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The putative RNA silencing protein ERL‐1 is involved in chloroplast ribosomal RNA processing in plants
Doctorate thesis submitted by DI Jutta Maria Helm March, 2011
I hereby declare that I have written this thesis independently. All results presented in the result section have been obtained by my own work, probes and plasmids have been shared by several lab members. Relevant results obtained by co‐workers are only presented in the supplementary result section with proper citations. All intellec‐ tual property used for the preparation of this work has been cited properly. The results obtained from transgenic N. benthamiana plants misexpressing ERL1 in‐ cluding crossing, light microscopy as well as photosynthetic analysis, and rRNA cloning experiments have been described and discussed by Heiko Schumacher in 2009. However, all experimental procedures and plant maintenance have been exe‐ cuted by me. The chloroplastic localization, analysis of chloroplast‐related transcripts and electron microscopy experiments have been executed independently by Heiko Schumacher and me to prepare replicates of the findings. March 2011, Vienna DI Jutta Maria Helm
Es‐ pecially I would like to thank… …Dr Kriton Kalantidis for being a valuable teacher and still leaving me a great free‐ dom for my work and development. …Sergia Tzortzakaki. Giorgos. Evguenia.Acknowledgements This work would not have been possible without the support of various people. I will keep in mind the nice times we had. for some I am especially proud to call them friends. …all the people who had been there and went the way in Crete along with me for a while. and for spending their holidays with me. …Dr Marie‐Theres Hauser for giving me the opportunity to learn in her lab and for all the support and good advice from far. …the members of the Plant Developmental Genetics Laboratory in Vienna for wel‐ coming me so friendly before and after my stay in Greece. …my brother Andreas for constantly solving all my computer problems in no time. for keeping contact by Skype. …my friends back at home and around the world who did not forget me even from a distance. Kallia and Ritsa who all provided valuable help. …my sister Martina for always being there! …Harald Zwilling for being Harald Zwilling! 5 . despite any local distance between us. …all members of the Plant Molecular Biology Laboratory in Crete for making it a place to remember. Eva Papadogiorgaki. …my parents for letting me go and for giving me the feeling that I can always count on them in case of need. email. …Heiko Schumacher for teaching me a lesson for life. etc. Kosmas Haralampidis and my students Andreas.
This mechanism is also used in the de‐ fense against pathogens that have therefore developed several strategies to counter‐ act it by expression of viral suppressors of silencing (VSRs). A putative ribonuclease has been proposed earlier to assist in chloroplastic rRNA processing in a mutant background of RIBONUCLEOTIDE REDUCTASE 1 (RNR1) in Arabidopsis thaliana which may be constituted by ERL1. 7 . mosaic green and white to complete loss of chlorophyll. In‐ deed it could be shown that 5S rRNA is downregulated after transient and constitu‐ tive overexpression of ERL1 and elongated by two nucleotides at its 3’‐end in a frac‐ tion of the analyzed samples. This finding is not surprising in this context since RNA silencing is restricted to the cytoplasm. In this work the plant homologue termed ERL1 (ERI‐1‐LIKE 1) is analyzed. The transgenic plant lines showed morphological and transcriptional altera‐ tions reminiscent of reported defects in chloroplastic ribosomal RNA biogenesis. The observed pheno‐ types reached from pale green. The result could be confirmed in Arabidopsis thaliana plants overexpressing ERL1. Also the Drosophila melanogaster homologue does not possess this function suggesting two functionally distinct groups of ERI‐1 homologues. The pro‐ tein localizes to the chloroplast and fails to exhibit any RNA silencing suppressor activity. A putative endogenous suppressor of silencing might be the 3’‐5’ exonuclease ERI‐1 (enhanced RNAi) and its homologues in various species. Constitu‐ tive overexpression of ERL1 in transgenic Nicotiana benthamiana plants manifested in variegated phenotypes characterized by a bleaching of the plants.Abstract During evolution eukaryotes have acquired a system using small RNA molecules (siRNAs) as negative regulators of endogenous and exogenous RNA sequences called RNA interference or RNA silencing. The severity of the bleaching corresponded to the expression levels of ERL1. In addition a fraction of 16S rRNA has been elongated by one nucleotide in Arabidopsis insertion mutants suggesting also a function in maturation of this chloroplastic rRNA. which specifically bind and degrade siRNAs. where they catalyze the final step in 5. Re‐ cently an additional conserved role of ERI‐1 homologues has been identified.8S rRNA processing. yellow.
da RNA‐ Silencing auf das Zytoplasma beschränkt ist. die spezi‐ fisch siRNAs binden und abbauen kann. die der Ribo‐ nukleotidreduktase 1 (RNR1) bei der Verarbeitung von Chloroplasten rRNA assis‐ tiert.Zusammenfassung Im Laufe der Evolution haben Eukaryonten ein System erworben. Das Protein wird in Chloroplasten geschleust und besitzt keine RNA‐Silencing‐ Suppressor‐Aktivität. bei denen ERL1 unterdrückt war. um diesem Mechanismus durch die Ausbildung von viralen Silencing Suppressoren (VSR) entgegen zu wirken. Es konnte tatsächlich gezeigt werden. deshalb haben diese verschiedene Strategien entwickelt. Diese sogenannte RNA‐Interferenz oder RNA‐Silencing wirkt auch als Immunsystem gegen Krankheitserreger. das kleine RNA‐ Moleküle (siRNAs) als negative Regulatoren von endogenen und exogenen RNA‐ Sequenzen verwendet. teilweise Verlängerungen um ein Nukleotid. über gelb. Das homologe Drosophila Protein zeigt ebenfalls dieses Verhalten. Dieses Ergebnis ist insofern nicht überraschend. Der Schwere‐ grad des Phänotyps entsprach der Überexpression von ERL1. In dieser Arbeit wurde das Pflanzen‐Homolog ERL1 (ERI‐1‐like 1) analysiert.8S ribosomaler RNA. Außerdem katalysiert ERI‐1 den letzten Schritt bei der Reifung von 5. wahrscheinlich existieren zwei unterschiedliche Gruppen von ERI‐1 Homologen in Eukaryoten. die an bereits be‐ schriebene Mängel in der Reifung ribosomaler RNAs in Chloroplasten erinnern. Außerdem zeigte auch die 16S rRNA in Arabidopsis‐Mutanten. Die transgenen Linien zeigten morphologische und transkritptionelle Veränderungen. ERL1 könnte die mutmaßliche Ribonuklease sein. dass die 5S rRNA Levels nach der Überexpression von ERL1verringert sind und am 3’‐Ende eine häufige Verlängerung um zwei Nukleotide besaßen. Überexpression von ERL1 in transgenen Ta‐ bak‐ und Arabidopsis‐Pflanzen resultierte in vielfältigen Phänotypen. Ein mutmaßlicher eukaryotischer endogener Silencing Suppressor könnte die 3ʹ‐5 ʹExonuklease ERI‐1 sein. 9 . eventuell besitzt ERL1 auch eine Funktion in der Reifung dieser rRNA. die durch ein Bleichen der Pflanzen gekennzeichnet waren: es reichte von blassgrün. grün und weiß gesprenkelt bis zu komplettem Chlorophyll‐Verlust.
pombe 11 . tumefaciens aa ACS ADAR Ago/AGO AP‐conjugate APS ARC Arg Asp ATP RLI2 att sites Aub BLAST bp BSA C C.Abbreviations % °C β‐ME μm μM μCi μg μJ μL 3’ 3’hExo 5’ 7mG A Å A. elegans/Ce CBC cDNA Chp1 Percent Degrees Celsius β‐Mercaptoethanol Micrometer(s) Micromolar Microcurie(s) Microgram(s) Microjoule(s) Microlitre(s) 3 prime 3 prime histone exonuclease 5 prime 7‐methylguanosine Adenosine Ångström(s) Arabidopsis thaliana Agrobacterium tumefaciens Amino acid(s) Acetosyringone Adenosine deaminase that acts on RNA Argonaute protein Alkaline phosphatase‐conjugate Ammonium persulfate ACCUMULATION AND REPLICATION OF CHLOROPLASTS Arginine Aspartate Adenosine triphosphate RNASE L INHIBITOR 2 Attachment sites Aubergine Basic Local Alignment Search Tool Base pair(s) Bovine serum albumin Cytosine Caenorhabditis elegans Cap‐binding complex Complementary DNA Chromodomain protein in S. thaliana/At A.
g.Abbreviations CHS Ci CLP CLSY1 cm cm² CTAB C‐terminus D D. CHALCONE SYNTHASE Curie(s) CASEINOLYTIC PROTEASE CLASSY 1 Centimeter(s) Square centimeter(s) Cetyltrimethylammonium bromide Carboxy‐terminus Aspartate Danio rerio Deoxyadenosine triphosphate DICER‐LIKE1‐4 De‐capping protein 2 Dicer Dicer Related 1/2 Deoxycytosine triphosphate Dideoxycytosine DAWDLE DiGeorge syndrome chromosomal region Deoxyguanosine triphosphate Dimethylsulfoxide Deoxyribonucleic acid DNA polymerase III epsilon subunit Deoxyribonuclease Deoxynucleoside triphosphate Days post‐infection/days post‐infiltration DOUBLE‐STRANDED RNA‐BINDING PROTEIN 4 DEFECTIVE IN RNA‐DIRECTED DNA METHYLATION 1 DOMAINS REARRANGED METHYLTRANSFERASE 2 Double‐stranded DNA Double‐stranded ribonucleic acid binding domain Double‐stranded RNA Dithiothreitol Deoxythymidine triphosphate Domain of unknown function Glutamate Escherichia coli exempli gratia 12 D. rerio dATP DCL1‐4 DCP2 DCR DCR‐1/2 dCTP ddC DDL DGCR8 dGTP DMSO DNA DnaQ DNase dNTP dpi DRB4 DRD1 DRM2 dsDNA dsRBD dsRNA DTT dTTP Duf E E. melanogaster/Dm Drosophila melanogaster . coli e.
relative centrifugal force Green Fluorescent Protein Glycine tryptophan repeat protein 182 Hour(s) Histidine Homo sapiens Water Helper component proteinase HUA ENHANCER 1 4‐(2‐hydroxyethyl)‐1‐piperazineethane‐sulfonic acid Horseradish peroxidase HYPONASTIC LEAVES 1 Id est INVOLVED IN DE NOVO METHYLATION 2 Intergenic non‐coding transcript Institute of Molecular Biology & Biotechnology Isopropyl‐β‐S‐1‐thiogalactopyranoside Lysine Kilobase(s) Knock‐down Kilodalton(s) Knock‐out KATANIN Kilovolt(s) 13 . EtBr EXOIII fmol For/F FoRTH FRY1 G g GFP GW182 h H H.Abbreviations EDTA eIF4E ELSS ERI‐1/Eri1 ERL1 EST et al. 5’‐BISPHOSPHATE NUCLEOTIDASE/INOSITOL POLYPHOSPHATE 1‐PHOSPHATASE Guanosine. sapiens H2O HC‐Pro HEN1 HEPES HRP HYL1 i.e. IDN2 IGN IMBB IPTG K kb KD kDa KO KTN kV Ethylenediaminetetraacetic acid Eukaryotic initiation factor 4E Extensive local silencing spread Enhanced RNAi 1 ERI‐1‐LIKE 1 Expressed sequence tag and others Ethidium bromide Exonuclease (III) domain Femtomole Forward Foundation for Research & Technology ‐ Hellas 3’(2’). Glycine Gramm(s).
tabacum/Nt NADP NAT nat‐siRNA NCBI NEP ng Ni‐NTA nm NPC NRPD1a/b nt N‐terminus O. sativa/Os OD600 Liter(s) Lysogeny broth/Luria broth/Luria‐Bertani broth LEThal‐7 Abnormal cell‐LINeage‐4/14 Loquacious Molar Mus musculus Milliampere(s) 2‐(N‐Morpholino)ethanesulfonic acid Methionine Milligram(s) Minute(s) MicroRNA MicroRNA passenger strand Milliliter(s) Millimolar Millimeter(s) MS/MES/acetosyringone 3‐(N‐Morpholino)propanesulfonic acid Messenger RNA Murashige & Skoog Normal Neurospora crassa Nicotiana tabacum Nicotinamide adenine dinucleotide phosphate Natural antisense transcript Natural antisense transcript‐derived siRNA National Center for Biotechnology Information Nucleus‐encoded polymerase Nanogram(s) Nickel‐Nitriloacetic acid Nanometer(s) Nuclear pore complex NUCLEAR RNA POLYMERASE D 1A/B Nucleotide(s) Amino‐terminus Oryza sativa Optical density at 600 nm 14 N. crassa N. musculus mA MES Met mg min miRNA miRNA* mL mM mm MMA MOPS mRNA MS N N. benthamiana/Nb Nicotiana benthamiana .Abbreviations L LB let‐7 lin‐4/14 LOQS M M.
associated with DCR‐2 Rapid amplification of cDNA ends Ras‐related nuclear protein Repeat‐associated short interfering RNA Ribulose bisphosphate carboxylase.N′‐bis(2‐ethanesulfonic acid) Piwi‐interacting RNA P element‐induced wimpy testes Peach latent mosaic viroid Picomole(s) Polynucleotide kinase Polymerase RNA polymerase II/IV/V Pentatricopeptide‐repeat Plum pox virus Precursor miRNA Piwi‐related gene 1 Primary miRNA PHLOEM SMALL RNA‐BINDING PROTEIN 1 Potato spindle tuber viroid Posttranscriptional gene silencing Quelling‐deficient 2 QDE‐2‐interacting protein Quantitative PCR Glutamine‐rich Two dsRNA‐binding domains. trichocarpa/Pt PAA PAGE PAP PAZ P‐body PCMP PCR PEP pH PIPES piRNA Piwi PLMVd pmol PNK Pol Pol II/IV/V PPR PPV pre‐miRNA PRG‐1 pri‐miRNA PSRP1 PSTVd PTGS QDE‐2 QIP qPCR Q‐rich R2D2 RACE Ran rasiRNA RBCL RdDM RDR1‐6/RdRP Rev/R Hydroxyl Populus trichocarpa Polyacrylamide Polyacrylamide gel electrophoresis poly‐A polymerase Piwi/Argonaute/Zwille Processing body Plant combinatorial and modular protein Polymerase chain reaction Plastid‐encoded polymerase pondus Hydrogenii/potentia Hydrogenii Piperazine‐N.Abbreviations OH P. large chain RNA‐directed DNA methylation RNA‐DEPENDENT RNA POLYMERASE 1‐6 Reverse 15 .
pombe S. Acinus and PIAS SILENCING DEFICIENT 3 SMALL RNA DEGRADING NUCLEASE Sodium dodecyl sulfate SERRATE SUPPRESSOR OF GENE SILENCING 3 Short interspaced element Small interfering RNA Small interfering ribonuclease Stem‐loop binding protein Short‐range local silencing spread Small nucleolar RNA Snipper Small nuclear RNA Small nuclear ribonucleoprotein Super optimal broth Species 16 .5‐bisphosphate carboxylase/oxygenase Second(s) Svedberg (sedimentation coefficient) Sorghum bicolor Schizosaccharomyces pombe Strongylocentrotus purpuratus Scaffold attachment factor SAF‐A/B. bicolor S. Nuclear restorer REGULATOR OF GENE SILENCING CALMODULIN‐LIKE RNA‐induced silencing complex RNA‐induced transcriptional silencing Ribonucleic acid RNA interference Ribonuclease RNase inhibitor RIBONUCLEOTIDE REDUCTASE 1 RNA polymerase II large subunit Rotations per minute RNA POLYMERASE SUBUNIT BETA RNA‐directed RNA polymerase family Ribosomal RNA Reverse transcription. Room temperature Ribulose‐1.Abbreviations Rf rgs‐CaM RISC RITS RNA RNAi RNase RNasin RNR1 RPB1 rpm RPOB RRF rRNA RT RuBisCo s/sec S S. purpuratus SAF SAP SDE3 SDN SDS SE SGS3 SINE siRNA siRNase SLBP SLSS snoRNA Snp snRNA snRNP SOB sp.
vinifera v/v VCS VSR W W w/v X.‐C TRBP Tris tRNA TRV U UBA UTR UV V V. mays/Zm Suppressor of Ty insertion 5 Sodium chloride/sodium citrate buffer Single‐stranded DNA Single‐stranded RNA Thymine Tris/Acetate/EDTA Thermus aquaticus TRANS‐ACTING SIRNA 3 Trans‐acting siRNA Tris/Borate/EDTA Tris‐EDTA Transmission electron microscopy Tetramethylethylenediamine Translocon at the inner/outer envelope membrane of chloro‐ plasts Melting temperature Trinucleotide repeat containing 6A. ‐B.‐B. laevis X‐Gal x‐ray XRN1‐4 Y Z. ‐C Transactivating response RNA‐binding protein Tris(hydroxymethyl)‐aminomethan Transfer RNA Tobacco rattle virus Unit(s) Ubiquitin‐associated Untranslated region Ultraviolet Volt(s) Vitis vinifera Volume per volume VARICOSE Viral suppressor of silencing Watt(s) Tryptophan Weight per volume Xenopus laevis 5‐Bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside Roentgen rays EXORIBONUCLEASE 1‐4 Tyrosine Zea mays 17 .Abbreviations SPT5 SSC ssDNA ssRNA T TAE Taq TAS3 tasiRNA TBE TE TEM TEMED TIC/TOC Tm TNRC6A.
.1 Plant cultivation.....................1....................1....................1 Materials .......................1.........4.....................2......... 88 2................... 61 1.................................. 44 1.......................................6 Gel extraction ...............................10 ERI‐1‐LIKE 1..............................2 Chemically competent cells .................2..................................................2.........................3 Bacterial strains..............5 Others .........1............................... 85 2................................................2.2............... 88 2..........1.......... 85 2.........................................4 Plasmid preparation..............2 miRNAs modulate the expression of endogenous sequences............................................... 76 2... the plant homologue of ERI‐1 ................................................................................................................................2................................................................1.....................2..........4 Plants possess a high diversity of siRNA molecules..............................................................................1 Enzymes....................... 85 2.....................Table of Content 1...2............ 86 2................. 41 1..5 Peculiarities of plant miRNAs ... 38 1. 69 2............6 Spreading of RNA silencing in plants resembles an immune system..2...........9 ERI‐1 is an example for an endogenous suppressor of RNA silencing.. 88 2.................................................................. 88 2...................................................... 85 2............2.1 Standard molecular biology methods ...................................................................................... 29 1.....1................................................................2. 84 2................... 51 1...................................................................................................................................................2 RNA silencing – new roles for the intermediate................1.......................1 RNA molecules and their life between DNA and protein ......................................1 Cis‐acting siRNAs mediate chromatin silencing in plants ...............................................................................8 Repressing the repressors ‐ endogenous suppressors of RNA silencing...........2......... 86 2....................4 Thesis objectives .................................... Materials and Methods .......................... 85 2...2....8 Ligation .....2 Trans‐acting siRNAs............................... 45 1...2 Chemicals ......1..................................... 89 19 ................. 66 2.......... 87 2..........................2 Specificities of chloroplastic RNAs ....2............ 32 1................ Introduction..............................2...................................................... 62 1........................................................1.........4 Solutions ....................2 Methods ....... 71 2........................2.................. 49 1.........................................................1.......3 Chloroplasts .. 36 1.........2.....3 Natural antisense siRNAs ...........................................3 Consumables & kits ..................... 25 1....................... 69 2.................................................2 Plant transformation techniques ...2............................ 42 1............. 27 1............1...................1 siRNA‐mediated gene silencing.....................3 Transformation ................................................................. 84 2....2 Size markers .......1...........2..2.. 46 1.......................2...................................... 88 2............................................................................4........ 56 1.............3......................2......... 64 1...............................................................................................................1...................2 Leaf‐Disc transformation........2.... 69 2..........................3 The piRNA pathway protects the germline from transposon activity.......2............................. 50 1................................................................... 88 2..........................................5 Agarose gel.......4.............................1.................2....................1 Instruments ..................1................................. 74 2..............................7 Digest ...5......2....5.........................................................5........................ 25 1...........................................................1 Photosynthesis ..7 Viral strategies to suppress RNA silencing in plants....2.3...1 Cultivation................
89 2..........3 Reverse Transcription (RT) ........3 Capillary blot ..........7..3..................................................................................................... 103 3...............1 Protein extraction ..5 Effect of ERL1 on silencing ......................2 Overexpression of ERL1 .....4.. 93 2................3...............2. 96 2..................3 Effect of ERL1 overexpression on the photosynthetic apparatus...................................7.......2..............7......................7.......................................................................3 Floral Dip...2...... 92 2............................. 93 2....... washes and developing .........................1 Self‐Ligation .............2.................................................................................3..........4 Transgenic Arabidopsis thaliana plants.....2 Denaturing agarose/formaldehyde gels...........................................5...4.....4 Polymerase Chain Reaction (PCR)........................6.. 91 2......7 rRNA cloning ...................................................Table of Content 2............. 94 2........................................................2.............................. 93 2................ 103 3...................................................................................................2..........................5.................. 115 3.....................2 RACE...................................................3 Hybridization................1 DNA extraction.......... 117 3.. 92 2....................... 94 2............... 112 3.............................................2.......................................................2..................2................ 96 2............... 101 3.................................... 107 3......... 119 20 ................ 94 2..3 PAGE northern .............1 Microscopy ....4 Semi‐dry blot................2 Southern analysis .........2...................... 110 3........................... 91 2...........4......2.....................5.....3..2................6................................2..........2............................3................................... 115 3.....................................................................................4................................8 Rapid Amplification of cDNA ends (RACE)...........................................................11 Preparation for microscopic analysis .......................2.......... 90 2....................2..................... 90 2.......................................... 92 2.................................................2 Overexpression of ERL1 .....................3..... 94 2.........2.......................................................................12 Fluorescence measurement............2 Linker‐Ligation ....2............................................3.............2..... 93 2.2............9 Quantitative real‐time PCR (qPCR) ........................ 99 3................................. 95 2... 97 2........ 97 3..........................................................................................................2 Effect of ERL1 overexpression on chloroplast mRNAs ............................ 91 2......................... 96 2........................................1 Knock‐down of ERL1... 89 2...........................................................................................4 Western detection.....................................................1 Random‐primed labelling .3 Transgenic Nicotiana benthamiana plants .................4.....................................................2.....6 Western analysis....................................................................................6..................... 90 2........................ 104 3........................2...2........................................2.....................................................................................................5 Hybridization..4.................................................... 92 2............2............ 93 2..............2 SDS‐PAGE ...2.................................2............2...............2.. 95 2..........2.......................3.............2..............................................................................................3 Southern analysis ......................................2 End‐labelling ...................6.2......1 Localization ......... 91 2...................................... Results ...............................................................................1 RNA extraction ....2......10 Protoplasts .. 94 2......................................... 99 3...........................................................................................................................................2........................................4 Agroinfiltration.........................3 Electro‐blot ...............................................4 Northern analysis .....2.............................1 Knock‐down of ERL1.............
Table of Content
Crosses .............................................................................................................. 119 LNA159 ............................................................................................................. 120 3.6 Effect of ERL1 on ribosomal RNA .................................................................... 121 3.6.1 rRNA blots........................................................................................................ 121 3.6.2 Linker ligations ................................................................................................ 123 22.214.171.124 Effect on 5.8S rRNA .................................................................................... 123 126.96.36.199 Effect on chloroplastic rRNAs ................................................................... 124 4. Discussion ...................................................................................................................... 129 4.1 Implications of plant ERL1 in RNA silencing processes ............................... 129 4.2 Involvements of ERL1 in chloroplast metabolism.......................................... 131 4.3 Severe phenotypic alterations after overexpression of ERL1 suggest an in‐ volvement in chloroplast development ........................................................................... 133 4.4 ERL1 is involved in chloroplastic ribosomal RNA processing..................... 135 4.5 Conclusions .......................................................................................................... 140 5. References ...................................................................................................................... 143 6. Supplements .................................................................................................................. 171 6.1 Supplementary methods .................................................................................... 171 6.1.1 Virus/viroid infections in Nicotiana sp. plants ............................................. 171 6.1.2 In vitro transcription........................................................................................ 171 6.1.3 Purification of recombinant ERL1 protein................................................... 171 6.1.4 In vitro assays for recombinant ERL1 protein............................................. 172 6.2 Supplementary results........................................................................................ 172 6.2.1 PSTVd‐derived siRNAs are suppressed upon ERL1 overexpression ..... 173 6.2.2 ERL1 fails to affect RNA silencing in Agrobacterium co‐infiltration assays 174 6.2.3 Exogenously induced silencing spread may be suppressed after ERL1 overexpression .............................................................................................................. 176 6.2.4 ERL1‐overexpressing plants are hypersensitive towards viral infection 177 6.2.5 In‐vitro experiments with ERL1..................................................................... 178 6.2.6 Preparation of a NbERL1 suppression construct and analysis of its effects after transient and transgenic expression ................................................................. 179 6.3 Oligonucelotides.................................................................................................. 181 6.4 Vector maps.......................................................................................................... 183 6.4.1 At‐ERL1‐GFP.................................................................................................... 183 6.4.2 At‐leader‐GFP .................................................................................................. 183 6.4.3 At‐ERL1‐over ................................................................................................... 184 6.4.4 Nt‐ERL1‐hp ...................................................................................................... 184 6.5 Sequences.............................................................................................................. 185 6.5.1 Newly identified sequences........................................................................... 185 6.5.2 Published sequences used for in silico analysis and primer design ......... 186 6.6 Curriculum vitae (March, 2011) ........................................................................ 196
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List of Figures: Figure 1.1: Ghildiyal & Zamore, 2009................................................................................. 28 Figure 1.2: MacRea et al., 2006 ............................................................................................. 28 Figure 1.3: Argonaute proteins............................................................................................ 29 Figure 1.4: Okamura et al., 2007........................................................................................... 31 Figure 1.5: Carthew & Sontheimer, 2009............................................................................ 35 Figure 1.6: Zamore, 2010 ...................................................................................................... 38 Figure 1.7: Ghildiyal & Zamore, 2009................................................................................. 40 Figure 1.8: Matzke et al., 2009 .............................................................................................. 39 Figure 1.9: Allen & Howell, 2010 ........................................................................................ 43 Figure 1.10: Kalantidis et al., 2008 ....................................................................................... 47 Figure 1.11: Cheng & Patel, 2004......................................................................................... 51 Figure 1.12: Alignment of published ERI‐1 homologs..................................................... 56 Figure 1.13: Alignment of ERL1 homologues in various plant species......................... 57 Figure 1.14: Winter et al., 2007 ............................................................................................. 57 Figure 1.15: Winter et al., 2007 ............................................................................................. 58 Figure 1.16: Campbell et al., 1996 ........................................................................................ 63 Figure 1.17: Stern et al., 2010 ................................................................................................ 64 Figure 3.1: Localization of plant ERL1 ............................................................................. 100 Figure 3.2: Alignments of Nicotiana sp. ERL1 sequence................................................. 101 Figure 3.3: Analysis of presumable ERL1 suppressor plants........................................ 101 Figure 3.4: Analysis of Nicotiana benthamiana plants overexpressing ERL1 ............... 104 Figure 3.5: TEM analysis of phenotypes of Nicotiana benthamiana plants overexpress‐ ing ERL1................................................................................................................................ 108 Figure 3.6: Phenotypic analysis by light microscopy of Nicotiana benthamiana plants overexpressing ERL1 .......................................................................................................... 107 Figure 3.7: Northern analysis of selected chloroplast‐related genes ........................... 109 Figure 3.8: Determination of photosynthetic parameters in Nicotiana benthamiana plants overexpressing ERL1 .............................................................................................. 111 Figure 3.9: Characterization of selected publicly available Arabidopsis thaliana ERL1 knock‐out plants .................................................................................................................. 116 Figure 3.10: Analysis of Arabidopsis thaliana plants overexpressing ERL1.................. 118 Figure 3.11: Effect of plant ERL1 on different molecules of the RNA silencing appara‐ tus .......................................................................................................................................... 117 Figure 3.12: Northern analysis of chloroplastic ribosomal RNAs and cytosolic 5.8S rRNA following overexpression of ERL1 ........................................................................ 122 Figure 3.13: Alignment of cytosolic 5.8S rRNA from Nicotiana benthamiana plants overexpressing ERL1 .......................................................................................................... 123 Figure 3.14: Alignment of chloroplastic rRNAs of Arabidopsis thaliana and Nicotiana benthamiana plants misexpressing plant ERL1 ................................................................ 126 Figure 4.1: Secondary structures of chloroplastic rRNA (predicted by RNAfold; Gru‐ ber et al., 2008). ..................................................................................................................... 135
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Figure 6.1: Comparative agro‐infiltration time course in systemically PSTVd‐infected tobacco. ................................................................................................................................. 174 Figure 6.2: Agrobacterium co‐infiltration assays in N. benthamiana line 16C to test ERL1 for RNA silencing suppressor activity ............................................................................. 175 Figure 6.3: Silencing of the ERL1 phenotype induced by agroinfiltration.................. 177 Figure 6.4: ERL1 overexpressor plants are hypersensitive towards infection by PPV ................................................................................................................................................ 178 Figure 6.5: Analysis of the suppression of ERL1 in Nicotiana benthamiana. ................ 180 List of Tables: Table 1.1: predicted localization of plant ERL1 homologs .............................................. 58 Table 3.1: Segregation of Nicotiana benthamiana T2 plant lines transformed with a hairpin construct designed for downregulation of ERL1.............................................. 104 Table 3.2: Summary of segregation and phenotypic pattern of Nicotiana benthamiana T1 plant lines overexpressing ERL1 ................................................................................. 107 Table 3.3: Summary of characteristics of Arabidopsis thaliana ERL1 knock‐down plant lines........................................................................................................................................ 117 Table 3.4: Summary of sequence alterations in chloroplastic rRNA after ERL1 misex‐ pression in Nicotiana benthamiana (Nb) and Arabidopsis thaliana (At) plants............... 125
2008). They possess three RNA poly‐ merases with Pol II being responsible for most protein coding genes and Pol I and Pol III transcribing genes for other RNA molecules. is a typical chicken‐and‐egg paradox. An RNA polymerase guides the transcription of a defined genomic stretch by catalyzing the formation of phosphodiester bonds between ribonucleo‐ tides. 1. One hypothesis trying to overcome this paradox considers RNA as the molecule both storing genetic information and catalyzing chemical reactions in primitive cells of this so‐called “RNA world”. This process.1 RNA molecules and their life between DNA and protein mRNA (messenger RNA) is the first molecule involved in the information flow from DNA to protein. The intermediary form in this process is RNA. however. since nucleic acids are required for protein synthesis while proteins are necessary for nucleic acid produc‐ tion. lack‐ ing both the stability and the flexibility of the above mentioned key players. DNA has then developed as a more suitable molecule for storage since its stability increased the maximum size of the hereditary molecules.. which are factors with an unlimited versatility for catalytic processes. Introduction Since its discovery DNA has been considered as a very stable molecule with the abil‐ ity to encode for an infinite number of proteins. This might have happened in parallel with the necessity for storage of increased amounts of genetic information after accumulation of additional protein catalysts (Alberts et al. It synthesizes a single‐stranded mRNA molecule in 5’‐to‐3’ direction and stops the elongation when reaching the terminator sequence. The polymerase recognizes a certain promoter sequence where it binds to the DNA strand. This general process is more complex in eukaryotes.1. The transcription initiation re‐ quires the action of general transcription factors facilitating the binding to the pro‐ moter sequence and forming a transcription initiation complex. The synthesized 25 .
The intronic sequence is circularized into a structure called lariat. the final length of this poly‐A tail is deter‐ mined by poly‐A‐binding proteins. Aberrant RNAs are degraded by the nuclear exosome consisting of 3’‐to‐5’ RNA ex‐ onucleases. Eukaryotic protein coding gene stretches contain expressed sequences (exons) and non‐coding intervening sequences (introns). The nucleotide sequence of the mRNA is translated into amino acids by codons con‐ sisting of three consecutive nucleotides. Only spliced mature mRNA with a 5’‐cap and a 3’‐poly‐A tail are ex‐ ported to the cytoplasm through the nuclear pore complexes (NPCs). U5 and U6) together with at least seven proteins forming the small nuclear ribonucleoprotein complex (snRNP). The polypeptide chain is synthesized by the stepwise addition of amino acids to its C‐terminal end that is activated by the binding to a tRNA molecule (which is then called peptidyl‐tRNA). The above described process takes place in the ribosomes. The necessary RNA molecules for this proc‐ ess are tRNAs (transfer RNAs) which are extensively structured L‐shaped adaptor molecules with unusual bases organized into three single‐stranded loops. The relevant amino acid is connected to the 3’‐end of the tRNA molecule by aminoacyl‐tRNA syntheta‐ ses. The poly‐A polymerase (PAP) then adds ap‐ proximately 200 adenosine nucleotides.1. Protein factors accompanying the transcribed mRNA molecule recognize the 3’‐end of the strand and lead to its cleavage. U4. Introduction mRNA strand is modified at its 5’‐end by addition of a cap of 7‐methylguanosine (7mG) which binds a protein complex called cap‐binding complex (CBC). The removal of the latter from the pre‐ cursor mRNA strand is performed by the spliceosome. U2. One of them contains the anticodon which binds to the mRNA sequence. They contain another type of RNA. excised and the ends of the exons are then joined together to a continuous coding sequence. a catalytic machinery con‐ sisting of ribosomal proteins and ribosomal RNAs (rRNA). the five small nuclear RNAs (snRNAs U1. 26 . They constitute up to 80 % of the total RNA of cells and are encoded in multiple rRNA genes which are often arranged in tandems and transcribed by Pol I.
stating that the genetic information only flows from nucleic acids to proteins. 5. The overexpression of CHALCONE SYNTHASE (CHS). They are often encoded in and further excised from intronic sequences. The eukaryotic 80S ribosome consists of a 60S subunit (with 5S. also known as small nucleolar RNAs (snoRNAs). 1987). where an endogenous gene is downregulated after strong overexpression of the same se‐ quence. The two ribosomal subunits are then exported to the cytoplasm where they join to form the mature ribosome.. was discovered by two groups trying to increase the purple pigmentation of petunia. This phenomenon called co‐suppression. an enzyme of the 27 . The importance of RNA had been boosted significantly by the finding.. Introduction Long rRNA precursor molecules are extensively modified at positions that are speci‐ fied by guide RNAs. Many other important observations which finally lead to the discovery of the RNA silencing mechanism had also been made in plants: after expression of antisense RNA the tobacco nopaline synthase was inhibited (Rothstein et al. The nucleolus is a distinct structure in the nucleus where rRNA processing and ribosome assembly take place. 1998).1. The major catalytic reactions are carried out by the rRNA molecules which can be considered as ribozymes (Alberts et al. The mature ribosome contains four binding sites for RNA. one for mRNA and three for tRNA: the latter are bound at the A‐site. that double‐ stranded RNA is a trigger of gene silencing and therefore providing a negative feed‐ back mechanism which is independent of protein synthesis (Fire et al.2 RNA silencing – new roles for the intermediate For many years the central dogma of molecular biology was postulated by Francis Crick. the amino acids are then connected together at the P‐site and the empty tRNA finally gets released at the E‐site. The first observation of an RNA silencing mechanism was reported in 1928 when newly emerging leaves of TRV‐infected Nicotiana tabacum plants were found to be free of infection symptoms (Wingard.8S and 23S rRNA) and a 40S subunit (with 18S rRNA). 1. 2008).. The prokaryotic 70S ri‐ bosome consists of a 50S subunit (with 5S and 23S rRNA) and a 30S (with 16S rRNA). especially from ribosomal proteins. 1928).
(B) In the miRNAs pathway a hairpin‐ shaped precursor molecule is exported into the cytoplasm and processed by Dicer... 1990. chromatin remodeling can occur in some species. 28 . (C) In the piRNA pathway a single‐stranded precursor RNA is cleaved by a PIWI‐protein into piRNAs which get methylated at the 3’‐end. Binding results in target repression either by translational arrest or by cleavage of the target. The major players in the RNA silencing mechanism are small interfering RNAs (siRNAs). the latter being restricted to the animal kingdom. In addition. members of the Argonaute/Piwi (AGO) protein families and the RNase III‐type protein Dicer function in all silencing pathways. Moreover.1. They may initiate the production of secondary piRNAs. They are incorporated into the RISC and lead to target cleavage. The miRNA is loaded into the miRISC and leads to translational repression of the target. 1990). resulted in a large fraction of plants with white petals (Napoli et al.1: Ghildiyal & Zamore. Several classes of small RNAs have been identified to result in various silencing pro‐ cedures. Figure 1. van der Krol et al. 2009 (A) The siRNA pathway is characterized by a dsRNA which is processed by Dicer into siRNAs. microRNAs (miRNAs) and Piwi‐interacting RNAs (piRNAs). Introduction anthocyanin pathway. Common features are the involvement of 18–35 nucleotide long small RNAs which are complementary to target RNAs.
. which is a dsRNA‐specific ribonuclease of the RNase III family (Bernstein et al. some members lack one or more of them. 2000. The PAZ [(Piwi/Argonaute/Zwille) (Cerutti et al. 1999) and animals (Zamore et al. 2000)] domain functions in binding of single‐ and double‐stranded RNA and DNA with a prefer‐ ence for single‐stranded RNAs molecules or single stranded 3’‐overhangs (Lingel et al.. In Arabidopsis four different Dicer‐like proteins have been identified [(Baulcombe.. Vertebrates and nematodes possess a single Dicer protein (Carmell & Hannon. 1999). 2004) (see chapter 1.2. 2001a). The N‐ terminus is comprised of a DExD helicase domain (Bernstein et al. Additional double‐stranded RNA binding domains (dsRBDs) may function in the binding of double‐stranded RNA. 2004). Hammond et al.. 29 .2) and DCR‐2 producing siRNAs (Lee et al.1 siRNA‐mediated gene silencing siRNAs were identified to direct endonucleolytic cleavage of the target RNAs in plants (Hamilton & Baulcombe. 2006).1. The family is classified into three classes with Dicer being a member of class III (Nicholson et al. 2003)... 1968). 2001b).. 2001).. the strands possess a 5’‐phosphate and a 3’‐hydroxyl (Elbashir et al. It usually consists of six distinct domains..4)].. 2006). 2000).2. Introduction 1. 2004a).. Characterization of the Dicer homologue of Giardia intestinalis revealed that a functional enzyme only requires the core protein consisting of PAZ and two RNase III domains which dimerize and use a two‐metal‐ion mechanism for RNA cleavage (MacRea et al.2. The number of Dicer proteins varies between species. 2006).. The length of the produced small RNA is determined by the distance between the PAZ and the RNase III domains (MacRea et al. They are produced from long dsRNA molecules by the enzyme Dicer.. They cleave their target approximately every 21 nucleotides into a double strand of 19 nucleotides and two nucleotide overhangs at the 3’ ends (Elbashir et al. Drosophila has two Dicer proteins with DCR‐1 being responsible for miRNA production (see chapter 1. It is directly connected to the endonucleolytic RNase III domain responsible for RNA cleavage (Robertson et al. 2001) and a do‐ main of unknown function (Duf283) that may be involved in strand selection (Dlakić.
