APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed

Altering PRC2 activity partially suppresses ddm1 mutant phenotypes in Arabidopsis

12 13 In plants and mammals, DNA methylation is a hallmark of transposable element (TE) 14 sequences that contributes to their epigenetic silencing. In contrast, histone H3 lysine 27 15 trimethylation (H3K27me3), which is deposited by the Polycomb Repressive Complex 2 16 (PRC2), is a hallmark of repressed genes. Nevertheless, there is a growing body of evidence for 17 a functional interplay between these pathways. In particular, many TE sequences acquire 18 H3K27me3 when they lose DNA methylation and it has been proposed that PRC2 can serve as 19 a backup silencing system for hypomethylated TEs. Here, we describe in the flowering plant 20 Arabidopsis thaliana the gain of H3K27m3 at hundreds of TEs in the mutant ddm1, which is 21 defective in the maintenance of DNA methylation specifically over TE and other repeat 22 sequences. Importantly, we show that this gain depends solely on CURLY LEAF (CLF), which 23 is one of two otherwise partially redundant H3K27 methyltransferases active in vegetative 24 tissues. Finally, our results challenge the notion that PRC2 can be a compensatory silencing 25 system for hypomethylated TEs, as the complete loss of H3K27me3 in ddm1 clf double mutant 26 plants was not associated with further reactivation of TE expression nor with a burst of 27 transposition. Instead, and surprisingly, ddm1 clf plants exhibited less activated TEs, and a 28 chromatin recompaction as well as hypermethylation of linker DNA compared to ddm1. Thus, 29 we have described an unexpected genetic interaction between DNA methylation and Polycomb 30 silencing pathways, where a mutation in PRC2 does not aggravate the molecular phenotypes 31 linked to TE hypomethylation in ddm1 but instead partially suppresses them

Polycomb mutant partially suppresses DNA hypomethylation–associated phenotypes in Arabidopsis

International audienceIn plants and mammals, DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the polycomb repressive complex 2, are considered as two specialized systems for the epigenetic silencing of transposable element (TE) and genes, respectively. Nevertheless, many TE sequences acquire H3K27me3 when DNA methylation is lost. Here, we show in Arabidopsis thaliana that the gain of H3K27me3 observed at hundreds of TEs in the ddm1 mutant defective in the maintenance of DNA methylation, essentially depends on CURLY LEAF (CLF), one of two partially redundant H3K27 methyltransferases active in vegetative tissues. Surprisingly, the complete loss of H3K27me3 in ddm1 clf double mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, ddm1 clf plants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared with ddm1. Thus, a mutation in polycomb repressive complex 2 does not aggravate the molecular phenotypes linked to ddm1 but instead partially suppresses them, challenging our assumptions of the relationship between two conserved epigenetic silencing pathways

Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons

International audienceMobilization of transposable elements (TEs) in plants has been recognized as a driving force of evolution and adaptation, in particular by providing genes with regulatory modules that impact their transcription. In this study, we employed an ATCOPIA93 long-terminal repeat (LTR) promoter-GUS fusion to show that this retrotransposon behaves like an immune-responsive gene during pathogen defense in Arabidopsis. We also showed that the endogenous ATCOPIA93 copy EVD, which is activated in the presence of bacterial stress, is negatively regulated by both DNA methylation and polycomb-mediated silencing, a mode of repression typically found at protein-coding and microRNA genes. Interestingly, an ATCOPIA93-derived soloLTR is located upstream of the disease resistance gene RPP4 and is devoid of DNA methylation and H3K27m3 marks. Through loss-of-function experiments, we demonstrate that this soloLTR is required for the proper expression of RPP4 during plant defense, thus linking the responsiveness of ATCOPIA93 to biotic stress and the co-option of its LTR for plant immunity

Specific interaction between APC10 and DRB4 occurs <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. DRB4 was fused to the binding-domain (BD) whereas APC10, CDC20-1/-2/-3/-4 and CCS52A1/A2/B were fused to the activation domain (AD). Empty BD and AD vectors were used as negative controls. (<b>B</b>) [³⁵S]methionine-labelled DRB4 was incubated with recombinant GST-APC10, GST-CDC20-1/-2/-3 or GST alone. After several washes, proteins were affinity-purified on glutathione-Sepharose beads, and loaded on an acrylamide gel. The pulled-down proteins were analyzed by autoradiography. The same result was obtained in three independent experiments. (<b>C</b>) Bimolecular fluorescence complementation showed APC10 and DRB4 interaction <i>in planta</i>. Recombinant YN-APC10 and YC-DRB1/2 or 4 were co-bombarded together with a NLS-CFP construct into 4 day-old mustard seedlings. Fluorescence was observed using an E800 fluorescence microscope. YN and YC alone were used as negative controls. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. A strong YFP signal was observed in the nucleus of 91% of examined cells (64/70). No fluorescence signal was obtained after bombardment with the following plasmid combinations YN-+YC-DRB4 and YN-APC10+YC- or with YN-APC10+YC-DRB1 and YN-APC10+YC-DRB2. (<b>D</b>) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. APC10 was fused to the BD and DRB1/2/3/4 and 5 were fused to the AD. Empty BD and AD vectors were used as negative controls.</p

Mapping of interaction domains between APC10 and DRB4.

<p>(<b>A</b>) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. −, no growth on selective media. (<b>B</b>) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28°C for 3 days on non-selective (−LW) or strong selection (−LWA) media. Empty AD and BD vectors were included as negative controls.</p

Reduced APC/C activity slightly affect polIV/polV-dependent heterochromatic siRNA accumulation.

<p>Northern blot analysis of polIV-dependent (siRNA02, TR2258) or polIV/polV-dependent (siRNA1003, simpleHAT) siRNA accumulation in Col-0, <i>drb4</i>, <i>dcl4</i>, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. miR173 accumulation is used here as a loading control. Values are normalized to miR173 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1.</p

Viral infection of <i>drb4</i> and DRB4 OE line.

<p>(<b>A</b>) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (<b>B</b>) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, <i>drb4</i> and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1.</p

RNA silencing is resistant to low-temperature in grapevine.

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species

Over-accumulation of DRB4 does not affect <i>trans</i>-acting siRNA biogenesis and their targets accumulation.

<p>(<b>A</b>) Northern blot analysis of various sRNA accumulation in Col-0, <i>drb4</i>, <i>dcl4</i>, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. <i>Trans</i>-acting siRNA and miRNA accumulation were detected using a <i>TAS1</i> tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (<b>B</b>) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against <i>APC10</i>, <i>APC6</i>, <i>DRB4</i> and three target genes of the tasiRNA pathway (<i>TAS1</i> target, <i>ARF3</i> and <i>ARF4</i>) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each.</p