Table 1.
Summary of all the siRNA sequences with their locations and the targeted genes, with the calculated ΔΔG.
siRNA are written as 5’ ->3’ sense strand: 5’->3’ antisense strand. The bold and underlined letters denote non-native nucleotides. Pos, Position on the cDNA.
Fig 1.
Sandpaper application of siRNA duplexes resulted in an RNAi phenotype.
A, Location of siRNAs targeting the CHLH and GFP genomic locations in the N. benthamiana wildtype and16C lines. Boxes and lines indicate the exons and introns, respectively. The 21 bp sequences with 2 nt 3’ overhangs were tested as wild type or with mutations shown in red. The top strand is the sense sequence, and the bottom is the antisense sequence of the duplex. Leaves were treated with the indicated siRNA duplexes by sandpaper abrasion and photographed under white (B) or blue light (C) 4 dpa. The phenotypic area was quantified with Image J and was graphed as percent of the treated leaf area; error bars represent standard error of the means. D, CHLH transcript levels were determined in leaves harvested 4 dpa; error bars represent standard error of the means. E, GFP protein levels were determined by western blot from leaf tissues collected 4 dpa. Means not followed by the same letter indicate statistical significance (α = 0.05).
Fig 2.
The influence of the 5’ nucleotide of the antisense strand of a 21nt siRNA duplex on GFP transgene silencing.
A, Illustration of interactions between the GFP mRNA and the AS strand of siRNA duplex. dsRNAs with different 5’ nucleotides for the antisense strand (paired with a complementary or identical (G) nucleotide at the sense strand) were tested. B, GFP silenced area was quantified and expressed as percent of the treated leaf area; error bars are standard error of the means. Means not followed by the same letter indicate statistical significance (α = 0.05).
Fig 3.
The influence of the 5’ nucleotide of a 21 bp dsRNA on silencing the CHLH gene.
A, Each 5’ AS nucleotide was tested both in a paired state and in an unpaired states with all other nucleotide possibilities. Leaves were harvested 4 dpa and photographed under white light. B, Phenotypic area was quantified and expressed as percent of the treated leaf area. Means not followed by the same letter indicate statistical significance (α = 0.05).
Fig 4.
Silencing of a GFP:CHLH-fragment fusion in protoplasts was used to measure the efficiency of silencing for sixteen CHLH siRNA variants.
A, Diagram of the siRNA reporter plasmid containing the GFP:CHLH fragment:3’ UTR cassette for gene suppression in protoplasts, the synthetic siRNAs, and the representative protoplast images captured by Oppereta imager of no plasmid, plasmid only, plasmid + CHL-1 UA, and plasmid + CHL-1 GC. B, siRNAs as described in the legend to Fig 3 were transfected together with the GFP:CHLH-fragment fusion, and GFP fluorescence was quantified at 525 nm with 488 nm excitation. Means not followed by the same letter indicate statistical significance (α = 0.05).
Fig 5.
siRNAs containing a 5’ G can be efficacious for gene silencing when the nucleotide on the antisense strand is the non-complementary G or when a U-A pair is substituted.
A, Location of three 21nt siRNAs in the CHLH genomic sequence in the N. benthamiana. Boxes and lines indicate the exons and introns, respectively. These siRNAs were synthesized in their native form as a G-C pairing at the 5’ end of AS strand or substituted with a G-G mismatch or U-A pairing. The nucleotides of 1st position of AS and 19th position of S strands are shaded in grey. B, Key properties for the siRNAs tested were compared (40, 41). C, siRNA duplexes were applied to N. benthamiana leaves with sandpaper abrasion, and treated leaves were photographed under white light 4 dpa.