The silencing component of Argonaute proteins can be subdivided into two groups: the AGO clade consists of members that are similar to AGO1 of Arabidopsis thaliana.. (B) Resolved crystal structure of the Giardia Dicer with the N‐terminal platform domain (blue).. 2006 (A) Domain organization of human Dicer with Helicase‐.. two RNase III and the dsRBD domains and the minimal Dicer of Giardia intestinalis with the PAZ and the two RNase III domains.. 2004). the PAZ domain which binds ssRNA (Lingel et al. 2003. which is also named slicing (Song et al. 2000). They bind small RNAs derived from dsRNA in the RISC and are expressed ubiqui‐ tously. 1997)] with an RNase H fold that functions as a ribonucle‐ ase and confers the cleavage of ssRNA. b): an N‐terminal domain.. 2003). In contrast the Piwi clade consists of three proteins which are primarily ex‐ 30 . 1995) and the Piwi [(P‐Element‐induced wimpy testis domain) (Lin & Spradling. The passenger strand is destroyed (Matranga et al. The double strand is separated depending on the relative thermodynamic stability of the two ends of the duplex (Khvorova et al. 2005.1.2: MacRea et al. which has a bridge (grey) to the RNaseIIIb domain (green). Introduction (A) (B) Figure 1.. It is characterized by the guide strand that is bound to an Argonaute protein and auxiliary proteins de‐ pending on the species and the type of small RNA (Hammond et al. 2003).. PAZ‐. Leuschner et al. the PAZ domain (orange) connected (red) to the RNase IIIa domain (yellow).3 a. Argonaute proteins contain four domains with partly unidentified functions (see Figure 1. Schwarz et al.. DUF283‐. 2006) and the guide strand is incorporated into the RNA‐induced silencing complex (RISC).. the middle domain with resemblance to the sugar‐binding domain of the lac repressor (Friedman et al.
pressed in gonadal tissues (see Figure 1.3 c): Piwi, the Drosophila P‐element‐induced wimpy testes protein (Lin & Spradling, 1997), Aubergine (Aub) first identified by its role in dorsoventral patterning (Schüpbach & Wieschaus, 1991) and Argonaute 3 (AGO3). The incorporated siRNA binds to its target sequence and the respective Argonaute protein cleaves the phosphodiester bond of the target between the tenth and eleventh nucleotide of the bound guide strand. The cleaved target is then released of the ma‐ ture RISC (Elbashir et al., 2001b). Humans possess eight Argonaute proteins but only Ago2 shows Slicer activity (Liu et al., 2004; Meister et al., 2004), Drosophila melanogaster has five Argonaute proteins and all of them possess the ability to slice (Miyoshi et al., 2005). Caenorhabditis elegans has the largest number of Argonaute proteins with 27 different members (Yigit et al., 2006). Arabidopsis thaliana possesses 10 Argonaute proteins [(Vaucheret, 2008) (see chapter 1.2.4)].
Figure 1.3: Argonaute proteins (A) Domain organization of human AGO2 with the N‐terminal PAZ‐domain, the Mid domain includ‐ ing the cap‐binding like MC domain and the C‐terminal cleavage‐competent PIWI domain (B) Crystal structure of the Argonaute of Pyrococcus furiosus including the siRNA (purple) and mRNA (turquoise) duplex. Active residues of the PIWI domain (a DDH motif) are shown in red. (Hutvagner & Simard, 2008) (C) Multiple sequence alignment revealed three clades of Argonaute proteins (Tolia & Joshua‐ Tor, 2007). 31
1.2.2 miRNAs modulate the expression of endogenous sequences
The first report of a microRNA gene was lin‐4 which had the ability to repress cell proliferation in C. elegans (Chalfie et al., 1981). Although encoded by a gene, it was later shown to be only transcribed into a non‐coding RNA with some complementar‐ ity to the 3’‐UTR of another mRNA transcript, lin‐14, which consequently became downregulated (Lee et al., 1993). First considered as a unique phenomenon some years later let‐7 was discovered to use the same mechanism (Reinhart et al., 2000). Subsequently it was shown that miRNAs are an abundant class of small RNA mole‐ cules responsible for many intracellular regulation processes (reviewed in Carthew & Sontheimer, 2009). Animal miRNAs are highly conserved between species which has also been used for their identification in the past (Ambros et al., 2003). Many new miRNAs have been identified by deep‐sequencing technologies with now 15172 entries in miRBase re‐ lease 16, Sept 2010 (Griffiths‐Jones et al., 2008). Genomic regions coding for miRNAs can be located in protein‐coding genes or in intergenic regions. It was shown that they contain standard promoter elements. Con‐ sequently they are transcribed by RNA Polymerase II (Pol II) (Lee et al., 2004b) into primary miRNA (pri‐miRNA) transcripts which are usually highly structured (Lee et al., 2002). The first maturation step is always located in the nucleus and executed by the RNase III endonuclease Drosha (Lee et al., 2003) assisted in the Microprocessor complex by a dsRNA‐binding protein [Pasha in flies (Denli et al., 2004) and DGCR8 in mammals (Gregory et al., 2004; Han et al., 2006)]. It results in the precursor miRNA (pre‐miRNA) which is comprised of an imperfect hairpin structure (Lee et al., 2002). In animals the molecule is subsequently exported into the cytoplasm by Exportin‐5 and the GTPase Ran (Yi et al., 2003). The second maturation step is executed by a Dicer enzyme (Grishok et al., 2001; Hutvágner et al., 2001; Ketting et al., 2001) again assisted by a dsRNA‐binding protein [in flies R2D2 for dsRNA (Liu et al., 2003) and LOQS for structured loci (Förstemann et al., 2005; Saito et al., 2005) and TRBP in mammals (Chendrimada et al., 2005; Haase et al., 2005)]. It generates a miRNA/
miRNA* duplex of approximately 21 nt and 2 nt overhangs at the 3’ end. Although resembling siRNAs in this step, they can be distinguished by the imperfect binding between the two strands (Lee et al., 2002). The duplex is loaded into the RISC and the miRNA* strand degraded by the respective Ago protein (Filipowicz et al., 2008). 5‐10 % of all miRNA genes with low expression are located in introns which fold into short hairpins. These so‐called mirtrons are first processed by the splicing machinery and then linearized by the lariat debranchase. They further fold into a hairpin similar to pre‐miRNAs which are processed as described above (Okamura et al., 2007; Ruby et al., 2007). Recently a distinct biogenesis mechanism has been discovered in mice (Cheloufi et al., 2010) and zebrafish (Cifuentes et al., 2010) for miR‐451 which possesses an un‐ usual secondary structure with a short stem of 17 nucleotides. The pre‐miR‐451 is cleaved by Ago2 and the resulting intermediates are polyuridylated and further processed by yet to be defined nucleases into the mature miRNA (Cheloufi et al., 2010; Cifuentes et al., 2010).
Figure 1.4: Okamura et al., 2007 The canonical miRNA pathway consists of a Pol II transcript which folds into a hairpin. This pri‐miRNA is processed by the Drosha complex into the pre‐ miRNA hairpin. In the mirtron pathway, a short in‐ tron is spliced and branched into a hairpin. The pre‐ miRNAs are exported to the cytoplasm by Eportin‐5 and cleaved by Dicer.
Animal miRNAs bind their targets in a different manner than siRNAs: only a seed region of approximately six nucleotides around the usual cleavage site requires per‐ fect complementarity to the 3’‐UTRs of their targets, the rest of the miRNAs pos‐ sesses mismatches and frequent non‐conventional base‐pairing (G:U wobbles) with the targeted mRNA. The exact mechanism of suppression, however, is still under investigation. The imperfect binding prevents target cleavage by Argonautes. One model proposes competition with the cap‐binding protein eIF4E and subsequent in‐ hibition of the translation initiation. Another hypothesis accounts for the fact that many miRNA‐regulated mRNAs are deadenylated. It proposes RISC to stimulate deadenylation and subsequent mRNA decay. A third model uses the finding that RISC has some binding affinity to the 60S ribosomal subunit and may therefore pre‐ vent the assembly of the ribosome (reviewed in Carthew & Sontheimer, 2009). Argonaute proteins involved in the miRNA pathway interact with GW182 proteins (Behm‐Ansmant et al., 2006). They contain frequent glycine (G) and tryptophan (W) repeats (Eystathioy et al., 2002) organized in three distinct regions. The N‐terminal GW‐repeat region is followed by a ubiquitin‐associated (UBA)‐like domain and a glutamine‐rich (Q‐rich) region. The middle‐ and a C‐terminal GW‐repeat region are separated by a RNA recognition motif (reviewed in Ding & Han, 2007; Eulalio et al., 2007). While there exist three paralogues in vertebrates (TNRC6A, ‐B and –C), fungi have no GW182 proteins and Drosophila possesses only one orthologue making it a good model for studying their function (Behm‐Ansmant et al., 2006). The C. elegans orthologues AIN‐1 and AIN‐2 contain only the N‐terminal GW‐repeat region but function also in miRNA silencing (Ding et al., 2005; Zhang et al., 2007). Binding of Argonaute by GW‐containing proteins has been also identified in plants (NRPD1b and SPT5‐like transcription elongation factor) (El‐Shami et al., 2007; Bies‐Etheve et al., 2009) and S. pombe [(Tas3) (Partridge et al., 2007; Till et al., 2007)]. GW182 proteins exhibit some intrinsic silencing activity (Eulalio et al., 2009a; Zip‐ prich et al., 2009). In addition loss of GW182 suppresses miRNA silencing (Rehwinkel et al., 2005), but it acts downstream of miRNA processing and loading into the RISC
2010).. which appears to be critical for miRNA‐mediated silencing (Huntzinger et al.1. 2009 miRNA‐dependent translational repression may be mediated by competition with cap or ribosome binding. 2009. 2009).5: Carthew & Sontheimer. 2009).... 2009a. Figure 1. All factors involved in 5’‐3’ exonucleolytic decay of mRNA.. They are not required for miRNA silenc‐ ing but can be formed as a consequence of it (reviewed in Eulalio et al. 2009b)]. 2006) and together with the Q‐rich region responsible for localization to the P‐bodies [(processing bodies) (Eulalio et al. including the decapping enzyme DCP2 and the main cytoplasmic 5’‐3’ exoribonuclease XRN1 co‐localize to the P‐bodies in the cytoplasm... Miyoshi et al. The N‐terminal region is required for the binding to AGO1 (Behm‐Ansmant et al. Alternatively deadenylation and subsequent mRNA degradation might be induced. circularization may be blocked. 2007). A con‐ served motif of approximately 40 residues inside the middle GW‐repeat region has recently been identified as a PolyA‐binding protein‐interacting motif (Fabian et al. or the ribosomes could drop‐off after translation initiation. They are cytoplasmic granules where translationally repressed mRNAs can concentrate. Zekri et al.. 35 . Introduction (Eulalio et al.
. 2008).. They have 2’‐O‐methylated 3’‐ends. they have a strong bias for a 5’‐uridine resi‐ due and a Dicer‐independent biogenesis pathway resulting in a larger size compared to the other small RNAs. small RNA species similar to rasiRNAs but not derived from repeat and transposon‐sequences. usually enriched for sequences antisense to transposons. Another control point is the regulation of miRNA processing.3 The piRNA pathway protects the germline from transposon activity In Drosophila melanogaster a large family of small RNAs with a length of 23‐26 nt was identified to map to repetitive heterochromatic regions and transposable elements. they were called repeat‐associated small‐interfering RNAs (rasiRNAs) (Aravin et al.. These antisense piRNAs are bound by Piwi and Aub whereas the sense‐orientated fraction interacts with AGO3. 2007).. 2006). The newly identified family of small RNAs was further called Piwi‐interacting RNAs [(piRNAs) (Malone & Hannon. 2008) and they in‐ teract with the Piwi‐related gene PRG‐1 (Wang & Reinke. 2008). 2008). 2008. they suppress transposon mobility (Das et al.. 2007. Under certain conditions the miRNA‐loaded RISC has been shown to activate trans‐ lation but the exact reason and mechanism is not clear (Vasudevan et al. Piwi and its orthologues were shown to bind rasiRNAs and. where many factors have been already identified. 2009)]. They all arise from chromosomal clusters and have a single‐ stranded RNA precursor (reviewed in Klattenhoff & Theurkauff. Ohara et al. A first report of exonucleases degrading miRNAs comes from plants (Ramachandran & Chen. The two classes 36 . 1.. 2010). in mammals.. elegans are also piRNAs since they contain a 5’ uridine (Ruby et al. There is evi‐ dence that the 21U‐RNAs of C. elegans (Chatterjee & Grosshans. or the regulation of the effector proteins of the miRISC (reviewed in Krol et al. Henke et al. 2001) and could later also be identified in zebrafish (Chen et al.. 2005). In Drosophila piRNA populations can be matched to transposons. Ørom et al. Introduction miRNAs themselves are very often under control of their targets in a negative feed‐ back loop.. 2009). 2007. In addition.1. which is deposited by an orthologue of the plant methyltransferase HEN1 (Kirino & Mourela‐ tos. 2008) and a homologue has also been identified in C.2.
4. This suggests a compartmentalization of piRNA bio‐ genesis and action (Lim & Kai.. 2007). Additional factors have been identified to be required for the piRNA pathway... It is initiated by primary antisense piRNAs which target the cleavage of transposon mRNA. suggesting processing by Piwi which had been shown to cleave its target ten nucleotides from the 5’‐end of the guide strand (Saito et al. 2007) and mice. However. Gunawardane et al. The reaction activates the RNA‐dependent RNA polymerase complex which generates further siRNAs. 2008) and the germ‐ line function (Wang & Reinke.. 2007). 2004). 2007). In the latter the tran‐ scription of centromeric repeats leads to siRNAs which direct the AGO orthologue to cleave target transcripts of this locus. The same can be observed after a loss of the putative helicases Armitage (Vagin et al. which is a germline‐specific perinuclear structure implicated in RNA processing.1. 2006) and Spindle E (Aravin et al. Parts of this cycle have also been detected in zebraf‐ ish (Houwing et al. The biogenesis and amplification of piRNAs follows the so called ping‐ pong cycle. This results in secondary sense piRNAs which are bound by AGO3 and direct the cleavage of antisense transposon sequences (Brennecke et al.2. 2007). 2006). although the cycle there seems to be initiated by sense piRNAs and is only present in the male germline (Aravin et al.. They are all part of nuage. 2009)..1). an additional pathway of piRNA function has been dis‐ covered recently in somatic ovarian follicle cells. The cycle shares similarities with the silencing of heterochromatic regions by RNA‐directed DNA methylation in plants and S. Finally these siRNAs direct the modification of histones (Moazed. reviewed in Klattenhoff & Theurkauff. 2007. elegans 21U‐RNAs are also required for fertility (Batista et al. muta‐ tions in the putative nucleases Zucchini and Squash disrupt piRNA production and release transposon silencing (Pane et al. Introduction of piRNAs have overlapping 5’‐ends separated by ten nucleotides (Brennecke et al. 2008). In Drosophila mainly Aub‐ and AGO3‐associated piRNAs take part in the ping‐pong cycle. 2007.. depending exclusively on Piwi and 37 . Gunawardane et al. C.. 2007. pombe (see chapter 1.. Piwi‐associated piRNAs may only comprise the primary piRNAs in the germ‐ line‐specific cycle. 2008).
discussed in Zamore. Armitage and the putative helicase/ tudor domain protein Yb are required for silencing of the gypsy transposon in the somatic cells.1. Arabidopsis thaliana has four Dicer‐like and ten Argonaute pro‐ teins with distinct molecular functions. Piwi accumu‐ lates in the cytoplasm in the absence of Zucchini. 2010 In somatic cells piRNAs are synthesized by a PIWI‐dependent linear pathway without amplification.6: Zamore. 2010. Figure 1. The latter proteins are both localized to cytoplasmic foci. In armitage mutants no piRNAs accumulate suggesting a function early in the pathway (Olivieri et al. Introduction the flamenco piRNA cluster (Malone et al.2. recent findings suggest the following proteins being indispensa‐ ble for the somatic piRNA pathway: Zucchini. which maybe confers shuttling be‐ tween the cytoplasm and the nucleus.. Although initially more factors had been identified. whereas in the germline the primary piRNAs are dependent on Aubergine and get amplified via the so‐called ping‐pong cycle and Ago3.. Every member of the DICER‐LIKE protein family in Arabidopsis has distinct functions although some redundancies have also been identified: DCL1 is the main Dicer in‐ 38 . 2009a.b). 1. 2010).4 Plants possess a high diversity of siRNA molecules Plants display an astonishing variety of siRNA types and proteins which are needed for their generation.
. It is itself regulated by a feedback mechanism through miR168 (Vaucheret et al. Moreover. AGO1 predominantly acts in the miRNA pathway but it can also bind several classes of siRNAs (Baumberger & Baulcombe.1.. AGO10 (originally termed PINHEAD/ZWILLE) is the closest paralogue of AGO1 and partly shows redundant function in development (Lynn et al. 2004). 2005b) and in the production of miR822 and miR839 (Rajagopalan et al. AGO1 is the founding protein for the whole Argonaute protein family and its pleio‐ tropic defects have first been described in 1998 (Bohmert et al... DCL4 is the main plant Dicer for the production of viral 21‐nt siRNAs (Dunoyer et al. In addition.. 2006). Xie et al.. Recent studies implicate AGO10 as a negative regulator of AGO1 (Mallory et al. DCL1 can process some nat‐siRNAs from endogenous inverted repeat sequences (Borsani et al. Deleris et al. 2005. It had later been identified to act in RNA silencing (Fagard et al.2)].. 2005. The former will be discussed in more details (reviewed in Vaucheret.. 2006. 1998). 2006). AGO5 is the third member of this group. 2006).. 2003). but it has been shown to preferentially interact with a 5’‐cytosine (Takeda et al. Yang et al. 2000). Katiyar‐Agarwal et al... 2005). Another clade of Arabidopsis AGO proteins contains AGO7 which is involved in the TAS3 biogenesis pathway [(Montgomery et al. 2002... DCL2 also participates in the latter function and gener‐ ates 22‐nt siRNAs from virus sequences (Xie et al. AGO2 39 . 1999). 2008). Reinhart et al. 2010). 2006). it is involved in tasiRNA metabo‐ lism (Gasciolli et al. Bouché et al.. 2002. 2008a) (see chapter 1. Papp et al. Its exact function is not clear.2. After processing by DCL proteins in plants the re‐ sulting small RNAs are methylated at their 3’‐ends by the S‐adenosyl‐dependent me‐ thyltransferase HUA ENHANCER 1 (HEN1) which protects them from uridylation and further degradation (Li et al.. DCL3 produces 24‐nt siRNAs which are mainly involved in chromatin silencing (Xie et al.. 2004). 2006) Plants contain a high number of Argonaute proteins with ten members in Arabidopsis thaliana and 19 members in Oryza sativa.. 2005. 2008).. Introduction volved in miRNA processing which cannot be compensated by the other members (Park et al.. 2005.4....
(C) natsiRNAs derive from overlap‐ ping transcripts which are processed into a dsRNA. Figure 1.7: Ghildiyal & Zamore. is still unknown.. AGO1 prefers uridine which is the predominant 5’ nucleotide of plant miRNAs.4. 2007). Since the AGO8 expression is very low it has been suggested to be a pseudogene (Takeda et al. The third clade is comprised of AGO4.1)]. but their similarity suggests a redun‐ dant function. The former is probably regulated by miR403 (Allen et al. RDR6 generates a dsRNA which is processed by DCL4 into tasiRNAs which associate with AGO1/7. 2005).2. the major protein involved in transcriptional gene silencing [(Zilberman et al. 2009 (A) In plants cis‐acting siRNA precursor molecules are transcribed by Pol IV and a dsRNA generated by RDR2. Introduction and AGO3 are highly similar proteins with currently unknown function. DCL3 cleaves the 24‐nt casiRNAs which associate with AGO4. (B) In the tasiRNA pathway a precursor molecule is subject to miRNA‐mediated cleavage. 2008). AGO8 and AGO9. The role of the other two proteins of this clade. AGO6 has a partial re‐ dundant activity with AGO4 (Zheng et al.1... 2008). It has been shown that the 5’‐nucleotide of the small RNA species acts as a sorting signal into the different AGO proteins in plants: AGO2 and AGO4 preferentially in‐ teract with adenosine. A dicer molecule then generates the natsiRNAs. 2003) (see chapter 1. 40 ... Finally AGO5 binds small RNAs with a terminal cytosine (Mi et al.
2008. 2009).8: Matzke et al. Introduction 1. Their largest (NRPD1 and NRPE1) and second largest subunits (the shared subunit NRPD2/NRPE2) are unique with the latter being also related to the largest subunit of POL II (RPB1) (Wierzbicki et al. After primary RdDM Pol IV transcribes the methylated template and downstream sequences which result in secondary RdDM..1 Cis‐acting siRNAs mediate chromatin silencing in plants RNA‐directed DNA methylation (RdDM) is an epigenetic siRNA‐mediated modifica‐ tion in plants (reviewed in Matzke et al. In addition RdDM requires two plant‐specific POL II‐related RNA poly‐ merases. 2010). They act in complexes Figure 1.1. 2008).4.. 2009. Ream et al.. POL IV and POL V (Pikaard et al. Pol IV transcribes the methylated DNA which is further copied by RDR2 into dsRNA.. 2008. In plants it is responsible for 30 % of de novo methylation of cytosines in heterochromatic and some euchromatic regions such as transposons (Cokus et al. 2008). 41 . Lister et al...2. 2008. 2009 dsRNA is processed by DCL3/HEN1 into 24‐nt siRNAs which are loaded onto AGO4... Law & Jacobsen. This methylation is deposited by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). Transcription by Pol V facilitates de novo methylation at the siRNA‐targeted site. Wierzbicki et al.
The action of RDR2 may lead to the production of secondary siRNAs and further me‐ thylation spreading (Daxinger et al.2... 2009).. consisting of three loci.. 2004. and TAS2 were the first identified TAS loci (Peragine et al.. The other is compromised by the TAS3 family which requires two miRNA‐binding sites for tasiRNA biogenesis (Allen & Howell. Recently POL II‐ dependent non‐coding transcripts have been identified to also recruit RdDM factors (Zheng et al. INVOLVED IN DE NOVO METHYLATION 2 (IDN2) may inter‐ act with the siRNA‐RNA duplex and recruit RDM2 (Ausin et al... 2006). 2009). 2010). The TAS1 family. The IGN transcripts are probably recog‐ nized by the AGO4‐bound siRNAs (Wierzbicki et al. They depend on the cleavage by miR173 (Yoshikawa et al. The initial POL II transcripts are bound by the miRNA with 42 . There exist four TAS families which can be further subdivided into two classes: one consists of TAS1. TAS2 and TAS4 which require one miRNA‐ binding site for tasiRNA biogenesis. 2009). whereas TAS4 depends on the cleavage by miR828 (Ra‐ jagopalan et al. 2008). 2009). These are bound by AGO4 [(or sometimes AGO6 (Zheng et al. siRNAs also appear to guide active de‐ methylation (Zheng et al. 2004).1.. POL V together with DEFECTIVE IN RNA‐DIRECTED DNA METHYLATION (DRD1) (Pikaard et al. 2008) identifies and maybe also transcribes low‐abundance intergenic non‐coding transcripts (IGN) (Wierzbicki et al. The RdDM mechanism is conserved in S.. 2008). 1. RNA‐DPENDENT RNA POLYMERASE2 (RDR2) produces dsRNA from the single‐ stranded POL IV‐dependent transcripts which are further processed by DCL3 into 24‐nt heterochromatic siRNAs (Mosher et al. Vazquez et al.. Introduction including SNF2‐like chromatin remodeling factors: POLIV together with CLASSY1 (CLSY1) is involved in the initiation of siRNA biogenesis by transcribing a long sin‐ gle‐stranded RNA... 2008). 2007)] which can interact with POL V through a con‐ served GW/WG motif (El‐Shami et al. 2005).4.2 Trans‐acting siRNAs Trans‐acting siRNAs (tasiRNAs) are a plant‐specific type of small RNAs generated from specific TAS loci.. 2008).. 2007). pombe as well where it leads to heterochromatinization (reviewed in Moazed.
Felippes & Weigel. 43 . Vazquez et al. 2009) which synthesizes the complementary strand from the 3’‐end towards the 5’‐cleavage site.. substitution of these mismatches abolishes the production of tasiRNAs (Montgomery et al..9: Allen & Howell. Introduction unusual mismatches in the seed region. 2009). 2010 In the first pathway miR173/828 guides the cleavage of the TAS transcript by AGO1. It can interact with the RNA‐DEPENDENT RNA POLYMERASE 6 (RDR6) (Kumakura et al. 2004).1. 2004) and bound by SUPPRESSOR OF GENE SILENCING 3 (SGS3) (Peragine et al. The cleaved transcript is synthesized by RDR6 into a dsRNA which is processed by DCL4 into the tasiRNAs.. 2004). probably by the redundant Figure 1. 2004. Vazquez et al.. The transcript is cleaved by AGO1 (Vazquez et al.. The dominant phasing pattern can also drift for one or two nucleotides.. 2008b.. In contrast in the second pathway miR390 binds twice to the TAS3 transcript and guides its cleavage by AGO7 at the 3’‐ site. The resulting dsRNA is further processed by DCL4 from the miRNA cleavage site into 21‐nt long siRNAs (Peragine et al. 2004.
. 2005.. 2004. 2004).. Williams et al. Although rather rare under physiological conditions. Vazquez et al..1. Katiyar‐Agarwal et al. 2008). 2007). The TAS3 family consists of three loci (Howell et al.. MYB transcription factors (Rajagopalan et al... 2008a). 2008). 2006. Introduction function of other DCL proteins (Howell et al.. 2005). Schwab et al. 2006).. since NAT pairs compromise more than 7 % of all transcriptional units in Arabidopsis thaliana (Henz et al. miR390 was shown to interact with AGO7. 2007)..3 Natural antisense siRNAs Natural antisense transcript‐derived siRNAs (natsiRNAs) have first been described as a result of salt‐stress in Arabidopsis (Borsani et al. 2006) and Auxin responsive factors (Allen et al. 2009. 2008). SGS3 and POL IV (Borsani et al. Factors required for the synthesis of the primary natsiRNAs are DCL2 and/or DCL1. Identified targets are Pentatricopeptide‐repeat proteins (Peragine et al..2. all characterized by two binding sites for miR390 which flank the tasiRNA‐producing sequence (Allen et al.4. 2005.. 2006. they may substantially contribute to the small RNA population during stress conditions. tasiRNAs can be considered as an amplification of an initial silencing signal. Fahl‐ gren et al. the binding of AGO7 to the 5’‐site is indispensable for tasiRNA produc‐ tion (Montgomery et al. 2006. 1. The 5’‐site possesses critical mismatches and it is not clear if it is also cleaved... Garcia et al. In contrast. The tasiRNAs are sorted into the corresponding Argonaute proteins according to their 5’‐nucleotide (Mi et al.. In addition they are non cell‐autonomous and can create a silencing gradient across neighboring cells (Chitwood et al. 2007). From these only the 3’‐site is cleaved and determines the resulting tasiRNAs. trans‐NATs are transcribed from different loci resulting in short and imper‐ fect complementarity of the double strand (Jin et al. 2005. 2008).. They guide the cleavage of the complementary transcript. 44 . While the cleavage at the 3’ site can be accomplished by another miRNA‐ AGO pair. Zhang & Trudeau. Adenot et al.. 2009). RDR6.... There exist two different types of natural antisense transcripts (NATs): cis‐NATs have a high sequence com‐ plementarity since they are transcribed from opposing strands of the same locus.
2009). The pri‐miRNAs are further processed by DCL1 into mature miRNAs in a two‐step process. Reinhart et al. The miRNAs are loaded into the Argonaute protein AGO1 of the RISC which slices the mRNA targets between the tenth and eleventh nucleotide (Rhoades et al.. 2005). Kurihara et al. 2008). therefore it is believed that they evolved independently in the two genera (Ax‐ tell & Bowman. Plant miRNAs consequently possess several special features compared to animal miRNAs (reviewed in Voinnet... Examples of evolutionary conserved miRNAs between animals and plants are rare. 2002). The first tran‐ sition to pre‐miRNAs takes place in nuclear processing centers (D‐bodies) where DCL1 interacts with the double‐stranded RNA‐binding protein HYPONASTIC LEAVES1 (HYL1) and the Zinc‐finger protein SERRATE (SE).1. 2006). It also binds other mRNAs and might be involved in siRNA biogenesis too (Yu et al... 2008). Later a mechanism of translational repres‐ sion similar to the miRNA mechanism in animals has been identified in miRNA‐ 45 . Yang et al. 2002. there exist more than 100 families of related miRNAs which are usually conserved between angiosperms (Ax‐ tell & Bowman. 2007. 2006). 2008) and partly also back to moss (Garcia. The strand folds into an imperfect hairpin which is probably sta‐ bilized by the RNA‐binding protein DAWDLE (DDL).. MIR genes are transcribed by POL II into pri‐ miRNAs which get capped at the 3’‐end and polyadenylated at the 5’‐end like mRNA transcripts.. The miRNAs are exported into the cytoplasm by the nuclear pore complex HASTY. 2002). After the second processing step by DCL1. 2008). Another group of miRNAs are evolutionary younger. the latter also having a role in mRNA splicing (Fang & Spector. the mature miRNA duplexes are stabilized by methylation at their 3’‐ends which is transferred by HEN1 (Li et al..5 Peculiarities of plant miRNAs The first plant miRNAs have been identified in 2002 (Park et al. Introduction 1. 2006). Most plant MIR genes are located in intergenic regions.. therefore less conserved and with a highly di‐ verse target range (Zhang et al. The first identified mode of action of plant miRNAs is characterized by extensive complementarity to their target mRNAs. 2005. al‐ though other export mechanisms cannot be excluded (Park et al.2.
1. 2005a) which show a high degree of transcrip‐ tion factor binding sites (Megraw et al. since they host a wide range of viruses and have a lifecycle and size more favorable for studying the effects of viral infections. Tissue‐specific differences in protein expression can account for altered miRNA action. however. Their activity is stimulated by the presence of aberrant RNA with missing 5’‐cap structures or 3’‐polyadenylation (Herr et al. 2005.2. Since viruses replicate rapidly and are able to spread throughout the whole plant..6 Spreading of RNA silencing in plants resembles an immune system In plants RNA silencing acts as sort of an immune system providing resistance to viruses. For studying this phenomenon A. Diaz‐Pendon et al. 2003. 2008). A major disadvantage.. thaliana is usually replaced by Nicotiana sp. as a model organism.. 2004). Introduction action deficient (mad) class III‐mutants. 2006. 2007). The initiation of silencing is mediated by the overexpression of exogenous RNA which is transformed into dsRNA by the action of RNA‐dependent RNA poly‐ merases [(RDRs) (Wassenegger & Krczal... 2006). Abundant transcription of short interspaced elements (SINE) RNA can compete for the essential factor HYL1 (Pouch‐Pelissier et al. Moreover single stranded miRNAs are also subject to degradation by members of the SMALL RNA DEGRADING NUCLEASE (SDN) family of exonucleases (Ramachandran & Chen.. Players of the miRNA path‐ way are frequently itself targets of miRNA regulation. is the missing genomic information of Nicotiana sp... the host needs a mechanism transmitting the initial immunity of RNA silenc‐ ing to other leaves. Qu et al. 2008) 46 . The extent of miRNA expression is regulated on different levels: MIR genes have their own promoters (Xie et al. suggesting a feed‐back regula‐ tion (Xie et al. it probably also requires the function of AGO1 as well as a microtubule‐severing enzyme KATANIN (KTN) and the P‐body component VARICOSE (VCS) which is required for mRNA decapping (Brodersen et al. 2008).1. 2006)]. Vaucheret et al.. 2008). High DCL3 levels compete with DCL1 for miRNA processing. RDR6 and RDR1 are the major players in antiviral si‐ lencing mechanisms (Schwach et al. 2007. resulting in 24‐nt products which are predominantly sorted into AGO4 and therefore not available for the RISC (Vazquez et al... Luo & Chen. 2008).
2001) and SUPPRESSOR OF GENE SILENCING 3 (SGS3)...10: Kalantidis et al. After initiation of silencing in a cell the signal is trans‐ 47 . 2008 Examples of silencing spread of a GFP transgene: (A) not silenced (B) spontaneous short‐range local silencing (C) induced short‐range local silencing (D) fully silenced (E) systemic silencing (F) extensive local spread (left) and systemic silencing (right). 2003.. 2005).. Introduction Figure 1. 2010) can influence the efficiency of silencing onset.. 2005... short‐range local spread (SLSS). The short‐range local silencing spread is not limited to exogenous RNA and can also affect endogenous sequences. Endogenous sequences cannot serve as substrates for RDRs (Himber et al. Schwach et al. 2009). a putative RNA heli‐ case (Dalmay et al. Exogenous factors such as temperature (Szittya et al. Silencing of DNA viruses and the RNA Tobacco rattle virus (TRV) additionally requires RDR2 (Donaire et al. Bleys et al. a coiled‐coiled domain protein (Mourrain et al.. The spreading of RNA silencing can be differentiated into three different stages.1. 2003) and light (Kotakis et al.. together with the co‐factors SILENCING DEFICIENT 3 (SDE3).. Kumakura et al. 2000. 2008). extensive local spread (ELSS) and systemic silencing.
2006). 2003.. Tournier et al... Schwach et al. Smith et al. 2006.. Nakazawa et al. 2005). AGO1. It is unclear which factors define the onset of extensive local spread. 1998)..1. It is accompanied by amplification of the initial signal as a result of an RDR6‐dependent mechanism (Himber et al. 2006) with a mobility comparable to soluble pro‐ teins of 27‐54 kDa (Kobayashi & Zambryski. Several other proteins of the various RNA silencing mecha‐ nisms have been shown to be a prerequisite for the short‐range spread including HEN1.. The signal transmitting the silencing spread of viral sequences is believed to be an RNA mole‐ cule (Jorgensen et al. 2003. Its action might be executed by a virus‐specific siRNA‐RISC (Lakatos et al.. Yang et al.. Several factors have been shown to be indispensable for the short‐range spread: loss of DCL4 abolishes the mechanism suggesting an involvement of 21‐nt siRNAs which are the products of DCL4 action (Dunoyer et al. Extensive local spread of silencing is characterized by the fact that the signal exceeds the limit of 10–15 cells but does not spread to the whole plant. DRB4. Dunoyer et al. 2007. This multitude of in‐ volved proteins suggests an intensive cross‐talk of the distinct RNA silencing mechanisms. 2003). 2001).. Other required factors appear to be DCL4 and the putative RNA helicase SDE3 (Dalmay et al. The least understood mechanism is systemic spread which is probably accompanied by the transport of the silencing signal through the phloem (Voinnet & Baulcombe. 2005. but it probably relies on passive diffusion through the plasmodesmata (Voinnet et al. 2006.. Adenot et al. 2002. 2006).. RDR2 and its presumable inter‐ acting protein CLSY1 (Hiraguri et al. it was proposed that a certain threshold has to be exceeded to trigger a stronger reaction.. Introduction ferred to 10–15 cells surrounding the initial source of silencing without amplification (Himber et al.. 1998.. Kalantidis et al. Himber et al. 2006). The exact nature of the signal could not be discovered yet.. 2007. Fagard & Vaucheret.. It is restricted to sink tissues which receive the signal from the source tissues of the leaves which had initially been chal‐ lenged with the exogenous RNA (Kalantidis et al.. Mlotshwa et al.. but it has been proven not to be of the size of siRNAs 48 . the POL IV subunit NRPD1a. 2005).. 2007). 2007). 1997. 2000.
1..7 Viral strategies to suppress RNA silencing in plants Viruses also possess strategies to counteract the RNA silencing mechanisms used for their clearance from the plant genomes.. 2006. However.. 2007. Several other virus‐encoded proteins have been shown to possess viral RNA silencing sup‐ pressor activity with yet unknown mechanisms. 2005. Vogler et al... 2004). Shiboleth et al.. 2006. 2007. so far no homologues of this PHLOEM SMALL RNA‐BINDING PROTEIN 1 (PSRP1) have been identified in Arabidopsis thaliana or Nicotiana sp. 2005) and prevents RISC assembly (Merai et al. 2002. 2007) and the p23 protein which controls viral RNA accumulation (Sat‐ yanaryana et al. Yang et al. 1. 2001). 2006. 49 ... 2003). Pazhouhandeh et al. Yu et al.. 2007). 2007) which has also been shown for p21 (Yu et al. 2006. 2007). 2007). 2006). 2007). the translational enhancer P6 (Love et al. More than 35 VSR families could be found in all plant virus types. They encode for proteins acting as viral sup‐ pressors of silencing (VSRs) in a very diverse manner. It interferes with methylation (Ebhardt et al.. The tobamoviral protein p126 contains methyltransferase and helicase do‐ mains and is present in a complex with p183 (Komoda et al..2. Another protein necessary for binding and facilitating the movement of RNA molecules between cells has been discovered in cucurbits (Yoo et al. 2004).. 2008). 2002. The poleoviral P0 is also a member of class I VSRs and appears to mediate the degradation of AGO1 which abolishes intracellular RNA silencing processes (Pfeffer et al.. Csorba et al.. class II VSRs suppress the local silencing spread and class III VSRs are the largest class suppress‐ ing the systemic spreading of silencing (reviewed in Díaz‐Pendón & Ding.. It binds duplex siRNAs and interferes with methylation by HEN1 (Blevins et al. Introduction (Mallory et al. they can be classified into three different categories: class I VSRs suppress the local silencing of the viral RNAs... Baumberger et al.. Lu et al. such as the transcription factor AL2/AC2/C2 (Trinks et al. The class I suppressor potyviral helper component proteinase (HC‐Pro) is a multi‐ functional protein whose performance partly depends on its silencing suppression activity (Kasschau & Carrington... Bortolamiol et al.
. 2005).2.. Most viral suppressors of silencing belong to class III... 2005. Ectopic overexpression of rgs‐CAM mimics the symptoms of HC‐ Pro‐containing viruses (Anandalakshmi et al. Some viral coat proteins also exhibit VSR function. 2000.. Ye et al. The related p14 also binds dsRNA molecules (Havelda et al. The potexviral p25 is an RNA helicase that interferes with the plasmodesmata (Bayne et al. 2006). 2007). Later it has been shown that upon simultaneous overexpression it reduces the amounts of siRNAs (Sarmiento et al. The sequestering of siRNA duplexes may pre‐ vent RISC functionality (Silhavy et al.. 2004).. Vargason et al.1. 2003. 2002). Omarov et al. Laka‐ tos et al... Merai et al.. 2002).. 2004. it can selectively inhibit DCL4 and also suppress 22‐nt siRNAs which are DCL2 products (Merai et al... It blocks the production of RDR1‐dependent sec‐ ondary viRNAs (Cao et al. A first report was the Ca2+ sensor protein REGULATOR OF GENE SILENCING CALMODULIN‐LIKE (rgs‐CAM) which became upregulated upon HC‐ Pro expression. Introduction Many viral movement proteins have been shown to be members of class II VSRs. 1.. 2005. the carmoviral p38 protein is able to replace p19 (Qu & Morris. Yaegeshi et al... 2000). 2006). The cytoplasmic exonuclease XRN4 has been proposed as an endogenous suppressor of silencing since in the mu‐ 50 ... RNase L inhibitor 2 (RLI2) has been found to be upregulated when transgenic plants are subject to RNA interference (Braz et al. p50 inhibits systemic spread of si‐ lencing (Yaegeshi et al. while p25 targets downstream of dsRNA synthesis (Voinnet et al. Moissiard et al. 2007). 2004). 2007).. 2007). The tomoviral p19 binds short dsRNA molecules with high affinity. 2005.8 Repressing the repressors ‐ endogenous suppressors of RNA silencing The power of the small RNA molecules and the presence of VSRs which specifically sequester small RNAs suggest that there also exist endogenous suppressors of RNA silencing. 2003. The cucumoviral 2b pro‐ tein may have a dual role since it weakly suppresses intracellular silencing in addi‐ tion to its potent inhibition of RNA silencing spread which allows long‐distance vi‐ rus movement (Guo & Ding. Pantaleo et al. The tymoviral p69 appears to target plant silencing upstream of RDR‐ dependent dsRNA synthesis (Chen et al. 2007).. Bayne et al. 2002. 2003.
