Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5’RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5’RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5’RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection.Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in  Nicotiana benthamiana plants. 5’RDGs can inhibit the accumulation of exogenous gene transcripts or viral RNA via the RNA decay pathway when RNA silencing is compromised. Therefore, RNA silencing and RNA decay are important defense mechanisms that coordinate to function against exogenous gene transcripts or viral RNA. It is well known that viruses have evolved to encode viral suppressors of RNA silencing (VSRs) that counteract RNA silencing for an invading virus to establish infection. We discover that two known VSRs of TuMV interact with 5’RDGs and the interactions subvert RNA decay. Taken together, this study provides new insights into the concerted function of RNA silencing and RNA decay in plant immunity against exogenous gene transcripts and viral RNA, and reveals a novel function of VSRs.RNA silencing and RNA decay are two essential pathways that determine the fate of cellular RNA molecules. RNA decay has been suggested to compete with RNA silencing for the RNA substrates and thus to suppress RNA silencing in plants. In this report, we show that the deficiency of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) rather than overexpression of them attenuates RNA silencing and facilitates viral RNA accumulation in turnip mosaic virus (TuMV)-infected  Among major infectious pathogens, viruses are the obligate intracellular agents that can infect all types of life forms from bacteria to plants and animals, and exclusively multiply in the host cells. The vast majority of known plant viruses are positive-sense single-stranded (+ss) RNA viruses that typically have a relatively small genome encoding no more than a dozen proteins. Since the genomic RNAs of +ss viruses are similar to the endogenous mRNAs, the regulation machinery of cellular RNA metabolism in the infected plants is unavoidably involved in viral infection.de novo synthesis by the endogenous RNA-dependent RNA polymerases (RdRps) [An essential RNA regulation mechanism is RNA silencing, which is an evolutionarily conserved and sequence-specific immunity response and triggered by double-stranded RNAs (dsRNAs) –3. In pl (RdRps) . These d (RdRps) ,5. As a Arabidopsis thaliana, DCP1, DCP2, DCP5, VARICOSE (VCS) and possibly DEA(D/H)-box RNA helicase 1 (DHH1) constitute the decapping complex [In addition to RNA silencing, RNA decay is another crucial pathway that regulates RNA turnover ,7. RNA d complex . Deadeny complex ,7. Arabi complex ,12. AtXR complex , whereasSaccharomyces cerevisiae single-gene deletion library and subsequent work revealed that XRN1p is involved in the degradation of tomato bushy stunt virus (TBSV) RNA in yeast [NbXRN4 in Nicotiana benthamiana, homologous to XRN1 in yeast, promotes viral RNA accumulation of tobacco mosaic virus (TMV) and TBSV [dcp2 mutant plants infected by a recombinant tobacco rattle virus (TRV) carrying the green fluorescence protein gene (GFP) is significantly higher than that in wild type plants [AtXRN4 from Arabidopsis and OsXRN4 from Oryza sativa in N. benthamiana suppresses cucumber necrosis virus (CNV) and TMV infection, respectively [Accumulated evidence suggests that RNA decay is also a major cellular antiviral mechanism . Since vin yeast ,15. Consand TBSV ,17. The e plants . In contectively . Then, hectively . For insectively –22. In tectively ,24.N. benthamiana as a model system to investigate the possible involvement of several essential cytoplasmic 5’–3’ RNA decay pathway genes (5’RDGs) in RNA silencing and RNA decay in virally infected plant cells. We found that knockdown of 5’RDGs but not overexpression of them suppresses PTGS induced by sense transcripts, and RNA silencing, compared to RNA decay, plays a predominant role in the degradation of foreign transcripts . VSRs could hijack the key components from these pathways to suppress plant defense. These data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and cooperative actions against foreign transcripts, and VSRs not only supress RNA silencing but also RNA decay to promote viral infection in plants.Despite recent progress in understanding the antiviral role of RNA silencing and RNA decay, many fundamental questions are yet to be unanswered. For instance, it is not clear if these two pathways are interlinked to operate against viral infection and whether they function simultaneously, sequentially or preferentially. The molecular mechanism underlying virus-mediated suppression of RNA decay remains poorly understood. This is particularly true in virus-plant interactions. In this study, we used turnip mosaic virus (TuMV) and 5’RDGs from N. benthamiana, including NbDCP1, NbDCP2, NbXRN4 and NbPARN. Sequence analysis revealed that the open reading frames (ORFs) of NbDCP1, NbDCP2, NbXRN4 and NbPARN contain 1113 nt (GenBank accession number: KY402210), 966 nt (GenBank accession number: KY402211), 2949 nt (GenBank accession number: KY402212), and 2056 nt (GenBank accession number: KY402213), respectively. The deduced amino acid sequences of NbDCP1, NbDCP2, NbXRN4 and NbPARN share 70.2%, 68.4%, 67.1% and 62% identity with their counterparts AtDCP1, AtDCP2, AtXRN4 and AtPARN from Arabidopsis, respectively. To document basic biology features of these 5’RDGs from N. benthamiana, we determined the expression pattern of these genes and subcellular localization of their encoded proteins. Quantitative real-time reverse-transcription PCR (qRT-PCR) was performed using total RNA isolated from different N. benthamiana tissues as template. All the four genes were constitutively expressed but their expression levels varied at different tissues . Overall, all the four genes showed the highest expression level in the root tissue, and the lowest in the stem tissue.We initiated this study by cloning and sequencing of four key N. benthamiana 5’RDGs, their coding regions were firstly introduced into pDNOR221 vector by BP reaction (Invitrogen), followed by recombination into in frame downstream of the coding sequence of yellow fluorescence protein (YFP) by LR reaction (Invitrogen). The chimeric genes were transiently expressed in leaves of transgenic H2B-RFP N. benthamiana plants, and fluorescence was examined in agroinfiltrated transgenic leaves at 32 hours post infiltration (hpi) by confocal microscopy . Western blotting using GFP (WB:GFP) antibody  detected the expected and specific band corresponding to the YFP-tagged NbDCP1, NbDCP2, NbXRN4 and NbPARN protein, confirming the presence of full-length recombinant proteins.To examine the subcellular localization of these four N. benthamiana plants and analyzed by live cell fluorescence microscopy at 32 hpi . Self as well as all mutual combinations of NbDCP1, NbDCP2, and XRN4 showed positive interactions. P3N-PIPO, which is a dedicated movement protein of TuMV [Fig 2A). The viral proteins P3N-PIPO and CI that interact to form a complex for viral cell-to-cell movement [S1 Fig). Interestingly, the NbDCP2 self-interacting complex was present in the cytoplasm as well in the nucleus. Except the NbDCP2 self-interacting complex and the NbDCP2-N-YFP and NbXRN4-C-YFP combination, both of which were evenly distributed in the cytoplasm, the remaining combinations all formed cytoplasmic foci, regardless of reciprocal fusion with an N- or C-YFP .To determine whether NbDCP1, NbDCP2 and NbXRN4 interact with each other, we performed bimolecular fluorescence complementation (BiFC) assays . The N-  of TuMV , was fusmovement  were useN. benthamiana plants, and confocal microscopy was performed at 32 hpi. We found that NbDCP1-CFP and YFP-NbDCP2 or YFP-NbXRN4 co-localized in the cytoplasm to form the bright granules . Such bright foci were also observed in the cytoplasm of cells co-expressing NbDCP2-CFP and YFP-NbXRN4 . Taken together, these data suggest that NbDCP1, NbDCP2 and NbXRN4 can form protein complexes through protein-protein interactions.We further conducted a co-localization assay to confirm whether theses interaction complexes are formed in the same protein complexes. C-terminal cyan fluorescent protein (CFP) fused NbDCP1 (NbDCP1-CFP) and N-terminal YFP fused NbDCP2 (YFP-NbDCP2) or NbXRN4 (YFP-NbXRN4), or C-terminal CFP fused NbDCP2 (NbDCP2-CFP) and YFP-NbXRN4 were co-expressed in the leaves of H2B transgenic N. benthamiana 5’RDGs also affect S-PTGS and IR-PTGS as those in Arabidopsis, Agrobacterium cultures expressing 35S-GFP, 35S-GF  and N-Myc-tagged NbDCP1, NbDCP2, NbXRN4 or NbPARN were co-infiltrated into N. benthamiana leaves. Leaves co-infiltrated with 35S-GFP, 35S-GF and empty vector (Vec) or the vector for expression of TBSV P19 (a well-known gene silencing suppressor) serve controls. Agroinfiltration of N. benthamiana leaves with 35S-GFP, 35S-GF and Vec induced GFP RNA silencing, and led to reduced GFP fluorescence under UV light at 5 days post infiltration (dpi) . While the intensity of green fluorescence increased substantially in leaf patches co-expressing GFP and P19, no obvious differences of fluorescence were observed among leaf patches co-expressing Myc-tagged NbDCP1, NbDCP2, NbXRN4 or NbPARN or co-infiltrated with the vector control . Therefore, transient expression of the four 5’RDGs was unable to suppress the sense RNA trigger (35S-GF)-induced GFP silencing. On the contrary, expression of these 5’RDGs led to the generation of more GFP-derived siRNAs and the reduced levels of GFP mRNA and protein, enhancing sense GFP-induced RNA silencing . As a positive control, P19 suppressed S-PTGS and IR-PTGS, and intensified GFP fluorescence, which was confirmed by immunoblot and RNA blot analyses . Taken together these data demonstrate that the four N. benthamiana 5’RDGs are likely involved in GFP-induced S-PTGS, but not in IR-PTGS.Several recent studies have reported that essential components of the cytoplasmic 5’–3’ RNA decay pathway including XRN4 and DCP2 can suppress sense RNA-induced PTGS (S-PTGS), but not inverted repeat RNA-induced PTGS (IR-PTGS) in Arabidopsis, suggesting that the RNA silencing and 5’–3’ RNA decay pathways are