PREreview of Ovule siRNAs methylate protein-coding genes intrans

This full PREreview is the result of a live-streamed preprint journal club organized and hosted by Plant Postdocs and PREreview on April 26, 2022.

Summary

In this preprint, the authors are investigating the siRNAs in siren loci in Brassica napa. This study nicely complements another recent study in which siRNAs produced in anthers can methylate male meiotic genes in trans. We are pleased that this study has utilized a more economically important crop, B. napa, a close relative of the model organism Arabidopsis thaliana. Unlike Arabidopsis, loss of RNA-directed DNA methylation (RdDM) leads to severe consequences for reproduction in B. napa. The authors check if these siren siRNAs are methylating genes in trans. As the title indicates, the authors discover that ovule siRNAs methylate protein-coding genes in trans. In ovules, it is shown that the majority of the 24-nt siren siRNAs map to non-TE regions. In fact, a large number of ovule siren loci encompass genes, pseudogenes or gene fragments. When the authors study the methylation patterns of siren siRNA generating gene fragments, they find that these gene fragments show elevated CHH and CHG methylation, while CG methylation is mostly unaffected. With this preprint, the authors present a new mechanism for RdDM influencing gene expression. Overall, we believe the authors’ conclusions are supported by the data they represented. However we list some issues that we hope may help the authors improve the manuscript.

Major issues and feedback

• The experiment reported in Figure 1D could not clearly address the issue of whether aligning to non-TE regions is particular to siren loci or to all 24-nt-generating loci from ovules. Instead of comparing data from leaves, the authors may consider mapping the alignment of siren loci and compare it to 24-nt producing loci.
• Reviewers found it contradicting that the 74-bp sequence in Persephone elements was claimed to be responsible for siren behavior in ovules, yet there were some elements with the 74-bp sequence that did not qualify to be a siren loci. Could there be a smaller region within the 74-bp that might be more essential? Is it possible for authors to check for a smaller motif?
• We recommend adding axes to sRNA read and methylation distribution plots in Fig. 3B, C and G. As it is presented now, it appears as the CG methylation is not affected in rdr2 ovule, but apparently it is. Adding descriptive axes could make interpretation easier.
• In Fig. 7B, it is stated that the two copies of NRPD1 are expressed differently. It would be nice to show that with a statistical test, if possible.

Minor issues and feedback

• In some figures where significance is indicated, I did not find information on what type of statistical test was applied. It would be better if the authors can share this information.
• I feel the authors should be a little bit clear in their introduction in the beginning paragraph about why DNA methylation or RNA Directed DNA methylation per se is important in plants and then further discuss the process and the mechanistic details.
• We believe that sufficient detail was provided to allow the reproduction and validation of the bioinformatics analyses. However, we noticed that versions of the tools and packages used in the analyses were not included in the methods. Version control is a key issue in reproducibility and it would be beneficial to have versions included. Overall, this preprint provided more details of bioinformatics analyses than most papers, which is a great indication for this team’s efforts on reproducibility.
• A custom script was used to count loci, but not having access to that could be an issue if someone wanted to reproduce the work. Authors should consider adding it to the supplementary materials.
• We deeply appreciated seeing the distribution of the data points represented in box plots in Figure 4.
• The heavy use of red and green colors in Figure 2 have concerned us. Authors are encouraged to pick other colors that may be more color-blind friendly. Tools such as i want hue or Viz Palette can help with choosing colors that are distinguishable by color-blind individuals.
• Similarly, when shifted to grayscale, some sections of the Figure 1 and 5 are indistinguishable. We encourage authors to check their color schemes in these figures as well.

We thank the authors for sharing their work as a preprint. We hope our feedback above will be helpful as they consider any revisions to the manuscript or future lines of work.