Unexpected variations in posttranscriptional gene silencing induced by differentially produced dsRNAs in tobacco...

In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.