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• W4248473821 abstract "Article10 July 2008free access Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4 Gabrielle Haas Gabrielle Haas Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Jacinthe Azevedo Jacinthe Azevedo Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Guillaume Moissiard Guillaume Moissiard Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Angèle Geldreich Angèle Geldreich Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Christophe Himber Christophe Himber Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Marina Bureau Marina Bureau Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Toshiyuki Fukuhara Toshiyuki Fukuhara Laboratory of Molecular & Cellular Biology, Tokyo University of Agriculture & Technology, Tokyo, Japan Search for more papers by this author Mario Keller Corresponding Author Mario Keller Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Olivier Voinnet Corresponding Author Olivier Voinnet Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Gabrielle Haas Gabrielle Haas Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Jacinthe Azevedo Jacinthe Azevedo Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Guillaume Moissiard Guillaume Moissiard Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Angèle Geldreich Angèle Geldreich Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Christophe Himber Christophe Himber Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Marina Bureau Marina Bureau Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Toshiyuki Fukuhara Toshiyuki Fukuhara Laboratory of Molecular & Cellular Biology, Tokyo University of Agriculture & Technology, Tokyo, Japan Search for more papers by this author Mario Keller Corresponding Author Mario Keller Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Olivier Voinnet Corresponding Author Olivier Voinnet Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France Search for more papers by this author Author Information Gabrielle Haas1, Jacinthe Azevedo1, Guillaume Moissiard1, Angèle Geldreich1, Christophe Himber1, Marina Bureau1, Toshiyuki Fukuhara2, Mario Keller 1 and Olivier Voinnet 1 1Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, Strasbourg, France 2Laboratory of Molecular & Cellular Biology, Tokyo University of Agriculture & Technology, Tokyo, Japan *Corresponding authors: Institut de Biologie Moléculaire des Plantes, CNRS UPR2353, Université Louis Pasteur, 12, rue du Général Zimmer, F-67084 Strasbourg Cedex, France. Tel.: +33 3 88 41 71 58; Fax: +33 3 88 61 44 42; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:2102-2112https://doi.org/10.1038/emboj.2008.129 Correction(s) for this article Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB419 August 2015 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-α-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function. Introduction In RNA silencing, homologues of the RNase-III enzyme Dicer process double-stranded (ds)RNA molecules into 21–24 nt RNAs, called short interfering (si)RNAs and micro (mi)RNAs. Four specialized Dicer-like (DCL) proteins with specifically sized small RNA products define multiple endogenous RNA silencing pathways in Arabidopsis thaliana: DCL1 catalyses processing of fold-back precursors into miRNAs that repress expression of cellular transcripts; DCL3 produces 24 nt siRNAs guiding heterochromatin formation, whereas DCL4 converts non-coding RNA precursors into 21 nt trans-acting (ta)siRNAs controlling developmental timing and organ polarity (reviewed in Brodersen and Voinnet, 2006). The size of DCL products is likely influenced by dsRNA-binding proteins (DRBs) that physically and specifically interact with Dicers: Arabidopsis DRB1, known as HYL1, facilitates DCL1-directed miRNA synthesis, whereas DRB4 enhances DCL4-mediated tasiRNA processing (Adenot et al, 2006; Nakazawa et al, 2007). Besides its regulatory roles, RNA silencing confers sequence-specific antiviral immunity to plants and invertebrates through the action of virus-derived (vi) short interfering RNAs (reviewed in Ding and Voinnet, 2007). In Arabidopsis, DCL4 is the primary Dicer of RNA viruses, and produces 21-nt-long viRNAs; DCL2 rescues antiviral silencing if DCL4 is genetically inactivated or suppressed, producing diagnostic 22-nt-long viRNAs (Bouche et al, 2006; Deleris et al, 2006; Fusaro et al, 2006). viRNAs are then thought to guide endonucleolytic cleavage (‘slicing’) of viral genomes/transcripts upon incorporation