How different helicase families with a conserved catalytic ‘helicasecore’ evolved to function on varied RNA and DNA substrates by diversemechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box proteinthat utilizes ATP to locally unwind dsRNA, to investigate helicase specificity andmechanism. Our results define the molecular basis for the substrate specificity of aDEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-bindingproperties and interacts with all four NTPs and both RNA and DNA. The efficiency ofunwinding correlates with the stability of the ‘closed-state’ helicasecore, a complex with nucleotide and nucleic acid that forms as duplexes are unwound.Crystal structures reveal that core stability is modulated by family-specificinteractions that favor certain substrates. This suggests how present-day helicasesdiversified from an ancestral core with broad specificity by retaining core closureas a common catalytic mechanism while optimizing substrate-binding interactions fordifferent cellular functions.DOI:http://dx.doi.org/10.7554/eLife.04630.001 Living cells store their genetic material as DNA, which can be copied to make anothermolecule called RNA. DNA consists of two strands that are wound around each other ina double helix. RNA is made in a similar way to DNA, but it is usually present as asingle strand that folds into a three-dimensional structure that is held in shape byregions of the molecule interacting with each other.Before DNA and RNA can perform their essential tasks in cells, enzymes calledhelicases must separate the interacting strands. A large group of helicases, known assuperfamily 1 and 2, are involved in virtually all aspects of the control of RNA andDNA structure. All of these helicases contain a region called the ‘helicasecore’, but they work in different ways. For example, some move along the DNAor RNA strand whilst they unwind it, while others can unwind RNA without moving. Itremains unclear how these helicases have evolved different ways to unwind DNA and RNAstructures using the same helicase core.Mallam et al. have now analyzed a helicase from yeast called Mss116, which belongs tosuperfamily 2. It is known from previous work that Mss116 binds to many different RNAmolecules and—unlike most other helicases—it does not require any extraproteins to help. This makes it an ideal model to study the properties of a helicasecore on its own.Helicases use the energy released from breaking down molecules called nucleotides topull apart the bonds that hold DNA and RNA strands together. The experiments foundthat for Mss116, a nucleotide called ATP is the best for providing the energy neededto unwind RNA but other nucleotides can work less efficiently. The experiments alsoshow that in addition to RNA, Ms116 is able to unwind double-stranded DNA moleculesthat have a certain shape.Using a technique called X-ray crystallography, Mallam et al. observed the structureof the Mss116 core when it is bound to RNA and DNA. While there are some sharedpoints of contact between the helicase and the DNA or RNA, there are more points ofcontact between Mss116 and RNA than between Mss116 and DNA.Mallam et al. propose that present-day helicases have diversified from enzymes thathad broad specificity for RNA and DNA, by optimizing interactions that favor thebinding of particular nucleotides and nucleic acids. These changes enabled thehelicases to become a versatile set of tools that control the structure of RNA or DNAin different ways.DOI:http://dx.doi.org/10.7554/eLife.04630.002 Helicases of superfamilies (SFs) 1 and 2 use ATP or other NTPs to bind, unwind, orremodel RNA or DNA in essentially all facets of nucleic acid metabolism . They coHere, we use the yeast DEAD-box protein Mss116  as a modIn this study, we determined the structural and biochemical factors that govern howanalogues of NTPs  and different nucleic acids  interact withthe helicase core. In this way, we identify the core–substrate interactions thatdictate the physiological specificity and mechanism of Mss116. Our results define thestructural and biochemical determinants for the substrate specificity of a DEAD-boxprotein. Furthermore, they demonstrate that Mss116 has surprisingly ambiguous substratebinding and unwinding properties. Considered in the context of other SF1 and SF2helicases, our findings show how small structural changes within conserved regions ofthese protein families can facilitate the emergence of specialized enzymes with newactivities and cellular functions.x, where N = A, C, G, or U, can promote theunwinding of a dsRNA. However, ADP-BeFx is the most efficient with atleast sixfold higher concentrations of C-, G-, or U-analogues required for RNA duplexunwinding  to use differentnucleotides to catalyze RNA unwinding. First, we measured the concentration ofdifferent NTP analogues required by the helicase core to unwind an RNA duplex underequilibrium conditions . This wax analogues form longer-lived, more stable complexes with RNAthan do the corresponding NTPs , while CDP-BeFx,GDP-BeFx, and UDP-BeFx promote formation of the closed stateonly at significantly higher concentrations of nucleotide analogue.Similarly, analytical size-exclusion chromatography (SEC) shows that a closed-statehelicase core with A10-RNA is maintained during elution for complexescontaining ADP-BeFx, CDP-BeFx, or GDP-BeFx but notthose containing UDP-BeFx, consistent with the latter complex having alower stability -labeledAtability . Togethe10-RNA forms at significantly lower concentrations ofADP-BeFx compared to AMP-PNP -bonds fromG128 and Q133 in the Q-motif to the base are absent in the complex withGDP-BeFx, and all of the direct interactions of the Q-motif with thebase are missing in the structures with CDP- or UDP-BeFx. The fewercontacts of all other bases relative to adenine and the less favorable stacking ofpyrimidine bases in the ATP-binding pocket explain the relative stabilities of theclosed-state complexes and reveal how the helicase core of Mss116 adapted to unwindRNA most efficiently using ATP.To investigate the structural basis for the difference in stability of the closedstate with different NTP analogs, we determined crystal structures of theclosed-state helicase core with Aectively . These sectively . Motif Ihosphate .Howeverhosphate , whereashosphate . Also, fhosphate . In partK1/2 = 400 nM) and A-DNA and B-DNA duplexes (Ki = 1700nM) (D2 of Mss116 (residues 342–597) functions as an RNA-duplex recognition domainin the open-state enzyme  Figure . To detectively) . Circulactively) . The B-D1700nM) . These r1700nM) . Our fin1700nM) . D2 of Mx . Notably, we did not observe any appreciableunwinding of the B-DNA duplex under these conditions (k1) of 0.46and 0.15 min−1, respectively, but does not unwind the B-DNA duplex and ssDNA (A10-DNA) substrates. Equilibriumfluorescence anisotropy assays in the presence of increasing concentrations ofADP-BeFx indicate that the closed-state complex forms with bothsubstrates, but at a much lower concentration of ADP-BeFx for ssRNA thanfor ssDNA  in a closed statecomplex and translocate with no observable unwinding or dsDNA , grown in ZYP-5052 auto-inducing medium for 24 hr at 22°C,and purified at 4°C, as described (The helicase core of Mss116 (D1D2) and separate domains D1 and D2 were expressed asN-terminal MalE fusions in escribed , 2012. Px using a gel-based fluorescence assay to monitor the formation of a closed-statecomplex containing a bound single-stranded substrate. Duplexes were labeled with afluorescent probe (FAM) and quencher (Iowa Black FQ) at the 5′ and 3′ends, respectively. These substrates gave a change in fluorescence upon unwinding andformation of a closed state  was preparedas described (x-Mg2+ (ranging from 0 to 20 mM) at 22°Cfor at least 1 hr in a reaction medium containing 20 mM Tris–HCl (pH 7.5), 100mM KCl, 10% glycerol, 1 mM DTT, 5 mM MgCl2, and 0.1 mg/ml of bovine serumalbumin. The protein concentration was chosen so that all of the duplex substrate isbound in the open state at equilibrium (x was quantified by using ImageJ and fit to a one-site bindingmodel to estimate the concentration of nucleotide at the midpoint(K1/2) of the unwinding reaction. In all cases,equilibrium was verified by additional assays for samples that were incubated forextended times (up to approximately 4 hr), which gave the same unwinding profiles asthose incubated for 1 hr.Equilibrium unwinding of 12-bp dsRNA, A-form DNA, and B-form DNA duplexes wasmeasured in increasing concentrations of NDP-BeFed state . NDP-BeFescribed .Measureilibrium . Samplesilibrium . The app2+ (5 mM), labeled duplex (125 nM),and unlabeled duplex (500 nM) at 22°C in a reaction medium containing 20 mMTris–HCl (pH 7.5), 100 mM KCl, 10% glycerol, 1 mM DTT, 5 mM MgCl2.Reactions were terminated at appropriate time points with 1 volume of stop buffer  and run in a non-denaturing 20% polyacrylamide at22°C for 60 min. The fluorescence signal of duplex substrate was quantified byusing a Typhoon imager  to measure the extent ofunwinding/re-annealing. The apparent fraction of unwound duplex at various timepoints was quantified by using ImageJ and (where appropriate) fit to a first-orderreaction to estimate an observed first-order rate constant(k1).Kinetic-unwinding assays of 12-bp dsRNA, A-form DNA, and B-form DNA duplexes by thehelicase core were performed with the same fluorophore–quencher labeled probes inthe p10-RNA and A10-DNA to D1D2 inincreasing concentrations of NDP-BeFx was measured by fluorescenceanisotropy using MBP-tagged protein to increase the change in anisotropy uponbinding. 