RNA-dependent RNA polymerase 6 (RDR6) and suppressor of gene silencing 3 (SGS3) form small interfering RNA (siRNA) bodies to synthesize double-stranded RNA, triggering RNA interference. Like viral RDRs, RDR6 not only has polymerase action but also has terminal nucleotidyltransferase activity that tails single-stranded RNA templates, causing their degradation. How the dual activities are controlled is unknown. Here we discovered that Arabidopsis Tousled (TSL) predominantly phosphorylates Ser65 and Ser68, located in the intrinsically disordered region of SGS3, to promote RNA interference. SGS3 phosphomimics trigger the formation of liquid-like siRNA bodies but have lower RNA-binding affinity, thereby enhancing RDR6 polymerase activity to synthesize double-stranded RNA for downstream processing by Dicer-like 2/4. Conversely, hypo-phosphorylated SGS3 forms larger and solid-like aggregates that hold RNA templates with stronger affinity, thereby prompting RDR6 terminal nucleotidyltransferase activity. Moreover, viral infection drives SGS3 hypo-phosphorylation and aggregation, suppressing viral siRNA production and host defence. Collectively, stoichiometric phosphorylation of SGS3 switches two distinct activities of RDR6 to tune RNA silencing and host anti-viral immunity. TSL phosphorylation of SGS3 boosts RDR6’s polymerase activity, promoting RNAi. Conversely, hypo-phosphorylation of SGS3 enhances RDR6’s terminal nucleotidyltransferase activity on RNA templates, weakening RNAi. This is a previously uncharacterized host–virus battlefront.
RNA silencing plays a central role in plant antiviral defence, in which viral RNA is processed into small RNAs (sRNAs) that guide Argonaute-mediated cleavage of viral transcripts. Among these, 22-nucleotide (nt) sRNAs are unique in their ability to trigger secondary sRNA production, thereby amplifying transitive silencing. In tomato, this 22-nt antiviral pathway is strongly activated upon virus infection through the upregulation of DCL2b, yet the underlying mechanism has remained unclear. miR6026, generated by DCL2b and targeting DCL2b itself, forms a feedback loop that fine-tunes this response. Here we show that tobacco mosaic virus (TMV) and tomato bushy stunt virus (TBSV) activate DCL2b transcription via the Dof16 transcription factor. Transcription of MIR6026 is also activated by both TMV and TBSV, while viral infection promotes DCL2b relocalization towards the cytoplasm, preventing pri-miR6026 processing into mature miR6026. This redistribution is associated with reduced phosphorylation of DCL2b at Tyr69, which weakens its interaction with importin-α and limits its nuclear localization. Notably, Arabidopsis DCL2 exhibits a similar virus-induced relocalization pattern, highlighting a conserved mechanism that enhances antiviral silencing during infection while minimizing off-target effects on host genes under normal conditions. The DCL2b-mediated 22-nt sRNA pathway is critical for tomato antiviral defence. Viral infection induces DCL2b transcription and promotes its cytoplasmic accumulation, enhancing antiviral activity while limiting nuclear miR6026 feedback.
