An endogenous, systemic RNAi pathway in plants

Abstract

Recent work on metazoans has uncovered the existence of an endogenous RNA-silencing pathway that functionally recapitulates the effects of experimental RNA interference (RNAi) used for gene knockdown in organisms such as Caenorhabditis elegans and Drosophila. The endogenous short interfering (si)RNA involved in this pathway are processed by Dicer-like nucleases from genomic loci re-arranged to form extended inverted repeats (IRs) that produce perfect or near-perfect dsRNA molecules. Although such IR loci are commonly detected in plant genomes, their genetics, evolution and potential contribution to plant biology through endogenous silencing have remained largely unexplored. Through an exhaustive analysis performed using Arabidopsis, we provide here evidence that at least two such endogenous IRs are genetically virtually indistinguishable from the transgene constructs commonly used for RNAi in plants. We show how these loci can be useful probes of the cellular mechanism and fluidity of RNA-silencing pathways in plants, and provide evidence that they may arise and disappear on an ecotype scale, show highly cell-specific expression patterns and respond to various stresses. IR loci thus have the potential to act as molecular sensors of the local environments found within distinct ecological plant niches. We further show that the various siRNA size classes produced by at least one of these IR loci are functionally loaded into cognate effector proteins and mediate both post-transcriptional gene silencing and RNA-directed DNA methylation (RdDM) of endogenous as well as exogenous targets. Finally, and as previously reported during plant experimental RNAi, we provide evidence that endogenous IR-derived siRNAs of all size classes are not cell-autonomous and can be transported through graft junctions over long distances, in target tissues where they are functional, at least in mediating RdDM. Collectively, these results define the existence of a bona fide, endogenous and systemic RNAi pathway in plants that may have implications in adaptation, epiallelism and trans-generational memory.

Figure 1

Small-RNA populations and DICER usage at endogenous IR loci. A representation of sequenced sRNAs derived from either the IR71 (A) or IR2039 (B) endogenous hairpins. Indicated are the number of each size class of sRNA and the number of reads compared with the total number of sequenced sRNAs derived from IR71 (A) or IR2039 (B). Shown are the location of the probes used in panel C and the location and orientation of the genes adjacent to the respective hairpins. The arrow indicates the predicted terminal loop of the folded RNA. (C) RNA gel blot analysis of DCL usage at IR71 (@IR71) and IR2039 (@2039). miR159 (@159) serves as a control for miRNA levels and trans-acting siRNA255 (@255) as a control for other siRNAs in this and subsequent blots.

Figure 2

Genetic requirements for siRNA biogenesis from exogenous and endogenous IR. (A) RNA gel blot analysis of the predicted full-length fold-back hairpin RNA corresponding to IR71 and IR2039 in Col-0 and triple dcl2/dcl3/dcl4 mutants, respectively. (B) Accumulation of siRNAs from endogenous IRs is largely unaffected in rdr, drb and xrn mutant backgrounds unlike in drb4 mutant background where 24-nt siRNAs strongly over-accumulate. The right panel shows similar affects of drb4 on SUL siRNA accumulation. (C) Northern analysis (left) showing the effect of dcl1 and hyl1 mutations on IR71 siRNA accumulation. The same analyses in a SUC:SUL background are shown in the right panel. (D) RNA blot analysis in hen1 mutants in Col-0 (IR71 and IR2039, left) and Ler (SUL, right). (E) Phenotype and northern analysis of SUL siRNA (@SUL) in plants heterozygous or homozygous for the hst1 mutation. The same analysis is shown on the right for the IR71 loci in Ler and hst1.

