Coral bleaching is a stress response of corals induced by a variety of factors, but these events have become more frequent and intense in response to recent climate-change-related temperature anomalies. We tested the hypothesis that coral reefs affected by bleaching events are currently heavily infested by boring sponges, which are playing a significant role in the destruction of their physical structure. Seventeen reefs that cover the entire distributional range of corals along the Mexican Pacific coast were studied between 2005/2006, and later between 2009/2010. Most of these coral reefs were previously impacted by bleaching events, which resulted in coral mortalities. Sponge abundance and species richness was used as an indicator of bioerosion, and coral cover was used to describe the present condition of coral reefs. Coral reefs are currently highly invaded (46% of the samples examined) by a very high diversity of boring sponges (20 species); being the coral reef framework the substrate most invaded (56%) followed by the rubbles (45%), and the living colonies (36%). The results also indicated that boring sponges are promoting the dislodgment of live colonies and large fragments from the framework. In summary, the eastern coral reefs affected by bleaching phenomena, mainly provoked by El Niño, present a high diversity and abundance of boring sponges, which are weakening the union of the colony with the reef framework and promoting their dislodgment. These phenomena will probably become even more intense and severe, as temperatures are projected to continue to rise under the scenarios for future climate change, which could place many eastern coral reefs beyond their survival threshold. Climate-driven coral bleaching has emerged as one of the greatest threats to coral reef ecosystems  , to 2°S, including the mainland coast of Ecuador and the southern Galapagos Islands  distributed along the Mexican Pacific coast Fig.  as “the The most important coral reefs in terms of their state of conservation and extension are in Oaxaca and Guerrero (southern region of Mexico)  harbors important coral reefs with at least 10 species of scleractinian corals, mainly belonging to the genus Pocillopora. The Careyeros fringing reef extended up to 1.5 km along the shore and occupied approximately 83 Ha before the 1997/98 ENSO  , which is representative of the central Mexican Pacific coral reef area. It was only possible to maintain this program in this reef because we were able to revisit it periodically . Thus, at the start of this study, we placed two HOBO Water Temperature devices at 6 m depth in the center of the reef, programmed to record water temperature every 6 h, as temperature loggers positioned within the marine communities are more representative of the water temperatures affecting the coral reef than satellite generated data  at approximately every 5-s of filming. Thus, a subset of approximately 40–50 random nonoverlapping images per transect were extracted, which represent approximately one image each 0.5 m interval. This method resulted in a coverage area of 10–12 m² per transect, which is enough to document changes in reef cover Haralson .Each image was analyzed using CPCe  software developed by the National Coral Reef Institute (FL), using 100 random dots. This program was specially developed to meet the needs of the video-transect method , a small fragment of the coral reef framework (CRF), and a complete branch of a live coral colony (LCC) (from the tip to the stem) from the colony closest to the transect, were collected at random, approximately every 2 m, yielding 75 pieces per transect (25 pieces of each category) .Differences in coral invasion by boring sponges over time were analyzed using a one-way analysis of variance (ANOVA) after verifying normality (Kolmogorov–Smirnov test)  had the lowest richness; 17 and 10 spp, respectively Fig. . HoweverCliona vermifera, which was found in 42% of the samples analyzed, followed by Thoosa mismalolli, present in 13% of the fragments, Cliona tropicalis in 9%, and Pione carpenteri in 8% , followed by C. tropicalis (17.6%), A. cryptica (15.6%), and P. carpenteri (11.3%).Regarding the abundance, the most important species was  8% Fig. . There wCliona vermifera is the most abundant and amply distributed species. As shown in Fig. C. vermifera does not show any preference of habitat, and it can live in the same way in coral colonies, unstable coral rubble, and coral framework. In contrast, there are others species, such as C. tropicalis that prefer to live in the basal stem of the coral colonies instead of in the coral rubble  that prefer to live inside of coral rubble or in the matrix  Fig. , Table 3Boring sponges account for 70% or more of bioerosional damage to skeletons of hermatypic corals in both the Western Pacific and Caribbean regions, accounting for 65–90% of total bioerosion occurring within the first 2 cm of coral skeleton (Highsmist 1981). We do not have similar information in the east Pacific coral reefs yet, but the fact that 46% of the fragments of the coral studied were invaded by boring sponges suggests that they are currently playing a significant role in the destruction of the physical structure of Mexican Pacific corals, which could extrapolate to the eastern Pacific Ocean, since the study area represents 46% of the coral distribution of the Eastern Pacific Ocean.Thus, the most important results of this study was the high diversity (20 species) and abundance of boring sponges (46% of the fragments were invaded) that we found in all coral reefs, especially in those that suffered the massive bleaching during El Niño 97/98, such as Careyeros, Antiguo Corral del Risco, and Cabo Pulmo Table . It is kOn the other hand, although sponge abundance has not increased over time, the high levels of sponge invasion recorded in almost all the reefs studied seem to support our hypothesis. Massive bleaching have been recorded in Mexican pacific coral reefs several years before our study, the main one after El Niño 1997/98  after the bleaching of 1987   after an important loss of coral cover by strong bleaching events in the central Great Barrier Reef  (Nava and Carballo Sponges are able to dissolve part of the carbonate during the bioerosion process (Rützler and Rieger 2-rich upwelling water masses at eastern Pacific area lead to naturally low pH and saturation states, that together with the associated decrease in coral calcification rate, as a result of lower aragonite saturation state, is likely to reduce the ability of reef builders to prevent and/or repair damage caused by bioeroders (Langdon et al. Modern scleractinian corals have existed on earth for over 240 million years Stanley , and cur


Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3′ end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation.Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces  Tomato bushy stunt virus (TBSV) replication in a yeast model host. Knock down of eEF1Bγ level in plant host also decreases TBSV accumulation. Moreover, the authors demonstrate that eEF1Bγ binds to the viral RNA and is present in the tombusvirus replicase complex. Functional studies revealed that eEF1Bγ promotes minus-strand synthesis by serving as an RNA chaperone. The authors also show that eEF1Bγ and eukaryotic translation elongation factor 1A, another host factor, function together to promote tombusvirus replication.RNA viruses recruit numerous host proteins to facilitate their replication and spread. Among the identified host proteins are RNA-binding proteins (RBPs), such as ribosomal proteins, translation factors and RNA-modifying enzymes. In this paper, the authors show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces  Plus-stranded (+)RNA viruses recruit numerous host proteins to facilitate their replication and spread Tomato bushy stunt virus (TBSV) is model plant RNA virus coding for two replication proteins, p33 and p92pol, which are sufficient to support TBSV replicon (rep)RNA replication in a yeast (Saccharomyces cerevisiae) model host pol are components of the membrane-bound viral replicase complex, which also contains the tombusviral repRNA serving not only as a template for replication, but also as a platform for the assembly of the viral replicase complex Various proteomics analyses of the highly purified tombusvirus replicase has revealed at least five permanent resident host proteins in the complex, including the heat shock protein 70 chaperones (Hsp70) The emerging picture from systems biology approaches is that eukaroyotic translation elongation factors (eEFs), such as eEF1A, play several roles during TBSV replication. Accordingly, eEF1A has been shown to facilitate the assembly of the viral replicase complex and stimulate the initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) in vitro replication assays reveal that eEF1Bγ is a component of the tombusvirus replicase and binds to the 3′-end of the viral RNA. Using a cell-free replication assay, we define that eEF1Bγ plays a role by enhancing minus-strand synthesis by the viral replicase. The obtained data support the model that eEF1Bγ opens up a ‘closed’ structure at the 3′-end of the TBSV (+)RNA, rendering the RNA compatible for initiation of (-)-strand synthesis. Moreover, we find that eEF1Bγ and eEF1A play nonoverlapping functions to enhance (-)-strand synthesis. Altogether, the two translation factors regulate TBSV replication synergistically by interacting with different portions of the viral (+)RNA and the replication proteins.In this paper, we characterize the function of eEF1Bγ in TBSV replication. Our approaches based on yeast and TEF3 and TEF4 nonessential genes in yeast TEF3(CAM1) or TEF4 reduced TBSV repRNA accumulation to ∼25% -stranded repRNA and excess amount of (+)-stranded progeny To obtain direct evidence on the involvement of eEF1Bγ in TBSV replication, we prepared cell-free extracts (CFE) from a yeast strain lacking the tef4Δ yeast supported only 29% of TBSV repRNA replication