Nicotiana benthamiana and Arabidopsis thaliana.Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.Leaves of N. benthamiana.The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in  RNA silencing is a sequence-specific gene regulatory mechanism involved in development, maintenance of genome integrity and cellular defense against virus infection in plants and animals Arabidopsis thaliana contains four DICER-like proteins (DCLs) faithfully generating size-specific siRNAs from both endogenous and exogenous dsRNA precursors trans-acting siRNAs (tasiRNAs) from RDR6-dependent dsRNA precursors The model plant Despite DCLs processing endogenous precursors to siRNAs, all four DCLs are involved in producing the virus-derived siRNAs (vsiRNAs) during virus infection A. thaliana were used with wild-type Col-0 and several dcl and rdr mutants to diagnose the function of different DCLs and RDRs Cucumber mosaic virus satRNA (satCMV) Recently, genome-wide screening and analyses of siRNAs derived from Bamboo mosaic virus (BaMV) belongs to the potexvirus genus and contains a single-stranded, positive-sense RNA genome that encodes five open reading frames (ORFs) in vivo and in vitroNicotiana benthamiana co-infected with BaMV N. benthamiana and Chenopodium quinoaTomato bushy stunt virus (TBSV) could enhance the silencing pathway on elevating the level of TBSV vsiRNAs and result in saturation of the viral silencing suppressor and a high level of unbound vsiRNAs Previously, we characterized two representative satBaMVs: BSL6 and BSF4 To analyze the profiles of viral and satellite siRNAs (satsiRNAs), we performed large-scale siRNA sequencing from BaMV-infected or satBaMV-co-infected plants followed by bioinformatics analyses. In plants co-infected with BaMV and BSL6, the level of siRNAs of BaMV was much less than that in plants infected with BaMV alone or co-infected with BSF4. In general, BaMV and satBaMV siRNAs are enriched in 21 and 22 nt of both strand polarity types and not evenly distributed in the viral or satRNA genome. The abundant region of siRNAs along BaMV and satBaMV genomes are altered in different infected hosts, which suggests that the DCL target site of the virus genome may be involved in the virus susceptibility of different hosts.N. benthamiana is highly susceptible to BaMV and satBaMV, which can move systemically and cause mosaic symptoms. To understand the association of the accumulation of BaMV and satBaMVs and their derived small RNAs, N. benthamiana was mechanically inoculated with BaMV and a noninterfering satBaMV, BSF4, or an interfering satBaMV, BSL6. The inoculated and systemic leaves were harvested at 8 and 20 days post-inoculation (dpi), respectively, for detection of the level of BaMV and satBaMV RNAs, vsiRNAs and satsiRNAs by northern blot analysis. As shown in N. benthamiana on co-inoculation with non-interfering BSF4 satBaMV; however, BaMV accumulation was greatly reduced on co-inoculation with interfering BSL6 satBaMV. In BaMV-inoculated or BSF4 co-inoculated N. benthamiana, BaMV siRNAs were barely detected in the inoculated leaves, and BaMV siRNAs were not detected in the BSL6 co-inoculated leaves  as the antiviral mechanism in plants d leaves . In contthamiana . The levthamiana , probablN. benthamiana inoculated with water (mock), BaMV, BaMV and BSF4, or BaMV and BSL6 were submitted for Solexa sequencing.To study the profiles of siRNAs, total RNA extracted from inoculated and systemic leaves of After trimming the sequences, only the 17- to 28-nt siRNAs were further analyzed. More than 2 million siRNA sequences of 17 to 28 nt, including endogenous and virus- or satRNA-derived siRNAs, were obtained from each sample  and 2. IN. benthamiana. A similar ratio of positive- to negative- strand satsiRNAs was observed  regardless of inoculated or systemic leaves, in all of the samples analyzed from observed . No compobserved .N. benthamiana  and 22 nt (33∼43%), whether in inoculated or systemic leaves of BaMV-infected or satBaMV-coinfected thamiana . SatsiRNthamiana . These rN. benthamiana, we compared the total siRNAs of 2–5 million reads. With mock inoculation, the sizes of siRNAs peaked at 22 nt, which represents the endogenous siRNAs. However, after BaMV inoculation or co-inoculation with satBaMVs, the profiles of total siRNAs changed. The population of 21-nt siRNAs increased and that of 22-nt siRNAs greatly decreased, whereas 24-nt siRNAs were induced to about 30%–40% of total siRNAs after BaMV inoculation and co-inoculation with satBaMVs, respectively -infected Dactylis glomerataTo further analyze whether any bias in size distribution of siRNAs occurs in mock and virus-infected or satRNA-co-infected ectively . Becauseectively , most ofN. benthamiana. The small RNAs were enriched and 5′-end labeled with [γ-32P]-ATP. The 5′-end labeled small RNAs separated by denaturing PAGE revealed that a stronger signal migrated at position of 24-nt RNAs in the BaMV-infected sample than that in the mock-inoculated control  region, which folds a conserved AHSL structure N. benthamiana. However, the GC % (mean 47.8%) of BaMV siRNAs in BaMV-inoculated leaves was lower than that of BaMV  of the virus-derived siRNAs. The GC % (mean 53.8%) of BaMV siRNAs was slightly higher than that of the BaMV genome (50.6%) in systemic leaves of BaMV-infected or BSF4-co-infected  of BaMV . MoreoveN. benthamiana.We then analyzed the 5′ terminal nucleotides of siRNAs. The bioinformatic data did not reveal any strong preference for the 5′ terminal nucleotide of BaMV- or satBaMV- specific siRNAs, whether derived from the (+) or (−) strand or were 21- or 22-nt siRNAs (data not shown). In summary, we found no nucleotide preference in the generation of BaMV and satBaMV siRNAs in infected A. thaliana, a non-natural host of BaMV, with BaMV and satBaMVs. The inoculated and systemic leaves were harvested at 7 and 15 dpi, respectively. No visible symptoms were observed even after 1-month BaMV infection. However, BaMV RNAs could be detected in inoculated leaves but not in systemic leaves by northern blot analysis  or satBaMV (0.1%) in BaMV- and satBaMV-co-infected thaliana  as compathamiana . This fithaliana . The posA. thaliana revealed the 24-nt siRNAs the most predominant N. benthamiana. Most of the siRNAs isolated from mock-inoculated N. benthamiana were 22 nt but decreased in level after BaMV inoculation or co-inoculation with satBaMVs, which was followed by accumulation of the 24-nt siRNAs  was comparable to that of the viral genome (50.53%), but that of satBaMV siRNAs (49.81%) was a bit lower.Regarding the distribution of vsiRNAs, most of the BaMV siRNAs from  regions . Howeverthaliana  and 7B. N. benthamiana at 20 dpi and inoculated leaves of A. thaliana at 7 dpi were hybridized with different probes by northern blot analyses. Although different probes may result in different intensities, the signals obtained in the northern hybridization  probe detected more than did the CP (−) probe, which indicates that vsiRNAs derived from the (−) strand of the CP region were indeed more abundant. All these results matched the findings for the siRNAs analyzed along the genome  and TBSV can activate PTGS N. benthamiana  and A. thaliana  (The interfering determinant region of BSL6 was previously mapped to the AHSL at the 5′ UTR of BSL6 ctively) , and 4. ctively) . These rN. benthamiana, the siRNAs derived from BaMV or satBaMVs were mainly 21 and 22 nt -infected N. benthamiana and A. thalianaN. benthamiana, 24 nt in A. thalianaBrassica junceaN. benthamiana, the DCL4-dependent 21-nt siRNAs and DCL2- and DCL3-dependent 24-nt siRNAs were actively produced after virus infection  Potato virus X (PVX) Peach latent mosaic viroid (PLMVd) Cauliflower mosaic virus (CaMV) Cucumber yellow virus and hypovirus-infecting fungi, showed an approximately equal ratio of positive- to negative-strand RNA N. benthamiana infected with BaMV alone or with non-interfering or interfering satBaMV  alone or co-inoculated with satBaMV transcripts (0.1 µg/leaf). All inoculated leaves were harvested at 7 dpi for northern blot analysis, small RNA northern blot analysis and small RNA sequencing.N. benthamiana plants or inoculated leaves at 7 dpi from A. thaliana. An amount of 2.5 µg total RNA of N. benthamiana or 5 µg total RNA of A. thaliana was used for BaMV and satBaMV detection by northern blot analysis.Total RNA was extracted with use of TRI Reagent  from 1 g inoculated leaves at 8 dpi and systemic leaves at 20 dpi from For northern blot analysis, total RNA was denatured with use of glyoxal. Electrophoresis was conducted in 1% agarose gels. RNA was transferred to a Hybond™-N nylon membrane  by the capillary method with 3 M sodium chloride and 0.01 N NaOH and immobilized by UV cross-linking.32P]CTP 32P-labeled BaMV-specific probe, L probe, was prepared by linearization of pBaHB with HindIII, then transcription with SP6 RNA polymerase, which is complementary to the 3′ ends of positive-strand BaMV RNA EcoRI-linearized pBSHE by use of T7 RNA polymerase Hybridization was performed as described with probes labeled with [32P]CTP-labeled ORF1 and CP probe were prepared as described previously 2CO3 with 1 mM NaHCO3, pH 10.2) at 60°C for 1 hr 30 min before hybridization.For BaMV and satBaMV small RNA detection, low-molecular-weight (LMW) RNA from 25 µg total RNA was enriched by 20% PEG-8000/3M NaCl. An equal volume of 50% deionised formamide with bromophenol blue was added to the LMW RNA and boiled at 95°C for 5 min, then placed on ice until being loaded into the 19% acrylamide/7 M urea gel. LMW RNA was transferred to a Hybond™-N nylon membrane by a semi-dry electroblotting system  and immobilized by UV cross-linking. Blots were prehybridized and hybridized at 42°C by use of ULTRAhyb®-Oligo (Ambion). The [32P]-ATP and 5 units of T4 polynucleotide kinase, incubated at 37°C for 30 min, and