FIG 5.

CLCuMuV V2^L76S abolishes TGS suppression function in plants. (A) 16c-TGS plants were agroinfected with PVX-HA-V2, PVX-HA-V2^L76S, or PVX-HA-nLUC. Photographs were taken under white light or UV light at 9 days postinfiltration (dpi). (B) RT-qPCR analysis of relative GFP mRNA levels in 16c-TGS plants infected with PVX-HA-V2, PVX-HA-V2^L76S, or PVX-HA-nLUC. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01 [Student’s t test]). The bars represent means ± SD. Data were obtained from three independent experiments. (C) Immunoblot assay of GFP protein using anti-GFP antibody in 16c-TGS plants infected with PVX-HA-V2, PVX-HA-V2^L76S, or PVX-HA-nLUC. RuBisCO was stained with Ponceau Red as a loading control. (D) The protein expression of V2, V2^L76S, and HA-nLUC was confirmed by immunoblot assays with anti-HA and anti-V2 polyclonal antibodies. (E) Percentage of methylated cytosine in CaMV 35S promoter sites in 16c-TGS plants infected with PVX-V2, PVX-V2^L76S, and PVX-HA-nLUC. The histogram represents the proportions of methylated cytosine residues in different sequence groups. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01 [Student’s t test]). The bars represent means ± SD. Data were obtained from three independent experiments. (F) Detection of V2^L76S protein in transgenic plants by immunoblotting. V2 protein was immunoblotted using an anti-V2 antibody. RuBisCO was stained with Ponceau Red as a loading control. (G) Percentage of cytosine methylation of N. benthamiana transposons Tnt1 and TE1 in 35S:HA-V2, 35S:HA-V2^L76S transgenic or mock plants. The histogram represents the proportions of methylated cytosines in different sequence groups. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01 [Student’s t test]). The bars represent means ± SD. Data were obtained from three independent experiments.