Caenorhabditis elegans SID-2 is required for environmental RNA interference

Abstract

In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi.

Fig. 1.

sid-2 worms are capable of spreading transgene-initiated RNAi. (A) Wild-type expression of GFP in the pharynx (ph) (myo-2::gfp) and body-wall muscle nuclei (bm) (myo-3::gfp) of an HC46 worm. (B) Expression of dsRNA (myo-2::gfp) in the pharynx causes incomplete silencing of pharynx and body-wall muscle GFP in the HC57 strain. (C) A sid-2(qt13) worm expressing the same transgenes as HC57 is capable of spreading transgene-initiated RNAi from the pharynx to the body-wall muscle. GFP images were taken at equal exposures. Anterior is upper left. (Scale bar, 0.1 mm.)

Fig. 2.

SID-2 is a predicted membrane protein conserved in C. briggsae and C. remanei, and SID-2::GFP is expressed in the luminal intestinal membranes. (A) Translated sequence of C. elegans sid-2 cDNA aligned with a conceptual assembly of the homologous C. briggsae and C. remanei genes. Identical amino acids are marked with an asterisk and similar amino acids are marked with a colon or period. The predicted signal peptide and transmembrane domain are underlined. The positions and amino acid sequence changes for five sequenced alleles are shown above the sequences (∗, stop). (B) Localization of SID-2::GFP expression to intestinal lumen. Fluorescent, white light, and overlay respectively at low (Upper) and high (Lower) magnification. (Scale bars, 0.1 mm and 10 μm.)

Fig. 3.

Injection of dsRNA bypasses sid-2 defects in nonneuronal cells. (A and B) Injection of gfp dsRNA (1 mg/ml) into the pseudocoelom (A) or anteriormost intestinal cell (B) of sid-2(qt13); sur-5::gfp adult hermaphrodites results in complete silencing of GFP in nonneuronal cells. (C and D) Injection into the pseudocoelom (C) or anterior intestine (D) of control sur-5::gfp worms yields comparable results. (E and F) sid-1(qt9); sur-5::gfp worms are completely resistant to RNAi after injection into the pseudocoelom (E) and show silencing in only the injected intestinal cell (arrow) (F). Animals were photographed 48 h after injection at 20°C. Insets are bright-field photomicrographs of the same worm. GFP images were taken at equal exposures.

Fig. 4.

Phylogenetic relationship of select Caenorhabditis species and systemic and environmental RNAi proficiency. Phylogeny from ref. . Only C. brenneri (31) was conclusively deficient for systemic RNAi, whereas only C. elegans and C. sp. 1 (SB341) were proficient at environmental RNAi. nt, not tested.