2009) 1. 2007). 2002) exhib‐ ited a similar phenotype to eri‐1 mutants... 2007). 2004). 2004). elegans the loss of the putative RNA‐directed RNA polymerase RRF‐3 lead to hypersensitivity to RNAi (Simmer et al. 2004). In the same screen the 3’. It partly degraded double‐stranded siRNAs with 2‐nt 3’‐overhangs in vitro. 2002).. Introduction tant background xrn4 RDR‐dependent silencing was increased (Gazzani et al. 2004). The mutants were viable and showed a weak phenotype except for sterility due to a defect in sperm function. 2008) and C. In a genetic screen eri‐1 null mutants were identified to possess enhanced sensitivity to dsRNAs throughout the whole organism. For miRNAs it has recently been shown that they are specifically downregulated by a family of exoribonucleases called SMALL RNA DEGRADING NUCELASE (SDN) in plants (Ramachandran & Chen. Both proteins were found in a screen for DCR‐1 interacting pro‐ 51 . elegans was the protein RRF‐3 which is similar to RNA‐dependent RNA polymerases RdRPs). Similar results have been obtained for the nuclear exonucleases XRN2 and XRN3 (Gy et al. In addition.. For none of the above described pro‐ teins a specific effect on siRNAs has been proven and secondary effects cannot be excluded. In C.5’‐bisphosphate nu‐ cleotidase/inositol polyphosphate 1‐phosphatase FIERY1 (FRY1) has been identified as a suppressor of virus‐ and transgene‐induced post‐transcriptional gene silencing (PTGS) (Gy et al. elegans (Chatterjee & Grosshans. The first identification of an endogenous inhibitor of silencing in C.1. 2001.2. overaccumulation of miRNA cleavage products could be detected (Souret et al. ERI‐1 is mainly localized in the cytoplasm of developing somatic gonads and in a subset of neurons... In addition eri‐1 mutants accumulated more siRNAs after ingestion of long dsRNAs or injection of siRNAs (Kennedy et al.9 ERI‐1 is an example for an endogenous suppressor of RNA silencing In Caenorhabditis elegans the neuronal cells are refractory to RNA interference.. It is an evolutionary conserved protein with nucleic acid binding properties (con‐ ferred by a SAP/SAF‐box domain) and a DEDDh‐like 3’‐5’ exonuclease domain.(2’). Simmer et al. including the enhanced RNAi phenotype (Timmons.. rrf‐3 mutants (Sijen et al.
. It can remove the 2nt overhangs and the first nucleotide of the double‐stranded region of the siRNAs in vitro (Yang et al. Asp298‐ and Met235 are indispensible for the enzymatic activity of 3’hExo. Deletion of the SAP domain abolished binding of 3’hExo to the stem‐loop RNA but the residual exonuclease exhibited enzymatic activity. It binds the 3’‐terminal ACCCA of the stem‐loop and degrades it. unless the histone mRNA is protected by the stem‐loop binding protein SLBP (Dominski et al.. They iden‐ tified a protein binding the highly conserved stem‐loop structure of metazoan his‐ tone mRNAs. Introduction teins.. 3’hExo contains a SAP (Kipp et al. Asp234. 2005). 3’hExo requires a terminal hydroxyl group and cannot process RNAs terminating with a phosphate group (Dominski et al. 2006). 2004).6 Å in the presence of Mg2+. The structure is similar to DnaQ‐like 3’‐5’ exonucleases which usually bind to DNA and produce hydrolytic products releasing a nucleotide 5’‐monophosphate and leaving a 3’‐hydroxyl on the penultimate nucleo‐ tide (Viswanathan & Lovett. The crystallographic structure of the exonuclease domain of 3’hExo bound to rAMP. A homologue of ERI‐1 had been described earlier by Dominski et al.. The conserved stem‐loop sequence of histone mRNAs is necessary for its selec‐ tive degradation and confers the same reaction when introduced to other mRNAs at their 3’ends. 2003. The 3’hExo‐GFP protein predominately accumulated in the cytoplasm and the nucleoli. 2003). 1999).. Mutation of the latter amino acid leads to global structural changes unable to bind the stem‐loop (Yang et al.. Only the long isoform of ERL‐1 (ERI‐1b) could be detected in DCR‐1 immunoprecipitates suggesting distinct molecular functions of the two iso‐ forms (Duchaine et al.1. It is composed of a six‐stranded. 2005). The re‐ placement of Arg105 in the SAP domain eliminated binding to the stem‐loop.. a reaction product of the enzyme. 2006). 2000) and a 3’‐5’ exonuclease do‐ main. The active site is comprised of the conserved 52 . has been resolved at a resolution of 1. They also showed an enhanced RNAi phenotype and promoted the DCR‐ 1/ERI‐1 interaction. In addition two newly identified genes eri‐3 and eri‐5 were interacting with DCR‐1. twisted β‐sheet which is bracketed by nine α‐helices (Cheng & Patel.
12). catalysis or product release.1. Acinus and PIAS) DNA/RNA‐binding domain positions the 3’‐overhangs within the active site (Cheng & Patel. It has a broad substrate specificity and de‐ grades in vitro single‐stranded and double‐stranded DNA and RNA with the re‐ quirement of a minimal 2‐5 nt 3’‐flank. The homologue of 3’hExo in Drosophila melanogaster is named Snipper (Snp). Introduction acidic DEDD motif which binds two magnesium ions. Snp is a more efficient nuclease than 3’hExo toward histone stem‐loop RNAs since it can also cleave the double‐ stranded stem portion. Snp shares 31 % sequence identity with ERI‐1 and has a characteristic DEDDh motif (compare Figure 1. 2004 The positioning of the rAMP substrate in the 3’hExo reaction center requires two Magnesium ions and is conferred by two hydrogen bonds.11: Cheng & Patel. 3’hExo and ERI‐1 share 38 % sequence identity and 60 % sequence similarity. It does not require a 2’‐OH for substrate rec‐ ognition. The exonuclease domain appears to lack a binding pocket accommodating RNAs longer than dinucleotides. Figure 1. a highly active and promiscuous 3’‐5’ exonuclease. 2004). These two together with the conserved Histidine are in direct contact with the monophosphate of rAMP. One reason may be the absence of the SAP domain which 53 . Probably the SAP (SAF‐box.
suggesting that in qip KOs the siRNAs had already been pre‐processed. 2006). it also does not play a major role in the clearance of apoptotic DNA in Drosophila (Kupsco et al. In QIP KOs siRNA levels were significantly higher than in wildtype. In fission yeast heterochromatin assembly requires the RNAi machinery and is initi‐ ated by siRNAs. its components are QDE‐2. dsDNA or ssDNA. pombe orthologue. an Argonaute protein associated with siRNAs and two Dicer proteins. It is required for efficient processing of siRNA duplexes.1. They are derived from heterochromatic regions and processed by the RNA‐induced transcriptional silencing (RITS) complex which contains Ago1. It degrades dsRNA with 2‐nt overhangs and the RNA moiety of RNA‐DNA hybrids. The hypothetical protein QIP co‐purified with QDE‐ 2 and contains a 3’‐5’ exonuclease domain belonging to the DEDDh superfamily. the single S. elegans ERI‐1. Loss of Eri1 which predominately localizes to the cytoplasm did not affect normal cellular growth but the overexpression of Eri1 caused a severe growth defect. QIP probably functions in the RISC activa‐ tion process by removing the nicked passenger strand from the siRNA duplexes (Maiti et al. 54 . Introduction might bind the stem in the other homologues and thereby protects it from degrada‐ tion. 2007).. Homozygous Snp mutants showed no increased RNAi function. Gene silencing was impaired in the absence of QIP. It contains conserved SAP and DEDDh exonuclease domains and shows more than 30 % identity to C. Snp can cleave DNA substrates and is also able to degrade the stem portion of the DNA hairpin. causes an increase in siRNAs associated with the RITS complex and enhances heterochromatic silencing. The minimum length of the 3’‐flank for association in mobility shift assays was found to be at least two nucleotides. unlike siRNA duplexes from qde KOs. but acting down‐ stream of QDE‐2. siRNA duplexes from qip KOs were less stable and single‐stranded at 57 °C.. In an electrophoretic mobility shift assay the SAP‐ domain efficiently bound dsRNA and RNA‐DNA hybrids but not ssRNA. In Neurospora crassa RNAi is essential for the dsRNA‐ or transgene (quelling)‐induced gene silencing. Deletion of Eri1. suggesting that QIP is essential for functional RNAi. Chp1 and Tas3.
Growth defects were also observed for cells cultured in vitro. Point mutations showed that the catalytically inactive D130 and E132 55 . Both ERI‐1 isoforms rescued the 5. Suppression of the mouse orthologue of ERI‐1 increased the effect of RNAi. suggesting a common ancestor for this function in animals and fungi. It is present in the cytoplasm. maybe leading to the observable rebound after the initial RNAi‐induced target suppression. 2006). In vivo substrates of ERI‐1 in Caenorhabditis elegans and Schizosaccharomyces pombe have long been poorly understood until the discovery that 5.8S rRNA in eri‐1 null mutant worms is longer than in wildtype. Introduction The amount of centromeric siRNAs was considerably greater than in wildtype cells (Iida et al. nucleus and slightly enriched in the nucleolus.. 2008). but only ERI‐1b was functional in RNAi rescue while it also failed to rescue the rRNA processing in vitro.8S rRNA were variable with 1‐ or 2‐nt 3’‐extensions. Newly generated siRNAs can also recruit heterochromatin proteins and initiate de novo silencing in trans. mouse ERI1 and ADAR1 (adenosine deaminases acting on RNA which convert adenosine into inosine) tran‐ script levels are increased. with maxima in spleen. In mice ERI1 is ubiquitously expressed. pombe eri‐1 KOs where the 5. but this in trans silencing is strongly inhibited by Eri1 (Bühler et al. elegans ERI‐1 (Gabel & Ruvkun. The birth weight of Eri1 KO mice is reduced and this remained significant in the 10 % surviv‐ ing adult mice. reminiscent of the histone mRNA stem‐loop and siRNA structures. After introduction of high amounts of exogenous siRNAs. Mutations in H317 and D321 completely disrupted the function of C. ERI1 was found to bind independently to each ribosome subunit (40S and 60S). thymus and testis.8S rRNA was able to co‐immunoprecipitate with endogenous ERI1. 2005). In Eri1 KO mice the 3’‐ends of 5.8S rRNA length in vivo. Most rRNA processing occurs in the nucleolus. At least one additional nucleotide at the 3’‐ end could be detected in all mutant worms. In the mature ri‐ bosome the 3’‐end of 5.8S rRNA had two to eight additional 3’ nucleotides. Under stringent lysis conditions only 5.1..8S is paired with the 5’‐end of the 25‐28S rRNA. The same was found for S. 2006). (Hong et al. a substantial fraction contained two to four.
laevis D. While the N‐ and C‐terminal re‐ gions vary .nlm. elegans 56 .10 ERI‐1‐LIKE 1. sapiens M. pombe Consensus C. rerio X. rerio X. The SAP and linker domains have supportive function in rRNA binding but are not crucial for 5. sapiens M. elegans H. The conserved DEDDH motif is indicated with asterisks (*).cgi. laevis D. The N. melanogaster S. Wildtype ERI1 was able to convert the abnormal 5. laevis D. BLAST search (http://blast. 1. musculus D. It codes for a 337 amino acid protein with 50 % similarity to the C. rerio X. but efficient processing involves interaction with other features of the ribosome (Ansel et al. elegans H. musculus D. laevis D. elegans H. melanogaster S. C. pombe Consensus (1) (1) (1) (1) (1) (1) (1) (1) (99) (83) (79) (69) (80) (20) (27) (101) (196) (176) (172) (164) (175) (113) (92) (201) (277) (256) (252) (244) (255) (205) (192) (301) (376) (331) (327) (319) (330) (274) (262) (401) 1 100 MSADEPSPEDEKYLESLRDLLKISQEFDASNAKQNDEPEKTAVEVESAETRTDESEKSIDIPREQQLLPSERVEPLKSMVEPEYVKKVIR--QMDTMTAE -------MEDPQSKEPAGEAVALALLESPRPEGGEEPPR--PSPEETQQCKFDGQET-----KGSKFITS----SASDFSDPVYKEIAITNGCINRMSKE -------MEDERGRE---RGGDAAQQKTPRPECEESRP---LSVEKKQRCRLDGKET-----DGSKFISS----NGSDFSDPVYKEIAMTNGCINRMSKE -------METKEKSR------------KPPNKTPQSEG-----DQEDQPCPDTSCEK-----NEDQEPSSP---KQGEFSDPVYKEIALANGAINRMNRE -------MEEQKENRP-LDTEDSVVEEDLCKKLSRNLD----LVGVKQRCRFDGQED-----NGTSTVSS----NTSDFSDPVYKEIAIANGCVNRMTKD ---------------------------------------------------------------------------------MALIKLARQLGLIDTIYVD --------------------------------------------------------------------------MESPVQILVWPFPCDEMNQKTPSTVE MED Q CR D E ISS SDFSDPVYKEIAI NG INRMTKE 101 * * 200 QLKQALMKIKVSTGGNKKTLRKRVAQYYRKENALLNRKMEPNADKTARFFDYLIAIDFECTCVEIIY---DYPHEIIELPAVLIDVREMKIISEFRTYVR ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESNFADS---YYDYICIIDFEATCEEGNPP--EFVHEIIEFPVVLLNTHTLEIEDTFQQYVR ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESSAGDS---YYDYICIIDFEATCEEGNPA--EFLHEIIEFPVVLLNTHTLEIEDTFQQYVR ELRAKCTELKLDTRGVNDVLRKRLKSYYKKQKLMHSPAAEGNSDM---YFDYICVVDFEATCEENNPP--DYLHEIIEFPMVLIDTHTLEIVDSFQEYVK ELKAKLVEHKLDTRGVKDVLRKRLKNYYKKQKLTHALHKDSNTDC---YYDYICVIDFEATCEAGNSL--DYPHEIIEFPIVLLNTHTLEIEDVFQCYVR GARPDPNNDPEESFNEDEVTEANSVPAKSKK-------SRKSKRLAMQPYSYVIAVDFEATCWEKQAPPEWREAEIIEFPAVLVNLKTGKIEAEFHQYIL EIRIALQELGLSTNG-----------------------NK---------R-YLLIVDVEATCEEGCGF--SFENEIIELPCLLFDLIEKSIIDEFHSYVR ELRAKL E KLETRGVKDVLRKRLKNYYKKQ D YYDYICIIDFEATCEEGN DF HEIIEFPVVLLNTHTLEIED FQ YVR 201 * * 300 PVRNPKLSEFCMQFTKIAQETVDAAPYFREALQRLYTWMRKFN-------------------LGQKNSRFAFVTDGPHDMWKFMQFQCLLSNIRMPHMFR PEINTQLSDFCISLTGITQDQVDRADTFPQVLKKVIDWMKLKE-------------------LGTK-YKYSLLTDGSWDMSKFLNIQCQLSRLKYPPFAK PEVNAQLSEFCIGLTGITQDQVDRADAFPQVLKKVIEWMKSKE-------------------LGTK-YKYCILTDGSWDMSKFLSIQCRLSRLKHPAFAK PVLHPQLSEFCVKLTGITQEMVDEAKTFHQVLKRAISWLQEKE-------------------LGTK-YKYMFLTDGSWDMGKFLHTQCKLSRIRYPQFAR PEINPQLSEFCVNLTGITQDTVDKSDTFPNVLRSVVEWMREKE-------------------LGSK-YKYAILTDGSWDMSKFLNMQCRISRLKYPRFAK PFESPRLSAYCTELTGIQQKTVDSGMPLRTAIVMFNEWLRNEMRARNLTLPKMN--------KSNILGNCAFVTWTDWDFGICLAKECSRKGIRKPAYFN PSMNPTLSDYCKSLTGIQQCTVDKAPIFSDVLEELFIFLRKHSNILVPSVDEIEIIEPLKSVPRTQPKNWAWACDGPWDMASFLAKQFKYDKMPIPDWIK P INPQLSEFCI LTGITQDTVDKA F QVLKKVIEWMR KE LGTK YKYAFLTDGSWDMSKFL QCKLSRIKYP FAK 301 * 400 -SFINIKKTFKEKFNGLIKGNGKSGIENMLERLDLSFVGNKHSGLDDATNIAAIAIQMMKLKIELRINQKCSYKENQRSAARKDEERELEDAANVDLTSV -KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHCGLDDSKNIARIAVRMLQDGCELRINEKMHAGQ---------------------LMSV -KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHSGLDDSKNIARIAVRMLQDGCELRINEKILGGQ---------------------LMSV -KWINIRKSYGNFYKVPRT---QTKLICMLENLGMEYDGRPHCGLDDSRNIARIAIHMLKDGCQLRVNECLHSGE---------------------PRSV -KWINIRKSYGNFYKVPRT---QTKLTTMLEKLGMTYNGRLHSGLDDSKNIARIAAHMLQDGCELRVNERMHAGQ---------------------LMTV -QWIDVRAIYRSWYKYRPCN-----FTDALSHVGLAFEGKAHSGIDDAKNLGALMCKMVRDGALFSITKDLTPYQ------------------------GPFVDIRSFYKDVYRVPRT-----NINGMLEHWGLQFEGSEHRGIDDARNLSRIVKKMCSENVEFECNRWWMEYE------------------------KWINIRKSYGNFYKVPRT QTKLT MLEKLGM YDGR HSGLDDSKNIARIAIKML DGCELRINEKL GQ L SV 401 473 DISRRDFQLWMRRLPLKLSSVTRREFINEEYLDCDSCDDLTDDKVKHLHSCDIYEIFDEKTSASFTDSKCLIC SSSLPIEGTPPPQMPHFRK-----------------------------------------------------SSSLPVEGAPAPQMPHSRK-----------------------------------------------------PISAPIEGAPAPQPPKKRD-----------------------------------------------------SSSLPFEGAPVPQNPHLKN----------------------------------------------------------------QLNPRFVL--------------------------------------------------------K-NGWIPNRSYPPYFAS----------------------------------------------------S P EG P PQ P R Figure 1. sapiens M. sapiens M.8S rRNA. musculus D.a higher level of conservation is present in the exonuclease domain.gov/Blast. 1997) identified a single homo‐ logue in Arabidopsis thaliana encoded on the third chromosome at the locus At3g15140. pombe Consensus C. rerio X.ncbi. elegans H. rerio X.8S rRNA interaction.12: Alignment of published ERI‐1 homologues The sequences share a total identity of 7 % and a similarity of 54 %. laevis D.1. the plant homologue of ERI‐1 So far no plant homologue of ERI‐1 has been characterized. melanogaster S. Introduction mutants still bound 5. melanogaster S.nih. 2008). musculus D. The naturally occur‐ ring 5.8S‐28S duplex is sufficient.8S rRNA of purified ribosomes in vitro. pombe Consensus C. elegans H. whereas linker region mutants K107 and K108 showed impaired binding.. Altschul et al. crassa QIP protein has been excluded due to higher sequence variability.2. melanogaster S. musculus D. pombe Consensus C. sapiens M..
trichocarpa (83) Consensus (101) A. bicolor (16) Z. but it lacks the N‐terminal SAP domain like the Drosophila melanogaster ERI‐1 homologue Snipper (Kupsco et al. thaliana (82) N. thaliana N. In addition the sequence is not annotated completely at the N‐terminus since it lacks a 57 . 1997).. sativa S. benthamiana V. trichocarpa Consensus (182) (188) (164) (181) (106) (176) (183) (201) (280) (286) (262) (248) (171) (274) (283) (301) Figure 1. 2006). mays P. vinifera O.nlm. According to plant nomenclature conventions the protein is termed ERI‐1‐ LIKE 1 (ERL1). Introduction protein. vinifera (64) O. trichocarpa Consensus A. The gene is currently annotated to result in one transcript assembled of six exons. bicolor homologue which possesses a deletion inside the exonuclease domain and also a shortened N‐terminus. vinifera O. mays P. mays (76) P. An excep‐ tion is the S. which is indicated with asterisks (*). mays P. Thierry‐Mieg & Thierry‐Mieg. benthamiana V. sativa (81) S. bicolor Z. Oryza sativa probably contains several ERL1 homologues. bicolor Z. The protein contains the characteristic conserved DEDDh ex‐ onuclease domain where it also shares higher similarity with the C. sativa S.gov/IEB/Research/Acembly/. thaliana N. bicolor Z.ncbi. vinifera O. The Sorghum bicolor sequence contains a prominent deletion of 33 amino acids inside the exonuclease domain.1. elegans ERI‐1. Highly conserved plant homologues of ERL1 could be identified in the fully se‐ quenced plant genomes and the EST collections of many other plant species. benthamiana V.13: Alignment of ERL1 homologues in various plant species The plant homologues show a very high degree of conservation in the C‐terminal exonuclease do‐ main. benthamiana (88) V. all amino acids of the DEDDh motif are present. but a second splice variant lacking the N‐terminal region cannot be ex‐ cluded (http://www.nih. A. trichocarpa Consensus (1) (1) (1) (1) (1) (1) (1) (1) 1 100 MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCN--------------SSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSEN-----ARWRP ------SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRK-ISISASRSTTEESTSSLIQPTPSR------TRWKP ---------MAFYRVSPFRYGSLS-S---LIPYVS-------------SP-----SSLSPPVRT-FTLSASISTPHPSPPSLLTASPKAS-----DRWRP ---------MALARVSPPAFSSPFLIHSLLRPFSSPSSVLRPR---------VTRVPHHRGFAIAAALSQASPLPSADGDGAVMEAPPRPS--SRRPWKP ---------------------------------------------------------------------------SAASSATVRASGSVG------------------MALARVSPSSLANLIPPLLQSFFRPFSSDFPIR-------------NSRRRSSPVAAAFSLTSQSAHAAREGLVMEAPRPSS---RYPWKP ----MSFPRIPLSRVPSYLHNSNN--CFHLLHPPFIPVSKTP------------SLPTYQTARTYTDFNSQTQTQPPLSLPSLIPSPPVNNPNATHRWKP MALARVSPF LI S SASS T AS VM SP RWKP 101 * * 200 MCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKY TCLYFTQGKCTKMDDPMHIDKFNHNCSLELMQNAAGLKNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGKY MCLYYTQGKCTKMDDPTHLETFNHNCSRELQVNAANFQHLQSQHLDFFLVLDLEGKIEILEFPVLMINAKTMDVVDLFHRFVRPSEMSEQRINEYIEGKY TCLYYTQGKCTMLNDTLHLEKFNHNLPTDLPVNYSAADKVKSQKLDYFLVLDLEGKVEILEFPVVMIDAQSMEFVDSFHRFVHPTAMSEQRIREYIEGKY ----------CHMNDAMHLEKFGHNLKMDLPVNASATDKFKPQKLEYFLILDLEGRVEILEFPVVMIDAQSTEFIDSFHRFVRPTAMSEQRTTEYIEGKY TCLYYTQGKCTMMNDAMHLEKFSHNLKMDLPVNASATDKSKPQKLEYLLILDLEGRVEILEFPVMMIDAQNREFVDSFHRFVRPTAMSEQRTTEYIEGKY MCLYHTHGKCTKIDDPVHVERFNHDCSRDFQVSAADFERKRPQDFDFFLVFDLEGKVEILEFPVLIIDAKTMGVVDLFHRFVRPTAMSEERVNEYIYNKY CLYYTQGKCTKMDDPMHLEKFNHNCS DL VNAAA DK K Q LDYFLVLDLEGKVEILEFPVLMIDAKTMEVVD FHRFVRPTAMSEQRINEYIEGKY 201 * * 300 GELGVDRVWHDTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWINLKDVYLNFYG--REARGMVSMM GKLGVDRVWHDTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQCKVAGTKLPPYFMEWINLKDVFLNFYK--RRAKGMLSMM GKLGVDRVWHDTSIPFKEVIQQFEAWLTQHHLWTKEMGGRLDQAAFVTCGNWDLKTKVPQQCKVSKMKLPPYFMEWINLKDVYLNFYK--RRATGMMTMM GKFGVDRVWHDTAIPFMEVLQEFEDWIEHHKFWKKEQGGALNSAAFITCGNWDLKTKVPEQCRVSKIKLPSYFMEWINLKDIYLNFYN--RRATGMMTMM GKFGVDRVWHDTAVPFKEVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKT-----------------------------------KATGMMTMM GKFGVDRVWHDTATPFKQVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKVPEQCKVSKINLPTYFMEWINLKDIYLNFYN--RRATGMMTMM GKFGVDRVWHDTALPFNEVLQQFESWLTQHNLWEKTRGGRLNRAAFVTCGNWDVKTQVPHQCSVSKLKLPPYFMEWINLKDVYQNFYNPRNEARGMRTMM GKFGVDRVWHDTAIPFKEVLQEFE WLGNHNLWKKE GG LNRAAFVTCGNWDLKTKVP QC VS I LPPYFMEWINLKDVYLNFY RRA GMMTMM 301 * * 358 RQCGIKLMGSHHLGIDDTKNITRVVQRMLSEGAVLKLTARRSKSNMRNVEFLFKNRIK RELQMPLLGSHHLGIDDAKNIARVLQHMLSDGALVQITARRNPHSPEKVEYLFEDRIV KELQIPLLGSHHLGIDDTKNIARVLQRMLADGALLQITARRNADSPENVEFLFKNRIR RELQMPIVGSHHLGIDDAKNIARVVQRMLADGAVMQITAKRQS-ATGDVKFLFKNRIR RELQLPIIGNHHLGIDDSKNIARVVQRMIADGAVIEITAKRQ-STTGNVKFLFKDRIR RELQLPIVGNHHLGIDDSKNIARVVQRMLADGAVIQITAKRQ-SATGDVRFLFKDRIR SQLKIPMVGSHHLGLDDTKNIARVLLRMLADGAVLPITARRKPESPGSVNFLYKNRIRELQIPLLGSHHLGIDDTKNIARVLQRMLADGAVL ITARRN S V FLFK RIR A. thaliana N. sativa S.
none = none of the above iPSORT BaCelLo (Bannai et (Pierleoni al. 29 % predict a mitochondrial localization. 2007).utoronto. in the reproductive organs pollen and stigmata as well as in imbibed seeds. mays S.. vinifera N. bicolor V.. thaliana P. trichocarpa and M. whereas in Populus it is detected in the seedlings (compare Figure 1. nucl = nuclear. the absolute ex‐ pression levels are higher than in Arabidopsis. truncatula. In Medicago the highest expression is also observed in the vegetative tis‐ sue. 2006) mito chloro mito mito mito mito mito chloro chloro chloro chloro nucl chloro chloro PCLR (Schein et al. The gene is found to be ubiquitously transcribed at very low levels (for comparison ALPHA‐ TUBULIN is expressed at 40 times higher values). Caption: mito = mitochondrial. Table 1.bar. Expression data is also available for P. Winter et al. but comparable in relation to ALPHA‐ TUBULIN. 2002) et al.14 & Figure 1. trichocarpa O.15)..1.14). chloro = chloroplastic.. 2004) chloro none mito chloro none mito mito WoLF PSORT (Horton et al. The highest ERL1 expression could be detected in the shoot apex.1).. The same is true for the poplar sequence but an upstream start codon could be identified possibly leading to the full length protein (compare Figure 1. Subcellular localization prediction algorithms revealed a potential chloroplast signal peptide of the individual plant ERL1 homologues. An exception is the Sorghum se‐ quence but it cannot be excluded to be targeted to the chloroplast as well because of its incomplete annotation (compare Table 1.ca/. 2001) chloro chloro chloro chloro none chloro chloro Predotar (Small et al.1: Predicted localization of plant ERL1 homologues Five prediction algorithms calculate a chloroplastic localization of plant ERL1 homologues in 60 % of all cases. benthamiana ERL1 expression levels have been obtained from publicly available microarray data of Arabidopsis thaliana (http://www. Introduction Methionine. 11 % predict other or no subcellular localization. 2007) chloro chloro chloro chloro chloro chloro mito A. In fully developed tissues the expression drops by a factor of ten (compare Figure 1. 58 ..13). sativa Z.
Introduction Figure 1. 59 . 2007 The developmental map of A. In P.1. trichocarpa ERL1 is mainly transcribed in etiolated and light‐grown seedlings..14: Winter et al. thaliana shows increased ERL‐1 expression in the shoot apex and im‐ bibed seeds.
ovaries and during early pollen development. 2007 A more detailed expression map of A. 60 .1.15: Winter et al. Introduction Figure 1. thaliana shows ERL1‐expression maxima in the shoot apical meristem..
During evolution gene transfer from the chloro‐ plast into the nucleus took place.. These are imported into the chloroplasts through the TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope mem‐ brane of chloroplasts) complexes (Inaba. Evolutionary evidence suggests their origin in photosynthetic bacteria which had been endocytosed by the eukaryotic cell. They possess their own genome coding for approximately 120 genes and protein synthesis machinery. They can serve as storage plastids for starch (amyloplasts) or lipids (elaioplasts). As a rule of thumb the chloroplastic ribosomal and transfer RNAs and photosyn‐ thetic proteins are still encoded in the chloroplasts. are the chloroplasts. 2008). storage of energy‐ containing molecules to metabolic tasks such as synthesis of nucleotides.3 Chloroplasts Chloroplasts develop from proplastids which are small precursor plastids with a double membrane present in all immature plant cells. which is able to convert to normal chlorophyll in the case of light exposure. Another plastid type are chromoplasts which store the pigments of flower petals or fruits. All of them contain the same genomic information but they are able to differentiate into different plastid types. They contain inner membrane structures with a yellow chlorophyll precursor. however. unlike chloroplasts. 2010). certain amino acids and fatty acids (Alberts et al. although they depend on the supply with energy and pre‐ cursor metabolites by the plant cell (Bräutigam & Weber. Introduction 1. but it is regulated in a way that the 61 .. Etioplasts are developed when a plant is grown in the dark. but the larger enzyme complexes and ribosomal proteins usually contain also nuclear proteins (Alberts et al.1. They are inherited from the ancestor plants into the zygote. Plastids have various functions ranging from photosynthesis for energy supply. can also be found in epidermal plant cells. The metabolic functions can also be performed by proplastids. therefore most of their proteins are now encoded in the nucleus and imported by a special import complex. The replication of chloroplastic DNA is independent from the S‐phase of the cells. 2008). 2009). Leucoplasts are plastids which. The most prominent plastids.
2008). The electrons residing in the primary electron acceptor are further trans‐ ported via several molecules to photosystem I.1. 2008). moreover the latter have a function in protecting chlorophyll from excessive light energy. The most prominent is chlorophyll a consisting of a light absorbing porphyrin ring and a hydrophobic phytol group anchoring the molecule in the membranes. together with chlorophyll b and caroti‐ noids the wavelength which can be used for photosynthesis is broadened.. The resulting protons can be used for ATP‐synthesis and in addition oxygen is re‐ leased. The division of chloroplasts is controlled by ARC (ACCUMULATION AND REPLICATION OF CHLOROPLASTS) genes (Marrison et al. To return back to its initial state it gets reduced by electrons originating from water which had been photolyzed.. the membranes themselves are organized in stacks which are called grana and contain the photosyn‐ thetic systems of the chloroplasts (Alberts et al. 1999). They form an internal compartment called the thylakoid space. The pri‐ 62 .1 Photosynthesis Photosynthesis consists of two different reaction types: the light reactions. In the development from proplastids their inner membrane folds and gets constricted leading to the thylakoid membranes inside the chloroplasts (Vothknecht & Westhoff. where the absorbed light energy has also resulted in oxidized chlorophyll a molecules which now get reduced. Both are comprised of a light‐harvesting complex formed by several hundred pigment molecules which chan‐ nel the absorbed light energy to a reaction center. where carbon dioxide is fixed into sugar molecules..3. Introduction number of chloroplasts has doubled before cell division to ensure a constant amount of plastids in the daughter cells (Alberts et al. In photosystem II the chlorophyll a molecules of the reaction center transfer their high energy electrons onto a primary electron acceptor. 2001). It has absorption maxima at 430 nm and 662 nm. The thylakoid membranes contain different pigments which are able to absorb light. where sunlight is transformed into electric energy and the consecutive synthesis reactions of the Calvin cycle. Photosynthesis is performed in two distinct photosystems. 1.
In the first phase of the subsequent Calvin cycle carbon dioxide is attached onto ribu‐ lose‐1. In a last step the carbon dioxide acceptor is regenerated (Campbell. 1996).5‐bisphosphate carboxylase. The reac‐ tion products function in the subsequent Calvin cycle which leads to the synthesis of sucrose. This reaction mechanism is called non‐cyclic electron transport resulting in equimo‐ lar amounts of ATP and NADPH. The energy gets transported via the cyto‐ chrome complex and Photosystem I to the NADP+ reductase for the production of NADPH.5‐bisphosphate by the enzyme ribulose‐1. The absorbed energy then leads to the chemiosmotic production of ATP. Figure 1. The in‐ stable product dissociates and gets further phosphorylated and reduced to glyceral‐ dehyde‐3‐phosphate which can be used for the production of sucrose and starch. 1996 Graphic representation of the photosynthetic reactions in the chloroplast. Alternatively photosystem I can also produce extra ATP by the cyclic electron trans‐ port where the primary electron receptor reduces the reaction center of the photosys‐ tem. Introduction mary electron acceptor of photosystem I leads to the reduction of NADP+ to NADPH. 63 .1.16: Campbell. Light energy is absorbed in photosystem II and leads to the production of ATP and O2.
1. 2010 Chloroplastic genes expression involves the transcription of a polycistronic precursor which gets processed at its 5’‐ and 3’‐ends.2 Specificities of chloroplastic RNAs The chloroplast genome of land plants encodes components required for chloroplast protein synthesis (rRNA. The chloroplastic rRNA genes are highly conserved between plant species and or‐ ganized in the ribosomal RNA (rrn) operon which is transcribed into a polycistronic molecule.3.. ribosomal proteins and RNA polymerase subunits) and for photosynthesis. tRNAs. 64 . Introduction 1. After intercistronic cleavage the resulting transcripts are edited by PPR proteins and spliced into functional RNAs. In land plants the transcript starts with the 16S rRNA. followed by two Figure 1.17: Stern et al.
Nishimura et al. 2000.. 23S rRNA. They 65 . Although there are reports for functions in mitochondria in other species. Lurin et al. The first processing of the primary precursor involves the excision of the tRNAs. Plants possess a large gene family with more than 450 members in Arabidopsis which encode for proteins with pentatricopeptide repeats (PPRs) It is a degenerate 35‐ amino‐acid motif which is often arranged in tandem of 2‐27 repeats per peptide (Small & Peeters. Unlike tetratricopeptide proteins from which their name was derived of.. Bisanz et al. 2004)..18 b). 1985... E+ and DYW stretch which is present in a large fraction of PPR proteins (Lurin et al.5S rRNA intermediate requires 3’‐maturation before the final processing into the monocistronic rRNAs takes place already in the ribosome. in plants they are mainly participating in chloroplast RNA metabolism in processes such as RNA trimming. 2005. 2010)... 2004)] and can be grouped into two subfamilies: classical PPR proteins with only the above described motifs and plant combinatorial and modular proteins (PCMPs) containing also short (31 amino acids) and long (35‐36 amino acids) motifs [(Lurin et al. Additional endonucleolytic cleavage generates the 16S and 5S rRNA. They are mostly targeted to organelles [(chloroplasts and mitochondria) (Small & Peeters.5S rRNA and finally 5S rRNA and another tRNA (Harris et al. 2004). Introduction tRNAs. pentatricopeptide proteins are able to bind RNA and DNA in a sequence‐specific manner (Saha et al.. PPR genes are usually short since most of them do not contain introns and they generally have a low expression rate (Lurin et al.. translation and editing (compare Figure 1. 2004). chloroplastic RNAs possess introns and pass through many RNA editing steps. 4..18 a)]. Bellaoui et al. Most of them are also influenced by heli‐ cal repeat proteins (reviewed in Stern et al. Also the other rRNA molecules need further endo‐ and exonucleolytic processing until the mature rRNAs are ready (Kössel et al. The resulting dicistronic 23S–4.. 2003. In contrast to the prokaryotic RNA metabolism... Another conserved feature is a C‐terminal E motif. 2003. 2007). 2004) (compare Figure 1.1. 2010). E and E+ or E. Bollenbach et al. 2000. Lurin et al. 1994). stabilization.
(B) Functions of PPR pro‐ teins in (1) Translation. (3) RNA splicing and (4) RNA stability (Schmitz‐Linneweber & Small. (2) RNA editing. 2007). 1. 2007). So far. 66 . therefore it should also be identi‐ fied in the course of this work. The in silico predicted chloroplastic localization of the Arabidopsis protein should be proven in vivo as well. only partial sequence in‐ formation was available for the Nicotiana homolog. (A) (B) Figure 1. no scientific publication describes this plant protein.. Introduction are involved in chloroplast translational control. The protein is further called ERI‐1‐LIKE 1. in vitro characterization of the Arabidopsis homologue is possi‐ ble and has been presented in chapter 1. ERL1.. In contrast. Due to the complete genomic sequence information. 2004). embryogenesis and organ develop‐ ment as well as the restoration of male fertility through modification or silencing of cytotoxic mitochondrial transcripts (Saha et al.4 Thesis objectives The initial aim of this work was the characterization of the plant homologue of the 3’‐ 5’ exonuclease ERI‐1 (enhanced RNAi) in the two model plants Arabidopsis thaliana and Nicotiana benthamiana.10.2. Rf‐homologous genes have been shown to generate siRNAs and might be under gene‐silencing regulation them‐ selves (Howell et al..18: PPR proteins (A) Domain organization of PPR and PCMP proteins (Lurin et al. Mitochondria‐encoded cyto‐ plasmic male sterility can be overcome by nuclear restorer (Rf) genes which are usu‐ ally PPR proteins (Schmitz‐Linneweber & Small.1. 2008). 2008).
Involvement of ERL1 in plant RNA silencing processes. Interestingly. the effect of ERL1 in plant RNA silencing processes should be investigated. the effect of ERI‐1 on plant ribosomal RNA mole‐ cules should have been examined in more detail. In vivo characterization of ERL1 in Nicotiana benthamiana.1. the chloroplast is be‐ lieved to be free of RNA silencing.8S rRNA should be explored in more details. Involvement of ERL1 in ribosomal RNA processing.8S rRNA had been identified as a new substrate of ERI‐1 and its homologues. Summarized the following question should be addressed: • • • • In vivo characterization of ERL1 in Arabidopsis thaliana. Hence. The effect of plant ERL1 on these and the homologous 5. 67 . but it contains four chloroplastic ribosomal RNA species. In the course of the work cytosolic 5. Introduction Since the nematode protein is suggested to suppress RNA silencing in Caenorhabditis elegans.
1. USA Shel Lab. Greece Owl B2 EasyCast. Bio‐Rad. USA SD20 Semi Dry Midi. Heraeus. Germany Kubota 5800. Germany Eppendorf 5810R. USA Brema Ice makers. Eppendorf. Heraeus.. Kabbalos. UK Mini Trans‐Blot® Electrophoretic Transfer Cell. Nikon. Nikon. 2. Herolab. Eppendorf. Belgium Coolpix 990. Kubota. Germany Biofuge Stratos. Japan JEM‐100C. UK UVT‐28 MP. alternative suppliers are possible in some cases. Hansatech Instruments. Germany Curix 60. Thermo Scientific. Germany Centrifuges Confocal microscope Developer Digital Camera Electron Microscope Fluorescence measurement Gel documentation system Hybridization bottles Hybridization oven Ice machine Eppendorf 5415D. USA Shel Lab Model 1004.1 Materials The used materials of this work are presented below together with their standard supplier. Cleaver Scientific. Italy 69 . Japan Coolpix 5600. Materials and Methods 2. JEOL Ltd. Germany Hybaid.2. Leica. AGFA. Japan Handy PEA. Thermo Scientific.1 Instruments Agarose gel electrophoresis Blotting Device A. Germany Biofuge 15R. Japan Leica TCS SP.
IKA® Werke. Qiagen. Germany Ρωμαίος. Germany Biocyt 180 NF X44201. USA DNA Engine PTC‐200 Gradient. Jouan GmbH. USA Spectrophotometer NanoDrop ND‐1000. Thermo Scientific. GE Healthcare. Gilson. USA Blak‐Ray B‐100AP/R. USA 70 .2. Perkin Elmer. USA Vortex‐Genie 2. Heraeus Instruments. UK Microcomputer Electrophoresis. Japan Local electronic supplier Mini‐Protean® 3. Nikon. Beckman. Germany Scintillation System LS 1701. UVP. USA Thermocycler Lambda 2. Bio‐Rad. Bio‐Rad. USA Laminar Flow Hood ESI Flufrance Ariane 18 UV. Scientific Industries. Germany qPCR machine Scintillation counter RG‐3000A Rotor‐Gene. Ger‐ many Magnetic stirrer/heater Microlitre pipettes Microscope (optical/fluorescent) Microwave oven PAA gel electrophoresis Power supply IKAMAG® RET. handheld Vortex mixer Stratalinker 1800. Germany UV crosslinker UV lamp. Germany Pipetman. Thermo Scientific. Bio‐Rad. Materials and Methods Incubator B‐5060. USA Eclipse E800. Greece PowerPac Basic. Germany Pharmacia ECPS 3000/150. Germany Forma Incubated Console Orbital Shakers. Agilent. Jouan GmbH. Renner GmbH.
Germany Sigma‐Aldrich. Germany Bristol‐Myers Squibb. Materials and Methods Water bath X‐ray cassettes Lauda A100. USA Perkin Elmer. USA Sigma‐Aldrich. USA Local pharmaceutical supplier Merck. Netherlands Merck. USA Perkin Elmer. Lauda Dr.S. Wobser GmbH & CO. R. Germany Sigma‐Aldrich. USA Merck. USA Merck. USA Lonza. Germany 5‐Bromo‐4‐chloro‐3‐indolyl‐β‐D‐galacto‐ Melford Laboratories. USA 2.B. England pyranoside (X‐Gal) [C14H15BrClNO6] Bromophenol blue [C19H9Br4O5SNa] Bovine serum albumine (BSA) Calcium chloride [CaCl2] Cefotaxime [C16H17N5O7S2] Cetyltrimethylammonium bromide (CTAB) [C19H42BrN] Chloroform [CHCl3] 71 Sigma‐Aldrich. Scientific Co. USA Sigma‐Aldrich. Germany . Switzerland Sigma‐Aldrich. USA Merck. KG.2. Germany Sigma‐Aldrich. Germany Merck. USA Merck. low‐melting [(C12H18O9) x] Ammonium nitrate [NH4NO3] Ammonium persulfate [(NH4)2S2O8] Ammonium thiocyanate [NH4SCN] Ampicillin [C16H19N3O4S] 6‐Benzylaminopurine (BAP) [C12H11N5] Boric acid [B(OH)3] Perkin Elmer.1. USA Duchefa.2 Chemicals [α32P]‐dATP [C10H16N5O12P3] [α32P]‐dCTP [C9H16N3NaO13P3] [γ‐32P]ATP [C10H16N5O13P3] Acetic acid [CH3COOH] Acetosyringone (ACS) [C10H12O4] Acrylamide [C3H5NO] Agar‐agar [(C12H18O9) x] Agarose [(C12H18O9) x] Agarose. Germany C.
fuming [HCl] 4‐(2‐hydroxyethyl)‐1‐piperazineethane‐ sulfonic acid (HEPES) [C8H18N2O4S] Kanamycin [C18H36N4O11] Magnesium chloride [MgCl2] Magnesium sulfate [MgSO4] Manganese chloride [MnCl2] β‐Mercaptoethanol [C2H6OS] Methanol [CH3OH] 72 Merck. absolute [C2H6O] Ethidium bromide (EtBr) [C21H20BrN3] Merck. USA Ethylenediaminetetraacetic acid (EDTA) Merck. Switzerland Merck. Germany Merck. Materials and Methods Coomassie Brillantblue G 250 [C47H48N3O7S2Na] Deoxyribonucleotide (dNTP) set Dimethylformamide (DMF) [C3H7NO] Dimethylsulfoxide (DMSO) [C2H6OS] Dithiothreitol (DTT) [C4H10O2S2] DNA oligos Ethanol. Germany MBI. USA Merck. USA Sigma‐Aldrich. Germany Merck. Germany Sigma‐Aldrich. Germany IMBB Microchemistry. Germany Merck. Germany Fluka. Germany [C10H16N2O8] Formaldehyde [HCOH] Formamide [CH3NO] Glucose [C6H12O6] Glycerol [C3H5(OH)3] Glycine [C2H5NO2] Guanidinium thiocyanate [C2H6N4S] Hydrochloric acid. Switzerland Fluka. USA Merck. Germany Merck. Germany Biomol GmbH. Germany . Germany Merck. Greece Metabion. Germany Biomol GmbH. Germany Sigma‐Aldrich. Lithuania Bioron. Germany Merck.2. Fermentas. Germany Sigma‐Aldrich. Germany Merck.
2. Materials and Methods
N,N’‐methylenebisacrylamid (BIS) [C7H10N2O2] 2‐(N‐Morpholino)ethanesulfonic acid (MES) [C4H8ONC2H4SO3H∙H2O] Merck, Germany Sigma‐Aldrich, USA
3‐(N‐Morpholino)propanesulfonic acid Merck, Germany (MOPS) [C7H15NO4S] Murashige & Skoog macroelements Murashige & Skoog microelements Murashige & Skoog vitamins α‐Naphthalene acetic acid (NAA) [C12H10O2] Phenol crystals [C6H5OH] Piperazine‐N,N′‐bis(2‐ethanesulfonic acid) (PIPES) [C8H18N2O6S2] Polysorbate 20 (Tween® 20) [C58H114O26] Polyvinylpyrrolidone (PVP‐40) [(C6H9NO)n] Potassium acetate [CH3COOK] Potassium chloride [KCl] Potassium hydroxide [KOH] Potassium nitrate [KNO3] Merck, Germany Merck, Germany Merck, Germany Sigma‐Aldrich, USA Sigma.Aldrich, USA Sigma‐Aldrich, USA Fluka, Schwitzerland Merck, Germany Duchefa, Netherlands Duchefa, Netherlands Duchefa, Netherlands Duchefa, Netherlands
Potassium phosphate, dibasic [K2HPO4] Merck, Germany Potassium phosphate, monobasic [KH2PO4] 2‐Propanol (isopropanol) [C3H8O] Rifampicin [C43H58N4O12] Silwet L‐77 Skimmed milk powder Sodium acetate [CH3COONa] Merck, Germany Merck, Germany Duchefa, Netherlands OSI Specialities, USA Local market Merck, Germany
2. Materials and Methods
Sodium carbonate [Na2CO3] Sodium chloride [NaCl] Sodium citrate [Na3C6H5O7] Sodium dodecyl sulfate (SDS) [C12H25NaO4S] Sodium hydroxide [NaOH] Sodium hypochlorite [NaClO] Sodium phosphate, dibasic [Na2HPO4] Sodium phosphate, monobasic [NaH2PO4] Sodium thiosulfate [Na2S2O3] Sorbitol [C6H14O6] Spectinomycin [C14H24N2O7] Sucrose [C12H22O11] Tetramethylethylenediamine (TEMED) [C6H16N2] Tris(hydroxymethyl)‐aminomethan (TRIS) [C4H11NO3] Triton® X‐100 [C14H22O(C2H4O)n] Tryptone Urea [(NH2)2CO] Water, nanopure Xylene cyanol FF [C25H27N2O6S2Na] Yeast extract Merck, Germany Sigma‐Aldrich, USA Merck, Germany Millipore Sigma‐Aldrich, USA Sigma‐Aldrich, USA Biomol GmbH, Germany Merck, Germany Sigma‐Aldrich, USA Sigma‐Aldrich, USA Sigma‐Aldrich, USA Merck, Germany Sigma‐Aldrich, USA Sigma‐Aldrich, USA Merck, Germany Merck, Germany Merck, Germany Merck, Germany Merck, Germany Merck, Germany
2.1.3 Consumables & kits
anti‐Rabbit AP‐conjugate Blotting paper Promega, USA Whatman 3MM, UK
Fermentas Maxima™ SYBR Green qPCR ThermoFischer Scientific, USA Master Mix (2X)
2. Materials and Methods
Gateway® LR Clonase® II Enzyme Mix, Invitrogen, USA HRP SuperSignal West Pico Chemilumi‐ ThermoFischer Scientific, USA nescent substrate Illustra MicroSpin™ S‐200 Spin Columns GE Healthcare, UK Laboratory film Membrane Pechiney ParafilmTM ‘M’, Pechiney, USA ProtranTM Nitrocellulose 0.22 μm, Whatman, UK NucleoBond® Xtra Midi Kit NucleoSpin® Extract II Pasteur glass pipettes Petri dishes ø 10 cm pGEM®‐T Easy Vector System Pipette tips Nylon 0.45 μm, Nytran® N, Whatman, UK Macherey‐Nagel, Germany Macherey‐Nagel, Germany Volac, Poulten & Graf Ltd., UK Sarstedt, Germany Promega, USA Sarstedt, Germany & Greiner Bio One, Germany Platinum® Taq DNA pol. High Fidelity Polypropylene tubes, 15/50 mL RadPrime DNA Labeling System Reaction tubes, 0.2/0.5/1.5/2 mL Gloves Invitrogen, USA Sarstedt, Germany Invitrogen, USA Sarstedt, Germany Digitil N powderfree, Hartmann, Ger‐ many Scalpel blades Syringe 1 mL Syringe filters TaKaRa 3’‐Full RACE Core Set TOPO® TA Cloning® Kit X‐ray films Fuji Super RX Paragon, UK HMD Healthcare Ltd., Horsham, UK Pall Corporation, USA TaKaRa, Japan Invitrogen, USA Fujifilm, Japan
2. Materials and Methods
30 % (w/v) acrylamide mix (29:1) 40 % (w/v) acrylamide mix (38:2) Binding Buffer Blocking buffer Church hybridization buffer Coomassie destaining solution Coomassie staining solution 40 % (v/v) methanol 10 % (v/v) acetic acid 50 % (v/v) water 0.1 % (w/v) Coomassie 15 % (v/v) ethanol 10 % (v/v) acetic acid 75 % (v/v) water 0.5 M phosphate buffer pH 7.2 1 % (w/v) BSA 1 mM EDTA 7 % (w/v) SDS 1x PBS 1 % (w/v) skimmed milk powder 0.05 % (v/v) Tween‐20 1x PBS 2 % (w/v) skimmed milk powder 0.05 % (v/v) Tween‐20 38 % (w/v) acrylamide 2 % (w/v) N, N’‐methylenebisacrylamide 29 % (w/v) acrylamide 1 % (w/v) N, N’‐methylenebisacrylamide
1 M cacodylate buffer 0.5 % (w/v) yeast extract 1 % (w/v) NaCl adjust pH to 7. Materials and Methods CTAB buffer 2x DNA loading dye 6x dNTP mix Fixing buffer High Salt Solution 2x LB 77 2 % (w/v) CTAB 100 mM Tris‐HCl pH 8.0 1. autoclave .6 60 mM EDTA 60 % (v/v) glycerol 0.8 M sodium citrate 1.4 with NaOH.2 M NaCl 1 % (w/v) tryptone 0.2.4 M NaCl 1 % (w/v) PVP‐40 (Mw = 40 000 g/mol) 10 mM Tris pH 7.0 20 mM EDTA pH 8.0‐7.03 % (w/v) bromophenol blue 10 mM dATP 10 mM dCTP 10 mM dGTP 10 mM dTTP 2 % (v/v) glutaraldehyde 2 % (w/v) paraformaldehyde 0.
7 with KOH. Materials and Methods LB‐agar LB Rif LB Rif‐agar MMA MS MS‐agar Laemmli Buffer 4x LDM Base MS 1x Murashige & Skoog vitamins 3 % (w/v) sucrose 78 LB 1.5 % (w/v) agar agar LB 120 μg/mL Rifampicin LB Rif 1.8 8 % (w/v) SDS 40 % (v/v) glycerol 20 % (v/v) β‐mercaptoethanol 0.5 % (w/v) agar agar MS 10 mM MES pH 5.7 200 μM acetosyringone 1x Murashige & Skoog macroelements 1x Murashige & Skoog microelements adjust pH to 5. autoclave MS 1 % (w/v) agar‐agar 250 mM Tris‐HCl pH 6.01 % (w/v) Bromophenolblue .2.
7 with KOH.8 mg/L BAP 0.1 M sodium acetate 10 mM EDTA adjust pH to 7. temper to 30 °C prior to use 0.0 79 0.8 % (w/v) agar adjust pH to 5.2.7 with KOH.7 with KOH. autoclave LDM Base 0. autoclave add the antibiotics after cooling to 55 °C LDM Base 250 μg/mL Cefotaxime 200 μg/μL Kanamycin adjust pH to 5.4 M MOPS 0. pH 7. low‐melting autoclave.0 with NaOH pellets .1 mg/L NAA adjust pH to 5. Materials and Methods LDM I LDM II LDM III Low‐melting agarose 1 % (w/v) MOPS 10x.7 with KOH. autoclave add the antibiotics after cooling to 55 °C 1 % (w/v) agarose. autoclave LDM I 250 μg/mL Cefotaxime 200 μg/μL Kanamycin adjust pH to 5.
4. Materials and Methods MOPS running buffer PAGE northern gel PBS 10x Protein extraction buffer Protein running buffer 5x Protoplast extraction buffer MS 0.2.4 M NaCl 27 mM KCl 100 mM Na2HPO4 18 mM KH2PO4 adjust pH to 7.4 M sucrose 2 mM CaCl2 80 1x MOPS 0.0375 % (w/v) APS 0.9 150 mM NaCl 0.5 % (w/v) SDS .2 % (w/v) PMSF 125 mM Tris 96 mM glycine 0.74 % (v/v) formaldehyde 8‐15 % (w/v) acrylamide mix (38:2) 8 M urea 1x TBE 0.075 (v/v) TEMED 1.5 % (v/v) Tween‐20 5 % (v/v) glycerol 1 % (v/v) protease inhibitors 0. autoclave 20 mM HEPES pH 7.
0 81 25 mM MES pH 5.4 M ammonium thiocyanate 0.7 1 % (w/v) cellulose 0.1 % (w/v) APS 38 % (v/v) phenol 0.0 5 % (v/v) glycerol 4x MOPS 31 % (v/v) formamide 0.03 % (w/v) bromophenolblue .1 M sodium acetate pH 5.01 % (v/v) TEMED 0. Materials and Methods RadPrime Buffer 2.27 % (v/v) formaldehyde 4 mM EDTA pH 8.2.5 % (w/v) macerozyme 125 mM Tris‐HCl pH 6.5 mM MgCl2 25 mM β‐mercaptoethanol 375 mM Tris‐HCl pH 8.8 12.0 20 % (v/v) glycerol 0.8 M guanidine thiocyanate 0.1 % (w/v) SDS 0.5x Resolving gel RNA extraction buffer RNA loading dye 5x Sequencing dye 2x 98 % (v/v) formamide 10 mM EDTA pH 8.8 10–15 % (w/v) acrylamide mix (29:1) 0.
03 % (w/v) xylene cyanol FF 2 % (w/v) tryptone 0.5 .5 % (w/v) yeast extract 10 mM NaCl 2.5 M NaCl 0.03 % (w/v) bromophenol blue 0.0 0.2 M HCl 1.5 M NaOH 0. Materials and Methods SOB medium Solution I Solution II Solution III Southern Denaturation Southern Depurination Southern Neutralization 82 0.2 M NaOH 1 % (w/v) SDS 3 M KCH3COO 11 % (v/v) glacial acetic acid 1.5 mM KCl 1 mM MgCl2 adjust pH to 7.0 and autoclave 50 mM glucose 10 mM EDTA 25 mM Tris pH 8.5 M NaCl 1 M Tris pH 7.2.
8 4 % (w/v) acrylamide mix (29:1) 0.05 % (v/v) Tween‐20 5 % (w/v) sucrose 0.2. sterilized by filtration .5 % (w/v) sodium hypochlorite 0.03 % (v/v) Silwet L77 2 M Tris 5.3 M sodium citrate adjust pH 7.71 % (v/v) glacial acetic acid 50 mM EDTA pH 8. Materials and Methods SSC 20x Stacking gel Sterilization solution Sucrose dipping solution TAE 50x TB TBE 10x 0.0 83 3 M NaCl 0.7 15 mM CaCl2 250 mM KCL 55 mM MnCl2 freshly prepared.0 10 mM PIPES pH 6.9 M boric acid 20 mM EDTA pH 8.1 % (w/v) SDS 0.1 % (w/v) APS 0.9 M Tris 0.01 % (v/v) TEMED 0.0 with HCl and autoclave 125 mM Tris‐HCl pH 6.
M HindIII. XbaI. pH 8.0 Transfer Buffer 1x Wash I Wash II Wash III Washing Buffer 1x PBS 0. UK 84 .. USA Restriction endonucleases: BamHI. PstI.5x SSC 0. NotI.5.5 Others 2.0 1 mM EDTA pH 8.1 Enzymes Cellulase DNase I Macerozyme Protease inhibitor cocktails (P9599) Proteinase K Duchefa.05 % (w/v) Tween‐20 0. EcoRI. KpnI.1 % (w/v) SDS 2x SSC 0. Switzerland Duchefa. XHoI Reverse transcriptase USA HT Biotechnology Ltd. USA Invitrogen. Netherlands Roche. inotech.1.2. Materials and Methods TE 1x.1. Greece & New England Biolabs. Netherlands Sigma‐Aldrich.1 % (w/v) SDS 1x SSC 0.0 2.1 % (w/v) SDS 25 mM Tris 150 mM glycine 20 % (v/v) methanol 10 mM Tris pH 8.
5. Materials and Methods RNase A RNase H RNase inhibitor (RNasin) Taq DNA polymerase T4 DNA Ligase T4 Polynucleotide kinase (PNK) T4 RNA Ligase Klenow Fragment 2. USA New England Biolabs.2 Size markers 1 kb DNA Ladder 100 bp DNA Ladder Lambda DNA.2.5. 85 .1.. USA HT Biotechnology Ltd. Greece 2. broad range (161‐0318) 2.2 Methods The following methods were used to obtain the results presented in chapter 3.1 Standard molecular biology methods 2. Greece Stratagene. USA Minotech. USA Invitrogen.1. USA New England Biolabs. Agilent. USA New England Biolabs. USA New England Biolabs. if necessary. Germany Qiagen. USA Invitrogen. Greece Promega.2. PstI‐digested Prestained SDS‐PAGE standard.3 Bacterial strains Agrobacterium tumefaciens C58C1 Escherichia coli DH5α® IMBB.1 Cultivation Unless stated otherwise bacteria were grown in LB medium. Germany Invitrogen. UK Minotech. Heraklion. 2. antibiotics were added as selective markers to a final concentration of 100 μg/ mL. Greece Bio‐Rad.1.2. USA Minotech.
2.3 Transformation • Escherichia coli DH5α® cells 50‐100 μL of chemically competent DH5α® cells were thawed on ice and mixed with the desired amount of DNA.2 Chemically competent cells • Escherichia coli DH5α® cells Chemically competent cells were prepared with an adapted protocol from Inoue et al. The pellet was resuspended gently in 1 mL ice‐cold 20 mM CaCl2 solution and aliquoted.6‐0. 2. When an OD600 of 0. nutrient plates contained 1. After another incubation on ice for 10 min the cells were aliquoted and quick‐frozen in liquid ni‐ trogen. The flask was left on ice for 10 min and the cells were sub‐ sequently harvested by centrifugation at 2500x g for 10 min at 4 °C. heat‐shocked at 86 . 1990.1.6‐0. The pellet was gently resuspended in 80 mL ice‐cold TB.2.8 had been reached. The pellet was resuspended in another 20 mL ice‐ cold TB and DMSO was added to a final concentration of 7 % (v/v). Materials and Methods Liquid cultures were grown in a vessel at least three times the culture volume on an orbital shaker. The mixture was left on ice for 20 min. The cells were then quick‐frozen in liquid nitrogen and stored at ‐80 °C.5 % (w/v) agar‐agar.8 had been reached the flask was left on ice for 10 min and the cells were subsequently harvested by centrifugation at 2500x g for 10 min at 4 °C. left on ice for 10 min and harvested as described above.. 250 mL SOB medium in a 2 L Erlenmeyer flask were inoculated with a single colony of recently streaked DH5α cells and incubated at 18 °C with vigorous shaking at 250 rpm.2.2.1. 2 mL of the starter culture were used to inoculate 50 mL LB Rif and the cells were grown as above until an OD600 of 0. • Agrobacterium tumefaciens C58C1 cells For the preparation of competent Agrobacterium tumefaciens cells 5 ml LB Rif were inoculated with a single colony of recently streaked C58C1 cells and incubated over night at 28 °C with vigorous shaking at 250 rpm. The cells could be stored at ‐80 °C for several months.
air‐dried and resuspended in water. 2001. The cell debris was pelleted by centrifugation and the supernatant precipitated with 0.2. 250 mL LB were added and the cells incubated for 45‐60 min at 37 °C with vigorous shaking at 250 rpm. 200 μL Solution II were added and mixed gently. The mixture was left on ice for 10 min. 2. If a higher purity was desired.7 volumes of isopropanol.2. Materials and Methods 42 °C for 45 sec and quick‐chilled on ice for 1 minute. • Agrobacterium tumefaciens C58C1 cells Agrobacteria cells were transformed with an adapted protocol from Höfgen & Willmitzer. Large‐scale plasmid preparations were performed with a NucleoBond® Xtra Midi Kit according to the manufacturer’s protocol. 1988. Bacterial cells of a 5 mL LB over night culture were pelleted by centrifugation at maximum speed and resuspended in 100 μL Solution I. 250 mL LB were added and the cells incubated for 4 h at 28 °C with vigorous shaking at 250 rpm. The cells were then streaked on selective LB‐agar plates and incubated over night at 37 °C. After 5 min incubation on ice 150 μL Solution III were added. The cells were then streaked on LB Rif‐agar plates containing the selective antibiotic and incubated for 2 days at 28 °C. The pellet was washed with 70 % (v/v) ethanol.1. mixed well and left on ice for another 10 min. 87 . quick‐frozen in liq‐ uid nitrogen for 1 min and afterwards incubated at 37 °C for 10 min. 100 μL of chemically competent C58C1 cells were thawed on ice and mixed with the desired amount of DNA. the supernatant was extracted prior to precipitation with an equal volume of phenol and then chloroform or in a single step with a phe‐ nol/chloroform 1:1 mixture.4 Plasmid preparation Plasmid DNA was extracted by alkalic lysis as described in Sambrook & Russel.
2.1. plants were further re‐ potted to fresh soil.2 Plant transformation techniques 2. perlite and fertilizer.1.1.5 Agarose gel 0. The bags were opened gradually to acclima‐ tize the seedlings to greenhouse humidity levels. 2.2.8 Ligation Vector and insert DNA sequences were cut by restriction enzymes.1. Nicotiana sp.1 Plant cultivation For selection purposes seeds were sterilized for 3 min in sterilization solution and subsequently washed three times with sterile water.0 % (w/v) agarose were melted in 1x TAE and 0. 2. Approximately 2 weeks post germination green seedlings were transferred onto pot‐ ting soil and covered with plastic bags.2. 2.2. containing peat moss.188.8.131.52.0 % (w/v) agarose gels in 1x TAE and extracted out of them as described above.2. The gels were run in 1x TAE at 20‐120 V depending on the size of the gel tank and the application.7‐2. Materials and Methods 2.01 % (w/v) ethidium bromide added before casting.7 Digest For site‐specific cleavage 1‐10 μg of DNA were incubated at 37 °C for 1‐2 hours with 1‐10 U of the respective restriction enzyme in its corresponding 1x buffer. seeds were spread on MS‐agar plates containing kanamy‐ cin at a final concentration of 50 μg/mL for Arabidopsis thaliana seeds or 200 μg/mL for Nicotiana benthamiana seeds. After addition of 250 μL 1 % (w/v) low‐melting agarose. The extracted samples were mixed in a molar ration of 1:3 of vector to insert and incu‐ bated with 1 U T4 DNA ligase in 1x ligase buffer at 16 °C over night. 88 . Nucleic acids were visualized under UV light.7‐ 2. separated on 0.6 Gel extraction DNA fragments separated on agarose gels in 1x TAE were cut under UV light and extracted with NucleoSpin Extract II according to the manufacturer’s protocol.
1985. 2. 5mL LB Rif and the appropriate selective antibiotic were inoculated with a single colony of recently streaked Agrobacteria and incubated at 30 °C with vigorous shak‐ ing. The overnight culture was further used to inoculate 300 mL LB medium in a 2 L Erlenmeyer flask and incubated again at 30 °C with shaking at 180 rpm. The leaf discs were rinsed in fresh MS and transferred to LDM I plates with the stomata facing towards the air. Materials and Methods 2. Shoots from independent calli were transferred to LDM III and after rooting in the presence of 200 μg/mL kanamycin plantlets were put in soil and moved to greenhouse conditions.2 Leaf‐Disc transformation Transgenic Nicotiana benthamiana plants were created by leaf disc transformation us‐ ing an adapted protocol from Horsch et al. After 2 days the leaf discs were transferred to LDM II plates and changed repeatedly to fresh plates once a week until shoot forma‐ tion started.2. 2. 1997. About 60 leaf discs were cut and soaked in the Agrobacteria suspension for 20 min. Young leaves were sterilized for 10 min in sterilization solution and consecutively washed three times with sterile water.2..3 Floral Dip Transgenic Arabidopsis thaliana plants were created by floral dip transformation based on Clough & Bent.2. were dipped into the solution for 2 min with slight shaking. The pellet was resuspended in sucrose dipping solution and the OD600 was adjusted to 0. 89 . The shoots of the plants. which had been prepared previously by removing all ripe siliques. 1998.2. The cells were harvested by centrifugation at 2500x g. They were har‐ vested by centrifugation at 2500x g for 10 min at 4 °C and resuspended in 20 mL MS medium..2. The plants were stored horizontally in the dark for 2 days and then stored under normal growth conditions until maturity. Agrobacteria were grown in LB Rif containing the appropriate selective antibiotic to an OD600 of approximately 0.2.7.2.8.4 Agroinfiltration The method is based on Schoeb et al.
Approximately 20 mg of finely ground plant tissue were resuspended in 200 μL 2x CTAB buffer by vigorous vortexing and incubated at 65 °C for at least 5 min. Prior to load‐ ing the sample volume was decreased by isopropanol precipitation. The cells were incubated at 28 °C with vigorous shaking of 250 rpm until an OD600 of ap‐ proximately 0. 2.25. After repeating this washing step twice. the samples were again vortexed vigor‐ ously and afterwards centrifuged at maximum speed. The samples (in 1x DNA loading buffer) were loaded on a 0.1 DNA extraction DNA was extracted with an adapted protocol based on Rogers’ and Bendich’s (1985) CTAB‐based protocol.3. the OD600 was adjusted to approximately 0. The gel was depurinated until the bromophenol blue of 90 .2.2 Southern analysis 2 μg (Arabidopsis thaliana) or 20 μg (Nicotiana benthamiana) of genomic DNA were di‐ gested with 10‐30 U restriction enzyme in its appropriate 1x buffer. For Southern Analysis the amounts were up‐scaled accordingly. Materials and Methods 5 mL LB Rif and the appropriate antibiotic were inoculated from a glycerol stock.3. The reactions were incubated at 37 °C for approximately 3 hours. The agrobacteria were harvested by centrifugation at 2500x g for 10 min at 4 °C. The ex‐ tracted DNA was treated with RNase A and used for PCR. 2. The clear supernatant was transferred to a new tube and precipitated with 0. The volumes could be adjusted according to the amount of necessary leaves. After addition of an equal volume of chloroform.2.2.7 % (w/v) agarose gel and run at ap‐ proximately 20 V over night. The pellet was resuspended in 5 mL of MMA and incu‐ bated in a shaker at 28 °C for at least 2 hours.7 volumes of isopropanol.7 was reached.3 Southern analysis 2. The solution was infiltrated with a syringe into the leaf through a previously made small puncture. After 2 hours a small aliquot was separated on a gel to test if the digestion had been already completed. The bacteria were pelleted by centrifu‐ gation as above and resuspended in 2 mL of 10 mM MgCl2.2.
2. The nucleic acids were crosslinked on the membrane by applying UV radiation of 100x 1200 μJ/cm2. The gels were pre‐run at 100 V in 1x MOPS 91 .2 % (w/v) agarose gels in 1x MOPS containing 0.4.7 % (v/v) formalde‐ hyde and 0. two more Whatman papers and a 10 cm thick tissue paper stack on top of the sandwich.2. 2.2.2. 200 μL chloroform were added to the cleared supernatant and the samples were again vortexed and incubated for 15 min at room temperature. After pelleting and removing cell debris. The blot was assembled by a Whatman paper submerged in 10x SSC acting as a bridge.4.1. 1987. Approximately 100 mg of finely ground plant tissue were resuspended in 1 mL RNA extraction buffer by vigorous vortexing and incubated at room temperature for at least 5 min.4 Northern analysis 2.2 Denaturing agarose/formaldehyde gels For detection of RNA molecules longer than 200 bases 10‐20 μg of total RNA were separated on 1. a gel‐sized membrane and four whatman papers were equilibrated in 10x SSC. the gel. then denatured for 30 min and finally neutral‐ ized with the corresponding solutions from chapter 2.01 % (w/v) ethidium bromide. The RNA pellet was washed with 70 % (v/v) ethanol. air‐dried and resuspended in water. The nucleic acids were blotted onto the membrane by applying constant pressure from top over night. by adding 1x high salt mixture to the isopropanol.3 Capillary blot The gel.2. 2. the upper phase was used for a modified isopropanol precipitation.4. The volumes of the liquids should be at least two times the gel volume to ensure complete covering. the membrane. The dried membrane was stored at room‐temperature until detection. 2. After separation of the two phases by centrifugation. Materials and Methods the loading buffer changed to yellow.1 RNA extraction Total RNA from plants was extracted using an adapted protocol from Chomczynski & Sacchi.3. two gel‐size Whatmans.
2.4. The RNA was boiled in 1x RNA loading dye and quick‐chilled before loading. 10 mM dTTP. 92 . Materials and Methods running buffer. The samples were run at 100 V for at least 3 hours. Gels were run at 10‐25 Watt for keeping a constant gel temperature of 50 °C.2. 10 mM dGTP. the blot consisted of five gel‐size Whatman paper soaked in 1x TBE. as well as 20 U Klenow Fragment. 3 μg random primers. 2.3 PAGE northern For detection of RNA molecules shorter than 200 bases 20‐50 μg of total RNA were separated on 8‐15 % (w/v) denaturing polyacrylamide (PAGE northern) gels. then the membrane facing the anode and another layer of five Whatman papers. 20 μCi α32P‐dATP and 20 μCi α32P‐dCTP. The wells were rinsed with 1x TBE to remove released urea prior to loading.2. The typical reaction volume was 50 μL.4.2.1 Random‐primed labeling 100 ng of purified DNA template were denatured by boiling for 3 min and subse‐ quent quick‐chilling. The gels were stained in ethidium bromide and equilibrated in 1x TBE until the transfer.4 Semi‐dry blot The RNA was blotted with a SD20 Semi Dry device. The dried membrane was stored at room temperature until detection. Prior to use the probe was denatured by boiling for 3 min and subsequent quick‐chilling. After polymerization the gels were pre‐run in 1x TBE until they reached a temperature of 50 °C. followed by the equilibrated gel facing the cath‐ ode.5. A constant amperage of 3 mA per cm2 membrane area was applied for 30 min and the RNA was subsequently crosslinked on the membrane by UV radiation of 100x 1200 μJ/cm2.5 Hybridization 2. 2. The RNA was boiled in 1x sequencing dye and quick‐chilled before loading. After an incubation of 60 min at 37 °C the labeled probe was purified from unincorporated nucleotides with a MicroSpin S‐200 column according to the manufacturer’s protocol. consisting of 1x Rad‐ Prime Buffer.2. 2. The gels were rinsed in water and used for capillary blotting as described above.
5 mL stacking gel were prepared with a BioRad casting device. The samples were 93 . The boiled probe was added and the membrane hybridized over night. washes and developing The membranes were pre‐hybridized for at least one hour in Church hybridization buffer.2.5 mL resolving gel and 2. sealed in plastic bags und subsequently exposed on X‐ray films. After 45 min the labeled probe was purified from unincorporated nucleotides with a MicroSpin S‐200 column ac‐ cording to the manufacturer’s protocol. After 30 min incubation on ice the sam‐ ples were centrifuged at full speed for 15 min at 4 °C.2. The clear supernatant was transferred to a new tube and stored at ‐20 °C.5. 2. The hybridization temperature depended on the length of probe and sample and was usually set to 42 °C for small RNAs <50 nt and 65 °C for long RNAs and DNA. 184.108.40.206.6 Western analysis 2. Materials and Methods 2. The membrane was rinsed at room temperature with Wash I and then washed at hybridi‐ zation temperature according to the following protocol: two washes for 15 min with excess Wash I.1 Protein extraction 100 mg finely ground leaf powder was mixed with at least four volumes of protein extraction buffer and vortexed thoroughly.2 SDS‐PAGE SDS‐Polyacrylamide gels of 10‐15 % (w/v) consisting of 7.6. Prior to use the probe was denatured by boil‐ ing for 3 min and subsequent quick‐chilling. 2.6.2 End‐labeling 8 pmol DNA oligo were end‐labeled by incubation with 20 U T4 PNK containing 40 μCi of γ32P‐ATP in 1x PNK reaction buffer at 37 °C. Signals could be detected by developing the film after 1‐10 day exposure at ‐80 °C depending on the amount of the targeted nucleic acid.2.2. Finally the membrane was rinsed in 2x SSC. two washes for 10 min with excess Wash II and in the case of random‐ primed probes two additional washes for 5 min with excess Wash III.3 Hybridization.
precipitated and further amplified in a second PCR reaction. 5S rRNA and 4. comprised of serum from ERL1‐injected rabbits (Vlatakis. It was again purified by 94 .7 rRNA cloning 2. 2010) diluted 1:20000 in binding buffer for one hour.. loaded and run at 70‐140 mA in 1x protein running buffer. The correct products were eluted from gel slices by incubation in 300 mM NaCl over night at 4 °C with constant rotation.2.7. 2. After three final washes the membrane was incubated with HRP substrate and exposed on X‐ray film until protein bands appeared.2. 2.3 Electro‐blot The gel as well as two sponges.4 Western detection The membrane was blocked in blocking buffer for at least one hour and washed three times for 5 min with washing buffer. 5 μg total RNA were self‐ligated with 20 U T4 RNA Ligase 1 in 1x RNA ligase buffer and reverse‐transcribed as described below.2. The obtained cDNAs were amplified by PCR and separated on 8 % (w/v) acrylamide gels in 1x TBE. The membrane was incubated with primary antibody. The released DNA was purified by phenol/chloroform extraction.2. 2005). four gel‐size whatman papers and the nylon mem‐ brane were equilibrated in 1x transfer buffer.6.8S rRNA.6.5S rRNA were determined by a modified circular RT‐PCR protocol as described before (Bollenbach et al.1 Self‐Ligation The precise 5’ and 3’ ends of 5. 2. The gels were stained with Coomassie staining solution and destained in de‐ staining solution until the protein bands were visible.2. Materials and Methods boiled in 1x Laemmli buffer. After three washes the membrane was incu‐ bated with secondary antibody anti‐Rabbit AP‐conjugate diluted 1:2500 in binding buffer for another hour. The blot was assembled by two Whatman papers followed by the gel facing the cathode and the membrane facing the anode and another two Whatman papers and one sponge on each side. The pro‐ tein was blotted by applying 90 mA constant amperage over night at 4 °C.
For a more specific amplification of the linker‐ligated RNAs a touch‐down protocol was used.220.127.116.11‐2. 200 μM of each dNTP.7. The products were cloned and sequenced using vector‐specific primers. 2. The cDNA was synthesized by supplementing the reaction with 1x RT buffer. It started with a high annealing temperature of the corresponding primer melting temperature plus five degrees Celsius (Tm + 5 °C) which was reduced gradu‐ 95 . Materials and Methods phenol/chloroform extraction and precipitated with isopropanol. A typical program consisted of an ini‐ tial denaturation step at 95 °C for 3 min.2.4 Polymerase Chain Reaction (PCR) For specific amplification of a DNA fragment a reaction containing 1x Taq buffer.2. 1. The reaction was carried out in 1x T4 RNA ligase buffer containing 10 % (v/v) DMSO and 1 U RNasin. 2.5 mM MgCl2. The reaction was terminated by a final incubation at 85 °C for 5 min. 1 U RNasin and 2 U reverse transcriptase and incubated at 42 °C for 45‐60 min. 1 mM of each dNTP. The purified PCR products were cloned and sequenced using vector‐specific primers. 1 U Taq polymerase and the appropriate amount of template DNA depending on the application were incubated in a thermocycler.3 Reverse Transcription (RT) 1‐5 μg total RNA were incubated with 1μM first‐strand primer at 70 °C for 10 min. The obtained cDNA was am‐ plified by PCR and gel‐extracted with NucleoSpin. 200 pmol 3’‐ddC‐modified linker were phosphorylated with 1 U PNK and ligated with 20 U T4 RNA ligase 1 to the 3’‐ends of total RNA. 2.2 μM forward and reverse primer. followed by 35 cycles of denaturation for 15‐ 30 sec at 95 °C.2 Linker‐Ligation To verify the position of the additional nucleotides of chloroplastic rRNA species.2. The product was then reverse‐transcribed with a linker‐specific primer. 0. annealing for 15‐30 sec at 45‐60 °C (depending on the nature of the primers) and extension for 30 sec per kb at 72 °C.
