Non-perfectly matching small RNAs can induce stable and heritable epigenetic modifications and can be used as molecular markers to trace the origin and fate of silencing RNAs

Abstract

Short non-coding RNA molecules (sRNAs) play a fundamental role in gene regulation and development in higher organisms. They act as molecular postcodes and guide AGO proteins to target nucleic acids. In plants, sRNA-targeted mRNAs are degraded, reducing gene expression. In contrast, sRNA-targeted DNA sequences undergo cytosine methylation referred to as RNA-directed DNA methylation (RdDM). Cytosine methylation can suppress transcription, thus sRNAs are potent regulators of gene expression. sRNA-mediated RdDM is involved in genome stability through transposon silencing, mobile signalling for epigenetic gene control and hybrid vigour. Since cytosine methylation can be passed on to subsequent generations, RdDM contributes to transgenerational inheritance of the epigenome. Using a novel approach, which can differentiate between primary (inducer) and secondary (amplified) sRNAs, we show that initiation of heritable RdDM does not require complete sequence complementarity between the sRNAs and their nuclear target sequences. sRNAs with up to four regularly interspaced mismatches are potent inducers of RdDM, however, the number and disruptive nature of nucleotide polymorphisms negatively correlate with their efficacy. Our findings contribute to understanding how sRNA can directly shape the epigenome and may be used in designing the next generation of RNA silencing constructs.

Figure 1.

TRV-based VIGS system used for studying the impact of mismatched small RNAs on silencing of the GFP transgene. (A) Schematic diagram of the CaMV 35S promoter driven GFP transgene in N. benthamiana 16c plant. 16c plants show green fluorescence under UV light. (B) Schematic diagram of virus induced TGS. Reproduction of RNA viruses, such as TRV results in the accumulation of double-stranded replication intermediates, which are processed into primary sRNAs by antiviral DICER-like (DCL) nucleases. sRNAs are then associated with and guide ARGONAUTE (AGO) proteins to nucleic acid targets by base-pair complementarity. If the target is chromatin, sRNAs can induce the methylation of cytosine residues. This process is referred to as RdDM. Consequently, inoculation of 16c plants with a recombinant TRV carrying the 35S promoter sequence (TRV-35S) can bring about RdDM, which results in TGS of the GFP reporter gene. GFP silenced 16c plants display red fluorescence under UV-light due the autofluorescence of chlorophyl in lack of GFP expression. It is not known whether RdDM of 35S is associated with the production of secondary (target-generated) sRNAs. sRNA-induced DNA methylation is indicated as red lollipop. RDR, RNA-dependent RNA polymerase (C) Schematic diagram of virus induced PTGS. Virus-derived primary sRNAs can also be loaded into AGO complexes to destroy target RNAs with complementary sequences. It is known as antiviral PTGS, which involves AGO-induced cleavage, destabilization, or translational inhibition. Infecting 16c plants with a recombinant TRV harbouring the GFP coding sequence (TRV-GFP) can result in PTGS of both TRV and GFP mRNAs. Intriguingly this process is associated with the generation of secondary (GFP-specific) sRNA. sRNA-induced cleavage is indicated as scissors. (D) Schematic diagram of the TRV VIGS vectors pTRV1 and pTRV2. A 120 nt fragment of the CaMV 35S promoter (-208 to -89 relatives to the transcription start site, yellow lines) or a 120 nt fragment of the GFP coding sequence (+364 to +483, green lines) was cloned into pTRV2 to induce TGS and PTGS, respectively. Single nucleotide substitutions (SNS; white boxes) were introduced into the 120 nt fragments at regular intervals of 20, 10 or 5 nucleotides, which produced sRNAs with one, two or four mismatches, respectively. SNS were introduced from position 10 in TRV-35S-1M_A and from position 20 in TRV-35S-1M_B. pTRV1 was used along with pTRV2 to generate functional TRV particles. Rz, self-cleaving ribozyme; MCS, multiple cloning sites; CP, coat protein; MP, movement protein; NOSt, NOS terminator. (E) Sequence alignment of the 120 nt fragment from CaMV 35S and its derivatives from (A). Substituted A, C, G, T nucleotides are highlighted with red, green, blue and yellow coloured circles, respectively. (F) Sequence alignment of the 120 nt fragment from GFP5 and its derivative from (A).

Figure 2.

