Intracellular RNA signalling in RNAi and its role in antiviral defence

RNA silencing (also known as RNA interference, RNAi) is a conserved and common cellular regulatory and defence mechanism in plants, animals, fungi and other eukaryotes. It plays an important role in regulating growth and development and maintaining genomic stability in response to biotic and abiotic stresses. RNA silencing operares in two forms, transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). It is an important mechanism in a variety of antiviral defence mechanisms in plants. RNAi is involved in mobile RNA signalling and various genetic factors. Although studies on how RNAi is transported from cells to systems are controversial, there are some indications that intracellular RNAi and RNA are transported between organelles. For example, there have been clear reports that intracellular RNA transport between the cytoplasm and chloroplasts may occur. However, how RNAi spreads from the cytoplasm to chloroplasts, mitochondria, and other organelles in plant cells and its biological relevance to plant antiviral defence remains to be investigated. On the other hand, intracellular RNA signalling in RNAi and the spread of RNAi within plant cells may be a new mode of gene regulation and defence mechanism that regulates the expression of organelle genes through small RNAs derived from nuclear genes. In plants, the combined function of DCL2 and DCL3 is essential for their concerted defence, while DCL4 is required for the nucleus replication of viral infectors. However, how RNA silencing targets and counteracts chloroplast replicating viruses remains largely unknown.
In this project, we transformed infectious RNA clones of ELVd into binary plasmids using Eggplant latent viroid (ELVd), a chloroplast replication viroid and a set of transgenic Nicotiana benthamiana (N. benthamiana) RNAi lines RDR6i and DCLi and initiated ELVd replication in wild-type and transgenic N. benthamiana plants using Agrobacterium infiltration. N. benthamiana knocking out various DCLs or RDR6 in infiltrated leaves, combined with high-throughput small RNA sequence analysis, I examined the effect of ELVd infection on microRNA and sRNA biogenesis of chloroplast-derived small RNAs in N. benthamiana leaves and the effect of RNA silencing on ELVd siRNA. The role of DCLs and RDR6 on the dynamic RNA silencing-mediated response to ELVd infection is revealed and extensive experimental data are reported. DCL1i, DCL2i, DCL3i and DCL4i enhanced ELVd RNA levels, but RDR6i had little effect on ELVd RNA accumulation in the early and late infection stages. DCL2 had little effect on the generation of 22nt ELVd siRNA. DCL3 is functionally redundant with DCL2 and targets ELVd RNA for 24nt chloroplast virus siRNA (cvd-siRNA) biogenesis. DCL4 is involved in the production of the less abundant 21nt cvd-siRNA. DCL1 and RDR6 did not contribute to the biogenesis of cvd-siRNA. However, ELVd infection reduced the microRNA processed by DCL1 in both wild-type and RNAi plants, suggesting that DCL1 may be indirectly involved in protecting plants from ELVd attack. These results also demonstrate that different processing processes may be responsible for the production of chloroplast small RNAs (csRNAs) in chloroplasts. csRNAs dynamics during ELVd infection suggest that csRNAs may be biologically relevant to chloroplast virus-host plant interactions.