Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins.
In plants, worms, and fungi, RNA-dependent RNA polymerases (RDRs) amplify the production of short-interfering RNAs (siRNAs) that mediate RNA silencing. In Arabidopsis, RDR6 is thought to copy endogenous and exogenous RNA templates into double-stranded RNAs (dsRNAs), which are subsequently processed into siRNAs by one or several of the four Dicer-like enzymes (DCL1-->4). This reaction produces secondary siRNAs corresponding to sequences outside the primary targeted regions of a transcript, a phenomenon called transitivity. One recognized role of RDR6 is to strengthen the RNA silencing response mounted by plants against viruses. Accordingly, suppressor proteins deployed by viruses inhibit this defense. However, interactions between silencing suppressors and RDR6 have not yet been documented. Additionally, the mechanism underlying transitivity remains poorly understood. Here, we report how several viral silencing suppressors inhibit the RDR6-dependent amplification of virus-induced and transgene-induced gene silencing. Viral suppression of primary siRNA accumulation shows that transitivity can be initiated with minute amounts of DCL4-dependent 21-nucleotide (nt)-long siRNAs, whereas DCL3-dependent 24-nt siRNAs appear dispensable for this process. We further show that unidirectional (3-->5') transitivity requires the hierarchical and redundant functions of DCL4 and DCL2 acting downstream from RDR6 to produce 21- and 22-nt-long siRNAs, respectively. The 3-->5' transitive reaction is likely to be processive over >750 nt, with secondary siRNA production progressively decreasing as the reaction proceeds toward the 5'-proximal region of target transcripts. Finally, we show that target cleavage by a primary small RNA and 3-->5' transitivity can be genetically uncoupled, and we provide in vivo evidence supporting a key role for priming in this specific reaction.
Moissiard
Guillaume
G
Institut de Biologie Moléculaire des Plantes, Strasbourg Cedex, France.
Parizotto
Eneida Abreu
EA
Himber
Christophe
C
Voinnet
Olivier
O
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D010942:Plant Viruses / Q000737:chemistry; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D054334:Viral Regulatory and Accessory Proteins; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; C504982:DCL4 protein, Arabidopsis; D012260:Ribonucleases; C511919:DCL2 protein, Arabidopsis; D043244:Ribonuclease III