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 re...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 silencingmediated 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 wildtype 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.展开更多
基金supported by grants from the Hangzhou Joint Fund of the Zhejiang Provincial Natural Science Foundation of China(LHZY24C140001)the Ministry of Science&Technology of China(Key International R&D Program 2017YFE0110900)+4 种基金the National Natural Science Foundation of China(31872636)the Ministry of Agriculture of the People’s Republic of China(National Transgenic Program of China 2016ZX08009001-004)Hangzhou Normal University(Sino-EU Plant RNA Signaling S&T Platform Initiative 2018PYPTL001)Chinese Academy of Tropical Agricultural Sciences(Central Public Interest Scientific Institution Basal Research Fund for Chinese 1630042019017)the Spanish Ministerio de Ciencia e Innovacion(BIO2017-83184-R,co-financed European Union FEDER funds).
文摘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 silencingmediated 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 wildtype 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.