then heat inactivated at 65°C for 10 min. The labeled small RNA were flowed into the G50 column  to remove the free [γ-32P]-ATP. The 5′-end labeled small RNAs were resolved on 10% acrylamide gel containing 7 M urea. RNA markers with 23 and 21 nt were 5′-end labeled by the same method.To label the 5′-end of the isolated total small RNAs, two µg of small RNAs were dephosphorylated with 2 units of alkaline phosphatase at 37°C for 1 h, followed by phenol/chloroform extraction, and then ethanol precipitation prior to kinase treatment 5′-GUUCAGAGUUCUACAGUCCGAC GAUC-3′) was first ligated to the RNA pool and a 40–60 base-pair fraction was gel purified. Then, the 3′ RNA adapter (5′- pUCGUAUGCCGUCUUCUGCUUGU-3′) was ligated to the isolated RNA, and the 60–100 base-pair fraction was excised and purified. Reverse-transcription PCR was used to create cDNA constructs based on the small RNAs with adapter molecules on both ends. The amplified PCR products were then gel purified. The purified PCR products were quantified on the Agilent DNA 1000 chip and sequencing was performed by use of an Illumina 1G Genome Analyzer .After total RNA was extracted from plants as indicated, small RNA libraries were generated following the manufacturer's protocol . Briefly, for each library, 10 µg of total RNA was size fractionated on a 15% tris-borate-EDTA urea polyacrylamide gel, and a 15–30 base-pair fraction was excised and purified. The 5′ RNA adapter (AF018156), BSF4 (AY205227) and BSL6 (AY205210) satBaMV, and only sequences with perfect matches were considered BaMV and satBaMV small RNAs.The Illumina 1G sequencing reads were first trimmed to remove the adaptor sequence. The exact matches of the adapter sequence in the reads were identified in 3 steps as follows: 1) The adapter sequence was used as a probe, which allows for identifying exact match inserts. 2) If no adapter sequence was found, the last bases of the reads were probed successively with the first five bases of the adapter (minimum 5 bases) until a match was found, thus identifying inserts up to 30 bases. 3) Finally, the remaining reads were searched for no exact matches of the adapter. The first 4 bases of the adapter were used as a probe; the following bases were used to validate the adapter presence, and 70% of them had to be identical to the adapter sequence. The trimmed sequencing reads were then blasted to the BaMV  are shown in red above or negative-strand (−) in green below the horizontal line. The X axis represents the length of the genome, and the Y axis represents the counts of the siRNAs. S: systemic leaves. I: inoculated leaves.The distribution of siRNAs on the BaMV (A) and satBaMV (B) genome from BaMV and BSF4- or BSL6-co-inoculated (7.52 MB TIF)Click here for additional data file.Figure S2N. benthamiana. The siRNAs derived from positive-strand RNA (+) are shown above or negative-strand RNA (−) below the horizontal line. The X axis represents the length of the 5′ UTR of satBaMV, and the Y axis represents the counts of the siRNAs. The secondary structures of 5′ UTRs of BSF4 and BSL6 satBaMV are shown in the right. The hypervariable (HV) regions folding into conserved apical hairpin stem loop (AHSL) are shown in red. S: systemic leaves. I: inoculated leaves.The distribution of siRNAs in the 5′ UTRs of BSF4 (A) and BSL6 (B) satBaMV from BaMV and BSF4- or BSL6-co-inoculated (10.07 MB TIF)Click here for additional data file.Figure S3N. benthamiana (Nb) and A. thaliana (At). The X axis represents different samples, and the Y axis represents the GC percentage of the siRNAs.The siRNA GC contents of BaMV (A) and satBaMV (B) genome from BaMV or BaMV and BSF4- or BSL6-co-inoculated (7.22 MB TIF)Click here for additional data file.Figure S4A. thaliana. The total siRNAs were isolated from BaMV or BaMV and BSF4- or BSL6-co-inoculated leaves. (A) siRNAs matched to positive-strand (+) BaMV (left panel) or negative-strand (−) BaMV (right panel). (B) siRNAs matched to satBaMV (+) or satBaMV (−) genome. The X axis represents the length of siRNAs, and the Y axis represents the counts of siRNAs. The relative percentages of siRNAs of 21 and 22 nt to total siRNAs are shown above the bars.Size distribution of siRNAs derived from BaMV or satBaMV-co-inoculated (5.47 MB TIF)Click here for additional data file.Figure S5A. thaliana. The X axis represents the counts of siRNAs and the Y axis represents the scores of the predicted host gene targeted by BaMV siRNAs.Scores of host genes predicted as targets for BaMV in BaMV or BaMV- and satBaMV-co-inoculated (3.72 MB TIF)Click here for additional data file.


Caenorhabditis elegans nervous system synapses are electrical, that is, gap junctions composed of innexins. The locomotory nervous system consists of several pairs of interneurons and three major classes of motor neurons, all with stereotypical patterns of connectivity that include gap junctions. Mutations in the two innexin genes unc-7 and unc-9 result in identical uncoordinated movement phenotypes, and their respective gene products were investigated for their contribution to electrical synapse connectivity.Approximately 10% of unc-7 encodes three innexin isoforms. Two of these, UNC-7S and UNC-7SR, are functionally equivalent and play an essential role in coordinated locomotion. UNC-7S and UNC-7SR are widely expressed and co-localize extensively with green fluorescent protein-tagged innexin UNC-9 in the ventral and dorsal nerve cords. A subset of UNC-7S/SR expression visualizes gap junctions formed between the AVB forward command interneurons and their B class motor neuron partners. Experiments indicate that expression of UNC-7S/SR in AVB and expression of UNC-9 in B motor neurons is necessary for these gap junctions to form. In Xenopus oocyte pairs, both UNC-7S and UNC-9 form homomeric gap junctions, and together they form heterotypic channels. Xenopus oocyte studies and co-localization studies in C. elegans suggest that UNC-7S and UNC-9 do not heteromerize in the same hemichannel, leading to the model that hemichannels in AVB:B motor neuron gap junctions are homomeric and heterotypic.C. elegans, and other factors must influence the determination of synaptic partners.UNC-7S and UNC-9 are widely expressed and contribute to a large number of the gap junctions identified in the locomotory nervous system. Proper AVB:B gap junction formation requires UNC-7S expression in AVB interneurons and UNC-9 expression in B motor neurons. More broadly, this illustrates that innexin identity is critical for electrical synapse specificity, but differential  innexin expression cannot account for all of the specificity seen in  Xenopus oocytes  molecules. Single gap junction channels are formed from half channels (hemichannels) contributed by apposing cells. The six subunits forming a hemichannel may be homomeric or heteromeric, and the hemichannels in one channel may be identical (homotypic) or not (heterotypic). Until recently it was thought that all vertebrate gap junction subunits are connexins, and invertebrate gap junction proteins are innexins  kDa mole. Althougexample, ,7,8).unc-7 gene is essential for coordinated locomotion in C. elegans  is unusual in the innexin family in having a predicted amino terminus extending 120 amino acids upstream of the canonical innexin translational start site  exon; mutations confined to intron 1  or weak (mn384) Unc-7 phenotype ; Affymax Research Institute, Palo Alto, CA, USA) including a stop site was inserted into the SalI site in exon 11 , resulting in deletion of 17 amino acids of the UNC-7 carboxyl terminus. This construct rescued forward locomotion when micro-injected at DNA concentrations ranging from 1–75 ng/μl. Derivatives of this basic construct included removal of the alternative exon 4 by deleting an NheI/ApaI fragment ; and introduction of a Met to Leu mutation  by PCR amplification with primers that included the sequence change  and included a frameshift generated by filling in the SalI site . To introduce GFP near TM4, a BamHI site was introduced at the desired position  and GFP sequences including a translational stop site were cloned into the new site, resulting in deletion of 94 amino acids from the carboxyl terminus of UNC-7.Most t Figure  begins at Figure  extendedunc-7 by various constructs was assessed by tapping transformed animals on the nose or tail and observing whether or not smooth sinusoidal waves were propagated. unc-7 mutants typically cannot move more than one full body-length forward before assuming a severely kinked posture; in the reverse direction they initially produce smooth waves  but quickly display sharp kinks and stop progressing. Forward locomotion was considered rescued if animals could progress forward smoothly for at least several body lengths before showing signs of kinking. Many animals showed fairly long periods of forward movement before suffering a bout of spastic kinking, followed by progressive forward movement. A more precise analysis of the variations involved in rescue of locomotion was beyond the scope of this work.Phenotypic rescue of Determination of the expression pattern of antibody- or GFP-labelled puncta (presumptive UNC-7-containing gap junctions) in various mutant backgrounds was based on examination of at least two  independently isolated transgenic lines for each condition. Mixed stage animal cultures were antibody-stained, and at least a dozen late larval stage animals  were examined for each line. Pictures used in figures show representative animals expressing constructs on extrachromosomal arrays for each condition.unc-9 gene from exon 4 to the polyadenylation site at nucleotide 12,661 of cosmid R12H7 -B) by virtue of introduced NheI, SpeI and SacII sites. The resultant construct is predicted to delete 138 amino acids from the carboxyl terminus of INX-4. To generate the long-range PCR GFP