Investigating locus-specific effects on transgene silencing and DNA methylation in Arabidopsis thaliana

The repression of transgene expression by RNA silencing can affect the development of genetically modified plants for both research and agronomical purposes. It remains unclear why some transgenes when introduced into plants undergo silencing whereas other identical transgenes do not. Additionally, some transgenes when silenced can support DNA methylation whereas others cannot. One possibility for this variation is that there are locus-specific effects influencing a transgene’s potential to undergo RNA silencing and/or DNA methylation. These locus-specific effects may be a result of either the site of transgene integration or be due to the properties of the transgene locus itself. This research has aimed to address these questions by generating and characterizing multiple independent single locus GFP transgenic Arabidopsis lines that have been triggered to undergo silencing by crossing with an amplicon trigger line (AMP243). The ability of these lines to undergo RDR6-dependent Post Transcriptional Gene Silencing (PTGS) and/or RNA directed DNA Methylation (RdDM) has been investigated in an attempt to correlate characteristics of the transgene in each independent line with RNA silencing outcomes. Additionally, epitope-tagged versions of RDR2 and RDR6 have been generated in order to investigate their potential recruitment to target loci. The data presented in this body of work supports there being no locus-specific effect on RNA silencing. For example, amplicon-triggered RDR6-dependent PTGS occurred in all the independent lines suggesting that RDR6 activity is not influenced by either genome location or locus structure. Despite the production of high levels of small interfering RNAs, not all lines supported detectable levels of RdDM in the F1 of the crosses with the trigger line. However, the results presented here clearly demonstrate that the levels of RdDM increase over a generation. Additionally, Chromatin Immunoprecipitation experiments have revealed that amplicon-triggered RdDM was not associated with changes in the histone marks H3Ac or H3K9me. This suggests that this non-canonical pathway of DNA methylation may not be linked with changes in histone modification, although testing will be required in order to strengthen this conclusion.