Non-perfectly matching virus-derived small RNAs can induce strong transcriptional gene silencing. (A) Systemic leaves of N. benthamiana 16c plants infected with recombinant TRV as indicated. Leaves were collected from independent plants. An uninfected 16c leaf is shown as a control (right). Leaves were photographed at 21 dpi under UV light. (B and C) Analysis of GFP expression and TRV accumulation in recombinant TRV-infected plants by qRT-PCR. RNA was extracted from the systemically infected leaves at 21 dpi. Error bars show the standard error of the mean (SEM) of three independent biological replicates. Asterisks indicate significant differences (Student's t-test, P < 0.01). No spontaneous GFP silencing was observed neither in 16c plants nor in plants infected with wild type TRV.

Figure 3.

Virus-induced TGS is not associated with the accumulation of secondary small RNAs. (A) Small RNA analysis of N. benthamiana 16c plants infected with TGS-inducing TRV-35S and TRV-35S-2M at 7 dpi and 21 dpi. sRNA reads were aligned to the 35S promoter or to 35S promoter-2M harbouring the corresponding SNSs. sRNAs from TRV-35S-2M infected plants were separated according to SNS content to yield primary (containing SNSs) and secondary sRNAs (lacking SNSs). The numbers of sRNAs mapping at each position of the plus strand are shown as positive values, to the minus as negative values, for 21, 22, 23 and 24 nt sRNAs separately. The target sequence is highlighted by dotted lines. (B) Small RNA analysis of N. benthamiana 16c plants infected with PTGS-inducing viruses TRV-GFP and TRV-GFP-2M at 7 dpi and 21 dpi. sRNA reads were aligned to the GFP coding sequence and to its variant GFP-2M containing the corresponding SNSs. sRNAs from TRV-GFP-2M infected plants were separated into primary and secondary sRNAs according to SNS content. Labelling as in (A).

Figure 4.

Non-perfectly matching small RNAs can efficiently induce DNA methylation in virus-infected plants. (A) Analysis of DNA methylation at the target CaMV 35S promoter (from -208 to -89) by bisulfite sequencing in TRV-infected N. benthamiana 16c plants. DNA was extracted from the systemically infected leaves at 21 dpi. The histogram shows the percentage of total methylated cytosine. Asterisks indicate significant differences (Student's t-test, P < 0.01). (B) Summary of bisulfite sequencing analysis. Red, blue and green bars indicate the percentage of methylated cytosine residues at CG, CHG and CHH sites, repetitively. Data presented in (A) and (B) were obtained from three independent biological replicates. Raw data are available in Supplementary Figure S6. Error bars represent a confidence interval with 95% confidence limits (Wilson score interval; see details in Supplementary Figure S6B).

Figure 5.

Non-perfectly matching small RNAs can induce transgenerational epigenetic silencing of the 35S:GFP reporter gene. (A) Progeny of recombinant-TRV-infected N. benthamiana 16c plants. Wild type N. benthamiana and uninfected 16c plants are shown as controls. Plants were photographed under UV light at 20 days after germination. (B) Representative plants displaying different degree of GFP silencing in the progeny of recombinant-TRV-infected N. benthamiana 16c plants: S+++, full GFP silencing; S++, GFP silencing in leaves and in one pair of petioles; S+, GFP silencing only in leaves; S-, no visible sign of GFP silencing. Plants were photographed under UV light at 22 days after germination. (C) Proportion of plants in each silencing category. Forty individual plants were analyzed from each line in two independent biological replicates. Bars represent average values. (D) Analysis of GFP expression in the progeny of recombinant TRV-infected plants by qRT-PCR. RNA was extracted from 21-day-old plants. Error bars show the standard error of the mean (SEM) of three independent biological replicates. Asterisks indicate significant differences (Student's t-test, P < 0.01). (E) Analysis of DNA methylation at the CaMV 35S promoter (from -208 to -89) by bisulfite sequencing in the progeny of recombinant-TRV-infected N. benthamiana 16c plants. DNA was extracted from 21-day-old plants. The histogram shows the percentage of total methylated cytosine. Asterisks indicate significant differences (Student's t-test, P < 0.01). (F) Summary of bisulfite sequencing analysis. Red, blue and green bars indicate the percentage of methylated cytosine residues at CG, CHG and CHH sites, repetitively. Results presented in (E) and (F) were obtained from three independent biological replicates. Raw data are available in Supplementary Figure S6. Error bars represent a 95% interval (Wilson score interval; see details in Supplementary Figure S6B).

Figure 6.

