A Genetic Screen for Impaired Systemic RNAi Highlights the Crucial Role of DICER-LIKE 2

Abstract

Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and lowabundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.

A Genetic Network for Systemic RNA Silencing in Plants

“…Moreover, DCL2 is 484 required to respond to mobile signals for systemic PTGS (Fig. 9), supported by a recent 485 report of a critical role of DCL2 in systemic PTGS in Arabidopsis (Taochy et al, 2017).…”

“…9). Taochy et al (2017) 494 have elegantly shown that Arabidopsis DCL2 is required in both the source rootstock and 495 recipient shoot tissue for RDR6-dependent systemic PTGS, although this latest discovery is 496 in contrast to their previous finding (Brosnan et al, 2007). as RNAi of DCL4 or DCL3 enhances systemic PTGS in distant young leaves (Fig.…”

“…This view is indeed supported by the requirement of DCL2 and DCL2-150 dependent mobile signals for intercellular antiviral silencing in N. benthamiana (Qin et al,151 2017). A genetic screen for impaired systemic RNAi revealed that DCL2 is crucial for RDR6-152 dependent systemic PTGS (Taochy et al, 2017). Nevertheless, it is not known whether, and 153 to what extent, DCL2 and other DCLs regulate systemic silencing.…”

“…Our findings also differ from early works showing that long-distance movement of 574 transitive silencing occurs in the absence of sRNAs in the rootstock (Mallory et al, 2001), 575 and that no specific sRNAs are associated with systemic silencing in Arabidopsis (Brosnan et 576 al., 2007;Taochy et al, 2017). Considering the transitivity and sequence-specificity of RNA 577 silencing along with the fact that various types of RNA molecules can trigger cell-578 autonomous silencing, we concede that various forms of transportable RNAs, if they can be 579 perceived and processed by DCL2 or a DCL2-dependent pathway in recipient cells, could 580 function as systemic silencing signals (Fig.…”

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

“…The DCL2 protein processes double-stranded RNA (dsRNA) molecules of exogenous origin and natural antisense small interfering RNAs (siRNAs) into 22-nt primary siRNAs (Borsani et al, 2005;Xie et al, 2004). DCL2 is mainly involved in antiviral defence; however, there is mounting evidence that its role is masked by DCL4 activity, as it has been clearly demonstrated that DCL2 is also involved in transitivity and the RNA decoy mechanism (Mlotshwa et al, 2008;Parent et al, 2015;Qin et al, 2017;Taochy et al, 2017;Zhang et al, 2015).…”