Research ArticlePLANT SCIENCES

DEAD-box helicases modulate dicing body formation in Arabidopsis

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Science Advances  28 Apr 2021:
Vol. 7, no. 18, eabc6266
DOI: 10.1126/sciadv.abc6266
  • Fig. 1 Plant homologs of Dhh1/DDX6 are novel components of D-bodies.

    (A) BiFC analyses of N. benthamiana cells coexpressed with NLS-mCherry. YFP signals (green) and mCherry fluorescent signals (red) were observed at 72 hours post-inoculation (hpi). Scale bars, 10 μm. (B) Immunofluorescence analyses performed in A. thaliana root tip cells in the elongation region of stable transgenic plants. The immunofluorescence of RH6-3FLAG/RH8-3FLAG appears green, and the immunofluorescence of HYL1-YFP appears red. The positive cell percentages in the RH6-3FLAG and RH8-3FLAG lines were 74% (n = 87) and 80% (n = 70), respectively. The blue signals from 4′,6-diamidino-2-phenylindole (DAPI) represent nuclei. Scale bars, 5 μm. (C) Colocalization of RFP-SE together with RNA helicases in N. benthamiana. Fluorescence was analyzed at 72 hpi. Scale bars, 5 μm. YFP-H2A was used as nuclear marker. (D to F) Co-IP assays in N. benthamiana showing interactions between RH12 and DCL1 (D), SE (E), or HYL1 (F). (G) Co-IP assays in pRH12::RH12-3FLAG transgenic A. thaliana plants showing interactions between RH12 with SE and HYL1. (H) Detection of the direct interactions between RH12 with SE and HYL1 by pull-down assays. GST, GST-HYL1, and GST-SE (top) were used as matrix-bound bait, where RH12 served as prey. WT, wild type.

  • Fig. 2 RH6/RH8/RH12 is required for D-body formation.

    (A) Representative images and fluorescence recovery curves of D-body components. Leaf epidermal cells of N. benthamiana (inoculated with DCL1-YFP/RFP-SE for 72 hours) and root tip cells (elongation region) of 1-week-old A. thaliana (pHYL1::HYL1-YFP) plants were analyzed. The region of interest is highlighted with a red circle. Scale bars, 2 μm. The intensity was normalized against the average prebleach fluorescence. n = 3 for each group. The green line indicates the half-recovery time. (B) Whole-plant images of 6-week-old plants. Scale bars, 0.5 cm. (C) Detection of DCL1 subcellular localization with anti-DCL1 antibodies. Left: One set of representative images. Right: The quantification analysis. The numbers 0 to 3 represent the number of DCL1 granules in each nucleus. In total, 150 to 200 nuclei were analyzed for each genotype. Scale bars, 1 μm. (D and E) Subcellular localization of HYL-YFP (D) and YFP-SE (E) in 3-week-old A. thaliana plants. Fifteen root tip cells (elongation region) for HYL-YFP and 115 to 125 leaf epidermal cells for YFP-SE were analyzed for each genotype. Scale bars, 5 μm. Photo credit: Ningkun Liu, Institute of Zoology, Chinese Academy of Sciences.

  • Fig. 3 RH6/RH8/RH12 drives D-body formation through LLPS.

    (A) Representative images and fluorescence recovery curves of the RNA helicase FRAP in N. benthamiana leaf epidemical cells or root tip meristematic cells of 1-week-old A. thaliana. Scale bars, 2 μm. The fluorescence intensity was normalized against the average prebleach fluorescence. n = 3 for each protein. The green dashed line indicates the half-recovery time. (B) In vitro phase separation of RH12 proteins with or without polyU (300 ng/μl) and/or 5 mM ATP. Scale bars, 20 μm. (C) In vitro phase separation of SE with/without RH12 under pre-miR172a (15 ng/μl). Scale bars, 20 μm. (D) Northern blot (left panels) and small RNA (sRNA) sequencing (right) analysis of miRNAs. U6 was used as a loading control. The relative abundance (RA) of miRNAs in amiR-RH6/RH8/RH12 transgenic plants compared to Col-0 WT plants is indicated. The sequencing data are shown as reads per million mapped reads. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of selected pri-miRNAs in 3-week-old plants. Elongation factor 1-α (EF-1α) was used as an internal control. Each error bar represents 1 SD of three biological replicates. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for the others, P ≥ 0.05. eGFP, enhanced GFP; ns, not significant.

  • Fig. 4 TuMV infection leads to decreased accumulation of D-bodies.

