Detection and investigation of transitive gene silencing in plants.

RNA-induced post-transcriptional silencing is a common tool in functional gene analysis and its application in crop improvement is widely investigated. However, its specificity might be impaired by off-target silencing as a result of transitivity. Generally transitivity is investigated by the detection of secondary siRNAs; however, these tests fail to demonstrate the siRNA's bioactivity.

Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target.

Post-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.

Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA.

Endogenes 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.

The frequency and efficiency of endogene suppression by transitive silencing signals is influenced by the length of sequence homology.

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.