when compared with the extract obtained from wt yeast (CFE obtained from in vitro was due to reduced (+) or (-)-strand synthesis, we measured the replication products under non-denaturing versus denaturing conditions (32P-labeled (-)RNA product hybridized with the input (+)RNA; lane 1, tef4Δ yeast in comparison with those products in the wt CFE (lane 3). Since the ratio of dsRNA and ssRNA did not change much in the CFEs . The TCV RdRp, unlike the E. coli-expressed TBSV p92pol or the closely-related Cucumber necrosis virus (CNV) p92pol RdRps, does not need the yeast CFE to be functional in vitroE. coli does not have a homolog), unlike the yeast or plant-derived tombusvirus replicase preparations, facilitating studies on the role of eEF1Bγ on the template activity of a viral RdRp. When we added various amounts of the highly purified recombinant eEF1Bγ to the TCV RdRp assay programmed with TBSV-derived SL3-2-1(+) RNA template, which is used by the TCV RdRp in vitro to produce the complementary (-)RNA product 32P-labeled RNA product, excluding that our eEF1Bγ preparation contained RdRp activity (not shown). Altogether, our data suggest that eEF1Bγ can stimulate in vitro activity of TCV RdRp on a TBSV (+)RNA template, confirming a direct role for eEF1Bγ in viral (-)RNA synthesis by a viral RdRp.To obtain additional evidence if eEF1Bγ could stimulate RNA synthesis by the viral RdRp, we used the in vitro RdRp activity was due to binding of eEF1Bγ to the (+)RNA template and/or to the TCV RdRp protein, we performed assays, in which the recombinant eEF1Bγ was pre-incubated with the TCV RdRp or the (+)RNA template prior to the RdRp assay. These experiments revealed that pre-incubation of the purified eEF1Bγ with the TBSV-derived SL3-2-1(+) RNA template prior to the RdRp assay led to a ∼4.5-fold increase in (-)RNA products RNA template. The mutation [SL3-2-1m(+)] opens up the closed structure in the promoter region that leads to increased template activity 32P-labeled regions of (+)repRNA that included known cis-acting elements involved in (-)RNA synthesis in vitro experiments with purified eEF1Bγ and  in vitro.To further define what sequence within SL3-2-1 is bound by eEF1Bγ, we used complementary DNA oligos to partially convert portions of SL3-2-1 into duplexes (RNA/DNA hybrids) as shown in in vitro. However, we cannot exclude that eEF1Bγ binding may be dependent on stabilizing effects of the GNRA tetraloop on the stem structure. The loop nucleotides may or may not be involved in protein-RNA contacts.Since eEF1Bγ is known to bind to A-rich single-stranded sequences in vitro RNA synthesis assay using a mutated SL3-2-1 carrying the ‘CUUG’ tetraloop instead of the wt ‘GAAA’ tetraloop sequence (in vitro.To examine if binding of eEF1Bγ to SL1 is important for stimulation of (-)-strand RNA synthesis by the viral RdRp, we performed an sequence . Unlike 6-Flag-tagged p33 (HF-p33) replication protein via Flag-affinity purification from the detergent-solubilized membrane fraction of yeast 6-tagged p33 (H-p33) that were also purified via the Flag-affinity procedure  fraction of yeast. Interestingly, the viral RNA was co-purified with eEF1Bγ from both fractions (To demonstrate that eEF1Bγ can indeed bind to the TBSV (+)repRNA in cells, we Flag-affinity-purified HisSince eEF1Bγ was found in association with the TBSV repRNA in the cytosolic fraction of yeast, it is possible that eEF1Bγ might affect the viral RNA recruitment from the cytosol into replication that takes place on the peroxisomal or ER membrane surfaces in vitro RdRp assays with purified eEF1A and recombinant eEF1Bγ as shown in Since both eEF1Bγ and eEF1A bind to the 3′-terminal region of the TBSV (+)RNA  and ref:Nicotiana bethamiana leaves via VIGS (virus-induced gene silencing). Efficient knocking down of eEF1Bγ mRNA level in N. benthamiana (Tobacco rattle virus (TRV) vector. The lethal necrotic symptoms caused by TBSV in N. benthamiana were also greatly attenuated in the eEF1Bγ knock-down plants  RNA  RNA . We founTMV) RNA . AccumulTMV) RNA  and systTMV) RNA  leaves owww.yeastgenome.org). For example, eEF1A, which is also a permanent member of the tombusvirus replicase, is known to interact with eEF1Bγ in vitro data RNA viruses, subvert a yet unknown number of host-coded proteins to facilitate robust virus replication in infected cells. The co-opted host proteins could be part of the viral replicase complexes and provide many yet undefined functions. Translation factors, such as eEF1Bγ and eEF1A, are among the most common host factors recruited for (+)RNA virus replication tro data . Indeed,tro data .in vitro by using a cell-free extract prepared from tef4Δ yeast that supported (-)-strand RNA synthesis ∼3-fold less efficiently than CFE from wt yeast -strand synthesis by binding to the viral (+)RNA template (in vitro (We confirmed a direct role for eEF1Bγ in RNA synthesis wt yeast . Moreovetemplate . Accorditemplate . On the in vitro . We propIn vitro initiation of (-)-strand synthesis by the viral RdRp requires the gPR promoter consisting of a short 3′-terminal single-stranded tail and a stem-loop (SL1) sequence in vitro assay due to the difficulty for the viral RdRp to recognize and/or open the ‘closed’ structure in vitro assay; (iii) stimulation of (-)-strand synthesis when eEF1Bγ was pre-incubated with the (+)-strand template, but not when eEF1Bγ was pre-incubated with the viral RdRp -strand synthesis could make the 3′-terminus of the (+)-strand RNA fold back into a ‘closed’ structure. This could prevent efficient re-utilization of the original (+)-strand template during TBSV replication, and the switch to efficient (+)-strand synthesis on the (-)RNA intermediate . This moOur finding of TBSV RNA binding by eEF1Bγ adds to the growing list of RNAs bound by eEF1Bγ. For example, the 3′ UTR of vimentin mRNA is bound by eEF1Bγ Turnip yellow mosaic virus (TYMV) Brome mosaic virus, and Turnip mosaic virusTranslation elongation factors seem to be important for replication of many RNA viruses. For example, EF-Tu and EF-Ts play a role in replication of bacteriophage Qbeta pol RdRp and the SL3 region of TBSV (+)repRNA The emerging picture on the functions of eEF1Bγ and eEF1A is that these translation elongation factors play different, yet complementary roles in TBSV replication as suggested in N. benthamiana  and/or locations in yeast cells. Silencing of eEF1Bγ in N. bethamiana showed even more inhibition of TBSV RNA accumulation than deletion of eEF1Bγ genes in yeast. This is likely due to the robust antiviral response  of the plant host, which could result in degradation of the small amount of viral RNA produced by the less efficient viral RNA replication in the presence of limited eEF1Bγ in the knock-down plants.The current work also provides evidence that eEF1Bγ is a key factor in TBSV replication in yeast  and in Nthamiana . Since eN. benthamiana also reduced the accumulation of the unrelated TMV  enhSaccharomyces cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the single-gene deletion strain of the TEF4-encoded form of eEF1Bγ (tef4Δ) were obtained from Open Biosystems . TKY680 strain in which both yeast encoded eEF1Bγ, TEF4 and TEF3 were deleted (MATa ura3-52 leu2Δ1 his3Δ200 trp1Δ101 lys2-801 tef3::LEU2 tef4::TRP1) and its isogenic wild type TKY677 (MATa ura3-52 leu2Δ1 his3Δ200 trp1Δ101 lys2-801) as well as the isogenic single deletion mutant strains, TKY678 (MATa ura3-52 leu2Δ1 his3Δ200 trp1Δ101 lys2-801 tef3::LEU2) and TKY 679 (MATa ura3-52 leu2Δ1 his3Δ200 trp1Δ101 lys2-801 tef4::TRP1) were published previously URA3 based pGBK-ADH- Hisp33/GAL1-DI72, pGBK-CUP1-HisFLAGp33/GAL1-DI-72, and pGBK-CUP1- Hisp33/GAL1-DI-72 plasmids were constructed by Daniel Barajas (unpublished result). The URA3 based, low copy-number plasmid, pYC-GAL1-Tef4 expressing non-tagged full-length Tef4 protein was constructed as follows: pYC/NT-C plasmid was digested with BamHI and XhoI restriction enzymes and then PCR product of the TEF4 gene was generated with primers #2089 (ccgcGGATCCATGTCCCAAGGTACTTTATAC) and #2320 (CGCCTCGAGTTATTTCAAAACCTTACCGTCAACAATTTCC) and digested with the same restriction enzymes, followed by ligation. The plasmid pYES-NTC2-GAL1-HisTef4 expressing His6-tagged Tef4p protein was created with the same restriction enzymes using pYES-NT-C2.HIS3-based pEsc-His/Cup-FLAG plasmid BamHI and XhoI restriction enzymes and then PCR product of the TEF4 gene was generated with primers #2089 and #2320 and digested with the same restriction enzymes, followed by ligationto obtain pEsc-His/Cup-FLAG-TEF4.HIS3 based pESC-GAL1-His33/GAL10-DI-72 and LEU2 based pGAD-CUP1-Hisp92 plasmids were transformed into tef4Δ strain. In the in vivo complementation assay, non-tagged Tef4p protein was expressed from URA3 plasmid pYC-GAL1-Tef4 and TEF4 mRNA was detected with a specific probe generated by the T7 transcription of the PCR product obtained with primers #2089 and #3788 (TAATACGACTCACTATAGGATTATTTCAAAACCTTACCGTCAACAATTTCC).tef3Δ/tef4Δ), the isogenic TKY679 (tef4Δ), TKY678 (tef3Δ) and wild type TKY677 yeast were transformed with plasmids pESC-GAL1-His33/GAL10-DI-72 and pCM189-TET-His92. Yeast was pre-grown at 23°C overnight in 3 ml synthetic complete dropout medium lacking the relevant amino acids containing 2% glucose and 1 mg/ml doxycyclin to suppress p92 expression by the inhibition of TET promoter and then TBSV replication was launched by replacing the media with 2% galactose without doxycycline. Cells were harvested at 48 h time point. Total RNA extraction from yeast cells and Northern blotting and Western blotting were done as previously described TKY680 (tef4Δ strain. Yeast was pre-grown overnight at 29°C in 2 ml synthetic complete dropout medium lacking histidine (SC-H- medium) containing 2% glucose. The volume of the media was increased up to 100 ml 16 h later and copper sulfate was added to a final concentration of 50 µM for induction of protein expression. Yeast was grown to 0.8 OD600 (∼4–6 h). Then, yeast cells were harvested and broken by glass beads in a FastPrep