5′ FAM-labeled A10-RNA or A10-DNA was incubated with protein (2 μM) and increasing concentrations ofNDP-BeFx  at 22°C for at least1 hr in a reaction medium containing 20 mM Tris–HCl (pH 7.5), 100 mM KCl, 10%glycerol, 1 mM DTT, 5 mM MgCl2, and 0.1 mg/ml of bovine serum albumin. Theobserved fluorescence anisotropy at increasing concentrations of protein was measuredby using an EnVision Microplate Reader  and was fit to aone-site binding model with a Hill coefficient to estimate theKd of single-stranded nucleic acid in the presence ofincreasing nucleotide. Equilibrium was verified by carrying out assays on samplesincubated for extended times up to 4 hr, which gave the same binding profiles asthose incubated for 1 hr. Equivalent experiments were performed to measure thebinding of A10-RNA to D1D2 in increasing concentrations of AMP-PNP or ADP(0–10 mM) and ADP + Pi (0–100 mM Pi in thepresence of 10 mM ADP).Equilibrium binding of A2, and 0.1 mg/ml of bovine serum albumin to stabilize the proteinat low concentrations. Samples were then analyzed in a non-denaturing 6%polyacrylamide gel run at 4°C for 60 min, and the fluorescence signal of thebound duplex substrate was quantified by using a Typhoon imager. The fraction ofbound duplex with increasing concentrations of MBP-tagged protein was quantified byusing ImageJ and fit to a one-site binding model with a Hill coefficient to estimatea Kd.Equilibrium binding of 12-bp RNA (A-form) and DNA (A-form and B-form) duplexes to D1or D2 was measured by EMSA using MBP-tagged proteins to increase protein solubilityas described .5′ FAM-Ki = 860 ± 40 nM) and of5′ FAM-dsRNA (250 nM) by unlabeled B-DNA duplex . In these cases, thefraction of free substrate was quantified and a Ki wasestimated from a one-site binding model.Competition assays were performed similarly by measuring the competitive displacementfrom MBP-D2 (500 nM) of 5′ FAM-B-DNA duplex (250 nM) by unlabeled dsRNA wereincubated at 22°C for 30 min in NDP-BeFx-Mg2+  and single-stranded  or duplex  nucleic acid and loaded onto a Superdex 75 column pre-equilibrated in a buffer containing 20 mM Tris–HCl (pH 7.5), 200 mM KCl,10% glycerol, 1 mM DTT, 5 mM MgCl2. The absorbance and elution volume ofthe protein complexes above the background signal of the buffer were measured at 260and 280 nm . The fod 280 nm . Control2 buffer using a thermostatically controlled0.01-cm path-length cuvette at 25°C and a Jasco J-815 spectrometer . Scans were taken between 200 and 325 nm at a scan rate of 0.5 nms−1 with 30 accumulations. Measurements were made on samples ofSEC-purified A-form DNA or B-form DNA duplexes (100 μM) in the absence orpresence of Mss116 D2 or MBP-D2 (120 μM).All measurements were performed in 20 mM Tris–HCl (pH 7.5), 100 mM KCl, 10%glycerol, 1 mM DTT, 5 mM MgCl10-RNA–NDP-BeFx complexes, protein(∼350 μM) was incubated with A10-RNA (600 μM),NDP-BeFx-Mg2+  andMgCl2 (1 mM) for 30 min on the desktop. Sitting drops were assembledusing 0.5 μl of complex and 0.5 μl of a well solution of 0.1 M HEPES, pH7.5, 2% tacsimate, pH 7.0, 20% PEG (polyethylene glycol) 3350 forD1D2–A10-RNA–ADP-BeFx; 0.2 M sodium malonate,pH 5.0, 20% PEG 3350 for D1D2–A10-RNA–CDP-BeFx;4% tacsimate, pH 8.0, 12% PEG 3350 forD1D2–A10-RNA–GDP-BeFx; and 0.1 M DL-malicacid, pH 7.0, 12% PEG 3350 forD1D2–A10-RNA–UDP-BeFx . Drops were stored at 22°C and plate-like crystals appeared within1–2 weeks. Crystals were removed from sitting drops and flash cooledimmediately in liquid N2. Crystals ofD1D2–A10-DNA–ADP-BeFx were obtained similarlyand drops were assembled with a well solution of 0.2 M ammonium acetate, 20% PEG3350.For the D1D2–ARwork and Rfree afterrefinement of molecular replacement solutions. Molecular replacement was performedwith Phaser (X-ray diffraction data were collected at the Advanced Light Source (ALS), LawrenceBerkeley National Laboratory . Details of data collection and refinement are in h Phaser , using th Phaser  and refih Phaser . Validath Phaser  and indi10-RNA–ADP-BeFx), 4TYY(D1D1–A10-RNA–CDP-BeFx), 4TZ0(D1D1–A10-RNA–GDP-BeFx), 4TZ6(D1D1–A10-RNA–UDP-BeFx), and 4TYN(D1D1–A10-DNA–ADP-BeFx).Coordinates and structure factors were deposited in the Protein Data Bank underaccessions 4TYW (D1D1–A reviewprocess). Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent , a Reviewing editor and 2reviewers.Thank you for sending your work entitled “Molecular insights into RNA and DNAhelicase evolution from the determinants of specificity for a DEAD-box RNAhelicase” for consideration at Leemor Joshua-Tor (Reviewing editor) and Ben Luisi (peer reviewer) have agreed to revealtheir identity.The Reviewing editor and the reviewers discussed their comments before we reached thisdecision, and the Reviewing editor has assembled the following comments to help youprepare a revised submission.Lambowitz and co-workers address an important question in RNA biology: how RNA helicaseshave become specific for RNA, even though they are structurally very similar to DNAhelicases. Using structural and biochemical approaches on the DEAD-box helicase Mss116that functions as a chaperone for the process of mitochondrial intron splicing, theyshow that this helicase can, in principle, bind both RNA and DNA, and a variety ofnucleotides, but with a significant preference for A over the others. Crystal structuresare presented of the Mss116 construct in complex with Berylium Fluoride and ADP, CDP,GDP and UDP. These structures go some way to delineate specific interactions that conferspecificity for the adenine base and RNA, and show that these specificities are causedby rather small tweaks in the helicase core. Their results also suggest that the gammaphosphate of ATP pays a critical role in maintaining a 'closed state'conformation when engaging the nucleotide and RNA. The authors also show that, inaddition to the known ability to bind duplex RNA, Mss116 is capable of binding to A-formdouble stranded DNA, but not DNA in the more common B-form, while the isolatedC-terminal RecA domain (D2) can bind the B-form. Crystal structures are also presentedof Mss116 in complex with A10 RNA and A10 DNA, showing a similar mechanism ofinteraction for both substrates.In general, the paper is well written and the methodology is sound, and the experimentalwork, encompassing X-ray crystallography and in vitro binding and unwinding assays, iscomprehensive. The Discussion is instructive and insightful. The figures are excellentand are very easy on the eye. Although the results are perhaps not ground breaking, thepaper provides new, and in some aspects surprising, insight for a large and ubiquitousfamily of enzymes with central biological roles. The manuscript will thus be of interestto a diverse readership. There are a number of minor comments on specific points thatwill hopefully be helpful for the authors to consider, outlined below.1) Is it clear that Mss116 functions in vivo in isolation, or is the enzyme'sactivity directed and dependent upon other partner proteins? How might this bear on thecentral conclusion of the Abstract and Discussion, regarding the apparentfamily-specific specificity? Many of the DExD-box proteins seem to have partners thatstrongly affect their activity.2) A surprising finding is the ability of Mss116 to unwind pure DNA duplexes, providedthese adopt A-form geometry. The unwinding is shown with ADP-BeFx, a non-hydrolysableATP analog. It would be important to check whether unwinding is also seen with ATP, asseveral recent papers suggest that DEAD-box helicases, which have previously beenconsidered as pure RNA helicases, might function on DNA.3) Unwinding of RNA with all four nucleotide diphosphate beryllium fluoride compounds isalso surprising. Here, again, it would be instructive to test unwinding with thenucleotide triphosphates. These results might be influenced by the affinity of bindingto the nucleotide rather than the ability of the helicase to hydrolyse a particularnucleotide and simultaneously unwind the duplex, for example. In addition, the differenttriphosphates have been tested in unwinding assays for many DEAD-box helicases, and itappears that no unwinding has been reported with NTPs other than ATP. It would thus beimportant to sort out whether one should have tested these NTPs over a largerconcentration range than examined, or whether the use of the diphosphate berylliumfluoride compounds somehow biases these experiments, as has been suggested, too. If abias were to be found, this would not invalidate any of the points made, but add afurther important facet to the story.d values for ADP-BeFx binding to Mss116 in themicromolar range. Binding of ADP-BeFx to Mss116 and RNA has recently been described byLiu et al. . Although no Kd valueswere reported there, the ADP-BeFx complex with Mss116 was very long-lived. It might beprudent to specifically verify that equilibrium was reached for the Kdmeasurements, even after incubation of 1h. In addition, the paper by Liu et al. shouldprobably be cited.4) The authors report K5) In 6) In 7) 8) Results section, “The structural basis for the ATP specificity of the helicasecore of Mss116”: “two side-chain hydrogen (H)-bonds from G128 andE133”. 9) Summary: 'core stability'. Perhaps change the wording here, because aswritten this might be misinterpreted as stability of the fold, which is clearly notintended.10) Results section  shows that a94 closed-state helicase…”), 11) Results section: the sentence “the effective concentration of the ATP gammaphosphate plays a key role in maintaining the closed-state structure” appears tobe repetitive with “the effective concentration of the ATP gamma phosphate iscritical for the stability of the closed-state”.12) 13) 14)  1) Is it clear that Mss116 functions in vivo in isolation, or is theenzyme's activity directed and dependent upon other partner proteins? How mightthis bear on the central conclusion of the Abstract and Discussion, regarding theapparent family-specific specificity? Many of the DExD-box proteins seem to havepartners that strongly affect their activity.Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18protein), to promote the splicing of a N. crassa group I intron  and may function as a transcriptionelongation factor for this enzyme . However, this function does notrequire the helicase activity  ofMss116.To address this comment, we have modified the Introduction to state that Mss116 bindsdiverse RNA substrates non-specifically and displays high RNA helicase activity in theabsence of partner proteins.2) A surprising finding is the ability of Mss116 to unwind pure DNA duplexes,provided these adopt A-form geometry. The unwinding is shown with ADP-BeFx, anon-hydrolysable ATP analog. It would be important to check whether unwinding is alsoseen with ATP, as several recent papers suggest that DEAD-box helicases, which havepreviously been considered as pure RNA helicases, might function on DNA.k1) of 0.46 and 0.15 min-1,respectively. However, we do not observe unwinding of the B-DNA duplex with ATP. This isthe same unwinding trend that we observed with ADP-BeFx. The kineticunwinding assays have been added to the Results  Unwinding of RNA with all four nucleotide diphosphate beryllium fluoridecompounds is also surprising. Here, again, it would be instructive to test unwindingwith the nucleotide triphosphates. These results might be influenced by the affinityof binding to the nucleotide, rather than the ability of the helicase to hydrolyse aparticular nucleotide and simultaneously unwind the duplex, for example. In addition,the different triphosphates have been tested in unwinding assays for many DEAD-boxhelicases, and it appears that no unwinding