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. 2021 Jul 13;12:4278. doi: 10.1038/s41467-021-24617-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Transgenic 16c N. benthamiana plants co-infiltrated with constructs to express GFP (35S-GFP) and Myc-V3, P19 (as positive control), or mock (empty vector, as negative control) at 4 dpi (upper panel) or 20 dpi (lower panel) under UV light. b Western blot showing the GFP accumulation in inoculated leaves from (a) at 4 dpi. The corresponding Ponceau S staining of the large RuBisCO subunit serves as a loading control. This experiment was repeated three times with similar results; representative images are shown. c Relative GFP mRNA accumulation in inoculated leaves from (a) at 4 dpi measured by qRT-PCR. Data are the mean of three independent biological replicates. Error bars represent SD. An asterisk indicates a statistically significant difference according to unpaired Student’s t test (two-tailed), * p < 0.5, *** p < 0.001. NbActin2 was used as the internal reference. d Transgenic 16c N. benthamiana plants co-infiltrated with constructs to express GFP (35S-GFP) and PVX, PVX-V3, PVX-βC1 (as positive control), or mock (infiltration buffer, as negative control) at 7 dpi (upper panel) or 20 dpi (lower panel) under UV light. e Western blot showing the GFP accumulation in inoculated leaves from (d) at 7 dpi. The corresponding Ponceau S staining of the large RuBisCO subunit serves as loading control. This experiment was repeated three times with similar results; representative images are shown. f, g Symptoms of 16-TGS N. benthamiana plants infected with PVX, PVX-V3, PVX-βC1 (as positive control), or mock-inoculated under white light or UV light at 10 dpi (f) and 28 dpi (g). h, i Western blot showing accumulation of GFP and PVX CP in systemically infected leaves from (f) and (i). The corresponding Ponceau S staining of the large RuBisCO subunit serves as loading control. This experiment was repeated three times with similar results; representative images are shown. j DNA methylation analysis by restriction enzyme digestion in V3-YFP transgenic N. benthamiana plants. Genomic DNA extracted from WT N. benthamiana or two independent V3-YFP transgenic lines (#4 and #5) was digested with the methylation-dependent restriction enzyme McrBC and the methylation-insensitive enzyme DraI. “Sham” indicates a mock digestion with no enzyme added. The positions of undigested input genomic DNA is indicated with an asterisk; the position of the McrBC-digested products is indicated with a hashtag. This experiment was repeated three times with similar results; representative images are shown. The original data from all experiments and replicates can be found in the Source data file.