    (A to F) Diffusion of the punctate signals of D-body components in the nucleus. (A) N. benthamiana plants were coinoculated with buffer (Mock)/TuMV with DCL1-YFP/RFP-SE. Fluorescence was observed at 72 hpi. (D) Three-week-old A. thaliana transgenic plants were inoculated with buffer (Mock)/TuMV. Fluorescence was observed at 10 days post-inoculation (dpi). The white arrowheads represent nucleus. Scale bars, 10 μm. (B, C, E, and F) Quantification analysis of the studies in (A) and (D). A total of 100 nuclei of root tip cells (elongation region) of five A. thaliana plants and 30 to 50 nuclei of N. benthamiana leaf epidermal cells of three leaves (one sample per leaf) were analyzed. (G and H) qRT-PCR analysis of selected pri-miRNAs in 6-week-old A. thaliana plants treated with Mock/TuMV. Systemic leaves were collected at 14 dpi. EF-1α was used as an internal control. (I and J) Quantitative detection of RH6/RH12 in nuclei after TuMV infection. H3 and PEPC: nuclear and cytoplasmic markers. Each error bar represents 1 SD of three biological replicates. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for the others, P ≥ 0.05.

  • Fig. 5 RH6/RH8/RH12 promotes the phase separation of VPg.

    (A) Confocal fluorescence microscopy images showing the localization change of RH6-HA-CFP, RH8-HA-CFP, and RH12-CFP upon viral infection. RH6-HA-CFP, RH8-HA-CFP, or RH12-CFP was coinoculated together with TuMV in N. benthamiana plants. 6K2-mCherry represents the localization of virus protein 6K2 in cells. Fluorescence signals were observed at 72 hpi. The white arrows indicate nuclei. Scale bars, 10 μm. (B) Localization change of RH12-eGFP in pRH12::RH12-eGFP transgenic A. thaliana plants upon viral infection. Scale bar, 10 μm. (C) Co-IP analysis of the interaction between RH12 and VPg in N. benthamiana leaves. (D) GST pull-down assays confirming the direct interaction between RH12 and VPg. GST and GST-VPg (top) were used as matrix-bound bait, whereas RH12 served as prey. (E) BiFC analysis of the interactions of RH6/RH8/RH12 with 6K2-VPg in N. benthamiana plants infected with TuMV. Reconstituted YFP fluorescence (green), mCherry-tagged viral protein 6K2 (purple), and chloroplast autofluorescence (red) were observed at 72 hpi. Scale bars, 5 μm. (F) In vitro phase separation of 8 μM VPg proteins with or without RH12 under polyU (50 ng/μl; without ATP). Fluorescence microscopy images present liquid droplets formed by phase-separated proteins. Scale bars, 20 μm.

  • Fig. 6 RH6/RH8/RH12 facilitates viral proliferation.

    (A) Visualization of rosette leaves of plants inoculated with TuMV::GFP or buffer (Mock). Images were taken at 14 dpi. In the pseudocolor images, green color refers to infected plant pixels, while red color refers to noninfected plant pixels. Scale bars, 0.5 cm. (B) Bar plot showing the ratio of infected area to the total area of Col-0 WT and mutant plants at 14 dpi. (C) Bar plot showing the rosette size at 14 dpi. Statistical analysis was performed between treatments in each genotype. The boxes with different letters are significantly different (n = 8, Tukey post hoc test, with α = 0.05). (D) TuMV-infected amiR-RH6/RH8/RH12 plants. Four-week-old A. thaliana plants with TuMV::GFP were examined, and the plants were imaged under ultraviolet (UV) light at 14 and 22 dpi. Scale bars, 0.5 cm. (E) Western blot analysis of the accumulation of CP, VPg, and HC-Pro proteins. Representative images and quantitative analysis are shown on the left and right, respectively. Photo credit: Ningkun Liu, Institute of Zoology, Chinese Academy of Sciences.

  • Fig. 7 Model of the formation of D-bodies driven by RH6/RH8/RH12.

    RH6, RH8, and RH12 interact with SE, the key D-body component, and drive the formation of D-bodies (left) under normal conditions. Upon infection with TuMV, RH6, RH8, and RH12 may be retained or translocated to the perinuclear globular structure and on chloroplast periphery, coupling with the formation of V-bodies and decreased accumulation of D-bodies (right).

Supplementary Materials

  • Supplementary Materials

    DEAD-box helicases modulate dicing body formation in Arabidopsis

    Qi Li, Ningkun Liu, Qing Liu, Xingguo Zheng, Lu Lu, Wenrui Gao, Yang Liu, Yan Liu, Shicheng Zhang, Qian Wang, Jing Pan, Chen Chen, Yingjie Mi, Meiling Yang, Xiaofei Cheng, Guodong Ren, Yao-Wu Yuan, Xiaoming Zhang

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