1. Volume 48, Issue 1, Pages 109-120 (October 2012)
Ago Hook and RNA Helicase Motifs Underpin Dual Roles for SDE3 in Antiviral Defense and Silencing of Nonconserved Intergenic Regions 
Damien Garcia, Shahinez Garcia, Dominique Pontier, Antonin Marchais, Jean Pierre Renou, Thierry Lagrange, Olivier Voinnet 
Molecular Cell 
Volume 48, Issue 1, Pages (October 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
The SDE3 CTD Is an Evolutionary Conserved and Plant-Specific GW Motif AGO-Binding Platform
(A) Alignment of motifs I and II of the RNA helicase domain and GW-rich CTD of angiosperm SDE3 homologs.
(B) The six GW repeat motifs in the SDE3 CTD.
(C) Alignment of the ultimate GW motif of true plant SDE3 homologs shows clustering in dicots and monocots.
(D) Binding of AGO1 and myc-AGO4 by an SDE3 GW motif hexamer (GW6) or its point mutant derivative (GF6). WT or myc-AGO4 Arabidopsis inflorescence extracts were applied to equimolar amounts of GST or GST-SDE3 GW/GF beads; bound proteins were detected by immunoblotting using anti-AGO1 or anti-myc.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
RNA Helicase and AGO Binding Are Respectively Required and Dispensable for PVX-GFP Silencing
(A) UV illumination revealing PVX-GFP levels in the first leaves of WT C24, sde3-1, and sde3-1 T2 SDE3-FHA.
(B) Western analysis of PVX-GFP and SDE3-FHA accumulation in T2 lines transformed with SDE3-FHA.
(C) Western analysis of protein levels in selected T2 lines expressing WT or mutant versions of SDE3-FHA.
(D) Western analysis of the SDE3 content of AGO1, AGO4, AGO2, or AGO9 IPs from plants expressing SDE3-FHA, as in (B). IPs were validated using antibodies against AGO proteins (lower panels). CoIP of AGO and SDE3-FHA was tested using anti-HA.
(E) Western analysis of AGO1 IPs from plants used in (C). Input (Inp), Flow-Through (FT) and Immunoprecipitated (IP) fractions are indicated in (D) and (E).
(F) UV illumination as an indicator of PVX-GFP levels plants used in (C).
(G) Complementation of PVX-GFP silencing in plants depicted in (C) and (F), assessed by western and Q-PCR analyses (mean of three technical repeats). Error bars: SD. RbC: RUBISCO small subunit, loading control.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
PVX-GFP Silencing Entails Genetic and Physical Interactions between SDE3 and Primary siRNA Loaded AGO1 and AGO2
(A) UV illumination revealing PVX-GFP levels in leaves of rdr6 (sde1-1), sde3-1, and double mutant plants at 52 days post germination (dpg).
(B) ImageJ-based quantification of green fluorescence (upper panel) and western analysis of GFP accumulation (lower panel) in plants depicted in (A). Error bars: SD; n = 3.
(C) UV illumination revealing PVX-GFP levels in various mutants introgressed into the original PVX-GFP line. Pictures of cotyledons at 14 dpg are shown for WT Col0, rdr6-15, sde3-5, ago1-27, WT Ler, and ago4-1. Pictures of the first leaves at 21 dpg are shown for WT Col0, sde3-5, and ago2-1.
(D) Upper panels: Western analysis of PVX-GFP accumulation in the mutants shown in (C). Lower Panel: Northern analysis of PVX-GFP siRNA levels in selected mutants. U6: RNA loading control.
(E) Northern analysis of PVX-GFP siRNAs extracted from AGO1, AGO2, or AGO4 IPs isolated from PVX-GFP sde1-1 plants. miR159 and REP2 transposon-derived siRNAs are used as positive controls for AGO1 and AGO4 IPs.
(F) AGO2 IPs in four distinct, weak ago1 alleles expressing SDE3-FHA. IPs were validated with anti-AGO2. CoIP between SDE3 and AGO2 was tested with anti-HA.
