Down-regulation of endogenes mediated by a transitive silencing signal

Abstract

Some RNA silencing systems in plants, nematodes, and fungi show spreading of silencing along target sequences, termed transitive silencing. Here, we address the question of whether endogenous targets can be silenced by a transitive silencing signal in plants. In transgenic Arabidopsis thaliana plants that harbored a silencing-inducing locus and a transgenic chimeric primary target, silencing of a secondary transgenic target occurred and the expression of the endogenous catalase genes was downregulated, coinciding with a knock-down phenotype. Strikingly, the efficiency of the catalase silencing appeared to be correlated with the zygosity of the primary target locus and, to a lesser extent, with that of the silencing-inducing locus. These data suggest that silencing of an endogene induced by transgenic secondary small interfering RNAs (siRNAs) might depend on the amount of primary target transcripts that can act as template for the production of an efficient transitive silencing signal.

The Frequency and Efficiency of Endogene Suppression by Transitive Silencing Signals Is Influenced by the Length of Sequence Homology

“…Here, we study in more detail the 3# to 5# spreading previously observed with the XYZ-silencing system (Van Houdt et al, 2003;Bleys et al, 2006a) by using primary targets Y 98 , Y 250 , and Y 500 in which 98 nt, 250 nt, and 500 nt of the CAT2 sequence, respectively, were inserted between the nptII-3#chs end targeted by the silencing inducer X 21 and the upstream region homologous to a transgenic gus target Z c . We demonstrate that the length of the CAT2 inserts influences the timing and efficiency of silencing induced by secondary CAT2-and gus-specific siRNAs originating from the primary target transcripts.…”

“…The production of plants harboring the silencing-inducing locus X 21 and locus Z c has been described previously (Bleys et al, 2006a). Locus X 21 is an IR transgene locus with two convergently transcribed neomycin phosphotransferase II (nptII) reporter genes (P35S-nptII-3#chs) that show posttranscriptional gene silencing.…”

“…We have now implemented this system in Arabidopsis (Arabidopsis thaliana) and showed that both transgenes and endogenes can be silenced by the secondary transitive signals (Bleys et al, 2006a). In this XYZ system, the primary target Y harbors a direct target region homologous to the silencing inducer X and an adjacent region homologous to the secondary target Z.…”

“…1; P35S-gusnptII-3#chs), which in turn is able to silence the transgenic gus target Z c ( Fig. 1; P35S-gus-3#nos; Bleys et al, 2006a). To assess whether the frequency of transitive gus silencing varies with an increasing distance between the direct and adjacent target sequences located in the primary target Y, we constructed three primary targets: Y 98 , Y 250 , and Y 500 .…”

Requirement for CRIF1 in RNA interference and Dicer-2 stability

“…Conversely, Drosophila and mammals neither have RdRPs encoded in their genomes nor have the capability to induce RNAi silencing in cells that do not themselves produce siRNAs. 10 Moreover, it appears that the RNAi machinery is not involved in the heterochromatic silencing of multiple copies of transgenes in mammalian cells. 11 While some of this diversity can be attributed to different components of the pathway in different species, some of the diversity may be due to different mechanisms of pathway regulation.…”