interlinked, possibly by sharing the same RNA substrate ,29. Consrvations –30, therN. benthamiana plants. Compared to the weak GFP fluorescence in N. benthamiana leaf patches infiltrated with 35S-GFP, 35S-GF and Vec, expression of dsNbDCP1, dsNbDCP2, dsNbXRN4 or dsNbPARN led to an increase in green fluorescence in co-infiltrated regions , indicating that silencing of NbDCP1, NbDCP2, NbXRN4 or NbPARN suppressed sense GFP-induced RNA silencing. Accordingly, qRT-PCR, RNA blot and protein gel blot analyses revealed that suppression of GFP silencing by dsNbDCP1, dsNbDCP2, dsNbXRN4 or dsNbPARN was accompanied by the increased levels of GFP mRNA and protein, and the reduced levels of GFP-specific siRNAs in the infiltrated leaves . As a control, a hairpin RNAi construct of GUS failed to suppress S-PTGS , indicating that knock-down of NbDCP1, NbDCP2, NbXRN4 and NbPARN specially inhibits S-PTGS, and expression of unrelated dsRNA does not overload and inhibit the silencing machinery. When 35S-GFP and 35S-dsGF were co-expressed with dsNbDCP1, dsNbDCP2, dsNbXRN4 or dsNbPARN in N. benthamiana, no GFP fluorescence was detected in the co-infiltrated areas, similar to the Vec-infiltrated leaf patches . In contrast, strong GFP signals were evidenced in the P19 co-expressing patches. GFP-specific siRNA blots showed that in comparison with Vec, silencing of NbDCP1, NbDCP2, NbXRN4 or NbPARN drastically supressed the production of secondary siRNAs (‘P’ siRNAs) during S-PTGS, but showed no obvious changes in the accumulation of primary siRNAs (‘GF’ siRNAs) during IR-PTGS . These data support that NbDCP1, NbDCP2, NbXRN4 or NbPARN participate in GFP-induced S-PTGS, but are not involved in IR-PTGS.To further determine whether NbDCP1, NbDCP2, NbXRN4 or NbPARN is involved in RNA silencing, three expression vectors including 35S-GFP, 35S-GF and a hairpin RNAi construct containing NbDCP1 (dsNbDCP1), NbDCP2 (dsNbDCP2), NbXRN4 (dsNbXRN4) or NbPARN (dsNbPARN) sequences under the control of the cauliflower mosaic virus (CaMV) 35S promoter, were agroinfiltrated into leaves of GFP induced S-PTGS is compromised [N. benthamiana leaves. At 5 dpi, the leaf patch infiltrated with 35S-GFP, 35S-GF and Vec still showed that strong GFP fluorescence owing to the suppression of S-PTGS in dsRDR6 N. benthamiana leaves compared to the corresponding patch in wild type (Wt) N. benthamiana leaves . Expression of NbDCP1, NbDCP2, NbXRN4 or NbPARN in dsRDR6 transgenic plants reduced GFP fluorescence in comparison with the Vec control . qRT-PCR, RNA and protein gel blot analyses revealed that the reduced fluorescence in leaf patches expressing NbDCP1, NbDCP2, NbXRN4 or NbPARN was accompanied by a reduction of both GFP mRNA and protein, but without an increase of GFP-specific siRNAs . These data suggest that in RDR6-deficient plants, NbDCP1, NbDCP2, NbXRN4 or NbPARN suppresses GFP expression through the RNA decay pathway. In addition, 35S-GFP and 35S-GF were also co-infiltrated with dsNbDCP1, dsNbDCP2, dsNbXRN4 or dsNbPARN in dsRDR6 leaf patches. Consistent with our data in Fig 4, much stronger fluorescence and more GFP RNA accumulations were observed when dsNbDCP1, dsNbDCP2, dsNbXRN4 or dsNbPARN was co-expressed with 35S-GFP and 35S-dsGF compared to Vec at 7 dpi . Taken together, these data suggest that all four 5’RDGs promote RNA silencing to target GFP RNA in Wt N. benthamiana plants, and degrade GFP transcripts through RNA decay in the RDR6-deficient plants where S-PTGS is blocked. Thus, RNA silencing compared to RNA decay has the priority to degrade GFP RNA.To analyze the role of NbDCP1, NbDCP2, NbXRN4 and NbPARN in RNA decay in the absence of S-PTGS, we used dsRDR6 transgenic plants, in which sense promised . Three e5’RDGs in TuMV-infected local and systemic leaves of N. benthamiana plants at 3 and 10 dpi by using qRT-PCR . We further checked whether TuMV replication proteins or replication vesicles are associated with P-bodies. The decapping protein DCP1 is a well-established marker for P-bodies in plants [Fig 6C). Possible co-localization between 5’RDGs and viral replication proteins was also tested using two TuMV infectious clones TuMV-6K2-mCherry and TuMV-CFP-NIb [Fig 6D). These suggest that TuMV infection upregulates the RNA decay pathway which is not apparently associated with the virus replication complex.To clarify the role of RNA decay in TuMV infection, we determined the expression levels of the four  qRT-PCR . The expn plants ,33. We c-CFP-NIb ,35. The N. benthamiana 5’RDGs affect TuMV RNA accumulation, TuMV-GFP was co-infiltrated into N. benthamiana leaves with NbDCP1, NbDCP2, NbXRN4, NbPARN or an empty vector (Vec). At 3 dpi, the leaf patches infiltrated with these N. benthamiana 5’RDGs showed weaker GFP fluorescence compared to the Vec control . As GFP originated from the recombinant virus, its fluorescent intensity could be considered to be an indicator of TuMV replication. qRT-PCR analyses confirmed the reduced viral RNA levels in leaf patches co-expressing NbDCP1, NbDCP2, NbXRN4 or NbPARN . Consistently, this reduction was accompanied with an increase of TuMV-siRNAs . We also checked if NbDCP1, NbDCP2, NbXRN4 or NbPARN affects TuMV infection in dsRDR6 transgenic N. benthamiana plants . Overexpression of NbDCP1, NbDCP2, NbXRN4 or NbPARN in dsRDR6 transgenic plants inhibited TuMV infection too, evidenced by attenuated GFP fluorescence and reduced levels of viral RNAs . Interestingly, remarkable amounts of TuMV-derived siRNAs were detected in the Vec-infiltrated control sample of dsRDR6 transgenic plants, possibly due to primary silencing induced by viral dsRNA  during robust viral replication . These data demonstrate that expression of 5’RDGs inhibits TuMV RNA accumulation in both Wt and dsRDR6 transgenic plants.To examine whether these N. benthamiana, we used a replication-deficient TuMV infectious clone, TuMV-GFP-ΔGDD [N. benthamiana suppressed TuMV-GFP-ΔGDD RNA accumulation and remarkably boosted the level of TuMV-siRNAs . In dsRDR6 transgenic plants, overexpression of NbDCP1, NbDCP2, NbXRN4 or NbPARN reduced the accumulation of TuMV-GFP-ΔGDD transcripts and supressed the generation of TuMV-GFP-ΔGDD transcripts-derived siRNAs . These data suggest that NbDCP1, NbDCP2, NbXRN4 or NbPARN may degrade TuMV RNA via the RNA decay pathway when PTGS is disrupted.To examine 5’RDGs-mediated antiviral defense without dsRNA-mediated primary silencing in Wt and dsRDR6 transgenic GFP-ΔGDD . This clS4A Fig). Overexpression of NbDCP1, NbDCP2, NbXRN4 or NbPARN, compared to Vec, reduced TuMV or TuMV-ΔGDD RNA accumulations. Overexpression of these RNA decay components also resulted in an obvious decrease of TuMV-derived siRNAs . Due to infiltration with a very lower concentration of agrobacterial culture, no detective viral siRNAs were found in TuMV-ΔGDD-infiltrated leaves. These results further suggest that during viral replication, siRNAs mainly derive from viral RNA replicative intermediates, which are proportional to viral RNA accumulations, rather than from RDR6-dependent secondary siRNAs. Taken together, these data support that 5’RDGs act as plant defense against TuMV RNA accumulation.To minimize the interference of input RNA, we also conducted this assay with a very low concentration of agrobacterial cultures (OD600 = 0.05) harboring TuMV or TuMV-ΔGDD. As expected, in TuMV-infiltrated Wt or dsRDR6 transgenic plants, viral RNA accumulation was significantly higher than that in the leaves infiltrated with TuMV-ΔGDD  as a control, NbDCP1, dsNbDCP1, dsNbDCP2, dsNbXRN4 and dsNbPARN. At 4 dpi, knock-down of NbDCP1, NbDCP2, NbXRN4 or NbPARN led to the increased levels of TuMV-GFP fluorescence, GFP proteins, and TuMV RNA, and a reduced level of TuMV siRNAs . Thus, silencing of NbDCP1, NbDCP2, NbXRN4 or NbPARN promoted TuMV RNA accumulations possibly through suppression of TuMV-derived siRNA production. To verify if this also holds true for TuMV systemic infection, a modified TRV VIGS vector carrying the partial sequence of GUS (as a control), NbDCP1, NbDCP2, NbXRN4 or NbPARN was pre-inoculated into N. benthamiana to knock down NbDCP1, NbDCP2, NbXRN4 or NbPARN expression . Silenced plants were then inoculated with TuMV-GFP. At 6 dpi, GFP signals started to appear along the veins in newly developed leaves under UV lamp in TRV-GUS-treated plants . However, all NbDCP1, NbDCP2, NbXRN4 or NbPARN-silenced plants developed much stronger GFP signals in the corresponding leaves . Consistently, higher levels of viral RNAs and GFP proteins were found in the 5’RDGs-silenced plants . It was also evidenced that silencing of 5’RDGs inhibited TuMV-derived siRNAs . In comparison with Wt Arabidopsis plants, all mutants showed enhanced susceptibility to TuMV infection by accumulation of higher levels of viral RNA . Clearly, 5’RDGs play an anti-TuMV role in both N. benthamiana and Arabidopsis species.To further examine if these 5’RDGs also inhbit TuMV infection in Arabidopsis, we obtained Arabidopsis knockdown mutants of Fig 9A and 9B, regardless of whether AD or BD fusions were used for the assay, positive interactions were only found between NbDCP2 and VPg, and between NbXRN4 and HC-Pro.To screen for possible protein-protein interactions between the four 5’RDGs and 11 TuMV proteins, we conducted yeast two-hybrid (Y2H) assays. All the tested proteins were fused with the GAL4 transcription activation domain (AD) and the GAL4 DNA binding domain (BD). As summarized in N. benthamiana expressing H2B-RFP as a nuclear marker . The NbDCP2 and VPg interaction was detected in the nucleus . Consistently, NbDCP2 interacted with NbDCP1 (serving as a positive control) and formed bright granules in the cytoplasm, and no interaction was found between NbDCP1 and VPg . The fact that NbDCP2 interacts with VPg in the nucleus, and with NbDCP1 in the cytoplasm to form the decapping complex that is required for 5’–3’ RNA decay [Fig 10A), suggesting that VPg indeed disrupts the NbDCP1 and NbDCP2 interaction.The interaction between NbDCP2 and VPg was further confirmed by BiFC in transgenic NA decay ,33 prompN. benthamiana leaves were co-infiltrated to transiently co-express VPg-YFP and NbDCP1-CFP or NbDCP2-CFP. No co-localization was observed for VPg-YFP and NbDCP1-CFP, whereas VPg-YFP co-localized