TRV-based VIGS system used for transcriptional silencing of the FWA endogene. (A) Schematic diagram of the FWA locus in Arabidopsis thaliana. FWA is a suppressor of flowering time. In wild-type A. thaliana (Col-0), the direct tandem repeats in the FWA promoter are hyper-methylated which results in transcriptional gene silencing of FWA. Repression of FWA is associated with early flowering phenotype. In the fwa-d epigenetic mutant, DNA methylation at direct tandem repeats is reduced and consequently FWA is expressed. Ectopic expression of FWA results in late flowering phenotype. (B) Experimental design to induce TGS of FWA by infecting fwa-d with a recombinant TRV harbouring a short and a long repeat sequence referred to as Bs (TRV-FWA-Bs). (C) Schematic diagram of the tandem repeats in FWA promoter (At4g25530) and the repeat-derived sequences used for VIGS. The short (38 nt) and long (198 nt) repeats are indicated as purple and pink arrows, respectively. A 239 nt fragment of the FWA promoter harbouring a single short and long repeat sequence referred to as FWA-Bs was cloned into pTRV2 to induce TGS. SNSs were introduced at regular intervals of 20 and 10 nucleotides to generate sRNAs with one or two mismatches, respectively. SNSs were inserted from position 10 in FWA-Bs-1M_A and from position 20 in FWA-Bs-1M_B. The full-length tandem repeat, referred to as FWA-B (32) or FWAtr (33) was used as a positive control. pTRV1 was utilised along with pTRV2 to generate functional TRV particles. (D) Sequence alignment of FWA-Bs and its derivatives from (A). Substituted A, C, G, T nucleotides are highlighted with red, green, blue and yellow coloured circles, respectively. The short and long repeat sequences are indicated as purple and pink arrows, respectively.

Figure 7.

Non-perfectly matching sRNAs targeting the FWA promoter can induce early flowering in the progeny of virus-infected Arabidopsis plants. (A) Analysis of virus accumulation in recombinant-TRV-infected Col-0 fwa-d plants by qRT-PCR. RNA was extracted from leaf tissues at 28 dpi. Error bars show the standard error of the mean (SEM) of three technical replicates. Individual plants are labelled with a combination of capital letters and numbers. (B) Proportion of early- (dark green), intermediate- (green), and late- (light green) flowering plants in the progeny of recombinant-TRV-infected Col-0 fwa-d plants (A). (C) Analysis of DNA methylation at the FWA promoter (full length FWA tandem repeats) by bisulfite sequencing in the progeny of recombinant-TRV-infected Col-0 fwa-d plants. DNA was extracted and analysed from three individual plants from each selected line 40 days after germination. The histogram shows the percentage of total methylated cytosine residues. Asterisks indicate significant differences (Student's t-test, P < 0.01). (D) Summary of bisulfite sequencing analysis. Red, blue and green bars indicate the percentage of methylated cytosine at CG, CHG and CHH sites, repetitively. Raw data are available in Supplementary Figure S8. Error bars represent a confidence interval with 95% confidence limits (Wilson score interval; see details in Supplementary Figure S8C).

Figure 8.

Small RNAs with transversal mismatches can also induce strong TGS. (A) Sequence alignment of the 120 nt fragment from CaMV 35S (from -208 to -89) and its derivatives. Substituted A, C, G, T nucleotides are highlighted with red, green, blue and yellow coloured circles, respectively. (B) Systemic leaves of N. benthamiana 16c plants infected with recombinant TRV as indicated. Leaves were collected from independent plants. An uninfected 16c leaf is shown as a control (right). Leaves were photographed at 21 dpi under UV light. (C and D) Analysis of GFP expression and TRV accumulation in recombinant TRV-infected plants by qRT-PCR. RNA was extracted from the systemically infected leaves at 21 dpi. Error bars show the standard error of the mean (SEM) of three independent biological replicates. Asterisks indicate significant differences (Student's t-test, P < 0.01). (E) Analysis of DNA methylation at the target CaMV 35S promoter (from -208 to -89) by bisulfite sequencing in TRV-infected N. benthamiana 16c plants. DNA was extracted from the systemically infected leaves at 21 dpi. The histogram shows the percentage of total methylated cytosine. Asterisks indicate significant differences (Student's t-test, P < 0.01). (F) Summary of bisulfite sequencing analysis. Red, blue and green bars indicate the percentage of methylated cytosine residues at CG, CHG and CHH sites, repetitively. Results presented in (E) and (F) were obtained from three independent biological replicates. Raw data are available in Supplementary Figure S10. Error bars represent a 95% interval (Wilson score interval; see details in Supplementary Figure S10B).