(G) Left panel: UV illumination revealing synergistic effects of mutations in ago1 and ago2 on PVX-GFP levels. Pictures of 44 dpg rosette leaves are shown for WT Col0, ago1-27, ago2-1, and double mutant. Right panel: Western analysis of PVX-GFP accumulation in the mutants shown in the left panel. RbC: RUBISCO small subunit, loading control.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Complementation of 2a3 Silencing in the sde3-6, by WT or Mutant SDE3-Myc Versions
(A) Upper panel: Western analysis of SDE3-Myc accumulation in T1 plants of sde3-6 transformed with Myc-tagged versions of SDE3. Lower panels: Northern analysis of NIA2 siRNA at 18 dpg and 36 dpg, respectively.
(B) Chlorotic phenotypes of the plants in (A) at 36 dpg.
(C) Proportions of WT versus chlorotic or dead phenotypes of T1 plants at 36 and 48 dpg.
(D) Phenotypes displayed by T1 SDE3-mHelic-myc and SDE3-ΔGW-myc in 2a3 sde3 background at 48 dpg.
(E) Analysis of a SDE3 version mutated in the Helicase (mHelic) and GW (GP6pm) domains. Upper panel: Western analysis of SDE3-Myc accumulation in T1 plants of sde3-6 transformed with Myc-tagged versions of SDE3. Lower panel: Northern analysis of NIA2 siRNA at 36 dpg. RbC: RUBISCO small subunit, loading control. U6: RNA loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5
Mutations in SDE3, AGO1, or AGO2 Affect Transcription and Methylation of SDE3 Endogenous Target Loci
(A) Semiquantitative RT-PCR analysis of expression of SDE3 targets in various silencing mutant backgrounds. ATSN1 is used as a control for RdDM pathway mutants.
(B) McrBC PCR analysis of the methylation levels at SDE3 target loci in sde3-5 and other mutants in the RDR6 pathway, as well as in the nerd mutant.
(C) McrBC PCR analysis of the methylation levels at NERD target loci in sde3-5 and other mutants of the RDR6 pathway.
(D) Semiquantitative RT-PCR analysis of the expression of AT1TE93275 (Helitron) in various silencing mutant backgrounds.
(E) DNA methylation status of AT5G35935 and AT1TE93275 loci as determined by McrBC PCR assays in WT, met1, drm1/2, cmt3, and ddc. The Actin and ATSN1 loci were used as unmethylated and methylated control loci, respectively.
(F) H3K4 trimethylation profile at AT5G35935, AT1TE93275, and AT5G35940 control loci, in WT, nerd-1, and sde3-5. In (B), (C), (E), and (F), means of two biological repeats ± SD are presented.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Silencing of AT5G35935 Requires the RNA Helicase and AGO Hook Functions of SDE3, which Display a Dual Cytoplasmic and Nuclear Localization
(A) Semiquantitative RT-PCR analysis of AT5G35935 expression in flowers of T1 transformants of sde3-5 with WT and mutant forms of SDE3-FHA.
(B) Western analysis of cellular fractionations from plants expressing SDE3-FHA. Cyto, Nucl: cytoplasmic, nuclear fraction, respectively. Histone H3 and UGPase antibodies provided positive controls for nuclear and cytoplasmic enriched fractions, respectively.
(C) Subcellular distribution of cytosolic mGFP5-ER and SDE3eGFP fusion, as detected under confocal microscope in Nicotiana benthamiana leaves 4 days after Agrobacterium-mediated transient delivery. pSDE3:SDE3eGFP and the mGFP5-ER control were coagroinfiltrated with P19 to allow detection of SDE3eGFP. Scale bars: 10 μm, nucleus close up; 50 μm, whole cell view. n: nucleus.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Model for SDE3 Action as a Facilitator of Weakly Induced S-PTGS
See text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions