Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells

Abstract

A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFP⌬CP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFP⌬CP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFP⌬CP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFP⌬CP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.RNA silencing, including gene quelling, RNA interference, and posttranscriptional gene silencing are sequence-specific RNA degradation mechanisms that operate in fungi, animals, and plants (6,7,30). RNA silencing is triggered by doublestranded RNA and requires a conserved set of gene products (1,13,14). The double-stranded RNA is processed into small interfering RNAs (siRNAs) of 21 to 25 nucleotides (nt), and these siRNAs become associated with an RNA-induced silencing complex that degrades specific target RNA sequences (3,11,12). RNA silencing plays a natural role in protecting fungi, plants, and animals against viral infection. To withstand the RNA-silencing defense, viruses across kingdoms have evolved diverse mechanisms either to avoid or actively suppress RNA silencing (22,35,40).One intriguing feature of RNA silencing is that it is not cell autonomous. In plants, RNA silencing can be induced locally and then spread to distal parts (16,(36)(37)(38). While RNA-silencing induction and RNA degradation have been elucidated in detail, much less is known about how RNA silencing moves from cell to cell and spreads systemically in plants. No mobile silencing signal has been characterized, although the sequence specificity of RNA silencing implies that nucleic acids, possibly siRNAs, may be a component of such an RNA-silencing signal (26). We have used Turnip crinkle virus (TCV) to explore the requirements for cell-to-cell and long-distance spread of RNA silencing in plants.TCV, a member of the Carmovirus genus, has a positive single-strand genomic RNA (4,053 nt), packaged in icosahedral capsids, which contains five major open reading frames (5). The p28 and p88 proteins are translated from genomic RNA, by readthrough of the p28 terminator, and are involved in viral RNA replication. Two overlapping proteins, p8 and p9, are expressed from subgenomic RNA1 and are required for cell-to-cell movement and systemic spread of the virus (10, 23).The 3Ј-proximal open reading frame encodes the 38,000-molecular-weight coat protein (CP), which also plays an essential role in cell-to-cell movement of TCV in Nicotiana benthamiana (8) and acts as an effective suppressor ...

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…It was shown that a short-distance spreading of RNA silencing, once initiated from a small group of cells, can extend over 10-15 cells independently of the presence of cognate transcripts (Himber et al, 2003). Short range silencing was observed for both transgenes and endogenous genes (Ryabov et al, 2004;Schwach et al, 2005;Kalantidis et al, 2006;Dunoyer et al, 2007;Smith et al, 2007).…”

A cucumber mosaic virus mutant lacking the 2b counter-defence protein gene provides protection against wild-type strains

“…Firstly, Fny-CMVD2b might induce highly localized, non-systemic RNA silencing against homologous sequences in the challenging virus. Highly localized RNA silencing can be induced by viral mutants lacking the gene for a silencing suppressor protein; for example, a P38 (a CP) deletion mutant of tobacco crinkle virus (Ryabov et al, 2004). Highly localized RNA silencing, without detectable accumulation of siRNAs, can also occur in certain lines of transgenic plants overexpressing the gfp reporter gene (Kalantidis et al, 2006).…”

Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing

“…We previously developed a Turnip Crinkle Virus (TCV)-based local silencing assay to investigate intraand intercellular VIGS in Nicotiana benthamiana (Qin et al, 2012;Ryabov et al, 2004;Shi et al, 2009;Zhou et al, 2008). TCV belongs to Carmovirus with a single positive-stranded RNA genome (Carrington et al, 1989).…”