has been reported with NTPs other thanATP. It would thus be important to sort out whether one should have tested these NTPsover a larger concentration range than examined, or whether the use of thediphosphate beryllium fluoride compounds somehow biases these experiments, as hasbeen suggested, too. If a bias were to be found, this would not invalidate any of thepoints made, but add a further important facet to the story.2+, as previous data indicates that the unwindingactivity of Mss116 increases at lower Mg2+ concentrations  The authors report Kdx to Mss116 and RNA has recently been described by Liu et al.. Although noKvalues for ADP-BeFx binding to Mss116 in the micromolar range. Binding ofADP-BeFdx complex with Mss116 was verylong-lived. It might be prudent to specifically verify that equilibrium was reachedfor the Kvalues were reported there, the ADP-BeFdmeasurements, even after incubation of 1h. In addition, the paper by Liu et al.should probably be cited.We had verified that equilibrium was reached in all our experiments by carrying outassays for extend times (up to approximately 4 h), which gave the same binding orunwinding profiles as those incubated for 1 h. This information has been incorporatedinto the Methods section. We note that the protein , RNA substrates, and reaction conditions differbetween the two studies. We have cited the paper by Liu et al. as example of thedifferent behaviors of ATP analogues.5) Ini and RNAit might be helpful to extend the cartoon to include the final step in theunwinding cycle where the ATP is hydrolysed, and then the helicase re-enters the openstate upon departure of the ADP, P.We have added this step final step in the unwinding cycle to 6) In, perhaps itwould be better to have the binding assay  first, then the unwinding assay(A).In the current version of the manuscript, the panels that display the unwinding  and bind7): Label G128 inFigure B? Is the Q133 oriented such that the carbonyl is forced to be pointed at theO6 of the GDP? This would be an unfavorable interaction.The O-O distance from the carbonyl of the Q133 side chain and the O6 of the GDP is 3.4Å in the GDP structure. This is compared to 2.4 Å in the ADP structure, eventhough the two distances look similar in the view in 8) Results section, “The structural basis for the ATP specificity of thehelicase core of Mss116”: “two side-chain hydrogen (H)-bonds from G128and E133”.has residue 133 labelled as Q. Which one is it? Also, can glycine really make aside chain H-bond?We have corrected this sentence to read: ‘two hydrogen (H)-bonds from G128 andQ133…”.9) Summary: 'core stability'. Perhaps change the wording here, becauseas written this might be misinterpreted as stability of the fold, which is clearlynot intended.We have changed ‘core stability’ to ‘complex stability’.10) Results section shows that a 94 closed-state helicase…”), Table1 and: do the elution volumes definitively demonstrate whetherthe state is open or closed? There is for example the formal possibility that theproteins are forming oligomers. Do the authors have access to SEC-MALS? This wouldgive the molecular weights fairly precisely, which can be used to confirm that thecomplexes are intact.260/A280, which is approximately 0.6 for freeprotein and >1 for protein-nucleic acid complexes, was used as an indicator of theformation of a closed-state complex that contains nucleotide and nucleic acid. Proteincores that remain mostly in the open state under the SEC conditions have aA260/A280 ratio much closer to that of the free protein. Theelution volumes are consistent with the complex formation and are used only inconjunction with the A260/A280 ratio as indicating the open orclosed state of the core.The ratio of A11) Results section: the sentence “the effective concentration of the ATPgamma phosphate plays a key role in maintaining the closed-state structure”appears to be repetitive with “the effective concentration of the ATP gammaphosphate is critical for the stability of the closed-state”.We have removed the sentence: “They also indicate that the effectiveconcentration of the ATP gamma phosphate plays a key role in maintaining theclosed-state structure”.12): the anglesfor the unit cell are not in Greek characters.We have changed the angles of the unit cell to Greek characters.13)appears to be missing.Apologies for the omission of this Figure supplement, it is now included.14): out ofcuriosity, what happens to the binding of the A-form DNA with D2, does it retainA-form CD spectral signature?We have now also measured the CD spectra of the A-form DNA duplex when bound to Mss116D2, and this duplex also retains A-form geometry upon binding. This is included as


Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles.RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30–50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs.With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.The online version of this article (doi:10.1186/s12862-016-0589-0) contains supplementary material, which is available to authorized users. Coincidentally, the very first nuclear specificity factor (CRR4) identified for a plant organelle RNA editing event 14 years later [ndhD mRNA of Arabidopsis thaliana. This and many other nuclear-encoded RNA editing factors affecting chloroplast or mitochondrial RNA editing events turned out to belong to a plant-specific subclade of RNA-binding pentatricopeptide repeat proteins [The discovery of C-to-U RNA editing in plant mitochondria –3 was sors later  also affproteins –12. Thesproteins .Arabidopsis thaliana, Nicotiana tabacum or Oryza sativa where some 400–500 mitochondrial sites coexist with only 30–40 such sites in chloroplasts [Physcomitrella patens where only two events of chloroplast editing [Physcomitrella a particular interesting plant model to study RNA editing. In fact, the moss has recently become the first organism with a completed mutual assignment of organelle editing sites to their nuclear PPR protein cofactors [One striking discrepancy of RNA editing in the two organelles, however, concerns the observed abundance of editing events in widely studied model plants such as roplasts . Interes editing  are cont editing . Its lowofactors –19.Selaginella uncinata [S. moellendorffii [The reasons for significantly higher frequencies of mitochondrial over chloroplast editing in plants remain unclear. Such a mitochondrial vs. chloroplast editing bias seems to disappear when organelle RNA editing ultimately reaches record frequencies, approaching or even exceeding 1,000 editing events per organelle. A new organelle RNA editing record with more than 3,500 editing sites in the chloroplasts of the lycophyte uncinata  even excndorffii .Arabidopsis, Nicotiana or Oryza. Our subsequent cDNA analyses confirm these expectations of high diversities and frequencies of cp editing in the basal angiosperms. The observations suggest a tremendous decrease of chloroplast RNA editing frequencies during flowering plant evolution, in line with previous reports describing losses of mitochondrial RNA editing on different levels of angiosperm diversification [Plant organelle RNA editing dominatingly serves to re-establish evolutionarily conserved amino acid codon identities in mRNAs. This feature makes this type of RNA editing predictable to a very reasonable extent. To this end, the PREPACT software has been developed, which in its version 2 allows to predict RNA editing for entire new organelle genomes based on a manually curated reference organelle trancriptome database , 23. Usification –28.Amborella and the model angiosperm Arabidopsis, for which chloroplast editing site specificity factors have already been characterized. Selecting two cases for pilot studies we found evidence for multiple independent losses of editing factor RARE1 simultaneously with its cognate RNA editing site accDeU794SL among angiosperms. In stark contrast, we find evidence for only one single loss of editing factor CRR28 addressing two editing sites simultaneously (ndhBeU467PL and ndhDeU878SL), perfectly correlating with the serial loss of both editing sites exclusively in chickpea (Cicer arietinum).The gain or loss of organelle RNA editing sites can be expected to be correlated with an accompanying gain or loss of nuclear RNA editing factors or, alternatively, a functional extension or restriction of editing factors acting on several editing sites simultaneously. We observed interesting cases of chloroplast RNA editing sites conserved between the basal angiosperm Amborella trichopoda, Calycanthus floridus, Ceratophyllum demersum, Chloranthus spicatus, Illicium oligandrum, Liriodendron tulipifera, Magnolia kwangsiensis, Nuphar advena and Zingiber spectabile. For example, in the case of Amborella trichopoda, the only living representative of the likely sister lineage to all other extant flowering plants, we found predictions of 90, 142 or 162 sites of C-to-U RNA editing to restore conserved codons at stringency thresholds of 90, 80 or 70 %, respectively, of the 17 references implemented in PREPACT 2.0  and could verify the absence of editing in these  in 46 chloroplast mRNAs of Adiantum , Ophioglaginella  or the hthoceros  . Particularly unexpected codon-changing “extra” edits are ccsAeU68TM and rpoBeU2926LF that cannot be explained from reference comparisons. It remains to be seen whether these are taxon-specific individual “orphan” edits that show up occasionally (such as chloroplast editing psbZeU50SL in Arabidopsis) or whether they are shared with at least some related taxa. Vice versa, we could not confirm 36 of the reasonably predicted chloroplast candidate RNA editing sites in Amborella. While a lack of editing at most of these sites may be explained by relaxed sequence conservation in other taxa, the lack of RNA editing events, rpl16eU310HY, rpoC1eU389SL, rpoC1eU617PL and rpoC1eU760LF, which have already been documented in other taxa, is surprising  may exist in Zingiber spectabile. Many more additional sites of RNA editing are predicted for the other basal angiosperm chloroplast genes, of which we could already verify more than 50, mainly in Chloranthus and Illicium (not shown). Once completed, the editomes of those taxa will become further additions to the PREPACT reference database. At present, it remains unclear to which extent the diversity of chloroplast RNA editing among the early angiosperms reflects independent gains or losses of editing in evolution. Should the latter dominate, one can assume the ancestral angiosperm chloroplast editome to comprise more than 200 RNA editing sites.An example, how RNA