Fig 2: In vitro reconstitution assays showed that FVE might not have significant enhancement effect on RDR6/SGS3 activity in vitro.(A and B) SDS-PAGE of purified recombinant proteins FVE-8, GST-6×His-SGS3, FVE (A), and GST-6×His-RDR6 (B). Bovine serum albumin (BSA) is a reference. (C) In vitro assays of RDR6 activity with different combinations of proteins. The reaction took place in 20 min with a range of [32P]-UTP dosage. At least three independent repeats were conducted with similar results. Signals were detected by phosphor imaging. (D) Michaelis-Menten model of RDR6 reaction velocity calculated from (C). The velocity was calculated from image quantification of (C) with ±SD (n = 3). Individual data points were shown. (E) Vmax, Km, and R2 values calculated with (D). (F) Western blot analysis of immunoprecipitated HA-tagged RDR6 prepared from N. benthamiana using an anti-HA antibody with mock treatment as a negative control. (G) Semi in vitro assays of RDR6 activity in a time course with ssRNA substrate. HA-RDR6 was incubated with and without different combinations of proteins for the indicated time points. Signals were detected by phosphor imaging. Note that sRNA products otherwise in red dashed squares in the FVE-8–treated reactions were shifted up compared with those in other reactions.

Fig 3: In vitro reconstitution assay showed that FVE promotes whereas FVE-8 inhibits activities of DCL2/4-DRB4 complexes.(A) LCI assay showed that FVE (top) and FVE-8 (bottom) interact with DRB4 in N. benthamiana. PAG1-nLUC/cLUC-SE is a positive control (39). See also fig. S7. (B) Co-IP validated the FVE-DRB4 interaction. IP was performed with an anti-Flag antibody with the protein extracts from transfected N. benthamiana. Western blot assay was performed with indicated antibodies. (C) Y2H assay validated the DRB4-SGS3 interaction. SE/DCL1 is a positive control. (D) LCI assay showed that SGS3 interacts with DRB4 in N. benthamiana. See also fig. S7. (E and F) DCL2 (E) and DCL4 (F) dicing activity with and without FVE/FVE-8 at the indicated time points. The positions of intact substrates, processed products, and RNA markers are shown. At least three independent repeats were conducted with similar results (see also fig. S7). (G and H) Statistics of image quantitation of DCL2 (G) and DCL4 (H) activity. Individual data points are shown. *P < 0.05; ***P < 0.001; t-test. (I) A proposed model for FVE function in transgene silencing. FVE promotes dimerization and launching of SGS3 on ssRNA substrate, which, in turn, recruits RDR6 to produce dsRNA in vivo. Subsequently, FVE/SGS3/dsRNA is translocated to DCL4/DRB4 complexes for siRNA production. By contrast, FVE-8 does not interact with SGS3 and inefficiently promotes RDR6/SGS3 association with ssRNA substrates. Furthermore, FVE promotes DCL4/DRB4 activity, while FVE-8 sequesters dsRNA to prevent DCL4/DRB4 function in producing siRNAs.

Fig 4: Isolation of fve-8 as a new mutant with attenuated RNA silencing through a genetic screening.(A) Schematic construct of E5-4 used for the EMS screening in this study. E4 and E5, the fourth and fifth exons of PHB, respectively; PAGO10, the native promoter of AGO10 locus; Nos Ter, nopaline synthase terminator. (B) The established mutations in miRNA and siRNA pathways increased LUC luminescence. Five-day seedlings of Ec5-4/se-2, Ec5-4/hyl1-2, and Ec5-4/rdr6-11 were photographed in bright field (top), under charge-coupled device (CCD) camera for LUC signal (middle), and under regular camera (bottom). The crossing lines of M17-1 with sgs3-1 displayed similar patterns (see also fig. S1A). Scale bars, 0.5 cm. (C) Mutation in FVE caused increased LUC luminescence. Five-day seedlings of ars1-1 (fve-8), E5-4, and fve-8; gFVE were photographed in bright field (top) and under CCD camera for LUC signal (middle). The signals were displayed by LightField software. Scale bars, 1 cm. Bottom: Six-week-old ars1-1/fve-8 and complementation lines displayed later and normal flowering phenotypes, respectively. Scale bars, 1 cm. (D) RNA blot analysis showed LUC transcript accumulated in ars1-1(fve-8) and E5-4. Ribosomal RNA is control. (E) Next-generation mapping (NGM) analysis of F2 mapping population delineated eight candidates in 1.3 Mb of chromosome 2. bp, base pairs. (F) Top: The gene structure of FVE includes untranslated regions (gray boxes), exons (black boxes), and introns (lines). The fve-8 mutation (triangle) is labeled. Bottom: The protein schematic shows a low complexity region (light gray boxes), 6 × WD40 domains (dark gray boxes), and putative nuclear localization signal (NLS; black box).

Fig 5: FVE interacts with SGS3 and promotes its homodimerization.(A) Y2H screening showed the FVE-SGS3 interaction. SE and ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) were used as positive control. AD, galactose-responsive transcription factor (GAL4) activation domain; BD, DNA binding domain; FVE5+6, truncated FVE with WD40 domains #5 and #6. (B) Co-IP validated the FVE-SGS3 interaction. IP was performed with an anti-Flag antibody. Western blot assay was performed with the indicated antibodies. The experiment was independently repeated with similar results. (C) Confocal imaging showed the interaction between FVE and SGS3. cYFP and nYFP without fusion proteins serve as negative controls. nYFP-SGS3 and cYFP-SGS3 serve as a positive control. Scale bars, 10 µm. (D) BiFC of YFP assays showed that FVE promotes homodimerization of SGS3. Box plot showed numbers of SGS3 dimer foci per cell. The line in the middle of the box refers to the median. The whiskers are drawn from minimum to maximum percentiles. Individual data points are shown. *P < 0.05; ***P < 0.001; t-test. (E) Y3H assay showed that FVE promoted the interaction between pBridge-SGS3 and AD-SGS3. The colonies of pBridge-SGS3-FVE-AD-SGS3, but not those of pBridge-SGS3-AD-SGS3, grew in the dilution of 10-2 without Met. SE and DCL1 serve as positive control. See also fig. S5B and Materials and Methods. (F) In vitro pull-down assay showed that FVE promoted SGS3 homodimerization. GST-ATXR5, His-SUMO, and FVE-8 serve as negative controls. Left: Input. Top right: Bead-recovered MBP-SGS3 by CBB staining. Bottom right: Western blot analysis using an anti-GST antibody (see also fig. S5C).