Transitivity, the spread of RNA silencing along primary target sequences, leads to the degradation of secondary targets that have no sequence homology to the initial silencing trigger. We demonstrate that increasing the distance between direct and adjacent target sequences in a transgenic primary target delays the onset of silencing of a secondary target gene. Silencing can spread in a 3# to 5# direction over a distance of at least 500 nucleotides (nt), but this requires consistently more time compared to a distance of 98 nt or 250 nt. The efficiency and frequency of transitive silencing of an endogene depends on the length of its sequence homology with the primary target. With a length of 500 nt, efficient silencing can eventually be established in all plants, whereas lengths of 250 nt and 98 nt homology result in less efficient and less frequent suppression. These results suggest that amplification of secondary small interfering RNAs (siRNAs) is a time-requiring process that gradually expands the population of siRNAs until a steady-state level is reached. Moreover, the length of the sequence homology in the primary target providing secondary siRNAs determines whether this steady-state level readily exceeds the threshold necessary for efficient silencing.
SUMMARYEndogenes rarely support transitive silencing, whereas most transgenes generally allow the spread of silencing to occur along the primary target. To determine whether the presence of introns might explain the difference, we investigated the influence of introns in the primary target on 3¢-5¢ silencing transitivity. When present in a transgene, an intron-containing endogene fragment does not prohibit the spread of silencing across this fragment, indicating that introns do not preclude silencing transitivity along endogenes. Also, a multiple intron-containing genomic gene fragment that had previously been shown not to support transitivity in an endogenous context could support transitivity when present in a transgene. Nevertheless, genomic intron-containing fragments delayed the onset and diminished the efficiency of transitive silencing of a secondary target compared with the corresponding cDNA fragments. Remarkably, transitivity was impaired proportionally with the length of the pre-mRNA, and not of the mRNA. The latter result suggests that the RNAdependent RNA polymerase-based spreading of silencing progresses along the non-spliced rather than the fully processed mature mRNA.
SUMMARYPost-transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target region and with de novo RNA-directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to the transgene. Our data indicate that RdDM is correlated with the in situ generation of secondary siRNAs, occurring in P35S-driven transgenes but not in most endogenes. We conclude that although both endogenes and transgenes are equally sensitive to transitive silencing, differences exist in their susceptibility to undergo secondary RdDM.
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