The resulting amount of fluorescence was recorded at 72 °C.10 Protoplasts Young Nicotiana benthamiana leaves were submerged in protoplast extraction buffer and cut into thin.5 μM Oligo(dT) re‐ verse primer. fol‐ lowed by 40 cycles of denaturation for 5 sec at 94 °C. The reaction was incubated for 10 min at 30 °C. 5 min at 95 °C and finally 5 min at 5 °C. Intact protoplasts floated on top and could be used directly for microscopic analysis.2. The samples were incubated for ap‐ proximately 5 hours at room temperature with slight shaking at 50 rpm. The melting curve was recorded by increasing the temperature to 94 °C in 1. annealing for 5 sec at 55 °C and extension for 12 sec at 72 °C. cloned and sent for sequencing. Materials and Methods ally until an annealing temperature of the corresponding primer Tm – 5 °C was reached. The samples were filled into long tubes with a small diameter and left for sedimentation at 4 °C for some hours or preferably over night. 2.2. 15‐30 min at 50 °C. 96 . 2. The program cycled at this temperature 25 times as described above. The RT reaction was performed as above with 2. A typical program consisted of an initial denaturation step at 94 °C for 10 min.2.9 Quantitative real‐time PCR (qPCR) Total RNA was treated with 10 U DNase I in 1x DNase buffer to remove traces of genomic DNA. The resulting curves were analyzed with the Rotor‐Gene Version 6 program by com‐ parison with a standard curve of known template amounts. 2. less than 1 mm wide slices.2. The reaction was then diluted one to ten and 5 μL were used for a qPCR with the Fermentas Maxima™ SYBR Green qPCR Kit according to the manufacturer’s documentation. 10 μL were used for PCR.8 Rapid Amplification of cDNA ends (RACE) 3’‐RACE was performed with 1 μg total RNA using the TaKaRa 3’‐Full RACE Core Set according to the manufacturer’s protocol. 81 °C and 84 °C.0 °C steps and holding each temperature for 2 sec. The cDNA was treated with 1 U RNase H to remove RNA/DNA hy‐ brids.
After analysis transverse leaf sections were stained with 1 % (w/v) toluidine blue and used for optical microscopy. 2. 97 . this humid chamber was then covered with aluminum foil. The results were analyzed with the BiolyzerHP3 software. The fixed tissue was washed.2.12 Fluorescence measurement Leaf discs of Nicotiana benthamiana plants were cut and left on wet Whatman papers in a petri dish.2. stained with 1 % (w/v) OsO4 and sec‐ tions prepared for electron microscopy.11 Preparation for microscopic analysis Nicotiana benthamiana leaves were submerged in fixing buffer and infiltrated by ap‐ plying vacuum. After a dark adaptation of 15 min the leaf discs were excited with light and the resulting fluorescence measured with a Handy‐PEA fluorometer.2. Materials and Methods 2.
when plain GFP was infiltrated.10 plant ERL1 sequences possess an N‐terminal sequence motif possibly leading to the import of the protein sequence into chloroplasts. pro‐ toplasts were prepared and analyzed with a confocal microscope. all expressed ERL1:GFP was targeted to the chloroplast (Figure 3.3.1 Localization As described in chapter 1.1 a). Nicotiana bentha‐ miana wildtype tissue was used as a negative control. This leader peptide is present in various plant varieties including mono‐ and dicotyledo‐ nous species (Figure 3. Plain GFP infil‐ trated into a different spot on the same leaf was used as a control for the specificity of chloroplast import. These organelles could be identified as chloroplasts by detection of the red auto‐fluorescence of chlorophyll in the second channel since both showed a perfect overlap. Results 3. but the latter sequence can be considered as incomplete since it starts with Ser‐ ine instead of Methionine. two exceptions are Medicago truncatula and Sorghum bi‐ color. In contrast. the green fluorescence was excluded from the chloroplasts and could only be detected in the cytoplasm (Figure 3.2. To verify the predicted chloroplastic localization of Arabidopsis thaliana ERL1. The cytoplasm and vacuole were free of GFP fluorescence.1 e). as has been 99 . As soon as the GFP expression could be observed as green fluorescence under a handheld UV lamp. Protoplast preparations of Nicotiana benthamiana line 16C with stably integrated GFP served as a positive transgenic GFP control.1 b). In protoplasts prepared from leaves of Nicotiana benthamiana line 16C strong green fluo‐ rescence could be detected throughout the Endoplasmatic Reticulum (Figure 3. its full‐ length cDNA was fused to the N‐terminus of GFP (ERL1:GFP) and transiently over‐ expressed by agroinfiltration into Nicotiana benthamiana leaves. Expression of the ERL1:GFP construct resulted in a strong GFP signal detectable in defined spots inside the cells.1 d). This corresponds to the localization of the integrated GFP in this line.
2007) therefore an accessible N‐ terminal leader sequence is indispensable for chloroplast import. (E) Protoplast preparations of N. To determine whether the predicted leader peptide was able to drive the import of proteins into chloroplasts.mays (1) (1) (1) (1) (1) (1) (1) (1) 1 100 MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCN--------------SSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSEN-----ARWRP ---------------------------------------------------------------------------------------------------------SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRK-ISISASRSTTEESTSSLIQPTPSR------TRWKP ---------MAIARVSPPAFS--SPFLIHSLLRPFSSPSSVL-------RPRVTRVPHHRGFAIAAALSQASPLPSADGDGAVMEAPPRPSSR--RPWKP ----MSFPRIPLSRVPSYLHNSNN--CFHLLHPPFIPVSKTP------------SLPTYQTARTYTDFNSQTQTQPPLSLPSLIPSPPVNNPNATHRWKP ---------------------------------------------------------------------------SAASSATVRASG-SVG-----------------MAFYRVSPFRYGSLS-S---LIPYVS-------------SP-----SSLSPPVRT-FTLSASISTPHPSPPSLLTASPKAS-----DRWRP ---------MALARVSPSSLANLIPPLLQSFFRPFSS-------------DFPIRNSRRRSSPVAAAFSLTSQSAHAAREGLVMEAP-RPSSR--YPWKP Caption: identical. 1999). (C) Fusion of the ERL1 leader sequence to GFP shows the same effect as above and identifies the leader to be capable of chloroplast import. Protoplast preparations of wildtype showed no detectable green fluorescence and confirmed the specificity of the detected sig‐ nals. 100 . M.trichocarpa S.benthamiana O. (A) A. The expression pattern was identical to the result obtained with the ERL1:GFP construct (Figure 3. benthamiana line 16C where the GFP pro‐ tein is localized in the Endoplasmatic Re‐ ticulum. bicolor ERL1 sequence is incomplete..3.truncatula N.1 c). (D) Infil‐ tration of plain GFP results in green fluo‐ rescence which is excluded from chloro‐ plasts and only detectable in the cyto‐ plasm. fusion of GFP to the 5’‐end of ERL1 resulted in strong GFP fluorescence in the cytoplasm (Eckhardt.thaliana M. While the S. Results described before (Voinnet et al.1: Localization of plant ERL1 (A) An alignment of the N‐terminal ERL1 protein regions reveals differences be‐ tween plant species. (B)—(C) Confocal micros‐ copy of protoplast preparations from leaves agroinfiltrated with GFP‐ containing constructs: (B) Fusion of ERL1 to GFP cDNA proves perfect co‐ localization of ERL1 with the chlorophyll of chloroplasts.sativa P. similar (B) GFP Chlorophyll Merge 10 μm 10 μm 10 μm (C) 10 μm 10 μm 10 μm (D) 10 μm 10 μm 10 μm (E) 5 μm 5 μm 5 μm Figure 3. trunca‐ tula ERL1 does not contain a chloroplast signal peptide. its cDNA was fused to GFP (leader:GFP) and infiltrated as above. In addition.vinifera Z.bicolor V.
corresponding to the length of the ESTs. (C) Alignment of the A. thaliana. 5’‐RACE could not be performed successfully. tabacum ESTs EB681897.1 as the corresponding EST.2 RACE Until now no complete sequence information is available for ERL1 of Nicotiana sp.1 and At_ERL1 37.2 % similarity on the protein level.3% identity and 76.1 together with A.harvard. Nb_ERL1 = ERL1 of N. benthamiana sequence on the length of the already pub‐ lished stretch.3.2% identity and 64. From the latter only the first 800 nucleotides are depicted. The obtained Nicotiana benthamiana sequences are presented in the supplementary result section (chapter 6. (B) The consensus of obtained 3’‐RACE sequences and its alignment with the later cloned N.2 %.dfci.2 % and BP529372. none of the cloned sequences corresponded to EST BP529372 at the 3’‐end. benthamiana 101 .1) both ESTs could be the result of transcription of two individual genes.1). whereas their 3’‐ends are more dissimilar. The latter is incomplete at the 5’‐end which can be concluded from a missing methion‐ ine.9% similarity. it has 98% identity with the newly identified N. thaliana ERL1 protein sequence and the translated N.2 a). benthamiana sequences identifies EST EB681897. Results 3. EB681897.2% similarity.cgi) with the ac‐ cessions EB681897 with 732 nt and BP529372 consisting of 632 nt. The obtained consensus sequence corresponded to EST EB681897. as well as 53. in the exonuclease domain (highlighted by an arrow) the sequences are even more related sharing 68.1 and BP529372. Since Southern analysis revealed two copies of the ERL1 gene in Nicotiana benthamiana (compare chapter 3. an alignment of the gained se‐ quences is provided in Figure 3. both ESTs have an identity of more than 70 % on a stretch of 30 % of the sequence (Figure 3. thaliana ERL1 cDNA.2: Alignments of Nicotiana sp.edu/tgi/cgi‐bin/tgi/Blast/index.5. 3’‐RACE identified an additional stretch of 362 nt.2 % identity and 64. To identify the ERL1 sequence in Nicotiana benthamiana RACE experiments were per‐ formed.2 % sequence identity with At_ERL1.3 % identity with Arabidopsis thaliana ERL1 on the nucleotide level.2 b and c: Figure 3. The two ESTs share 75.3. but the sequence comparison with Arabidopsis thaliana ERL1 cDNA suggests a nearly com‐ plete 5’‐end of the ESTs. Caption: At_ERL1 = ERL1 of A. They have an iden‐ tity of 75 % with their 5’‐ends being almost identical. ERL1 sequence (A) Alignment of the published N. The sequences share 53. There exist two published ESTs from Nicotiana tabacum in the DFCI Tobacco Gene Project (http://compbio.1 shares 32. The resulting product showed 55. bentha‐ miana cDNA. When aligned with Arabidopsis thaliana ERL1 cDNA.
1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 EB681897.1 Nb_ERL1_1 Nb_ERL1_2 (1) (1) (1) (1) (1) (100) (101) (101) (1) (200) (201) (201) (48) (300) (301) (301) (148) (400) (401) (401) (248) (500) (501) (501) (348) (600) (601) (601) (448) (700) (701) (701) (548) (733) (801) (801) (648) (733) (901) (901) (748) (733) (1001) (1001) 1 100 ----------------------------------------------------------------------------------------------------GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAAGGAGCCGTTTAATTCCAATGGCTACGGGATTTT GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTT GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTT 101 200 ---------------------------------------------------------------------------------------------------GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCT GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCG GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCG 201 300 -----------------------------------------------------CCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATCTACTCCTTCCCTAATTCAGCCCACAACTTCCCGTACCCGTTGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATTTACTTCTTCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATCTACTTCTTCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT 301 400 AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCTTGAGCTTATGCAAAATGTTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT 401 500 TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTCGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT 501 600 CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT 601 700 ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGTGAACGTCAATTGTGGAGAAATGAACTGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG 701 800 CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAA CCTTTGTTACTTGTGGGAACTGGGATCTGAAGA------------------------------------------------------------------CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAA CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAACGGATTAA 801 900 TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT ---------------------------------------------------------------------------------------------------TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT 901 1000 GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCATTCTCCTG ---------------------------------------------------------------------------------------------------GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAACCCTCATTCTCCTGA GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCATTCTCCTG 1001 1097 AAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTTCTTCTGAACCATTTTGTTATCACCTAAACATTTTTAGAAAAAAAAAAAAAAA ------------------------------------------------------------------------------------------------AAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTT----------------------------------------------------AAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTT---------------------------------------------------- 102 .1 1 100 (1) --------AGTTCCCAGTCCCTGTACTCGAAAGGA-AGATCTTCATCTTCAATCTTCATGCTAATCGACGAAAATGGCGTCCGCATTCTCTGCATTTAGG (1) ----------------------------------------CTTCTTCAGGAAAACTCATAC----CTCAGAGA--GCCGTTCAATTCCAATGGCTAT-GG (1) GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAAC-TCACTC----CTCAAAGGA-GCCGTTTAATTCCAATGGCTAC-GG 101 200 (92) GTTTCGTTGTCCAGAATCAGTCCTTTCCGTGATACCCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAAT-CATGTGCAACTCTTCG (54) GATTT----TCTAGGGTCC--CCTTGCTGCGG----CGTTTCCTTGGTATCTCCTC--CGGTACTACCTTCTTCGTACTCACTTCAGGCCCAACCGTAAA (94) GATTT----TGTAGGGTCC--CCTTGCTGCGG----CGGTTCCTTG-TATCTCCGC--CGGTACTACCTTTTTCGTACTCACTTCA-GCCCAGCCGTAAA 201 300 At_ERL1_cDNA (191) CATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGTGAAA BP529372.1 EB681897.1 (312) GGAGTTTATGCAAAATGCTGCGGGACTTGAGAATTTGCGGAAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAG EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (350) TGAGCTTATGCAAAATGTTGCGGGACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAG 501 600 At_ERL1_cDNA (491) TTTCCTATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGACTTATTCCACAGGTTTGTAAGACCCACCAAAATGAGCGAGCAAGCAATTAACAAAT BP529372.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (142) ATGAATATCTCAGCCTCTCTTTCTACCACCGAAGAATCTACTTCTTC--C-----C---TAATTCAGCCCACACCTTCCCGT--------ACCCGT---EB681897.3.1 (180) ATCAGTATCTCCGCCTCTCTTTCTACCACCGAAGAATCTACTCCTTC--C-----C---TAATTCAGCCCACAACTTCCCGT--------ACCCGT---301 400 At_ERL1_cDNA (291) ATGCAAGGTGGAGACCCATGTGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGATGATCCTGCCCATTTGGAGATTTTTAACCACGATTGTTCAAA BP529372.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897. Results (A) At_ERL1_cDNA BP529372.1 (648) GGTGAACGTCAATTGTGGAGAAATGAACTGGGCGGCTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGA--------------- (B) 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (600) GC-------GCCTTTTGGT-TCTATATGAAGAATTGCATG-----------------------------------------------------------EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (412) TTTCCAGTTCTCCTCTTTGATGCTAAAACCATGGATGTGGTTGACTTGTTCCATAGGTTTGTGAGGCCAACAAAAATGCACGAAGAAAGAATAAACGAAT EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (258) --------TGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATGATCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCT 401 500 At_ERL1_cDNA (391) GGAACTTCGAGTGGCTGCTGCTGATCTTGAGAGAAAGAAGTCACAAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGAAAAGTTGAGATTCTTGAG BP529372.1 At_ERL1_cDNA BP529372.1 (550) ATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGATA--CAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTG 701 800 At_ERL1_cDNA (689) GCTGAGCATGACTTGTGGGATAAAGATACAGATTGGGGTCTGAACGATGCAGCTTTTGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTCCTGAGC BP529372.1 (512) ATATAGAAGGGAAATATGGAAAACTAGGAGTTGATCG-GTATGGTATCTAATTCAGTAGTTCGAAATA--CAA----TTCAGCAGTTGGAACTT-----A EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 (220) --------TGGAAGCCAACGTGTCTCTACTTTACTCAAGGTAAGTGCACCAAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCGCT EB681897.1 (450) TTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTTTTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAAT 601 700 At_ERL1_cDNA (591) ACATCGAAGGCAAGTATGGGGAACTCGGGGTTGATCGTGTGTGGCATGACA--CAGCTATTCCATTTAAGCAAGTTGTTGAGGAGTTTGAAGTTTGGTTA BP529372.
Five out of these six lines had a segrega‐ tion pattern most likely corresponding to a single insertion (compare Table 3. This con‐ struct was supposed to downregulate the endogenous Nicotiana sp. then at a very late (A) (B) (C) wt – 103 Figure 3. ERL1 mRNA by RNA silencing. The plants showed resistance to kanamycin and slight bleaching in a young stage (Figure 3. Results (C) At_ERL1 Nb_ERL1 At_ERL1 Nb_ERL1 1 100 (1) ---------MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSENARWRPMCLYYTHGKC (1) SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQ---PSRKISISASRSTTEESTSSLIQPTPSRTRWKPTCLYFTQGKC 101 200 (92) TKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGVDRVWH (98) TKMDDPMHIDKFNHNCSLELMQNAAGLKNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGKYGKLGVDRVWH EXOIII 201 300 At_ERL1 (192) DTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWINLKDVYLNFYGREARGMVSMMRQCGIKLMGSHH Nb_ERL1 (198) DTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQCKVAGTKLPPYFMEWINLKDVFLNFYKRRAKGMLSMMRELQMPLLGSHH 301 346 At_ERL1 (292) LGIDDTKNITRVVQRMLSEGAVLKLTARRSKSNMRNVEFLFKNRIK Nb_ERL1 (298) LGIDDAKNIARVLQHMLSDGALVQITARRNPHSPEKVEYLFEDRIV Caption: identical. In addition. This phenomenon reverted to a wildtype‐like phenotype after some weeks (Figure 3.3 b). conserved.1). four out of the six lines did not produce any seeds or if. not only macroscopically but also when observed with the light and electron microscope (data not shown).3. Exonuclease domain 3.3. 3.3: Analysis of presum‐ able ERL1 suppressor plants (A) Slight bleaching in young plants (B) wildtype‐like phenotype in adult leaves (C) Southern analy‐ sis revealed the same ERL1 copy number in wildtype (wt) and the transformed plants (–) suggesting no presence of the ERL1 hairpin. similar.3 Transgenic Nicotiana benthamiana plants Agrobacterium tumefaciens‐mediated leaf disc transformation was used to produce transgenic Nicotiana benthamiana plants which were misexpressing ERL1.1 Knock‐down of ERL1 Six individual lines were created by transforming Nicotiana benthamiana leaves with a hairpin designed from Nicotiana tabacum EST EB681897 (Schumacher. The stable integration was used to study the effect of ERL1 during the whole lifecycle of the plants. .3 a). 2009).
Table 3. The observed phenotype is not considered as a result of downregulation of ERL1. Results stage and only in small amounts. that the construct designed by a prior lab member was not correct. From this data the segregation ratio could be calculated (compare Table 3.0 1:4.5 1:18.3.5 1:5. qPCR analysis showed the same ERL1 expression level as in wildtype leaves (data not shown).3 c).1: Segregation of Nicotiana benthamiana T2 plant lines transformed with a hairpin construct designed for downregulation of ERL1 line ERL1 KD1 ERL1 KD2 ERL1 KD3 ERL1 KD4 ERL1 KD5 ERL1 KD6 segregation 1:4. In total. however. RNA of 344 plants was analyzed and revealed a direct relation of the ERL1 expression level and the severity of the 104 . The pres‐ ence of the ERL1 hairpin. Northern analysis failed to detect the hairpin or small RNAs resulting from it. In the resulting twelve lines the plants showed already in the first leaves multi‐ faceted phenotypes which were sometimes not easy to distinguish from susceptibil‐ ity to kanamycin. Crossing of these lines with Nicotiana benthamiana wildtype in both directions showed. Finally Southern hy‐ bridization with an ERL1 probe showed the same two copies of the ERL1 gene as in wildtype (Figure 3.2).5 3. that the male sexual organs were not functional. Further sequence analysis of the original vector proved. could not be proven.2 Overexpression of ERL1 an Arabidopsis thaliana ERL1 cDNA construct driven by the constitutive CaMV 35S promoter was used to transform leaves of Nicotiana benthamiana. The plants were therefore considered as transgenic.7 1:3.8 1:4. Therefore all plants were transferred to soil and their ERL1 expres‐ sion levels determined by Northern analysis. but there was probably no downregulation of ERL1 present.3. whereas the plants produced seeds when pollinated with wildtype pollen.
It is possible. Only the strongest bleach plants of distinct lines showed this behavior but the phenomenon was inheritable and appeared in consecutive generations.4 f). The reversion of the bleach phenotype could also be observed which may have been triggered by environmental factors. These plants contained com‐ pletely white and green tissue which could be collected independently.3. the macroscopic phenotypes could be subdivided into four distinct groups: • The term “Bleach” was used for a phenotype varying from plants with leaves which were slightly brighter than wildtype to plants with an almost complete loss of chlorophyll and white leaves. The size of the plants and their life cycle showed no difference when com‐ 105 . it cannot be distinguished whether the green patches are a result of endogenous ERL1 transgene silencing or possessed wildtype characteristics. The latter showed a stunted growth with matching small sized leaves. • Some bleach plants with a strong phenotype entered a pathway of ERL1 transgene silencing and reverted to a wildtype‐like phenotype. but they are considered to be a direct consequence of ERL1 overexpression.4 b). Compared to wildtype they reached the maturity state strongly delayed and produced only few seeds (Figure 3. Most transgenic lines showed more than one of the different phenotypes. bleach plants appeared to have a prolonged lifecycle compared to wild‐ type.4 d and e). Therefore they cannot be attributed solely to the number and location of the insertions into the Nicotiana benthamiana genomes. that the endoge‐ nous ERL1 expression is also silenced in this line. • “Mosaic” plants had a phenotype with speckled leaves of green and white patches. In general. Its progression was comparable to systemic silencing spread. The line was further named “Self‐ silencer” (Figure 3. but not tested. Results phenotype (Figure 3. In addi‐ tion.
6 = mosaic. 1 = wildtype.4 a and f).3.8 kDa 28. in self‐silenced tissue the signal was very weak. These plants showed an ERL1 expression comparable to mosaic and bleach plants. therefore the phenomenon was attributed to positional effects (Figure 3. 2‐4 = agroinfiltrated ERL1+FLAG of an unsuccessful pull‐down experiment. 5 = no phenotype (NP) . In mosaic and white tissue the strong signal might result from two bands as in Arabidopsis thaliana transgenic lines. The degraded signal in self‐silencing lines could be due to silencing of the ERL1 mRNA (G) Western analysis of plants overexpressing ERL1 lead to a strong band of approximately 42 kDa. but nei‐ ther the morphology nor the life‐cycle of the plant differed to wildtype. • The ”No phenotype” (NP) line was the exception to the above mentioned rela‐ tion of ERL1 transgene expression and the bleaching phenotype.9 kDa M 1 2 3 4 5 6 7 8 Figure 3. Caption: M = Bio‐Rad Prestained SDS‐PAGE standard. Results pared to wildtype although they showed strong overexpression of ERL1 (Figure 3. Coomassie staining is depicted as a loading control. 7 = self‐silencer green tissue.4 c). E) Self‐silencing phenotype (F) Northern analysis of transgenic plant lines revealed strong overexpression of ERL1 in most individuals. (A) (B) (C) (D) (E) (F) mosaic (G) bleach no phenotype self‐silencer 49.4: Analysis of Nicotiana benthamiana plants overexpressing ERL1 (A) Mosaic phenotype (B) Bleach phenotype (C) No phenotype (NP) (D. There was only one line that showed this behavior (in contrast to the other pheno‐ types).1 kDa 34. In consecutive generations individuals of this line exhibited mild bleaching of some leaves. 8 = self‐silencer white tissue 106 .
4 1:2.4 1:0.3 no data 1:1. In 107 . self‐silencing mosaic. self‐silencing no overexpression bleach mosaic. bleach mosaic. self‐silencing no overexpression bleach.8 1:3.6 no data 1:11. Wildtype chloroplasts are oval‐shaped organelles with intercellular membranes (thy‐ lakoids) which are arranged in stacks (grana) and responsible for photosynthesis. The morphology of a wildtype leaf (as depicted in Figure 3.7 1:5.3 1:3.7 3.3. • the mesophyll which is assembled of two distinct chloroplast‐rich cell types: the upper palisade layer with elongated cells arranged in parallel and the lower spongy layer with rounder cells and larger intercellular spaces.3.1 1:1.1 Microscopy To better describe the macroscopically observed phenotypes leaf sections were pre‐ pared and the organelle structure was analyzed by transmission electron microscopy (TEM).8 1:0. one layer of chloroplast‐free cells covering the upper and the lower side of the leaves.2. The spongy layer also contains the vasculature cells.2: Summary of segregation and phenotypic pattern of Nicotiana benthamiana T1 plant lines overexpressing ERL1 line ERL1 over1 ERL1 over2 ERL1 over3 ERL1 over4 ERL1 over5 ERL1 over6 ERL1 over7 ERL1 over8 ERL1 over10 ERL1 over11 ERL1 over12 ERL1 over13 phenotype bleach bleach bleach. bleach segregation 1:8. Results Table 3. Some of these sections were then stained with toluidine blue and the leaf morphology further analyzed by light microscopy.6 a) can be described as following: • the epidermis. bleach no phenotype bleach.
Usually there are no starch granules detectable. Leaves from bleach plants were also thinner compared to wildtype (Figure 3. Results (A) cyt (B) cw st pg gra cw 0.3. Caption: cw = cell wall. lower) and green tissue (right cell.5 a). but the number of plastoglobuli appears to be normal.8 μm 0. cyt = cytoplasm. An even more dramatic effect could be observed in the electron micro‐ 108 .7 μm pla chl cyt (C) (D) pg cyt chl st cw chl gra pg cyt 0. chl = chloroplasts. (C) Chloroplasts in green tissue of self‐silencing plants possess roundish chloro‐ plasts filled with grana stacks. (D) Mosaic plants show the characteristics of white tissue (left cell.5: TEM analysis of phenotypes of Nicotiana benthamiana plants overexpressing ERL1 (A) In wildtype plants chloroplasts are oval‐shaped and contain storage molecules packed into starch granules and plastoglobuli. st = starch granules. pl = plasma membrane addition. up‐ per) in neighboring cells.5 μm cw pla gra cyt Figure 3. The palisade cells were less elongated and lost their parallel organiza‐ tion pattern. they contain lipid droplets named plastoglobuli and starch granules which serve as storage compound for excess energy (Figure 3. Up to ten thylakoid membranes are packed per granum. White tissue caused by strong ERL1 overexpression showed a slightly disordered transverse leaf structure when observed in the light microscope.6 b). which are assembled by more than ten thylakoid membranes. (B) Undifferenti‐ ated proplastids in bleach plants show only unorganized intercellular membrane structures and no storage molecules. gr = granum. In ERL1‐overexpressing transgenic plants several structural alterations could be ob‐ served which did not only influence the leaf architecture but distorted the whole or‐ ganization of the chloroplasts.
(D) Mo‐ saic plants show a mixture of white (margins) and green (middle) characteristics. chl = chloroplast 109 . pal = palisade mesophyll. no starch granules could be detected as there was no excess energy present (Figure 3.3. (B) In bleach tissue the organization of the mesophyll is lost. In addition the leaf diameter is significantly decreased compared to wildtype. The leaf thickness was similar to wildtype (Figure 3. Green tissue in areas with silenced ERL1 expression resembled wildtype sections when observed in the light microscope. Caption: epi = epidermis (lower and upper).6: Phenotypic analysis by light microscopy of Nicotiana benthamiana plants overexpress‐ ing ERL1 (A) In wildtype two layers of epidermal cells without chloroplasts cover the mesophyll cells with many chloroplasts aligned along the cell walls. vasc = vasculature. which could also be observed macroscopically when a green patch developed within a white leaf. Consistent with the inability for proper photosynthesis. When examined in greater detail (A) pal chl spo mes epi (B) epi mes epi 50 μm (C) pal epi chl mes spo vasc epi 50 μm 50 μm vasc (D) pal chl 50 μm epi epi spo mes epi Figure 3. The palisade cells had the usual elongated shape with sometimes slightly enlarged spaces between them. mes = mesophyll. Results scope. a normal number of chloroplasts and a leaf diameter comparable to wildtype. The cells possessed only few plastids which resembled undifferentiated pro‐ plastids of meristematic tissues.6 c). They contained only undeveloped thylakoid mem‐ branes which were dispersed inside the stroma without organization. (C) In green tissue the mesophyll organization is partly restored with more elongated palisade cells. there is no palisade layer detectable and the cells contain no chloroplasts. spo = spongy mesophyll.5 b). It consists of the upper palisade layer with tightly packed elongated cells arranged in parallel and the spongy layer with rounder cells without organiza‐ tion.
in the electron microscope one could observe some differences between wildtype and green tissue. They contained a comparable amount of chloroplasts with drastically enlarged grana in the latter, consisting of more than double the amount of thylakoid membranes than in wildtype. Since green and white tissues were collected from the same leaf, the increased amount of photosynthetic compartments in green areas probably compensates for the white sectors. The fact that green tissue is able to re‐ generate from white tissue supports the hypothesis of plastids in white areas being meristematic proplastids which can further differentiate into chloroplasts when nor‐ mal ERL1 levels are restored (Figure 3.5 c). Mosaic tissue combined both of the above described phenomena. In green patches the leaves were organized normally whereas in white patches they showed the dis‐ torted palisade mesophyll. Chloroplasts from green tissue had enlarged grana stacks next to cells containing chloroplasts with randomly dispersed thylakoids (Figure 3.6 d and Figure 3.5 d). 18.104.22.168 Effect of ERL1 overexpression on chloroplast mRNAs The strong effect of ERL1 overexpression on chloroplasts was further investigated by Northern analysis of chloroplast‐related gene transcripts. For this purpose green and white tissue was again collected independently and used for RNA extraction. The mRNA levels were compared to Nicotiana benthamiana wildtype total RNA, in addi‐ tion, some of the genes were also investigated in wildtype plants after infiltration and consecutive transient ERL1 overexpression. As negative controls for transient overexpression served wildtype material infiltrated with GFP or the erroneous ERL1 hairpin construct (see chapter 3.3.1), as negative control for transgenic plants and constitutive gene misexpression served a line which had also been transformed with the wrong ERL1 hairpin construct (compare chapter 3.3.1). ERL1 overexpression was investigated in wildtype, green and white tissue; from these only the latter showed detectable expression of ERL1. Several other genes which are also encoded in the nucleus and transcribed by Pol II had been investi‐ gated for their transcript levels (Figure 3.7 a). The Nicotiana gene PFTF codes for the
homologue of the metalloprotease FTSH2 from Arabidopsis thaliana which is respon‐ sible for the turnover of photosystem II components by proteolytic degradation (Summer & Cline, 1999). The expression of the gene was slightly increased in white tissue but even further upregulated in green tissue. This increase could correspond with the higher amount of photosynthetically active membranes in green tissue (compare Figure 3.5 c). In contrast, its antagonist CLPC showed comparable expres‐ sion in green and wildtype tissue, but slight upregulation in tissue overexpressing ERL1. Misexpression of unrelated genes lead to the same upregulation of CLPC, therefore this result was not considered as specific. Finally the level of the nucleus‐
Figure 3.7: Northern analysis of selected chloroplast‐related genes ERL1 (A) Chloroplast‐related proteins (A) transcribed in the nucleus by RNA PTFT Nucleus‐encoded Polymerase II (POLII). All three POLII transcripts genes of this group tested here CLPC (PTFT, CLPC and NEP) showed a slightly stronger expression in NEP white tissue compared to wildtype. There was no clear trend in green (B) CLPP tissue. (B) mRNA levels of chloro‐ Plastid‐encoded plastic genes transcribed by the NEP transcripts PEP chloroplast‐imported nuclear‐ encoded RNA polymerase (NEP) (C) RBCL were strongly upregulated in white Plastid‐encoded tissue, all other samples showed an PEP transcripts PSB expression comparable to wildtype. (C) The mRNA levels of two genes (RBCL and PSB) transcribed by the rRNA plastid‐encoded RNA polymerase (PEP) were strongly impaired in white tissue. Since chloroplastic rRNAs were almost missing (com‐ pare the rRNA loading control), translation of the upregulated PEP transcript had been low and PEP‐ dependent genes were probably not 1 2 3 4 5 6 7 transcribed normally. Due to the problems with the transgenic plants supposedly suppressed for ERL1 (see text) the tissue from these plants is regarded as control. Caption: 1 = infiltrated GFP overexpression in Nicotiana benthamiana wildtype plants, 2 = infiltrated ERL1 hairpin in Nicotiana benthamiana wildtype plants, 3 = presumable transgenic ERL1 suppressor plants, 4 = Nicotiana benthamiana wildtype plants, 5 = self‐silencer green tissue, 6 = self‐silencer white tissue, 7 = infiltrated ERL1 overexpression in Nicotiana benthamiana plants 111
encoded polymerase (NEP) which is transcribed and translated outside of the chloroplast and then imported therein was assessed as well. It was marginally upregulated in white tissue but the same amount of upregulation could also be de‐ tected in plants being transgenic for an unrelated gene; therefore this result was not considered as specific for constitutive ERL1 overexpression. NEP‐dependent transcripts were also investigated (Figure 3.7 b), one of them is the plastid‐encoded polymerase (PEP) which is the second active RNA polymerase in the chloroplast. Its expression was strongly upregulated in white tissue when compared to wildtype. Another gene, CLPP, which is coding for a protease subunit considered as indispensible for chloroplast development (Shikanai et al., 2001), showed an inter‐ esting transcription pattern in white tissue: instead of a single band a second larger one could be detected with both signals being stronger than in all other analyzed tis‐ sues which showed approximately equal expression. Finally PEP‐dependent transcripts were also investigated (Figure 3.7 c). These tran‐ scripts are mainly compromised of photosynthesis‐related genes and showed a se‐ verely reduced expression in white tissue. All other samples had a comparable ex‐ pression with the only exception of green tissue showing upregulated levels of the Ribulose‐1,5‐bisphosphate carboxylase oxygenase (RuBisCO) being responsible for the first step of carbon fixation in the chloroplasts . The fact that chloroplastic ribosomal RNAs are nearly absent in white tissue resulted in reduced total protein levels in these lines (compare Figure 3.4 g where in lane 8 the otherwise prominent RuBisCO protein band of 52.7 kDa is almost missing). 22.214.171.124 Effect of ERL1 overexpression on the photosynthetic apparatus Since overexpression of ERL1 in Nicotiana benthamiana plants macroscopically leads to a loss of chlorophyll and microscopically results in chloroplast alterations, its ef‐ fect on the photosynthetic apparatus was investigated in more detail. Leaf discs of Nicotiana benthamiana ERL1 overexpressors and wildtype plants were cut and dark‐ adapted in a humid chamber. After at least 15 minutes the fluorescence of chloro‐
Figure 3.8: Determination of photosynthetic parameters in Nicotiana benthamiana plants overex‐ pressing ERL1 The values are normalized to wildtype tissue, which is depicted as the circle in black. (A) The meas‐ ured fluorescence values of green tissue almost correspond to wildtype tissue except for TF(max), which is significantly lower. (B) In contrast, no phenotype tissue shows more differences, the most promi‐ nent being Sm , N and PI(csm). (C) White tissue shows no similarity with wildtype tissue, with major peaks for TF(max), F0/Fm, PHI(D0) and DI0/CS0. (D) Mosaic tissue looks like a mixture of white and green or wildtype tissue which is not easy to interpret, but the most prominent parameter is PI(csm) which is almost not existent.
phyll a was measured with a Handy‐PEA fluorophotometer. The results are shown in Figure 3.8; only the altered parameters are discussed explicitly (for further infor‐ mation please refer to Busotti et al., 2007). Parameters resulting from wildtype tissue were used as a reference and defined as value 1.0. As expected, white tissue from a self‐silencing plant did not share any
3. Bleach and mosaic did not show any normal photosynthetic behavior. Most prominent was the parameter PI(csm) which had been completely lost. Interestingly. Together with the results from above (see Figure 3. All parameters differed signifi‐ cantly and it was therefore assumed that no part of the photosynthetic apparatus was functional in white tissue (Figure 3.8 d). Not only the basic fluorescence F0 but also the maximal fluorescence Fm were slightly decreased.8 a). this extended period must have been the result of an‐ other intrinsic factor. Since the maximal fluorescence Fm and the number of chlorophyll a molecules (which could be concluded from parameter F0. Summarized it can be stated that the photosynthesis was less efficient in NP plants overexpressing ERL1 than in green tissue with silenced ERL1 expression. the number of reactions until the maximal fluorescence is reached.8 b). the fluorescence in the dark‐adapted state) were comparable to wildtype. green tissue of the same leaf showed an almost wildtype‐like photosyn‐ thetic behavior. described by the parameter TF(max) (Figure 3.8 c).7) it can be stated that the chlorophyll a molecules of green sectors show an enhanced photosyn‐ thetic activity compared to wildtype. 114 . The latter even showed an enhanced photosynthetic activity. The photosynthetic performance was lower than in wildtype. with one significant exception: the maximal fluorescence was reached later than in wildtype. this parameter describes the photosynthetic performance per leaf area (Figure 3. No phenotype (NP) tissue. although macroscopically not distinguishable from wild‐ type tissue. the energy which is needed for a complete reduction of all reaction centers and N. showed more differences than the reverted green tissue (Figure 3. Results similarity with photosynthetic behavior of wildtype. were higher than in wildtype. Mosaic tissue had been analyzed as well but its results were difficult to interpret its result since it contains a mixture of white cells with chaotic fluorescence parameters and green tissue with probably normal photosynthetic behavior. Sm.
Presumable knock‐out lines had been ordered from public seed collections and the overexpressing lines were created by floral dip transformation. Results 3.. which also corresponds to the promoter of the next gene. None of these lines showed any macroscopic phenotype compared to their back‐ ground line Col‐0. Line CS834430 contains the T‐DNA in an intron of the ERL1 gene and segregated with a ratio of 1:5. 115 . The two SALK lines (Alonso et al. Line CS834430 (McElver et al. but lines SALK_579265 and SALK_544378 revealed increasing si‐ lencing of the kanamycin resistance gene.5. Line SALK_544378 has the insertion in an exon of the ERL1 DNA sequence. a selective marker providing resistance to the herbicide BASTA®. this line did not segregate at all. Since this effect prevented the segregation on a selective medium seeds of these transgenic lines were grown on plates without an‐ tibiotics and selected by PCR analysis of the insertion site.7. observable as bleaching of the first leaves and consecutive death of the seedlings. the European Arabidopsis Stock Centre.4. a selective marker providing resistance to the antibiotic kana‐ mycin.3). homozygosity was further veri‐ fied by PCR analysis in the surviving plants.1 Knock‐down of ERL1 Three individual T‐DNA insertion lines (named CS834430.3. The T‐DNA insertions reside in different positions of the ERL1 sequence and showed a distinct segregation behavior: in line SALK_579265 the insertion lies in the pro‐ moter of the ERL1 gene and showed a segregation of 1:2. 3.4 Transgenic Arabidopsis thaliana plants Arabidopsis thaliana plant lines misexpressing ERL1 were used to confirm the results which had been obtained before in Nicotiana benthamiana plants (compare chapter 3.. 2003) contain the NPTII gene coding for neomycin phosphotransferase. SALK_579265 and SALK_544378) generated by vacuum infiltration of A. 2001) contains the BAR gene coding for phosphinothricin acetyl transferase. thaliana ecotype Columbia (Col‐0) were received from NASC. Line CS834430 was germi‐ nated on soil and transgenic individuals were selected by spraying the seedlings with 100 μg/ml of BASTA® on two consecutive days.
SALK_544378 and wildtype DNA probed for the NPTII gene. line SALK_544378 three insertions and line CS834430 eight insertions (com‐ 116 .3. quantitation curve and standard curve are shown.99894.9: Characterization of selected publicly available Arabidopsis thaliana ERL1 knock‐out plants (A) Southern analysis of lines SALK_579265. in the case of TUB9 the standard curve has an R2 value of 0. SALK_544378 and CS834430 with a probe specific for the respective resistance gene. There was a single line of background hybridization detectable in wildtype which is considered as plasmid contamination. there was no contamination of BAR DNA ob‐ servable in wildtype.3. 378 = SALK_544378. the probes were specific for the corresponding resistance‐providing genes. Exact quantitation is provided in the text and Table 3. Caption: 265 = SALK_579265. The left picture shows lines SALK_579265.99924. melt‐ ing curve. In contrast. In line SALK_579265 one additional band can be detected. Line SALK_544378 shows three strong bands corresponding to three copies of the kanamycin resis‐ tance gene. In the right. there is a possible second band but due to the segregation pattern this is unlikely. line CS834430 and wildtype DNA are probed for the BAR gene. Based on this analysis line SALK_579265 contains a single in‐ sertion. In the mutant plant eight bands could be detected while the wildtype DNA is free of background here. The high copy number does not correspond to the detected segregation rate of 1:5. In the case of ERL1 the standard curve possesses an R2 value of 0. 430 = CS834430. In the case of kanamycin resistance a contaminating band could be detected in wildtype. Results (A) (B) 265 378 wt 430 wt ERL1 expression TUB9 expression Figure 3. for the further determination of the copy number this band was subtracted in these two mutant lines.5 (B) Quantitative real‐time analysis of ERL1 expression in mutant and wildtype plants compared to the expression of TUB9. Due to the very low expression rate of ERL1 the threshold had to be set very high. wt = wildtype Southern analysis was used to determine the copy number of the inserted T‐DNA sequences.