into an RNA-induced silencing complex (RISC) that likely contains AGO1 because viRNAs immunoprecipitate with AGO1 (Zhang et al, 2006). This model for antiviral silencing is supported by the fact that most plant viruses produce proteins called viral silencing suppressors (VSRs) thought to target many steps involving DCL, RISC or small RNA activities (reviewed in Ding and Voinnet, 2007). Nonetheless, clear-cut examples of interactions between VSRs and host silencing components are scarce. The tombusviral P19 protein forms a viRNA calliper preventing RISC loading (Vargason et al, 2003), whereas the Cucumber mosaic virus (CMV) 2b protein interacts with AGO1 to inhibit slicing (Zhang et al, 2006). AGO1 was also characterized as a direct target of the polerovirus F-Box-like protein P0 (Baumberger et al, 2007; Bortolamiol et al, 2007). More recently, the geminiviral protein V2 was shown to interact with SGS3, involved in amplification of antiviral silencing. The only two available examples of genetic, rather than physical, interactions linking viral proteins to RNA silencing components were provided by studies of VSR-deficient carmoviruses and cucumoviruses (reviewed in Ding and Voinnet, 2007). DNA caulimoviruses are sensitive to the four Arabidopsis DCLs, with a prevalent role for DCL4 and DCL3 (Blevins et al, 2006; Moissiard and Voinnet, 2006). The dsDNA genome of Cauliflower mosaic virus (CaMV), type member of the Caulimovirus genus is replicated by RNA reverse transcription, and is transcribed by RNA PolII into the 35S and 19S RNAs. The 35S RNA 5′ end forms an extensive fold-back structure known as translational leader, ensuring ribosomal shunting required for expression of all CaMV ORFs (Ryabova and Hohn, 2000). The leader is a major source of CaMV-derived viRNAs, which accumulate as 24-nt (DCL3-dependent) and 21-nt (DCL4-dependent) species (Moissiard and Voinnet, 2006). Among the CaMV-encoded proteins, the ORF6 product, called P6, is a symptom and host range determinant that was recently found to suppress transgene RNA silencing, suggesting that it is a VSR (Love et al, 2007). P6 is mandatory for translational transactivation of the 35S RNA, and is, therefore, vital for CaMV accumulation in single cells (Kobayashi et al, 1998). The most part of P6 aggregates into large, amorphous cytoplasmic bodies called viroplasms (Kobayashi et al, 1998; Haas et al, 2005), considered so far essential for virus assembly, replication, protein synthesis and, possibly, silencing suppression. Nonetheless, a small fraction of P6 is also found in the nucleus of infected cells, but its mode of import and function(s) into this organelle are unknown (Haas et al, 2005). Using a combination of cell biology, genetics and biochemistry, we address these questions and show that the small nuclear fraction of P6 is essential for CaMV infection, independently of its roles in translational transactivation or viroplasm formation. Our results indicate that nuclear P6 is a VSR that genetically and physically interacts with DRB4, a nuclear protein that interacts with, and facilitates the activity of the antiviral enzyme DCL4. Results Two nuclear localization signals are cooperatively required for nuclear import of monomeric P6 molecules through the importin α pathway Use of N-terminal green fluorescent protein fusions (GFP–P6; Figure 1) shows that P6 shuttles between the cytoplasm and the nucleus of bombarded BY-2 cells and forms nuclear speckles in authentic infection context (Haas et al, 2005). Nonetheless, most of GFP–P6 is normally found in cytoplasmic, perinuclear bodies structurally identical to the viroplasms found in CaMV-infected cells (Figure 1A, top panel; Haas et al, 2005). This prevalent cytoplasmic P6 distribution is contributed by the N-terminal domain (domain A, aa 1–111), which contains a nuclear export signal (NES) overlapping with residues required for P6–P6 intermolecular interactions and viroplasm formation (Haas et al, 2005). To address the mechanisms of P6 import and its possible role(s) in the nucleus, we used GFP–P6ΔA, which localizes exclusively to the nucleus owing to a deletion of domain A (Figure 1A, middle panel; Haas et al, 2005). The molecular mass of GFP–P6ΔA (85 kDa) is incompatible with a passive diffusion across nuclear pores, suggesting that the region between aa 112 and 520 contains at least one nuclear localization signal (NLS) for P6 active import. Scanning P6 sequence, we found that the KYLPKKVKDAVKRFR motif between aa 314 and 328 defines a bipartite basic NLS frequently found in plant proteins, and shares homologies with the NLS of human ribosomal protein L22 (Figure 1A, middle panel; Shu-Nu et al, 2000). Deleting this motif in GFP–P6ΔA indeed caused cytoplasmic redistribution and partial loss of nuclear import of the resulting protein GFP–P6ΔAΔNLS (Figure 1A, lower panel). Figure 1.