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

Abstract

Abstract22 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.

Barriers to Efficient Foliar Uptake of dsRNA and Molecular Barriers to dsRNA Activity in Plant Cells

“…These 22 bp siRNAs also generated a greater silencing phenotype in the leaf to which they were applied compared to 21 bp siRNAs for a variety of targeted genes. Importantly, 22 bp siRNAs, but not 21 bp siRNAs, were also able to trigger systemic silencing for the GFP transgene in N. benthamiana (Dalakouras et al, 2016;Hendrix et al, 2020), which is discussed further below.…”

“…In addition to containing sequences that match the target gene, the specific dsRNA size and structure is important for successful local or systemic down-regulation of a plant gene product from topically applied dsRNA. Down-regulation of targeted genes using topically applied RNAs has been observed with dsRNAs from 21 to ∼150 bp, but 22 bp siRNAs produced the strongest silencing phenotypes (Dalakouras et al, 2018;Hendrix et al, 2020;unpublished Bayer Crop Science data). It is hypothesized that 22 bp siRNAs provide the greatest silencing efficiency, compared to the other dsRNAs tested, because they can induce production of secondary (transitive) siRNAs for the targeted mRNAs, which could increase the number of active silencing siRNAs for that gene target (Chen et al, 2010;Cuperus et al, 2010).…”

“…It is hypothesized that 22 bp siRNAs provide the greatest silencing efficiency, compared to the other dsRNAs tested, because they can induce production of secondary (transitive) siRNAs for the targeted mRNAs, which could increase the number of active silencing siRNAs for that gene target (Chen et al, 2010;Cuperus et al, 2010). Topically applied 22 bp siRNAs induced production of secondary siRNAs homologous to the target mRNA for all targeted genes examined, although the amount of secondary siRNAs varied by gene (Hendrix et al, 2020). These 22 bp siRNAs also generated a greater silencing phenotype in the leaf to which they were applied compared to 21 bp siRNAs for a variety of targeted genes.…”

“…Systemic Silencing Using Topical dsRNA Has Only Been Documented for the GFP Transgene in N. benthamiana Systemic RNAi has been observed for the GFP transgene in N. benthamiana line 16C (Ruiz et al, 1998) using topical RNA with dsRNAs that are 124 bp or 22 bp, but not 21 bp (Dalakouras et al, 2016;Schwartz et al, 2019;Hendrix et al, 2020; unpublished Bayer Crop Science research). The ability of 22 bp siRNAs to cause systemic silencing is likely related to their ability to induce production of secondary sRNAs (Chen et al, 2010;Cuperus et al, 2010;Dalakouras et al, 2020;Hendrix et al, 2020). Activity by longer dsRNAs to induce systemic silencing may result from generation of 22 bp siRNAs by dicerlike enzymes in plant cells.…”

“…A number of endogenous genes were down-regulated with topically applied siRNAs in N. benthamiana, but systemic silencing was not observed (unpublished Bayer Crop Science research). Endogenous genes that have been targeted in N. benthamiana included magnesium cheletase subunit H (CHL-H), magnesium cheletase subunit I, GUN4, and phosphoribosylanthranilate transferase (Hendrix et al, 2020;and unpublished Bayer Crop Science research). Visible local silencing and/or reduced mRNA levels were observed after targeting each of these gene products in N. benthamiana, but no systemic phenotypes were detected.…”