Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis

Abstract

The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of posttranscriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes.

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

“…The seven selected fragments were checked by BLAST to confirm the absence of stretches of 14 nt or more of identity in the lettuce genome to minimize the possibilities of off-target effects (Elbashir et al, 2002;Jackson et al, 2003) on lettuce gene expression. The B. lactucae gene fragments were amplified and cloned as inverted repeats of the RNAi trigger sequences fused to a 400bp fragment of the UidA gene ( Figure S1, Wroblewski et al, 2014). The UidA fragment served as a reporter for silencing using A. tumefaciens-mediated transient b-glucuronidase (GUS) assays, which allowed us to select transgenics exhibiting high levels of silencing (Wroblewski et al, 2007(Wroblewski et al, , 2014.…”

“…The B. lactucae gene fragments were amplified and cloned as inverted repeats of the RNAi trigger sequences fused to a 400bp fragment of the UidA gene ( Figure S1, Wroblewski et al, 2014). The UidA fragment served as a reporter for silencing using A. tumefaciens-mediated transient b-glucuronidase (GUS) assays, which allowed us to select transgenics exhibiting high levels of silencing (Wroblewski et al, 2007(Wroblewski et al, , 2014.…”

“…Figure S1 Schematic of the interfering hairpin RNA (ihpRNA) construct in modified binary vector pGOLLUM (Wroblewski, 2007;Wroblewski, 2014). Figure S2 Microscopic Z-images of the hypersensitive resistance response in lettuce cv.…”

“… Figure S1 Schematic of the interfering hairpin RNA (ihpRNA) construct in modified binary vector pGOLLUM (Wroblewski, ; Wroblewski, ).…”

The Whys and Wherefores of Transitivity in Plants

“…In contrast to organisms supporting transitivity outside of the plant kingdom, the spread of the silencing signal is bi-directional in plants, with secondary sRNAs originating up- and downstream of the target region ( Vaistij et al, 2002 ). Yet, the expansion of the silencing signal in plants shows a clear tendency toward the 3′ region of the transcript ( Braunstein et al, 2002 ; Petersen and Albrechtsen, 2005 ; Bleys et al, 2006b ; Haque et al, 2007 ; Moissiard et al, 2007 ; Aregger et al, 2012 ; Dadami et al, 2014 ; Wroblewski et al, 2014 ; Han et al, 2015 ; de Felippes et al, 2020 ). Another interesting aspect of transitivity in plants is the apparent susceptibility of transgenes in becoming template of RDR6.…”