• TrendMD is the leading scholarly content discovery solution.
  • Sign-up for PNAS eTOC Alerts

Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

  1. David C. Baulcombe1
  1. Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom

  1. Edited by Steven E. Jacobsen, University of California, Los Angeles, CA, and approved November 18, 2014 (received for review July 10, 2014)

  1. Fig. 1.
    View larger version:
    Fig. 1.

    Establishing VIGS of FWA. (A, Upper) Northern blot with TRV RNA species (Fig. S1B) and (Lower) sRNA Northern blot with FWAtr sRNAs in TRV:FWAtr-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants, 14 dpi. FDH and U6 were probed as loading controls, respectively. TRV:FWAtr is marked with a red asterisk. The black asterisks represent the 20-nt and 30-nt sRNA markers. For the sRNA Northern blot analysis, TRV:FWAtr-infected samples 7–12 were run on a separate gel as indicated by the white separating line between samples 6 and 7. (B, Upper) RT-PCR of TRV and TRV:FWAcds in TRV:FWAcds-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants, 14 dpi. FDH was assayed as an internal control. (Lower) sRNA Northern blot with FWAcds sRNAs in the same plants. U6 was probed as a loading control. The black asterisks represent the 20-nt and 30-nt sRNA markers. (C) FWA expression in TRV:FWAtr- (tr), TRV:FWAcds- (cds), TRV- (T), or mock (M)-infected Col-0(FWAC) plants, 14 dpi and 45 dpi. Each sample is represented by a black diamond and the average of all samples per treatment is represented by the red horizontal line. A two-tailed Student t test suggested FWA expression was repressed by TRV:FWAtr (*P < 0.05) and TRV:FWAcds (**P < 0.01), 14dpi. (D) Proportion of early- (black), intermediate- (dark gray), and late- (light gray) flowering V1 progeny from TRV:FWAtr-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants (A), compared with Col-0(FWACme) and Col-0(FWAC). Lines with a ratio of <1 (early):3 (late) are marked with a black asterisk.

  2. Fig. 2.
    View larger version:
    Fig. 2.

    VIGS-RdDM of FWA induces early flowering in V1 progeny plants. (A) DNA methylation (McrBC-qPCR) at FWAtr in V1 progeny of Col-0(FWAC) plants infected with TRV:FWAtr, TRV, or mock conditions compared with Col-0(FWACme) and Col-0(FWAC) plants. DNA from early- and late-flowering progeny from TRV:FWAtr-infected lines 6 and 12 (Fig. 1A) were assayed. Each independent sample is represented by a black diamond and the average of all samples per treatment is represented by the red horizontal line. (B) Bisulfite sequencing analysis of DNA methylation at FWAtr in early- and late-flowering V1 progeny plants from A. Col-0(FWACme) and Col-0(FWAC) were included as a comparison. The position of C residues along the FWAtr region (black arrows) is represented by the ticks on the x axis and the context of methylation is represented by the different colors: CG is in black, CHG is in blue, and CHH is in red.

  3. Fig. 3.
    View larger version:
    Fig. 3.

    VIGS-RdDM of FWA requires PolV and DRM2 and is enhanced in a dcl2/4 mutant. (A) Proportion of early- (black), intermediate- (dark gray), and late- (light gray) flowering V1 progeny from Col-0(FWAC), poliv(FWAC), rdr2(FWAC), dcl3(FWAC), ago4(FWAC), polv(FWAC), drm1/2(FWAC), dcl2/4(FWAC), and dcl2/3/4(FWAC) mutants infected with TRV:FWAtr (blue diamond), TRV (red diamond), or mock (orange diamond) conditions. (B) DNA methylation (McrBC-qPCR) at FWAtr in early- (E) and late- (L) flowering V1 progeny from two independent lines of poliv(FWAC), dcl3(FWAC), dcl2/4(FWAC), and dcl2/3/4(FWAC) mutants. Each independent sample is represented by a black diamond and the average of all samples per treatment is represented by the red horizontal line. (C) Proportion of different size classes of sRNAs that map to FWAtr in Col-0(FWAC) and dcl2/4(FWAC) TRV:FWAtr-infected V0 plants. The proportion of 21-nt (red), 22-nt (orange), 23-nt (light blue), and 24-nt (dark blue) sRNA reads was determined from the actual read count, corrected for multiple mapping. Three independent samples per genotype were assessed (Fig. S4 A and C).

  4. Fig. 4.
    View larger version:
    Fig. 4.

    VIGS-RdDM at FWA is established before or during gametogenesis. (A) Proportion of early- (black), intermediate- (dark gray), and late- (light gray) flowering F1 progeny from reciprocal crosses between mock- and TRV:FWAtr-infected Col-0(FWAC) plants, compared with self-fertilized Col-0(FWACme), Col-0(FWAC), mock-, and TRV:FWAtr-infected Col-0(FWAC) plants. (B) DNA methylation (McrBC-qPCR) at FWAtr in intermediate- (I) and late- (L) flowering F1 progeny from reciprocal crosses between mock- and TRV:FWAtr-infected Col-0(FWAC) plants (A), compared with self-fertilized control plants. Each independent sample is represented by a black diamond and the average of all samples per treatment is represented by the red horizontal line.

  5. Fig. 5.
    View larger version:
    Fig. 5.

    FWAtr is methylated in pollen of TRV:FWAtr-infected plants. Bisulfite sequencing analysis of DNA methylation at FWAtr in pollen from mock- and TRV:FWAtr-infected (A) Col-0(FWAC) and (B) dcl2/4(FWAC) plants. Data for two independent TRV:FWAtr-infected lines per genotype are presented. Col-0(FWACme) and dcl2/4(FWACme) plants were assayed as a comparison. The position of C residues along the FWAtr region is represented by the ticks on the x axis and the context of methylation is represented by the different colors: CG is in black, CHG is in blue, and CHH is in red. The four CG residues that are demethylated in the VN by DME (24, 25) are represented by an asterisk.

Online Impact