(A) Schematic of P6 and its functional domains (coloured boxes). NES: nuclear export signal; domain A: P6–P6 interaction; mini-TAV: minimal domain for translational transactivation; ssRNA: single-stranded RNA binding; dsRNA: double-stranded RNA binding. The various GFP–P6 alleles were transiently expressed into BY-2 tobacco cells and observed under confocal microscope 18 h post-bombardment. Images on the left show GFP detection and those on the right are composite images of green fluorescence and DIC. Position and amino-acid sequence of the putative basic bipartite NLS are indicated. Similarities with the NLS of human ribosomal protein L22 are shown in yellow. (B) P6 regions (a–c) showing homology with non-conventional, viral NLSs (red: influenza virus 1 NP protein; yellow and green: Borna disease virus P10 protein). The effects of their deletion (singly or in combination) on P6ΔA distribution are depicted on the right. (C) Cellular localization of GFP–P6m1 (leucine substitution in red) and P6m1ΔNLSΔa. (D) Cellular localization of GFP–GUS in the absence/presence of individual P6 NLS, or combination thereof. (E) GST pull down of P6 and rice importins. Radiolabelled P6 was incubated either with importin α (GST-Imp α), β (GST-Imp β) or GST alone (GST). Following SDS–PAGE, P6 was detected by autoradiography (upper panel) and total proteins by Coomassie blue staining (lower panel). Download figure Download PowerPoint The residual nuclear GFP signal in GFP–P6ΔAΔNLS-bombarded cells suggested that additional sequences are required for complete P6 import, as often observed for viral nuclear proteins. Further inspection of P6 identified three regions with homologies to known, non-conventional NLSs of viruses. NLS-a (spanning aa 219–234) contains the GTKR-S motif recruited by importin α1 for nuclear import of influenza A virus NP protein (Wang et al, 1997). The second and third sequences (NLS-b and NLS-c, respectively) display similarities with the importin α-dependent NLS of Borna disease virus P10 protein (Figure 1B; Wolff et al, 2002). To test the contribution of NLS-a, -b and -c to P6 import, their individual deletions or combinations thereof were engineered into GFP–P6ΔA, and subcellular distribution of the resulting proteins monitored in bombarded BY-2 cells (Figure 1B). Deletion of NLS-a alone caused a nucleo-cytoplasmic redistribution resembling that of GFP–P6ΔAΔNLS, except for the nucleolus (Figure 1B, panel 1). Nuclear import of GFP–P6ΔA, remained, however, unaltered by NLS-b or NLS-c deletions (Figure 1B, panels 2 and 3), although nucleolar localization was partially prevented. Neither the NLS-b nor the NLS-c deletion caused a change in the nucleo-cytoplasmic distribution of GFP–P6ΔAΔNLS (Figure 1B, panels 5 and 6). By contrast, its nuclear localization was abolished by the NLS plus NLS-a deletion (GFP–P6ΔAΔNLSΔa; Figure 1B, panel 4). Collectively, the results suggest that, among the three predicted non-conventional NLSs, only NLS-a cooperates with the bipartite NLS for GFP–P6ΔA import. Additional experiments further indicated that P6 molecules are likely imported into the nucleus as monomers (Supplementary data). To rule out that the large deletion in domain A artificially influenced localization of GFP–P6ΔA and its derivatives, we used construct GFP–P6m1 in which functionality of the NES within domain A is strongly reduced, albeit not eliminated, by three point mutations in essential leucine residues (Haas et al, 2005; Figure 1C). Consequently, GFP–P6m1 localizes to the cytoplasm and the nucleus. Deleting the bipartite NLS and NLS-a in GFP–P6m1 (leading to GFP–P6m1ΔNLSΔa) abolished nuclear localization, reinforcing the idea that both sequences are indeed necessary for P6 import. We then measured the ability of each sequence, or combination thereof, to promote nuclear import of the unrelated and strictly cytoplasmic GFP–GUS fusion protein (120 kDa; Figure 1D, top panel). Each sequence individually contributed to GFP–GUS import, but their effect was only partial and consistently stronger with NLS-a (Figure 1D, middle panels). By contrast, GFP was strictly nuclear when both sequences were introduced (Figure 