cell disruptor followed by Flag-affinity purification of FLAG-Tef4p protein 6-tagged Tef3 protein from plasmid pTKB523 was performed as described in ref: pEsc-His/Cup-FLAG-TEF4 plasmid was transformed into in vitro was prepared as described 32P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.5x Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85°C for 5 min in the presence of 50% formamide Yeast extract capable of supporting TBSV replication in vitro activity of Tef4p, different concentrations (26 and 13 pmol) of purified FLAG/His6-Tef4p was added to 0.25 µg (4 pmol) DI-72 (+)repRNA transcript and incubated in the presence of yeast cell-free extract and reaction buffer for 10 minutes at RT followed by the addition of MBP-p33 and MBP-p92 along with the rest of the reaction components. The reaction was performed at 25°C for 3 h and analyzed as above.To test the E. coli or Flag-affinity purified HF-Tef4p obtained from yeast) were added to the reaction at the beginning or as indicated in the text and 32P-labeled RNA products were analyzed by electrophoresis in a 5% PAGE/8 M urea gel TAATACGACTCACTATAGGACACGGTTGATCTCACCCTTC) and #1190 (GGGCTGCATTTCTGCAATG) for SL3-2-1(+), #1662 and #4390 (GGGCTGCACAAGTGCAATGTTCCGGTTGTCCGGT) for SL3-2-1cuug(+). SL3-2-1m(+) RNA was generated with T7 transcription on PCR products amplified with primers #1662 and #1190, on a plasmid template harboring GGGCU nucleotide-deletion in SL3 region as described GTAATACGACTCACTATAGGGCTACTTCCGGTTGTCCGGTAGTGCTTCC) and # 4362 (CGGTTGATCTGACCCTTCGG). For hybridization, equal amounts of both RNAs were mixed in 1X STE buffer [0.1 M NaCl 10 mM Tris-HCl (pH 8.0) 1 mM EDTA (pH 8.0)] followed by treatments: 94°C for 15 s, 70 cycles with gradually lowering the temperature by 1°C at each cycle for 30 s and finally 20°C for 30s.The TCV RdRp reactions were carried out as previously described for 2 h at 25°C tef4Δ strain with anti-FLAG M2-agarose affinity resin. Different concentrations  of HF-Tef4p protein was used for incubation with 0.2 pmol of 32P-labeled SL3/2/1(+) RNA or mutated RNAs at 25°C in a binding buffer . Samples were incubated at 25°C for 15 min, then resolved in 4% nondenaturing polyacrylamide gel E. coli (not shown).For EMSA, 6xHis-Flag tagged Tef4p was purified from a yeast tef4Δ strain was co-transformed with pGBK-ADH-Hisp33/GAL1-DI72, pGAD-CUP1-Hisp92 and pESC-CUP1-HisFLAG-Tef4. The pESC-CUP1-FLAGHis-Tef4 plasmid was replaced with the pESC plasmid in the control experiment. Yeast was pre-grown overnight at 29°C in 2 ml SCULH- medium containing 2% glucose and 5 µM copper sulfate. The volume of the media was increased to 20 ml after 16 h for an additional 10 h (OD600 of ∼0.8), then the cultures were transferred to 20 ml SCULH- medium containing 2% galactose to induce TBSV DI-72 RNA transcription at 23°C. The transcription of DI-72 RNA was stopped by changing to the media containing 2% glucose after 8 h. The cultures were diluted to 200 ml and copper sulfate was added to a final concentration of 50 µM to induce the expression of Flag-tagged Tef4 protein. After incubation at 23°C for 24 h, the samples were centrifuged at 3000 rpm for 4 min. Cells (∼1 g) were re-suspended in 2 ml TG Buffer  supplemented with 0.5 M NaCl and 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma) and RNase inhibitor (Ambion). Yeast cells were broken by glass beads in a FastPrep cell disruptor  4 times for 20 sec each at speed 5.5. Samples were removed and incubated 1 min in an ice-water bath after each treatment. The samples were centrifuged at 500 ×g for 5 min at 4°C to remove glass beads, unbroken cells and debris then supernatant was moved into fresh pre-chilled tubes. After being centrifuged again at 500 ×g for 5 min at 4°C supernatant transferred into fresh pre-chilled tubes and soluble (SU) and membrane (ME) fractions containing the viral replicase complex were separated with centrifugation at 35,000 ×g for 15 min at 4°C. The SU fraction was applied on 0.1 ml anti-FLAG M2-agarose affinity resin (Sigma) and Tef4 protein tagged with 6xHis- and FLAG affinity tags was purified. Before applying ME fraction on the anti-FLAG M2 resin, solubilization of the membrane-bound replicase was performed in 1 ml TG buffer with 0.5 M NaCl, 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma), and 2% Triton X-100 via rotation for 2 hours at 4 °C. The solubilized membrane fraction was centrifuged at 35,000 ×g at 4°C for 15 min and the supernatant was added to the resin pre-equilibrated with TG buffer supplemented with 0.5 M NaCl and 0.5% Triton X-100, followed by gentle rotation for 2 h at 4°C. The unbound proteins were removed by gravity flow, and the resin was washed two times with 1 ml TG buffer supplemented with 0.5 M NaCl, 0.5% Triton X-100 and once with 1 ml TG buffer, 0.5% Triton without NaCl. The bound proteins were eluted with 150 µl TG buffer without NaCl, 0.5% Triton X-100, supplemented with 150 µg/ml flag peptide and 1% yeast protease inhibitor cocktail via gentle tapping the column occasionally for 2 h at 4°C. After centrifugation at 600 ×g 2 min at 4°C, semi-quantitative RT-PCR was performed to detect TBSV repRNA co-purified with eEF1Bγ using primers, #359 (GTAATACGACTCACTATAGGAAATTCTCCAGGATTTC) and #1190, amplifying full length (+)repRNA.For the co-purification of TBSV DI-72 repRNA and eEF1Bγ protein, the yeast tef4Δ strain was transformed with pGBK-CUP1-HisFLAGp33/GAL1-DI-72, pGAD-CUP1-Hisp92 and pYES-GAL1-HisTef4. In the control experiment, 6xHisp33was expressed from pGBK-CUP1-Hisp33/GAL1-DI-72. Yeast cultures were grown in SC-ULH- media containing 1% raffinose and 1% galactose with 5 µM copper-sulfate for 4 days with increasing the volume of the culture from 2 ml to 100 ml to a final OD600 of∼ 1.0. After harvesting of cells, co-purification of 6xHis-tagged Tef4p with HF-p33  was conducted by using anti-FLAG M2-agarose affinity resin as described above (in the section: FLAG-affinity purification of eEF1Bγ-TBSV repRNA complex), with the exception that only solubilized ME fraction was loaded on the column. Proteins bound to affinity resin were eluted by incubation with 150 µl buffer containing FLAG peptide and precipitated with Trichloroacetic acid (TCA) To test if eEF1Bγ is present in the viral replicase, yeast N. benthamiana was done as described N. benthamiana using the following pair of primers: #2993 (CGCGGATCCAAAGGTTTCTGGGACATGTATGA) and #2994 (CGCCTCGAGACACGCTCCTTCTGTGATTCATC) and inserted into the corresponding (BamHI/XhoI) restriction sites of pTRV2 plasmid.Virus-induced gene silencing (VIGS) in N. tabacum eEF1Bγ gene (GenBank: ACB72462.1) was derived via a BLASTP search based on the C- terminal (translation elongation factor) domain (aa 252–412) of the Saccharomyces cerevisie Tef4 protein. The selected sequence (TC64920) from the Solanaceae Genomics Resource (www.tigr.org) gave 98% identity with N. tabacum EF1Bγ -like gene (GB#: EU580435.1).The sequence of the N. benthamiana, we performed RT-PCR amplification with primer pairs: #2952 (CGCGGATCCGGAAAGGTTCCTGTGCTTGA) and #2992 (CGCCTCGAGGTCCAGAAGTATCTCTCTACATGTGG) on total RNA extract of pTRV2- EF1BγNt and pTRV2empty agro-infiltrated N benthamiana plants. PCR conditions were as follows: 27 cycles of 94°C 20sec, 60°C 30sec, 68°C 30 sec with HiFi Taq polymerase. Tubulin mRNA control from the same total RNA samples was detected by RT-PCR using primers #2859 (TAATACGACTCACTATAGgaACCAAATCATTCATGTTGCTCTC) and #2860 (TAGTGTATGTGATATCCCACCAA) th day after silencing 32P-labeled complementary RNA probes specific for the 3′-ends of the viral genomic RNAs based on T7 transcription. To obtain the PCR templates for the probes, we used the following primers for TBSV: #1165 (AGCGAGTAAGACAGACTCTTCA) and #22; for TMV: #2890 (TCTGGTTTGGTTTGGACCTC) and #2889 (GTAATACGACTCACTATAGGGATTCGAACCCCTCGCTTTAT).To confirm the silencing of the EF1Bγ gene in in vitro RNA recruitment reaction was performed according to 32P-labeled DI-72 (+)repRNA were used and rCTP, rUTP, 32P-labeled UTP, and Actinomycin D were omitted from the assay. As a negative control, p33 and p92 were omitted from the reaction to detect DI-72 binding nonselectively to host proteins present in the membrane.The TBSV viral RNA is recruited to the membrane from the soluble fraction with the help of TBSV replication proteins and host factors present in the yeast CFE. The Figure S1In vitro binding assay with purified recombinant eEF1Bγ (Tef3). The TBSV (+)RNA templates were the four noncontiguous segments of the TBSV (+)RNA that are present in defective interfering RNAs, including DI-72 repRNA used in this study. RI(+) represents the 5′-UTR, RII(+)-SL is an internal highly conserved sequence that binds to p33 replication protein, RIII(+) is ashort conserved sequence closed to the 3′ end, andSL3-2-1(+), which contains the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The assay contained 32P-labeled free ssRNA (as shown), plus 0.6 pmol purified recombinant eEF1Bγ, respectively. The bound RNA-protein complexes were separated on nondenaturing 5% acrylamide gels. Quantification of the free (unshifted) RNA was done with ImageQuant. (B) RNA gel shift analysis shows SL3-2-1(+) RNA binds competitively to eEF1Bγ. The RNA templates representing the 3′ end of the TBSV RNA and the deleted nucleotides are shown schematically. The cold competitor was SL3-2-1(+) RNA, which represents a large portion of the 3′-UTR RNA. (A) e 3′-UTR . The eEF(EPS)Click here for additional data file.Figure S2in vitro. Purified recombinant p33/p92 and 32P-labeled DI-72 (+)repRNA and eEF1Bγ (affinity purified recombinant Tef3) were added to a whole cell extract (CFE), followed by centrifugation/washing to remove the 32P-labeled repRNA that is not bound to the membrane. Then the membrane-bound RNA was analyzed in a denaturing PAGE gel. Note that the repRNA binds to the cellular membrane fraction nonspecifically (∼20% level) in the absence of the viral replication proteins.eEF1Bγdoes not affect the template recruitment step (EPS)Click here for additional data file.


Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.In  The RNA interference (RNAi) pathway utilizes small non-coding RNAs to silence gene expression. In insects, RNAi regulates endogenous genes and functions as an RNA-based immune system against viral infection. Here we have uncovered details of how RNAi is triggered by RNA viruses. Double-stranded RNA (dsRNA) generated as a replication intermediate or from transcription of the RNA virus can be used as substrate for the biogenesis of virus-derived small interfering RNAs (vsiRNAs). Unlike other dsRNAs, virus RNA processing involves Dicer but not its canonical partner protein Loqs-PD. Thus, vsiRNA biogenesis is mechanistically different from biogenesis of endogenous siRNAs or siRNAs derived from other exogenous RNA sources. Our results suggest a specialization of the pathway dedicated to silencing of RNA viruses versus other types of RNAi silencing. The understanding of RNAi mechanisms during viral infection could have implications for the control of insect-borne viruses and the use of siRNAs to treat viral infections in humans. Drosophila, exogenous and endogenous dsRNAs are processed into siRNAs by Dicer-2 (Dcr-2) in association with the PD isoform of Loquacious (Loqs-PD) RNA interference (RNAi) utilizes small non-coding RNAs in association with an Argonaute (Ago) protein to regulate gene expression in virtually all eukaryotes Drosophila, Ago2, R2D2 and Dcr-2 mutant individuals exhibit increased sensitivity to infection by several viruses Drosophila S2 cells infected with Flock house virus (FHV) generate 21-nucleotide (nt) vsiRNAs that preferentially map to the 5′ region of both RNA segments of the viral genome The siRNA pathway is a major arm of the antiviral response in plants and invertebrate animals Drosophila to characterize the siRNA responses triggered by two RNA viruses, Sindbis virus (SINV) and VSV. SINV belongs to the Togaviridae family and has a positive RNA genome, while VSV belongs to the Rhabdoviridae family and has a negative RNA genome. We chose SINV and VSV because they have distinct strategies of replication, allowing us to uncover common and unique features of each antiviral response. Our results indicate that biogenesis of siRNAs from viral RNA is mechanistically distinct from siRNA biogenesis from endogenous or exogenous sources of dsRNA. We propose a mechanism whereby dsRNAs generated during viral replication and transcription are sources of vsiRNAs, and viral transcripts are major targets of RISC-mediated silencing.Here, we utilize wildtype and mutant Drosophila adults by injecting either SINV or VSV into their hemocoelic cavities. We monitored viral RNA genome levels for three days post-infection (dpi), and observed significantly higher levels of SINV and VSV genomes in Dcr-2 and R2D2 mutants, compared to wildtype  to facilitate the direct comparison between viruses, since GFP expression faithfully reflects replication levels for both VSV and SINV loqs R2D2 double mutants compared to R2D2 single mutants  and normalized to the levels detected at 2 hpi. Wildtype embryos experienced a 500-fold increase in SINV genome levels between 2 and 24 hpi . We found that Dcr-2 mutants had virtually no VSV vsiRNAs when compared to wildtype  with SINV Dcr-2 null mutants, A500VDcr-2 mutants showed increased levels of SINV replication  and 3.9-fold for VSV (p = 0.001)  . This grp = 0.49) or VSV (p = 0.48). If there was direct targeting of genomes and antigenomes, we predicted that the antigenome RNA levels would be more strongly affected. This is because vsiRNA levels from both strands are equivalent but the level of genome RNA greatly exceeds the level of antigenome RNA  that impairs slicer activity of the protein Ago2 null mutants  mediates the bulk of the inhibitory effect by slicing viral target RNAs. However, it is possible that cleavage of viral RNA by Dcr-2 is the major inhibitory mechanism, and Ago2 simply acts as a sink to drive the cleavage reaction. To distinguish between these mechanisms, we assayed an  mutants . These rDrosophila. The major difference between the two pathways seems to lie in the mechanism of siRNA biogenesis. In the antiviral pathway, virus dsRNA can be processed by Dcr-2 without Loqs-PD. In contrast, the canonical pathway relies upon Dcr-2 and Loqs-PD to process exogenous and endogenous dsRNAs. Downstream of processing, the two pathways appear to merge. vsiRNAs, exo-siRNAs, and endo-siRNAs are all sorted by a Dcr-2/R2D2 complex and loaded onto Ago2. These siRNA-Ago2 complexes inhibit target gene expression by a RNA slicing mechanism. Our results are consistent with other studies. Han et al loqs mutant had normal vsiRNA production and antiviral defense against FHV infection. However, it was possible that residual Loq-PD activity in the mutant rescued an antiviral function for the gene. We found that complete loss of Loqs-PD has no effect on antiviral silencing. Obbard et al Ago2, R2D2 and Dcr-2 are among the fastest evolving genes in the Drosophila genome. Since many host defense and pathogen genes co-evolve in a genetic arms race, rapid evolution of Ago2, Dcr-2 and R2D2 is possibly related to their antiviral functions loqs gene shows no sign of rapid evolution.Our results indicate the existence of a siRNA pathway dedicated to antiviral defense that is distinct from the one triggered by endogenous and exogenous dsRNA in Drosophila genome, and two of these were found to interact with Dcr-2, but they are unlikely to mediate a global antiviral response since their expression is restricted to the male testis R2D2 has been identified as rapidly evolving as Dcr-2 and Ago2In vitro studies have demonstrated that purified Dcr-2 protein efficiently processes dsRNA substrates and does not require a cofactor for its processing activity in vitro processing activity of Dcr-2 in vivo is still unknown. A fourth explanation is that the Dcr-2/R2D2 heterodimer recognizes and processes virus dsRNA, although processing is not affected by the absence of R2D2. If virus dsRNA is processed by Dcr-2/R2D2, then vsiRNA products could be directly loaded onto Ago2 and avoid loading competition with endogenous siRNAs. This might enhance the antiviral response.There are at least four possible interpretations of our results. First, Dcr-2 could process virus dsRNA in partnership with a dsRBP cofactor other than Loqs-PD. We have ruled out R2D2 as a potential substitute. Several other dsRBPs are encoded in the in vitro synthesized SINV RNA into cells also show no effect of the loqs mutant on RNA replication (data not shown). Therefore, it is unlikely that Dcr-2 recognizes chemical modifications of SINV RNA as the distinguishing feature. If Dcr-2 does not recognize modified features of virus RNA, what is the nature of the signal? RNA virus transcription and replication are typically sequestered into ribonucleoprotein “factories” that contain concentrated levels of RNA and enzymes in vitro, is also necessary for Dcr-2 to recognize non-canonical ends of dsRNA duplexes If Dcr-2 acts on virus RNA without the need of a dsRBP cofactor, then how does the enzyme recognize virus RNA as different from other types of dsRNA? Purified SINV RNA injected into cells replicates over time in a manner that is unaffected by Loqs-PD. Thus, it is not virion structure or mode of entry that signals Dcr-2 to differentially recognize virus RNA. Instead, it indicates that Dcr-2 specifically recognizes something intrinsic to the virus RNA or its intermediates. Preliminary experiments injecting Our work also addresses the origin of vsiRNAs. Others have suggested that viral replication intermediates are the exclusive substrates for vsiRNA production What is the mechanism by which vsiRNAs inhibit viral replication? Some have proposed that Dcr-2 mediated processing of viral dsRNA is primarily responsible for the reduction seen in viral RNA levels Drosophila mutants analyzed were: L811fsXDcr-2 and A500VDcr-21/R2D2S165fsXR2D2loqsf00791/loqsKOloqsKOR2D2S165fsX/loqsf00791R2D21Ago2414Ago2V966MloqsKO PB and PD rescue lines Wolbacchia, which has been shown to influence Drosophila antiviral defense 8 pfu/mL and 4×1010 pfu/mL, respectively.All mutant alleles used in this study were previously described. The different To avoid possible complications related to differences in background, microbiota or rearing, we crossed heterozygous animals bearing mutant alleles to each other, and we infected their mutant and heterozygous wildtype offspring at the same time. These then served as mutant and wildtype control samples for each experiment. We utilized a microinjector to inject 50 nl of a PBS solution containing the viruses into the thorax of 2–4 day old female adults. Animals were injected with 5,000 pfu of VSV and 20,000 pfu of SINV in all experiments.t test was used to statistically analyze differences in median survival between groups.For the survival analysis, three groups of 20 adults of each genotype were injected separately and survival was monitored daily. Each experiment was repeated at least three times. Survival graphs and median survival were plotted and calculated using Prism (GraphPad). A two-tailed student t test was used to statistically analyze differences in viral RNA transcript levels between control and mutant animals. For strand specific qPCR, 2 pmols of primers for one strand were used during reverse transcription. Reverse transcription reactions were performed in the absence of primers or enzyme as negative controls for qPCR to ensure the identity of the products. Oligonucleotides designed in this study for RT and qPCR are described in Rpl32, GFP and Actin5 were used as normalization standards as described previously Total RNA from adults was extracted using Trizol reagent according to the manufacturer's protocol (Invitrogen). 1 µg of total RNA was reverse transcribed using 250 ng of random primers, 500 ng of anchored oligo dT primers or 2 pmol of gene and strand specific primers per reaction. The resulting cDNA was used as template for qPCR reaction containing Sybr Green (Invitrogen) and primers specific for the amplification of the genes of interest. The relative amount of the indicated RNAs normalized to an internal control  was calculated using the delta Ct method. A two-tailed student 4 pH 7.8, 5 mM KCl solution for the injections. Female and male adults of a given genotype were placed inside an egg collecting cage, and eggs were collected every hour at 25°C, dechorionated, and injected within the next 45 minutes. SINV RNA was injected at the posterior end of eggs with a volume of ∼100 pL. After injection, embryos were incubated at 23°C under halocarbon oil in an oxygenated chamber, and harvested after 2 or 24 hours post injection. They were directly put into 100 µl of Trizol for RNA extraction. An average of 30 embryos were pooled per sample per time point. RNA was reverse transcribed using anchored oligo-dT primers, and qPCR reactions were performed with SINV-specific primers at the 3′ end of the SINV sense genome  and SINV (J02363.1) genomes deposited on NCBI using the Small RNA Analysis Pipeline Tool (Rna2Map) available from Applied Biosystems. Briefly, Rna2Map uses the Mapreads program from Applied Biosystems to simultaneously align reads to a reference and to filter out contamination from sequencing adaptors. Mapping was done allowing up to one mismatch in color space for the overall alignment. Reads aligning to the Drosophila and viral genomes were retained, whereas reads aligning to sequencing primers were removed from further analysis.For the construction of the small RNA libraries, total RNA was isolated from adults at 48 h post injection of virus using Trizol (Invitrogen). Low molecular weight (LMW) RNA was prepared from total RNA, and small RNAs between the 18–34 nt size range were PAGE purified from the LMW RNA as described previously Drosophila reference genome were further analyzed as described previously Drosophila miRNAs from Flybase v5.18 and searching the genomic alignment for reads overlapping these coordinates. The same process was repeated for mRNAs and transposons, where mRNA coordinates were taken from Flybase and transposons coordinates were taken from version dm3 of the UCSC RepeatMasker annotations. Reads mapping to rRNA sequences were determined by filtering transposon, miRNA, and mRNA reads from the