product, upstream sequences covering nucleotides 26,894-13,521 of F26D11 were amplified, digested with KpnI , the appropriate 8-kb fragment purified and ligated to KpnI-digested inx-4::gfp plasmid, and the final 14.6-kb product amplified from the ligation mix. This fragment (20 ng/μl) was co-injected with 50 ng/μl RIp16 for transformation rescue.For ScaI/BamHI fragment from cosmid F09B12 containing the 3' end of the unc-7 gene, inserting GFP amplified from pPD114.24 (provided by A Fire) into the SalI site near the 3' end of the unc-7 coding region, and then inserting the 5' coding region with a BamHI/SgrAI fragment obtained from the pCS2+U7S construct used for expression in Xenopus ooctyes. The resultant plasmid therefore includes the cDNA sequence for the coding region of UNC-7S plus native 3' untranslated region sequences. The only introns present are those found in the GFP sequences. For all promoter fusions the first intron/exon splice site (or S1 trans-splice site) of the heterologous gene was included on the PCR-amplified promoter fragment.A cassette for driving expression of UNC-7S::GFP from heterologous promoters was made by blunt-end cloning a 5.3-kb unc-7 animals, a 2-kb presumptive promoter region of UNC-7S (Punc-7S) was PCR-amplified (primers 5'-AAGGATCCTCACATATTCAAATTCAAGAGATC-3' and 5'-AACCGCGGAACAAATATCAGGAAACTGCGCTC-3') and inserted as a SacII/BamHI fragment into the cassette; the construct (Punc-7S::unc-7S::GFP) rescued after micro-injection at a concentration of 25 ng/μl into unc-7(e5) animals. A translational fusion of Punc-7S in the GFP expression vector pPD95.67 (provided by A Fire) was injected at a concentration of 50 ng/μl and found to express in various head neurons but not in ventral nerve cord motor neurons.To show that this construct could rescue forward locomotion in sra-11 (Psra-11::unc-7S::gfp), inserted as a 5.1-kb BamHI fragment (amplified with primers 5'-AGACTTCGACAGTTCAAGCTCTCGG-3' and 5'-GTTGTCATGGATCCTAGCTGAAATATAAGGG-3') and micro-injected at 10–100 ng/μl; acr-5 , inserted as a 4.4-kb SacII/BamHI fragment (primers 5'-GAGAAAGAGAAGCGCCGCGGCTCAG-3' and 5'-ACTCTGCTACCGGATCCACAGGAGC-3') and micro-injected at 10–25 ng/μl; and cex-1 (Pcex-1::unc-7S::gfp), inserted as a 1-kb SacII/BamHI fragment (primers 5'-CCGCGGTTTGTCGTGTTCCAAACAGAAGC-3' and 5'-TGCAACAACCAATTCTGAAAGTATAAGATTTGACTG-3') and micro-injected at 100 ng/μl.Other presumptive promoters tested included that for acr-5 promoter (Pacr-5::unc-9) was achieved by PCR-amplifying the unc-9 coding region (primers 5'-CTAGTCTTTGCAGAGCAAGTGAGG-3' and 5'-CCGTCACGACATGACTTAGGATGAG-3') and cloning this 4.5-kb product into the EcoRV site of pBS in an orientation allowing for insertion of SacII/BamHI heterologous promoter fragments. An unc-9::gfp expression cassette was made by introduction of GFP (afmx) from the partially rescuing unc-9::gfp genomic construct (above).Expression of UNC-9 under control of the col-19::gfp construct expressed in adults, thus allowing for examination of unc-7::gfp localization in larval stages.For many of the heterologous promoter fusions, the plasmid JA-1  was usedBamHI/EcoRI-digested pCS2+ methionine . The in vitro-translated products were separated by 12.5% SDS-PAGE and the dried gel imaged with a Phosphoimager .unc-124(hs10) was originally isolated as a cold-sensitive (cs) unc mapping to the left arm of chromosome X between dpy-8 and unc-10, at approximately 2.4 cM. unc-93(e1500) confers a gain-of-function rubberband phenotype that is suppressed by the recessive sup-10 (mn219) mutation. The focus of action of the e1500 rubberband phenotype is the body wall muscles – when the sup-10(+) free duplication mnDp3 is present in body wall muscles, the animals have a rubberband phenotype due to unc-93. If in a rare mitotic event mnDp3 is lost in the lineage giving rise to body wall muscles, animals will be wild type due to recessive suppression by sup-10. unc-3 animals are severe Unc coilers, and unc-3 is epistatic to unc-9; the focus of action of unc-3 is widely distributed in the ABp lineage. The focus of action of osm-1 is in sensory neurons associated with amphids and phasmids that also derive from AB. We used SP800 to screen for rare non-Unc-3 non-Unc-93 non-Osm-1 animals. These animals are predicted to maintain mnDp3 in the AB lineage (non-Unc-3 and non-Osm-1 phenotypes) but will have lost mnDp3 in the P1 lineage . Further verification of the loss of mnDp3 from P1 is the absence of mnDp3 from any of the progeny . We screened approximately 10,000 animals and identified 7 hermaphrodites that were non-Unc-3 non-Unc-93 non-Osm-1 and gave rise to broods that showed only Unc-3 progeny (osm-1 was not scored in the broods). None of these seven animals displayed an Unc-9 phenotype, suggesting that the focus of action of unc-9 is not in the P1 derived lineage, and that mnDp3 maintained in the AB lineage of these animals rescued the unc-9 mutation.Mosaic analysis of unc-9(e120) daf-6(e1377) sup-10(n983); mnDp3 [unc-9(+) daf-6(+) sup-10(+)]. The gain-of-function sup-10(n983) allele [daf-6 is in the amphid and phasmid sheath cells that derive from ABp [daf-6 animals are defective in the dye-filling (Dyf phenotype) of sensory neurons associated with these structures. This strain gives rise to wild-type (carrying mnDp3) and Unc-9 Rubberband (lacking mnDp3) progeny. If the unc-9 focus of action is derived from AB, we expected to find rare Unc-9 non-Rubberband animals arising from loss of mnDp3 only in the AB lineage. Such animals were expected to be non-Rubberband because mnDp3 complements the sup-10 mutation in the body wall muscles, dye-filling defective because of the ABp focus of daf-6, and to give rise to both wild-type and Unc Rubberband progeny because of maintenance of mnDp3 in the germline derived from P1 descendants. Of approximately 15,000 animals screened, 2 were identified that met the criteria of being Unc-9 non-Rubberband Dyf and giving rise to both wild-type and Unc Rubberband progeny. Additionally, six were identified as Unc-9 non-Rubberband non-Dyf, giving rise to both wild-type and Unc Rubberband progeny. Five of the latter were classified as exhibiting a weaker Unc-9 phenotype. We interpret the latter group as potentially representing losses of mnDp3 within the ABa lineage, or representing a more complex pattern of mosaicism within the ABp lineage that maintains mnDp3 in the amphid and phasmid sheath cells.The second strain employed was of genotype ) allele  confers ) allele ; the focfrom ABp , and dafunc-9 cannot be precisely determined from these mosaic experiments, the results are consistent with the interpretation that loss of unc-9(+) function from the P1 lineage can be tolerated without apparent effects on locomotion, and that the loss of unc-9(+) function from within the AB lineage gives rise to the Unc-9 phenotype.Although the focus of action of cs: cold-sensitive; EM: electron micrograph; GFP: green fluorescent protein; RACE: rapid amplification of cDNA ends.The authors declare that they have no competing interests.C. elegans and contributed to writing of the manuscript, IMS and JX carried out electrophysiology experiments in Xenopus oocytes and JX contributed to writing of the manuscript, BJN participated in design and coordination of electrophysiological studies and contributed to writing of the manuscript, and JES participated in design and coordination of C. elegans experiments and contributed to writing of the manuscript.TAS carried out all experiments involving unc-7 mutantsAnti-UNC-7 staining of . Anti-UNC-7 staining of unc-7 mutants.Click here for fileinnexin::gfp constructsExpression of other . Tabulated neuronal innexin expression data.Click here for fileGj∞/Gj0)/Vj relationsBoltzmann fit parameters of (. Tabulated data of Boltzmann fit parameters.Click here for fileSDS-PAGE analysis of UNC-7 isoforms in rabbit reticulocytes. Western blot analysis of UNC-7 isoforms translated in rabbit reticulocytes.Click here for fileUNC-7S mosaic analysis. Genetic mosaic analysis of rescue of forward locomotiion by UNC-7S.Click here for file


Bats of 2 species harbor group 1 coronaviruses. Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People’s Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription–PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in  Coronaviridae, genus Coronavirus). Other coronaviruses have also jumped to new host species and caused emerging diseases. For example, porcine epidemic diarrhea virus emerged in European pigs from an unknown host species during the late 1970s and caused severe enteric disease (Emerging diseases are frequently zoonoses caused by RNA viruses (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) in live animal markets  from different locations in southern People’s Republic of China and the Hong Kong Special Administrative Region were found to be infected with SARS-like CoVs, and some of the bats had antibodies to these newly recognized coronaviruses  and 1 Brazilian free-tailed bat (Tadarida brasiliensis) were captured in mist nets as they emerged from a roost in a building in Mancos in Montezuma County in southwestern Colorado (site 1) at dusk on August 19. This species was previously thought to be conspecific with the little brown bat (M. lucifugus) that is common throughout North America (Eptesicus fuscus) were sampled at a roost in a building in Fort Collins in Larimer County in north-central Colorado (site 2) on August 7. Other bats (n = 27) were sampled at sites 3 and 4 incidental to ongoing, unrelated bat faunal surveys. One western small-footed myotis (M. ciliolabrum) and 1 long-eared myotis (M. evotis) were captured in mist nets over water on August 8 at Soapstone Prairie Natural Area in Larimer County (site 3). Four big brown bats, 3 long-eared myotis, 8 occult myotis, 1 Brazilian free-tailed bat, 7 long-legged myotis (M. volans), and 2 silver-haired bats (Lasionycteris noctivagans) were trapped in mist nets during the nights of August 14–20 as they drank or foraged near open water at 2 sewage treatment lagoons (9 km apart) (site 4) in Montezuma County, Colorado. Bats were captured under authority of a Colorado Division of Wildlife Scientific Collection License following procedures approved by the Institutional Animal Care and Use Committee of the US Geological Survey, Fort Collins Science Center. Typically, each bat was sampled within 5–10 minutes of capture and then released.Bats were sampled at 4 sites in the Rocky Mountain region in August 2006. At sites 1 and 2, bats of 2 species were sampled in colonies inhabiting 2 buildings 480 km apart on opposite sides of the continental divide of the Rocky Mountains. Eight occult myotis  following the manufacturer’s instructions. Extracted RNA was eluted in 50 μL of RNase-free water and stored at –80°C. We used Moloney murine leukemia virus reverse transcriptase  with random hexamers in a 20-μL reaction to generate cDNAs by using 10 μL of RNA as a template according to the manufacturer’s instructions. All samples were extracted and analyzed in triplicate. RT products were stored at –20°C.1b was used to screen the cDNA samples , and 0.2 μmol/L of primers 1 and 2: 5′-GGTTGGGACTATCCTAAGTGTGA-3′ (primer 1) and 5′-CCATCATCAGATAGAATCATCATA-3′ (primer 2) by using the following PCR program: 15 min at 95°C; 45 cycles for 1 min at 95°C, 1 min at 48°C, and 1 min at 72°C; and 10 min at 72°C.All samples were screened by PCR and nested PCR. On the basis of previous reports, PCR with a pair of consensus primers that target a highly conserved region of coronavirus gene For nested PCR, 5 μL from each PCR was amplified in a 50-μL reaction with primer 2 and primer 3 (5′-GTTGTACTGCTAGTGACAGG-3′), an internal primer based on nucleotide sequences of the PCR amplicons by using 40 cycles of the same PCR program. All RT-PCRs were conducted in an enclosed nucleic acid workstation equipped with a UV light  in a room separate from the main laboratory. Water controls in all RT-PCRs did not show false-positive results. To overcome possible PCR inhibitors in fecal samples, PCR was performed both on the cDNA and on a 1:10 dilution of the cDNA. Amplicons were analyzed by agarose gel electrophoresis. For each positive specimen, amplicons from 2 independent RT-PCRs were sequenced on an ABI 3730 DNA sequencer  at the University of Colorado Health Science Center Cancer Center DNA Sequencing and Analysis Core. Numbered specimens were then correlated with lists of bat samples.http://workbench.sdsc.edu). Phylogenetic trees were constructed by using the neighbor-joining method in the program PAUP* version 4.0  rooted with porcine respiratory and reproductive syndrome virus (GenBank accession no. NC_001961). Sequences used for alignment were AF304460 (HCoV-229E), AY567487 (HCoV-NL63), DQ648858 (BtCoV 512), AY594268 (BtCoV HKU2), DQ249224 (BtCoV HKU6), DQ249226 (BtCoV HKU7), and DQ249228 (BtCoV HKU8). The deduced sequences from this study were deposited in GenBank under accession nos. EF544563–EF544568.Viral sequences were analyzed and aligned by using ClustalW  were collected from 57 bats of 7 species in 4 locations in the Rocky Mountain region during a 2-week period in August 2006 . PCR amps tested . None ofDespite the small number of bats sampled, there was a high prevalence of coronavirus RNA shedding in fecal samples of 2 species of bats. Five (50%) of 10 fecal samples from occult myotis and 1 (17%) of 6 fecal samples from big brown bats were positive for coronavirus in screening tests. The 1 coronavirus-positive sample from big brown bat (bat sample 65) was from feces of 1 (33%) of 3 big brown bats sampled at site 2 in north-central Colorado, whereas the positive samples from the occult myotis  were from sites 1 and 4 in southwestern Colorado, ≈480 km from site 2 . Most of1b was amplified by RT-PCR from the 6 positive samples. Analysis of nucleotide sequences of these amplicons showed that all 6 RM-Bt-CoVs are members of coronavirus group 1  of 10 occult myotis and 1 (17%) of 6 big brown bats tested contained low levels of coronavirus RNA in feces. No coronavirus RNA was detected in the oral or anal region swabs tested. Similarly in Asian bats, coronavirus RNA was found in a higher percentage of fecal samples than saliva samples  (No bat of any species occurs in both the Eastern and Western Hemispheres (yotinae) . RhinoloIn our small, initial study of coronaviruses in North American bats, samples were restricted in size, location, and variety of bat species, and we found only group 1 coronaviruses. When larger numbers of bats and additional bat species in North America are studied, additional bat coronaviruses with complex phylogenetic attributes, biogeographic patterns, and perhaps epizootiologic attributes may be discovered. For example, determining if North American bat coronaviruses are species-specific will provide useful information. In Asia, different species of bats roosting in the same cave host different coronaviruses (A recent analysis of genome sequences of coronaviruses of bats, other animals, humans, and birds suggested that bats may be the original hosts from which all coronavirus lineages were derived (It is possible that another epidemic caused by an emerging coronavirus could occur in the future. As in the SARS epidemic, bats could play a role in future emergence of coronaviruses in humans or other species. Isolation of infectious bat coronaviruses and elucidation of their host ranges, receptor specificities, and genetic diversity will greatly aid in our understanding of their potential for emergence.


The structural studies, coupled to structure-directed biochemical analysis, together with other thermodynamic and kinetic studies, provide insights into Argonaute with implications for the mechanisms of RNA silencing in eukaryotes.Argonaute is the principal protein component of the mechanisms of RNA silencing, providing anchor sites for the small guide RNA strand and the 'slicer' activity for cleavage of target mRNAs or short passenger RNA strands. Argonaute is the core constituent of the silencing effector complexes RISC (RNA-induced silencing complex) and the RITS complex , interacting directly or indirectly with Dicer proteins, R2D2/Loquacious/TRBP and GW182 family proteins in the former, and Chp1 and Tas3 in the latter. In a breakthrough series of papers, Patel  Caenorhabditis elegans and outliers, which include the prokaryotic Argonautes , though the latter category displays some similarity to the Piwi subfamily. Structural studies have revealed the molecular functions of Argonaute, showing that Argonaute is 'Slicer' : RNA-induced initiation of transcriptional gene silencing [complex]; siRNA: small interfering RNA; TtAgo: Thermus thermophilus Argonaute.AfPiwi: The author declares that they have no competing interests.


The majority of viruses infecting animals and plants today are RNA viruses cis-acting signals in the viral RNA template have been implicated in regulating the relative levels of genomic and antigenomic RNAs.For many (+)RNA viruses, RNA replication requires viral enzymes such as RdRp, helicase, capping enzymes, and NTPase, as well as non-enzymatic proteins that participate in the assembly of the viral replication complex. Host cell proteins often play essential roles in (+)RNA virus replication as well. (+)RNA virus replication is asymmetric: the synthesis of (+)RNA, the one that is packaged into the progeny virus, is up to ∼100 times greater than that of (−)RNA Host membranes play an important role in (+)RNA virus replication, as the RNA replication complexes released from membranes during purification generally lose the ability to catalyze true RNA replication, although a limited activity may be retained. In general, the intracellular membranes of cells infected with (+)RNA viruses rearrange to form anchor sites for viral RNA replication complexes Although little is known about the structure of the membrane-associated RNA replication complexes, (+)RNA virus RdRps have been well characterized to have an overall right-handed shape with three domains, the fingers, palm, and the thumb RNA template is selected from the dsRNA genome during transcription. A 5′-cap binding site, identified on the reovirus polymerase, provides a neat solution for some dsRNA viruses of high eukaryotic hosts (−)RNA viruses have a helical-shaped, protein-coated genome, also known as the ribonucloeprotein complex (RNP), which functions as the sole template for viral RNA replication and transcription . Newly mThe composition of the RNA replication machinery differs between segmented and non-segmented (−)RNA viruses. The influenza A virus, a segmented (−)RNA virus, contains eight vRNAs, each packaged as a double helical-shaped RNP with multiple nucleoproteins (NPs) and a heterotrimeric polymerase  that is bound to the conserved vRNA termini . Each NPRNA replication of non-segmented (-)RNA viruses, such as the rabies virus and the vesicular stomatitis virus (VSV), requires an intricate interplay of the nucleoprotein N, the large protein L, a non-enzymatic P, and the N-enwrapped genome, also called the nucleocapsid Different replication strategies are used by (+), (−), and dsRNA viruses to limit dsRNA exposure, to regulate (+)RNA versus (−)RNA synthesis, and to modulate transcription versus replication throughout the infection cycle. The differences in the functionalities of the replication complexes are reflected in their structural composition, their subcellular localization, and in the interaction of the complex with viral RNA templates. It is expected that studies of RNA virus replication machineries will have a large impact on antiviral drug development due to their specific activities in virus replication.