1D, lower panel), indicating that the bipartite NLS and the non-conventional NLS-a are sufficient to cooperatively mediate full nuclear import. NLS-dependent nuclear import in plants occurs mostly through importin α (Smith and Raikhel, 1998). To address this point, we used GST fusions of rice importin α1 and β1 immobilized onto glutathione-coupled sepharose (GST–importin (Imp) α and GST–Imp β, respectively). A P6 variant carrying an N-terminal hexapeptide allowing its phosphorylation was expressed in Escherichia coli and radiolabelled in vitro with 32P-γ-ATP. Previous work showed that P6 is specifically labelled among the proteins produced in this recombinant E. coli strain (Leh et al, 2000). Labelled P6 was incubated with sepharose beads coupled to GST alone, GST–Imp α or GST–Imp β, and protein complexes resolved on SDS–PAGE. As shown in Figure 1E, the P6 signal (62 kDa) and a fainter signal corresponding to P6 homodimers (∼120 kDa) were only detected in fractions incubated with GST–Imp α. Therefore, nuclear import of P6 by the bipartite NLS and the non-conventional NLS-a is likely through the importin α pathway. A minor part of P6 import could also occur through interactions between the miniTAV domain (Figure 1A) and the L13, L18 and presumably other ribosomal proteins. However, this process causes P6 retention within the nucleolar, as opposed to the nucleoplasmic compartment (Supplementary data). A nuclear function of P6 distinct from translational transactivation is essential for CaMV infectivity An essential function of P6 in CaMV biology is to transactivate translation of the polycistronic 35S RNA and its derivatives through the mini-TAV domain (Pooggin et al, 2000; Figure 1A). To get insights into possible nuclear role(s) of P6 in this process, we created additional alleles of GFP–P6ΔA carrying point mutations in conserved amino acids of the non-conventional NLS-a. Some, or all of the GTKRPS residues were replaced by valines or alanines in constructs GFP–P6ΔAVVV and GFP–P6ΔAVVVVAV, respectively (Figure 2A). Compared to the strict nuclear localization of GFP–P6ΔA (Figure 1A, middle panel), GFP–P6ΔAVVV had a nucleo-cytoplasmic pattern, whereas nuclear import was nearly completely abolished with GFP–P6ΔAVVVVAV (Figure 2A), resembling GFP–P6ΔAΔNLSΔa distribution (Figure 1B, panel 4). We then re-inserted the N-terminal A domain into the above P6 alleles to generate P6VVV and P6VVVVAV and test their ability to re-initiate translation of a bicistronic GUS reporter gene, using a transactivation assay in Nicotiana plumbaginifolia protoplasts (Pooggin et al, 2000). In this assay, a second, monocistronic reporter gene encoding the chloramphenicol acetyl transferase (CAT) is used as an internal transfection and translation control. Transactivation values obtained with wild-type (WT) P6, arbitrarily set to 100%, served as a reference for the activity of P6 variants. In two independent experiments, neither P6ΔNLSΔa nor P6VVVVAV could transactivate GUS expression, whereas transactivation by P6VVV was 82% that of WT P6 (Figure 2B). Figure 2.(A) Effect of point mutations in NLS-a (red) on GFP–P6ΔA localization. (B). N. plumbaginifolia protoplasts were co-transfected with constructs expressing P6, CAT and GUS, under the control of the CaMV 35S promoter. The GUS ORF cloned downstream of the CaMV ORF VII is used to monitor translational transactivation. CAT expression is an internal control for translation and transfection efficacy. The effects of P6, or mutated version thereof, on GUS expression are represented by histograms. GUS activity in the presence of WT P6 was arbitrarily set to 100%. The data are from two independent experiments. (C) Schematic of the pGH recombinant viral vector. ORF VI variants can be inserted owing to the SacI/KpnI restriction sites. The indicated P6 variants were cloned in pGH and inoculated to Arabidopsis upon linearization with SalI. Plants were monitored for symptom formation over an 18-day period, upon which tissues were collected for analysis of the CaMV coat protein P4. The ratio of infected to inoculated plants is shown. Coomassie blue staining shows equal protein loading. (D) Schematic of the GFP–P6 and GFP–P6m3 proteins and analysis of their localization in BY-2 cells 18 h post-bombardment. Both alleles were introduced into pGH and infections were monitored as in (C). Download figure Download PowerPoint To test the three P6 alleles in infection contexts, we engineered a DNA-based viral vector (pGH) allowing insertion of ORF VI