with NbDCP2-CFP in the nucleus ( two top rows). Moreover, co-expression of VPg remarkably suppressed the formation of the NbDCP1/NbDCP2 granules in the cytoplasm, in comparison with the vector control . Subsequently, we considered the possibility that the VPg-NbDCP2 interaction might negatively regulate the distribution of NbDCP2 in the cytoplasm to inhibit the formation of NbDCP1/NbDCP2 cytoplasmic granules. To test this assumption, we extracted the cytoplasmic and nuclear proteins separately, and performed Western blot analysis to determine the accumulation of NbDCP2-CFP in the cytoplasm and nuclear. Indeed, when Myc-VPg was co-expressed, the amount of NbDCP2-CFP obviously decreased in the cytoplasm but remarkably increased in the nucleus . These data suggest that TuMV VPg interferes with the interaction between NbDCP1 and NbDCP2 by targeting NbDCP2 from the cytoplasm to nucleus.We further observed the subcellular co-localization of VPg, NbDCP1 and NbDCP2. N. benthamiana. Expression vectors YN-HC-Pro, YC-HC-Pro, YN-NbXRN4 and YC-NbXRN4 were generated to express HC-Pro or NbXRN4 fusions with YN or YC, respectively. Pairwise expression of YN-HC-Pro and YC-NbXRN4 or YN-NbXRN4 and YC-HC-Pro by agroinfiltration resulted in strong YFP fluorescence signals in the cytoplasm at 32 hpi . No YFP fluorescence was detectable in the leaf sample co-expressing HC-Pro and NbDCP1, which serves as a negative control . We also examined the subcellular localization of HC-Pro and NbXRN4 in N. benthamiana leaf epidermal cells co-expressing CFP-tagged HC-Pro (HC-Pro-CFP) and YFP-tagged NbXRN4 (YFP-NbXRN4). We found that HC-Pro-CFP localized in the cytoplasm and nucleus, and some formed granules in the cytoplasm . HC-Pro-CFP co-localized with NbXRN4-CFP to form a bright dot in the cytoplasm . These results confirm that TuMV HC-Pro interacts with NbXRN4.The interaction between HC-Pro and NbXRN4 was verified by BiFC assays in Arabidopsis. At first, we confirmed that TuMV HC-Pro indeed interacts with AtXRN4, the ortholog of NbXRN4 in Arabidopsis by conducting Y2H assays in yeast  and BiFC assays in N. benthamiana . Then, we obtained an Arabidopsis xrn4 mutant and generated transgenic Arabidopsis plants expressing HC-Pro and AtXRN4. Interestingly, the phenotype of the xrn4 mutant mimicked the mild phenotype of HC-Pro transgenic Arabidopsis plants , which displayed serrated leaf edges. Since the P-body is the RNA decay site, and HC-Pro interacts with XRN4 in the cytoplasmic granules resembling the P-body , we speculated that expression of HC-Pro might suppress AtXRN4 function in RNA decay. To test this idea, three potential substrates of AtXRN4 in RNA decay including AtEBF1, AtRAP2.4 and AtNMT [xrn4 mutant Arabidopsis plants and decreased in the XRN4 overexpression transgenic plants . Overexpression of HC-Pro significantly enhanced the mRNA accumulation of AtEBF1, AtRAP2.4 and AtNMT, which was antagonized by co-overexpression of AtXRN4 . Consistently, co-overexpression of AtXRN4 partially remedied the typical phenotype induced by overexpression of HC-Pro  and the expression levels of AtEBF1, AtRAP2.4 and AtNMT were similar in HC-Pro-expressing Wt (Col-0) and xrn4 mutant plants . Moreover, in TuMV-infected plants, the level of these XRN4 substrates was elevated , possibly due to HC-Pro. Taken together, these data suggest that HC-Pro interacts with XRN4 and the interaction inhibits XRN4 activity.To assess the biological relevance of the HC-Pro and XRN4 interaction, we used the model plant nd AtNMT  were anaN. benthamiana . These data suggest that 5’RDGs seem to play an additive role to S-PTGS in N. benthamiana. Our data are consistent with a recent report that the poly(A) tail of mRNA blocks RDR6 from converting canonical mRNAs into substrates for gene silencing and AtRDR6 has an intrinsic preference for poly(A)-less mRNAs over polyadenylated mRNAs as templates in Arabidopsis [A. thaliana, and it degrades predominantly the 5’ uncapped mRNA intermediates as well 3’ mRNA intermediates resulting from miRNA and possibly siRNA-mediated cleavage [N. benthamiana may degrade RNAs incompletely, generating RNA fragments, which facilitate RDRs-dependent dsRNA synthesis. Clearly, the interplay between RNA decay and RNA silencing is very complicated. The finding from this study may represent another example that not all findings from Arabidopsis can be simply extrapolated to other plant species such as N. benthamiana. The molecular mechanism by which the RNA decay pathway functions differently in relation to S-PTGS in Arabidopsis and N. benthamiana needs further study.In this study, we found that overexpression of any of four essential RNA decay components 5’RDGs including DCP1, DCP2, XRN4 and PARN failed to suppress GFP-induced S-PTGS and production of siRNAs, and instead enhanced S-PTGS in bidopsis . Howeverbidopsis –31. For bidopsis ,39, possbidopsis –30. The bidopsis –30. AtXRcleavage ,40. MoreN. benthamiana plants [GFP expression in dsRDR6 plants, and this was not concomitant with an increment of GFP siRNAs . Therefore, cytoplasmic RNA decay pathways are involved in the deadenylated or/ and decapped GFP RNA degradation. It is well known that silencing of RDR6 enhances transgene expression such as GFP [5’RDGs in RDR6-defective plants further improves GFP expression , suggesting that RNA silencing and RNA decay may coordinate against the over-accumulation of exogenous transcripts. Taken together these data demonstrate that 5’RDGs from N. benthamiana may process GFP RNA to facilitate them into RNA silencing for degradation, and may directly degrade GFP RNA via the RNA decay pathway when S-PTGS pathway is interrupted. Thus, RNA silencing compared to RNA decay seems to play a predominant role in the degradation of GFP RNA, while both of them constitute an important defense against exogenous RNA.RDR6 is required for S-PTGS by conversion of single stranded RNA into dsRNA, and GFP-induced S-PTGS is compromised in RDR6-defective a plants . In thish as GFP ,41. In t5’RDGs in both the inoculated leaves, and systemically infected leaves of N. benthamiana plants . TuMV replication-related proteins or TuMV replication complex were not co-localized with P bodies , suggesting that these 5’RDGs probably have no access to the virus replication site.P bodies are the RNA decay sites, which have been implicated in infection by a number of RNA viruses –22,42. IN. benthamiana such as TMV, TBSV, CNV and rice stripe virus infection [GFP RNA accumulation, overexpression of the four 5”RDGs including XRN4 supressed TuMV RNA accumulation and silencing of XRN4 and the other three 5’RDGs genes promoted TuMV infection in N. benthamiana . In addition, we also found that the orthologs of these 5’RDGs in Arabidopsis had the similar antiviral function . Therefore, we conclude that RNA decay is an antiviral pathway to TuMV in both Arabidopsis and N. benthamiana.As briefly discussed in Introduction, the cytoplasmic XRN4 inhibits infections by several RNA viruses in nfection –17,19. WFig 9). Moreover, we found that the VPg/NbDCP2 interaction disrupted the formation of the NbDCP1/NbDCP2 complex possibly through targeting the cytoplasmic NbDCP2 to the nucleus . Since the DCP1/DCP2 complex is essential for RNA decay, the VPg/NbDCP2 interaction would affect RNA decay-mediated TuMV RNA degradation. It is worth to mention that VPg is the virus-encoded protein that is covalently linked to the 5' end of the viral genome. Therefore, targeting DCP2 to the nucleus may inhibit the interaction of DCP2 with the genome-linked VPg in the cytoplasm, which can further supresses viral RNA degradation by RNA decay.To understand if and how TuMV counteracts the RNA decay machinery for viral infection, we screened possible interactions between 5’RDGs and TuMV encoding proteins. We found that NbDCP2 interacted with VPg, and NbXRN4 interacted with HC-Pro . Arabidopsis xrn4 mutant plants displayed serrated leaf edges, a developmental defect similar to HC-Pro transgenic plants, and overexpression of AtXRN4 could restore developmental defects in the HC-Pro plants , suggesting that the HC-Pro/AtXRN4 interaction may also contribute to the developmental defects through disruption of AtXRN4 activity to host gene expression. This assumption was further supported by the observation that overexpression of HC-Pro increased the level of XRN4 substrates likely via inhibition of XRN4 activity, mimicking the xrn4 mutant, and by the finding that ectopic overexpression of AtXRN4 mitigated the HC-Pro-induced phenotype and decreased the level of XRN4 substrates . Taken these data together, HC-Pro, as a highly efficient VSR of potyviruses, not only suppresses the PTGS and miRNA pathways, but also interferes with the XRN4-mediated 5’–3’ RNA decay pathway.Transgenic Arabidopsis plants expressing P1/HC-Pro, a potyvirus-encoded silencing suppressor, causes severe developmental anomalies such as stunting and serrated leaf edges. Initially, defects in both siRNA and microRNA (miRNA) pathways were through to account for the phenotype –45. SubsFig 12). In brief, TuMV genomic RNA is deadenylated, decapped (interfering with or removing VPg) and degraded by XRN4-mediated 5’–3’ decay or exosome-mediated 3’–5’ degradation. Deadenylated, decapped, or XRN4-partially degraded RNAs facilitate RDR6 to transform ssRNA into dsRNA that triggers the PTGS pathway. Typical potyviruses encode two known VSRs: HC-Pro and VPg. HC-Pro interacts with XRN4 to inhibit its slicing activity. Here, HC-Pro as a major virus-encoded suppressor of RNA silencing is not included for discussion, which has been discussed in several reviews [Based on the above discussion, we propose a model that in addition to RNA silencing, the RNA decay pathway is another important component of antiviral immunity and viral VSRs function to counteract RNA decay , and Michael M. Goodin , respectively. Arabidopsis plants were grown under the similar conditions described above for N. benthamiana. The Arabidopsis mutants: dcp1 (SALK_014408C), dcp2 (SALK_00519), xrn4 (SALK_014209) and parn  were obtained from the Arabidopsis Biological Resource Center (ABRC) at the Ohio State University. All mutants were confirmed by PCR essentially as previously described [escribed . AtXRN4 escribed  