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. 2024 Nov 7;25(12):5780–5809. doi: 10.1038/s44319-024-00304-5

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) EVD copy number analysis by qPCR in NRPD1-EVD and NRPE1-EVD lines, in both WT (gray) and mutant (colored) backgrounds, at generations F2, F4, and F6 derived from three independent F1s (biological replicates), using the EVD-GAG sequence as target. (B) qPCR analysis of shGAG expression normalized to ACT2 in NRPD1-EVD and NRPE1-EVD lines, in both WT (gray) and mutant (colored) backgrounds, at generations F2, F4, and F6 derived from three independent F1s (biological replicates). In (A) and (B), biological replicates (bulks of 8–10 plants each) are individually represented by dots. Error bars show standard error of the mean in (A) and (B). p-values for two-sided t-test between indicated samples are shown. Differences are considered statistically significant if p < 0.05 (5.00e−2) or non-significant (ns) if p ≥ 0.05. (C, D) RNA blot analysis of EVD siRNAs against GAG and LTR in the F6 generation of 3 independent WT and mutant lines of NRPD1-EVD (C) and NRPE1-EVD (D). WT Col-0 and nrpd1 or nrpe1 with no reactivated EVD are shown as negative control for EVD activity. siR1003 probe is used as control for NRPD1 and NRPE1 mutations, miR171 and snoRNA U6 are shown as loading controls. (E, F) % methylated cytosines (C) by bisulfite-PCR DNA methylation analysis at EVD-LTR sequences in the F6 generation of NRPD1-EVD (E) and NRPE1-EVD (F) lines, in both WT (darker shade) and mutant (lighter shade) backgrounds. Col-0, nrp1d and nrpe1 were used as controls. n: number of clones analyzed. Error bars represent 95% confidence Wilson score intervals of the % of methylated cytosines (C) in each context (CG, CHG, CHH). Source data are available online for this figure.