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

Abstract

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.

RNAi‐mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing

“…The sequence specificity of RNAi‐mediated gene inactivation allows silencing of individual genes as well as multiple members of a multigene family (Miki et al ., ). Transitivity, the spread of RNA silencing along primary target sequences, is an aspect of RNAi that has not been well understood (Bleys et al ., ). Endogene‐derived mRNAs can become a production source of secondary siRNAs that correspond to regions of the target gene outside the original trigger (Sanders et al ., ; Sijen et al ., ).…”

“…In plants, spreading of the RNAi signal has been shown to occur in both the 3′–5′ and the 5′–3′ directions along transgene RNAs in a primer‐dependent and primer‐independent manner (Bleys et al ., ; Braunstein et al ., ; Himber et al ., ; Klahre et al ., ; Kościańska et al ., ; Miki et al ., ; Petersen and Albrechtsen, ; Vaistij et al ., ; Van Houdt et al ., ). Silencing induced by 3′ fragments spread only for a limited distance of up to 332 nt, with a possible limit of 600 nt, while gene silencing in the 5′–3′ direction has been shown to spread over a distance of at least 1000 nt (Petersen and Albrechtsen, ; Vaistij et al ., ).…”

Vector-initiated transitive RNA interference in the filamentous fungus Aspergillus oryzae

“…We observed that white-spored transformants were obtained more frequently using the plasmid containing the wA inverted repeat when compared to transitive RNAi constructs (see Tables 2 and 3). This difference in silencing efficiencies has also been observed in Arabidopsis (Bleys et al, 2006;Filichkin et al, 2007). Similarly in A. oryzae, this decrease in transitive silencing would be expected if initiation of transitive silencing is reliant on initial processing of siRNAs within the hph-IR prior to amplification and silencing of IR flanking sequences.…”

RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants

“…Deep sequencing analysis revealed that the rdr1 and rdr6 mutants exhibit significantly reduced levels of small RNAs (Qi et al, 2009). The efficiency of gus silencing is correlated with the amount of secondary siRNA suggesting that the amplification of secondary siRNAs is required to exceed the threshold for efficient silencing (Bleys et al, 2006). Taken together, these results indicate that RDRs are required for the amplification of secondary siRNAs and efficient gene silencing.…”

“…This result may be due to several factors such as efficient GFP-siRNA production from the long hairpin RNA containing full-length GFP sequence, TGS induced by the CaMV35S promoter, which is used for both GFP and GFP-RNAi expression, and/or by co-suppression effects of the GFP gene expression. The efficiency and frequency of transitive silencing of an endogenous gene depends on the length of its sequence identity with the primary target (Bleys et al, 2006). Gene silencing mediated by promoter homology occurs at the level of transcription (Park et al, 1996).…”