Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing

Abstract

The term 'gene silencing' refers to transcriptional and post-transcriptional control of gene expression. Related processes are found across kingdoms in plants and animals. We intended to test whether particular RNA constituents of a silenced plant can induce silencing in an animal. We generated Nicotiana benthamiana lines that expressed green fluorescent protein (GFP) from a transgene. Plants in which GFP expression was spontaneously silenced showed siRNAs characteristic of post-transcriptional gene silencing (PTGS). RNA extracts prepared from silenced plants were injected into a GFP-expressing strain of Caenorhabditis elegans, where they induced RNA interference (RNAi). Extracts from non-silenced plants were inactive. This directly demonstrates a relationship and a mechanistic link between PTGS and RNAi. Controls confirmed that the silencing agent was an RNA. Size fractionation on denaturing gels revealed that an RNA of approximately 85 nt was most active in inducing silencing in the worm. Northern blot analysis of the region in question did not detect a prominent GFP-specific RNA of sense or antisense polarity, indicating that the RNA species which induced silencing was present only in low concentration or did not hybridize due to formation of an intramolecular double strand. In view of its high activity, it is possible that this agent is responsible for the systemic spread of silencing in plants and it might represent the aberrant RNA, a previously postulated inducer of silencing.

Figure 1

Analysis of GFP expression in different transgenic lines. (A) Nicotiana benthamiana leaves from five different plants under UV light. Leaf 1 is expressing GFP strongly, leaves 2–4 express GFP with degreasing intensity; leaf 5 is from a silenced plant. (B) Micrographs under UV light of a leaf of a non-transgenic (wt), a silenced (sil.) and a GFP-expressing plant (GFP) are shown; the green fluorescence of GFP expression is best seen in the close-up of a trichome. (C) Northern analysis of the GFP mRNA on a denaturing 1.2% formaldehyde agarose gel. The level of GFP expression correlates with the visual impression of (A). Leaf 5 does not contain detectable GFP mRNA; M, an in vitro synthesized GFP sense marker transcript of ∼750 bases. (D) Northern analysis of the same RNAs on a 12% polyacrylamide gel. Leaf 5 contains siRNAs. M, marker; the lower signal is a radiolabeled synthetic 22mer RNA.

Figure 2

Silencing potential of plant RNAs on GFP expression in C.elegans. (A) Representative examples of micrographs of nematodes taken under UV illumination for quantification of GFP expression. The treatments are indicated. It should be noted that the differences in GFP expression seen between non-injected and the buffer-injected animal (top) are within the variation limits amongst individuals. To remove this variability, 10 pictures were analyzed as described in Materials and Methods to determine the silencing data in each of the columns of (B)–(E), which indicate percent silencing compared with the non-injected control; standard deviation is indicated by bars. (B) Silencing data from analytical extracts from a non-silenced (N.S.) and silenced (Sil.) plant. Injections of buffer (Con) and of GFP dsRNA are indicated. The samples correspond to the pictures displayed in (A). (C) Silencing data for preparative extracts of silenced and non-silenced plants, the crude fractions and the LiCl-soluble fractions. The right three columns show the LiCl-soluble fraction after treatment with RNase, DNase and phosphatase. (D) Silencing data for the excised fractions in Figure 3A; a–c refer to the zones given there; nucleotide numbers are given. (E) Silencing data for the excised fractions in Figure 3B. The assumed size range of the extracted RNAs is indicated in nucleotide numbers.

Figure 2

Silencing potential of plant RNAs on GFP expression in C.elegans. (A) Representative examples of micrographs of nematodes taken under UV illumination for quantification of GFP expression. The treatments are indicated. It should be noted that the differences in GFP expression seen between non-injected and the buffer-injected animal (top) are within the variation limits amongst individuals. To remove this variability, 10 pictures were analyzed as described in Materials and Methods to determine the silencing data in each of the columns of (B)–(E), which indicate percent silencing compared with the non-injected control; standard deviation is indicated by bars. (B) Silencing data from analytical extracts from a non-silenced (N.S.) and silenced (Sil.) plant. Injections of buffer (Con) and of GFP dsRNA are indicated. The samples correspond to the pictures displayed in (A). (C) Silencing data for preparative extracts of silenced and non-silenced plants, the crude fractions and the LiCl-soluble fractions. The right three columns show the LiCl-soluble fraction after treatment with RNase, DNase and phosphatase. (D) Silencing data for the excised fractions in Figure 3A; a–c refer to the zones given there; nucleotide numbers are given. (E) Silencing data for the excised fractions in Figure 3B. The assumed size range of the extracted RNAs is indicated in nucleotide numbers.

Figure 3

Electrophoretic separation of RNA extracts on a denaturing 5% polyacrylamide gel. Visualization was with ethidium bromide, however, the negative is displayed to increase contrast. (A) Separation of the crude extract (Ex) and the LiCl-soluble fraction, which was loaded in five lanes. The positions of the bulk of the tRNA and the larger tRNA^Leu are indicated. Lane 1, synthetic 22mer RNA; lane 2, 100 bp DNA ladder; lane 3, pBR322 × HinfI (which is incompletely denatured). The left side indicates the origin of the gel (O) and the three zones (a–c) excised from the five lanes separating the LiCl-soluble material. (B) Separation of the crude extract (lane Ex). This lane was part of a preparative gel, in which several lanes were run in parallel, similar to the LiCl-soluble fraction in (A). Gel slices corresponding to different size classes were excised. After each of the five excisions the gel was photographed. The panel shows one particular lane after each round of excision, as indicated at the top of the lane. The boxes in excision lane 1 illustrate the excised zones. The numbers on the left refer to the sizes of the DNA fragments, as in (A). The positions of 5S and 5.8S rRNA are indicated (corresponding to 120 and 170 bases). The signal slightly larger than 100 bases represents 4.5S chloroplast rRNA.

Figure 4

Northern blot analysis of extracts after separation on a denaturing 12% polyacrylamide gel. The left panel shows hybridization with a GFP antisense probe, the right with a GFP sense probe. Lane M, pBR322 × HinfI; 22, an end-labeled 22mer RNA; G, extract from a GFP-expressing (non-silenced) plant; S1 and S2, extracts from two plants with silenced GFP. Extract of plant S2 had been shown to be active in inducing silencing in C.elegans and had been used for the fractionation in Figure 2B. As expected, the silenced plants showed siRNAs with both probes. The silenced plants contained less GFP sense RNA than the non-silenced control. The higher molecular weight signals detected with the GFP sense probe may be non-specific, due to the low stringency conditions applied for detection of siRNAs. However, in neither of the silenced plants was a specific sense and antisense signal detected in the size range 81–90 nt, indicating that the active RNA was present only in low concentration.