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.dsRNA ͉ transmembrane ͉ intestine lumen T ranslocation of nucleic acids across cellular membranes is associated with viral infection, bacterial conjugation, and transport of nuclear encoded tRNAs into mitochondria (1-3). In these cases, specific machinery acts to translocate a specific RNA or DNA across one or more membranous barriers. However, observations in plants suggest the routine intercellular transport of cellular mRNAs as well as processed, likely short, dsRNAs associated with RNAi-related phenomena that mediate systemic virus resistance (4, 5). In the nematode Caenorhabditis elegans, RNA-induced gene silencing is also systemic, spreading from the site of injection or expression to silence the targeted gene throughout the animal and in its progeny (6, 7). Furthermore, RNAi can be initiated by soaking animals in solutions of dsRNA or feeding worms bacteria expressing dsRNA (8, 9). Thus dsRNA can enter the animal from the environment. RNAi triggered by environmental exposure to dsRNA has also been documented in planaria, moth, tick, hydra, and numerous parasitic nematodes (10-15), suggesting that many invertebrates possess mechanisms to transport sequence nonspecific dsRNA into and between cells.To identify cellular components required for systemic RNAi in C. elegans, we isolated systemic RNAi defective (Sid) mutants defective for spreading of RNAi (7). The first characterized gene, sid-1, encodes a transmembrane protein expressed in all cells sensitive to systemic RNAi and is required for uptake of dsRNA (7, 16). Furthermore, SID-1 expressed in Drosophila S2 cells is sufficient to mediate passive uptake of dsRNA from the growth media, indicating that SID-1 most likely acts as a channel for diffusion of dsRNA into cells (16). These observations provide strong support for the notion that dsRNA is systemically transported in C. elegans.A SID-1::GFP fusion reporter construct that rescues sid-1 mutant worms was expressed at highest levels in cells directly exposed to the environment (7). This observation suggested that environmental dsRNA might enter the animal via these sid-1 expressing cells. Here we describe sid-2, a gene specifically required for uptake of silencing information (hereafter assumed to be dsRNA) from the environment...

In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence. Here, we report the identification and characterization of SID-5, a C. elegans endosome-associated protein that is required for efficient systemic RNAi in response to both ingested and expressed double-stranded RNA (dsRNA). SID-5 is detected in cytoplasmic foci that partially colocalize with GFP fusions of late endosomal proteins RAB-7 and LMP-1. Furthermore, knockdown of various endosomal proteins similarly relocalizes both SID-5 and LMP-1::GFP. Consistent with a non-cell-autonomous function, intestine-specific SID-5 expression restored body wall muscle (bwm) target gene silencing in response to ingested dsRNA. Finally, we show that sid-5 is required for the previously described sid-1-independent transport of ingested RNAi triggers across the intestine. Together, these data demonstrate that an endosome-associated protein, SID-5, promotes the transport of RNAi silencing signals between cells. Furthermore, SID-5 acts differently than the previously described SID-1, SID-2, and SID-3 proteins, thus expanding the systemic RNAi pathway.

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SUMMARYIn plants, as in all eukaryotic organisms, microtubule-and actin-filament based structures play fundamental roles during cell division. In addition to the mitotic spindle, plant cells have evolved a unique cytoskeletal structure that designates a specific division plane before the onset of mitosis via formation of a cortical band of microtubules and actin filaments called the preprophase band. During cytokinesis, a second plantspecific microtubule and actin filament structure called the phragmoplast directs vesicles to create the new cell wall. In response to intrinsic and extrinsic cues, many plant cells form a preprophase band in G 2 , then the preprophase band recruits specific proteins to populate the cortical division site prior to disassembly of the preprophase band in prometaphase. These proteins are thought to act as a spatial reminder that actively guides the phragmoplast towards the cortical division site during cytokinesis. A number of proteins involved in determination and maintenance of the plane of cell division have been identified. Our current understanding of the molecular interactions of these proteins and their regulation of microtubules is incomplete, but advanced imaging techniques and computer simulations have validated some early concepts of division site determination.

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