variants (Figure 2C, diagram). Arabidopsis plants inoculated with pGH:P6, carrying the WT P6 allele, showed typical chlorotic and leaf curling symptoms. Moreover P4 (coat-protein) accumulation in systemically infected tissues was the same in pGH:P6 as it was in CaMV-infected plants, confirming infectivity of the pGH:P6-derived virus (Figure 2C). By contrast, plants inoculated with pGH carrying either of the three P6 mutant alleles remained symptomless, and P4 was undetectable in systemic tissues (Figure 2C). This was notably the case for pGH:P6VVV, carrying a transactivation-proficient P6 allele (Figure 2B). Thus, partial loss of P6 nuclear localization caused by the GTK → VVV substitution in NLS-a (Figure 2A) was sufficient to abolish CaMV infectivity without significantly altering transactivation. Lack of infectivity was also observed with pGH:P6m1 (Figure 2D, right panel), carrying a triple mutation that compromises P6 nuclear export (Figure 1C). Unlike mutations affecting P6 nuclear import (Figures 1 and 2), m1 also alleviates viroplasm formation (Figure 1C). Viroplasms normally contain the vast majority of P6 molecules and have been so far considered essential for viral replication and particle morphogenesis or storage (Haas et al, 2002). Thus, to discriminate which impaired function (viroplasm formation or nuclear export) accounted for the lack of pGH:P6m1 infectivity, we used P6m3, carrying the EKI → AAA amino-acid substitution in the NES motif of domain A (Figure 2D, left panel). GFP–P6 fusions carrying this mutation (GFP–P6m3) display intact nuclear export but fail to form viroplasms in BY-2 cells (Haas et al, 2005; Figure 2D). In contrast to pGH:P6m1, pGH:P6m3 was fully infectious in Arabidopsis despite complete absence of detectable viroplasms in infected tissues (Figure 2D; Haas et al, in preparation). Therefore, although viroplasms are apparently dispensable for CaMV infection, P6 nuclear export is essential for this process. We conclude from the compared analysis of its import- and export-deficient alleles that nucleo-cytoplasmic shuttling of P6 is mandatory for infection, and that P6 exerts in the nucleus one or several essential functions distinct from its role in translational transactivation. Nuclear import is required for RNA silencing suppression by transgenic P6 Recently, P6 was found to suppress transgene silencing (Love et al, 2007). Therefore, we set out to determine whether P6 nuclear localization is required for its VSR function, which could explain the loss of infectivity of pGH variants expressing nuclear import-deficient P6 alleles (Figure 2C and D). WT P6 was cloned under the CaMV 35S promoter and transformed into the Arabidopsis AMPLICON (AMP) line, in which a transgene expressing GFP-tagged and replicating PVX RNA (PVX–GFP; Figure 3A) is both an initiator and a target of RNA silencing (Dalmay et al, 2000). In the AMP line (ecotype C24), PVX–GFP RNA levels are vastly reduced, plants appear uniformly red under UV illumination, owing to chlorophyll fluorescence (Figure 3B, upper panel); and viral-derived siRNAs remain undetectable (Figure 3B, lane 2). Figure 3.(A) Schematic of the PVX–GFP transgene in the Arabidopsis AMP line. (B). Restauration of GFP accumulation upon transgenic expression of P6 and P38 in the AMP line (left panel). The red colour is from chlorophyll autofluorescence under UV and signifies GFP silencing. High molecular weight (HMW) RNA and siRNA were extracted and detected using a GFP-specific probe (right panel). rRNA: ethidium bromide staining of ribosomal RNA; gRNA and sgRNA: genomic and subgenomic PVX–GFP RNA respectively. (C) Leaves of AMP plants expressing P6, P6Δa or P6ΔaΔNLS, under UV light. (D) Accumulation of P6 or its variants was detected by western blot analysis using a P6 antiserum (P6, upper panel). Prot: Coomassie blue staining of total protein. PVX–GFP-derived siRNA were detected as in (B) (lower panel). (E) HMW RNA analysis of PVX gRNA and sgRNA in AMP lines expressing P6 and its variants, as in (B). Download figure Download PowerPoint As a positive control, we used line AMP-P38 (Moissiard et al, 2007) in which green fluorescence, PVX–GFP and 21-nt-long viral siRNA accumulation are all restored by expression of the DCL4 antagonist P38 VSR of Turnip crinkle virus (Figure 3B, middle panel, lane 1). Whereas P38, similar to P6 (see below), strongly reduces DCL4-dependent tasiRNA accumulation, its paradoxical enhancing effect on the levels of AMP-derived siRNA is likely due to the dramatic