From these the lines named 1.3: Summary of characteristics of Arabidopsis thaliana ERL1 knock‐down plant lines Line SALK_579265 SALK_544378 CS834430 Segregation 1:2. The plants showed resistance to kanamycin which was determined by germination and consecutive segregation on selective plates and PCR analysis of the NPTII gene.4. 2 and 4 were further propagated. therefore it was not excluded from further analysis. Plants of the T1 generation of the 16 individual Arabidopsis thaliana lines suppos‐ edly overexpressing ERL1 were pooled for RNA extraction. Lines SALK_579265 and CS834430 were analyzed for the exact amount of downregu‐ lation of ERL1 by qPCR. but is not analyzed yet.10 a).9 b). Slightly unequal loading could be observed in the former 117 . An ERL1‐hairpin‐ expressing line has been created by floral dip transformation at the end of this work.9 a). Cross‐hybridization cannot be excluded in the latter case since the line was subject to segregation. Table 3. seven lines showed weak overexpression and five lines were con‐ sidered as strong overexpressors (Figure 3. Therefore the obtained lines only provided a reduced ERL1 activity. The overexpression levels of ERL1 were determined by Northern and Western analy‐ sis.2 Overexpression of ERL1 Agrobacterium tumefaciens‐mediated floral dip transformation was used to produce 16 individual transgenic Arabidopsis thaliana lines constitutively overexpressing ERL1 with the strong CaMV 35S‐promoter.10 c). 15 μg of total leaf RNA were separated on a gel and investigated by Northern analysis. but no complete knock‐out (Figure 3.7 ‐ 1:5. Out of these 16 lines four did not show any detectable ERL1 expression.5 Insertions 1 3 8 Downregulation 13 % of wt no data 6 % of wt 3. the former having a remaining ERL1 expression of 13 % and the latter 6 % compared to wildtype. radioactively labeled ERL1 DNA served as a probe. Few individuals out of three lines exhib‐ ited some bleaching and a slightly stunted growth when compared to wildtype (Figure 3.3. Results pare Figure 3.
The trans‐ genic overexpression of ERL1 resulted in two distinct protein bands. as a loading control served Ethidium bromide‐staining of 28S rRNA. but both had also shown the above described phenotype and were therefore chosen as well for further analysis. 1‐1 & 1‐2 T2 lines of 1. The progeny of these three lines was again selected for overexpression and the char‐ acteristics of ERL1 overexpression further identified by Western analysis (Figure 3. (C) A sample plant of Arabidopsis thaliana line 1 overexpressing ERL1 shows slight bleaching in the leaf tips (left plant. These two bands most likely correspond to plant ERL1 before and after maturation. as a loading control served Coomassie staining of the gel after transfer. (B) Lines 1. 4‐1 & 4‐2 T2 lines of 4. Transgenic overexpression of ERL1 resulted in two bands detectable in all lines. M = Bio‐ Rad Prestained SDS‐PAGE standard 118 . The strongest overexpression could be detected in line 1. Line 1 was found to be the strongest overexpressor. a major band of approximately 42 kDa and a minor band of approximately 35 kDa.3. lower). arrows) and a delayed growth compared to wildtype (right plant). 2 and 4 were propagated to the T2 generation and their ERL1 expression level was investigated with Western blotting.10 b). which showed approximately the same expression levels.9 kDa 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 (C) wt 1‐1 1‐2 2‐1 2‐2 4‐1 4‐2 M Figure 3. The latter line was still seg‐ regating since there was no expression of ERL1 detectable in plant 4‐1.1 kDa 34.10 a.10: Analysis of Arabidopsis thaliana plants overexpressing ERL1 (A) Northern analysis of 16 Arabidopsis thaliana lines overexpressing ERL1 revealed detectable ERL1 levels in twelve lines. consequently containing or missing the approxi‐ mately 8 kDa leader peptide. Results two when compared to the other 14 lines (compare Figure 3.8 kDa 28. followed by line 2 and 4. (A) (B) 49. Caption: 01‐16 = T1 lines. 2‐1 & 2‐2 T2 lines of 2. a major band of approximately 42 kDa and a minor band of approximately 35 kDa with less than half of the expression of the other band.
. 2008).11: Effect of plant ERL1 on different molecules of the RNA silencing apparatus.5 Effect of ERL1 on silencing The Caenorhabditis elegans homologue of ERL1 was first described to have an effect on RNA silencing. was published recently (Ramachandran & Chen. (A) Two individual crosses of ERL1 overexpressing plants with the metastable GFP‐expressing line 6.4 that had been created and described earlier by our lab (Kalantidis et al. G = green tissue of “Self‐silencer” plants. 3. Neuronal cells which are usually RNAi‐recalcitrant could be sub‐ jected to RNA silencing in eri1 mutants. (A) (B) wt G W A Figure 3. ERL1‐overexpressing Nicotiana benthamiana plants were crossed with the GFP‐expressing line 6. A = tissue collected after transient ERL1 overexpression by agroinfiltration.8S cytoplasmic rRNA is depicted as a loading control. systemic silencing spread could be observed leading to a completely silenced plant in the background of the right picture with a single branch in the foreground still expressing GFP.9 of the introduction).3. in addition. hence its name ERI1 (enhanced RNAi 1) (compare chapter 1.1 Crosses To test the involvement of ERL1 in RNA silencing processes.2. Therefore it can be used as a reporter line for testing the effect of other factors on RNA silencing. the effect of exonucleases on the degradation of miRNAs. therefore ERL1 was investigated for both phenomena. Spontaneous silencing onset (red spot in the left picture) was not influenced. The slightly weaker signal in green tissue was not considered as specific since there was high resistance in this part of the gel. W = white tissue of “Bleach” plants.4 shows frequent spontaneous silencing of its GFP transgene which usually also leads to sys‐ temic GFP silencing spread. Line 6. Caption: wt = wildtype.4 revealed that there was no effect of ERL1 overexpression on siRNA‐mediated RNA silencing in plants. EtBr staining of 5. Results 3. In addition.5. 2006). another mole‐ cule of the silencing pathways. 119 . probably leading to the smeary signal which can be observed above the detected band. (B) Northern analysis of 20 μg total RNA and hybridization with miRNA‐159 LNA showed a similar signal in all analyzed plant lines.
In the “green” tissue miR159 lev‐ els appear to be marginally reduced compared to wildtype.. Schumacher. The introduction of ERL1 into the reporter line did not alter its silencing characteristics. In F2 double homozygous plants were selected and monitored for their GFP silencing behavior. did not result in suppression of transgene silencing. Figure 3.11 b. three of them already in early stages.4 before cross‐ ing with ERL1 overexpression. benthamiana plants of line 6. Out of five N. The results are depicted in Figure 3. total RNA of plants overexpressing ERL1 was investigated for the expression level of miR159.4) were performed and their F1 generation was selected for expression of both transgenes. 2002). therefore the gene was considered to have no effect on endogenous gene silencing in plants. an abun‐ dant miRNA family repressing MYB‐domain containing transcription factors (Mette et al. in another experiment overexpression of ERL1 was able to quench the amounts of viroid‐related siRNAs (see supplementary results.3. Interestingly. but the low quality of the hybridization signal for this sample does not allow drawing safe conclusions. Results Two individual crosses in both directions (lines 6. three of them already in early stages. The same result had been observed after transient overexpression in co‐ infiltration assays. All plants of the six double‐homozygous F2 plant crosses showed onset of GFP silencing. 2009) 3.4 x ERL1+ and ERL1+ x 6. the expression was comparable in white tissue constitutively overexpressing ERL1 and wildtype tissue transiently overex‐ pressing ERL1 by agroinfiltration.5.4 (without in‐bred ERL1) which were grown in parallel.2 LNA159 To test for the possibility of ERL1 having an effect on miRNAs. in contrast to the identified viral silencing suppres‐ sor P19. where ERL1.11 a shows two examples of GFP silencing identical to line 6. 120 . In both overexpressing tissues miR159 expression seemed to be even a bit stronger than in wildtype. all showed onset of GFP silencing.
12 a.1 rRNA blots In a first approach the expression levels of small plant rRNAs were assessed by com‐ paring Northern hybridizations of Nicotiana benthamiana wildtype tissue with tissue overexpressing ERL1 (green tissue of self‐silencing plant lines. white tissue of bleach plants and wildtype tissue after transient overexpression of ERL1). how‐ ever. 3. One should note that in the case of white tissue the amount of loaded RNA was reduced by an empiri‐ cally determined arbitrary factor of 0. Two different approaches were used: the immediate effect of tran‐ sient ERL1 overexpression was detected by Northern analysis.12 a. middle & c).6. Since it was reported that homologues of ERI1 take part in 5. Chloroplastic 4. left & b). right & d). Results Summarized it can be stated that ERL1 overexpression did not have a significant negative effect on the expression of miR159.7. In addition ribosomal RNA of transgenic plants misexpressing ERL1 were analyzed by cloning analysis. 3.5S rRNA was significantly reduced in white tissue but did not show any alterations in expression levels for the other tissues when compared to wildtype (Figure 3. was not only almost lost in white tissue but also showed decreased expression levels in tissue where ERL1 was transiently overexpressed by agroinfiltration (Figure 3.8S rRNA did not show any effect after transient ERL1 overexpression (Figure 3. preventing an excessive loading of the resid‐ ual RNAs due to the almost total lack of chloroplastic RNA.3. Cytoplasmic 5. Chloroplastic 5S rRNA.6 Effect of ERL1 on ribosomal RNA In bleach plants strongly overexpressing ERL1 an impaired ribosomal RNA pattern could be detected. while it was unaffected after ERL1 overexpression (Figure 3.12 e). other plant miRNAs had not been tested since there was no effect expected either.12 a. As the expression after ERL1 overexpression was comparable on the 121 . 16S rRNA appeared to have been lost after GFP expression.8S rRNA ripening in Caenorhabditis elegans and Mus musculus this effect was also inves‐ tigated in plants. Since overexpression of GFP should not have an effect on chloroplastic 16S rRNA this result was not consid‐ ered as specific.
Caption: D1 = first day after agroinfiltration. in the lower panel 28S mitochondrial RNA is used as a loading control. D5 = fifth day after agroinfiltration.8S D1 D5 GFP (F) D1 D5 D1 D5 ERL1 GFP 16S D1 D5 D1 D5 ERL1 GFP 23S Figure 3. A = overexpression of ERL1 by agroinfiltration 122 .5S A wt (C) G 5S W A wt (D) G W 5. unequal loading is visible in the loading control.8S A D1 D5 D1 D5 ERL1 GFP 4. (A) Total RNA of tissue overexpressing ERL1 was separated on a Northern gel and probed for the corresponding small rRNA. In the left ERL1 overexpression. (A) wt (B) G W 4. therefore it is considered to be unaffected.5S (E) D1 D5 ERL1 5S D1 D5 GFP D1 D5 ERL1 5. W = white tissue of self‐silencer and bleach plants. (B) – (F) Agrobacteria overexpressing ERL1 or GFP (acting as a control construct) were infiltrated into Nico‐ tiana tabacum leaves and tissue was collected after one and five days. (D) Cytosolic 5.5S rRNA was not affected by the tran‐ sient overexpression of any construct. an effect of ERL1 on 16S rRNA is unlikely.5S and 5S rRNA. 23S rRNA seemed slightly upregulated after ERL1 overexpression which can probably be attributed to unequal loading of these samples (Figure 3. White tissue showed no expression of chloroplastic 4. (C) 5S rRNA had a decreased expression level on the fifth day after transient ERL1 overexpression.3. However. G = RNA of green tissue of self‐silencing plants. (E) Chloroplastic 16S rRNA was not affected after transient ERL1 overexpression but could not be detected after GFP overexpression which was probably due to a problem in Northern hybridi‐ zation.12 f). in addition wildtype tissue after transient overexpression of ERL1 had decreased levels of 5S rRNA.12: Northern analysis of chloroplastic ribosomal RNAs and cytosolic 5. wt = RNA of wild‐ type Nicotiana benthamiana tissue. in the right GFP overexpression is depicted: (B) Chloroplastic 4. (F) Chloroplastic 23S rRNA showed upregulation after overexpression of ERL1. Results first and fifth day after infiltration.8S rRNA showed no effect after transient overex‐ pression.8S rRNA following overexpression of ERL1 In the upper panel the hybridization with the respective rRNA probe is depicted.
This linker was then specifically ligated to the 3’‐ends of total rRNA extracted from Nicotiana benthamiana and Arabidopsis thaliana leaves.8S rRNA as a substrate it was analyzed by self‐ligation and subsequent cloning with specific primers for this ribosomal RNA. Ansel et al.8S rRNA was analyzed by self‐ligation of total RNA of N.8S rRNA Although ERL1 was found to be targeted to the chloroplasts.3.6.8s-01t Nb_ERL1-OE_5. For this reason two different approaches were used: to test for a general effect the total RNA was self‐ligated and the resulting product was cloned and sequenced to see an extension at the ends.8S rRNA from Nicotiana benthamiana plants overexpressing ERL1 5.8s-04 Nb_wt_5.8s-06 Nb_ERL1-OE_5.8s-03 Nb_wt_5. To test if plant ERL1 has 5.8s-02t Nb_ERL1-OE_5.2. the role of its ortholo‐ gous genes in animals in cytoplasmic 5. benthamiana. To specifically determine the 3’‐location of the exten‐ sions linker fragments were designed and synthesized using non‐plant sequences (in this case the bilbo transposon sequence from Drosophila melanogaster). 2008. 2008) prompted us to address this issue also in plants. benthamiana wildtype and overexpressing ERL1 plants and subsequent cloning. Caption: Nb_wt = N.8s-03t Consensus (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 98 CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT Figure 3.8S rRNA metabolism (Gabel & Ruvkun. 1 Nb_wt_5.8s-05 Nb_wt_5.2 Linker ligations To determine alterations in the sequence of ribosomal RNAs.13: Alignment of cytosolic 5. Nb_ERL1‐OE = overexpression of ERL1 in N.8s-02 Nb_wt_5. There was no effect detectable in seven sequences derived from plants either transiently or consti‐ tutively overexpressing ERL1 (compare Figure 3.13).8s-01c Nb_ERL1-OE_5.8s-01 Nb_wt_5. t = transient overexpression 123 . It was modi‐ fied at its 3’‐end with dideoxy‐Cytosin to protect this end from ligation. Neither the sequence of the mutant with a severe phenotype nor the sequence cloned after transient overexpression showed any differences compared to wildtype.1 Effect on 5. c = constitutive overexpression.8s-02c Nb_ERL1-OE_5.8s-04c Nb_ERL1-OE_5.8s-03c Nb_ERL1-OE_5. Results 3..6. benthamiana wildtype. it had to be cloned without imposing bias on the cloned ends (as would be the case through direct PCR). 3.
It should be noted that the published 16S rRNA sequence contains two extra Thymidines at its 3’‐end. Overexpression of ERL1 showed stronger effects than the knock‐down of ERL1 which only had consequences on 16S rRNA processing.4. 5S rRNA sequences could be roughly divided into two groups: one with additional nucleotides derived from the 5S precursor sequence which were presumable not trimmed properly and one with additional novel nucleotides. Interest‐ ingly.14 c).1) it is concluded that the plant homologue is unlikely to possess the same substrate as in the animal kingdom. Several sequence alterations could be detected. but cloning of 16S rRNA from Nicotiana benthamiana and Arabidopsis thaliana total RNA of wildtype plants proved that the correct 16S rRNA sequence in our experimental setup did not contain these two Thymidines at its 3’‐end. This was due to the fact that Arabidopsis thaliana plants overex‐ pressing ERL1 and Nicotiana benthamiana plants suppressing ERL1 were not available at the time of the analysis.2. There was no effect in ERL1‐KD Arabidopsis 124 .1 the strongest effect of ERL1 overexpression could be detected in 5S chloroplastic rRNA (Figure 3. The obtained results are summarized in Table 3.6.14 b).2 Effect on chloroplastic rRNAs Since plant ERL1 is targeted to the chloroplast its effect on all four chloroplastic rRNAs was tested. predominantly in chloroplastic 5S rRNA. overexpression of ERL1 did not have any effect on 16S chloroplastic rRNA (compare Figure 3.3. Results Since 5. Transient overexpression was used as well in order to prevent the detection of artifacts due to the detrimental effects of strong consecutive overexpres‐ sion of ERL1 in Nicotiana benthamiana. this corresponded to 33 % of the sequences (Figure 3. Two out of six 16S rRNA sequences showed an extra Thymidine at their 3’‐end compared to the cloned wild‐ type sequences.12 e). 3.6. whereas the effect of ERL1 suppression could only be assessed in Arabi‐ dopsis thaliana plants. the effect of ERL1 overexpression was only assessed in Nicotiana bentha‐ miana plants.8S rRNA is located in the cytoplasm and the enzyme is supposed to be tar‐ geted to the chloroplast (see chapter 3. As already described in chapter 3.
Results thaliana plants detectable. The summarized description of this sequence alteration is an 125 . comprised of an extra –ACC motif. In one case there were three extra nucleotides detected.5S 5S 16S 23S In three cases this was a clear extra –AC. in ones case it could not be concluded from the sequencing data whether the first of the additional nucleotides was an Adenosine or a Cytosine. Table 3. In Nicotiana benthamiana plants constitutively overexpress‐ ing ERL1 five out of eight sequences showed an addition of novel nucleotides at the 3’‐end of the rRNA.3.4: Summary of sequence alterations in chloroplastic rRNA after ERL1 misexpression in Nicotiana benthamiana (Nb) and Arabidopsis thaliana (At) plants rRNA Type of ERL1 Species species misexpression constitutive overexpression transient over‐ expression constitutive knock‐down constitutive overexpression transient over‐ expression constitutive knock‐down constitutive overexpression transient over‐ expression constitutive knock‐down constitutive overexpression transient over‐ expression constitutive knock‐down Total: Nb Nb At Nb Nb At Nb Nb At Nb Nb At Number of Type of Number of Ratio analyzed alteration alterations clones 5 4 4 8 12 9 10 no data 6 4 no data 6 68 none none none extra – A/C C (C) extra –CC extra –GA none none no data extra ‐T deletion of –CT no data none 0 0 0 5 1 3 0 0 no data 2 1 no data 0 12 0 % 0 % 0 % 63 % 8 % 25 % 0 % 0 % no data 33 % 25 % no data 0 % 18 % 4.
In 23S chloroplastic rRNA only one sequence from constitutively overexpressing ERL1 plant tissue showed a sequence alteration by missing two nucleotides at the 3’‐ end compared to the wildtype sequence (Figure 3. Since one of them is a wildtype sequence. Nb_ERL1‐OE = overexpression of ERL1 in Nicotiana benthamiana. In addition.14 d).5s-01 (1) ------------------------------------------------TAGGCATCCTAACAGACCGGTAGACTTGAAC 126 .14 a).5s 14 113 4.5s-04 Nb_wt_4. AT_ERL1‐KD = knock‐down of ERL1 in Arabidopsis thaliana. three wildtype sequences were analyzed and showed a two nucleotide deletion compared to the published sequence. (C) Six sequences with down‐ regulated ERL1 and ten sequences overexpressing ERL1 were analyzed for chloroplastic 16S rRNA.5s-01 At_ERL1-KD_4. Compared to this sequence two sequences with downregulated ERL1 pos‐ sessed an extra Thymidine at their 3’‐end. From twelve plants transiently overexpressing ERL1 four sequences possessed an extra two‐nucleotide motif which was either –CC or –GA. Figure 3. In total 68 cloned sequences had been analyzed and showed sequence alterations in 18 % of all cases. Results extra –A/C C (C).3.5s-03 At_ERL1-KD_4.5s-02 At_ERL1-KD_4. (A) Four sequences with downregulated ERL1 and nine sequences overexpressing ERL1 were ana‐ lyzed for chloroplastic 4. Of the analyzed plants with downregulated ERL1 none showed a sequence alteration com‐ pared to wildtype. this deletion is not considered as specific for ERL1 misex‐ pression.5S rRNA (Figure 3. t = transient overexpression (A) 4. From twelve sequences derived after transient overexpression of ERL1 four exhibited sequence alterations.5s+linker (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAACATCGTCACAACAAATGGCATC (14) (14) (14) (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC At_ERL1-KD_4. One of the sequences showed the same behavior as the constitutively overexpressing plants by containing an extra –CC mo‐ tif. (B) Five wildtype sequences.14: Alignment of chloroplastic rRNAs of Arabidopsis thaliana and Nicotiana benthamiana plants misexpressing plant ERL1 Chloroplastic rRNAs of wildtype plants as well as plants misexpressing ERL1 were ligated to a modi‐ fied linker sequence at their 3’‐end. Nb_wt = Nicotiana benthamiana wildtype. nine sequences with downregulated ERL1 and twenty sequences over‐ expressing ERL1 were analyzed for chloroplastic 5S rRNA. (D) Six sequences with downregulated ERL1 and four se‐ quences overexpressing ERL1 were analyzed for chloroplastic 23S rRNA and showed a single se‐ quence alteration compared to wildtype. From eight plants constitutively overexpressing ERL1 five sequences showed an extra –A/C C (C) sequence motif. For better visu‐ alization only the last 100 nucleotides of the sequences are depicted. Two sequences showed a single deletion. They are compared to the published sequences of the respective rRNA plus the linker sequence (named according to the corresponding rRNA+linker). There was no effect of plant ERL1 detectable on the processing of chloroplastic 4. One plant constitutively overexpressing ERL1 had a two nucleotide deletion at the 3’‐end. Caption: At_wt = Arabidopsis thaliana wildtype. c = constitutive overexpression. The other three altered sequences possessed an extra –GA which corresponds to the precursor sequence of 5S chloroplastic rRNA.5S rRNA and did not show any sequence alterations compared to wildtype.
5s_04t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC (B) 5s 19 119 5s+linker (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT---ATCGTCACAACAAATGGCATC (6) (15) (9) (9) (17) (17) (17) (16) (1) ACTTGGTGGGTTAAACTCTATCTGCGGTGACGATGCTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGCGTCGACGCTAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGAAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTKGGTTAAACTCTA-CTGCGGTGACGATACTGTCGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT-------------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--- At_ERL1-KD_5s-01 At_ERL1-KD_5s-02 At_ERL1-KD_5s-03 At_ERL1-KD_5s-04 At_ERL1-KD_5s-05 At_ERL1-KD_5s-06 At_ERL1-KD_5s-07 At_ERL1-KD_5s-08 At_ERL1-KD_5s-09 Nb_wt_5s-01 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGA---Nb_wt_5s-02 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCG-AAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-03 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-04 (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-05 (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-01c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-02c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATMCNb_ERL1-OE_5s-03c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-04c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-05c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGA---Nb_ERL1-OE_5s-06c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACC Nb_ERL1-OE_5s-07c (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGAGAGGTCCTGCGGAAAAATAGC-TCTACGCCAGGAT--Nb_ERL1-OE_5s-08c (16) ACTTGGTGG-TTAAACTCTA-CT-CGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-01t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-02t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-03t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-04t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-05t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-06t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCTGGATCCNb_ERL1-OE_5s-07t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TTGACGCCAGGAT--Nb_ERL1-OE_5s-08t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-09t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-10t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-11t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-12t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGAGACTGTAGGGGAGGTCCCGCGGAAAAATAGC-TCGACGCCAGGAT--- (C) 16s 182 281 16s+linker (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTTATCGTCACAACAAATGGCATC (174) (182) (161) (182) (182) (162) (182) (182) (182) (182) (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTGCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTGCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGNGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGTCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGGAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGCAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT— At_ERL1-KD_16s-01 At_ERL1-KD_16s-02 At_ERL1-KD_16s-03 At_ERL1-KD_16s-04 At_ERL1-KD_16s-05 At_ERL1-KD_16s-06 At_ERL1-OE_16s-01 At_ERL1-OE_16s-02 At_ERL1-OE_16s-03 At_ERL1-OE_16s-04 At_ERL1-OE_16s-05 At_wt_16s-01 (166) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-Nb_wt_16s-01 (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-Nb_wt_16s-02 (95) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT— Nb_ERL1-OE_16s_01c Nb_ERL1-OE_16s_02c Nb_ERL1-OE_16s_03c Nb_ERL1-OE_16s_04c Nb_ERL1-OE_16s_05c (182) (182) (161) (125) (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGGAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-- (D) 23s 198 297 23s+linker (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCTATCGTCACAACAAATGGCATC (198) (198) (198) (198) (198) (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGGATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT At_ERL1-KD_23s-01 At_ERL1-KD_23s-02 At_ERL1-KD_23s-03 At_ERL1-KD_23s-04 At_ERL1-KD_23s-05 At_ERL1-KD_23s-06 Nb_wt_23s-01 (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT Nb_ERL1-OE_23s_01c Nb_ERL1-OE_23s_02c Nb_ERL1-OE_23s_03c Nb_ERL1-OE_23s_04c (198) (198) (198) (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGACGAGTGCTCTC-CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT Caption: identical.5s_03t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4. conserved. Results Nb_ERL1-OE_4.5s_04c (1) ---------------------------------------------GCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_01c (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.3. linker 127 .5s_02c (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_03c (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_01t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_02t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_05c (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.
Iida et al. This hypothesis was initially supported by several hints (Schumacher.. One of the first observations had been the significant reduction of viroid‐derived siRNAs in PSTVd‐infected plants after over‐ expression of ERL1.2. supplementary results 6.4). Therefore a similar function in plant RNA silencing processes has been impli‐ cated. They follow various strategies.4. So far no plant homologue of ER1‐1 has been described in the literature. which has been termed ERI‐1_3’hExo_like EXOIII domain. This implicated a specific sequestering of siRNAs by ERL1. 2009. 2006.. The proteins are characterized by an exonuclease domain with a functional DEDDh motif. Although there have been several reports of endogenous suppressors of silencing (compare chapter 1. 2004. Yang et al. one cannot exclude the possibility that the strong overexpression level which prevails after agroinfiltration would have the same effect with any other ex‐ 129 . the observed effects upon their loss‐of‐ function are mainly secondary and not the result of a specific loss of the suppression of RNA silencing suppression. 6. A single lo‐ cus could be identified in Arabidopsis which shares significant homology to the al‐ ready described ERI‐1‐like proteins and contains the ERI‐1_3’hExo_like EXOIII do‐ main. An exception presents the protein ERI‐1 from C. Discussion 4..1 Implications of plant ERL1 in RNA silencing processes In the past years RNA silencing mechanisms have proven to be involved in the regu‐ lation of numerous cellular pathways.2. elegans and its homologues which have been shown to specifically degrade siRNAs in vitro (Kennedy et al. Kupsco et al. So far only viral sup‐ pressors of silencing have been identified to specifically counteract the silencing mechanism of their hosts in order to circumvent its function as antiviral immune sys‐ tem. one being the specific sequestering of siRNAs. 2006) and therefore suppressing the extent of silencing in diverse tissues in vivo. This ubiquitous involvement certainly raises the question about the endogenous control of RNA silencing.3 & 6.. However.2.8 of the introduction).2.1. 2006.
therefore pro‐ viding a more sensitive system to investigate any mild silencing suppressor effects. Another finding had been the fact that N. Therefore another approach has been used by crossing ERL1‐overexpressing N. 2009). supplementary re‐ sults 6.2). Despite these findings several other results contraindicated an implication of plant ERL1 in RNA silencing: agroinfiltration assays have earlier been described as a method for the description of RNA silencing suppressor activity (Brigneti et al. the additional overexpression of a GFP transgene into a GFP‐expressing plant line leads to co‐suppression by using the in‐ trinsic RNA silencing pathways. One has to note. The onset of this GFP silencing is probably characterized by overcoming a threshold of aberrant transgene RNA. Discussion onuclease.5. 2009. In the case of ERL1 no effect could be detected after its transient overexpression by co‐ infiltration with the respective GFP construct (Schumacher. 130 .. that these assays are rather insensitive and have a low tempo‐spatial resolution.2. Two distinct mechanisms are exploited. The strongest result impli‐ cating an effect of plant ERL1 on RNA silencing processes is the fact that the systemic silencing of the ERL1‐overexpression by introducing an ERL1‐hairpin by agroinfiltra‐ tion is only successful in 50 % of the plants.1).4 which is de‐ scribed by initiation of spontaneous GFP silencing and subsequent systemic GFP si‐ lencing spread (Kalantidis et al. however. nei‐ ther the initial onset. benthamiana plants overexpress‐ ing ERL1 showed a hypersensitivity to infection by Plum pox virus which usually has only mild symptoms in planta. did not show any differences in GFP silencing. nor its spread including the systemic spread of silencing are negatively influenced by the overexpression of plant ERL1 (compare chapter 3. The rest fails to maintain systemic silenc‐ ing and reverts back to the bleach phenotype typical of ERL1‐overexpression (Schumacher. The resulting crosses.4. benthamiana plants with line GFP 6. Taking into account the weak constitution of the se‐ vere ERL1‐overexpressing plants this hypersensitivity might not specifically be a re‐ sult of ERL1‐overexpression but rather a secondary effect. The use of a GFP‐suppressing hairpin circumvents the necessity of RNA silencing amplification by RDR‐dependent mechanisms. 1998).. 2006). however.
The SAP domain. which confers binding to nucleic acids. 2008). is the distorted systemic silencing in ERL1‐overexpressing plants. It can possibly be accounted to the fact that the levels of DCL‐1 and DCL‐4 transcripts are. Interestingly. the Arabidop‐ sis homologue would also associates with the group II proteins. Up to now there is no clear explanation whether this is a specific result of ERL1 overexpression or a secondary effect of the poor constitution of the plants. severely downregulated in plants which overexpress ERL1 (Schumacher. In this group I all members with described effects of ERI‐1 on RNA silencing are included. sea urchin and humans (Tomoyasu et al. elegans.g. 2008). therefore it probably also possesses a function apart from RNA silencing (Kupsco et al. in contrast to other nuclear mRNAs. maybe explaining the fact that it is not involved in plant RNA silencing processes. The second group (group II) is character‐ ized by the Drosophila Snipper protein. Snipper has not proven to be involved in RNA silencing processes in vivo.4. In conclusion. Discussion The only result pointing towards an involvement of ERL1 in RNA silencing in plants. 4.2 Involvements of ERL1 in chloroplast metabolism Due to the low expression of plant ERL1 a minor regulatory impact in vivo is imagin‐ able. Interestingly. Both groups contain the ERI‐1_3’hExo_like EXOIII domain with a high degree of conservation. it can be stated that the protein likely does not have an effect on sup‐ pression of RNA silencing in plants but rather a distinct function compared to ERI‐1 in other species. 2009).. since some transcripts may always escape the 131 . humans and also a yet to be analyzed protein of sea urchin (Strongylocentrotus purpuratus). In general.. 2006). from the already published ERI‐1 homologues also Snipper in Drosophila did not show an enhanced RNAi phenotype upon its loss‐of‐ function. is only present in a subset of ERI‐1 homologues. e. a complete loss of a gene is hard to accomplish either with insertion mutants or with hairpin constructs.. Based on phylogenetic analysis two distinct subclasses of ERI‐1 homologues have been identified in a study for RNAi‐related proteins in Tribolium castaneum (Tomoyasu et al. other yet to be analyzed homologues origi‐ nate from Tribolium. homo‐ logues which are found in C.
2005). they code for photo‐ synthesis‐related proteins. 2006). In the case of low gene transcription a detectable effect of this knock‐down is hardly obvious (compare chapter 3. The gene expression retained many prokaryotic character‐ istics leading to polycistronic transcripts which are mainly regulated post‐ transcriptionally.. Therefore our approach used the overexpression of the gene in addition to the incomplete knock‐out. In addition. The corresponding proteins are imported into the chloro‐ plasts by several import mechanisms and take part in their regulatory pathways. Discussion downregulation.1 of the result section). This has been proven by pre‐ paring a carboxy‐terminal fusion to GFP followed by confocal microscopy.4.3. Ever since the endosymbiotic incorporation of photosynthetically active prokaryotes. during evolution chloroplasts have transferred most of their genes to the nucleus.. but first conclusions to the influenced pathways can be drawn. After constitutive overexpression the resulting transgenic plants showed a distortion in the chlorophyll levels.. chloroplasts still contain a fully functional gene expression machinery which is mostly encoded in the chloroplast itself. In addition. in order to identify possible targets of the protein. Due to its chloroplastic localization and its exonuclease activity it is likely that ERL1 is a player in this post‐transcriptional editing of chloroplastic RNAs. Strong effects following overexpression of ERI‐1 homologues have been reported earlier (Bühler et al. Almeida et al. Therefore this chloroplastic localiza‐ tion is considered as an additional proof for ERL1 having a function apart from RNA silencing in plants.4. Secondary effects of this overexpression cannot be excluded of course. chloroplasts contain many eukaryotic proteins independent of the chloro‐ plast prokaryotic origin. 132 . Interest‐ ingly the chloroplast is considered as an intracellular compartment which is free of RNA silencing processes (Hegeman et al. Subsequent in silico analysis of the protein revealed a possi‐ ble chloroplast targeting signal at its amino‐terminus. Nevertheless.2 of the introduction). The maturation of chloroplastic transcripts includes several endo‐ and exonucleolytic cleavage steps (compare chapter 1. 2005.
trichocarpa contain a chloroplast targeting signal. Although this may represent a functional diversification of plant ERL1 proteins. therefore making pure coincidence unlikely. In the case of P. 2003).13 of the introduction) they were probably added independently after splitting into the different plant lineages. There‐ fore it cannot be excluded that ERL1 proteins from sorghum and poplar serve dis‐ tinct functions. The variegation pattern had been shown to be influenced by environmental factors such as light and temperature (re‐ viewed in Sakamoto. In the case of sorghum this is already obvious from the fact that an amino‐ terminal methionine is missing from the sequence. In addition. incomplete annotation cannot be excluded. 133 . this behavior could also be seen in our experiments al‐ though due to the greenhouse conditions it cannot be stated if intense light or higher temperature or both caused the more intense bleaching in the summer months. Discussion From published EST collections probably all plant ERL1 sequences except S. bicolor and P. The resulting leaves contain nor‐ mal green sectors and white sectors with non‐canonical chloroplasts resembling un‐ differentiated proplastids. cells usually contain either canonical or dis‐ torted plastids in the variegated phenotypes. the up‐ stream region of the annotated gene was analyzed and a possible chloroplasts target‐ ing signal immediately upstream of the annotated ERL1 sequences could be identi‐ fied. This variegation has been described before as a result of mutations in photosynthesis‐related and other plastid genes. it is interesting that both major plant lineages of monocoty‐ ledons and dicotyledons contain members which possess a predicted chloroplastic localization of ERL1. Since chloroplasts are not synthesized from scratch after plant cell division but rather divide.4. 4. Due to the fact that the chloroplast targeting signals show no conservation be‐ tween the different plant species (compare Figure 1. However. trichocarpa this is not the case but since the complete genomic sequence of poplar is available.3 Severe phenotypic alterations after overexpression of ERL1 suggest an involvement in chloroplast development The overexpression of ERL1 resulted in various phenotypes with a slow and stunted growth. all phenotypes were characterized by the partial or full bleach‐ ing of the plants.
since it suggests that chloroplast development is only blocked reversibly in white tissue and not lost in these cells. There‐ fore there is an accumulation of PEP transcript detectable.3.2. Discussion The analysis of ERL1 transcript levels in the mutant plant lines revealed that the se‐ verity of the bleaching clearly corresponds to the amount of ERL1 present in the plants. The commonly observed phenomenon of phenotype reversion back to wild‐ type phenotype was accompanied by the degradation of the ERL1 mRNA (compare Figure 3. responsible for tran‐ scription of the second polymerase. green tissue ap‐ pears to have an enhanced photosynthetic activity (compare chapter 3.. Bol‐ lenbach et al. The two sequences do not share an identical stretch of 21‐nt which would result in siRNAs able to silence the endogenous sequence too. it is not clear whether the endogenous ERL1 of N. However. immature rRNAs are considered to be non‐functional for ribosome assembly. comparable to systemic silencing of a trans‐ gene (compare chapter 1. 2003.. Rodio et al..4. It has to be noted that PEP‐dependent tran‐ 134 . In addition to the morphological phenotype the transcription of chloroplastic genes is distorted in the mutant plant lines. accompanied by a lack of ribosomal RNAs which are themselves a product of PEP transcription. In addition. Interestingly. in the case if endogenous ERL1 is silenced as well.3. Bellaoui & Gruissem.2 and 3. amongst others.7 of the results). the hyperactive photosynthetic apparatus might be a result of loss of ERL1.3 of the results section).2. 2005. Inter‐ estingly.4 & Figure 3. either to compensate for the residual plant which is still characterized by white leaves or. Bisanz et al. benthamiana is silenced as well in this phenotype. The transcription by the former is independent of chloroplastic molecules whereas the translation of the gene products depends on the ribosomes of the chloroplasts. Two individual polymerases are responsible for chloroplastic gene transcription: the nucleus‐encoded plastid RNA polymerase (NEP) is imported into the chloroplasts and. The pos‐ sible reversion of the phenotype is also interesting. 2007).6 of the introduction). distorted chloroplastic rRNAs have previously been reported to cause varie‐ gated phenotypes (Barkan. 1993.2. since the Arabidopsis sequence had been used for the preparation of the overexpressing plants. 2004. the plastid‐encoded RNA polymerase (PEP).
. 1997. One line presented an exception to the direct connection between the severity of the phenotype and the ERL1 overexpression levels. Discussion scription is not completely lost in the overexpressing tissue.7 of the result section) (Hess et al. De Santis‐MacIossek et al. 2000)]. In the beginning it did not show any phenotype albeit having a high expression level of ERL1. benthamiana [(compare Figure 3. Another example of the observed phenotype is the infection of peach with a variant of the peach latent mosaic viroid (PLMVd). Literature search revealed several examples of mutants with the observed morpho‐ logical phenotypes [(compare figure Figure 3. 2008. S. ERI‐1 knock‐ out mutants of C. The PC‐C40 strain leads to peach calico. These symptoms are associated with a stable sec‐ ondary structure which is comprised of a 3’‐stem loop. a severe chlorosis of the leaf lamina. This fact maybe accounts for the observation that white tissue is able to recover when the ERL1 expression is silenced by the plant. 4.. 1993. Allison et al.4 ERL1 is involved in chloroplastic ribosomal RNA processing In addition to their involvement in RNA silencing processes ERI‐1 homologues have later shown to be involved in the 3’‐processing of cytosolic 5... It is believed that the SAP assists in the interaction. 1996. 2008). Another scenario could be a mutation in the chloroplast leader signal preventing an import into the plastids. Intriguingly. 2002)]. as well as the transcriptional alterations which could be detected after constitutive overexpression of ERL1 in N. Gabel & Ruvkun. Wang et al. this was explained by posi‐ tional effects. Babiychuk et al. musculus frequently showed a 2‐nt ex‐ tended version of the ribosomal RNA (Ansel et al. However.5 & Figure 3.8S rRNA. 1994. Zubko & Day.4. but when a mutant version of ERI‐1 135 ..6 of the result section) (Reiter et al... 2007).. Chatterjee et al. 1999. in subsequent generations the line showed an increasing amount of bleaching reminiscent of the other lines with high transgene expression. It belongs to the group of Avsun‐ viroidae which replicate in the chloroplast. 1996. pombe and M. a disturbance in chloroplastic rRNA generation has been attributed to be the cause of the phenotype (Rodio et al. elegans..