RNA silencing has been implicated in virus symptom development in plants. One common infection symptom in plants is the formation of chlorotic tissue in leaves. Chlorotic and healthy tissue co-occur on a single leaf and form patterns. It has been shown that virus levels in chlorotic tissue are high, while they are low in healthy tissue. Additionally, the presence of siRNAs is confined to the chlorotic spots and the boundaries between healthy and infected tissue. These results strongly indicate that the interaction between virus growth and RNA silencing plays a role in the formation of infection patterns on leaves. However, how RNA silencing leads to the intricate patterns is not known.Here we elucidate the mechanisms leading to infection patterns and the conditions which lead to the various patterns observed. We present a modeling approach in which we combine intra- and inter-cellular dynamics of RNA silencing and viral growth. We observe that, due to the spread of viruses and the RNA silencing response, parts of the tissue become infected while other parts remain healthy. As is observed in experiments high virus levels coincide with high levels of siRNAs, and siRNAs are also present in the boundaries between infected and healthy tissue. We study how single- and double-stranded cleavage by Dicer and amplification by RNA-dependent RNA polymerase can affect the patterns formed.This work shows that RNA silencing and virus growth within a cell, and the local spread of virions and siRNAs between cells can explain the heterogeneous spread of virus in leaf tissue, and therewith the observed infection patterns in plants. RNA silencing is an evolutionary conserved mechanism in eukaryotes that has a major role in gene regulation, development, transposon control and defense against viruses.Antiviral RNA silencing is induced by virus double-stranded RNA (dsRNA) or by specific single-stranded RNA (ssRNA) structures. Double- or single-stranded RNA is cleaved into small interfering RNA (siRNA) by RNase III-like enzymes such as Dicer and Dicer-like ,2. siRNAThe antiviral role of RNA silencing is extensively studied in plants ,6,7. VirPreviously we developed a mathematical model of RNA silencing and its interaction with viral growth within a cell . We founWe here study how RNA silencing can explain the development of leaf patterns resulting from viral infection. To this end we use a detailed modeling approach in which we combine an intra-cellular model of viral growth and the RNA silencing pathway with inter-cellular tissue dynamics.We observe that RNA silencing and virus growth on a tissue can result in a discontinuous spread of the virus: the virus reaches high levels in some cells, while it is suppressed in other cells. We study the conditions for different type of patterns. These patterns could be the basis of plant symptom development. We elucidate the mechanisms leading to these patterns and how increased silencing efficiency, siRNA movement and the occurrence of a secondary response relate to the pattern formation.To investigate the formation of infection patterns in plants we model an area of plant tissue on a grid. Each grid point represents a plant cell. Within each cell we calculate virus levels and levels of RNA silencing proteins with a detailed model. Virions and siRNAs can move from cell-to-cell. A schematic representation of the model is shown in Figure The number of molecules in each cell is calculated with our previously described model of the antiviral RNA silencing pathway and a replicating plus-strand RNA virus .The intracellular model consists of coupled differential equations, representing viral plus- and minus-strand RNA, dsRNA, virions, RdRP, siRNA targeting plus or minus-stranded RNA, and free and active RISC. The virus replication cycle starts with the translation of plus-stranded RNA into a poly-protein. After auto-cleavage one of the products is RNA-dependent RNA polymerase (RdRP) that associates with plus-strand RNA to synthesize a complementary strand. The formed dsRNA separates into a plus- and minus-strand that can both associate with RdRP again. We assume that the minus-strand is the preferred template for dsRNA synthesis. Semi-conservative synthesis of multiple plus-strands from a single minus-strand template is incorporated in the model, resulting in a biased plus-to-minus ratio. The virus produces virions that consist of plus-stranded RNA and coat proteins. We simplify here by using the number of plus-stranded RNA instead of modeling a separate coat protein.Viral double- or single-stranded RNA is degraded by host encoded Dicer into siRNAs, that have a plus- or minus-strand polarity. siRNAs cleaved from dsRNA have a 50% chance of targeting either the plus or the minus strand. siRNAs cleaved from a ssRNA hairpin automatically target strands with the opposite polarity. Via RISC the siRNAs cause degradation of either plus- or minus-stranded viral RNA.Secondary siRNAs can be synthesized through the amplification pathway that involves synthesis of dsRNA by host encoded RDR. We implement unprimed, primed and guided amplification. Each type of amplification can be studied separately.All equations are integrated using a timestep of 3.6 seconds. Simulations run for 300 hours, unless indicated otherwise. The equations can be found in the Appendix.To study RNA silencing and viral infection in a tissue we use a spatial model. Each grid point represents a cell for which the intracellular dynamics are calculated with the model described above.Viruses encode movement proteins that enable the movement of virions from cell-to-cell through plasmodesmata. We implement the movement of virions to the four neighboring cells in our model. Virions can be unpacked in each cell into naked plus-strand RNA. We chose a 4 neighborhood because cells share almost no surface area with the diagonal neighbors.For movement we shift to a particle based system, because we do not want incomplete particles to trigger a reaction in a neighboring cell. Movement of particles occurs every timestep (3.6 seconds). A fraction of the total number of virions in the cell is evenly distributed among the four neighbors, and excess virions are distributed randomly among the neighbors. When the number of moving virions is smaller than one, we draw a random number to decide if one virion moves to a random neighbor. With this method we underestimate the heterogeneity of viral spread as compared to Brownian motion. This method is therefore a good worst-case scenario for the study of heterogeneous spread of virus particles.The silencing response is able to spread from cell-to-cell with a short range silencing signal, most likely siRNAs . We implWe vary Dicer cleavage rates as representative for silencing strength to study the effect of RNA silencing on the spread of virus particles over the tissue.Within the cell three different behaviors can be observed . High DiWe first study virus spread without silencing present. We initialize the tissue with healthy cells and infect one cell in the center with 10 viral plus strands. After initiation the virus starts to produce virions that spread from cell-to-cell. We fix the fraction of virions leaving a cell to 1% per hour.The virus spreads rapidly over the entire area and 75 hours post infection (hpi) a circular area of the grid has become infected with the virus. In Figure When RNA silencing is active, we initialize the cells with RISC and Dicer. We first assume that Dicer is only capable of cleaving siRNA from viral dsRNA, and that siRNAs cannot move from cell to cell. Even without siRNA movement RNA silencing slows down virus spread over the area Figure . In the As shown above, without siRNA movement the infection spreads homogeneously over the area. When siRNAs do move from cell to cell, they can limit virus growth in neighboring cells, resulting in viral growth in some cells and suppression in others. This results in patterns that can spread over the entire area or stay localized to the area around the inoculated site. We observe somewhat different patterns for low and intermediate Dicer cleavage rates and we will discuss results from both possibilities.We first study the effects of a low Dicer cleavage rate for all possible siRNA movement rates. Varying the rate of siRNA movement we observe that the virus does not spread uniformly over the area and that patterns are formed.Low siRNA movement results in a circular pattern that is shown in Figure In the time-series Figure  it can bFor a higher siRNA movement rate a different pattern occurs Figure . The cirFor the maximum siRNA movement rate (10% of the available siRNAs in a cell) the infection is confined to the area around the inoculated site Figure .Concluding, siRNA movement creates silenced cells in which the virus is suppressed, and cells in which the virus grows to high values. Increasing siRNA movement increases the number of silenced cells, rather than decreasing virus load in all cells.When Dicer cleavage rate is intermediate, similar patterns can be observed Figure . HoweverWhen siRNA movement increases concentric circles are formed as is the case for the lower silencing efficiency Figure . The numWith still higher siRNA movement the thin bands in which the virus reaches high levels break, and a growing ice crystal-like pattern is observed Figure . The patWith even higher siRNA movement rate the protrusive waves are reduced to a local spot, with the possibility of one or two small outbreaks Figure . For lowAs shown in the previous section, virus levels in the spatial model can reach two different equilibria, a high and a low one, while in the intracellular ODE model only a single equilibrium exists. The patterns disappear when siRNA movement is stopped, therefore siRNA movement maintains the silenced equilibrium. To analyze the influence of the tissue dynamics on the cellular dynamics mathematically we add in- and efflux of siRNAs and virions to the intracellular model. Efflux is fixed to the movement parameters used in the tissue model. We measure average virion and siRNA influx in the equilibrium for 4 cells from the center of the tissue and use these as influx values in the intracellular model.As an example case we take the parameters and measurements for the mosaic-like pattern for low and intermediate silencing strength shown in Figure We calculate bifurcation diagrams as a function of the siRNA influx. In Figure In the spatial model both virion and siRNA influx change over time. Therefore not the final siRNA influx but the combined virion and siRNA influx during the growth phase of the virus determine which equilibrium is reached. Once a stable state is reached a change in siRNA influx has no effect on equilibrium values. Only when siRNA influx is dramatically lowered or when a very large amount of virions would be introduced it would be possible to pass the bifurcation point and move from the silenced to the high state.For intermediate silencing strength the bifurcation diagram is similar, however due to the lower siRNA efflux and the stronger silencing response within the cell, it is shifted toward lower siRNA influx. The high and the low equilibrium are connected, meaning that an increase in siRNA influx results in a shift from the high equilibrium to the low one. This is the case in the speckle pattern: all cells initially expand to the high equilibrium, but some cells decline to the silenced state later in infection.To indicate the measured siRNA influx from the patterns shown in Figure Concluding, the effect of siRNA influx from neighboring cells is two-fold. Firstly, siRNA influx creates two coexisting equilibria; secondly, siRNA influx during the initial growth phase of the virus largely determines which equilibrium is reached.It has been proposed that Dicer can cleave ssRNA in addition to dsRNA . On the The complex feedbacks between the intracellular and the spatial model result in an unexpected effect of ssRNA cleavage in the spatial model. While keeping total Dicer cleavage rate constant, increasing ssRNA cleavage by Dicer shifts the silenced equilibrium to higher RNA levels. An increase of virus levels in the silenced equilibrium results in less pronounced patterns and it can even result in disappearance of the silenced state.In Figure In Figure These results are explained further in the bifurcation diagram in Figure This means that, although combined single- and double-stranded RNA cleavage by Dicer can clear the virus at lower Dicer cleavage rate , the sliAmplification of silencing through RDR can decrease viral levels in plants -22. It aTo study the effect of the secondary response on pattern formation and virus levels we add amplification to the model. At the cellular level RNA silencing with unprimed amplification can clear the virus for much lower silencing strength. It has to be noted however, that unprimed amplification can be triggered by any RNA, and will lead to responses against host RNA. Therefore, a mechanism has to be included to protect the host against auto-immunity ,26. PrimConcluding, for low silencing strength amplification has a similar effect to increasing silencing strength or siRNA movement.New patterns can be observed when the virus is cleared after an initial growth peak at the cellular level. Because the virus can expand and produce virions initially, it is able to spread over the tissue. However, in each cell the virus will be completely degraded by the RNA silencing response. We therefore observe transient patterns that leave healthy tissue behind. These patterns are shown in Figure We have shown that RNA silencing causes local differences in virus accumulation that can be the basis of different virus symptoms developed in plants. We have studied all qualitatively different patterns that can occur for different parameter values.When siRNAs spread from cell to cell, we observe patterns that can spread over the entire tissue and consist of alternating healthy and infected tissue. When siRNAs are able to spread quickly we observe localized spots around the infection site. The presence of a secondary response can result in transient patterns that leave healthy tissue behind. In accordance with our results, it has been shown that in absence of a secondary response tissue can get fully infected, while with secondary response patterns develop ,24. The In plants a variety of chlorotic patterns caused by viral infection have been observed. Chlorotic tissue contains high virus levels . Some ofWhen silencing is stronger we observe a mosaic-like pattern that resembles symptoms observed for many mosaic viruses. A difference with the experimentally observed patterns and our simulated patterns is the scale. We only observe larger scale mosaic when silencing strength is low. When silencing strength is high or when the secondary response is active we observe a small scale mosaic pattern. Our model, however, does not include growth of the tissue, and it has been shown that the healthy spots in mosaic-like patterns come about from cell division of single cells or a cluster of a few cells (reviewed in ). DeveloIn our model local spots occur when siRNA movement and silencing strength are sufficiently high. The virus infection stays localized to the area around the initially infected cell. We observe two types of spots: spots that are completely infected and ring spots that have a circle of low virus levels in between high levels. Lesions and ring spots are very common local symptoms after inoculation.Ring patterns similar to our concentric rings have been observed for Tomato ringspot virus . RingspoWe have used a detailed model of both intra- and inter-cellular dynamics of virus replication and RNA silencing. Nevertheless we were able to analyze mathematically (by bifurcation diagram) the intracellular dynamics that lead to alternative equilibria underlying the formation of infection patterns in plants. We have shown that siRNA movement is the driving force behind the pattern formation observed. Data of Hirai et al.  on siRNAIn conclusion, we have shown that the interplay of RNA silencing and virus growth within a cell, and the spread of virions and siRNAs between cells can explain the variety of viral infection patterns observed in plants.MG and PH conceived and designed the models, and wrote the paper. MG performed the numerical computations. All authors read and approved the final manuscript.Intracellular dynamicsThe entire intracellular model:The biological meaning of the variables is mentioned to the left of the equations. Multiple RdRPs can bind to minus-strand RNA, we refer to these as 'active RdRPs'. +RISC and -RISC are RISC loaded with siRNA with a plus- or minus-strand polarity. The abbreviation 'sec.' stands for secondary and is used to indicate siRNA that is produced through a secondary amplification pathway. Secondary RISC is loaded with secondary siRNA.All parameter values can be found in Table The complex formation :De, Dp, Dm, P and M. The Dicer cleavage functions, one for cleaving dsRNA and one for ssRNA, are saturated according to the ratio between single- and double-stranded RNA cleavage (q) by Dicer:Dicer can cleave double-stranded and single stranded RNA and is saturated for The amplification terms are:De). This dsRNA is degraded into secondary siRNAs with a plus- or minus-strand polarity; Sisp and Sism respectively.Where For the intracellular dynamics we use the parameters previously described . ParametThe extra parameters of the spatial model are the percentages of siRNAs and virions that move out of a cell (0 to 10% per hour), and virion unpacking rate.


