Comparison of different methods for the establishment of RNA silencing in plants

RNA interference (RNAi) occurs naturally in plants and is involved in developmental gene regulation as well as in transposon silencing and defence against viruses. RNAi is initiated by small double-stranded RNAs (dsRNAs) of different origins, eventually leading to the silencing of genes with complementary sequences. Besides its use as a basic tool to study gene function, RNAi has widely been exploited for the generation of pathogen resistant crops and for modifying plant metabolism. For the elucidation of gene functions, silencing-inducing dsRNA is often delivered transiently by means of agroinfiltration. This method is independent of plant regeneration and allows rapid testing of multiple silencing constructs. To get more insight into the applicability of transient gene silencing, we compared a variety of different transient and stable approaches to induce silencing of the b-glucuronidase (gus) transgene by the expression of gus hairpin (hpGus) transcripts. While stable expression of the hairpin always resulted in specific gene silencing, transiently expressed hpGus transcripts could only trigger silencing when the hairpin construct was introduced simultaneously with or prior to the target gene