Figure 3

IR-derived siRNAs are loaded into cognate AGO proteins and can function at both post-transcriptional gene silencing and RNA-directed DNA methylation levels. (A) Immunoprecipitation experiments were conducted using either an AGO4- or AGO1-specific antibody. The presence of either AGO1 or AGO4 in each IP was confirmed by protein blot analysis (upper panels). Total RNA extracted from the respective IPs was subjected to northern analysis using the indicated probes. (B) Sequencing and molecular confirmation of siRNAs from Col-0 and a T-DNA insertion line at the IR71 locus. siRNAs sequenced from the aforementioned genotypes, including the predicted terminal loop (arrow), the number of IR71 reads compared with the total number of reads and the location of probes used in the gel blot analysis. Also shown is the location of the T-DNA insertion (triangle), with the boxed region representing the predicted region of the IR fold-back structure that would be disrupted by the insertion. (C) A schematic representation of the 35S:GFP sensor used to assay the post-transcriptional silencing ability of AGO1-loaded IR71-derived siRNAs. A recognition sequence (blue) for the highly AGO1-loaded siRNA (red) was inserted three bases after the stop codon of GFP at the start of the 3′UTR. The middle panels show the GFP sensor fluorescence after transformation into either a dcl2/dcl3/dcl4 triple mutant (left) or Col-0 plant (right). Northern blot analysis (bottom panel) confirms the strong GFP mRNA decrease and the presence of IR71-derived siRNAs in silenced Col-0 plants, and the converse for non-silenced dcl2/dcl3/dcl4 plants. (D) Analysis of DNA methylation induced by IR71-derived siRNAs. A schematic representation of the predicted regions of methylation within a 300-nt portion of the IR71 fold-back disrupted by the T-DNA insertion, including the location of the primer and restriction sites used. sqPCR analysis (@IR71) of DNA extracted from the indicated genotypes after digestion with the methylation-sensitive enzyme AluI or Sau96I. Equal input of DNA was confirmed by amplification of a region of actin-2 (@Act2) lacking either restriction site. Quantitative real-time PCR analysis (right) confirmed the results of the semi-quantitative approach.

Figure 4

Rapid evolution and regulated expression of endogenous IR loci. (A) Northern analysis of IR71- and IR2039-derived siRNAs in various Arabidopsis accessions representing the genetic diversity within the A. thaliana species. Also shown are miRNA (@159) and trans-acting siRNA (@255) in each accession. (B) A schematic representation of the DCL2 mRNA showing the domain structure and genomic region with the indicated SNP and indel in ecotypes Ms-0 and Kas-1 (boxed). The right panel shows that the mutations in the DCL2 genomic sequence do not affect mRNA accumulation (@DCL2) in the indicated accessions as assessed by RNA gel blot analysis. (C) Alignment of DCL2 amino acid sequence covering both the predicted amino acid substitution and deletion (marked ^*) between a representative sequence present in most Arabidopsis accessions (except Ms-0 and Kas-1) and the distinct species A. lyrata. (D) Northern analysis of virus-infected Col-0 plants probed with either IR71- (@IR71) or virus siRNA (vsRNA)-specific probes. NI, non-infected and I, infected with either CaMV (Cauliflower mosaic virus) or TCV (Turnip crinkle virus). (E) GUS staining of leaves from promoter::GUS fusions of two independent transgenic lines containing ∼1.5-kb upstream promoter regions from either IR2039 (left) or IR71 (middle). The right panel shows GUS staining of a whole plant representative of the IR71 promoter::GUS fusion lines.

Figure 5

IR71-derived siRNAs are mobile and functional over long distances. (A) A schematic representation of the genotypes Col-0 and IR71 T-DNA (referred to subsequently as IR71−/−), and grafting used to detect the long-distance movement of IR71-derived siRNAs. (B) Sequencing of siRNA populations in the roots of Col-0, IR71−/− and grafted roots as represented in panel A. The inset boxes show a close-up comparison of the siRNA populations in the IR71−/− line and the siRNAs received in the IR71−/− line once grafted onto the donor Col-0 line. Blue, green and red correspond to 21-, 22- and 24-nt size classes respectively. (C) In planta biolistic delivery of ALEXA555-labelled 21- and 24-bp siRNA duplexes. The top four panels show fluorescence 1 hpb for the indicated siRNA size at locations distal (left two panels) or proximal (right two panels) to the leaf veins. The same is shown 20 hpb in the lower four panels. Note that both 21- and 24-bp siRNAs, when delivered proximal to veins (V), have the ability to enter the vasculature and potentially move over longer distances. (D) Northern analysis of the genotypes shown in panel A confirms the sequencing results shown in panel B, with two IR71-specific probes (@IR71 probe 1 and @IR71 probe 2) as well as controls for miRNA accumulation (@173) and heterochromatic siRNA accumulation (@1003). (E) Semi-quantitative (top panel) and quantitative (bottom panel) methylation-sensitive PCR of Col-0, IR71−/− and grafted root tissue. Genomic DNA extracted from the indicated genotypes was digested with the methylation-sensitive enzyme Sau96I and amplified using the primers as shown in Figure 3D. Actin-2 serves as a loading control.