increase in PVX–GFP replication and subsequent dicing, by DCL4, of highly abundant viral dsRNA (Moissiard et al, 2007). Independent T2 AMP-P6 lines were isolated and P6 expression was confirmed by western blot analysis. The results in Figure 3 are representative of several independent P6 lines showing comparable expression levels. In all cases, AMP-P6 plants had visual and molecular phenotypes resembling those of AMP-P38 plants (Figure 3B, lower panel, lane 3), indicating that transgenic P6 suppresses AMP silencing nearly to the same extent as transgenic P38. To test if P6 nuclear import is required for silencing suppression, we transformed the AMP line with the P6Δa or P6ΔNLSΔa alleles, expected to be partially and completely excluded from the nucleus, respectively (Figure 1). Transgenic lines were selected to express similar levels of P6 and its variants (Figure 3D). Of note, only two lines expressing the P6ΔNLSΔa allele to significant levels could be obtained in multiple transformation attempts, suggesting cellular toxicity. The silencing suppression phenotype of AMP-P6Δa lines was consistently less pronounced than in AMP-P6 lines: green fluorescence was less extensive in plant tissues (Figure 3C, middle panel compared to left panel) and AMP-derived siRNAs accumulated slightly less than in AMP-P6 plants (Figure 3D, lane 3 compared to lane 1). Accordingly, reactivation of PVX–GFP replication was less pronounced in AMP-P6Δa compared with AMP-P6 plants (Figure 3E, lanes 1 and 3). The AMP-P6ΔNLSΔa lines remained completely red fluorescent (Figure 3C, right panel), whereas siRNAs and PVX–GFP replication products were below detection limit (Figure 3D and E, lane 4). We conclude that silencing suppression by transgenic P6 requires its nuclear import. P6-mediated suppression of the tasiRNA pathway requires nuclear import, but none of the previously characterized features of the protein Viral symptom expression, viroplasm formation and dsRNA binding through the mini-TAV domain are verified or suspected biochemical features of P6 that could all contribute to its VSR function (discussed in Ding and Voinnet, 2007). To address this issue, we established transgenic lines in WT Arabidopsis (ecotype Col-0) expressing either P6, P6ΔdsR, P6m3, P6Δa or P6ΔNLSΔa from the CaMV 35S promoter, and tested the effect of these various alleles on accumulation of endogenous small silencing RNA. These experiments were not carried out in the AMP background because high PVX–GFP replication caused by silencing suppression (Figure 3) results in the accumulation of the PVX-encoded VSR protein P25, which can alter endogenous silencing pathways (Moissiard et al, 2007). Plants expressing WT P6 displayed strong stunting, leaf serration and a ‘silvering’ chlorosis resembling CaMV-elicited symptoms (Figure 4A–C). This agrees with work in ecotype Ler (Love et al, 2007) and confirms that P6 is a major symptom determinant. By contrast, none of the transgenic lines expressing the various P6 mutant alleles displayed these phenotypic defects (Figure 4D), despite accumulation of comparable P6 levels (Figure 4E, left panel). Figure 4.(A, B) P6 transgenic Arabidopsis ecotype Col-0 (B) are stunted, chlorotic and exhibit leaf serration compared with their non-transgenic counterparts (A). (C) Magnified view of (B) showing the ‘silvery’ chlorosis developing on older leaves. (D) None of the P6 mutants induce developmental anomalies when expressed transgenically in the Col-0 ecotype. (E) Upper panel: western blot analysis of P6 accumulation in plants depicted in (A–D), as in Figure 3D. Lower panel: LMW RNA analysis in the corresponding genotypes using probes for tasiRNA255, miR173 and miR156. Download figure Download PowerPoint We then measured accumulation of endogenous tasiRNAs in the various transgenic lines. In tasiRNA biogenesis, the initial miRNA-guided cleavage of primary tasiRNA transcripts sets a defined point for RDR6-directed complementary strand synthesis, followed by a phased DCL4-dependent processing reaction that generates mature tasiRNAs (Yoshikawa et al, 2005; Adenot et al, 2006). Accumulation of tasiRNA255 from the TAS1 locus was decreased but still detectable in the P6Δa transgenic lines compared with non-transgenic plants (Figure 4E, left panel, lanes 3 and 6). It was strongly" @default.
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• W4248473821 title "Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4" @default.
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