shown for a duplicate gel that was blotted to one membrane, cut into sections corresponding to the indicated viral suppressors, and hybridized with an RNA probe specific for that suppressor; otherwise, grouped lanes are all from the same gel, blot, and exposure. (C) RNA gel blot analysis is shown for progeny of crosses between the silenced L1 GUS line and lines expressing the indicated viral suppressor. All procedures and designations are as described in the legend to Figure 1 except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262140&req=5

pone-0001755-g004: P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.(A) Gel blot analysis of HMW and LMW RNA is shown for progeny of crosses between the ΔGUS-SUG hairpin line 306-1 and lines expressing the indicated viral suppressors of silencing. All procedures and designations are as described in the legend to Figure 2. Grouped lanes are all from the same gel, blot, and exposure. (B) RNA gel blot analysis is shown for lines that express the indicated viral suppressors and are transgenic for both the ΔGUS-SUG hairpin and 6b4 GUS expressing locus. Controls that carry 6b4 GUS alone (lane 10) or ΔGUS-SUG alone (lanes 1, 2, 9) are also included. All procedures and designations are as described in the legends to Figures 2 and 3. A longer exposure of the loop band in lanes 7–9 is also shown. Accumulation of viral suppressor mRNA in lanes 1–6 is shown for a duplicate gel that was blotted to one membrane, cut into sections corresponding to the indicated viral suppressors, and hybridized with an RNA probe specific for that suppressor; otherwise, grouped lanes are all from the same gel, blot, and exposure. (C) RNA gel blot analysis is shown for progeny of crosses between the silenced L1 GUS line and lines expressing the indicated viral suppressor. All procedures and designations are as described in the legend to Figure 1 except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.
Mentions: Both of these suppressors restored accumulation of GUS mRNA and eliminated accumulation of GUS siRNAs in progeny of crosses with the sense transgene silenced line L1 (Figure 4C), similar to the effect of TEV P1/HC-Pro on sense transgene-induced silencing in tobacco. Neither suppressor, however, enabled accumulation of high levels of the full-length ΔGUS-SUG transcript in progeny of crosses with the hairpin-transgenic line 306-1, although accumulation of the full-length transcript was increased (Figure 4A, lanes 1–5). In contrast to the full-length transcript, high levels of loop mRNA accumulated in both the P1/HC-Pro and P38 lines (Figure 4A, lanes 1–4). Thus, P1/HC-Pro and P38 have little or no inhibitory effect on processing the dsRNA stem of the hairpin, but they suppress the secondary siRNA-dependent pathway responsible for degradation of the loop. Consistent with this interpretation, P1/HC-Pro and P38 reduced accumulation of secondary (loop) siRNAs (Figure 4A, lanes 1–5). Interestingly, the suppressors also increased accumulation of primary (stem) siRNAs (Figure 4A, lanes 1–5). The relatively minor accumulation of full-length transcript in the P1/HC-Pro and P38 lines suggests that although most ΔGUS-SUG transcripts are degraded via processing of the dsRNA stem into primary siRNAs, some are degraded as a result of being targeted by primary siRNAs and that P1/HC-Pro and P38 block this latter pathway. The increase in primary siRNA accumulation in the viral suppressor lines compared to wild type plants is consistent with the idea that some full-length hairpin transcripts are degraded by transitive silencing in wild type plants, thereby reducing production of primary siRNAs. Alternatively, the suppressors might increase primary siRNA stability, perhaps by binding siRNA duplexes [41], [42].

Bottom Line: Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs.Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis.We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

Show MeSH
Related in: MedlinePlus