and screAtDCP1, AtDCP2, AtXRN4 or AtPARN and N. benthamiana NbDCP1, NbDCP2, NbXRN4 or NbPARN were retried from public domains and obtained by RT-PCR. The amplified fragments were subcloned into pDONR221 entry vector and confirmed by DNA sequencing, and then transferred into different gateway-compatible vectors, including pGADT7-DEST (AD), pGBKT7-DEST (BD) [NbDCP1 separated by an Arabidopsis intron was produced by overlapping PCR. A fragment of the NbDCP1 sense sequence was amplified using primers A-NbDCP1-F and A-Intron+NbDCP1-R, and primers B-NbDCP1+Intron-F and B-Intron-BamHI-R. The overlapping products were cloned into pCHF3 between the SacI and BamHI sites to produce pCHF3-35S:NbDCP1-intron. The corresponding antisense NbDCP1 fragment was amplified using primers C-NbDCP1-F and C-NbDCP1-R and subsequently cloned into pCHF3-35S:NbDCP1-intron between the BamHI and SalI sites to produce the RNAi construct pCHF3-35S:NbDCP1. The similar strategy was used to generate the RNAi constructs of GUS, NbDCP1, NbCP2, NbXRN4 and NbPARN. A partial fragment of NbDCP1, NbDCP2, NbXRN4 or NbPARN was cloned into pTRV2 vector [NbDCP1, NbDCP2, NbXRN4 or NbPARN.Full-length coding sequences of Arabidopsis EST (BD) , pEarleyEST (BD) , pBA-FLAEST (BD) , pEarleyEST (BD) , to geneEST (BD) ,54,55. A2 vector  to constS1 Table. pCHF3:P19 and PTGS suppression assays were described previously [The coding sequence of full-length (1–711 bp), or N-terminal (1–340 bp) of mgfp5-ER was cloned into pCHF3 to generate pCHF3-35S-GFP or pCHF3-35S-GF. The antisense fragment of mgfp5-ER  was cloned into pCHF3-35S-GFP to generate pCHF3-35S-dsGF. The detailed primers and restriction endonuclease sites were given in eviously ,57,58.GenBank accession numbers for the genes analyzed in this study are as follows: NbDCP1 (KY402210), NbDCP2 (KY402211), NbXRN4 (KY402212), NbPARN (KY402213), AtDCP1 (NM_113710), AtDCP2 (NM_131203375), AtXRN4 (AF286718) and AtPARN (AB223028).Agrobacterium-mediated transient expression assays and viral infections in Wt N. benthamiana and H2B or dsRDR6 transgenic N. benthamiana plants were performed essentially as described [Agrobacterium cultures harboring an empty vector. The PTGS suppression assay and TuMV infection assay were repeated at least three times.escribed ,59. The Agrobacterium cultures harboring pTRV1 and one of the pTRV2 vectors including TRV2-GUS (control), TRV2-NbDCP1, TRV2-NbDCP2, TRV2-NbXRN4 and TRV2-NbPARN were mixed at a 1:1 ratio before infiltration.For the TRV-VIGS assay, Y2H, BiFC and subcellular localization experiments were performed essentially as described ,55,59.+ nylon membranes (Amersham Pharmacia Biotech) by capillary transfer. Membranes were hybridized at 45°C to specific probes labeled with digoxigenin (Roche). For siRNA blotting, LMW RNAs were enriched from total RNA, transferred to a Hybond-N+ membrane and hybridized for detection of siRNAs as described previously [For RNA blot analysis, total RNA was extracted from the infiltrated leaf patch, or virus-infected leaves with Trizol. High molecular weight (HMW) and low molecular weight (LMW) RNA analyses were conducted using 20 and 50 μg total RNA, respectively. HMW RNA was transferred to Hybond Neviously ,57,58. BNbActin or AtActinII was used as an internal control for N. benthamiana and Arabidopsis, respectively. All primer information used in qRT-PCR was given in S1 Table.For qRT-PCR analysis, total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) and treated with DNase I (Thermo Fisher Scientific) following the manufacturer's instructions. cDNA synthesized from reverse transcription of RNA samples was used to determine the mRNA levels of target genes as well as for quantification of TuMV accumulation levels. The procedures of cDNA synthesis and qRT-PCR assays were described previously ,55. NbAcN. benthamiana leaves as described previously [Total protein was extracted from infiltrated leaf patches or TuMV-infiltrated or systemically infected eviously . ImmunobS1 Table(DOCX)Click here for additional data file.S1 FigN. benthamiana leaves at 32 hpi. The YFP halves (YN and YC) were fused with P3N-PIPO and CI. The interaction of P3N-PIPO and CI brought the split YFP halves in close proximity to restore yellow fluorescence (green). H2B-RFP is indicated by red. Bars = 25 μm.P3N-PIPO and CI serve as a positive control for the protein-protein interaction assay in H2B transgenic (TIF)Click here for additional data file.S2 FigA) GFP fluorescence in N. benthamiana. Leaf patches were agroinfiltrated with three expression vectors including 35S-GFP, 35S-GF and one of the following vectors: an empty vector (Vec), dsGUS, dsNbXRN4, and P19. The representative picture was taken at 6 dpi under UV light. (B) Relative accumulation of GFP mRNAs analyzed by specific qRT-PCR in the infiltrated leaves shown in (A) at 6 dpi. NbActin serves as an internal standard. Values represent the mean ± SD (n = 3). Double asterisks indicate a highly significant difference compared to 35S-GFP+35S-GF+Vec . ((TIF)Click here for additional data file.S3 FigA) GFP fluorescence in RDR6-deficient N. benthamiana. Leaf patches were agroinfiltrated with three expression vectors including 35S-GFP, 35S-GF and one of the following vectors: an empty vector (Vec) as a control, dsNbDCP1, dsNbDCP2, dsNbXRN4, and dsNbPARN. The representative picture was taken at 7 dpi under UV light. (B) Relative accumulation of GFP mRNAs analyzed by specific qRT-PCR in the infiltrated leaves shown in (A) at 7 dpi. NbActin serves as an internal standard. Each mean value was based on three independent experiments (n = 3 samples). Values represent the mean ± SD. Double asterisks indicate a highly significant difference compared to 35S-GFP+35S-GF+Vec/dsRDR6 .((TIF)Click here for additional data file.S4 Fig(A) qRT-PCR analyses of TuMV RNA levels. In this experiment, a very lower concentration of agrobacterium cultures (OD600 = 0.05) harboring TuMV or TuMV-ΔGDD was used. RNA was extracted from the infiltrated patches with TuMV-GFP or TuMV-ΔGDD and one of the following vectors: Vec, NbDCP1, NbDCP2, NbXRN4 or NbPARN at 3 dpi. Each value was normalized against NbActin transcripts in the same sample. Error bars represent SD (n = 3). Double asterisks indicate a highly significant difference compared to the treatment of Vec . (B) Accumulation of TuMV siRNAs in the infiltrated patches as described in (A) at 3 dpi. Northern blotting was performed using DIG-labeled DNA probes complementary to the TuMV genome. U6 serves as a loading control for siRNA blot, respectively.(TIF)Click here for additional data file.S5 FigN. benthamiana leaves co-infiltrated with TuMV-GFP and one of the following vectors including an empty vector (Vec), NbDCP1, dsNbDCP1, dsNbDCP2, dsNbXRN4 and dsNbPARN at 4 dpi. Coomassie blue staining of the large subunit of Rubisco and U6 serve as a loading control for immunoblot, mRNA blot and siRNA blot, respectively. The values of GFP siRNAs/U6 were quantified by ImageJ software and then were normalized against the mean value corresponding to the Vec treatment, which was set to 1.00.Accumulation of GFP protein and TuMV siRNAs in (TIF)Click here for additional data file.S6 Fig(A) The phenotypes of TRV-GUS, TRV-NbDCP1, NbDCP2, NbXRN4, or TRV-NbPARN -treated plants at 14 dpi. A cDNA fragment of NbDCP1, NbDCP2, NbXRN4, or NbPARN was cloned into RNA2 of the TRV VIGS vector. N. benthamiana plants at the 4–5 leaf stage were infiltrated with Agrobacterium cultures carrying pTRV1 and pTRV2-GUS, or pTRV1 and TRV-NbDCP1, TRV-NbDCP2, TRV-NbXRN4, or TRV-NbPARN. (B) Silencing of target genes was confirmed in newly emerged leaves 14 dpi by qRT-PCR. **, P < 0.01, student’s t test.(TIF)Click here for additional data file.S7 FigA, B, C, D) Confirmation for homozygous dcp1 (A), heterozygous dcp2 (B), homozygous xrn4 (C) and heterozygous parn (D) T-DNA insertion lines. PCR was conducted using genomic DNA from dcp1 (SALK_014408C), dcp2 (SALK_00519), xrn4 (SALK_014209), parn  mutant and wild type (Col-0) Arabidopsis plants. Gene-specific primers (LP+RP) were used to detect the wild type DNA from Col-0 plants. A T-DNA specific primer and a gene-specific primer (LBb1.3+RP) were used to amplify a single PCR fragment which represented the inserted DNA. LP, left genomic primer; RP, right genomic primer; LBb1.3, left border primer of the T-DNA insertion.((TIF)Click here for additional data file.S8 FigA) Symptoms of TuMV-infected wild type (Col-0), dcp1, dcp2, xrn4 and parn mutant Arabidopsis plants. Images were taken at 16 dpi. Mock means that plants were inoculated with buffer and TuMV means that plants were inoculated with TuMV infectious clones. (B) Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from TuMV systemically infected leaves at 16 dpi. The values are shown as means ± SD (n = 3) relative to Col-0 and AtActinII were used as the internal reference. Data were analyzed using Student’s t test .((TIF)Click here for additional data file.S9 FigN. benthamiana leaves at 32 hpi. H2B-RFP is indicated in red. Bars = 10 μm.The YFP halves (YN and YC) were fused with NbDCP2 and VPg. The interaction of NbDCP2 and VPg was present in the nucleus (green) in H2B transgenic (TIF)Click here for additional data file.S10 FigA) qRT-PCR analysis of AtXRN4 expression in Col-0 and transgenic plants carrying 35S:Myc-AtXRN4-1, 35S:Myc-AtXRN4-2, 35S:HC-Pro-CFP-1/35S:Myc-AtXRN4-1 or 35S:HC-Pro-CFP-1/35S:Myc-AtXRN4-2. (B) Detection of Myc-AtXRN4 in 35S:Myc-AtXRN4, and 35S:HC-Pro-CFP/35S:Myc-AtXRN4 transgenic plants by immunoblotting. (C) qRT-PCR analysis of HC-Pro expression in Col-0 and transgenic plants carrying 35S:Myc-AtXRN4-1, 35S:Myc-AtXRN4-2, 35S:HC-Pro-CFP-1/35S:Myc-AtXRN4-1 or 35S:HC-Pro-CFP-1/35S:Myc-AtXRN4-2. (D) Confirmation of the protein expression from 35S:HC-Pro-CFP and 35S:HC-Pro-CFP/35S:Myc-AtXRN4 transgenic Arabidopsis plants by Western blot. Anti-Myc (B) or anti-GFP (D) polyclonal antibodies were used, respectively. Coomassie brilliant blue (CBB)-stained Rubisco large subunit was used as a loading control. 35S:HC-Pro-CFP/35S:Myc-AtXRN4 plants were obtained by genetic crosses between 35S:HC-Pro-CFP-1 and 35S:Myc-AtXRN4-1 Arabidopsis plants. T2 generation plants were used in the above experiments.((TIF)Click here for additional data file.