Drosophila genome alignment and matching the remaining reads against rRNA sequences from Flybase v5.18. Ad hoc perl scripts were used in all steps, including the calculation of read size/strand distributions. The numbers of reads (>16 nt) mapping to rRNA, mRNA, miRNA and TEs for each sample are detailed in Drosophila genome was used to normalize the libraries to allow for comparison between different libraries. Abundance of specific Drosophila small RNAs was plotted as the number of reads in a thousand reads from the total number of reads each library described Reads mapping to the Reads mapping to the VSV and SINV genomes were first normalized to the total size of the library as described above. Viral genome RNA levels were then used to normalize vsiRNA numbers to allow comparison between the different experimental samples. Importantly, viral genome RNA levels were determined by strand specific PCR in the total RNA extracted from the same animals that were used to make the small RNA libraries.The sequencing datasets were deposited on the Gene Expression Omnibus website at the NIH. Accession numbers are: GSE36449 GSM893954 GSM893955 GSM893956 GSM893957 GSM893958 GSM893959 GSM893960.For all the subsequent analyses , we separated only 21-nt reads mapped against the virus reference.http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/ngs-data-analysis-software/software-community.printable.html).In order to compare our sequencing results to the results of other groups using different platforms http://hannonlab.cshl.edu/fastx_toolkit/index.html). In both cases, reads below the minimum quality threshold were discarded. In the second step, adapters were removed using the cutadapt software (http://code.google.com/p/cutadapt/). In the third step, the remaining reads were mapped against the virus genome references using SHRiMP Reads from Illumina were filtered using fastx-toolkit (http://weblogo.threeplusone.com/create.cgi).Nucleotide probability cartoons for small RNAs were generated using Weblogo 3  from R software to investigate whether the read starting positions from the same strand are correlated. The 95% cutoff line for positive correlation or negative correlation (the null hypothesis is that the correlation = 0 at each lag) is shown in the plots. If the auto correlation at a given lag exceeds the cutoff line it can be regarded as significant .Let k from position j to j+k−1 (sense strand position). This implies that there are no reads starting at position j−20 to j+k−1 on the sense strand, and no reads starting at positions j to j+k−1+20 on the antisense strand. Suppose we observe a total of T1 and T2 reads from the sense and antisense strands respectively and the length of entire region is n. Under the null hypothesis, that is, the reads are evenly distributed across the entire region and the reads distribution on the two strands are independent, then we can use a Poisson distribution with mean T1/n and T2/n respectively to approximate the reads count distribution at each position given n is very large. Let p1(0) and p2(0) be the zero-probabilities under the two Poisson distributions. The probability that we observe a gap of exact length k is given by p(k) = p1k+20(0)p2k+20(0)(1−p1(0) p2(0))2 . The p-value of a gap of length k, defined as the probability to observed a gap of length k or even longer is given by p_value = ∑mk≥p(m) for integer m. m is the summation index, i.e. sum over integer m≥k+20. The expected value  thus is approximately n×p_value where n is the length of the entire genomic region.We defined the occupancy of any given position as the total number of reads (sense+antisense) that cover this position. A gap is defined as a region that is not covered by any reads from the sense or antisense strand. Suppose we observe a gap of length R2D2 or loqs mutants, we used a relatively larger bin size of 50 nt. For comparing the two mutants we used a bin size of 20 nt instead. The entire region contains five different transcripts with the start and end positions as follows: start =  end = . We tested the difference for each bin on each strand within each transcript sequentially. For a given transcript, suppose we observe a total of T1 and T2 tags on one strand for the two conditions respectively. For a particular bin under testing, let X and Y be the number of reads that start in this bin. We consider a Poisson distribution to approximate the sampling distribution of X and Y, the mean parameters of which are denoted as λ1 and λ2. The null hypothesis is, the reads distribution pattern everywhere is the same between these two conditions. Therefore the mean parameters λ1 and λ2 are proportional to the total number of reads observed in the two conditions. Given the observed total number of reads, the null hypothesis can be stated as:To test whether two conditions have different distribution patterns of reads count along the entire region for wildtype and mutant samples, we first divided each mRNA transcript into bins. Due to the very small sample size, when we comparing widltype to either X|(X+Y), which is known as a binomial distribution:We can test this hypothesis based on the conditional distribution of p-value is calculated based on this conditional distribution for each bin. In the plot, we plotted the log10 of the p-value with a “+” or “−” sign attached as follows. If P(X≥x|X+Y)≤0.5, which indicates the observed x is in the right tail, we attach a “+” sign, and otherwise a “−” sign. A “+” sign essentially means condition 1 has more reads than expected under the null, and “−” sign means the opposite.A two-sided Figure S1Survival of animals after infection. Survival of heterozygous wildtype, R2D2, loqs, and loqs R2D2 mutant animals after treatment. Animals were untreated (solid circles), mock injected (solid squares), SINV injected (hollow triangles), and VSV injected (hollow squares). The means and standard deviations for at least three independent experiments are shown.(TIF)Click here for additional data file.Figure S2The distribution of small RNAs matching the Drosophila genome is not affected by R2D2 and Loqs. The binned numbers of sequenced small RNAs that map to the second and third chromosomes of wildtype (wt), R2D2 and loqs mutants infected with VSV (A) or SINV (B). The number of reads is in log10 scale for reads on the positive strand (red) and negative strand (orange).(TIF)Click here for additional data file.Figure S321-nt small RNAs derived from the Drosophila genome are dependent on Dcr-2, R2D2 and Loqs. Frequency distribution of small RNAs derived from the Drosophila genome displayed by RNA length. Shown are samples prepared from wildtype (wt), Dcr-2, R2D2 and loqs mutants infected with VSV (A) or SINV (B).(TIF)Click here for additional data file.Figure S4Transcription promoters of VSV showing gaps in vsiRNA coverage. Shown are the regions in the VSV genome that surround the promoters for the P, M, G, and L genes. Non-transcribed promoters are defined by the blue and pink vertical lines in each plot. Also displayed are regions in which no vsiRNAs were detected by high-throughput sequencing. These gaps in vsiRNA coverage are scaled to the genome. The probability that each gap did not occur by chance is shown as the inverse expected value  on a log10 scale. The horizontal line in each plot represents a significance cutoff of p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Gaps are present in samples from R2D2 (A) and loqs (B) mutant infected animals.(TIF)Click here for additional data file.Figure S5Analysis of gaps in vsiRNA coverage over the VSV genome as detected by independent sequencing experiments. Shown are the regions in the VSV genome in which no vsiRNAs were detected by high-throughput sequencing performed by Mueller et al Ago2 mutants) and Sabin et al. p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Note that the L gene promoter most consistently shows significant gaps in vsiRNA coverage.(TIF)Click here for additional data file.Figure S6Phasing analysis of vsiRNAs derived from the positive and negative strands of SINV and VSV. Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from VSV (A) and SINV (B) positive (+) and negative (−) strands, as indicated. The samples were generated from infected wildtype (wt), R2D2, and loqs mutants. ACF values above the dotted line are statistically significant (p<0.05).(TIF)Click here for additional data file.Figure S7Phasing analysis of vsiRNAs derived from the SINV genome after infection of mosquitoes and Drosophila. Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from SINV positive (+) and negative (−) strands. Shown are all vsiRNAs mapping to the 5′-most 1000 nts of the relevant strand. Samples were from our R2D2 mutant Drosophila , mosquitoes from Myles et al C,D), the cell line Aag2 , and cell line U4.4  from Vodovar et al p<0.05).(TIF)Click here for additional data file.Table S1Statistical analysis of the differences in median survival with viral infection of different Drosophila mutants.(PDF)Click here for additional data file.Table S2Summary of the raw data from the sequencing of the small RNA libraries described.(PDF)Click here for additional data file.Table S3Percentage of reads in each feature class for the sequenced libraries.(PDF)Click here for additional data file.Table S4Statistical analysis of C-enrichment and U-depletion at the first position of 21-nt vsiRNA sequence reads(PDF)Click here for additional data file.Table S5Pairwise correlation of vsiRNA density along viral genomes(PDF)Click here for additional data file.Table S6Comparative analysis of the SOLiD and Illumina libraries from this work and other published studies.(PDF)Click here for additional data file.Table S7Pairwise correlation of vsiRNA density along the VSV genome between the libraries in this work and Mueller et al. .(PDF)Click here for additional data file.Table S8Oligonucleotides used in experimental analysis.(PDF)Click here for additional data file.


Some cellular editing functions can restrict the replication of some viruses but contribute to completion of the life cycle of others. A recent study has identified an isoform of the adenosine deaminase acting on RNA type 1 (ADAR 1) as required for embryogenesis, and as a restriction factor for a number of important RNA virus pathogens [ Exon 1A is under the control of IFN-α and generates the p150 isoform while expression from 1B is constitutive and gives rise to the p110 isoform. In an elegant approach, a targetting vector was designed for the specific elimination of the IFN-inducible p150 isoform of ADAR 1, leaving intact the constitutive expression of the p110 isoform. It was known that inactivation of ADAR 1 leads to embryonic lethality through uncontrolled apoptosis and liver disintegration [ADAR 1 were generated and interbred to obtain embryos in which the two copies of the genomic region encoding p150 were disrupted. Embryos which were negative for p150 could not complete development but they were the source for the mouse embryo fibroblast (MEF) cells for further study. Wild type and p150-deficient MEF cells were engineered to express the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). Comparison of infectious progeny production and cytopathology indicated an anti-MV activity of the p150 isoform of ADAR 1. Thus, the main methodological novelty of the study, the selective elimination of p150, demonstrated that the absence of this isoform was associated with embryonic lethality and susceptibility to infection by MV and other paramyxoviruses [in vivo, as well as to investigations on viral proteins that may antagonize the ADAR 1 antiviral activity.A recent publication by M.B. Oldstone and colleagues at the Scripps Research Institute of La Jolla has documented that one of the isoforms of the adenosine deaminase acting on RNA type 1 (ADAR 1) is a restriction factor for replication of measles virus (MV), other members of the Paramyxoviridae family, and influenza virus, but not for other RNA viruses such as lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus or reovirus [egration ,3. Mice 2.ADAR 1–3) identified in mammals (reviewed in [The ADARs are a family of adenosine (A) to inosine (I) editing enzymes that act on double-stranded RNA, encoded by three genes (iewed in ). RNA ediewed in ,6. One oiewed in ,8. The AXenopus laevis embryos as a