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Publishable summary

RNA silencing and jasmonate (JA) signalling are key pathways that regulate plant development and defence against pests and disease. Evidence indicates that these pathways interact with one-another and, moreover, with multiple other hormone signalling pathways. However, the full extent and nature of the interactions are unknown. An accurate understanding of these interactions is crucial to inform crop modification strategies that improve plant defence and growth. RNA silencing targets specific genes using small (s)RNAs. It controls expression of these genes through processes such as DNA methylation, which affects binding of transcription factors (TFs) to promoters, and mRNA degradation. We hypothesised that hormone signalling may influence DNA methylation via RNA silencing; JA signalling is inhibited by disruption of the RNA silencing pathway. In this project, we are conducting a targeted dissection of hormone signalling pathway interactions and examining potential roles of DNA methylation in regulating TF activity. In the outgoing host laboratory, we have been investigating dynamic changes in DNA methylation at a genome-wide scale. We have also been studying the in planta regulatory targets of tens of TFs that regulate both RNA silencing and JA signalling, by chromatin immunoprecipitation sequencing (ChIP-Seq).

- Investigate whether regulation of RNA silencing by key TFs is also a mechanism by which JA signalling is regulated.
- Investigate the roles of known RNA silencing components in regulating JA signalling.
- Examine the potential role of epigenetic gene silencing in regulation of JA responses by characterizing 
changes in methylation of the A. thaliana genome in response to JA.
- Investigate changes in target sequence binding of known JA-signalling regulatory TFs, when subjected to a 
JA stimulus.
- Examine the feedback into RNA silencing by JA signalling, by identifying novel JA-responsive miRNAs 
and siRNAs, and their targets.

Key results
We generated tens of Arabidopsis TF-epitope fusion lines using recombineering methodology to achieve native expression responses and patterns and responses. A core ChIP-Seq protocol was optimised and found to be most successful using the GFP tag in comparisons using numerous antibodies. We subsequently identified thousands of high-confidence in planta target genes for three of the four EDF family members. Amongst these there was an over-representation of genes involved in various hormone signalling pathways, suggesting that our TF family regulates or is involved in multiple hormone responses. Moreover, the data indicated that there are both unique and shared target genes amongst the EDF TFs. Analysis of TF knockout lines by RNA-Seq yielded two important results. Firstly, the overlap between genes differentially expressed in wild-type versus knockout seedlings and direct binding targets (identified by ChIP-Seq) was very high (35% of transcripts, p<1x10-10), indicating that the ChIP-Seq was successful. Secondly, 85% of differentially regulated transcripts were down-regulated, indicating that these TFs are likely transcriptional repressors, in concordance with in vitro data. These data were integrated with published ChIP-Seq from EIN3, the master-regulatory TF of ET responses, and a time series ET response transcriptome. This was achieved using a dynamic time series modelling approach and revealed contracting roles of the primary (EIN3) and secondary (EDF) TFs.

We assessed in vivo targets of further primary hormone responsive TFs (those directly activate by a hormone stimulus) followed by the secondary TFs that these target for activation. This was achieved through a combination of in planta ChIP-Seq studies and in vitro TF binding preference assays (protein binding microarrays). The results facilitated several interesting conclusions, for example that primary TFs tend to preferentially target their cognate signalling pathway (Fig. 1). Moreover, it revealed details about the role of the EDF TFs in responses to hormones other than JA.

All of the fellow’s training objectives were completed (conduct and analysis of ChIP-Seq, sRNA-Seq, RNA-Seq, methylC-Seq). As part of this training, the he was able to collaborate with other lab members in a high impact publication (Schmitz et al., 2011. Transgenerational epigenetic instability is a source of novel methylation variants. Science, 334, 369–373). During the course of the project studies in the host laboratories indicated that hormone-responsive changes in DNA methylation might occur in a cell specific manner. As a result analysing such changes using the whole plant approach we previously proposed would have been unsuccessful. Consequently, we initiated a collaboration to investigate dynamic intracellular signalling that regulates DNA methylation with the potential to influence TF activity, which might be more readily detected by our approach. This collaboration is in keeping with our initial proposal to investigate the interaction of TF activity and dynamic DNA methylation, and has yielded exciting results. A publication is prepared and ready for submission.

Main results and impact
Our studies have already resulted in three high-impact publications (Schmitz et al., 2011. Science, 334, 369–373; Schweizer et al., 2013. The Plant Cell, DOI: 10.1105/tpc.113.115139; Weirauch, et al., 2014. Cell, DOI: 10.1016/j.cell.2014.08.009) and have been presented at many national and international meetings. Two further publications are in preparation. The fellow also trained and established collaborations with members of the return host laboratory, which enabled effective knowledge transfer. Several downstream studies based upon our results are currently being conducted in both outgoing and return host laboratories. Moreover, we have established several national and international collaborations with external laboratories (US, UK, Canada, Australia) to provide resources and expertise that enables other groups’ studies. In sum these investigations will give an extensive, network-oriented view of transcriptional regulation of hormone responses and the interface of RNA silencing and hormone signalling. This information will greatly aid crop modification and optimization strategies aimed at tackling the existing food crisis. The results will have broad-reaching implications for the understanding of how plants co-ordinate multiple defensive and developmental programs, and will be made publically available by peer-reviewed publication and release of an associated website.

(See also attached figures)