editing reconstitutes conserved codon identities is exemplarily shown for the heavily edited ene Fig. . Only fous Table . On top Cucumis sativus) is substantially larger than previously reported [Cucumis was among the now confirmed sites, we used the opportunity to also check upon the orthologous site in Arabidopsis on which conflicting reports had been published [Arabidopsis, congruent with the 2001 report by Lutz and Maliga. We found editing petLeU5PL in Amborella reported previously [Arabidopsis, 20 exclusively with Amborella and 16 jointly with both these species .In the course of our studies we found reason to believe that the chloroplast editome of cucumber  is affected by RARE1 [The set of chloroplast editing sites shared between ana Fig.  includesor CRR28 , 38 wherby RARE1 .Amborella, Arabidopsis and Cucumis . We identified unequivocal CRR28 orthologues in all taxa including those where the one or the other of the two editing sites is lost  and the related Cicer species C. pinnatifolium for the ndhB and ndhD sites and could perfectly confirm the absence of both RNA editing sites in all three Cicer samples resulting from conversion of the affected cytidine positions into thymidines. Since no complete plastome sequence is hitherto available for Cajanus cajan (pigeon pea) we investigated the ndhB and ndhD sites individually on DNA and cDNA level. We observed loss of editing event ndhBeU467PL owing to a genomic C-to-T conversion but retention of the ndhDeU878SL editing event, hence exactly as in Glycine, Phaseolus and Vigna . This editing is absent in 30 of the 65 sampled angiosperms, either owing to conversion to a genomic thymidine or by loss of the entire accD gene from the chloroplast DNAs altogether. In the light of the current insights on flowering plant phylogeny, this suggests at least 14 independent losses . One key issue here may be the ever increasing use of RNA-seq and downstream bioinformatic pipelines that need careful adaptation to properly detect plant organelle RNA editing events. Editome information will continuously be updated with future versions of the references implemented in PREPACT .The plethora of chloroplast RNA editing initially predicted and subsequently confirmed in the basal angiosperms and the here presented case of many additional chloroplast RNA editing sites confirmed in Arabidopsis thaliana that we now found to be shared with Amborella trichopoda, hence spanning an evolutionary separation of more than 100 million years between the two extant flowering plants. RARE1 and CRR28 show highly different patterns of evolution among angiosperms that seem to correspond excellently to their known functionality.The presence of organelle RNA editing sites can be expected to be correlated with the presence of their corresponding nuclear specificity factors. The ever increasing amount of genome and transcriptome data offer a cornucopia of data to study this co-evolution of the nuclear and organelle genomes in the plant cell. We here present two cases of known editing factors, CRR28 and RARE1, affecting editing sites in accD gene in our sampling of 65 angiosperms. Numerous coinciding losses of the accD editing event and RARE1 have occurred independently during flowering plant evolution and suggest a surprisingly quick loss of RARE1 once it is obsolete owing to conversion of the cytidine to be edited into a thymidine or the loss of the accD gene from the chloroplast genome altogether. With this likely order of events it will be interesting to investigate further taxa branching phylogenetically close to the now identified loss events. We would predict to identify species representing “intermediate” cases of evolution where the accD RNA editing site has already been lost while RARE1 is still present, either in a functional or in an only mildly pseudogenized form. Such evolutionary scenarios have already been found for CRR4 and CRR21 on lower taxonomic levels (among Brassicaceae) once their cognate editing sites have vanished [RARE1 was found to perfectly co-exist with a requirement for its only known cognate RNA editing site accDeU794SL in the chloroplast vanished .Adiantum and Ophioglossum) and in Cycas taitungensis [Cathaya argyrophylla and Pinus thunbergii. Hence, the phylogenetic history of RARE1 may reach deep into the vascular plant lineage. A complicating issue is that the unrelated editing factor VAC1 [Arabidopsis. ECB2/VAC1, however additionally also targets editing site ndhFeU290SL, another editing event now found to be shared with Amborella , Calycanthus floridus (NC_004993), Ceratophyllum demersum (NC_009962), Chloranthus spicatus (NC_009598), Cucumis sativus (NC_007144), Illicium oligandrum (NC_009600), Liriodendron tulipifera (NC_008326), Magnolia kwangsiensis (NC_015892), Nuphar advena (NC_008788) and Zingiber spectabile (NC_020363) were done using PREPACT [http://www.prepact.de. Entire plastomes were used as input for the BLASTX mode of PREPACT to simultaneously identify protein coding genes and potential RNA editing events predicted from comparison to the 17 chloroplast editome references implemented in PREPACT 2.0. Thresholds were set to predictions from minimally 8 and at least 70 % of the references in the commons output  and ginger (Zingiber spectabile) material was obtained commercially from a local grocery store. Total nucleic acid preparation was done using the CTAB method [https://www.bioinfodata.org/Blast4OneKP/home) revealed that many editing events, including ones that we find efficiently edited here, are not represented in the assembled Amborella transcripts, likely owing to transcriptome assembly strategies. Conversely, four exceptional cases of editing identified there but not in our study are highlighted in Additional file B method , 70. RNAArabidopsis thaliana protein sequences of RNA editing factors CRR28 (NP_176180.1) and RARE1 (NP_196831.1) were used as queries to search for homologues in other angiosperm genomes both by standard protein BLASTP and by TBLASTN of whole genome shotgun or transcribed RNA sequences at the NCBI BLAST server available at http://blast.ncbi.nlm.nih.gov/Blast.cgi [Amborella orthologue, were encountered. For clarity, only the copy more similar to other orthologues was retained for species where duplicates likely originating from alternative gene models were present. The angiosperm protein sampling excluded more exotic taxa for which no plastome sequences have as yet been assembled , taxa for which genome sequence assembly qualities are insufficient at present to reliably identify protein orthologues  and, for clarity, very closely related proteins from species of the same genus . Two species each were included, however, for genera Arabidopsis, Cucumis, Nicotiana and Solanum that include more widely investigated RNA editing model organisms.Sequence handling and analyses was mainly done using the MEGA alignment feature . Arabidolast.cgi , 73. Givhttps://tree.opentreeoflife.org/opentree/opentree3.0@64078/Magnoliophyta and references therein). Phylogenetic trees were created and edited using the MEGA Tree Explorer feature.Protein sequence alignments and phylogenetic analyses were done using MEGA. Given the repetitive nature of the PPRs, careful manual alignment correction was needed subsequent to automatic alignment by CLUSTAL as implemented in MEGA. Phylogenetic tree construction of the CRR28 and RARE1 alignments was done with the Maximum Likelihood (ML) model using the JTT + Γ + I + F and the JTT + Γ + I model, respectively, which were selected as best-fitting models of sequence evolution. Only alignment positions with at least 90 % coverage were included resulting in 517 and 775 alignment positions included for CRR28 and RARE1 phylogenetic tree constructions, respectively. Node supports were determined with 100 bootstrapping replicates. A cladogram representing a wide selection of angiosperms for which high-quality genome data are available was created by manually editing a NEWICK file based on generally accepted insights of angiosperm phylogeny, as for example reflected in the recently realized “Open Tree of Life” project  section


Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription. Most cis-encoded sRNAs are located within 5′ untranslated regions (UTRs) of mRNAs and are transcribed in the antisense orientation to the corresponding mRNA. Cis-encoded sRNAs can expose or block a ribosome-binding site (RBS) by adopting different conformations in response to various environmental cues, thereby regulating translation. On the other hand, trans-encoded sRNAs are located in intergenic regions (IGRs). They share only limited complementarity with their target RNAs and are thought to regulate translation and/or stability of these RNAs 70 complex and regulates transcription by altering RNAP’s promoter specificity during stationary phase During infection, pathogenic bacteria must adapt to diverse and dynamic environments imposed by their host and regulate synthesis of a variety of molecules  needed to colonize, replicate and persist. This kind of regulation must be rapid, metabolically inexpensive and efficient. There is growing evidence that post-transcriptional control mediated by small RNAs (sRNAs) plays a significant role in bacterial regulation Coxiella burnetii, the causative agent of Q fever, is classified as a Gram-negative obligate intracellular γ-proteobacterium. Human Q fever is generally a zoonosis acquired by inhalation of contaminated aerosols and can present either as an acute or chronic disease. An acute case of Q fever typically ranges from an asymptomatic infection to an influenza-like illness accompanied by high fever, malaise, atypical pneumonia, myalgia and hepatitis. In approximately 2–5% of cases, chronic Q fever occurs and manifests as endocarditis, especially in patients with predisposing valvular defects Coxiella enters alveolar macrophages by endocytosis and generates a phagolysosome-like vacuole termed a parasitophorus vacuole (PV). The PV interacts with autophagosomes for bacterial nutrition C. burnetii encounters various and sudden changes in environmental conditions during its life cycle, including a rapid upshift in temperature upon transmission from contaminated aerosols to the human lung, and a downshift in pH and an increase in reactive oxygen intermediates (ROIs) in the PV. All of these events are relevant to rapid, sRNA-mediated regulation Legionella pneumophilaStreptococcus pyogenesStreptococcus pneumoniae, Salmonella spp., Yersinia pseudotuberculosis and Listeria monocytogenesC. burnetii. Thus, the aim of our study was to identify sRNAs associated with the bacterium’s developmental cycle and host cell infection. Here, we describe a set of 15 novel Coxiella sRNAs identified by high-throughput sequencing of RNA (RNA-seq) isolated from distinct life stages and culture conditions. We also characterized the 6S sRNA of C. burnetii in an effort to elucidate the function of one of the sRNA’s identified. We found that 6S RNA specifically binds to Coxiella’s RNA polymerase (RNAP), reaches its highest concentration in SCVs, and its expression is markedly increased during intracellular versus axenic growth.The sRNAs of intracellular bacterial pathogens are poorly characterized, and there are no reports on sRNAs of C. burnetii Nine Mile phase II  was propagated in African green monkey kidney (Vero) fibroblast cells  grown in RPMI medium (Invitrogen Corp.) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Bacteria were purified from host cells using differential centrifugation, as previously described C. burnetii was also cultivated axenically in ACCM2 at 37°C in a tri-gas incubator  with continuous shaking at 75 RPM C. burnetii RNA from infected Vero cells, LCVs were prepared as above and treated with 40 µg/ml RNase A in RNase A digestion buffer  to reduce host cell RNA contamination. SCVs were prepared as above and used directly. Total RNA used in deep-sequencing was purified from LCVs and SCVs with a Ribopure kit (Ambion). Resulting RNAs were treated with excess DNase I to remove trace amounts of residual DNA using a DNA-free kit, as instructed by the manufacturer (Applied Biosystems). RNA was precipitated with 100% ethanol and enriched for bacterial RNAs by sequential use of MICROBEnrich (Ambion), MICROBExpress (Ambion) and Ribo-Zero (Epicentre) kits to increase the relative level of C. burnetii RNA derived from Vero cell-propagated organisms and to exclude rRNAs, respectively. RNA from C. burnetii cells cultured in ACCM2 was done as for infected Vero cells, however, the MICROBEnrich (Ambion) step was omitted. RNA was quantified using a NanoPhotometer (Implen) and checked for integrity using a 2100 Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared with a TruSeq RNA sample preparation kit (Illumina). Libraries were sequenced on an Illumina HiSeq 2000 (76 cycles) at the Yale Center for Genome Analysis . Two independent samples were sequenced from all conditions, and sequencing statistics are given in To isolate C. burnetii Nine Mile Phase I (RSA 493) genome (NC_002971.3) using BWA software 70 consensus promoters and rho-independent terminators was predicted using BPROM Sequencing reads were mapped on the C. burnetii grown in Vero cells or ACCM2 were isolated by sequential use of Ribopure (Ambion) and DNA-free (Applied Biosystems) kits and then precipitated with 100% ethanol. For quality control purposes, RNA samples were occasionally analyzed on denaturing acrylamide gels to check for RNA integrity. RNA degradation was not observed in samples used in the study (data not shown). RNA  was electrophoresed through 1.5% agarose-formaldehyde gels and blotted onto positively-charged BrightStar-Plus nylon membranes (Ambion). Membranes were then UV-cross-linked or chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma-Aldrich), as previously described in vitro transcription of PCR products using a MEGAscript kit as instructed (Ambion), in the presence of biotin-labeled UTP . Finally, membranes were developed with a BrightStart BioDetect kit (Ambion) following the manufacturer’s protocol, and visualized using a LAS-3000 imaging system (Fujifilm). Densitometry was performed using ImageJ software Northern blots were carried out using a NorthernMax kit (Ambion) as per manufacturer’s instructions. Briefly, total RNAs of C. burnetii’s rpoS gene for generation of a growth curve showing genome numbers as a function of time C. burnetii’s 6S RNA encoding gene (ssrS) were designed using Beacon Designer 7.5 software . qRT-PCR data were obtained with a 6S RNA primer set and normalized to corresponding C. burnetii genome numbers.  supplemented with protease inhibitor . RNasin Plus (Promega) was added to a final concentration of 1 U/µl and bacteria were lysed by five alternating freeze-thaws cycles in liquid nitrogen and a 37°C water bath (5 min each). The resulting lysate was clarified by centrifugation , and the supernatant was used for further analysis.A mixed population (11 dpi) of Escherichia coli RNAP core polyclonal antibody , a corresponding rabbit pre-immune serum or rabbit anti-Coxiella Com1 polyclonal antibody. Antibodies were incubated with 100 µl PAS-Net2 at a 1∶50 dilution for 16 h at 4°C with gentle agitation. PAS-antibody conjugates were then washed five times with 400 µl cold Net2 buffer as above. C. burnetii extract (25 µl) was added to each PAS-antibody conjugate and incubated for 2 h at 4°C with rocking. IP reactions were separated by centrifugation, and PAS beads and supernatants were retained for further analysis. PAS beads were washed five times as above, and the final pellet resuspended in Net2 buffer (200 µl). Approximately 20% of this IP suspension was used for protein analysis and 80% for RNA analysis.Protein A Sepharose (PAS) beads  were swelled (2 mg PAS in 100 µl Net2 buffer) for 30 min at room temperature and washed three times with 400 µl cold Net2 buffer followed by centrifugation (400×g for 30 sec). IPs were carried out using rabbit anti-IP beads and supernatants were mixed with equal volumes of 2X Laemmli sample buffer, boiled for 5 min and centrifuged 1 min at 16,000×g. The resulting supernatants were resolved on a 10% acrylamide SDS-PAGE gel. The gel was immediately blotted onto a nitrocellulose membrane (0.45 µm pore size) and blocked for 2 h at room temperature in blocking buffer [5% nonfat dry milk in TBS-T ] with rocking. Blots were subsequently probed for 16 h with a 1∶2000 dilution of anti-RNAP antibody in antibody binding buffer (TBS-T containing 1% nonfat dry milk) followed by 5 washes of 10 min each in TBS-T. Blots were then incubated for 1 h at room temperature with rocking in a 1∶2000 dilution of peroxide-conjugated goat anti-rabbit IgG antibodies (Sigma) in antibody binding buffer, followed by 5 washes (10 min each) in TBS-T. Finally, blots were developed using a chemiluminescent substrate as instructed by the manufacturer  and visualized using a LAS-3000 imaging system (Fujifilm).Total RNA from IP beads and supernatant was isolated by extraction with phenol:chloroform:isoamyl alcohol  (Invitrogen) followed by ethanol precipitation. Purified RNA was processed using an RNase Protection assay (RPA) III kit (Ambion) as per manufacturer’s instructions. Specifically, 43 ng of RNA and 4.3 pg of probe were used in each reaction, except in the IP from the anti-Com1 antibody, where 22.8 ng RNA was used. The 6S RNA probe prepared for Northern blot analysis was also used in RPAs. RPA reactions were resolved on gels , transferred to BrightStar-Plus nylon membrane (Ambion) and UV-cross-linked. RPA blots were developed using a BrightStar BioDetect kit as instructed (Ambion) and visualized with a LAS-3000 imaging system (Fujifilm) [Please see C. burnetii and to identify potential sRNAs, we first isolated RNA from LCVs and SCVs co-cultured in Vero cells as well as those cultured axenically in ACCM2 medium. cDNAs prepared from these RNAs were subjected to Illumina sequencing. This deep sequencing analysis resulted in roughly 23 to 32 million reads from RNA isolated from C. burnetii cultured axenically, and ∼47 to 81 million reads from total RNA isolated from Vero co-cultures. On the whole, sequencing reads obtained from C. burnetii cultured in ACCM2 mapped well to the genome (97%). On the other hand, sequencing reads from total RNA isolated from bacteria cultured in Vero host cells mapped 76% and ∼72.5% to the genome, respectively   .Coxiella and most sRNAs were highly conserved within six available C. burnetii genomes  with ≥97% sequence identity. The exception was CbSR 8, which was only found in C. burnetii strains RSA493, RSA331 and Cb175 Guyana. Regions immediately upstream of all sRNAs possessed predicted σ70 consensus promoters , and group III includes sRNAs that are ORF-derived . A majorromoters , and intromoters , suggestC. burnetii LCV or SCV morphotypes . First, in the case of CbSR 3, a longer transcript (∼300 nt) was observed, which could represent a primary transcript that is cleaved to give a mature sRNA of ∼200 nt, as obtained by RNA-seq. A similar processing has been previously described for sRNAs of other bacteria Northern blot signal intensity of most CbSRs corresponded to the MEVs obtained by RNA-seq , with a C. burnetii cultured in Vero host cells to those cultured axenically in ACCM2  of C. burnetii’s ygfA locus   , a linkaC. burnetii cultured in Vero cells and in ACCM2. Northern blot analyses were also performed on total RNA isolated from SCVs at 14 dpi and 21 dpi to compare 6S RNA levels at early and late stationary phase. Blots were then probed with a biotinylated RNA designed from the 5′ UTR of C. burnetii’s ygfA locus. The resulting Northern blot validated the identity of 6S RNA and the size was observed to be ∼185 nt, which we later confirmed by RNA-seq. Furthermore, 6S RNA was found to accumulate in SCVs relative to LCVs, irrespective of growth conditions . When IP products were analyzed on western blots, two bands (∼154 kDa and ∼43 kDa) were observed that likely correspond to β/β’ and α subunits of C. burnetii’s RNAP, respectively (E. coli IPs using the same antibody Coxiella Com1) or the corresponding pre-immune rabbit serum  as compared to stationary phase (SCV). A similar observation has been reported in S. pyogenes, where transcript levels of most sRNAs are abundant at exponential and early stationary phase as compared to late stationary phase Analysis of sRNA libraries generated from total RNA isolated from Coxiella grown in host cells versus axenic medium, eight sRNAs were found to be at higher levels during intracellular growth. Of these, CbSR 12 is particularly striking with regards to its up-regulation in the host cell, and is a current focus of research in our lab. The role of sRNAs in controlling pathogenesis and virulence has been reported in a number of bacteria, including L. monocytogenesSalmonella typhimuriumVibrio choleraeWhen we compared the transcript levels of CbSRs obtained from C. burnetii lacks an apparent Hfq; an RNA chaperone that modulates translation of many mRNAs and also stabilizes interactions of sRNAs with target RNAs. Previous reports have shown that hfq null mutants of pathogens that normally possess Hfq show decreased