Transgene silencing system and step-wise establishment and spread of RdDM.

<p>The two-component transgene silencing system consists of a target locus (T) containing a minimal promoter (dotted box) fused to an upstream enhancer (black box), which drives the expression of a <i>GFP</i> reporter gene in shoot and root apical meristem regions (SAM and RAM, respectively) (white arrows, image T). <i>GFP</i> expression in the hypocotyl region is also seen at this stage of development (yellow arrow, image T). The silencer locus (S) contains an inverted repeat of upstream enhancer sequences under the control of the cauliflower mosaic virus 35S promoter. The resulting hairpin RNA (blue) is processed by DCL3 into 24-nt primary siRNAs (blue dashes), which induce methylation of the target enhancer and silencing of the <i>GFP</i> gene (image T+S). In a current hypothetical model, the presence of primary methylation provokes a polymerase switch such that Pol IV replaces Pol II in transcribing a non-coding ‘nascent’ RNA (black arrow) that initiates in the target enhancer region (T+S) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025730#pone.0025730-Daxinger1" target="_blank">[13]</a>. The nascent Pol IV transcript is cleaved by an AGO protein (most likely AGO4; this work), producing RNA fragments that provide substrates for RDR2. The resulting dsRNA is processed by DCL3 into 24-nt secondary siRNAs (red dashes), which foster spreading of methylation into the downstream region (shaded gray box; red m). Primary (blue) and secondary (red) RdDM both require the action of the Pol V complex and the <i>de novo</i> methyltransferase DRM2, as well as either AGO6 in RAM and SAM regions (this study) or the redundant action of AGO4 and AGO6 in leaves <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025730#pone.0025730-Daxinger1" target="_blank">[13]</a>.