Scientific Reports 10.1038/s41598-018-22300-1, published online 02 March 2018Correction to: The Acknowledgements section in this Article is incomplete.P. aeruginosa PA14 and pEX18Gm . We thank Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U for beamtime allowance. We thank the staffs of the National Center for Protein Sciences Shanghai (NCPSS) beamlines BL-18U and BL-19U and SSRF, Shanghai, People’s Republic of China, for assistance during data collection.”“We thank Matthew R Parsek for the gifts of should read:P. aeruginosa PA14 and pEX18Gm . We thank Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U for beamtime allowance. We thank the staffs of the National Center for Protein Sciences Shanghai (NCPSS) beamlines BL-18U and BL-19U and SSRF, Shanghai, People’s Republic of China, for assistance during data collection.”“The work was supported by National Natural Science Foundation of China (Grant Nos. 81670008 and 81501787) and National Key Research and Development Plan (Grant No. SQ2016YFJC040104). We thank Matthew R Parsek for the gifts of


Gene expression in plant chloroplasts and mitochondria is affected by RNA editing. Numerous C-to-U conversions, accompanied by reverse U-to-C exchanges in some plant clades, alter the genetic information encoded in the organelle genomes. Predicting and analyzing RNA editing, which ranges from only few sites in some species to thousands in other taxa, is bioinformatically demanding.Here, we present major enhancements and extensions of PREPACT, a WWW-based service for analysing, predicting and cataloguing plant-type RNA editing. New features in PREPACT’s core include direct GenBank accession query input and options to restrict searches to candidate U-to-C editing or to sites where editing has been documented previously in the references. The reference database has been extended by 20 new organelle editomes. PREPACT 3.0 features new modules “EdiFacts” and “TargetScan”. EdiFacts integrates information on pentatricopeptide repeat (PPR) proteins characterized as site-specific RNA editing factors. PREPACT’s editome references connect into EdiFacts, linking editing events to specific co-factors where known. TargetScan allows position-weighted querying for sequence motifs in the organelle references, optionally restricted to coding regions or sequences around editing sites, or in queries uploaded by the user. TargetScan is mainly intended to evaluate and further refine the proposed PPR-RNA recognition code but may be handy for other tasks as well. We present an analysis for the immediate sequence environment of more than 15,000 documented editing sites finding strong and different bias in the editome data sets.We exemplarily present the novel features of PREPACT 3.0 aimed to enhance the analyses of plant-type RNA editing, including its new modules EdiFacts integrating information on characterized editing factors and TargetScan aimed to analyse RNA editing site recognition specificities.The online version of this article (10.1186/s12859-018-2244-9) contains supplementary material, which is available to authorized users. Nearly 30 years after the discovery of C-to-U RNA editing in plant mitochondria –3 and quThe arrays of PPRs are key to specifically recognizing the RNA sequences upstream of cytidines targeted for conversion into uridines via deamination. PPR proteins serving as editing factors have a unique makeup of alternating P-, L- and S-type PPRs with distinct amino acid conservation profiles. Moreover, PPR proteins characterized as editing factors carry carboxyterminal protein domain additions, minimally “E” (extension) domains, frequently followed by the so-called “DYW” domain. The latter in particular is of fundamental interest owing to its significant similarity to cytidine deaminases, which likely provides the biochemical activity for C-to-U conversion –23.Intriguingly, DYW-type PPR proteins that were previously believed to be plant-specific, have recently been identified in very distant evolutionary lineages of eukaryotes where their presence likewise seems to be connected to mitochondrial RNA editing of the C-to-U type –27.A PPR-RNA recognition code has been proposed , which iThe PREPACT WWW service developed in our group , 39 aimeOne core component of PREPACT’s functionality is a set of mitochondrial and chloroplast genomes with curated and standardized RNA editing site information . A user-Amborella trichopoda [Aegilops tauschii [Cocos nucifera [Cucumis sativus [Gossypium hirsutum [Phalaenopsis aphrodite [Spirodela polyrhiza [Vigna radiata [Ginkgo biloba [Apopellia endiviifolia [Selaginella uncinata [Equisetum hyemale [Ophioglossum californicum and Psilotum nudum [Ginkgo, the early-branching angiosperm Amborella and the jungermanniid liverwort Apopellia fill important taxonomic gaps. Similarly, the horsetail Equisetum and the club moss Selaginella are important addendums as taxa representing the full range of a taxon lacking chloroplast editing altogether [We have added several new organelle editome references with reliably determined editing site identifications Table . The 14 ichopoda , Aegiloptauschii , coconutnucifera , cucumbe sativus , 43, cothirsutum , the orcphrodite , the ducolyrhiza  and the  radiata , the gymo biloba , the livviifolia , the lycuncinata , the hor hyemale  and the um nudum  have beeum nudum . The gymtogether  and the together .Table 1NOphioglossum californicum and Psilotum nudum are particularly valuable additions as the first fern mitochondrial editomes [Liriodendron tulipifera [Amborella trichopoda [Cocos nucifera [Acrasis kona where two events of plant-type C-to-U editing have recently been identified [Among the mitochondrial references, editomes . The edilipifera , Amborelichopoda  and Coconucifera  are inteentified  as a furHevea brasiliensis mitochondria [Glycine max [Utricularia reniformis chloroplast [Saccharum officinarum [Cycas organelle editomes [Ipomoea batata [Deschampsia antarctica [Elaeis guineensis [In some cases we refrained from adding further reference data owing to an evident lack of documented editing sites in the editomes at this stage like in the case of the rubber tree chondria , the soycine max  and the oroplast  as well icinarum  and Cycaeditomes , 61. Lika batata , Deschamtarctica  or Elaeiineensis . AltogetThe enhanced query interface of PREPACT 3.0 has several new options Fig. . SearcheChaetosphaeridium and in the liverwort Apopellia, which can be taken to further corroborate the likelihood of editing in the query (here Wollemia nobilis KP259800). Similarly, in the case of a leucine codon in rps18 that is widely conserved in plants and algae and which requires editing from a serine codon in several references, a phenylalanine codon (F) is present in the Poales Oryza and Zea  and conservation of a genomically encoded tyrosine in other taxa  codon in algae, liverworts, mosses and ferns  but not in the angiosperms, which lack editing and retain the genomically encoded serine codon. Editing event rps2eU134TI converting a threonine into an isoleucine codon is among those now to be added to the Atropa chloroplast editome (and the ones of Oryza and Zea) upon our re-investigation of cDNAs , remains to be seen. While edit ndhGeU332SL in Anthoceros . Examples like these emphasize the importance of taxonomically diverse editome data sets.In few cases, codon changes introduced by RNA editing in the chloroplast seem erratic. The case of the “orphan” editing psbZeU11AV introducing a valine codon exclusively in the ipt Fig.  is an exdhG Fig. . Whetherros Fig.  could bella Fig. , this edatp9 gene for a phylogenetically wide sampling as an example for the alignment prediction mode  in the heavily editing lycophytes and gymnosperms. RNA editing frequencies vary significantly in each plant clade. The Physcomitrella patens RNA editing event atp9eU92SL affecting atp9 codon 31, which we will also discuss below in the context of the new TargetScan feature, is shared by all other mosses, the hornworts, lycopyhtes, three gymnosperms and the two angiosperms in our example  or “orphan” RNA editings ccb452eU925PS in Arabidopsis and ccmFCeU706PS and ccmFCeU707SF in Nicotiana.If a characterized factor is known for a given editing site, the PREPACT output of that editing event is now highlighted with italic font and underlining, and dynamically cross-linked to the respective EdiFacts entry as exemplarily demonstrated for the two tes Fig. . This alple Fig.  also illA new module TargetScan has been added to allow position-weighted querying of the PREPACT organelle reference data sets or of user-uploaded sequences for sequence motif matches Fig. . The TarPhyscomitrella patens editing factor PPR_98  with the above settings  still identifies the atp9eU92SL editing site as the top-ranking target  re-inspection for RNA editing at such candidate alternative target sites, (ii) testing for different binding weights of the different PPR motifs and target positions and (iii) subsequent testing with recombinant proteins in model systems like Physcomitrella and Arabidopsis. Evidently, any TargetScan query motif has a different a priori probability to identify top-scoring hits in different organelle genomes depending on nucleotide composition (mainly GC content). However, any results with identical scores identified in different genomes should indicate equally good matches, e.g. as likely targets for a given PPR protein like in the example discussed above.With the simple arbitrary weights outlined above, the correct RNA editing target of PPR_98 is identified with a top score of 1385  set apart from the second-best hit (the ccmFCeU103PS editing site scoring 945) when scanning upstream of all tes Fig. . Such a get Fig. . HoweverSelaginella uncinata plastome, the Selaginella moellendorffii chondrome and the available chloroplast editomes of bryophytes and ferns, here considering C-to-U and U-to-C editing events separately , one can quickly obtain sorted results for each nucleotide identity as exemplarily shown for position − 1 in the angiosperm plastome example  in position − 4 and the dominance of A (67.5%) vs. C (6.4%) in position + 1 for the angiosperm chloroplast editomes Fig. . These pSelaginella  allows to distinguish the P-, L- and S-type PPRs of plant PLS-type editing factors specifically after carefully reconsidering the domains of plant-type PPR proteins [The manual identification of potentially relevant amino acid positions from the loosely conserved P-, L- and S-type repeats of PPR proteins is tedious and cumbersome. The TPRpred tool served as a publicly available bioinformatic service for de novo identification of tetratricopeptide (TPR) and pentatricopeptide (PPR) repeats . A new Wproteins . An autoOver the recent years, research on plant-type RNA editing has extended to the characterization of the specific, RNA-binding pentatricopeptide repeat (PPR) protein factors addressing individual editing sites in the endosymbiotic organelles. We have accordingly extended our WWW service PREPACT to include information on PPR-type editing factors in an additional database module EdiFacts. As a further extension of PREPACT, the new TargetScan module allows to search for position-weighted motifs in the now extended reference editome set of PREPACT or in user-defined references. The novel feature now implemented in version 3 of PREPACT should be of use to integrate information of RNA editing sites and corresponding specificity factors and help to further elucidate how PPR-type editing factors recognize their respective RNA targets.Amborella trichopoda mitochondrial DNA. The reference tabs of the BLASTX output  and jQueryUI (https://jqueryui.com/) libraries as well as additional jQuery extensions “File Upload” (https://blueimp.github.io/jQuery-File-Upload/) and “Add Clear” (https://github.com/skorecky/Add-Clear).The user interface has been improved mainly on the sequence upload/handling side via integration of additional JavaScript features with the help of jQuery  previously not reported not to be edited were rechecked in selected cases Additional file 2:Table of re-checked edits. Verification of additional RNA editing events previously overlooked in editome references. (DOCX 45 kb)Additional file 3:Alignment prediction output. An example for the output of a multiple-query alignment for different references. (DOCX 79 kb)Additional file 4:TargetScan of Editing environment. An example illustrating the use of TargetScan to identify nucleotide bias in the immediate environment of editing sites in position -4 to +3 as discussed in the text. (DOCX 87 kb)


