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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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Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (Figures 2–3) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. 32P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for DCL2 (lane 1) and in dcl2-1 mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with 32P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.
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pone-0001755-g001: Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (Figures 2–3) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. 32P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for DCL2 (lane 1) and in dcl2-1 mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with 32P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.

Mentions: To study sense transgene-induced silencing, we used the L1 transgenic line, which carries a direct repeat of the T-DNA insert and is silenced for the uidA gene encoding β-glucuronidase (GUS) [33]. Silencing of the GUS transgene in line L1 initiates at about the time the plant bolts and is characterized by very low accumulation of GUS mRNA and concomitant accumulation of GUS siRNAs that are RdR6-dependent [31] and 21- and 22-nucleotides (nt) in length (Figure 1A and 1B, lanes 5–6 and 11–12; [31]). These siRNA size classes have previously been attributed to the activity of DCL4 and DCL2, respectively [21]–[24]. About equal amounts of the two size classes are detected using a probe to the 3′-most region of the transcript, whereas only 21-nt siRNAs are detected with a probe to the central region, and no siRNAs of any size class can be detected using a probe to the 5′-most region (Figure 1B, lanes 5–6 and 11–12). This drop-off in accumulation of siRNAs at the 5′ end of the L1 GUS transcript was not seen in an earlier study [31], possibly because the 5′ probe used by that group extended 258-nt further 3′ than ours.


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)

Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (Figures 2–3) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. 32P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for DCL2 (lane 1) and in dcl2-1 mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with 32P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001755-g001: Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (Figures 2–3) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. 32P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for DCL2 (lane 1) and in dcl2-1 mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with 32P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.
Mentions: To study sense transgene-induced silencing, we used the L1 transgenic line, which carries a direct repeat of the T-DNA insert and is silenced for the uidA gene encoding β-glucuronidase (GUS) [33]. Silencing of the GUS transgene in line L1 initiates at about the time the plant bolts and is characterized by very low accumulation of GUS mRNA and concomitant accumulation of GUS siRNAs that are RdR6-dependent [31] and 21- and 22-nucleotides (nt) in length (Figure 1A and 1B, lanes 5–6 and 11–12; [31]). These siRNA size classes have previously been attributed to the activity of DCL4 and DCL2, respectively [21]–[24]. About equal amounts of the two size classes are detected using a probe to the 3′-most region of the transcript, whereas only 21-nt siRNAs are detected with a probe to the central region, and no siRNAs of any size class can be detected using a probe to the 5′-most region (Figure 1B, lanes 5–6 and 11–12). This drop-off in accumulation of siRNAs at the 5′ end of the L1 GUS transcript was not seen in an earlier study [31], possibly because the 5′ probe used by that group extended 258-nt further 3′ than ours.

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