RNA silencing response in chloroplast-replicating viroid siRNA biogenesis in plants

RNA silencing represents a cellular regulatory and defence mechanism in eukaryotes across kingdoms. In plants, a combined functionality of DCL2 and DCL3 is crucial for their synergistic defence against whilst DCL4 is required for nucleus-replicating viroid. However, how RNA silencing targets and fights against chloroplast-replicating viroid remains unknown. Here, utilizing eggplant latent viroid (ELVd), a chloroplast replicating viroid and a suite of transgenic Nicotiana benthamiana RNAi lines RDR6i and DCLsi , we reveal that DCLs and RDR6 partake a dynamic RNA silencing-mediated response to ELVd infection. DCL1i , DCL2i , DCL3i , and DCL4i enhance, whilst RDR6i seems to have little impact on, ELVd accumulation. Through small RNA profiling, we unravel that DCL2 plays an essential role in generating 22 nt ELVd siRNA. DCL3 is functionally redundant to DCL2 and targets ELVd for biogenesis of 24 nt chloroplastic viroid siRNA (cvd-siRNA) in the absence of DCL2 . DCL4 accounts for the less abundant 21 nt cvd-siRNA production. DCL1 does not contribute to the cvd-siRNA biogenesis. However, ELVd infection reduces DCL1-processed miRNAs in both wild-type and RNAi plants, suggesting that DCL1 might indirectly participate in protection of plants from ELVd attack. RDR6 imposes no influence on the 21–24 nt size profile of cvd-siRNA, but affects cvd-siRNA abundance at the late stage of ELVd infection. Our results also demonstrate that a different processing might be responsible to produce chloroplastic small RNAs (csRNAs) in chloroplasts. The dynamic changes of csRNAs during ELVd infection suggest that csRNAs might be of biological relevance to chloroplastic viroid-host plant interactions.