growth rates, increased sensitivity to stress conditions and impaired virulence C. burnetii is unclear. Either Coxiella’s sRNAs are Hfq-independent, similar to many Gram-positive bacteria Borrelia burgdorferiInterestingly, using in silico analysis we also discovered that rnpB), tmRNA (encoded by ssrA) and 6S RNA by RNA-seq. RNase P RNA and tmRNA are well-studied sRNAs that are conserved among all bacteria. Studies have shown that RNase P RNA is the ribozyme component of RNase P that is involved in processing of 4.5S RNA and tRNA precursor molecules E. coliB. subtilisL. pneumophilarelA and ppGpp synthesis during stringent response C. burnetii’s intracellular niche, these functions would be clearly beneficial. This encouraged us to further investigate the biology of 6S RNA in C. burnetii.In addition to identification of 15 novel sRNAs, we also identified the bacterium’s RNase P RNA , compared to 3 dpi (LCV), and then dropped ∼2 fold at 21 dpi (SCV). However, this drop was not observed in C. burnetii grown axenically. It is possible that, in Vero co-cultures, some SCVs are still intracellular at day 14 (i.e. some Vero cells are extant), whereas by day 21 all the host cells are dead, SCVs are extracellular and 6S RNA falls to a background level. Taken as a whole, our observations are suggestive of 6S RNA’s involvement in regulating genes related to C. burnetii’s stress response, especially during intracellular growth. In order to specifically identify genes whose transcript level is altered by 6S RNA, we are currently examining an ssrS mutant and a 6S RNA-overexpression strain of C. burnetii. Analysis of the transcriptomes of these strains will yield important clues regarding the 6S RNA regulon.When we examined the transcript levels of 6S RNA in nditions . These rnditions . This inC. burnetii’s 6S RNA associates specifically with RNAP. This was demonstrated by IP experiments using a C. burnetii lysate and an antibody that recognizes core RNAP. Western blotting was performed to confirm that the antibodies were specific to C. burnetii’s RNAP .Our studies have also shown that, similar to other bacteria, i’s RNAP . An RPA i’s RNAP . This cos) is classically the major starvation/stationary phase sigma factor, but it serves as the dominant sigma factor during exponential growth of C. burnetiis is thought to be due to the stressful conditions encountered by Coxiella in the PV. With this in mind, we were curious about the potential targets of Coxiella’s 6S RNA. Eight positively-charged amino acids have been shown to create a surface that is required for binding of 6S RNA to the 4.2 region of E. coli’s RpoD (σ70) Coxiella’s RpoS and RpoH 4.2 regions indicates that they each possess only five positively-charged amino acid residues  shares 100% identity with 30 amino acid residues of the E. coli σ70 4.2 region with all eight positively-charged amino acids present . Various computational and experimental approaches have been employed in order to identify these targets C. burnetii’s physiology and pathogenesis. Further investigations are required to determine the exact role played by each CbSR to help C. burnetii transition between the two different cell morphotypes and adapt to the intracellular niche.In conclusion, this study is the first step towards elucidating sRNA-mediated regulation of Table S1Sequencing statistics.(DOCX)Click here for additional data file.Table S2PCR primers used to make probes.(DOCX)Click here for additional data file.Table S3qPCR and qRT-PCR primers.(DOCX)Click here for additional data file.Table S4Probes used in Northern blots and RPAs.(DOCX)Click here for additional data file.


SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments.Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that  Small non-coding RNAs constitute an important element of post-transcriptional gene control in many biological systems. In eukaryotes, different classes of 20–24 nt RNAs are derived from double stranded RNA (dsRNA) via cleavage by Dicer ribonucleases. Small RNAs join proteins of the ARGONAUTE (AGO) family to form RNA induced silencing complexes (RISCs). RISCs use base pairing to target complementary mRNA for repression, in plants either via AGO-catalyzed transcript cleavage (‘slicing’) or via translational repression –5. Two msiRNA production may also result from amplification loops in which a target transcript of a primary small RNA becomes a substrate for RNA dependent RNA polymerase (RDR) resulting in production of dsRNA that is in turn diced into new waves of siRNAs, termed secondary siRNAs . Severalxrn4 mutants are exceptionally prone to RDR6-mediated transgene silencing , but in this system, the RNA exosome and Rrp6 are also required for degradation of 5′-cleavage fragments , ski2–5 ), SKI3 ) ) were ordered from the Nottingham Arabidopsis Stock Centre. For all seedling analyses, plants were grown for 16 days on 1xMurashige and Skoog medium in petri dishes under a 16 h light/8 h darkness regime. For analysis of inflorescences, seedlings were germinated on 1xMS plates, transferred to soil and grown in a Percival growth chamber at 70% relative humidity under a 16 h light (21C)/8 h darkness (16C) regime.The GFP171.1 line is in accession C24 and carries the  in RDR6 . mad7/sk012876))  and SKI8ski2–4 (Sau3A) and sde1-1 (StyI). Primer sequences are listed in Supplementary Table S4.T-DNA insertion lines were genotyped with two chromosomal primers flanking the insertion site for presence of wild type allele, and an outward left border primer and a flanking chromosomal primer for the T-DNA allele. CAPS markers were used for genotyping SKI2 and RDR6 mutant and wild type alleles, ski2–4/rdr6 was crossed to C24 wild type. Kanamycin selection was applied in F2 to select for the GFP171 transgene, and all mutant combinations were identified by polymerase chain reaction (PCR) in F2 using genotyping assays described above. Mutant genotypes were confirmed by PCR in F3. ski2–4/rdr6/GFPnomiR was generated by crossing ski2–4/rdr6/GFP171.1 to SKI2/rdr6/GFPnomiR (ski2–4 and rdr6 point mutations were detected by PCR and restriction digest as described above.To obtain double homozygous combinations of ski2–4/rdr6 in GFP171.1 was crossed to rdr6–3 (accession Ler). GFP northern blots of 12 individuals of two F2 families showed that the segregation of the presence of the 5′ cleavage fragment of GFP mRNA was consistent with a single recessive mutation in the mixed ecotype background. 414 kanamycin resistant seedlings were transferred to soil, and RNA was extracted from inflorescences of individual F2 plants and analyzed by northern blot with a GFP probe synthesized by random priming from a template amplified with the primers GFP1 and GFP4 from genomic DNA extracted from GFP171 (see Supplementary Table S4 for primer sequences). Genomic DNA from each of the 99 individuals selected to be mutant by northern blotting was prepared, and an equal amount of DNA from each sample was mixed. The pool of DNA was sequenced as 2 × 101 bp paired-end reads on an Illumina platform. Illumina reads were mapped to the Arabidopsis Col-0 reference genome using bowtie version 0.12.3 (http://1001genomes.org/data/MPI/MPISchneeberger2011/releases/2010_08_17/strains/C24/) (n 0.12.3 . SHOREman 0.12.3 . The marns/C24/) , and re-For preparation of total protein extracts, 100 mg of finely ground tissue was mixed with 500 μl NuPAGE LDS sample buffer , heated for 10 min at 70ºC and centrifuged to pellet plant cell debris. The supernatant was transferred into a new tube and 10 to 20 μl of the lysate were subjected to SDS-PAGE. GFP antibodies, a kind gift from David Gilmer, have been described  according to the manufacturer's instructions. The RNA was dissolved in 50% formamide for northern analysis.TM Plus Hybridization buffer (Sigma) for 1 h at 65ºC, the radioactively labeled probe  was hybridized overnight at 65ºC. Sequences for primers used for amplification of probe templates for GFP and endogenous miRNA targets are listed in Supplementary Table S4. After hybridization, the membrane was washed 3 times in 2xSSC , 0.1% SDS at 65ºC and developed by phosphorimaging. For Terminator (Epicentre) reactions, 10 μg of total RNA was incubated with 1 U Terminator (Epicentre) for 1 h at 30ºC. The reaction was terminated by phenol extraction and subsequent ethanol precipitation. The RNA was resuspended in 50% formamide and loaded with untreated RNA as control on a 1% denaturating agarose gel. Poly(A) RNA was purified from 10 μg of total RNA using Dynabeads Oligo(dT)25 (Life Technologies) according to the manufacturer's instructions.For cleavage fragment analysis, 20 μg of total RNA was loaded onto a 1% denaturing agarose gel and separated for 3–5 h at 120V. The RNA was blotted to an Amersham Hybond-NX nylon membrane  and UV-crosslinked (254 nm). After pre-hybridization in PerfectHybTM Plus Hybridization buffer (Sigma) at 42ºC overnight. Membranes were washed 3x with 2xSSC, 2% SDS at 42ºC for 15 minutes, and developed by phosphorimaging.10 μg RNA from total extracts was denatured at 95ºC for 10 minutes in sRNA loading buffer , and separated in a denaturing 18% polyacrylamide gel. The RNA was blotted on an Amersham Hybond-NX nylon membrane  and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 2 h at 60ºC . Radioac3′-ends of GFP171 cleavage fragments were identified as described in . BrieflyFor preparation of small RNA cDNA libraries for Illumina sequencing, 2 μg of total inflorescence RNA was used. All libraries were generated using the NEBNext Small RNA Library Prep Set (Multiplex) (New England Biolabs) according to manufacturer′s instruction with the following minor changes. After final PCR amplification, 30 nmol of DNA from each library with distinct barcodes was pooled and purified in a 3% metaphor agarose (LONZA) gel stained with ethidium bromide. A gel piece in the expected size range was cut out and DNA was purified using MinElute Gel extraction kit (Qiagen). The purified DNA was analyzed using an Agilent Bioanalyzer and sequenced on an Illumina platform.Arabidopsis thaliana genome (version TAIR9 from www.arabidopsis.org) and to analyze the quality of deep sequencing globally. Unmapped reads were aligned against the GFP171.1 sequence using the Bowtie program  was constitutively expressed in an rdr6 mutant background to avoid miR171-triggered transitivity  and miRNA action deficient (mad1-mad6) classes  was selected upon continued screening based on GFP reactivation in seedlings, and caught our interest, because northern blots showed the appearance of a shorter GFP RNA in addition to the full-length transcript