The principal structural component of a retrovirus particle is the Gag protein. Retroviral genomic RNAs contain a ‘packaging signal’ (‘Ψ') and are packaged in virus particles with very high selectivity. However, if no genomic RNA is present, Gag assembles into particles containing cellular mRNA molecules. The mechanism by which genomic RNA is normally selected during virus assembly is not understood. We previously reported  that at  A retrovirus particle is assembled from ~1500–3000 molecules of the Gag protein, together with RNA , as wellIn mammalian cells expressing Gag in the absence of Ψ-containing RNA, the protein assembles into virus-like particles (VLPs) structurally indistinguishable from immature virions; these particles contain roughly the same amount of RNA as wild-type particles, but this RNA is a nearly random sample of cellular mRNA molecules . SimilarIn an effort to understand the selective packaging of Ψ-containing RNA, we recently measured the affinity of recombinant HIV-1 Gag protein (lacking the p6 domain at its C-terminus) for different RNAs . We founTo explain how Ψ-containing RNAs are selectively packaged, despite the fact that Gag binds any RNA tightly at physiological ionic strength and any RNA can support assembly, we proposed that binding to Ψ leads to initiation of assembly more efficiently than binding to other RNAs . We now Gag has been suggested to bind specifically to several distinct sites in the 5’ region of HIV-1 RNA . These iOne important unresolved question is the exact sequence(s) which define Ψ. We measured binding affinities using, where not specified otherwise, the methodologies described earlier , except To test the effects of these changes upon the specific and non-specific binding by Gag, we titrated Gag into these RNAs, monitoring binding by the quenching of the fluorophore as described , either As discussed above, we have proposed that genomic RNA is selectively packaged because binding to Ψ is particularly efficient at initiating VLP assembly . Thus, iDs for MST and FCS were 14 and 17 nM, respectively, while at 0.45 M NaCl they were 256 and 302 nM. In all cases the Hill coefficient was greater than 1.0. These data show that MST is able to recapitulate our original FCS results , MBSM second Generation (Gen), and Reverse Complement (Rev Comp) RNAs, respectively. Specifically, it is evident that nearly all of each RNA is bound at 1–2 µM Δ16–99 Gag, significantly below the levels used in the assembly experiments . Results of this experiment for Ψ, MBSM second Gen, and Rev Comp RNAs are shown in Finally, we compared different RNAs with respect to their ability to support assembly of Δ16–99 Gag into VLPs. Different amounts of Δ16–99 Gag were added to 61 nM solutions of the Cy5-labeled RNAs; VLP assembly was monitored by the shift of the RNA into large, rapidly sedimenting structures, and was confirmed by negative-stain electron microscopy . AlthougWe also determined the distribution of the Δ16–99 Gag protein in these gradients, by performing immunoblotting on dot blots of aliquots of the gradient fractions . We founIn order to quantitatively assess the level of VLP assembly in each of the reactions, we summed the amount of RNA between fractions 10 and 18. The results of this analysis are shown in The results reveal a striking difference between Ψ RNA and either MBSM second Gen or Rev Comp RNA: particularly at the lower protein levels, Ψ supports assembly far more efficiently than the other RNAs. For example, at 11.25 μM Δ16–99 Gag, approximately ⅘ of the Ψ RNA has been shifted into the bottom half of the gradient, while only about half of the MBSM second Gen or Rev Comp RNA has undergone a similar shift.These results are in complete concordance with our hypothesis that binding to a packaging signal nucleates assembly with particularly high efficiency . Simulatst-generation MBSM was derived from HIV-1 Ψ 200 by replacement of G224, G226, G240, C243, G241, G270, G272, G273, C274, G275, G289, G290, G292, G310, C312, G318, G320, G328, and G329 with adenines . Samples were incubated 20 min at 22°C after loading into measuring capillaries. All experiments were done with temperature control set to 22°C. LED power was 90% for Ψ and second generation MBSM RNAs and 50% for Reverse complement RNA. MST power was 20% for all measurements with 5 s fluorescence read before MST laser on, 20 s MST laser switched on and 5 s fluorescence read after MST laser off. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Jeremy Luban (Reviewer #3).Thank you for submitting your article "Efficient support of virus-like particle assembly by the HIV-1 packaging signal" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper, submitted as a Research Advance, presents additional information about the interactions between the HIV-1 genomic RNA and Gag protein constructs. The work extends the earlier results demonstrating both specific and nonspecific RNA binding activity by Gag, and the need for either high salt or high competitor RNA concentrations to reveal the specific binding.  Based on the earlier work, most readers might have come to the conclusion that in vivo, high competitor RNA levels were present and could have explained the high efficiency of viral RNA packaging. However, the authors here provide additional support for their proposal that the HIV-1 RNA triggers virion assembly more efficiently than other RNAs and that this explains the higher abundance of viral RNA in the final virion preps. Here the binding curves are similar to before . The newer part is the sedimentation of assembled Gag constructs in the presence of RNA, measuring the rapid sedimentation resulting from coassembly . The specific packageable RNA is shown to induce particles at lower concentration than the nonspecific RNAs, and analysis of the Hill equation for binding suggests about a three-fold lower K value. While the electron micrographs of particles are underwhelming, the RNA results  seem to confirm the claim of selective assembly. The punch line should be of interest to the community. The new RNA mutants also provide some new information about what is required for the enhanced assembly.One reviewer had some specific issues that need to be addressed. Their review is included in its entirety below. Their points are valid and deserve as detailed a response as possible.Reviewer #2:eLife manuscript by Comas-Garcia et al. is a Research Advance submission based on the previous publication of Comas-Garcia et al., entitled "Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA." The stated advances of the current manuscript are support for the hypothesis that encapsidation signal positive (Ψ+) RNAs initiate particle assembly more efficiently than other RNAs, and that unpaired G residues are important elements of the Ψ signal. The degree to which these observations are novel enough to support eLife publication, rather than publication in a more specialized journal, is a matter of question. The certainty of the results, especially as regards particle assembly initiation, also is a concern. Specific comments are as follows:The d values differing by >10% from the consensus values were discarded." It would be nice to know how many such experiments were discarded, and why they were deemed inaccurate.1) Figures 1 and 2A: The results concerning the potential importance of unpaired G residues to RNA binding appear reasonably solid, but have been reported in other experimental systems already . I also am troubled by the statement in the legend to Figure 1 that results are the means of only two independent experiments, and that "experiments giving K2) MST experiments: My understanding is that MST experiments were not performed in the initial studies. Because of this, presenting some of the raw data on controls is of interest, as would be showing results with additional RNA substrates, and giving the actual Hill coefficients.3) Ψ+ RNA effects on assembly: The hypothesis that Ψ+ RNA triggers particle assembly is a logical one, and has received support elsewhere. With respect to the current manuscript, the proof for the hypothesis has not been explained clearly. As I understand it, the crux of the proof here is that the Ψ 2 200 RNA binds Δ16-99 Gag in assembly buffer with the same affinity as the other RNAs , but partitions more efficiently into higher S value fractions when assembled with higher concentrations of Δ16-99 Gag . The approach and results raise some questions and concerns:a) Why did the authors have to use Δ16-99 Gag rather than the otherwise wild type Δp6 Gag?b) Why doesn't Δ16-99 Gag quench the fluorophore in FCS experiments?c) The Δ16-99 Gag protein binds significantly less well to RNAs at 150 mM NaCl than the Δp6 Gag protein . What happens when the ionic strength is raised, or yeast RNA is added?d) Figure 3: Please show deviations on the graphs. Please also state how many experiments were excluded because the positions of the peaks were different from the consensus profiles.e) Figure 3—figure supplement 1: This should be in the paper. I also see lots of spots in A, things that look like tiny rods in B, and a clean background in C. The particles in C also look smaller than in A and B. Please explain.f) Figure 3—figure supplement 2: This should be in the paper. It also would be useful to see the Gag profiles for the incubations with all the higher concentrations of Gag. It would have been nice to have seen a shift in Gag.g) Figure 3: What happens when an excess of yeast tRNA is added? One reviewer had some specific issues that need to be addressed. Their review is included in its entirety below. Their points are valid and deserve as detailed a response as possible.Reviewer #2:The eLife manuscript by Comas-Garcia et al. is a Research Advance submission based on the previous publication of Comas-Garcia et al., entitled "Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA." The stated advances of the current manuscript are support for the hypothesis that encapsidation signal positive (Ψ +) RNAs initiate particle assembly more efficiently than other RNAs, and that unpaired G residues are important elements of the Ψ signal. The degree to which these observations are novel enough to support eLife publication, rather than publication in a more specialized journal, is a matter of question. The certainty of the results, especially as regards particle assembly initiation, also is a concern. Specific comments are as follows:1) Figures 1 and 2A: The results concerning the potential importance of unpaired G residues to RNA binding appear reasonably solid, but have been reported in other experimental systems already .