Anthoceros formosae and the fern Adiantum capillus-veneris and in mitochondrial genomes of the angiosperms Arabidopsis thaliana, Beta vulgaris and Oryza sativa, to gain an understanding of the question of how RNA editing originated.The C↔U substitution types of RNA editing have been observed frequently in organellar genomes of land plants. Although various attempts have been made to explain why such a seemingly inefficient genetic mechanism would have evolved, no satisfactory explanation exists in our view. In this study, we examined editing patterns in chloroplast genomes of the hornwort nd and 1st codon positions, 2) editing affected codons that resulted in larger hydrophobicity and molecular size changes much more frequently than those with little change involved, 3) editing uniformly increased protein hydrophobicity, 4) editing occurred more frequently in ancestrally T-rich sequences, which were more abundant in genes encoding membrane-bound proteins with many hydrophobic amino acids than in genes encoding soluble proteins, and 5) editing occurred most often in genes found to be under strong selective constraint.We found that 1) most editing sites were distributed at the 2These analyses show that editing mostly affects functionally important and evolutionarily conserved codon positions, codons and genes encoding membrane-bound proteins. In particular, abundance of RNA editing in plant organellar genomes may be associated with disproportionately large percentages of genes in these two genomes that encode membrane-bound proteins, which are rich in hydrophobic amino acids and selectively constrained. These data support a hypothesis that natural selection imposed by protein functional constraints has contributed to selective fixation of certain editing sites and maintenance of the editing activity in plant organelles over a period of more than four hundred millions years. The retention of genes encoding RNA editing activity may be driven by forces that shape nucleotide composition equilibrium in two organellar genomes of these plants. Nevertheless, the causes of lineage-specific occurrence of a large portion of RNA editing sites remain to be determined.This article was reviewed by Michael Gray (nominated by Laurence Hurst), Kirsten Krause (nominated by Martin Lercher), and Jeffery Mower (nominated by David Ardell). RNA editing refers to the general phenomenon of post-transcriptional modification of gene transcripts, including insertion, deletion and substitution of nucleotides ,2. In plPrevious studies have shown genome-wide occurrence of RNA editing in both chloroplasts and mitochondria ,10-15, rArabidopsis thaliana and Anthoceros formosae .Another issue that is not generally considered is the effect that editing may have on the efficiency of translation of an edited mRNA, not just its translatability This is an interesting fact to consider. However, only seed plants have a mix of 'native' mtDNA-encoded tRNAs inherited vertically from a bacterial ancestor, 'chloroplast-like' tRNAs expressed from pieces of chloroplast genome that have become incorporated into the mitochondrial genome, and imported (nucleus-encoded cytosolic) tRNAs. The mitochondrial genomes of bryophytes lack any tRNA genes from the chloroplast genome, yet RNA editing almost certainly already occurs in the moss Physcomitrella patens and the hornwort Megaceros aenigmaticus mitochondria . Whether the mixed population of tRNAs has anything to do with RNA editing remains to be investigated, but sufficient data are not available at present.Just as Covello and I considered that the RNA editing machinery (whatever it is) would have emerged under neutral conditions, we imagined that editable sites would have emerged in the same way, perhaps as a result of the GC-drive I mentioned earlier. In our model, we defined "editable positions or sites at the level of DNA not only as sites that are capable of being edited , but also those that already correspond to the edited sequence. In the case of plant organelles in which C→U editing occurs, editable sites can be recognized at the RNA level by the editing machinery and contain either a T or a C in the DNA at the position that is actually edited. The mutations in Step 2 would be between Ts and Cs at these editable positions in the DNA." Thus, not every C position in a sequence is an editable site, only those that can be recognized by the RNA machinery. Importantly, we also argued that "Step 1, the appearance of RNA editing activity, has the very important effect of making these transitions neutral." We actually did say that it "is possible to argue for a selective advantage of RNA editing, perhaps as a result of the different nucleotide composition of DNA and RNA (i.e. higher GC content in kinetoplastid and plant organellar DNA)", although admittedly we went on to say that "we do not think this suggestion can easily be supported." Given the data now available and considering some of the arguments Jobson and Qiu make, I would be more inclined now to accept that possibility, although again I would point out that the distinction between 'origin' and 'maintenance' becomes quickly blurred in these models.In regard to the authors' statement, "If mutations at editable sites were fixed through genetic drift, one would expect their distribution patterns to be completely random when examined against codon positions, types of codons, and kinds of genes", I would argue that's not necessarily the case if, in fact, one accepts that (i) editable sites, by definition, are non-random because they have to be capable of being recognized by the editing machinery (otherwise non-editable T→C mutations would quickly be eliminated by purifying selection), and (ii) the three codon positions are not equally C-rich .Regarding point (i): Once the structural and functional aspects of the editing machinery are solved we will be in a better position to answer this question. Does the machinery have to recognize a single site? If it utilizes a sequence motif, then the conversion will depend on the nucleotide composition of the motif.Jobson and Qiu present an analysis of RNA editing sites in two chloroplast genomes and, three mitochondrial genomes of land plants. With this, they aimed at improving our understanding of how the two editing reactions (C to U and U to C conversion) that exist in the plant organellar genomes could have evolved.I have two major (I. and II.) and two minor problems (III. and IV.) with the way this work is presented and interpreted:I. The authors present data that were by and large known before, but they offer a different interpretation of these data. The focus of their manuscript is, therefore, more towards presenting and justifying a new hypothesis that may help to unravel the mystery of how RNA editing in plant organellar genomes evolved, than towards a gain of actual and reproducible knowledge. Unfortunately, the authors were quite biased in the presentation of their arguments. They amply refer to the important paper of Covello and Gray of 1993 on the evolution of RNA editing while the thorough and much more recently published broad scale analysis by Tillich et al. (2006) is almost unmentioned. If this manuscript is to present a valuable reference for future research, the authors should take a broader approach in their discussion and name all the pros and cons of the different views on a still undecided question. In my opinion, the study by Tillich et al. (2006) has taken such a broad approach already with an investigation of the plastid genomes of several taxa that were similar or even identical to those used in the present study, including also a discussion on plant mitochondrial genomes. The authors should mention this more explicitly here and point out where their own analysis deviates from this previous work.We agree that the Tillich et al.,  represents the increase or decrease of T relative to the composition of A. We have described our reasons for its use in the paper. As the reviewer suggested, we have reduced the size of Figures p. 5, 2nd para: For chloroplasts, quite a few other species have been analyzed for editing, such as Phalaenopsis, Pisum, Solanum, Oryza and Saccharum.We have cited the Saccharum and Phalaenopsis papers. The Phalaeopsis paper contains a table of all known angiosperm chloroplast edit sites.On the other hand, only a small fraction of Zea mitochondrial genes has been evaluated for editing, so it should not be listed as complete.We have made this clear by including "partially" as suggested by the reviewer – "and partially for Zea mays [Zea mays ."/p. 10,According to the hydrophobicity scales below, hydrophobic differences between Pro and Ser residues constitute a decent shift in hydrophobicity.- Kyte-Doolittle hydrophobicity scale: Pro = -0.8, Ser = -1.6- Hopp-Woods hydrophobicity scale: Pro= 0.0, Ser= 0.3In light of the Grantham [ discrepancy we have included the citation for Kyte-Doolittle [ to support our statement.Grantham  discrepaoolittle  to suppoAnthoceros ribosomal subunits + polymerases , CRR (Q+R) codons, and ratios of compositional distance between nucleotide pairs T-A, and C-G, at 1st, 2nd, 3rd, and total codon positions. Analyses were performed across 68 chloroplast protein-coding genes from Anthoceros formosae (Af), and Adiantum capillus-veneris (Acv), and 30 mitochondrial protein-coding genes from Beta vulgaris (Bv). Significant two-tailed Pearson correlations are signified by * at the 0.05 level, and ** at the 0.01 level. 'a' = cannot be computed because at least one of the variables is constant. 'na' = not applicable. Relevant data were used in Figures Correlation analyses of editing frequency versus nucleotide composition. Table of correlations for forward (C→U) and reverse (U→C) editing frequency at 1Click here for filedN and dS analyses , alignment (matrix) length, and total editing frequency for each gene. Information is divided into mitochondrial and chloroplast data-sets. Soluble proteins are shown in italics.Evolutionary analysis of total editing frequency across genes. Input trees used for chloroplast and mitochondrial gene specific s Figure  are presClick here for filedN and dS analyses (Figure Taxonomic and genome information. The data provide a full description of taxon names for those abbreviated in Figure Click here for file


WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability.A variety of pathways target CDKI p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit β-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.CD437 regulates cell growth in part by regulating stability of p21 WAF1/CIP1, an important cell-cycle regulatory molecule, was originally identified as a gene regulated by the tumor suppressor protein p53 UTP , [α-32P]CTP  or a combination of both. The 12 nt RNA oligonucleotide (5'-UGUGGUGGCACA-3') was labelled using T4 polynucleotide kinase and [γ32P]ATP  as described [Plasmids pBKS-WAF (581-2110) and pBKS-GADD (10-55) were linearized with NotI and HindIII, respectively, and the T methods . In addiescribed . Radiolaescribed . The rad2, 1.25 mM EDTA, 1.25 mM DTT, 155 mM KCl and 10% glycerol in a total reaction volume of 9 μl. In some cases, proteinase K , calf intestinal alkaline phosphatase , or 2 μl of ~100 ng/μl unlabelled sense strand RNAs were included in the pre-incubation step. The specific competitor RNAs consisted either a160 nt long RNA derived from plasmid pBKS-WAF (1540-1700) or an ~1500 nt RNA derived from plasmid pBKS-WAF (581-2110). The nonspecific competitor RNA consisted of an ~400 nt 3'-UTR subfragment of c-myc mRNA containing an additional 100 nt poly A sequences derived from in vitro transcription of HindIII-digested plasmid pMycSD3, essentially as described [HBC and leukemia cell cytoplasmic as well as nuclear protein extracts were prepared according to procedures described before . HBC cel-WAF 581-10. The nCD437: ; HBC: Human Breast Carcinoma; CDKI: Cyclin-dependent Kinase Inhibitor; ActD: Actinomycin D; CHX: Cycloheximide; ER: Estrogen Receptor; UTR: Untranslated region; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; nt: nucleotides; bp: base pairs; Kb: kilobase pairs; kD: kiloDalton.The authors declare that they have no competing interests.LZ and AKR carried out the majority of experiments including cloning of chimeric RBG constructs, RNA preparations, half-life determinations, northern blots, and analysis of RNA-protein interactions. AW, MID and JAF participated in the design and discussion of various experiments. AKR is the corresponding author responsible for experimental design and coordination, as well as writing of the manuscript and formatting of all the figures. All authors read and approved the final manuscript.


Pseudomonas occupying remarkably diverse ecological niches enabled to gain insights into their adaptative assets. The objective of this study was to analyze the complete genetic repertoires of metal transporters  from four representative Pseudomonas species and to identify metal transporters with "Genomic Island" associated features.The availability of complete genome sequences of versatile Pseudomonas species: P.putida (Ppu) KT2440, P.aeruginosa (Pae) PA01, P.fluorescens (Pfl) Pf-5 and P.syringae (Psy)pv.tomato DC3000 using TIGR-CMR and Transport DB. Genomic analysis of essential and toxic metal ion transporters was accomplished from the above inventory. Metal transporters with "Genomic Island" associated features were identified using Islandpath analysis.A comparative metal transporter inventory was built for the following four Psy DC3000, 48% of metal transporters showed strong GI features while it was 45% in Ppu KT2440. In Pfl Pf-5 and Pae PA01 only 26% of their metal transporters exhibited GI features.Dataset cataloguing has been executed for 262 metal transporters from the four spp. Additional metal ion transporters belonging to NiCoT, Ca P-type ATPase, Cu P-type ATPases, ZIP and MgtC families were identified. In Pseudomonas spp is the complete suite of metal transportomes analysed till date in a prokaryotic genus. This study identified differences in the basic composition of metal transportomes from Pseudomonas occupying diverse ecological niches and also elucidated their novel features. Based on this inventory we analysed the role of horizontal gene transfer in expansion and variability of metal transporter families.Our comparative inventory of 262 metal transporters from four versatile  Metal ions play a variety of critical roles in the prokaryotic metabolism. Biological management of metal ions is accomplished by a complex interplay between metal ion transporters  and their regulatory components . In the Pseudomonas is one of the most versatile and ecologically significant groups of bacteria on the planet that thrives well in metal rich biotopes. In recent studies, the unexpected capacity of Ppu KT2440 and Ppu CD2 genomes to endure exposure to heavy metals has been unraveled [Ppu KT2440 metal transporters acquired through HGT were also reported [Pseudomonas species. The availability of complete genome sequences for saprophytic P.putida (Ppu) KT2440, commensalistic P.fluorescens (Pfl) Pf-5, pathogenic P.aeruginosa (Pae) PA01 and P.syringae pv. tomato (Psy) DC3000 [The genus nraveled . In Ppu ) DC3000  providedPseudomonas spp Ppu KT2440, Pae PA01, Pfl Pf-5 and Psy DC3000 employed in this study were selected from TIGR-CMR database [The genome sequence information of the four sequenced database . Transpodatabase . In ordedatabase  and PFAMdatabase . Transmedatabase . GenomicEco and FeoBPae.CLC protein workbench has been employed to analyse the metal transporter proteins . GenchekPhylogenetic analysis was performed with the characterised prototypes using CLC protein workbench . For thiMetal transporters located in sequence clusters, were identified by cluster viewer from PEDANT database . ClusterPseudomonas species with island path software (IPA version 1.0 tool) for the identification of those transporters located in GI's or exhibiting GI associated features like anomalous %G+C, dinucleotide bias above 1 STD DEV, presence of RNA genes  and mobility genes . A putative GI can be identified with certainty by the presence of eight or more consecutive ORF's with dinucleotide bias alone or dinucleotide bias plus a mobility gene in proximity.Island Path Analysis was used for the detection of metal transporters acquired through HGT . After gPseudomonas genomes we could draw a comparison among the genome size, number of genes, transporter proteins and total number of metal transporters  while Psy has the smallest . Other pathogenic species, Pae has more number of genes and transporter proteins but relatively less metal transporters  than Ppu .Based on the global features of the four rs Table . Among t+, K+, Ca2+, Mg2+), transition metals  and heavy metals  in Pseudomonas  Ion-channels, Secondary transporters and Unclassified. A comparative analysis depicting the basic differences in the composition of transporter classes and metal transporter families from the four Pseudomonas species is represented in Table Based on the TransportDB  we compiPseudomonas databases identified additional metal transporters belonging to Ca, ZIP, NiCoT, MgtC, ABC (copper), P-type ATPase (copper) families. Distinct features of transporters belonging to CPA3, CaCA, ZnuABC, MntABC, ZntA, Nramp, FeOB, OFeT, NikABC, Chr, PbrT, RND, CDF families and ABC super family were also reported.Apart from executing the dataset cataloguing, our analysis of Pseudomonas using two characterized members (SP1551 and SP1623) of S.pneumoniae [Pae alone with no reciprocal BLAST hits from other three species.Search for primary ATP-dependent calcium pumps in Pseudomonas were retrieved using the experimentally characterized transporter ZupTEco [Psy and Pae have single entities PA4467 and PSPTO_2053 while paralog expansion is seen for Ppu  and Pfl .ZIP family of transporters in  ZupTEco . Psy andEco of NiCoT subfamily 2 [Pseudomonas databases, we identified two new efflux members PP_2968 (RcnAPpu) and PSPTO_4280 (RcnAPsy).Using the experimentally characterized efflux member RcnAfamily 2  as a BLAPae (PA4635) [Ppu), PFL_4077, PFL_2871 (Pfl), PA4635 (Pae). No hits were identified for this family in Psy.The experimentally characterized MgtC family transporter (PA4635)  was usedPae uniquely harbours an ABC copper transporter PA3393 that is related to periplasmic copper binding protein NosD in Rhizobium meliloti whose function was presumed to insert copper into the exported reductase apoenzyme (NosZ) [Pseudomonas  were found to be the best BLAST hits for experimentally characterized CopAEhi, while the other group of CuPATP2 transporters  did not show significant BLAST identities with the characterized proteins of CopA, CopB.e (NosZ) . All the+ efflux system are present in all the three species except in Pae where this component is involved in sodium ion homeostasis. Ppu (PP_2225 to PP_2230) and Pfl Pf-5 (PFL_2606 to PFL_2611) have six component K+ efflux systems while it is a five-component system in Psy.CPA3 family members constituting multicomponent KPfl and Psy (PFL_0722 and PSPTO_0764) are annotated as Ca2+/H+ (ChaA) antiporters that exhibit significant homology to ChaA of E.coli [2+/Ca2+exchangers (YrbG) [Ppu (PP_0928), Pfl Pf-5 (PFL_0891) and Psy (PSPTO_4477). Pae PA01 lacks a distinct homologue for CaCA transporter. Representative Zinc transporters of Pseudomonas species are presented in table 2+ as their substrate and ABC manganese transporters (MntABC) that recognize Mn2+/Zn2+, were included in the phylogenetic profiling to study their overlapping specificities  or six TMD's  were identical to ZntA. The other CadA proteins with seven TMD's  did not show any significant identity in BLAST analysis (less than 24% identity) and appear to be unrelated to ZntA.CaCA transporters i.e. secondary calcium transporters in f E.coli . Protein5 PFL_089 and Psy  ZntAEco  resultedPfl while Pae has two members MntH1 (PA0809), MntH2 (PA4334). Psy was found to possess three distinct members MntH1 (PSPTO_2464), MntH2 (PSPTO_2499) and MntH3 (PSPTO_5377) in this family. No conserved motifs characteristic for Nramp super family were observed for PSPTO_5377. However, phylogenetic analysis with Nramp family members confirmed its position in this family  of Psy showed that PSPTO_5377 is the only metal transporter located in its LSR's (LSR no: 42).Only one Nramp member, MntH PFL_2262) was observed for 262 was oPseudomonas species with the restriction of FeOB transporter (PA4358) to Pae and OFeT transporter (PFL_3255) to Pfl  were identified basing on the characterised prototype YcdNEco [Borrelia burgdorferi and Treponema pallidum while we reported substantial number of iron transporters for the pathogenic Pae and Psy (six and four respectively).Three types of iron transporters  were found in the orter PA458 to Paegure Eco  highly corter PA458 