8S rRNA in vivo binds to 28S rRNA leading to a double‐stranded stem fol‐ lowed by a 2‐nt overhang. From initial experiments with probing for the rRNA levels after transient overex‐ pression of ERL1 by agroinfiltration. 2003. only 5S rRNA showed a detectable downregula‐ tion already after some days of ERL1 overexpression. Drosophila Snp has also been shown to process substrates with 3’‐overhangs of minimum two nucleotides (Kupsco et al. A more detailed picture could be obtained after mapping the exact ends of the rRNAs by a cloning approach (com‐ pare Table 3. chloro‐ plastic ribosomal RNAs have been considered as a likely substrate for the processing activity of ERL1. Indeed. alterations at the 3’‐ends could be de‐ tected in 18 % of all analyzed samples..4. This structure resembles the structure of siRNAs.8S rRNA. Discussion without SAP domain has been overexpressed in mice the enzyme was still able to interact with ribosomal proteins (Ansel et al. Despite apparent differences the two identified in vivo substrates share a major simi‐ larity: 5. 2005). Since this function of ERI‐1 seems to be conserved.. interestingly only after ERL1 overex‐ 136 . Unfortunately there are no re‐ sults available for the transient suppression of ERL1. it is localized in the cytoplasm and in addition it shows a slightly different folding compared to its animal counterpart with an extended 3’‐ overhang in plants. 5.. albeit not being verified in vivo. Taken together with the result that plant ERL1 localizes to the chloroplasts.. As expected from the initial transient assays the strongest effect could be seen in 5S rRNA. 2006). probably does not provide a suitable sub‐ strate for plant ERL1. 2008). Another identified substrate. is the short 3’‐terminal stem‐ loop of histone mRNA which is processed by human 3’hExo (Dominski et al.4 of the result section). in contrast to other species. Dominski et al. an involvement of ERL1 in similar plant pathways has been consid‐ ered as likely.1). In silico prediction of their secondary structures reveals that only 5S rRNA has a precursor structure comparable to other substrates of ERI‐1 homologues. The other chloroplastic rRNAs either possess extended overhangs or do not fold into a 3’‐terminal stem at all (compare Figure 4.
. 137 . Discussion (A) 3’ (B) 3’ (C) 3’ (D) 3’ Figure 4. 2008) (A) 4.1: Secondary structures of chloroplastic rRNA (predicted by RNAfold. Gruber et al. Again only the last 150 nt are depicted. For visualization only the last 161 nt are depicted. (B) 5S rRNA ends with a 3’‐terminal stem leaving any additional precursor nucleotides available for ERL1 processing. (D) 23S rRNA possesses only a two nucleotide double‐strand before the 3’‐end probably being too short for a presumable substrate of ERL1. a full length 16S structure prediction gives a similar result.4. (C) 16S rRNA has an extended single‐stranded 3’‐end.5S rRNA does not possess a 3’‐terminal stem.
The authors by then proposed an unknown 3’‐5’ exonuclease to act redundantly in the 3’‐end maturation of these rRNAs (Bollenbach et al. even in the mutant background more than half of all sequences corre‐ spond to the correct wildtype versions of the ribosomal RNA. So far. this type of extension could only be de‐ tected after transient overexpression. there is no explanation why overexpression of ERL1 would result in addi‐ tional nucleotides of 5S rRNA. 2000).. in two thirds of all extended 5S rRNA versions the extension corresponded to an untemplated extra –A/C C (C) mo‐ tif. In addition. Surprisingly. This may mean that misexpression of ERL1 first influences the canonical processing of 5S rRNA. In 45 % of all cases an addition of two nucleotides had been present. 138 . This suggests that ERL1 probably only acts redundantly to other proteins in 5S rRNA maturation. Intriguingly. However. It can be hypothesized that the additional nucleotides are added after the normal proc‐ essing of the 5S rRNA precursor has been disturbed. The rnr‐1 mutant shows a strong phenotype with extensions in 4.4. with the first base being comprised of either adenosine or cytosine. all altered sequences corresponded to templated extensions coming from the precursor sequence. these extensions belonged to two distinct groups: in one third of the altered sequences the extension corresponded to the precursor of 5S rRNA. 2004) and mitochondrial transcripts in maize (Williams et al. After constitutive overexpression only the non‐templated extension could be detected.. the second base being always cytosine. 2005). The scenario with transient overexpression of ERL1 represents the early steps of the pathway.5S and 5S chloroplastic rRNA parallel to wild‐ type versions of these molecules. Discussion pression but not upon its suppression in transgenic A. It can only be speculated how additional ERL1 might interfere with 5S rRNA processing. A similar effect had been observed earlier in loss‐of‐function mutants of the RIBONUCLEOTIDE REDUCTASE 1 (RNR1) in Arabidopsis. in rare cases a third cytosine could occur. untemplated addition of a similar motif has been reported for tobacco chloroplast transcripts (Zandueta‐Criado & Bock. thaliana plants.
If the level of the PPR transcript is influ‐ enced in the mutant line. In two out of six of the tested sequences they possessed an extra thymidine compared to the wildtype sequences. the genomic relation of the ERL1 and PPR gene are an interesting aspect. Although this behavior would be in accordance with the expected function of ERL1 by showing a deletion in the case of overexpression of the exonuclease. Sharwood et al. this function could be rescued by the PPR protein. However. since the relative distance of genes might also be an indicator for a function in similar processes. In the case of 23S rRNA a 3’‐deletion could be detected in one case. However. PPR proteins are implicated in editing of chloroplastic transcripts (compare chapter 1. it is impossible to argue that the elongated 16S rRNA is specific for the suppression of ERL1.. In the Arabidopsis genome the ERL1 gene lies imme‐ diately upstream of a sequence coding for a PPR protein whose promoter is part of the ERL1 sequence. it is hard to interpret at this time point since. so far. The extra –T cor‐ responds to the 16S rRNA precursor sequence.4. It has to be noted at this point that the sequenced wildtype clones in our experimental setup showed a two‐nucleotide deletion at their 3’‐ends compared to the published sequence. it cannot be excluded that in 139 . 2011). it is imaginable that ERL1 might exert this function. it had not been observed repeatedly. 16S rRNA was the only molecule with sequence altera‐ tions in the Arabidopsis erl1 suppression background.2 of the introduction). It can therefore not be excluded that a mutation could influence both transcripts. Discussion Recently. there‐ fore making it an unlikely substrate of ERL1. ERL1 misses a motif providing binding to RNA.3. Since this could be detected in all wildtype and most mutant sequences this shortened version had been considered as the wildtype setup. have proposed a dsRNA‐specific ribonuclease to destabilize 5S rRNA in the rnr‐1 mutant background (Sharwood et al. Nevertheless. 5S rRNA was not the only chloroplastic rRNA molecule showing sequence alterations at their 3’‐ends. The secondary structure predicts one of the deleted nucleotides to be bound in a double‐stranded stem. The fold of the 16S rRNA precursor does not suggest it to be a suitable substrate for ERL1 and a side effect of the inser‐ tion mutant cannot be excluded.
a mature protein without the leader peptide should therefore be expressed. especially 5S rRNA are not purely random but a specific result of ERL1 misexpression. The leader signal might interfere with substrate bind‐ ing and processing in the in vitro experiments. they do not show a clear phenotype. It will be important to create and analyze plant lines with minimal residual ERL1 expression.5S rRNA has not been changed in any of the tested sequences suggests that the observed sequence alterations in the other chloroplastic rRNAs.5 Conclusions Many hints point towards a contribution of plant ERL1 in the processing of chloro‐ plastic 5S rRNA. Preliminary results with the mature protein in shifting assays show binding to dsRNA with 2‐nt overhangs (Vlatakis. In addition. which specifically support an involvement of plant ERL1 in RNA silencing processes. Several possible reasons for this can be ad‐ dressed: one may be that the full length protein has been used for protein production and purification. It is very likely that it works in cooperation with a protein providing binding to nucleic acids. Reports from other species suggest an involvement of ERI‐ 1 homologues in rRNA processing and 5S rRNA is clearly influenced by the misex‐ pression of ERL1 in plants. a major disadvantage is the unavailability of data of complete knock‐outs of the gene. The proposed substrate should also be proven by in vitro assays. since it lacks a SAP motif. unpub‐ lished results). This sequence possesses a chloroplastic leader signal which will be cleaved in vivo after the import. The protein is likely to localize to the chloroplast in vivo. In addition. So far all attempts with plant ERL1 have been unsuccessful. These proteins might be indispensable for its func‐ tion also in vitro and need to be identified by immuno‐coprecipitation. The analyzed insertion mutants of Arabidop‐ sis thaliana still possess a detectable residual ERL1 transcript level. 4.4. ERL1 might require other factors for its function. The fact that 4. There have been no results obtained. Homologues 140 . the specific elongation of chloroplastic 16S rRNA still needs to be proven by analysis of the PPR transcript levels. Discussion the mature ribosome additional double‐stranded RNA stems could form between different RNA species. However.
it was not possible to regenerate these lines (Vamvaka. A last question to address is. 141 . 2005) and with ribosomal proteins in mice (Ansel et al. Discussion of ERI‐1 have been shown to interact with Dicer in C. Another approach had been the generation of transgenic plants overexpressing the mature protein without the leader peptide. 2008). It will be interesting to address if this is repeatable and therefore maybe a toxic effect of the overexpression of ERL1 in the cytoplasm... whether plant ERL1 would have the ability to degrade siRNAs if it did not localize to the chloroplast? This could be tested in the above mentioned in vitro assays with the respective co‐factors. 2010).4. elegans (Duchaine et al. However.
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1.7. 6. coli strain JM109.1. Leaves of Nicotiana sp. 100 mg tomato leaf material infected with Potato spindle tuber viroid (PSTVd) strain PH106 were ground in 1 mL of 1 % K2HPO4. 2007). The leaves were rinsed with water and grown under standard greenhouse conditions. 8 mM NTP mix.1.3 Purification of recombinant ERL1 protein ERL1 cDNA containing an N‐terminal His‐tag was cloned into pET‐15b and ex‐ pressed in E. plants Young Nicotiana sp. The cells were grown at 37 °C in selective LB to an OD600 of approximately 0. The cells were harvested by centrifugation at 171 . After a final DNase I treatment for 15 min the in vitro transcribed RNA was purified by phe‐ nol/chloroform extraction and precipitated with isopropanol. Alternatively 100 mg tobacco leaf mate‐ rial infected with Plum pox virus (PPV) was ground in 1 mL 50 mM phosphate buffer.1 Virus/viroid infections in Nicotiana sp. Supplements 6. Kalantidis et al.2 In vitro transcription For the production of small rRNA species T7 promoter‐containing oligonucleotides were used to create dsDNA which served as template for in vitro transcription. pH 8. 10 U RNase inhibitor in 1 x T7 RNA poly‐ merase reaction buffer were incubated at 37 °C for 1 h. plants were infected as described before (Tabler & Sänger. 1 μg dsRNA.. Alternatively 50 ng of in vitro transcribed viroid could be used. Full systemic infection could be ob‐ served after 3‐7 weeks post‐infection. 6. 10 mM DTT. 1984.1 Supplementary methods 6.6. Subsequently 250 U T7 RNA polymerase were added and the mixture was incubated at 37 °C for 2 h.0. were pretreated with carborundum and mechanically inoculated with 100 μL of the before created infectious sap per leaf. Protein expression was induced by addition of 0.5 mM IPTG and incubation at 37 °C for 3 h.
thaliana was conducted by several lab members as part of their Master and PhD theses.5 % glycerol (Iida et al. The reactions were run on PAA gels and exposed to X‐ray films.1. benthamiana and A. Evgenia – In vivo functional analysis of the plant 3ʹ exonuclease ERL1[original title: Βαμβάκα. 0..5 mM MgCl2. 6. 0. 2010)] 172 . 2009) Vamvaka. After two washes with washing buffer (protein purification buffer containing 20 and 30 mM imidazole) the protein was eluted with elution buffer (protein purification buffer containing 400 mM imidazole). 2010)] Vlatakis.4 In vitro assays for recombinant ERL1 protein Synthetic siRNAs or in vitro‐transcribed rRNAs were radio‐labeled and purified. Heiko – Involvements of the Plant 3’‐5’ Exonuclease ERL1 in Chloro‐ plast Ribosomal RNA Biogenesis and RNA Silencing Pathways (Kassel University.0. 2007) Schumacher. 6. The cells were lyzed with 1 mg/ml lysozyme on ice for 1 h and pelleted at 15000x g at 4 °C for 10 min.2 Supplementary results The project of characterizing ERL1 in N. For additional information please refer to the following theses: Eckhardt. 2006)] at room tempera‐ ture for empirically determined incubation times. απομόνωση και προσπάθεια ανεύρεσης του φυσικού υποστρώματός της (University of Crete. Therefore relevant additional results for this thesis are presented below and partly cited literally. Ioannis – In vitro study of AtERI‐LIKE1: purification and trying to discover its natural substrate [original title: Βλατάκης. The supernatant was loaded on a pre‐equilibrated Ni‐NTA column. Supplements 2500x g at 4 °C for 20 min and resuspended in protein purification buffer. Ιωάννης – In vitro μελέτη της AtERI‐ LIKE1 : καθαρισμός. 50 fmol were incubated with recombinant ERL1 protein in cleavage buffer [10 mM Tris pH 8. Stephanie – Functional Analysis of ERI‐1 (Kassel University.6. 27 mM KCl. Ευγενία – In vivo λειτουργική ανάλυση της Φυτικής 3’ εξωνουκλεάσης ERI1 (University of Crete.
1. Infiltrations with an empty binary vector served as controls. ruling out an un‐ specific effect due to aging of the plant or differential progression of the PSTVd infec‐ tion over the course of the experiment. left panel). In non‐treated samples of the same plant PSTVd siRNA levels remain constant over time (Figure 6.1. Nicotiana plants produce large amounts of 21‐24 nt siRNA‐like RNAs derived from the viroid sequence. right panel). tabacum. Georgios – Functional analysis of plant ERI1: ERL1 (ERI1‐LIKE1) [original title: Λαγιώτης. middle panel). These PSTVd siRNAs are easily detectable in northern hy‐ bridisations and hence present a suitable reporter system to study possible effects of ERL1 misexpression on siRNA steady‐state levels. 2009)] 6. Agro‐infiltration with a control plasmid simi‐ larly had no measurable effect on PSTVd siRNA levels (Figure 6. left panel). This experiment shows that the steady‐state levels of siRNAs produced from PSTVd upon infection of N. 22 and 24 nt) equally (Figure 6. and small RNA fractions were subsequently electrophoresed on 20 % PAA gels. To study PSTVd siRNA steady‐state levels over time. tabacum are being negatively affected by ectopic agro‐ infiltration‐mediated Arabidopsis ERL1 overexpression. if this reduction in siRNA levels is caused by an siRNA‐degrading 173 . and analysed by hybridisations with PSTVd‐specific probes.1. It cannot be undoubtedly de‐ duced. but are oth‐ erwise symptom‐free. samples were collected every 4th day. The reduction seems to affect the different siRNA size classes (21. Comparative agro‐infiltration time course experiments were conducted by overex‐ pressing Arabidopsis ERL1 in systemically PSTVd‐infected N. Γεώργιος – Λειτουργική Ανάλυση της Φυτικής ERI1: ERL1 (ERI1‐ LIKE 1) (University of Crete.1. The results of this experiment are summarised in Figure 6.1 PSTVd‐derived siRNAs are suppressed upon ERL1 overexpression Upon infection with Potato spindle tuber viroid (PSTVd).6. Non‐infiltrated samples of the same plant were used as time points zero. however.2. 8 days postinfiltration PSTVd siRNA levels are reduced approximately 4‐ fold in samples that were treated with ERL1 overexpression (Figure 6. Supplements Lagiotis. northern‐blotted.1.
benthamiana line 16c that is characterised by strong and stable GFP ex‐ pression (Ruiz et al. 1998).2 ERL1 fails to affect RNA silencing in Agrobacterium co‐infiltration assays In plant viruses. tumefaciens strain 174 . Figure 6... Ectopic GFP expression triggers the co‐suppression pathway and leads to RNAi‐mediated silencing of the transgenically produced GFP. 2004).6. tumefaciens strain C58C1. 2009). GFP silencing is induced by overexpression of GFP in an already GFP‐expressing plant. Supplements activity of ERL1. In this assay. To create ERL1 overexpression (35S‐AtERL1gen) and sup‐ pression (35S‐NtERL1hp) constructs. 1992) and trans‐ formed into A. 1998). viral genes are tested for silenc‐ ing suppressor activity by assaying their effects on sense‐induced GFP silencing in N. To determine. essentially as described before (Lu et al. were cloned in the binary vector pART27 (Gleave. the genomic sequence of the Arabidopsis ERL1 gene and a hairpin construct derived from a tobacco ERL1 EST (EST ID EB681897). Traditionally. benthamiana plants. if ERL1 shows RNA silencing suppressor activity. pathogenicity determinents have been shown to act as RNA silenc‐ ing suppressors (Brigneti et al. 2009) 6. or if this is the result of a secondary effect (literally cited from Schumacher. respectively.1: Comparative agro‐infiltration time course in systemically PSTVd‐infected tobacco.2. Agrobacterium co‐infiltration assays were performed. In the left panel ERL1 overexpression reduced the number of PSTVd siRNAs significantly 8 dpi whereas after infiltration of a control plasmid (middle panel) and in untreated plants (right panel) remain constant (result and picture by Schumacher. Bona fide suppressors of silencing are able to inhibit or delay the initiation of RNA silencing when expressed along with the silencing inducer. The assays were per‐ formed in N.. Equal volumes of an A.
The Agrobacterium mixtures were used to agro‐infiltrate dis‐ tinct patches on 16c leaves. 2004)] served as negative and positive controls. 2009). Kalantidis et al. which is visible by the formation of a red ring around the green spot of GFP infiltration.2 a). Figure 6.2 a).6. 2003... 1997)] serving as the silencing inducer were then mixed with Agrobacteria carrying the plasmids for ERL1 overexpression or ERL1 suppression. The final concentrations of all strains were ad‐ justed to 0. (B) Induction of GFP silencing by the infiltration of a GFP hairpin construct is not affected by overex‐ pression of ERL1 identical to the control constructs (results and picture by Schumacher. Upon co‐expression of ERL1 and GFP (Figure 3.. and GFP expression and silencing initiation were moni‐ tored over time using a handheld Blak‐Ray® long‐wave UV lamp. Supplements carrying a 35S‐GFP construct [pBIN 35S‐mGFP4 (Haseloff et al.. respectively. The red ring is not only indicative of RNA silencing initiation but also shows that SLSS was not affected by ERL1 overexpression or suppression. 1:1 mixtures with an empty binary plasmid or with a con‐ struct expressing the silencing suppressor P19 of Cymbidium ringspot virus [35S P19‐ CymRSV (Havelda et al. Under the condi‐ tions tested.2 a). ERL1 was not able to suppress the onset of GFP silencing (Figure 6.2: Agrobacterium co‐infiltration assays in N. the red ring of local GFP silenc‐ ing spread (Himber et al. ERL1 suppression with a hairpin construct similarly had no effect on the RNA silencing time course (Figure 6. 2008) appeared at the same time as in the empty plasmid negative control (Figure 6.25 at OD600. 2003..2).4 a). exemplified by strong GFP signal in the infiltrated patch and the absence of the red ring of local GFP silencing spread over the course of the experiment (Figure 6. Lakatos et al. 175 . Co‐expression of P19 on the other hand was able to suppress RNA silencing initiation. benthamiana line 16C to test ERL1 for RNA silencing suppressor activity (A) In contrast to the verified silencing suppressor protein P19 (upper left spot) ERL1 overexpression (upper right spot) does not result in suppression of GFP silencing similar to the control constructs (lower panel).
young ERL1‐ overexpressing plants with a mild bleach phenotype (i. 2009).7 f. 176 .e. Supplements In an equivalent experiment. which suggests that systemic RNA silencing may to some extent be suppressed in ERL1 overexpressor plants. however. red arrowhead indi‐ cating a newly emerging leaf void of RNA silencing‐type spreading of green tissue).6. Several days postinfiltration wildtype‐like green tissue started spreading from the veins of systemic leaves.3 Exogenously induced silencing spread may be suppressed after ERL1 overexpression To confirm that white sector formation is a direct result of ERL1 overexpression.7 f). It was observed that in about 50 % of the cases. The remaining 50 % of plants continued to systemically silence the ERL1‐dependent bleaching phenotype and regained wildtype appearance until senescence (literally cited from Schumacher. point of infiltra‐ tion indicated by black arrowhead).2. these assays show no ability of ERL1 to negatively affect RNA silenc‐ ing.7 f. pale green leaves) were agroinfiltrated with an Arabidopsis ERL1 hairpin construct (35S‐ AtERL1hp) to induce RNA silencing of the transgene (Figure 3. be too weak to be detected in Agrobacterium co‐infiltration assays that require rather robust silencing suppressors like P19 to counter agro‐infiltration‐induced RNA silencing (literally cited from Schumacher. systemic spread of induced ERL1 si‐ lencing did not propagate more than a few leaves (Figure 3. 2009). Given its low expression levels and predominantly chloroplastic localisation a possible role of ERL1 in negative RNA silencing regulation may.2 b). strongly resembling the phenotype of systemic GFP silencing spread (Figure 3. 6. ERL1 overexpression and suppression also failed to af‐ fect GFP silencing when a GFP hairpin construct was used to induce silencing (Figure 6. Taken together.
Under the conditions tested. however. 12 weeks postinfection total RNA was extracted from wildtype N.3: Silencing of the ERL1 phenotype induced by agroinfiltration Systemic ERL1 silencing in a bleach‐type plant. with mild mosaic symptoms developing after 1‐2 weeks that persisted until senescence (Figure 6.4 a). wildtype N. A growth/fertility defect as pronounced as in the PPV‐infected individuals. benthamiana. The slow growth and reduced fertility may to some extent be ex‐ plained by ERL1 overexpression itself.4 c). Only approximately 33 % of the PPV‐infected ERL1‐overexpressing plants survived the infection. ERL1 overexpressor plants accumulate approximately 3‐5x higher virus titres. The red arrowhead (in the middle of the picure) shows a newly emerging leaf void of systemic silencing‐type spread of wildtype‐like tissue (result and picture by Schumacher. such a high lethality rate implies a hypersensitivity of ERL1 overexpressor plants towards PPV infection. Supplements Figure 6. benthamiana and ERL1 overexpressor plants were infected with Plum pox virus (PPV).6. benthamiana and a surviving ERL1 overexpressor plant and tested for the respective viral loads in north‐ ern hybridisations (Figure 6. Given that PPV infections are typically non‐lethal in Nicotiana plants. ERL1 overexpressor plants in contrast developed much stronger symptoms with severely crippled leaves (Figure 6.4 b). it was investigated how plants with different ERL1 backgrounds behave upon viral infection. The surviving ERL1 overexpressor plants remained dwarves with crippled leaves until senescence and produced only few underdeveloped flowers that failed to pro‐ duce any seeds. has never been observed in the specific bleach‐type ERL1 overexpressor line used in the infection experiments. The increase 177 . benthamiana plants showed a typical pro‐ gression of PPV infection.4 ERL1‐overexpressing plants are hypersensitive towards viral infection Since antiviral defense constitutes one of the major functions of RNA silencing in plants. 2009). induced by agroinfiltration with an ERL1 hairpin construct (the black arrowhead in the lower part of the picture indicates the point of agro‐ infiltration). In comparison to wildtype N.2. To this end wildtype N. 6. while the remaining 67 % had died until six weeks postinfection.
4: ERL1 overexpressor plants are hypersensitive towards infection by PPV (A) Symptoms of PPV infection on bleach plants which show crippled leaves and a stunted growth. 6. (B) The symptom of PPV infection on N. the described hypersensitivity towards PPV infection may be caused by a reduction in DCL1‐4 production and hence not constitute a primary effect of ERL1 overexpression. namely DCL1 and DCL4 (Figure 6. however. Supplements in viral load may explain the observed aggravated symptoms. 5S. gives leeway to a possible connection between ERL1 function and RNA silencing pathways (literally cited from Schumacher. Northern analyses of variegated ERL1 overex‐ pressor tissues showed a significant decrease in the steady‐state mRNA levels of two of the four DICER‐LIKE proteins. (C) ERL1 overexpressor plants accumulate a higher viral load 12 weeks post‐infection. 4.6. Figure 6. as well as extracted natural rRNAs. The fact that at least DCL1 and DCL4 are downregulated in ERL1‐overexpressing tissue. in vitro transcribed 5.8S. (D) DCL1 and DCL4 transcript levels are downregulated in white tissue whereas they remain normal in green tissue overexpressing ERL1 (result and picture by Schumacher.2. 2009). benthamiana wildtype plants is a mild mosaic phenotype. not be determined in this experiment. 2009). Increasing amounts of recombinant ERL1 were incubated with the following radio‐labeled sub‐ strates: synthetic 21‐nt siRNA.5S rRNA and several of their natural processing intermediates. Since the DICER‐LIKE proteins are crucial core components of RNAi‐mediated antiviral de‐ fense in plants. Whether this hyper‐ sensitivity is accountable to an siRNA‐degrading activity of ERL1 could. however.4 d).5 In‐vitro experiments with ERL1 Several attempts have been conducted to characterize plant ERL1 in vitro. The sam‐ 178 .
could be detected in these assays.5 b). The results were compared with plants constitutively overexpressing ERL1 and showing a mo‐ saic phenotype. Neither any shortened processing products nor a shift of the substrates. In addition wildtype plants served as a control. The analysis of transgenic plants suppressing ERL1 started with three chloroplast‐ related genes and was compared with the transient suppression.6 Preparation of a NbERL1 suppression construct and analysis of its ef‐ fects after transient and transgenic expression For the analysis of ERL1 suppression in Nicotiana benthamiana a hairpin construct was prepared from the NbERL1 sequence with approximate length of 3000 bp. In contrast.2. In contrast to the results described before. Lagiotis. Finally RBCL was strongly downregulated in ERL1 overexpressing tissue and a bit less downregulated in ERL1 suppressing tissue com‐ pared to wildtype (Figure 6. 2010). 2010)].6. when the NbERL1 overex‐ pression construct was infiltrated.5 a). 179 . NEP and PSBA were not influenced by the misexpression of ERL1. The expression of CLPC. The effect of NbERL1 suppression on chloroplast‐related transcripts has been inves‐ tigated by transient expression of the construct and consecutive analysis. 2009. After co‐ infiltration with an NbERL1 overexpression construct of approximately 1000 bp an immediate suppression could be observed. which would be a result of binding to ERL1. Vlatakis. 6. its degradation by the plant started after the sec‐ ond day (Figure 6. the downregulation of PFTF and PEP could not be repeated in the transgenic plants and their expression levels were comparable to transgenic plants overexpressing ERL1 [(Figure 6. In the case of PFTF a slight reduction could be detected in the suppressing tissue in contrast to overexpressor tissue where the expression was comparable to wildtype. PEP and CLPP were downregulated in suppressing tissue while an upregulation could be ob‐ served in overexpressing tissue. This suggests no ERL1 processing activity at the chosen re‐ action conditions (Eckhardt.5 c) (Vamvaka. Supplements ples were then analyzed on PAA gels. 2007.
6. Supplements (A) (B) 3000 bp 1000 bp (C) Figure 6. (C) Control of the earlier identified influenced transcripts in transgenic ERL1 suppressor plants fails to detect specific expression alterations after knock‐down of ERL1 (results and pictures by Vamvaka. The overexpressing construct is easily detectable on the first day and vanishes over the time course. 180 . (A) Constructs for overexpression and suppression of ERL1 were infiltrated into N. (B) Analysis of chloroplast‐ related transcripts in wildtype N. benthamiana and their expression followed over four days. mosaic plants overexpressing ERL1 and after transient suppression of ERL1 by agroinfiltration.5: Analysis of the suppression of ERL1 in Nicotiana benthamiana. The co‐infiltration of the overexpression and hairpin con‐ structs prevents the detection of the overexpression from the beginning. benthamiana. 2010).
3 Oligonucelotides The following primers were used during the course of the work for the respective cloning purposes. Supplements 6.6. mutant analysis and for probe preparation: Primers used for analysis of Arabidopsis thaliana KD plants GABI_KAT LB3_SAIL Lbb1_SALK N544378 F N544378 R N579265_For N579265_Rev N834430_For N834430_Rev clpP Probe NEP For NEP Rev Nt‐CLP‐Bam‐F Nt‐CLP‐Not‐R psbA For psbA Rev rbcL For rbcL Rev rpoB For rpoB Rev Tob‐Pftf Forward Tob‐Pftf Reverse At‐Eri‐Bam‐F At‐Eri‐Not‐R Ath‐Eri qPCR F Ath‐Eri qPCR R Eri FLAG Rev Eri no Stop+TC Rev Leader+TC Rev mature+Bam+start Nbe eri FOR Nbe eri REV ATATTGACCATCATACTCATTGC TAGCATCTGAATTTCATAACCAATCTCGATACAC GCGTGGACCGCTTGCTGCAACT ACACCTTGTAAGCCATCAACG TTTCAAACATCAATCTTCCGC CCAATCTCGTTCAGACATTCC TTTCTTGGACCGGGTTTTAAG TTTACGTTCATTTCCTGAGGC TTCCATGGTTTTGGCATCTAC CCTTGTGAGGGTTTCACGCAGTTTCAGCAGTTCTTCCGCTTCCAG TTTGATGCGCACTCATGGATCTA GCAAATTCAAGGATTCCTCGACA GGATCCGAAAGGAGGCCGTCGTATAGGTT GCGGCCGCATCTGAGGGAGTTCCGCTAGTGC TAACCATGAGCGGCTACGATGTT GTAGCTTGTTACATGGGTCGTG TGCGAATCCCTCCTGCTTATGTT CCAAGCTAGTATTTGCGGTGAAT CTTCCAGCTACTTTATCGCCTAC CTTATATGCCGTGGGAGGGTTAC GGATCCCCCGAGAGGTTTACCGCAGTG GCGGCCGCGGTGTCCTCATGGCTATAACTT GGATCCATGGCGTCCGCATTCTCTGC GCGGCCGCTTACTTGATCCTGTTCTTGAAG CAAAATGAGCGAGCAAGCAATTA ATCCCAGTTTCCACAGGTTACAA GCGGCCGCCGATGAATAGTAGTAGTAGGAACATTAGGTATTGAT CCTGTTCTTGAAG GCGGCCGCCTCTTGATCCTGTTCTTGAAGAGA GCGGCCGCCTACTTGTTTCCATTAACGAAAAAG GGATCCATGGAAAATGCAAGGTGGAGACCCATG AGCAGTCACAAGACTCGC GGTGATAACAAAATGGTTCAG 181 Primers used for detecting chloroplast‐related transcripts Primers used for the cloning and detection of ERL1 .
6.8s rRNA start For 5.8S‐circular R 5S Probe 5S+link For 5S‐circular F 5S‐circular R linker mod linker REV Miscellaneous 35S pART7&27 F 35S pART7&27 R Ath‐UBQ_For Ath‐UBQ_Rev Intron For Intron Rev Nico‐UBQ_For Nico‐UBQ_Rev Racer dT AAGTTTGGCTGGGTGAACGTC AAGGCCCTCCTCTTGTAGAAG TGCTCGCTGGAGTTTATGCAA ACTTGAGAATTTGCGGAAGC GTGTGGGCTGAATTAGGGA TTGGGCCTGAAGTGAGTACGA GGCCTCACAAACCTATG TCATGGAGAGTTCGATCCTGGCT ATCCTCACCTTCCTCCGGCTTATCA CAAAAACCCGTCCTCAGTTC TCAACCATAAACGATGCCGACCA GCGTGCGGCCCAGAACATCTAA ACAGGTCTCCGCAAAGTCGTA GCCCTTCCAGAGTGGTATCTCA CCACCTGGGGCTGTAGTATG ATCCTGGCGTCGAGCTATTTTTCCGCAGGACCTCCCCTAC CGAGACGAGCCGTTTATCAT AGGCATCCTAACAGACCGGTAG TCCACTTGACACCTATCGTAATG GCAACGGATATCTCGGCTCT TAATGGCTTCGGGCGCAACT CGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATAC CGGATATCTCGGCTCT CGCCCGAAGCCATTAGG CGATGGTTCACGGGATTC GGATGCCTCAGCTGCATACATCACTGCACTTCCACTTGAC ACCAATCCATCCCGAACTT TAAACTCTACTGCGGTGAC CAAGTTCGGGATGGATTGG ATCGTCACAACAAATGGCATddC GATGCCATTTGTTGTGACGAT TATAGAGGAAGGGTCTTGCGAAGG CAACAAAGGATAATTTCGGGAAAC TAACCCTTGAGGTTGAATCATCC AACTCCTTCTTTCTGGTAAACGT CTTGGAGCAGAACATGAGATTCG ATTGAGCCAGGGCATTTACCTC GAGGTTGAATCTTCCGACACAAT AACATAGGTGAGCCCACACTTAC GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTT TTTTTTTT 182 Primers used for the detection and cloning of ribosomal RNAs .5S‐circular F 4.5S Probe 4.8s probe Rev 5.8S‐circular F 5.8s rRNA 5. Supplements Nbe‐eri new‐qRT F Nbe‐eri new‐qRT R Nt‐eri_RACE A1 Nt‐eri_RACE A2 Nt‐eri_RACE S1 Nt‐eri_RACE S2 Nt‐eri_RT‐primer 16S For 16S Rev 16S+link For 18S For 18S Rev 23s_For_new 23s_Rev_new 23S+link For 4.5S‐circular R 5.5S+link For 4.8s probe FOR 5.
Spec).4 Vector maps 6.6. 2002) GFP 0 att B2 ERL1 leader att B1 35S 183 . Supplements 6.4.4. Spec) .1 At‐ERL1‐GFP (11243 bp. based on pB7FWG2 (Karimi et al. based on pB7FWG2 (Karimi et al. 2002) GFP 0 att B2 ERL1 cDNA att B1 35S 6.2 At‐leader‐GFP (10451 bp...
2009 0 At‐ERL 1 genomic 35S Restriction sites: 963 (NotI). 963 (NotI). 5072 (NotI). 3684 (HindIII). 5774 (BamHI) 6. 2009 0 Nt‐ERL1 sense spacer Nt‐ERL1 antisense 35S Restriction sites: 960 (PstI).4. 3021 (PstI). 3713 (EcoRI). Schumacher. Supplements 6. Kan/Spec). 2823 & 2956 (BamHI). 1761 (EcoRI). 1026 (PstI). 2410. 3895 (HindIII). 5563 (BamHI). 5283 (NotI). 3023 & 3672 (EcoRI). 1747 (BamHI). 5728 & 8266 (PstI) 184 .4 Nt‐ERL1‐hp (15776 bp.4. Schumacher. Kan/Spec).6.3 At‐ERL1‐over (15987 bp.
benthamiana_ERL1_protein (incomplete) SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRKISIS ASRSTTEESTSSLIQPTPSRTRWKPTCLYFTQGKCTKMDDPMHIDKFNHNCSLELMQNAAGL KNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGK YGKLGVDRVWHDTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQ CKVAGTKLPPYFMEWINLKDVFLNFYKRRAKGMLSMMRELQMPLLGSHHLGIDDAKNIARVL QHMLSDGALVQITARRNPHSPEKVEYLFEDRIV >A.thaliana_ERL1_leader_cDNA ATGGCGTCCGCATTCTCTGCATTTAGGGTTTCGTTGTCCAGAATCAGTCCTTTCCGTGATAC CCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAATCATGTGCAACTCTT CGCATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTTCT TCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGT >A.thaliana_ERL1_mature_cDNA GAAAATGCAAGGTGGAGACCCATGTGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGA TGATCCTGCCCATTTGGAGATTTTTAACCACGATTGTTCAAAGGAACTTCGAGTGGCTGCTG CTGATCTTGAGAGAAAGAAGTCACAAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGA AAAGTTGAGATTCTTGAGTTTCCTATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGA CTTATTCCACAGGTTTGTAAGACCCACCAAAATGAGCGAGCAAGCAATTAACAAATACATCG AAGGCAAGTATGGGGAACTCGGGGTTGATCGTGTGTGGCATGACACAGCTATTCCATTTAAG CAAGTTGTTGAGGAGTTTGAAGTTTGGTTAGCTGAGCATGACTTGTGGGATAAAGATACAGA TTGGGGTCTGAACGATGCAGCTTTTGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTC CTGAGCAATGCGTAGTTTCAAACATCAATCTTCCGCCATATTTTATGGAGTGGATCAATCTC AAAGACGTCTACTTGAATTTCTATGGCCGTGAGGCAAGAGGAATGGTGTCAATGATGAGGCA 185 .benthamiana_ERL1_cDNA (incomplete) GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCC TCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTTGTAGGGTCCCCTTGCTGCGGCGGT TCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGT ATCTCCGCCTCTCGTTCTACCACCGAAGAATCTACTTCTTCCCTAATTCAGCCCACACCTTC CCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATG ATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCG GGACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAA AGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACT TTTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAA GGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGATACAGCTATCCCATTTGGAGA AGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCG GCTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCT CAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAATTTGAA GGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAAC TCCAGATGCCTTTGTTAGGGAGTCATCACCTTGGAATAGATGATGCAAAAAACATAGCAAGA GTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCA TTCTCCTGAAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTTCTTCTGAAC CATTTTGTTATCACCTAAACATTTTTAGAAAAAAAAAAAAAAA >N.1 Newly identified sequences >N.5 Sequences 6.5.thaliana_ERL1_leader_protein MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSS SSPSTFSLMETS >A. Supplements 6.6.
