Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

22 nt miRNAs or siRNAs have been shown to specifically induce production of transitive (secondary) siRNAs for targeted mRNAs. An abrasion method to deliver dsRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus . We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein ( GFP ) transgene in treated leaves of N. benthamiana , but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for 3 endogenous genes: magnesium cheletase subunit I ( CHL-I ), magnesium cheletase subunit H ( CHL-H ), and GUN4 . However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs, but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also had more transitive siRNAs produced in response to 22 nt siRNAs, but the response varied from weak ( CHL-I ) to strong ( CHL-H ). 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for GFP, CHL-H and GUN4 in N. benthamiana . The special activity of 22 nt siRNAs in producing a greater local phenotype and induction of elevated levels of transitive siRNAs was also shown in A. cruentus for the CHL-H gene. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.