to Paeter PFL_35 to Pfl Ppu and Psy there are high affinity nickel uptake systems (Nik) of ABC family whose PBP's (PP_3342 and PSPTO_3088) belong to nickel/peptide/opine ABC transporter family. Two IMP's were identified for each of the two Nik systems, in NikPpu  and NikPsy . They displayed a characteristic transmembrane topology of five  and six TMD's .In Pseudomonas species  except Psy. PbrT members conferring lead resistance are also present in all the four Pseudomonas species . As an exception Psy possess two PbrT systems, PbrT1 and PbrT2 . PbrT2Psy did not show the conserved motifs found in other lead transporters of this group. Uniquely there is one ABC family member for Pb in Ppu (PP_5165) with no existing sequence homology to PbrTPpu.Chromate transporters of Chr family mediating chromate resistance are present in all the three R.metallidurans have eight HME-RND proteins on chromosome. In comparison, Ppu has six RND chromosomal determinants , Pfl and Pae have two determinants each  while no RND determinant was identified in Psy. Pfl Pf-5 has a maximum of four CDF transporters  while they are three in Pae PA01  and two in Ppu KT2440 . However Psy DC3000 has only one distinct ortholog (PSPTO_4790) in this family.The best-characterized heavy metal resistant strains Analysis of ABC super family members from the above inventory unravelled a distinct transmembrane topology for their integral membrane proteins (IMP's) that varied with the metal ion specificity of their associated PBP's. Accordingly, five/six TMD's were observed for nickel transporters, twelve for iron transporters, seven for zinc, eight for manganese and five for molybdenumPseudomonas species has multiple paralogs for most of the metal ion transporters that are briefed out in Additional table 5. Some of the significant features of paralog distribution are highlighted in this section.Sodium ion transporters constitute the bulk of secondary transporters and has maximum number of paralogs distributed in different families Table .+ transporters are present in Pseudomonas species among which Kdp system (in Pfl), VIC super family (in Pae and Psy), CPA2 family  and Trk family (in Ppu) possessed paralogs.A multitude of KPseudomonas species were found to possess paralogs for PBP's of ABC Zn2+ and Mn2+ transporters. Psy alone has paralogs for PBP of ABC Mo2+ transporter.All the four Ppu has two paralogs for CitMHS family, which are presumed to facilitate transport of Citrate-Mn2+ complex while in Pfl this family has three paralogs with specificity for Mg2+/citrate.Pseudomonas species have paralogs for MIT family and P-type ATPases. Except for Psy all the three species possessed paralogs for RND and CDF families.All the four Psy and Pfl have paralogs for ILT family, mediating lead transport alone in the former while both ferrous iron and lead transport are mediated in the later.Ppu ArsB family has two paralogs that are part of separate arsRBCH operons while in Pfl out of the two paralogs  only former is located within an arsRBCH operon.In Pseudomonas spp we identified metal transporters with GI associated features from our inventory  in Psy, nineteen (26%) in Pfl and seventeen (26%) in Pae.In view of the mosaic nature of genomes of the Pae, NhaB family, NiCoT family in Ppu, NSS, MScS and CitMHS families in Psy. ABC transporter for Molybdenum was found to have GI features in the three Pseudomonas except for Pfl. Strong GI features were observed only for certain paralogs of metal transporter families indicating that paralog expansion could have occurred through HGT , ABC family (Cu transporter), P-type ATPase (Cu transporter) in Pseudomonas species. This is the first comprehensive genomic comparison of metal transporters, providing potentially important insights into the fundamental molecular aspects and novel facets of Pseudomonas metal transportomes.In this article we performed a dataset cataloging for 262 metal transporters from four representative Our comparative inventory identified and analyzed novel metal transporters belonging to the following families: NiCoT , Ca P-type ATPase (PA1429), Cu P-type ATPases , ZIP  and MgtC .Psy possessing least number of metal transporters showed maximum percentage (48%) of transporters with strong GI features. Our data is substantiated by the previous observations where majority of Psy ORF's shared features with horizontally transferred genes [Ppu has 45% of its transporters possessing strong GI features. On the contrary, Pfl and Pae have only 26% of their metal transporters exhibiting GI features.ed genes . Ppu hasPseudomonas community in mining large and heterogeneous data sets obtained from metagenome projects to identify new biologically relevant patterns of metal transporters resembling those in this study. A combinatorial approach of transcriptomics and functional genomics will aid in deducing the functions of these diverse metal transporters and assembling a complete picture of metal homeostasis.This comparative inventory can therefore provide a window for Ppu/PP/Ppu: P.putida KT2440; Pae/PA/Pae: P.aeruginosa PA01; Pfl/PFL/Pfl: P.fluorescens Pf-5; Psy/PSPTO/Psy: P.syringae DC3000; TMD's: Transmembrane domains; aa: Amino acid residues; bp: Base pairs; T.C.No: Transporter classification number; Me2+ transporter: Hypothetical metal transporter; MFS: Major facilitator super family; IMP's: Integral membrane proteins; HGT: Horizontal gene transfer.The authors declare that they have no competing interests.insilico analysis and wrote the draft of the manuscript.  AR contributed to critical evaluation of the work and improvement of the manuscript organization.  PMM conceived the study, guided the complete work and corrected the manuscript.AH performed the complete P.putida KT2440 metal transporters.   Catalogue of Description: This catalogue was compiled for the entire complement of alkali/alkaline earth metal transporters , transition metal transporters  and heavy metal transporters  from P.putida KT2440. Information provided in this catalogue includes the class/family/subfamily, Transporter classification number (T.C.No),  TIGR  locus,  protein  name,  genomic  location, orientation,  length  of  the  protein, Transmembrane  domains  (TMD’s), substrate and the predicted role of all the metal transporters.   Click here for fileP.aeruginosa PA01 metal transporters.Catalogue of    Description: This catalogue was compiled for the entire complement of alkali/alkaline earth metal transporters , transition metal transporters  and heavy metal transporters  from P.aeruginosa PA01. Information provided in this catalogue includes the class/family/subfamily, Transporter classification number (T.C.No),  TIGR  locus,  protein  name,  genomic  location, orientation,  length  of  the  protein, Transmembrane  domains  (TMD’s), substrate and the predicted role of all the metal transporters.   Click here for fileP.fluorescens Pf-5 metal transporters.   Catalogue of Description: This catalogue was compiled for the entire complement of alkali/alkaline earth metal transporters , transition metal transporters  and heavy metal transporters  from P.fluorescens Pf-5. Information provided in this catalogue includes the class/family/subfamily, Transporter classification number (T.C.No),  TIGR  locus,  protein  name,  genomic  location, orientation,  length  of  the  protein, Transmembrane  domains  (TMD’s), substrate and the predicted role of all the metal transporters.   Click here for fileP.syringae DC3000 metal transporters.Catalogue of    Description: This catalogue was compiled for the entire complement of alkali/alkaline earth metal transporters , transition metal transporters  and heavy metal transporters  from P.syringae DC3000. Information provided in this catalogue includes the class/family/subfamily, Transporter classification number (T.C.No),  TIGR  locus,  protein  name,  genomic  location, orientation,  length  of  the  protein, Transmembrane  domains  (TMD’s), substrate and the predicted role of all the metal transporters.   Click here for filePseudomonas spp.Sequence Cluster Analysis for detection of paralogs from four    Description:  This data provides evidence for the paralogous nature of metal transporters from various metal transporter families.   Click here for fileP.putida KT2440. Metal transporters exhibiting GI associated features from   Description: This table lists out the metal transporters with GI associated features from P.putida KT2440 and provides evidence for the metal transporters with strong and weak GI features.   Click here for fileP.aeruginosa PA01.Metal transporters exhibiting GI associated features from     Description: This table lists out the metal transporters with GI associated features from P.aeruginosa PA01 and provides evidence for the metal transporters with strong and weak GI features  Click here for fileP.fluorescens   Pf-5.Metal transporters exhibiting GI associated features from    Description: This table lists out the metal transporters with GI associated features from P.fluorescens Pf-5 and provides evidence for the metal transporters with strong and weak GI features.   Click here for file P.syringae DC3000Metal transporters exhibiting GI associated features from.  Description: This table lists out the metal transporters with GI associated features from P.syringae DC3000 and provides evidence for the metal transporters with strong and weak GI features.   Click here for filePseudomonas. Role  of  HGT  in  paralogous  expansion  and  acquisition  of  novel  transporter families in   Description:  This  table  demarcates  the  role  of  HGT  in  paralogous  expansion  and acquisition of novel transporter families in each of the four Pseudomonas species.   Click here for file


The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account.rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain.The role of Hfq in the maturation and degradation of the rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome transcription pauses and to prevent preliminary transcript release.The data obtained suggest that alteration of  Templatescribed . The lac32P] UTP . 5S rRNATotal RNA separation on 1% agarose formaldehyde gel or acrylamide/urea gel and Northern blotting were previously described ,35. RNA JLD, IVB and EH performed all presented experiments, IVB and EH conceived the study and analyzed the data. IVB, PR and EH wrote the manuscript. All authors read and approved the final manuscript.