in this mutant . On the other hand, inflorescences of ski2–4/rdr6 maintained elevated GFP levels compared to the parental GFP171.1 line, and since an otherwise identical, but non-targeted GFP line (GFP nomiR) did not overaccumulate GFP protein in ski2–4/rdr6 inflorescences compared to SKI2/rdr6 segregants, we conclude that a mutation in the ski2–4/rdr6 line affects repression of GFP171.1 expression by miR171 . GFP171 classes . mad7 , AGO1 (miR168), MYB33 (miR159) and CSD2  was within 65000 bp of the peak, and a G-A transition was identified in this gene, altering the deeply conserved Asp residue in the DEVH box to Asn  and ski2–5 (SALK_118579)) also showed clear accumulation of 5′ cleavage fragments of several endogenous miRNA targets  and SKI8 (AT4G29830) in degradation of RISC 5′ cleavage fragments. SKI2, SKI3 and SKI8 co-purify from Arabidopsis tissues suggesting that a SKI2–3–8 complex also exists in Arabidopsis , and analyzed accumulation of RISC 5′ cleavage fragments in these mutants in the RDR6 wild type background (accession Col-0). Similar to ski2–4/rdr6, the single mutants ski2–5, ski3–5 and ski8–1 showed clear overaccumulation of several endogenous 5′-cleavage fragments  . The free energy calculations systematically underestimate the strength of base pairing between 3′-cleavage fragment and 5′-half of the miRNA, because they do not take into account that nucleotides 2–5 of AGO-bound miRNA assume an ordered, helical conformation, thus reducing entropy loss of the miRNA strand upon base pairing (ski2 mutants were not among the 173 transcripts with significantly elevated amounts of siRNAs in ski2–4/RDR6 compared to SKI2/RDR6 (Supplementary Table S2). We conclude that on many miRNA targets, RDR action results in production of low-abundant siRNAs either 5′ or 3′ to the cleavage site in ski2–4. We also conclude that the production of siRNAs correlates poorly with detection of stable 5′-cleavage fragments, and that the strength of cleavage fragment base pairing is a good predictor of the direction of siRNA spreading.Mapping of transitive siRNAs onto individual miRNA target transcripts showed that they tended to accumulate on the 5′ side of miRNA target sites, that they were predominantly 21 nt in size, and that in all, but two cases, they were RDR6-dependent Figure . Transit Figures  and 3D.  Figures . Interes pairing ,47. For in planta since the first observations of miRNA-guided mRNA cleavage  promotes reiterated amplification of these siRNAs to produce highly abundant siRNAs, possibly still dependent on RISC. This distinction between a trigger wave and amplification waves is consistent with two genetic observations that remain unexplained. First, the helicase SDE3 is required for transgene silencing in systems strongly dependent on siRNA amplification via RDR6 (trans-acting siRNAs (tasiRNAs), a class of phased siRNAs that must result from a single amplification wave of a miRNA-guided cleavage fragment, because reiterated amplification would disrupt their phased pattern of accumulation  leads to production of AGO-dependent, low-abundant secondary siRNAs that map close to the piRNA cleavage site. These siRNAs are amplified via the action of the nuclear RNAi pathway that includes dedicated AGO proteins to produce abundant siRNAs along the entire target transcript (In the experiments reported by Zhang et al., RDR6-dependent siRNAs were only described in aneously . In mostvia RDR6 ,58,59. Imulation . Second,mulation . They maanscript . Thus, aSmall RNA sequence data summarized in Figures


We propose a formal instantiation of the model and, using computer simulations, examine how the sizes of the predicted primacy and recency effects are affected by (a) variability in the quality of the options; (b) the number of options presented (sequence length); (c) the level of choice inertia ; and (d) whether choice inertia dynamically increases over the sequence. We find that recency effects are reduced and primacy effects are increased with variability in quality as compared to without, and that this holds regardless of sequence length. A sizeable primacy effect occurs only with relatively short sequences or rather high levels of choice inertia. Dynamic inertia increases primacy effects and reduces recency effects, and the impact increases with higher inertia levels. We relate these results to empirical findings and derive novel predictions from the model.People have a stronger preference for options encountered earlier or later in a sequence than for options in the middle of the sequence. To account for these primacy and recency effects,  A prominent feature of experience is that it unfolds serially, at least to our minds. Due to constraints in attention, we process events and objects one after the other . Does thprimacy effect, defined as a tendency to favor the first option in a sequence over options in the middle: candidates listed first received more votes than other candidates. In addition, recency effects, defined as a tendency to favor the last option in a sequence over options in the middle: contestants in song competitions received higher ratings the later they appeared in the sequence. Given the constructive nature of preference, it is not surprising that the serial position in which an option is presented appears to impact how it is evaluated. For instance, pairwise-competition model (PCM)—to account for the recency and primacy effects in preference. In a nutshell, the model assumes that when encountering options sequentially, the decision maker conducts a repeated pairwise comparison process in which each new option is compared with the current favorite (see below for details).choice inertia , which may be linked to expertise, on the model’s predictions. Third, we formalize and test Our goal in this article is to propose a formal implementation of the PCM that accommodates all of the components proposed verbally by The PCM combines two mechanisms that have previously been proposed to underlie serial-position effects in preference construction: repeated pairwise comparison  and a biAccording to Given that choice inertia can be expected to reduce the recency effect, Note that the PCM does not attribute serial-position effects in preference to primacy and recency effects in memory, and some authors have indeed argued that memory may not play a key role. Due to its simplicity, the PCM offers an elegant account of how people construct preferences among sequentially encountered options. Moreover, it poses only moderate demands on working memory and is computationally simple, making it a psychologically plausible model of preference construction see . One limL options are sequentially encountered and each new option in the sequence is compared with the current favorite. Per default, the first option in the sequence is chosen as the initial favorite. Choice inertia was implemented as a parameter representing the probability π (0 ≤ π ≤ 1) that the current favorite is preferred over the new option irrespective of its quality. With probability 1–π, the current favorite’s quality is compared with that of the new option; at this stage, the probability that the new option is chosen as the new favorite follows from the options’ qualities. Acknowledging the probabilistic nature of choice . Overall, the probability that the new option becomes the new favorite is therefore determined as follows:In the computer simulation, we implemented the PCM such that f choice , we usedf choice . Specifip(new). The inertia parameter π was varied on four levels, π = {0;0.3;0.6;0.9}.The old favorite remains with probability 1–Q (truncated between 0 and 10) with a mean of M = 5 and a standard deviation of δ = 5. In the conditions without quality variability, δ was set to 0. At each run of the simulation, L options were drawn randomly from the respective distribution and distributed randomly across the L positions in the sequence. We tested the PCM’s predictions at six sequence lengths: L = {2; 4; 6; 10; 12; 20}. Quality variability was implemented as a normal distribution of i, πi, is defined as (see d as see :1 is the level of choice inertia at the first position in the sequence. The parameter c (0 ≤ c ≤ 1) governs the degree to which choice inertia increases over the positions in the sequence, with higher values indicating a stronger increase. In our simulations, we set the c parameter to either c = 0 (static choice inertia) or c = 0.3 (dynamic choice inertia), implementing a moderate increase of π.where πL) would emerge as the final favorite.For each of the 2  × 6 (sequence length) × 4 (choice inertia) × 2 (static/dynamic choice inertia) = 96 conditions, the model determined the favorite option at the end of the sequence. There were 100,000 runs per condition. For each condition, we determined the probability  that an option presented at a given position in the sequence  and 20 (panels in the right column). The black line represents the preference pattern without quality variability; the gray line represents the preference pattern with quality variability. As a reference, the dashed line represents the preference pattern under random choice . As can be seen, the recency effect is substantially smaller when there is quality variability than when not. The reason for this result is that, with quality variability, the probability that the object with the highest quality has already been sampled increases at later positions in the sequence. As a consequence, the probability of a new object becoming favorite decreases. Further, the difference between the preference patterns with and without quality variability diminishes at higher levels of inertia. This is due to the fact that at higher levels of inertia, quality differences play an ever weaker role.We first focus on the conditions assuming static choice inertia. Does variability in the quality of the options affect the degree to which the PCM predicts recency and primacy effects? Figure 1A shows that also the occurrence of a primacy effect is affected by quality variability; it is more pronounced when there is quality variability than when not. As for the recency effect, the difference between the preference patterns with and without quality variability decreases at higher levels of choice inertia. Figure 1A also reveals that the primacy effect emerges only if there is a substantial amount of choice inertia. This holds regardless of the existence of quality variability; without choice inertia , there is no primacy effect at all. These findings clarify that it is choice inertia—rather than the PCM’s “first-is-best” bias . As can