The Arabidopsis active demethylase ROS1 cis-regulates defence genes by erasing DNA methylation at promoter-regulatory regions

Essential revisions:

1) Subsection “ROS1 directs promoter demethylation of flg22-induced ROS1 targets, mostly by antagonizing DCL2 and/or DCL3 functions”: The authors state that there were 219 Flg22-responsive candidate genes that are associated with regions that are hyper-methylated in the ros1-3 mutant, of which 102 were "less- induced" and 115 were "less-repressed". These numbers don't add up though: 102 + 115 = 217. Furthermore, the hyperDMRs at these 219 (or 217?) genes are reported to be within the gene bodies plus 2Kb upstream/downstream regions. It would be helpful if the authors could show these data for the 115 less-repressed and 102 less-induced genes separately, and indicate the distribution of ROS-dependent methylation between gene bodies, 2 Kb upstream sequences and 2 Kb downstream sequences.

The number of flg22-responsive candidate genes is indeed 217. This mistake has been corrected. To address reviewers’ suggestions, we have added the distribution of ROS1-dependent demethylation regions between gene bodies, 2 Kb upstream sequences and 2 Kb downstream sequences. Furthermore, we now provide the hyperDMRs location for each ROS1 target from the 115 less-repressed and 102 less-induced gene sets.

This information has been added in the Results section as well as in Supplementary file 1:

“Among them, 102 less-induced (promoter regions: 44 DMRs; gene bodies: 33; downstream regions: 25) and 115 less-repressed (promoter regions: 67 DMRs; gene bodies: 27; downstream regions: 21) genes were recovered (Figure 3A, Supplementary file 1).”

2) Related to the above point, could the authors comment on how ROS1-dependent DNA demethylation may control the 115 genes showing less flg22-induced repression in the ros1-3 mutant? For these genes, one would expect that ROS1-dependent DNA demethylation facilitates binding of repressive TFs. Are the promoters of these Flg22-repressed genes also associated with methylation-sensitive W box elements?

To address this point, we performed a GAT analysis on 28 less-repressed ROS1 targets, which exhibit dense hyperDMRs at their promoter regions in ros1-3 mutants. By doing this analysis, we found that WRKYs enrichment was detected at only 2 candidate demethylated promoter regions. These data indicate that the flg22-triggered repression of these genes occurs mostly independently of WRKYs DNA binding. In addition, we noticed that other classes of TFs can bind to the demethylated promoter regions of flg22-repressed ROS1 targets. As proposed by the reviewers, ROS1 might direct demethylation of these promoters to facilitate binding of various repressive TFs, thereby ensuring a proper repression of these genes during PTI. We have now included a new figure presenting these results (see Figure 4—figure supplement 2). Furthermore, we have added the following sentences in the Results and Discussion sections of the revised manuscript to describe and discuss these new results:

“By contrast, only 2 out of 28 less-repressed stringent targets displayed WRKY TFs enrichment at their demethylated promoter regions (Figure 4—figure supplement 2)”.

“It is noteworthy that the proposed mechanism might also hold true for other TF families, which exhibit in vitro binding at the demethylated promoter regions of flg22-induced ROS1 targets (Figure 4—figure supplement 1). […] Consistent with this hypothesis, different classes of TFs were found to preferentially bind to the demethylated promoter regions of the flg22-repressed ROS1 targets (Figure 4—figure supplement 2).”

3) Using their DAP-qPCR system, the authors convincingly demonstrate in Figure 5 that WRKY18-GST and WRKY40-GST have lower affinity to the methylated W-box within the RLP43 promoter of the ros1-3 mutant. Is this also true for the W-box element in the promoter of the RMG1 gene?

The available DAP-seq datasets revealed a weak in vitro binding of WRKY TFs at the demethylated region of RMG1, which was difficult to reproducibly detect using DAP-qPCR analysis. Furthermore, the presence of W-box cis-elements in the close vicinity of the RMG1 hyperDMR is expected to complexify the interpretation of DAP-qPCR results as the primers flanking this region also amplify W-box sequences that are not subjected to ROS1-dependent demethylation. By contrast, the DAP-seq datasets at the RLP43 demethylated promoter region revealed robust in vitro binding of several WRKY TFs, which was convincingly detectable by DAP-qPCR analysis. Furthermore, only a single –and functional– W-box cis-regulatory element is present right in the middle of the RLP43 hyperDMR, which is devoid of W-box in its surrounding. The RLP43 promoter is therefore the most appropriate candidate to investigate the relevance of demethylation in WRKY TFs binding using our DAP-qPCR system. In the revised manuscript, we are now providing an explanation for why we made this choice. In the future, we intend to conduct DAP-seq analyses in Col-0 versus ros1 mutants, which should address reviewers’ comments at RMG1 but also at other ROS1 targets showing similar features. This approach will also determine the extent to which ROS1-directed demethylation facilitate WRKY TFs binding at the whole genome level.

The following sentence has been added in the Results section of our revised manuscript:

“The rationale for using RLP43 promoter for this specific analysis was based on (1) previous DAP-seq and ChIP-seq data showing robust binding of different WRKY TFs at the ROS1-directed demethylated RLP43 promoter (Figure 4A-C, Figure 4—figure supplement 1), (2), the presence of a single W-box cis-element located right in the middle of this genomic region (Figure 4B, C) and, (3) the central role of this W-box in the flg22-responsiveness of the RLP43 promoter (Figure 4D-F).”