Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

Abstract

ORCID ID: 0000-0001-9582-3533 (R.K.S.)The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here, we revealed two broad mechanisms that can initiate silencing independently: identity-based and expression-dependent silencing. We found that identity-based silencing is targeted by 21-to 22-nucleotide or 24-nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome. By transforming exogenous TEs into Arabidopsis thaliana, we circumvented identity-based silencing, allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We found that several siRNAgenerating mechanisms all trigger de novo expression-dependent RNA-directed DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed by introgression into the wild type does not result in the same level of silencing as direct transformation into the wild type. This demonstrates that the plant genotype during a narrow window of time at TE insertion (or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing.

Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons

“…The ATCOPIA93-LTR::GUS fusion was consistently found to be unmethylated in various transgenic lines in the wild-type Col-0 background, concordant with LTR::GUS reactivation in response to PtoΔ28E. This lack of de novo methylation upon transformation may be explained by weak LTR transcriptional activity in untreated plants, thus preventing expression-dependent RNA-directed DNA methylation (Fultz & Slotkin, 2017) and/or by low levels of CHH methylation/siRNAs at ATCOPIA93 (Fig 1A; Mirouze Marí-Ordóñ ez et al, 2013), thus preventing identity-based silencing in trans (Fultz & Slotkin, 2017). Similarly, in wild-type plants, the ATCOPIA93-soloLTRs appear usually to be constitutively unmethylated ( Fig EV5A), in particular the soloLTR-5 described in detail here.…”

Constant conflict between Gypsy LTR retrotransposons and CHH methylation within a stress‐adapted mangrove genome

“…4d). Experimental data have demonstrated that silencing of active TEs occurs quickly, within a few host generations (Teixeira et al, 2009;Mari-Ordonez et al, 2013;Fultz & Slotkin, 2017). It is thus very likely that old Gypsy elements have been silenced at least once and then reactivated more recently.…”

Arabidopsis RNA Polymerase IV generates 21-22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes

“…Therefore, Pol IV's overall function in plant biology is to generate the siRNAs necessary to reinforce heterochromatic marks and maintain euchromatin/heterochromatin boundaries [10]. A secondary role is to generate an siRNA defense against any new or active TEs that share sequence homology [11].…”

“…Therefore, Pol IV's overall function in plant biology is to generate the siRNAs necessary to reinforce heterochromatic marks and maintain euchromatin/heterochromatin boundaries [10]. A secondary role is to generate an siRNA defense against any new or active TEs that share sequence homology [11].Pollen is the male gametophytic generation of flowering plants, and contains two sperm cell gametes encapsulated in a larger vegetative cell, which directs the delivery of the sperm cells upon pollination. Pollen has a known broad activation of TE expression resulting in steadystate TE mRNAs, which occurs from the nucleus of the pollen vegetative cell [12,13].…”