be seen, relative to random choice, the PCM predicts the recency effect to increase and the primacy effect to decrease with longer sequences. This holds both with and without quality variability, though the effects (relative to the pattern under random choice) are smaller with quality variability than without.How is the occurrence of primacy and recency effects affected by sequence length? Figure 1B  contrasts the predicted preference pattern with and without quality variability for the conditions with dynamic choice inertia. As can be seen, the impact of quality variability  also manifests under this condition; likewise, the impact of sequence length and level of choice inertia play out as under static inertia. Nevertheless, dynamic choice inertia does influence the choice pattern relative to static inertia. A comparison of Figure 1B with Figure 1A reveals that dynamic choice inertia decreases recency effects and increases primacy effects.Finally, The aim of our analysis was to examine, based on a formal implementation of the PCM, the extent to which the model’s predictions of primacy and recency effects in preference are affected by various features of the decision context—such as quality variability . With longer sequences the recency effect is increased, but less so with quality variability than without. The impact of quality variability also holds under dynamic, increasing choice inertia, though primacy effects are more pronounced and recency effects are weaker.Our results showed that although the model predicts primacy and recency effects also under conditions of quality variability, the size of the predicted effects is clearly affected. Specifically, recency effects are reduced and primacy effects are increased when the options vary in quality. Further, choice inertia substantially reduces recency effects and increases primacy effects, and recency effects disappear altogether at high levels of inertia. In fact, our results highlight that choice inertia is the crucial variable driving primacy effects in the PCM: without choice inertia, the model does not predict primacy effects to occur  should display mainly recency effects, and decision makers with low knowledge (and high inertia), mainly primacy effects. Moreover, these differences between decision makers with low and high inertia are predicted to be more pronounced without quality variability than with.As mentioned earlier, different levels of choice inertia may be related to different levels of knowledge—a key issue in The findings of our analysis highlight that to test the descriptive validity of the model and identify ways in which it may require further development, future studies should specifically test the predicted sensitivity of recency and primacy effects to differences in quality variability, choice inertia, and sequence length. Evidence from several existing studies seems to be consistent with the model’s predictions as elaborated in our simulations. For instance, The results of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.Rice black-streaked dwarf virus (RBSDV) belongs to the genus  Laodelphax striatellus Fallén), that carries RBSDV after feeding on viruliferous rice or other RBSDV hosts. Unlike the rice stripe virus (RSV) that can be transmitted through SBPH ovary to the offspring [Rice is one of the major cereal crops in the world and is important for global food security. Rice black-streaked dwarf virus disease is a destructive viral disease, caused by rice black-streaked dwarf virus (RBSDV), which mainly infects rice and maize plants in East Asian countries ,2,3. Thiffspring , RBSDV cffspring ,7.S1–S10 [S5, S7 and S9 segments each contain two open reading frames (ORF), and the others  each contain a single ORF [S1, S2, S3 and S8, respectively [S4 and S10, respectively [The RBSDV genome is 29,141 bp in length and consists of 10 linear dsRNA segments, which are named S1–S10 ,9. S5, Sngle ORF ,12. The ectively ,14. The ectively ,15. P6, ectively . P6 funcectively ,18,19. Pectively . The P9-ectively . P10 proectively ,22.N gene with the conserved TIR-NBS-LRR domain is the first virus resistance gene to be cloned from Nicotiana tabacum [2 and sw-5 antiviral proteins, both containing a conserved CC-NBS-LRR domain, function in resistance to tomato mosaic virus (ToMV) and tomato spotted wilt virus (TSWV), respectively [STV11-R) and five quantitative trait loci  conferring resistance to rice stripe virus (RSV) have been cloned and/or characterized [In the process of long-term interaction with pathogens, plants have evolved a variety of complex and effective resistance mechanisms against virus infection . Virus r tabacum . A signa tabacum . Tomato ectively ,27. Howecterized ,29. Howecterized . An incrcterized .pC3 and pC4 genes showed an enhanced resistance to RSV [RNA silencing is an evolutionarily-conserved natural defence mechanism against viruses in a diversity of plants . In plane to RSV . Pns6- encoding four important proteins, including RNA polymerase (P1), putative core protein (P2), RNA silencing suppressor (P6) and coat protein (P10). We expected that targeting these proteins would both inhibit viral replication and enhance the host antiviral silencing, resulting in robust and durable viral resistance. Indeed, transgenic rice plants expressing this multi-target hpRNA showed high and durable resistance to RBSDV. Our results demonstrate the feasibility of simultaneously targeting multiple viral genes using hpRNA transgenes for achieving high levels of virus resistance in rice and perhaps other plant species.This study attempts to create rice germplasm with a stable and durable resistance to RBSDV using the hpRNA technology. As RBSDV is a multipartite virus consisting of 10 genomic segments, we developed an hpRNA construct targeting four genomic segments (AtFAD2 intron (0) transgenic plants and their T1, T2 and T3 progeny showed no visible difference to the wild-type Kitaake plants in growth period, plant height and other traits, such as number of tillers and grain yield.We generated rice plants containing an hpRNA construct designed to target four genes, encoding P1 (RNA-dependent RNA polymerase), P2 (a putative core protein), P6 (RNA silencing suppressor) and P10 (major outer capsid protein) of RBSDV, which play an important role in the pathogenicity and multiplication of the virus . The hpR2 intron . The pri1, T2 and T3 populations. We first investigated the disease incidence rate in the natural field nursery. The efficiency of RBSDV infection was relatively low in the natural field, with only ~11%–22% plants of the susceptible variety Huaidao 5 showing disease symptoms in 2012 and 2013 , nor the non-target S9 RNA were detected in the three transgenic rice lines showing both hpRNA expression and a high level of RBSDV resistance after inoculation with SBPH carrying RBSDV  for the presence of the hpRNA transgene. Of the 31 progeny of Line #2 genotyped, 24 and seven showed presence and absence of the hpRNA transgene sequence, respectively  for the hpRNA-derived siRNAs , which ipC5 gene or the movement protein pC6 gene of the rice grassy stunt virus (RGSV) showed resistance against RGSV infection [In recent years, rapid development in plant biotechnology has provided a diverse variety of feasible methods for generating antiviral rice germplasm . Previounfection . Howevernfection . In thisRT-PCR analyses showed that all four target viral RNAs were efficiently suppressed in the transgenic rice plants with strong resistance to RBSDV, suggesting that the four viral gene fragments in the hpRNA are all processed efficiently by Dicer-like enzymes into siRNAs. This was confirmed by the small RNA deep sequencing analysis, which showed that abundant siRNAs were detectable from all four sequences of the hpRNA. hpRNA in plants is processed primarily by DCL4 and DCL2 into 21- and 22-nt siRNAs ,48,49. CIt is difficult to evaluate the resistance level of RBSDV in rice by using the method of natural field nursery, because of the difficulties in controlling the density and percentage of the insect vector SBPH carrying RBSDV . In our FAD2 of Arabidopsis) to form the hpRNA construct (Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method. Transformation of rice (Oryza sativa Japonica variety Kitaake) was performed following the Agrobacterium-mediated transformation method using calli derived from mature seed and hygromycin B as the selective agent [We first amplified the individual gene fragments  separately based on the sequence information from NCBI  and thenonstruct . The intve agent .0) were grown in a growth chamber  with slow-release fertilizer added to the soil. Once recovered, transgenic plants were transplanted into bigger pots under field growth conditions for sample collection and seed harvesting. The presence of the hpRNA transgene in the T1 or T2 generation was confirmed by PCR, and positive transgenic plants were selected for producing subsequent generation. Plants of each generation were self-pollinated. For analysis of RBSDV resistance and for other molecular analyses, all plants (transgenic rice and wild-type Kitaake) were grown in a paddy field under natural light and temperature conditions.Primary transgenic rice plants  approach was used to detect RBSDV in SBPH , and theS1, S2, S6 and S10 RNA of RBSDV were designed to avoid amplification of the hpRNA fragments from transgenic plants  and treated with RNase-free DNase I . cDNA was synthesized using PrimeScript™ reverse transcriptase from total RNA according to the manufacturer’s instructions . The primers for detecting c plants . Actin 1Leaves of three independent transgenic lines  and wild-type Kitaake were collected at the tillering stage and used for total RNA extraction using the TRIzol Kit according to the manufacturer’s instructions . Small RNA sequencing libraries were prepared from the total RNA samples using the TruSeq Small RNA Sample Preparation Kit  and sequenced using the Illumina Genome Analyzer II . The CLC Genomics Workbench5 software was used for analysing the small RNA sequence data.In this study, we successfully generated transgenic rice lines with high-level resistance to RBSDV by targeting simultaneously four viral genes using the hpRNA transgene technology. Our results showed that hpRNA transgenes targeting multiple viral genes can be effectively used to generate RBSDV-resistant rice germplasm, which can provide alternative genetic resources for breeding RBSDV resistance. It can be anticipated that the same approach can be applied to engineering resistance to other viruses in other plant systems.