double-stranded RNA-unwinding activity, later identified as an A→I deaminase [ADAR genes evolved from the ADAT upon acquisition of a region that encodes a double-stranded RNA-binding domain. This would be yet another illustration of modular evolution of cellular proteins, a form of functional expansion and diversification prominent also in virus evolution.The ADARs were discovered in eaminase ,10. The eaminase . An inteThe A→I RNA editing mediated by ADAR proteins affects mainly non-coding RNA sequences: untranslated regions, introns, repetitive RNA elements, and precursors of some microRNAs (miRNAs), among other targets . Double-Editing activities can promote viral replication, and not only in the form of hypermutated MV genomes found in SSPE brains. Hepatitis delta virus (HDV) has a negative sense RNA genome (of opposite polarity to that needed for expression of the encoded proteins) and replicates via a positive sense RNA termed anti-genome. HDV requires hepatitis B virus (HBV) as helper virus that supplies the surface protein. Editing by ADAR occurs in the nucleus and allows synthesis of two forms of the protein encoded by HDV  from the same m-RNA. The S version is necessary for genome replication and L for virion assembly. Their expression is temporally regulated since L may inhibit S-mediated viral replication. Part of the benefits of IFN-α against HDV infection may be due to an increase of L HDV protein expression, resulting in a decrease of viral replication. Thus, cellular editing functions that play key functional roles in the cell can restrict the replication of some viruses, but can also contribute to the completion of the life cycle of other viruses.3.Several cellular functions may modulate the susceptibility of cells to viral infection, through a variety of mechanisms referred to as innate immunity. Among classical examples are the effects of murine proteins Fv1 and Fv4, which are the products of expression of endogenous retroviruses, on the retroviral life cycle. Fv1 blocks the entry into the nucleus of retroviral preintegration complexes, and Fv4 acts as a non-functional env protein that competes with the authentic viral env protein for receptor binding . Other eEditing may have promoted evolutionary adaptations of viral genomes. The polymerase of the Paramyxovirinae (the subfamily of Paramyxoviridae that includes MV) stutters in a controlled manner to edit the viral phosphoprotein gene mRNA. It has been suggested that these viruses may have evolved a genome of polyhexameric length  to prevent the deleterious effects of illegitimate or uncontrolled editing that may result in hypermutagenesis . EnhanceE.coli bacteriophage Qβ. This is a multi-subunit enzyme with one phage-coded protein and three bacterial proteins: the ribosomal protein S1, and translation elongation factors EF-Tu and EF-Ts [Yet, essential cellular proteins are, paradoxically, also key players in the life cycle of viruses. A classical example is the participation of host proteins in viral polymerases. The first documented case was the RNA-dependent-RNA polymerase (RdRp) of nd EF-Ts . Other vnd EF-Ts ). ViroloHow can this paradoxical interplay of cellular functions participating either in innate cellular immunity or in virus replication be interpreted from an evolutionary point of view? In terms of ADAR, why the same editing functions can be a restriction factor for MV and at the same time promote genetic changes that result in virus persistence in the brain? Hypermutation enhanced MV cell-to-cell spread within the infected host eventually allowing the virus to invade the CNS, as opposed to normal MV which spreads by budding particles. Almost one quarter of a century ago, François Jacob wrote an influential article entitled “Evolution and tinkering” , based o


Pterois volitans/miles) on Bahamian coral reef patches. We compared the extent of concealment and activity levels of lionfish at dawn and midday on 16 coral reef patches off Eleuthera, The Bahamas. Eight of the patches had been subjected to regular daytime removals of lionfish by spearing for two years. We also estimated the distance at which lionfish became alert to slowly approaching divers on culled and unculled reef patches. Lionfish on culled reefs were less active and hid deeper within the reef during the day than lionfish on patches where no culling had occurred. There were no differences at dawn when removals do not take place. Lionfish on culled reefs also adopted an alert posture at a greater distance from divers than lionfish on unculled reefs. More crepuscular activity likely leads to greater encounter rates by lionfish with more native fish species because the abundance of reef fish outside of shelters typically peaks at dawn and dusk. Hiding deeper within the reef could also make remaining lionfish less likely to be encountered and more difficult to catch by spearfishers during culling efforts. Shifts in the behaviour of hunted invasive animals might be common and they have implications both for the impact of invasive species and for the design and success of invasive control programs.As a result of being hunted, animals often alter their behaviour in ways that make future encounters with predators less likely. When hunting is carried out for conservation, for example to control invasive species, these behavioural changes can inadvertently impede the success of future efforts. We examined the effects of repeated culling by spearing on the behaviour of invasive predatory lionfish ( Being hunted can change the behaviour of animals. Individuals exposed to hunting pressure spend more time being vigilant, flee from humans more readily, and are more likely to change the location or timing of their activities than individuals not exposed to hunting Hunting or culling is sometimes carried out for conservation purposes, such as during the eradication and control of eruptive or invasive species e.g., . HoweverPterois volitans/miles). The rapid spread of Indo-Pacific lionfish onto coral reefs throughout the western Atlantic and Caribbean since the 1980s has generated great concern for the native reef fish and invertebrates of the region Here, we investigate the effects of control efforts on the behaviour of invasive predatory lionfish , and covered by a research permit to the Cape Eleuthera Institute by The Bahamas Department of Marine Resources.2 to 204 m2. There was no difference in patch area between culled  and unculled reefs (111.3±30.3 m2) . The shortest distance between two adjacent patches was 300 m, which exceeds the inter-patch movement of 90% of lionfish that were tagged and resighted in our study area . This suggests that the patches studied were independent samples.We conducted lionfish surveys on 16 reef patches in Rock Sound, Eleuthera Island, The Bahamas, in January 2013. Over the past two years, as part of an experiment investigating the impacts of lionfish predation, eight of these patches have been subjected to lionfish removals  by divers with non-projectile, three-prong paralyzer-tip pole spears, while no removals have occurred on the remaining eight patches. The last cull of lionfish was conducted at all eight removal sites in December 2012, three weeks prior to our study. During this cull, divers removed as many lionfish as they could, which resulted in the capture of 30-100% of lionfish initially sighted on each reef. Patches in different treatments were interspersed across the Sound . All patWe used three measures to assess lionfish wariness: (1) extent of concealment at first sighting; (2) tendency towards crepuscular activity; and (3) alert distance , which is similar to the recommended searching effort for lionfish-focused surveys .Three observers  recorded lionfish behaviour. On each patch, a pair of observers searched carefully for lionfish that were swimming, hovering above the reef or hidden in refuges. Each observer investigated the periphery of the reef first and then swam in an S-shaped pattern over the top of the reef, searching all crevices and overhangs for lionfish. Our search time was approximately 12 minutes per 100 mP<0.001; where Kappa  =  0 indicates results that were entirely generated by chance and Kappa  =  1 indicates perfect agreement; a significant P-value rejects the null hypothesis of random observations; see For each lionfish sighted, we estimated total length (to nearest 5 cm), categorised its behaviour at first sight as either inactive  or active . The method we used was similar to that used by Gotanda et al. t-tests, with reef patch as the unit of analysis. Alert distances of lionfish on culled and unculled patches were also compared using independent-samples t-tests, but with lionfish as the unit of analysis for two reasons: (1) it was not possible to find testable lionfish on some of the culled reefs because of low densities and inaccessibility, and (2) we had no reason to expect differences among reefs within treatment.We calculated the proportion of active lionfish, mean hiding score observed, and the proportion of high hiding scores  for each of the four surveys for each reef. We then averaged values for the two daytime surveys and, separately, the two crepuscular surveys conducted on each reef. Because the proportion of active lionfish and of high hiding scores can be unduly influenced by the number of lionfish present on a reef  and the number of lionfish per reef varied greatly , we weighted these variables by the reef-specific proportion of total lionfish present across the 16 reefs, separately for day and dawn observations. Thus reefs with more lionfish contributed more to the overall means. Mean proportion of active lionfish (weighted), mean hiding scores, and mean proportion of high hiding scores (weighted) were then compared between culled and unculled patches using independent-samples t-test for unequal variances, t7.14 = 1.93, p = 0.09). We found no difference in any of the lionfish behavioural measures between four patches that were culled every 3 months and four patches that were culled every 6 months , thus patches culled under both regimes were combined for further analyses.There were, on average, four times more lionfish on control than on culled patches, although this difference was not statistically significant . However, there were no differences in activity levels or hiding scores at dawn , but there was no detectable difference in length between the lionfish tested on culled  and control reefs .During the day, a lower proportion of lionfish were active , where fishing is prohibited, and adjacent fished areas. In general, fishes within MPAs have shorter flight initiation distances (FID) than those in unprotected areas, suggesting they are less wary than fishes exposed to fishing pressure It is not clear whether the behavioural changes observed here, and more generally in hunted populations, are learned or evolutionary responses. The perceived threat presented by a diver may be influenced by prior experience and/or social learning The differences in behaviour exhibited by lionfish on hunted patches have potential ecological implications. Lionfish foraging concentrated during crepuscular periods would result in a potentially greater encounter rate with many more native fish species. The abundance of exposed reef fish typically peaks at dawn and dusk Our results also have implications for lionfish control efforts. Targeted removals effectively reduce lionfish densities and have demonstrated benefits for the recovery of native fish species Identifying whether learning or selection explains the differences documented here is important because different mechanisms have different management consequences. If the behavioural differences stem from learning, a key question is how long-lasting these effects are. Control efforts will remain cost-effective only if culling frequency exceeds the memory retention time of lionfish, but long culling intervals might jeopardise the ecological effectiveness of these efforts in terms of recovering native prey populations


Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection.NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation.In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3′ untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis.The results show a significant decrease in BaMV RNA accumulation when the expression level of The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis. An RNA genome of c. 6.4 kb with a 5′-cap and a 3′-poly(A) tail contains five open-reading frames  was cloned into pTRV2 using EcoRI site to generate pTRV2/ACCA3 for virus-induced gene silencing (VIGS). The primer set designed for cloning the coding region of the ortholog gene from N. benthamiana is based on N. tabacum C-7 mRNA sequence. The forward primer is Nb_C-7/BamHI/F, 5′-GGATCCATGGCTGACGAAGTTGTCC-3′ (BamHI site is underlined); and the reverse primer is Nb_C-7/XhoI/STOP/R, 5′-GTTACTCGAGGGCAATGCCCCACTTTTG-3′ (XhoI site is underlined). The PCR product was cloned into pGEM-T easy vector and sequenced. The restriction sites BamHI/SalI and BamHI/XhoI flanking the coding region were used for cloning to pBImGFP and pGEX-41 for plant and E. coli expression, respectively. For pNbGSTU4/S13A construction, the mutant fragment was amplified from pNbGSTU4 using BamHI-NbGSTU4+1, 5′-GGATCCATGGCTGACGAAGTTGTCCT-3′, and NbGSTU4-54 mutant, 5′-CCTCATCCCAAACATGGCTACATAGGTATCCAA-3′, to synthesize a 60-bp fragment which was than used as a megaprimer for a second PCR with primer mGFP-31, 5′-GTTCTTCTCCTTTACTAGTCAGATC-3′. The amplified DNA fragment was gel purified and cloned into pGEM-T easy for sequencing. A correct recombinant clone was digested with BamHI and SpeI, and inserted into the corresponding region of pNbGSTU4. For measuring the knockdown efficiency of various NbGSTs in plants, we used real-time PCR with the primer sets: NbGSTU1 (F: 5′-GATGGCAGAAGTGAAGTTG-3′ and R: 5′-GCTCCTAGCCAAAATSCCA-3′); NbGSTF1 (F: 5′-GGCTTCAAGATTAACCTGGGA-3′ and R: 5′-GCCAARATATCAGCACACC-3′); NbGSTU2 (F: 5′-GTAGGCATAAAACCAGCTGTAGT-3′ and R: 5′-GTGAGTACATTGAWGAAGTTTGG-3′); and NbGSTU3 (F: 5′-GCATAAGAATAAAACCAACTAGTAA-3′ and R: 5′-GTGAGTACATTGAWGAAGTTTGG-3′).An ACCA3 cDNA fragment (200 bp) screened from cDNA-AFLP (Cheng NbGSTU4) were transformed into the Agrobacterium tumefaciens C58C1 strain by electroporation. To knock down the expression level of NbGSTU4 in N. benthamiana, the A. tumefaciens C58C1 containing pTRV1, pTRV2/Luc or pTRV2/ACCA3 was cultured to OD600 = 1 at 30°C before induction by the addition of 130 μM acetosyringone in 10 mM MgCl2 for 3 h at room temperature. The pTRV2/Luc- or pTRV2/ACCA3-containing A. tumefaciens C58C1 was mixed with pTRV1-containing A. tumefaciens C58C1 at a 1 : 1 volume ratio. The 1st and 2nd leaves were infiltrated with the mixed broth at the four-leaf stage (seedlings with two cotyledons and two leaves). Then 200 ng of BaMV, PVX, CMV or TMV virion RNA was inoculated onto the 6th leaf when it was mature. Total RNAs and proteins were extracted from the leaves at 3 dpi and measured for the mRNA and viral coat protein levels, respectively. For the protoplast inoculation assay, protoplasts prepared from the 6th leaf were transfected with 1 μg of BaMV virion RNA. The levels of NbGSTU4 mRNA and viral coat proteins were measured at 24 h post-inoculation.The control plasmid, pTRV2/Luc, containing a portion of Luciferase gene was constructed. Plasmids pTRV2/Luc and pTRV2/ACCA3 ethanesulphonic acid pH 5.7) at 25°C overnight. The mesophyll cells were isolated and transfected with 2 μg of virion RNA (c. 4 × 105 cells for each sample) with the help of polyethyleneglycol. Finally, the transfected protoplasts were incubated under constant light (15 μmol m−2 s−1) at 25°C for 24 or 48 h. For Northern blotting analysis, the total RNA was extracted from protoplasts (2.5 × 105 cells), glyoxalated, electrophoresed through a 1% agarose gel and transferred to a Zeta-Probe membrane (Bio-Rad), as described previously  and RNase-free water to a final volume of 20 μl. Cycling conditions began with an initial hold at 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s. Reactions were carried out in a RotorGene 3000  with data acquisition at 72°C on the channel with excitation at 470 nm and detection at 585 nm using a high-pass filter for both plus- and minus-strand. All samples were run at least twice, and the reaction without template or reverse transcriptase was performed as a negative control, and actin gene were taken for normalization.The qRT-PCR technique was used to detect both BaMV plus- and minus-strand genomic RNAs. The cDNA synthesis reaction was performed according to the manufacturer's instructions using ImProm-II™ Reverse Transcriptase (Promega) with the primers Oligo dT(25T), and BaMV+1 (5′-GAAAACCACTCCAAACGAAA-3′) for the plus- and minus-strand, respectively. qPCR for quantifying the accumulation of BaMV genomic RNA and minus-strand RNA, primers BaMV+1766 (5′-CACATCCGGCACTTACCA-3′) and BaMV-2002 (5′-ATGTATCACGGAAATAAGAGTT-3′) were used in the reaction containing a 1000× dilution of SYBR green I . qPCR was performed in 0.2-ml PCR tubes with 0.6 mM primer, 0.2 mM of each deoxynucleoside triphosphate, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 2 mM DTT, 5% glycerol), and irradiated with a 254-nm-wavelength UV lamp  on ice for 10 min. After irradiation, the samples were treated with 40 μg of RNase A for 30 min at 37°C, boiled in 1X SDS sample buffer, and electrophoresed on a 12% or 14% sodium dodecylsulphate polyacrylamide gel. GSH was added at a different concentration  in the UV-crosslinking reaction.The purified proteins (GST and GST-NbGSTU4) after dialysis against the buffer containing 20 mM Tris-HCl pH 7.6, 20 mM NaCl, 2 mM DTT, 5% glycerol were incubated with labeled r138/40A (the 3′ UTR of BaMV) or Ba-77 (the 3′-end of BaMV minus-strand) individually for 20 min on ice in a binding buffer , 4.8 mg ml−1 of bentonite, 10 mM dithiothreitol, 3 mM MgCl2, and 50 ng of RNA template and incubated at 30°C water bath for 1 h. The RNA products were extracted with phenol-chloroform and precipitated with ethanol. For test the effects of different redox agents, 50 mM of GSH, GSSG, DTT, and H2O2 were added in the reaction. Each reagent was dissolved in 50 mM Tris buffer and adjusted pH value to 7.0 before used.The replication complex of BaMV used for an N. benthamiana following BaMV infection was identified by using the cDNA-AFLP technique in our previous study -based virus-induced gene silencing (VIGS) technique  in N. benthamiana showed that the accumulation levels of the BaMV coat protein in the knockdown plants was reduced to 51% of that of the control plants. A similar reduction level was also found in another member of the Potexvirus, Potato virus X (PVX) (Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) when inoculated into the knockdown plants . The amino acid sequence of this cDNA has 87% identity to the GST C-7 of N. tabacum . To examine whether knocking down the expression of NbGSTU4 by using VIGS would also reduce the expression of other homologous genes in N. benthamiana, an inspection, using real-time PCR with specific primers, of the mRNA levels of NbGSTU1, NbGSTU2, NbGSTU3 and NbGSTF1in the NbGSTU4-knockdown plants was required. The results indicated that the expression levels of the other homologous genes tested in the NbGSTU4-knockdown plants were insignificant unlike those of the control plants  a,b. Howen plants a,b. The l plants c. These l plants c. The reNbGSTU4 expression, a protoplast assay was performed to exclude cell-to-cell movement. Northern blot analysis of RNA samples, derived from NbGSTU4-knockdown protoplasts transfected with the BaMV for 48 h, showed that the accumulation level of the BaMV genomic RNA was reduced compared to that of the control protoplasts . The addition of GSH at 4 and 6 hpi caused a significant enhancement in the accumulation of both BaMV plus- and minus-strand RNA   . HoweverN. benthamiana. The effect of the GSH on the replication of the BaMV is suggested to be associated with minus-strand RNA synthesis. Further validation of this hypothesis required performing an in vitro RdRp assay with exogenous RNA templates in different concentrations of GSH  RNA, but also binds to the TBSV p33 replication protein and acts as a co-factor assisting in viral RNA replication  have been reported to be involved in fungal infections , was specific in N. benthamiana  the BaMV RNA ; (2) theBaMV RNA ; and (3)BaMV RNA . These re of GSH .et al., et al., NbGSTU4 upregulated by virus infection is to provide an antioxidation environment for virus replication. The results derived from the in vitro RdRp assay indicate that providing GSH or DTT can enhance minus-strand RNA synthesis, but not plus-strand RNA synthesis. The likely role of NbGSTU4 is to bring GSH to the replication site and provide anti-oxidative conditions for minus-strand RNA synthesis. Because the concentration of GSH in cells is c. 1–20 mM, it might not provide a sufficiently strong reduced environment for viral RNA replication. The interaction of viral RNA with GST  induced by virus infections (Fodor This is the first study to examine the involvement of a new plant tau-class GST protein, NbGSTU4, in BaMV viral RNA minus-strand RNA synthesis. Through the binding of NbGSTU4 to the 3′ UTR of the genomic RNA, either by providing an antioxidative condition or by changing the redox status of the replicase complex, the minus-strand RNA can be synthesized efficiently. The discovery of a plant GST protein involving the replication of a plant virus could help us to gain insights into the relationship between viral RNA replication and the GST metabolic pathway.


CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs.Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved.We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the  Arabidopsis, DCL2, DCL3 and DCL4 produce 22-, 24- and 21-nt siRNAs, respectively [Arabidopsis, small RNA-mediated cleavage can trigger conversion of the targeted RNA to dsRNA by RNA-dependent RNA polymerase 6 (RDR6), which is then cleaved into 21-nt phased siRNAs by DCL4. These siRNAs can include those termed trans-acting siRNAs (tasiRNAs), which silence other gene(s) in trans[RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. RNA silencing of genes is induced by the presence of double-stranded RNA (dsRNA) homologous to the genes. The dsRNAs are processed into small RNAs, especially 21- to 24-nulceotide (nt) short interfering RNAs (siRNAs), by a dsRNA-specific ribonuclease, Dicer or Dicer-like (DCL) proteins [ectively . Transcrectively -8. Like ectively . Small R in trans-12. Smal in trans,14. A re in trans.chalcone synthase-A (CHS-A) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator causes the production of white sectors or completely white flowers in transformed petunia (Petunia hybrida) plants [CHS-A transgene and endogenous CHS-A gene was induced, so that the event was termed cosuppression [CHS-A silencing system [CHS-A silencing in petunia is a model system to study RNA silencing [CHS-A gene, thereby produced a plant that does not carry a transgene but has altered traits [Overexpression of the ) plants ,17. Thispression . The prog system ,19. Becailencing . Based oilencing . In our d traits ,22.CHS-A genes resemble those in nontransgenic varieties [CHS-A cosuppressed plants, the flower color pattern in Red Star was in fact demonstrated to be due to sequence-specific degradation of the CHS-A RNA in the white sectors [Some of the flower-color patterns observed in transgenic petunias having cosuppression of the arieties . One suc sectors . Petunia sectors . The gen sectors . Similar sectors , and for sectors , soybean sectors -29, maiz sectors  and dahl sectors .