+] and KD, and could not have detected the change in the proteins’ binding properties as the salt concentration is raised. We have now pointed out their prior results in the revised manuscript .The reviewer notes that previous publications have also highlighted the importance of unpaired G residues to RNA binding, as we mentioned in the original manuscript. However, we would point out that the previous studies involved binding of free NC protein (not Gag) to RNA  and RNA packaging in vivo . We previously showed that both MA and CA domains make important contributions to RNA-binding , so that binding by Gag is not equivalent to binding by NC protein. Effects on packaging in vivo could, of course, be mediated or influenced by any cellular constituent; in contrast, our results show directly that the G’s are important in the salt-resistance of the binding of Gag to Ψ and in the efficiency with which Ψ supports in vitro assembly, in the absence of any other reactants. In addition, Webb et al., 2013 did report that the binding of Δp6 Gag to a Ψ construct was less salt-resistant if 12 bases, mainly G’s, were changed to A’s; this “Psi-12M” is similar to our “MBSM” RNA. While the general conclusions about binding in Webb et al. are similar to those in our Figure 1D, we would also note that Webb et al. used a different experimental approach from ours: rather than assaying binding directly at different ionic strengths, they monitored dissociation of preformed protein-RNA complexes as the ionic strength was raised. As we discussed briefly in our 2017 paper, this approach assumes a linear relationship between [Nad values differing by >10% from the consensus values were discarded." It would be nice to know how many such experiments were discarded, and why they were deemed inaccurate.I also am troubled by the statement in the legend to Figure 1 that results are the means of only two independent experiments, and that "experiments giving Knd generation MBSM RNA at 0.4M NaCl, no binding was detected, in stark contrast to other experiments. We suspect that the target RNA had been degraded. The other was a competition experiment attempting to measure binding to RevComp RNA in the presence of tRNA. This experiment was discarded because the binding curve failed to reach a plateau.The reviewer also asks about experiments that were discarded, as mentioned in the legend to Figure 1. Only two experiments were discarded. In one, an attempt to measure binding of GagΔp6 to 22) MST experiments: My understanding is that MST experiments were not performed in the initial studies. Because of this, presenting some of the raw data on controls is of interest, as would be showing results with additional RNA substrates, and giving the actual Hill coefficients.The reviewer asks that more data be included on MST, which was used in the present manuscript to measure binding of protein to RNAs but was not used previously. We have added Figure 2—figure supplement 1, which presents in panels A and B, the raw MST time-trace data for binding of Δ16-99 Gag to ψ RNA in 0.15 and 0.5M NaCl; in panel C, the superimposed cross-sectional profiles of the fluorescence in the MST capillaries, where the symmetrical curves indicate that nothing is sticking to the walls of the capillaries; and in panel D, the profiles of the individual capillaries (containing RNA + different amounts of protein); the near-identity of these profiles shows that the capillaries all contained same amount of fluorescent RNA. The results of the MST measurements are now summarized in Table 1.3) Ψ + RNA effects on assembly: The hypothesis that Ψ + RNA triggers particle assembly is a logical one, and has received support elsewhere. With respect to the current manuscript, the proof for the hypothesis has not been explained clearly. As I understand it, the crux of the proof here is that the Ψ 2 200 RNA binds Δ16-99 Gag in assembly buffer with the same affinity as the other RNAs , but partitions more efficiently into higher S value fractions when assembled with higher concentrations of Δ16-99 Gag . The approach and results raise some questions and concerns:a) Why did the authors have to use Δ16-99 Gag rather than the otherwise wild type Δp6 Gag?We are sorry that we did not explain this in the original submission. We found many years ago  that the VLPs formed when nucleic acid is added to GagΔp6 are far smaller than authentic HIV-1 virions, and thus cannot have the same architecture as authentic immature particles. In contrast, Δ16-99 Gag forms particles of the correct size in the presence of nucleic acid, and extensive studies from several labs have shown that the overall architecture of these VLPs is an excellent facsimile of that in immature particles . We have added a brief explanation to the revised manuscript .b) Why doesn't Δ16-99 Gag quench the fluorophore in FCS experiments?We do not know. As we discussed in the original 2017 manuscript, the quenching of Cy5 by GagΔp6 represents stabilization of a non-fluorescent isomer of Cy5. We have also observed that free NC protein does not quench Cy5-labeled RNAs. These observations suggest that the quenching involves the MA domain in GagΔp6, but we have no further information on this. We have noted this in the fourth paragraph of the Results and Discussion.c) The Δ16-99 Gag protein binds significantly less well to RNAs at 150 mM NaCl than the Δp6 Gag protein . What happens when the ionic strength is raised, or yeast RNA is added?We are now investigating the role of the MA domain in specific and non-specific RNA-binding. We have found that the binding of Δ16-99 Gag to Ψ RNA, as well as to the control RNAs, is strongly depressed at high ionic strengths. We have not yet tested the effect of competitor tRNA. As mentioned above, we have added a supplementary figure and a Table with the MST data for binding of Δ16-99 Gag to the RNAs.d) Figure 3: Please show deviations on the graphs. Please also state how many experiments were excluded because the positions of the peaks were different from the consensus profiles.We have added the deviations to Figure 3  as requested by the reviewer. Two experiments were discarded because the labeled RNA barely moved from the top of the gradient. We assume that the RNA in these samples was degraded.e) Figure 3—figure supplement 1: This should be in the paper. I also see lots of spots in A, things that look like tiny rods in B, and a clean background in C. The particles in C also look smaller than in A and B. Please explain.The reviewer asks that the electron micrographs, which were Figure 3—figure supplement 1 in the original manuscript, be moved to the body of the paper. We have now made them Figure 3. The reviewer also asks for a little more information about what is seen in the electron micrographs. Our intention was just to demonstrate that the protein successfully assembles into VLPs under the conditions of these experiments. We have also added an inset to each of the 3 panels, presenting a VLP at higher magnification for greater clarity. Finally, the reviewer asks about the miscellaneous structures in these images which are not spherical VLPs. It is obvious that a wide variety of structures are formed and we have noted that in the revised manuscript . Finally, the reviewer mentions a “clean background” in C. We have not investigated this systematically as yet, but the reviewer may well be correct that fewer structures other than intact VLPs are formed on Reverse Complement RNA. We have noted this possibility in the aforementioned paragraph.f) Figure 3—figure supplement 2: This should be in the paper. It also would be useful to see the Gag profiles for the incubations with all the higher concentrations of Gag. It would have been nice to have seen a shift in Gag.The reviewer asks that Figure 3—figure supplement 2, the profile of Gag in the sucrose gradients, be included in the paper. We have now made it Figure 5. The reviewer also asks about the profiles obtained with higher Gag concentrations, where Gag is in even greater excess over the RNA. In fact the amounts of Gag here were so high that it is not trivial to obtain quantitative data on Gag in these gradients. The vast majority of Gag remains at the top of the gradient; this does not seem like useful information to us and we would prefer to omit these profiles.g) Figure 3: What happens when an excess of yeast tRNA is added?The reviewer asks about adding competitor RNA, such as yeast tRNA, in the assembly experiments. We agree that this is an interesting suggestion but have not tried it as yet.


During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as “riboregulators” that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and “riboregulatory” RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions. Dianthovirus. Also, xrRNAs had been found only in the 3′ untranslated regions (3′UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5′ ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known.Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus  Single-stranded positive-sense RNA viruses include pathogens that infect a wide range of animal and plant hosts, with significant human health, agricultural, and economic impact. During infection, these viruses must generate many copies of their full-length genomic RNA, but many also produce one or more infection-critical subgenomic RNA species (sgRNA). sgRNAs can have different functions: they can encode and serve as the template for production of specific viral proteins or act as noncoding “riboregulators” that interact with and influence the cellular and viral machinery to benefit viral infection –6. Most –7–F) sequences revealed two classes; class I (xrRNAF1) is exemplified by mosquito-borne flaviviruses, whereas class II (xrRNAF2) is found in diverse flaviviruses  fold containing an interwoven pseudoknot stabilized by conserved secondary and tertiary interactions; this creates an unusual ring-like conformation that protectively wraps around the 5′ end of the RNA structure  elements were first identified in mosquito-borne flaviviruses , where they protect the genome’s 3′ untranslated region (3′UTR) from degradation . The res16–iviruses . Extensitructure , 25. Alt unknown .F2, the structures of recently reported xrRNAs from most other viral clades remain unsolved  function to produce a noncoding RNA derived from the viral 3′UTR , which enables screening of massive data sets of DNA sequences for conserved RNA secondary structure patterns with poor sequence conservation   and sugarcane yellow leaf virus . With these sequences added to the alignment, subsequent searches identified >40 candidates within the public repository of all available sequences for Tombusviridae and Luteoviridae, demonstrating how powerful this tool is for computationally identifying putative functional elements in viral RNAs (To search for new xrRNAervation . Because [CRSV]) , we exparal RNAs .D-like elements revealed that their predicted secondary structures contain the same elements as were found in the known xrRNAD. Specifically, the assertion of formation of helices P1 and P2 and a predicted pseudoknot is supported by covariation data, which reveal little sequence conservation (R-scape  was added and the cDNA was purified on a magnetic stand according to the manufacturer’s instructions. The cDNA was eluted in 11 µl ROX-HiDi and analyzed on a model 3500 Genetic Analyzer (Applied Biosystems) for capillary electrophoresis. A Sanger sequencing (ddNTP) ladder of the undigested RNA was analyzed alongside each degradation product as a reference for band annotation.To determine the Xrn1 stop site at single-nucleotide resolution, 30 µg All data are available from us.


Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.Recently, CRISPR‐Cas  system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from  Viral invasions often reduce the fitness of their host, sometimes leading to host death  and the type VI‐A CRISPR/Cas effector from Leptotrichia shahii (LshCas13a) or Leptotrichia wadei (LwaCas13a), have been reported to target RNA in vivo /CRISPR‐associated (Cas) 9 (CRISPR/Cas9) systems comprises sequence‐specific RNA‐directed endonuclease complexes that bind and cleave nucleic acids  and tobacco mosaic virus (TMV), both are positive‐sense RNA plant virus that can infect many species  driven by an enhanced 35S promoter and a single‐guide RNA (sgRNA) driven by an AtU6 promoter  with five open reading frames (ORFs), and the 3′ untranslated regions (3′ UTRs) of the three genomes present very high nucleotide sequence similarity Figure . To idenAgrobacteria containing a given experimental vector and the control vector together with CMV agro‐infectious clones into individual 25‐day‐old N. benthamiana leaves. The CMV agro‐infectious clones were injected into the top part of the leaf and the experimental vector and the control vector were injected into separate areas of the bottom part of the leaf  could target RNA, which presented the possibility of developing RNA virus resistance using genome editing technology  virus, the hepatitis C virus (HCV), within hepatoma cell lines. Transient expression of Cas9 and sgRNA complexes targeted to either the 5′ or 3′ untranslated regions of the HCV genome resulted in a 50–60% reduction in viral protein expression relative to the control. In our study, the F. novicida CRISPR/Cas9 system was expressed both transiently and stably in plants, and the plants were inoculated with CMV or TMV to evaluate the feasibility of using CRISPR/Cas9‐mediated interference against RNA viruses. We selected 23 target sites for CMV and three sites for TMV, all of them could inhibit virus accumulation by 40–80% compared with the nontargeting control, showing that the F. novicida CRISPR/Cas9 system has similar or even better virus interference efficiency in plants as in mammalian cells.The CRISPR/Cas9 system has been developed as a powerful tool for genome editing in the past few years , along with its guide RNA, could bind and cleave single‐stranded RNA in the presence of an exogenous PAM oligonucleotide, and newly identified a Class 2 type VI CRISPR‐Cas system endonuclease LshCas13a and LwaCas13a, as an RNA‐targeting CRISPR effector  gene sequence including a 3×Flag tag fused to the N‐terminus was designed with codon optimization for plants . The FnCas9 gene was linked to an enhanced 35S promoter and NOS terminator, while the sgRNA was linked to the Arabidopsis U6 promoter. These two expression cassettes were inserted into a binary vector derived from pCAMBIA1300 , in which the original BsaI site was removed. The final vector was named pCR01 and was used for transient and stable expression of the F. novicida CRISPR/Cas9 system in dicot plants.An Appendix . This geFnCas9 genes were constructed using a Fast Mutagenesis Kit  to generate FnCas9_D11A/H969A and FnCas9_R59A. All the three modified FnCas9 genes were substituted for the FnCas9 in pCR01 to produce pCR01_NLS, pCR01_D11A/H969A and pCR01_R59A, respectively. The β‐Glucuronidase (GUS) gene was substituted for the FnCas9 to produce pCR01_GUS. The pCR01 and its variant vectors were digested by BsaI and ligated with the synthesized sgRNA oligos  to a final OD600 nm of 1.0. Four true leaves of 4‐week‐old N. benthamiana plants were chosen and injected with the agrobacteria. One day later, CMV or TMV‐GFP infectious clones with a final OD600 nm of 0.6 were injected into the bottom two previously infected leaves.EHA105 strains containing all pCR01–sgRNA vectors or the pCR01 vector were grown in YEP medium with kanamycin and rifampicin overnight. The agrobacteria were harvested by centrifugation and diluted in inoculation buffer  were screened for resistance to hygromycin (20 μg/mL). The surviving seedlings were tested for transgenic expression of FnCas9 by RT‐qPCR. Two selected lines of each construct were further propagated for CMV attacking by mechanical inoculation.® Premix Ex Taq™ II . Three replicates were conducted for each treatment, and three technical RT‐qPCR replicates were analysed for each biological replicate. The tobacco PP2A or Arabidopsis ACTIN8 gene was used as reference. Primers for RT–qPCRs are shown in Table The injected or systemic leaves were picked, and the RNA was extracted with TRIzol . For viral RNA quantification, one‐step RT‐qPCRs were carried out in a CFX96 Touch™ real‐time PCR detection system  using HiScript II Q RT SuperMix . For FnCas9 transgenic and GFP expression measurement, cDNA was synthesized from total RNA using the oligo(dT) primer and reverse transcriptase (Takara). RT‐qPCRs were carried out in the same instrument using SYBRArabidopsis were isolated for analysis, and three replicates were conducted for each treatment.The CMV titre was also quantified using the triple antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) following the standard procedures. Systemic leaves (100 mg) of infected tobacco or N. benthamiana leaves inoculated with pCR01‐sgRNA vectors and infected with CMV as indicated. The immunoprecipitated proteins were confirmed by Western blotting. Total RNA was extracted from the precipitated fraction using an RNeasy Mini Kit  according to the manufacturer's instructions, and the CMV genomic RNA and sgRNAs was further detected by RT‐PCR using a HiScript II One Step RT‐PCR Kit (Vazyme).Anti‐Flag immunoprecipitation was performed according to the manufacturer's instructions  from T.Z. and G.Z conceived the project. T.Z., H.A. and G.Z. designed the research. T.Z., Q.Z., X.Y., Y.Z. and S.M. performed the research. T.Z., X.Y., H.A. and G.Z. analysed the data. T.Z. and G.Z. wrote the article with contributions from all the authors.T.Z. and G.Z. have filed a patent application in China .Figure S1 Transient transformation of the sgRNA–FnCas9 system to develop CMV or TMV resistance in N. benthamiana.Figure S2 Structure of pCR01 and its variants used in the transient assay.Table S1 sgRNA target site IDs, sequences and oligonucleotides for cloning into pCR01.Table S2 Primers used in this study.Appendix S1 Sequence of the sgRNA and FnCas9 expression cassettes in pCR01.Click here for additional data file.


Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA. To design ssRNAs that efficiently replicate by Qβ replicase with minimum dsRNA formation, we previously proposed the “fewer unpaired GC rule.” According to this rule, ssRNAs that have fewer unpaired G and C bases in the secondary structure should efficiently replicate with less dsRNA formation. However, the validity of this rule still needs to be examined, especially for longer ssRNAs. Here, we analyze nine long ssRNAs that successively appeared during an in vitro evolution of replicable ssRNA by Qβ replicase and examine whether this rule can explain the structural transitions of the RNAs. We found that these ssRNAs improved their template abilities step-by-step with decreasing dsRNA formation as mutations accumulated. We then examine the secondary structures of all the RNAs by a chemical modification method. The analysis of the structures revealed that the probabilities of unpaired G and C bases tended to decrease gradually in the course of evolution. The decreases were caused by the local structural changes around the mutation sites in most of the cases. These results support the validity of the “fewer unpaired GC rule” to efficiently design replicable ssRNAs by Qβ replicase, useful for more complex ssRNA replication systems. The replication of genetic information is a fundamental function of living systems. Single-stranded RNA (ssRNA), one of the possible genetic information carriers, is used as a genomic molecule in some viruses and also experimental models of primitive life-forms in the hypothetical RNA or RNA–protein world . The synTo date, important information regarding ssRNA replication has been obtained from studies using Qβ replicase, the RNA replicase of bacteriophage Qβ . One of Escherichia coli encapsulated in microscale water-in-oil droplets (8), which presumably covers all possible point mutations. We believe that a series of RNAs that appeared in the course of the evolutionary experiment would be ideal samples to evaluate the importance of the rule for ssRNA replication.To examine the validity of the rule for a longer RNA from a different aspect, here we analyzed mutations introduced during an in vitro evolution of replicable ssRNAs. Through in vitro evolution, mutations should be successively introduced in the order of effectiveness to improve replication if population size is sufficiently high. Therefore, if the introduced mutations during the evolution follow the rule, it should strongly support the primary importance to obey the “fewer unpaired GC rule” for efficient replication. We recently performed an in vitro evolution of an artificial genomic ssRNA that encodes Qβ replicase (2125 nt) in a reconstituted translation system of droplets . During In this study, we focused on one of the main evolutionary routes, consisting of nine distinct genotypes that successively accumulated 11 mutations. First, we found that the replication ability increased step-by-step by decreasing the probability of dsRNA formation. We then examined secondary structures of all the nine ssRNAs using a structure-dependent chemical modification data. From the structural data, we found that the numbers of unpaired G and C bases were gradually decreased as mutations accumulated. These results support the importance of the fewer unpaired GC rule for efficient ssRNA replication by Qβ replicase.Escherichia coli . In this evolutionary route, the nine RNA genotypes successively accumulated 11 types of mutations, including point mutations, an insertion, and two deletions, by one or two mutations at each transition  that encoded the catalytic subunit of Qβ replicase in a reconstituted translation system of hia coli . In thishia coli A left, mhia coli . In thisansition B. The 1132P]-UTP incorporation assay followed by agarose gel electrophoresis and autoradiography (The RNAs that appeared during the evolutionary experiment have two types of replication ability: the ability of the encoded replicase (replicase ability) and the ability to be replicated as a template (template ability). In the previous study, we measured these activities for some RNAs that finally dominated the population and found that the template ability, rather than the replicase ability, selectively improved during the evolution . To evaliography A. In alliography B as mutaiography B,C. ThesSupplemental Fig. S2). The average SHAPE value and standard deviation for each RNA are shown in the Supplemental information.To investigate structural changes in the nine RNAs, we performed selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis . First, Supplemental Fig. S3 for direct comparison between the unpaired GC values and template abilities). These results indicate that the improved template abilities of the nine RNAs can be understood based on the fewer unpaired GC rule.To investigate the validity of the “fewer unpaired GC rule,” we used these average SHAPE values to obtain partition functions of the nine clone RNAs by using RNAstructure software , which pSupplemental Text. In the following part of this study, we analyzed these MEA structures.Next, to investigate how the structures changed to reduce unpaired GC values, we predicted the maximum expected accuracy (MEA) structures from the partition functions by using the same RNAstructure software . The basSupplemental Figure S4.We next investigated how each introduced mutation contributes to the reduction in the unpaired GC value by examining the local structures around mutation sites. Notably, mutations introduced in the nine RNAs are located in four local structural regions, named regions A , B , C , and D (1454–1606) , which cWe then examined each transition of these local structural regions in detail. At the transition from RNA #1 to #2, the introduced mutation (A186G) induced base-pairings around the mutation site, which decreased the probability of unpaired G and C bases in region A, and hence reduced unpaired GC value A. At theIn this study, we analyzed the step-by-step evolution of RNA structures for ssRNA replication by Qβ replicase. Focusing on nine previously obtained RNAs, we first showed that the RNAs gradually improved their template abilities with decreasing dsRNA formation as mutations accumulated . Since tSupplemental Fig. S5, correlation coefficients are −0.76 and 0.58, respectively), compared to the correlation between the ssRNA synthesis or dsRNA ratio and the unpaired GC values .One may think the unpaired GC value also correlates with other biophysical features of the RNA, such as overall folding stability. However, this should not necessarily be the case based on the suggested mechanism of dsRNA formation during replication  that unpUnderstanding the rule to efficiently design replicable long ssRNAs is particularly important to design synthetic ssRNAs that encode arbitrary genes. SsRNAs have been utilized as genomes of experimental models in the era of the RNA–protein world , which pThis study also provides insights into the requirements for replicable RNAs by Qβ replicase. The effect of RNA structure on the replication by Qβ replicase has been extensively studied for decades , but strE. coli that contains approximately 3 µM of the S1 ribosomal subunit , indicating that these regions were the primal target for improvement during the evolution. The accumulation of mutations in these regions is reasonable because this region is the intersection of two different RNAs. We constructed RNA #1 by inserting the Qβ replicase subunit gene into a small (222 base) replicable RNA, MDV-1 .The nine RNAs were prepared by in vitro transcription using corresponding plasmids as previously described . The pla32P]-UTP in a solution as previously described (RNA replication was performed with a template RNA (100 nM) and purified Qβ replicase (20 nM) at 37°C for 20 min in the presence of [escribed . The reaSupplemental Information file.SHAPE data was obtained as previously described  with theThe partition function of each RNA was calculated using RNAstructure software  with theSupplemental material is available for this article.