The employment of this simple module gives rise to repression that can be relieved by degradation or arrest of biogenesis of the miRNA. In plants, single-round amplification produces a diagnostic phased pattern of siRNA accumulation resulting from consecutive dicing of dsRNA with a well-defined end produced by RISC-catalyzed slicing 9β Regardless of the exact mechanism of secondary small RNA biogenesis, the decision to employ small RNA amplification represents a major checkpoint of genetic control because it determines the resulting type of regulation.
In plants, three fundamentally different outcomes are possible depending on whether and how small RNA amplification is employed. First, in the total absence of amplification, silencing is reversible and dependent on the continued production of the primary small RNA species. In addition, in the case of miRNAs, the regulation is typically cell-autonomous or restricted to spreading over a few cells by cell-to-cell movement 12β15 , although important examples of miRNA action over long distances via phloem transport have also been reported 16 , Second, if a single round of secondary siRNAs is produced, gene regulation remains reversible and dependent on the primary trigger 18 , but the regulatory siRNAs move efficiently from cell to cell This phenomenon has profound biological ramifications as it may result in formation of concentration gradients and consequent action of siRNAs as morphogens during development 19 , 20 , and in vesicle-mediated siRNA-transfer into fungal and oomycete pathogens as part of immune responses 21 , The typical example of such small RNAs resulting from single amplification rounds is trans- acting siRNAs tasiRNAs , whose accumulation pattern in phase with a miRNA-directed cleavage site is proof of single-round amplification 3 , 7 , 8.
Third, if reiterative amplification is produced, silencing is effectively irreversible as it becomes independent of the primary trigger and maintains the target silenced. Endogenous transcripts are typically subjected to the first two reversible outcomes of silencing, while the third option is used to silence foreign RNA, for example transgenic, transposon or viral RNA 23β Thus, the employment of the RdRP amplification module represents a central decision in gene regulation and is tightly linked to the fundamental problem of distinguishing self- from non-self RNA.
Although the RdRP RDR6 was the first factor required for small RNA-guided transgene silencing to be identified in plants 23 , 24 , only few fundamental questions regarding its engagement in production of dsRNA substrates for Dicers have been answered.
On the other hand, while it is now clear that nt, but not nt, miRNAs tend to trigger RDR6-dependent, secondary siRNA production 30β32 , there is no clear explanation for why the two different size classes of miRNAs exhibit this fundamentally different behavior.