′ capping are used as a template for RDR6 to produce dsRNA, thereby triggering PTGS [Cosuppression has been thought to be caused by a couple of mechanisms. It can be induced when multiple transgenes are integrated into the same site in the genome in an inverted orientation and fortuitous read-through transcription over the transgenes produces dsRNA homologous to an endogenous gene in the genome, a pathway termed inverted repeat (IR)-PTGS. When sense transcripts from a transgene trigger cosuppression through RNA degradation, the pathway is referred to as sense (S)-PTGS . A modeling PTGS . An alteing PTGS .CHS-A cosuppression is induced by a high level of transcription of the CHS-A transgene, shown by the fact that CHS-A cosuppression is induced when the CHS-A transgene is transcribed by the CaMV 35S promoter but not when the transcription from the promoter is repressed by epigenetic changes involving spontaneous cytosine methylation of the promoter [CHS-A transgene [CHS-A cosuppression can be triggered when a particular RNA, e.g., CHS-A primary transcripts or some other RNA molecule(s) derived from them, exceed a certain level. However, neither the RNA molecule(s) nor the sensing mechanism(s) of the threshold is known.Our previous data indicated that promoter . These opromoter . This noCHS-A cosuppression in petunia has been suggested on the basis of a deep sequencing analysis of CHS-A siRNAs [CHS-A mRNA cleavage and initiate the generation of phased siRNAs, leading to cosuppression. On the other hand, CHS-A siRNA profiles in another cosuppressed transgenic line having inverted repeat T-DNA [CHS-A cosuppressed line is common to different CHS-A cosuppressed lines or CHS-A naturally silenced lines is not known. Moreover, no insight into a general mechanism(s) of cosuppression in terms of siRNA production has been presented in any plant species.A potential trigger for A siRNAs . Two abuat T-DNA  or a petat T-DNA  indicateCHS-A siRNA populations from silenced and nonsilenced tissues of a transgenic line having CHS-A cosuppression and a non-transgenic variety Red Star in detail. We show that multiple abundant siRNAs from CHS-A exon 2 are produced in the silenced tissues in both silenced lines. We also found profound commonality in siRNA production in the silenced tissues of the cosuppressed line and Red Star, which suggests the presence of a common mechanism of RNA degradation that likely depends on an evolutionary conserved feature in exon 2 of the CHS-A gene.To address these questions, here we analyzed CHS-A gene in the white and pigmented portions of petal tissues of petunia plants that have cosuppression or naturally occurring RNA silencing of the CHS-A gene. The CHS-A cosuppressed line contains a single copy of the CHS-A transgene and produces petals with a white and purple pattern. The size of the white portions is variable, but they are invariably centered on the junctions between petals; hence, the pattern is called the junction pattern [CHS-A mRNA was barely detected . The position and abundance of the 21-nt to 24-nt siRNAs mapped in the CHS-A gene region are shown in Figure CHS-A gene region  detected in the white portions of J-type plants were found within the 24 most abundant siRNAs (in 469 siRNA species) detected in the white portions of Red Star flowers Figure a. Similars = 0.723, P < 0.01; for the antisense strand, rs = 0.852, P < 0.01)  of the production of phased siRNAs, including the sites of RNA cleavage to initiate phased siRNA production, is considerably conserved between these plants for both sense and antisense strands.CHS-A transcripts. They may cleave CHS-A RNA with AGO orthologue(s) to trigger secondary siRNA production. Alternatively, these abundant siRNAs can be a product of DCL cleavage of dsRNAs synthesized by an RDR6 orthologue(s) from the nascent transcripts or AGO-cleaved transcripts , and a causative relationship between RNA silencing and the presence of the two copies of the CHS-A gene has not been presented.Among the cases of naturally occurring RNA silencing so far reported, a triggering mechanism has been suggested for only a few cases, all of which involve production of dsRNA either through read-through transcription of duplicated and rearranged genes ,47,48 or mutants ,29. In poduction . A corre petunia . HoweverCHS-A cosuppressed petunias are consistent with this notion [CHS-A, phy-siR1 and phy-siR2, in a CHS-A cosuppressed petunia line and proposed that these siRNAs may trigger subsequent degradation of CHS-A transcripts [CHS-A cosuppressed lines, although circumstantial evidence indicates that RNA cleavages with these siRNAs can induce phased siRNA production [CHS-A transcripts are cleaved at multiple, conserved positions in both J-type and Red Star plants. The siRNAs that guide these cleavages may include a potential trigger of RNA silencing. Whether a single cleavage of RNA can lead to extensive RNA degradation through RNA silencing pathways in these silencing systems is an issue to be addressed.For sense RNA-mediated silencing such as cosuppression in transgenic plants, a threshold sensing model, in which aberrant single-stranded RNA that accumulates beyond a critical level triggers its copying into dsRNA, has been suggested . In facts notion ,35. Meannscripts . On the oduction . The reaCHS-A siRNA was present in pigmented portions in both J-type and Red Star plants at a low level. On the other hand, an extremely low level (only 2 reads) of CHS-A siRNA was detected in 16,651,540 total reads for line V26 (data not shown), a wild-type plant that produces completely purple flowers and was used to produce J-type plants through the introduction of the CHS-A transgene. Therefore, the presence of CHS-A siRNAs in the pigmented petal tissues in J-type plants is associated with cosuppression that occurred in other cells of the petal tissue.We found that CHS-A siRNA in pigmented cells is that RNA is degraded at a low rate in the pigmented cells. Alternatively, the siRNAs may migrate from cells that underwent PTGS through plasmodesmata. In either case, these results raise a novel possibility that a threshold level of CHS-A siRNAs might be associated with extensive RNA degradation in addition to the previous idea that an aberrant CHS-A primary transcript level constitutes such a threshold level. It would not be surprising that, taking into account the observed commonality in siRNA profiles between these two silencing systems, they share a common sensing mechanism for trigger RNAs.A likely explanation for the presence of CHS-A gene between cosuppression in transgenic plants and naturally occurring silencing in nontransgenic plants of petunia. In both silencing systems, 21-nt and 22-nt siRNAs were the first- and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that CHS-A RNA is degraded through processes including cleavage and secondary siRNA production in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, whose ranks, according to the number of siRNAs in these plants, were correlated with each other. Highly abundant siRNAs were produced from multiple sites, many of which were common to the two silencing systems. Phased siRNAs were detected in multiple phases, and some of the ends of the regions that produced phased siRNAs were conserved. These results indicate mechanistic similarity between cosuppression and naturally occurring RNA silencing of the CHS-A gene, especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs, which presumably depend on the nucleotide sequence and/or structural features of exon 2 RNA.The present study revealed common features in siRNA production of the Petunia hybrida variety Red Star  and a transgenic petunia line that produces junction-type flowers (J-type) [CHS-A transgene. The white and the pigmented petal tissues of these plants were analyzed separately. Petal tissues were used at the developmental stage when the mRNA level of the CHS-A gene is highest [(J-type)  were use(J-type) ,51 and c highest .CHS-A gene, 4246 (5′-GGCGCGATCATTATAGGTTC-3′) and 5003 (5′-TTTGAGATCAGCCCAGGAAC-3′); for the α-tubulin gene, tub 125 F (5′-CAACTATCAGCCACCAACTG-3′) and tub 267R (5′-CACGCTTGGCATACATCAGA-3′).Isolation of total RNA from flower tissues, cDNA synthesis, and RT-PCR were done as described previously . The folCHS-A siRNAs were detected by Northern blot analysis using a digoxigenin-labeled probe essentially as described by Goto et al. [N-lauroyl sarcosine; an RNA probe specific for CHS-A antisense RNA was labeled by in vitro transcription of the plasmid carrying a 0.44-kb region of the CHS-A gene [Low-molecular-weight RNA was isolated, and o et al. . The folS-A gene  using DICHS-A gene region (EMBL/GenBank/DDBJ database accession X14591), allowing only perfect matches. Nucleotide positions in this study correspond to those on this sequence. The secondary structure of CHS-A sense and antisense RNAs was predicted by m-fold software [CHS-A gene in 21 different phases. Calculation of phasing scores and assignment of scores to cycle position were done according to Howell et al. [Low-molecular-weight RNA was extracted from the petal tissues of flower buds before the buds opened (~4.5 cm long for J-type and ~5.0 cm long for Red Star). Tissues were frozen with liquid nitrogen and extracted with RNA extraction buffer containing 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 1% (w/v) SDS. After extraction with phenol/chloroform, high-molecular-weight RNA was precipitated by mixing the aqueous phase with 1/3 volume of 8 M LiCl. After the solution was kept on ice overnight, the solution was centrifuged, and the nucleic acids in the supernatant were precipitated with ethanol. After centrifugation, the pellet was dissolved in water, and an equal amount of 20% polyethylene glycol (MW = 8000) was added to the solution to separate high-molecular-weight nucleic acids. The solution was held on ice for 1 h, then centrifuged, and low-molecular-weight RNA in the supernatant was precipitated with ethanol. After centrifugation, the pellet was dissolved in water and used for the following reactions. Low-molecular-weight RNA was ligated to 5′- and 3′-RNA adapters, reverse transcribed, and amplified by PCR using a Small RNA Sample Prep Kit  according to the manufacturer’s protocol except that we separated small RNAs by electrophoresis on a 3% agarose gel instead of an acrylamide gel. Nucleotide sequence of the amplified cDNA was analyzed using an Illumina Genome Analyzer. The adapter sequence was trimmed from the raw short-read data, and the resulting short reads 15–45 nt) were mapped to the nucleotide sequence of the  nt were l et al. . NucleotAGO: Argonaute; CaMV: Cauliflower mosaic virus; CHS: Chalcone synthase; DCL: Dicer-like; dsRNA: Double-stranded RNA; IR: Inverted repeat; J-type: Junction type; miRNA: microRNA; NOS: Nopaline synthase; PTGS: Posttranscriptional gene silencing; RDR: RNA-dependent RNA polymerase; S-PTGS: Sense-PTGS; siRNA: Short interfering RNA; tasiRNA: Trans-acting siRNA; TGS: Transcriptional gene silencing.The authors declare that they have no competing interests.AK conceived and planned the study. MK did the experiments including RNA isolation, RT-PCR, Northern blot analysis, and preparation of small RNA cDNA library. HM, KY and RT carried out deep-sequencing of small RNAs. AT classified and mapped small RNA reads onto the gene sequence. MK and AK characterized small RNAs and drafted the manuscript. All authors read and approved the final manuscript.Highly abundant 22-nt siRNAs in white portion of J-type and Red Star petals. The siRNAs of sense (a) and antisense (b) strands were ordered according to the number of reads in deep-sequencing analysis. J-w and R-w refer to the siRNAs detected in white portions of J-type and Red Star petals, respectively. The 10 most abundant siRNAs in J-w are colored, and the siRNAs of the same sequence in Red Star are colored the same. Note that most of the highly abundant siRNAs in J-type plants are also highly abundant in Red Star plants. Click here for fileCHS-A transcripts or dsRNAs produced by RDR6 orthologue(s) from the nascent transcripts. These siRNAs then cleave the CHS-A RNA at the target site with AGO, which triggers RDR6-mediated dsRNA production and subsequent DCL cleavage that produces phased siRNAs. RNA cleavage at various sites that initiate production of siRNA can be a feature of siRNA production common to cosuppression and naturally occurring RNA silencing of the CHS-A gene. siRNAs are produced from DCL cleavage of secondary-structured nascent Click here for fileCommonality of the siRNA hot spots between J-type and Red Star petals. Abundant siRNAs in the white tissues of J-type and Red Star petals are mapped on a secondary structure (a) and antisense (b) strands predicted by m-fold. Close ups of major hot spots are shown in windows, in which positions of nucleotides corresponding to siRNAs are marked by circles. Darker colors represent more total reads of siRNAs that contain the nucleotide. Mapped positions of siRNAs often overlapped, so that neighboring nucleotides had different colors. Nucleotide positions of abundant siRNAs mapped in each region are listed in the corresponding windows. Ranks of siRNA according to read number (see Figure Click here for file