PTGS is dispensable for the initiation of epigenetic silencing of an active transposon in Arabidopsis

Transposable elements (TEs) are largely repressed in plants through transcriptional gene silencing (TGS), which is maintained by heritable epigenetic silencing marks such as DNA methylation. However, the mechanisms by which silencing is installed in the first place remains poorly understood in plants. Small interfering (si)RNAs and post-transcriptional gene silencing (PTGS) play a role in the initial response by reducing mRNA and protein levels of active TEs and are believed to mediate the initiation of TGS by guiding the first deposition of DNA methylation. To determine how this silencing installation works, we took advantage of EVADE (EVD), an endogenous retroelement in Arabidopsis, which can be used to recapitulate true de novo silencing with a well-established sequence of PTGS followed by a TGS phase. To test whether PTGS is a prerequisite for TGS, active EVD copies were introduced into RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) mutants lacking an essential PTGS component. EVD activity and silencing were monitored across several generations. Unexpectedly, even in the absence of PTGS, TGS and silencing of EVD were still achieved through installation of RNA-directed DNA methylation (RdDM) at EVD regulatory sequences without any prior DNA methylation at its coding sequence. Hence, our study shows that PTGS is dispensable for de novo EVD silencing. Although we cannot rule out that PTGS might facilitate the initiation of TGS, or control TE activity until then, initiation of epigenetic silencing can take place in its absence.