Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro

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

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

Two Plant Viral Suppressors of Silencing Require the Ethylene-Inducible Host Transcription Factor RAV2 to Block RNA Silencing

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro

P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figure 2</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g003" target="_blank">3</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g001" target="_blank">Figure 1</a> except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.</p

Sense Transgene-induced Silencing is Impaired in <i>dcl2</i> Mutant Plants, but Enhanced in <i>dcl4</i> Mutants.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figures 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g003" target="_blank">3</a>) 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 <i>DCL2</i> (lane 1) and in <i>dcl2-1</i> 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.</p