Seq.elegans_ERI‐1_protein (NCBI Ref.1) METKEKSRKPPNKTPQSEGDQEDQPCPDTSCEKNEDQEPSSPKQGEFSDPVYKEIALANGAI NRMNREELRAKCTELKLDTRGVNDVLRKRLKSYYKKQKLMHSPAAEGNSDMYFDYICVVDFE ATCEENNPPDYLHEIIEFPMVLIDTHTLEIVDSFQEYVKPVLHPQLSEFCVKLTGITQEMVD EAKTFHQVLKRAISWLQEKELGTKYKYMFLTDGSWDMGKFLHTQCKLSRIRYPQFARKWINI RKSYGNFYKVPRTQTKLICMLENLGMEYDGRPHCGLDDSRNIARIAIHMLKDGCQLRVNECL HSGEPRSVPISAPIEGAPAPQPPKKRD >X. NP_741293. Supplements GTGTGGAATAAAACTCATGGGAAGCCACCATCTGGGCATTGATGACACAAAGAACATCACGA GGGTGGTGCAACGGATGCTCTCAGAAGGTGCAGTTCTCAAGCTCACAGCTCGAAGGAGCAAA TCCAATATGAGAAACGTCGAGTTTCTCTTCAAGAACAGGATCAAGTAA >A.2) MEDERGRERGGDAAQQKTPRPECEESRPLSVEKKQRCRLDGKETDGSKFISSNGSDFSDPVY KEIAMTNGCINRMSKEELRAKLSEFKLETRGVKDVLKKRLKNYYKKQKLMLKESSAGDSYYD YICIIDFEATCEEGNPAEFLHEIIEFPVVLLNTHTLEIEDTFQQYVRPEVNAQLSEFCIGLT GITQDQVDRADAFPQVLKKVIEWMKSKELGTKYKYCILTDGSWDMSKFLSIQCRLSRLKHPA FAKKWINIRKSYGNFYKVPRSQTKLTIMLEKLGMDYDGRPHSGLDDSKNIARIAVRMLQDGC ELRINEKILGGQLMSVSSSLPVEGAPAPQMPHSRK >D. Q7TMF2. Seq.6. Seq.sapiens_3’hExo_protein (NCBI Ref. Seq. NP_699163.thaliana_ERL1_mature_protein SENARWRPMCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLE GKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGVDRVWHDTAIPF KQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWIN LKDVYLNFYGREARGMVSMMRQCGIKLMGSHHLGIDDTKNITRVVQRMLSEGAVLKLTARRS KSNMRNVEFLFKNRIK 6.rerio_Eri1_protein (NCBI Ref.1) MSADEPSPEDEKYLESLRDLLKISQEFDASNAKQNDEPEKTAVEVESAETRTDESEKSIDIP REQQLLPSERVEPLKSMVEPEYVKKVIRQMDTMTAEQLKQALMKIKVSTGGNKKTLRKRVAQ YYRKENALLNRKMEPNADKTARFFDYLIAIDFECTCVEIIYDYPHEIIELPAVLIDVREMKI ISEFRTYVRPVRNPKLSEFCMQFTKIAQETVDAAPYFREALQRLYTWMRKFNLGQKNSRFAF VTDGPHDMWKFMQFQCLLSNIRMPHMFRSFINIKKTFKEKFNGLIKGNGKSGIENMLERLDL SFVGNKHSGLDDATNIAAIAIQMMKLKIELRINQKCSYKENQRSAARKDEERELEDAANVDL TSVDISRRDFQLWMRRLPLKLSSVTRREFINEEYLDCDSCDDLTDDKVKHLHSCDIYEIFDE KTSASFTDSKCLIC >H.musculus_Eri1_protein (NCBI Ref.5.2 Published sequences used for in silico analysis and primer design >C. Seq.laevis_Eri1_protein (NCBI Ref.1) MEEQKENRPLDTEDSVVEEDLCKKLSRNLDLVGVKQRCRFDGQEDNGTSTVSSNTSDFSDPV YKEIAIANGCVNRMTKDELKAKLVEHKLDTRGVKDVLRKRLKNYYKKQKLTHALHKDSNTDC YYDYICVIDFEATCEAGNSLDYPHEIIEFPIVLLNTHTLEIEDVFQCYVRPEINPQLSEFCV NLTGITQDTVDKSDTFPNVLRSVVEWMREKELGSKYKYAILTDGSWDMSKFLNMQCRISRLK YPRFAKKWINIRKSYGNFYKVPRTQTKLTTMLEKLGMTYNGRLHSGLDDSKNIARIAAHMLQ DGCELRVNERMHAGQLMTVSSSLPFEGAPVPQNPHLKN 186 .2) MEDPQSKEPAGEAVALALLESPRPEGGEEPPRPSPEETQQCKFDGQETKGSKFITSSASDFS DPVYKEIAITNGCINRMSKEELRAKLSEFKLETRGVKDVLKKRLKNYYKKQKLMLKESNFAD SYYDYICIIDFEATCEEGNPPEFVHEIIEFPVVLLNTHTLEIEDTFQQYVRPEINTQLSDFC ISLTGITQDQVDRADTFPQVLKKVIDWMKLKELGTKYKYSLLTDGSWDMSKFLNIQCQLSRL KYPPFAK >M. NP_001089554. NP_001018450.
XP_002282697.1) MALARVSPPAFSSPFLIHSLLRPFSSPSSVLRPRVTRVPHHRGFAIAAALSQASPLPSADGD GAVMEAPPRPSSRRPWKPTCLYYTQGKCTMLNDTLHLEKFNHNLPTDLPVNYSAADKVKSQK LDYFLVLDLEGKVEILEFPVVMIDAQSMEFVDSFHRFVHPTAMSEQRIREYIEGKYGKFGVD RVWHDTAIPFMEVLQEFEDWIEHHKFWKKEQGGALNSAAFITWFRILVEQELWKILEVRVD >S.vinifera_ERL1_protein (NCBI Ref. XP_002436970. Supplements >D. Seq. Seq. Seq. Seq.1) MALARVSPSSLANLIPPLLQSFFRPFSSDFPIRNSRRRSSPVAAAFSLTSQSAHAAREGLVM EAPRPSSRYPWKPTCLYYTQGKCTMMNDAMHLEKFSHNLKMDLPVNASATDKSKPQKLEYLL ILDLEGRVEILEFPVMMIDAQNREFVDSFHRFVRPTAMSEQRTTEYIEGKYGKFGVDRVWHD TATPFKQVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKVPEQCKVSKINLPTYF MEWINLKDIYLNFYNRRATGMMTMMRELQLPIVGNHHLGIDDSKNIARVVQRMLADGAVIQI TAKRQSATGDVRFLFKDRIR 187 .1) MESPVQILVWPFPCDEMNQKTPSTVEEIRIALQELGLSTNGNKRYLLIVDVEATCEEGCGFS FENEIIELPCLLFDLIEKSIIDEFHSYVRPSMNPTLSDYCKSLTGIQQCTVDKAPIFSDVLE ELFIFLRKHSNILVPSVDEIEIIEPLKSVPRTQPKNWAWACDGPWDMASFLAKQFKYDKMPI PDWIKGPFVDIRSFYKDVYRVPRTNINGMLEHWGLQFEGSEHRGIDDARNLSRIVKKMCSEN VEFECNRWWMEYEKNGWIPNRSYPPYFAS >N. NM_001187847.sativa_ERL1_protein (NCBI Ref. NP_611632.3) MALIKLARQLGLIDTIYVDGARPDPNNDPEESFNEDEVTEANSVPAKSKKSRKSKRLAMQPY SYVIAVDFEATCWEKQAPPEWREAEIIEFPAVLVNLKTGKIEAEFHQYILPFESPRLSAYCT ELTGIQQKTVDSGMPLRTAIVMFNEWLRNEMRARNLTLPKMNKSNILGNCAFVTWTDWDFGI CLAKECSRKGIRKPAYFNQWIDVRAIYRSWYKYRPCNFTDALSHVGLAFEGKAHSGIDDAKN LGALMCKMVRDGALFSITKDLTPYQQLNPRFVL >S. NP_001131746.pombe_Eri1_protein (NCBI Ref.mays_ERL1_protein (NCBI Ref.bicolor_ERL1_protein (NCBI Ref. Seq.1) MEDEQFMQQLRNLTCQTVDWSGFDPSKWRDEDINDWDISDDAQGDEDDNYASDASILSARHL DPFNVKPATRPHHTGPTSLRIEDVTDEQEEYRDASDLENISWPEVSVEQGEIDPITELFTPW RMVLEYPNLFVGKRNGARARPLFTLESLHENRIWDLFYLYRPSNEGNNNPLIFVPTYQMQHL LDVINRKLDVEFTFPRGHQDMFAMPFGQSNTAKPRFLGRSRSAEEWKQLTNNVPARKPGDTS ENAPFLAKQELTRRLNSIFSIQDKSKKTKNNQYKRSNLHRAWGKNIKRVQRYLGLRRRVLSD PEVSSYTPLDLTQPTGIQPEKSVVFVAIDLEAYELDQSIITEVGLAILDTAEITNVAPGEGS KNWFDFIKARHIRVKEFSWAQNSRHVQGRAEYFDFGESEFIEVAKIASVLKETIEGESSIGG EGAKRPVVLVFHDQSQDLKYIRMLGYDVASADNILEVVDTREMYQYLSRSNNASKLSNVCGY LDIPWKNMHNAGNDAVYTLQAMMGLAIDMRQKSLERAAAKASKANTSNDGYVTYSEFTATKE DVDEGWISTGELSDGGEPSLVMAASTVPNSVVETTVCENWEL >V.crassa_Qip_protein (GenBank: ABQ45366.1) MAFYRVSPFRYGSLSSLIPYVSSPSSLSPPVRTFTLSASISTPHPSPPSLLTASPKASDRWR PMCLYYTQGKCTKMDDPTHLETFNHNCSRELQVNAANFQHLQSQHLDFFLVLDLEGKIEILE FPVLMINAKTMDVVDLFHRFVRPSEMSEQRINEYIEGKYGKLGVDRVWHDTSIPFKEVIQQF EAWLTQHHLWTKEMGGRLDQAAFVTCGNWDLKTKVPQQCKVSKMKLPPYFMEWINLKDVYLN FYKRRATGMMTMMKELQIPLLGSHHLGIDDTKNIARVLQRMLADGALLQITARRNADSPENV EFLFKNRIR >O.melanogaster_Snp_protein (NCBI Ref.6. Seq.1) SAASSATVRASGSVGCHMNDAMHLEKFGHNLKMDLPVNASATDKFKPQKLEYFLILDLEGRV EILEFPVVMIDAQSTEFIDSFHRFVRPTAMSEQRTTEYIEGKYGKFGVDRVWHDTAVPFKEV LQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKATGMMTMMRELQLPIIGNHHLGID DSKNIARVVQRMIADGAVIEITAKRQSTTGNVKFLFKDRIR >Z. XP_001713129.
6.tabacum _5S_rRNA (NCBI Ref.1) 188 .trichocarpa_ERL1_protein (NCBI Ref.1) MCLYHTHGKCTKIDDPVHVERFNHDCSRDFQVSAADFERKRPQDFDFFLVFDLEGKVEILEF PVLIIDAKTMGVVDLFHRFVRPTAMSEERVNEYIYNKYGKFGVDRVWHDTALPFNEVLQQFE SWLTQHNLWEKTRGGRLNRAAFVTCGNWDVKTQVPHQCSVSKLKLPPYFMEWINLKDVYQNF YNPRNEARGMRTMMSQLKIPMVGSHHLGLDDTKNIARVLLRMLADGAVLPITARRKPESPGS VNFLYKNRI >P. Seq.8S_rRNA (GenBank: AJ492448. Seq.1: 32084‐32329) MSFPRIPLSRVPSYLHNSNNCFHLLHPPFIPVSKTPSLPTYQTARTYTDFNSQTQTQPPLSL PSLIPSPPVNNPNATHRWKP >A. Seq.1) MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSS SSPSTFSLMETSENARWRPMCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQ EFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGV DRVWHDTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNI NLPPYFMEWINLKDVYLNFYGREARGMVSMMRQCGIKLMGSHHLGIDDTKNITRVVQRMLSE GAVLKLTARRSKSNMRNVEFLFKNRIK >N.thaliana_ERL1_protein (NCBI Ref. translated (NCBI Ref.thaliana_ERL1_cDNA (NCBI Ref.trichocarpa_putative ERL1_chloroplast_leader. NM_112377. AC217034.5S_rRNA (NCBI Ref.1) AGTTCCCAGTCCCTGTACTCGAAAGGAAGATCTTCATCTTCAATCTTCATGCTAATCGACGA AAATGGCGTCCGCATTCTCTGCATTTAGGGTTTCGTTGTCCAGAATCAGTCCTTTCCGTGAT ACCCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAATCATGTGCAACTC TTCGCATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTT CTTCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGTGAAAATGCAAGGTGGAGACCCATG TGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGATGATCCTGCCCATTTGGAGATTTT TAACCACGATTGTTCAAAGGAACTTCGAGTGGCTGCTGCTGATCTTGAGAGAAAGAAGTCAC AAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGAAAAGTTGAGATTCTTGAGTTTCCT ATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGACTTATTCCACAGGTTTGTAAGACC CACCAAAATGAGCGAGCAAGCAATTAACAAATACATCGAAGGCAAGTATGGGGAACTCGGGG TTGATCGTGTGTGGCATGACACAGCTATTCCATTTAAGCAAGTTGTTGAGGAGTTTGAAGTT TGGTTAGCTGAGCATGACTTGTGGGATAAAGATACAGATTGGGGTCTGAACGATGCAGCTTT TGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTCCTGAGCAATGCGTAGTTTCAAACA TCAATCTTCCGCCATATTTTATGGAGTGGATCAATCTCAAAGACGTCTACTTGAATTTCTAT GGCCGTGAGGCAAGAGGAATGGTGTCAATGATGAGGCAGTGTGGAATAAAACTCATGGGAAG CCACCATCTGGGCATTGATGACACAAAGAACATCACGAGGGTGGTGCAACGGATGCTCTCAG AAGGTGCAGTTCTCAAGCTCACAGCTCGAAGGAGCAAATCCAATATGAGAAACGTCGAGTTT CTCTTCAAGAACAGGATCAAGTAAAGCTCTCAAGGAAAAATGACAAACCCAACAAGCTCCAT GATCCATAAATTAGTTACTTAGTCAAGTCTTTTGAGTATTAAGATGATAACTCTTAGAAGAC AATGTGGTTACGCCTAAATGTGATGTGGTGAGAAGGTTTTGTAGATTCACATGTTTCAGAGC TATGATATTTAGATTACG >A. Supplements >P. AC217034. Seq. NP_566502. NC_001879: 109242‐109344) GAAGGTCACGGCGAGACGAGCCGTTTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGAT GTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC >N. Seq.tabacum _5.tabacum _4. NC_001879: 109601‐109721) TATTCTGGTGTCCTAGGCGTAGAGGAACCACACCAATCCATCCCGAACTTGGTGGTTAAACT CTACTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGCTCGACGCCAGGAT >N. Seq.
NC_001879: 102762‐104252) TCTCATGGAGAGTTCGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAG TCGGACGGGAAGTGGTGTTTCCAGTGGCGGACGGGTGAGTAACGCGTAAGAACCTGCCCTTG GGAGGGGAACAACAGCTGGAAACGGCTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGA ATCCGCCCGAGGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAGGC GATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCC GCGTGGAGGTAGAAGGCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGT ATCTGGGGAATAAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGATGCAAG CGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTTAAGTCCGCCGTCAAAT CCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCAAGCTGGAGTACGGTAGGGGCAGAG GGAATTTCCGGTGGAGCGGTGAAATGCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCAC TCTGCTGGGCCGACACTGACACTGAGAGACGAAAGCTAGGGGAGCGAATGGGATTAGATACC CCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGACCCGTGCAGTGCT GTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAA TTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAAGCGAAGAACCT TACCAGGGCTTGACATGCCGCGAATCCTCTTGAAAGAGAGGGGTGCCTTCGGGAACGCGGAC ACAGGTGGTGCATGGCTGTCGTCAGCTCGTGCCGTAAGGTGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCGTGTTTAGTTGCCATCGTTGAGTTTGGAACCCTGAACAGACTGCCGGTGAT AAGCCGGAGGAAGGTGAGGATGACGTCAAGTCATCATGCCCCTTATGCCCTGGGCGACACAC GTGCTACAATGGCCGGGACAAAGGGTCGCGATCCCGCGAGGGTGAGCTAACCCCAAAAACCC GTCCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGC CGGTCAGCCATACGGCGGTGAATTCGTTCCCGGGCCTTGTACACACCGCCCGTCACACTATG GGAGCTGGCCATGCCCGAAGTCGTTACCTTAACCGCAAGGAGGGGGATGCCGAAGGCAGGGC TAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCC TTT >N. Supplements GCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGA ATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCC GAGGGCACGTCTGCCTGGGCGTCACGCATCGCGTCGCCCCCCGCAC >N.tabacum _16S_rRNA (NCBI Ref.6. NC_001879: 106331‐109140) TTCAAACGAGGAAAGGCTTACGGTGGATACCTAGGCACCCAGAGACGAGGAAGGGCGTAGTA ATCGACGAAATGCTTCGGGGAGTTGAAAATAAGCATAGATCCGGAGATTCCCGAATAGGGCA ACCTTTCGAACTGCTGCTGAATCCATGGGCAGGCAAGAGACAACCTGGCGAACTGAAACATC TTAGTAGCCAGAGGAAAAGAAAGCAAAAGCGATTCCCGTAGTAGCGGCGAGCGAAATGGGAG CAGCCTAAACCGTGAAAACGGGGTTGTGGGAGAGCAATACAAGCGTCGTGCTGCTAGGCGAA GCAGCCCGAATGCTGCACCCTAGATGGCGAAAGTCCAGTAGCCGAAAGCATCACTAGCTTAT GCTCTGACCCGAGTAGCATGGGGCACGTGGAATCCCGTGTGAATCAGCAAGGACCACCTTGC AAGGCTAAATACTCCTGGGTGACCGATAGCGAAGTAGTACCGTGAGGGAAGGGTGAAAAGAA CCCCCATCGGGGAGTGAAATAGAACATGAAACCGTAAGCTCCCAAGCAGTGGGAGGAGCCAG GGCTCTGACCGCGTGCCTGTTGAAGAATGAGCCGGCGACTCATAGGCAGTGGCTTGGTTAAG GGAACCCACCGGAGCCGTAGCGAAAGCGAGTCTTCATAGGGCAATTGTCACTGCTTATGGAC CCGAACCTGGGTGATCTATCCATGACCAGGATGAAGCTTGGGTGAAACTAAGTGGAGGTCCG AACCGACTGATGTTGAAGAATCAGCGGATGAGTTGTGGTTAGGGGTGAAATGCCACTCGAAC CCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAGTTGACTGGACATCTAGGG GTAAAGCACTGTTTCGGTGCGGGCCGCGAGAGCGGTACCAAATCGAGGCAAACTCTGAATAC TAGATATGACCTCAAAATAACAGGGGTCAAGGTCGGCTAGTGAGACGATGGGGGATAAGCTT CATCGTCGAGAGGGAAACAGCCCGGATCACCAGCTAAGGCCCCTAAATGATCGCTCAGTGAT AAAGGAGGTAGGGGTGCAGAGACAGCCAGGAGGTTTGCCTAGAAGCAGCCACCCTTGAAAGA GTGCGTAATAGCTCACTGATCGAGCGCTCTTGCGCCGAAGATGAACGGGGCTAAGCGATCTG CCGAAGCTGTGGGATGTAAAAATACATCGGTAGGGGAGCGTTCCGCCTTAGAGAGAAGCCTC CGCGCGAGCGGTGGTGGACGAAGCGGAAGCGAGAATGTCGGCTTGAGTAACGCAAACATTGG 189 .tabacum _23S_rRNA (NCBI Ref. Seq. Seq.
6. BY14266) CTTCTTCAGGAAAACTCATACCTCAGAGAGCCGTTCAATTCCAATGGCTATGGGATTTTCTA GGGTCCCCTTGCTGCGGCGTTTCCTTGGTATCTCCTCCGGTACTACCTTCTTCGTACTCACT TCAGGCCCAACCGTAAAATGAATATCTCAGCCTCTCTTTCTACCACCGAAGAATCTACTTCT TCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTACTTTACTCA AGGTAAGTGCACCAAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCGC TGGAGTTTATGCAAAATGCTGCGGGACTTGAGAATTTGCGGAAGCAGGAGTTGGAATACTTT TTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTCTTTGATGC TAAAACCATGGATGTGGTTGACTTGTTCCATAGGTTTGTGAGGCCAACAAAAATGCACGAAG AAAGAATAAACGAATATATAGAAGGGAAATATGGAAAACTAGGAGTTGATCGGTATGGTATC 190 .105O08F. Supplements TGAGAATCCAATGCCCCGAAAACCTAAGGGTTCCTCCGCAAGGTTCGTCCACGGAGGGTGAG TCAGGGCCTAAGATCAGGCCGAAAGGCGTAGTCGATGGACAACAGGTGAATATTCCTGTACT GCCCCTTGTTGGTCCCGAGGGACGGAGGAGGCTAGGTTAGCCGAAAGATGGTTATCGGTTCA AGAACGTAAGGTGTCCCTGCTTTGTCAGGGTAAGAAGGGGTAGAGAAAATGCCTCGAGCCAA TGTTCGAATACCAGGCGCTACGGCGCTGAAGTAACCCATGCCATACTCCCAGGAAAAGCTCG AACGACTTTGAGCAAGAGGGTACCTGTACCCGAAACCGACACAGGTGGGTAGGTAGAGAATA CCTAGGGGCGCGAGACAACTCTCTCTAAGGAACTCGGCAAAATAGCCCCGTAACTTCGGGAG AAGGGGTGCCTCCTCACAAAGGGGGTCGCAGTGACCAGGCCCGGGCGACTGTTTACCAAAAA CACAGGTCTCCGCAAAGTCGTAAGACCATGTATGGGGGCTGACGCCTGCCCAGTGCCGGAAG GTCAAGGAAGTTGGTGACCTGATGACAGGGGAGCCGGCGACCGAAGCCCCGGTGAACGGCGG CCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGCACGA AAGGCGTAACGATCTGGGCACTGTCTCGGAGAGAGGCTCGGTGAAATAGACATGTCTGTGAA GATGCGGACTACCTGCACCTGGACAGAAAGACCCTATGAAGCTTCACTGTTCCCTGGGATTG GCTTTGGGCCTTTCCTGCGCAGCTTAGGTGGAAGGCGAAGAAGGCCTCCTTCCGGGGGGGCC CGAGCCATCAGTGAGATACCACTCTGGAAGGGCTAGAATTCTAACCTTGTGTCAGGACCTAC GGGCCAAGGGACAGTCTCAGGTAGACAGTTTCTATGGGGCGTAGGCCTCCCAAAAGGTAACG GAGGCGTGCAAAGGTTTCCTCGGGCCGGACGGAGATTGGCCCTCGAGTGCAAAGGCAGAAGG GAGCTTGACTGCAAGACCCACCCGTCGAGCAGGGACGAAAGTCGGCCTTAGTGATCCGACGG TGCCGAGTGGAAGGGCCGTCGCTCAACGGATAAAAGTTACTCTAGGGATAACAGGCTGATCT TCCCCAAGAGCTCACATCGACGGGAAGGTTTGGCACCTCGATGTCGGCTCTTCGCCACCTGG GGCTGTAGTATGTTCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGTACGTGAGCTGGGTTC AGAACGTCGTGAGACAGTTCGGTCCATATCCGGTGTGGGCGTTAGAGCATTGAGAGGACCTT TCCCTAGTACGAGAGGACCGGGAAGGACGCACCTCTGGTGTACCAGTTATCGTGCCCACGGT AAACGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCAC CCCAAGATGAGTGCTCTCCT >N. KP1B.060116T7) GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCT CAAAGGAGCCGTTTAATTCCAATGGCTACGGGATTTTGTAGGGTCCCCTTGCTGCGGCGGTT CCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTA TCTCCGCCTCTCTTTCTACCACCGAAGAATCTACTCCTTCCCTAATTCAGCCCACAACTTCC CGTACCCGTTGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATGA TCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCTTGAGCTTATGCAAAATGTTGCGG GACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAA GTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTT TTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAG GGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGATACAGCTATCCCATTTGGAGAA GTTATCGAGCAGTTTGAAGTTTGGCTGGGTGAACGTCAATTGTGGAGAAATGAACTGGGCGG CTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGA >N. tobacco (EB681897.tabacum _similar to UniRef100_A7P1T2 Cluster: Chromosome chr19 scaffold_4 (BP529372.tabacum _similar to UniRef100_A7P1T2 Cluster: Chromosome chr19 scaffold_4.
1) ATGGCTACTTCATCAGTATGCATAGCAGGAAATAGTTTGTCTACTCATAGAAGGCAGAAAGT TTTCAGGAAGGACATTTATGGCAGGAAAATTTTATTTTCCTCAAATCTTCCATCGTCTAGTA AAACATCGAGAATAGCTGTAAAAGCATCCCTTCAGCAAAGGCCAGATGAAGGAAGAAGAGGT TTTCTCAAATTATTGCTTGGAAATGTTGGGCTTGGAGTACCGGCTTTGTTAGGTGATGGAAA AGCCTACGCTGATGAGCAAGGTGTGTCTAACTCAAGGATGTCGTATTCTAGATTTTTGGAGT ATTTGGATAAGGATAGGGTGCAAAAAGTAGATTTGTTTGAAAACGGAACCATAGCTATTGTT GAGGCTATATCTCCAGAATTAGGAAACCGGGTTCAGAGGGTTCGGGTACAACTACCTGGGCT CAGCCAGGAACTCCTTCAGAAGTTGCGAGAAAAGAACATTGACTTTGCTGCTCACAATGCCC AAGAGGACTCGGGTTCTTTCCTATTCAACTTGATTGGGAATCTGGCATTCCCGCTTATTTTG ATTGGTGGTCTTTTCCTGCTATCAAGGCGGTCTCCCGGAGGAATGGGAGGTCCTGGTGGGCC TGGTAACCCATTAGCATTTGGTCAATCAAAGGCTAAATTCCAAATGGAGCCAAACACTGGTG TAACATTTGATGATGTTGCTGGTGTAGATGAAGCAAAACAAGATTTTATGGAGGTCGTAGAA TTTTTGAAGAAGCCCGAGAGGTTTACCGCAGTGGGGGCTCGTATTCCAAAAGGTGTTCTTCT TGTTGGTCCTCCTGGTACTGGGAAGACCCTGCTAGCAAAGGCAATTGCTGGTGAAGCGGGTG TTCCATTTTTCTCAATTTCAGGTTCAGAATTTGTTGAGATGTTTGTTGGTGTAGGAGCCTCT CGAGTCCGTGATCTTTTCAAGAAGGCCAAGGAAAATGCTCCCTGCATTGTATTTGTTGATGA AATTGATGCTGTTGGGCGGCAAAGAGGGACTGGAATTGGAGGAGGGAATGATGAAAGGGAAC AGACCCTGAACCAACTATTGACAGAAATGGACGGTTTCGAAGGAAATACTGGTATAATAGTT GTTGCGGCAACCAATCGTGCAGATATTCTTGACTCTGCTTTGCTGAGACCAGGGCGATTTGA TAGACAAGTAAGTGTGGATGTTCCAGATATCAAGGGAAGAACAGAGATCTTAAAGGTTCACG CGGGCAACAAGAAGTTCGATTCTGATGTTTCTCTTGAAGTTATAGCCATGAGGACACCCGGT TTTAGTGGTGCAGATCTTGCTAACCTCTTAAATGAAGCAGCCATTCTTGCTGGTCGGCGTGG TAAGACAGCAATCGCATCCAAAGAGATTGATGATTCAATTGATAGGATAGTGGCTGGAATGG AAGGAACAGTCATGACTGATGGCAAGAGCAAGAGTCTGGTGGCATACCACGAAGTTGGACAT GCCATCTGTGGAACTCTCACTCCAGGGCATGATGCTGTTCAAAAGGTCACATTAATCCCACG TGGTCAGGCAAAAGGTTTGACCTGGTTCATTCCTGCAGATGATCCAACCTTAATATCCAAGC AGCAACTCTTTGCTAGAATTGTCGGAGGACTTGGGGGAAGAGCTGCAGAGGAAGTTATCTTT GGTGAACCTGAGGTGACCACTGGTGCTGCAGGCGATTTGCAGCAGATCACCGGTTTGGCAAA ACAGATGGTTGTCACTTTTGGGATGTCTGAACTTGGCCCATGGTCACTCATGGATTCTTCTG CCCAAAGTGGTGATGTAATCATGAGAATGATGGCTAGGAATTCTATGTCAGAAAAGCTAGCT GAAGACATTGATGGTGCTGTGAAGAGGCTTTCAGACAGCGCATATGAGATTGCATTGACCCA TATCCGCAACAACCGTGAAGCAATTGATAAGATTGTGGAAGTCCTCCTTGAAAAGGAGACGA TGACCGGAGATGAATTCCGCGCTATTCTCTCAGAATTTGTTGAAATTCCTGCTGAAAACCGA GTTGCTCCTGTTGTACCTACCCCAGCAACTGTATAA >ATP‐dependent Clp protease Nicotiana tabacum homologue (NtGI: TC85451) CATTGAGAAAGATCCTGCGTTGGAGAGGAGGTTCCAACCAGTTAAAGTCCCTGAACCTACTG TGGATGAAACCATACAGATCTTGAAAGGGCTTCGGGAGAGATATGAGATTCATCACAAGCTC CGTTACACCGATGAGGCATTAGAAGCTGCTGCCCAGCTTTCTTATCAGTACATCAGTGACCG TTTTCTGCCTGATAAAGCAATTGATTTGATTGATGAAGCTGGTTCCCGTGTTAGACTACGCC ATGCACAGCTCCCTGAGGAAGCAAGAGAGCTCGAGAAAGAACTTCGTCAGATTACAAAGGAG AAAAATGAAGCTGTGCGAGGTCAAGATTTTGAAAAGGCGGGGGAACTGCGTGATAGAGAAAT GGATCTTAAGGCACAGATCTCAGCCCTGATAGACAAAAACAAAGAGATGAGCAAGGCTGAAT CCGAGGCTGGAGATACAGGTCCACTCGTTACAGAGGCAGATATTCAGCACATTGTGTCTTCA TGGACTGGCATCCCTGTCGAGAAGGTTTCAACAGATGAATCTGATCGCCTCTTAAAAATGGA AGAAACACTTCACACCAGAATCATTGGCCAGGATGAAGCTGTGAAAGCCATTAGTCGCGCTA TCCGACGTGCTCGTGTTGGGCTCAAGAATCCCAACCGACCTATTGCCAGTTTCATCTTTTCT GGTCCAACTGGTGTTGGGAAATCAGAACTGGCCAAGGCTTTAGCAGCGTACTACTTTGGTTC 191 . complete cds (GenBank: AF117339.tabacum _FtsH‐like protein Pftf precursor (Pftf) mRNA. nuclear gene encoding chloroplast protein. Supplements TAATTCAGTAGTTCGAAATACAATTCAGCAGTTGGAACTTAGCGCCTTTTGGTTCTATATGA AGAATTGCATG >N.6.
tabacum_ mRNA for chloroplast RNA polymerase. Supplements TGAAGAAGCAATGATCCGGCTTGATATGAGTGAGTTTATGGAGAGACACACTGTCTCTAAAC TCATTGGTTCACCCCCTGGTTATGTTGGTTACACTGAAGGTGGTCAACTGACTGAAGCTGTG AGGCGTCGACCTTACACTGTTGTGCTCTTTGATGAGATTGAGAAGGCTCATCCTGATGTCTT CAACATGATGCTTCAAATTCTTGAAGATGGAAGATTGACAGACAGCAAGGGCAGAACTGTCG ACTTCAAGAATACACTTCTCATCATGACATCGAATGTCGGAAGCAGTGTGATAGAGAAAGGA GGCCGTCGTATAGGTTTTGATCTAGATTATGACGAGAAGGATAGCAGTTACAACCGTATCAA GAGCTTGGTGACTGAGGAGTTGAAACAGTACTTTAGGCCAGAGTTCTTGAACAGATTGGATG AGATGATTGTATTCCGTCAGCTCACTAAGTTAGAGGTGAAGGAGATAGCTGATATCATGCTT AAGGAGGTCTTTGAGAGGTTGAAAAATAAGGAGATAGAACTTCAAGTGACGGAGAGGTTTAG AGACAGGGTGGTTGACGAAGGGTACAACCCAAGCTACGGAGCAAGACCGTTGAGGAGAGCTA TTATGAGACTGCTGGAAGACAGCATGGCCGAGAAGATGCTTGCAGGTGAGATCAAAGAAGGT GATTCAGTAATTGTGGACGTGGACTCTGATGGTAATGTGACCGTCCTCAATGGCACTAGCGG AACTCCCTCAGATCCAGCTCCTGAGCCTATCCCTGTGTAGATCCGCTCTGCTGCTTGCTTTA GCTCTGCAAATTTGTTGTTTGTAATGTTGCTTTCATTTGTCTTGGCCACTAAGCTCTCCTGG GGTTATGAAGCAACTTGTGAGTAATTTATGGGGTCATTGGGTGATGAAATTCTGCCAAGTTG AGAAGGGTAGCACCTCCATTATATCAGACCTTTCAGGATTACACACTATATACTGAATTTCT TTAAGATTGTAGTGACCTCGCAAGATAGTGTAAAATTGCGACGAAGACTATGTAGAACC >N.6. rpoT3‐tom gene (GenBank: AJ416570.2) CTGGCTTCCACAGCTTCTTACTCTCCAAGTCCAACATCTCAATGGAGAACTCAAAAACTCCC AAAAAGATTCAATTTTTATGTCATTCACAATCAAGAATTTGGAAAATTATCCCAAAGTTCAT CACTTTCCACTTCTTCATTTCCCAAAACTCTTAAATTACCTGTAATTCATATGCCAATTAAT AATATTCAGTCCCAAACAACAGTGTGTGTATCCACTGATGAGAATCTTGAAGAATTGGTGAA TTTGCAGAAAATCCCAAATGGGTTTTTGAATAAAGAATCAAATAAGAGAGTTTTTATTCAAG ACCCACCTTGGGTTTCTTCACTTTTTATGAATAGTTTGTTTGTTAGAGCTAAACAGGTTCAA GGGGTGAGAAGGGAATTTAGGGAAATTGAGAGAAGGAGAAGGTATGCTATGTTGAGGAGGAG ACAAATAAAGGCGGAAACTGAGGCTTGGGAGCAAATGGTGGAGGAATATAGGGAGTTGGAGA GGGAAATGTGTGAGAAGAAACTAGCACCCAATTTGCCTTATGTTAAAAAGCTGTTATTGGGT TGGTTTGAGCCATTGAGACAAGCTATAGAGAAGGAGCAGAACGCTGAGACGACCGTGAAACA TAGGGCGGCGTTTGCGCCACATATTGATTCTTTACCTGCTGATAAAATGGCTGTGATTGTGA TGCATAAGTTGATGGGATTGTTGATGATGGGTGGTAAAGAAGAGAGATGTGTTCAGGTCGTC CAAGCTGCGGTGCAGATTGGCATGGCAGTCGAGAATGAAGTTAGGATTCATAATTTCTTGGA GAAAACAAAGAAACTCCAGAAACATATGACTGGAGCTCAAAGTCAAGAAGATATGAGTAAGG AGACAATGATTCTAAGGAAACGGGTCAAAAGCTTGATTAAAAGGAATCGAGTAGTTGAGGTG AGAAAGCTGATGAAAAGTGAAGAACCCGAGTCTTGGGGTCGGGATACACAGGCTAAGTTAGG ATGCCGGCTTTTAGAATTATTAACAGAAACAGCTTATGTGCAACGTCCAGTGGATCAGTCTG CTGATACTCCTCCTGATATTAGGCCTGCGTTCAGGCACGTATTCAAAATTGCTACAAGAGAT CCGGGGAAGAACATTGTCAAGAAGTATGGTGTCATTGAATGTGATCCATTGGTTGTTGCGGG AGTTGACAGAACAGTGAAACAGATGATGATTCCTTATGTGCCTATGTTGGTGCCACCCAAAA AATGGAGAGGGTATGACAAAGGCGGATACTTATTCTTGCCCTCCTATTTGATGCGCACTCAT GGATCTAGGAGGCAACAAGATGCTGTAAGAGGTGTTCCCACGAAACAAATGCAGCAAGTTTA TGAGGCCTTGGATACCTTAGGAAGCACTAAATGGAGAGTGAATAAAAGGATACTAAATGTGG TTGAGAGTATTTGGGCTGGAGGAGGAAATATTGCTGGCCTAGTGGATCGCAAAGATGTTCCC ATACCGGAGTTGAGCAACTCCGATGATATAATGGAAGTGAAAAAGTGGAAATGGAAAGTGAG AAAATCCAAGAAAATCAACCAAGAGTTGCATTCCCAAAGATGTGACACAGAGCTCAAGCTTT CAGTTGCTCGGAAATTGAAAGATGAGGAAGGATTTTATTATCCTCACAATCTTGATTTTCGA GGACGTGCATACCCTATGCATCCTCATTTGAATCACTTGAGCTCTGATCTCTGTCGAGGAAT CCTTGAATTTGCGGAAGGACGACCACTAGGAAAGTCAGGATTGCGTTGGCTGAAAATACATT TAGCAAGTCTTTATGCAGGGGGGGTAGAGAAGCTCTGCTACGATGCACGCCTTGCATTTGTA GAAAACCACATTCATGACATATTAGATTCAGCAAACAATCCTCTAAATGGAAATCGATGGTG GTTAAATGCTGAGGATCCTTTTCAGTGCTTAGCAGCTTGCATCAACCTATCAGAAGCTTTAA 192 .
1) ATGCTCGGGGATGGAAATGAGGGAATATCTACAATACCTGGATTTAATCAGATACAATTTGA AGGATTTTGTAGGTTCATTGATCAAGGTTTGACGGAAGAACTTTATAAGTTTCCAAAAATTG AAGATACAGATCAAGAAATTGAATTTCAATTATTTGTGGAAACATATCAATTGGTCGAACCC TTGATAAAGGAAAGAGATGCTGTGTATGAATCACTCACATATTCTTCTGAATTATATGTATC CGCGGGATTAATTTGGAAAAACAGTAGGGATATGCAAGAACAAACAATTTTTATCGGAAACA TTCCTCTAATGAATTCCCTGGGAACTTCTATAGTCAATGGAATATATAGAATTGTGATCAAT CAAATATTGCAAAGTCCCGGTATTTATTACCGATCAGAATTGGACCATAACGGAATTTCGGT CTATACCGGCACCATAATATCAGATTGGGGAGGAAGATCAGAATTAGAAATTGATAGAAAAG CAAGGATATGGGCTCGTGTAAGTAGGAAACAAAAAATATCTATTCTAGTTCTATCATCAGCT ATGGGTTTGAATCTAAGAGAAATTCTAGAGAATGTTTGCTATCCTGAAATTTTTTTGTCTTT TCTGAGTGATAAGGAGAGAAAAAAAATTGGGTCAAAAGAAAATGCCATTTTGGAGTTTTATC AACAATTTGCTTGTGTAGGTGGCGATCCGGTATTTTCTGAATCCTTATGTAAGGAATTACAA AAGAAATTCTTTCAACAAAGATGTGAATTAGGAAGGATTGGTCGACGAAATATGAACCGAAG ACTGAACCTTGATATACCCCAGAACAATACATTTTTGTTACCACGAGATATATTGGCAGCCG CCGATCATTTGATTGGGCTGAAATTTGGAATGGGTGCACTTGACGATATGAATCATTTGAAA AATAAACGTATTCGTTCTGTAGCAGATCTTTTACAAGATCAATTCGGATTGGCTCTGGTTCG TTTAGAAAATGTGGTTCGGGGGACTATATGTGGAGCAATTCGGCATAAATTGATACCGACAC CTCAGAATTTGGTAACCTCAACTCCATTAACAACTACTTATGAATCCTTTTTCGGTTTACAC CCATTATCTCAAGTTTTGGATCGAACTAATCCATTGACACAAATAGTTCATGGGAGAAAATT AAGTTATTTGGGCCCTGGAGGACTGACAGGGCGCACTGCTAGTTTTCGGATACGAGATATCC 193 . complete cds (GenBank: U32397. Supplements AAAGCTCGTCACCACATACTGTCATCTCCCATCTGCCTATTCATCAGGATGGTTCATGCAAT GGCCTACAGCACTATGCTGCTCTGGGGAGAGATAGCATGGAGGCCGCAGCCGTCAACTTAGT TGCTGGAGAGAAACCAGCTGATGTTTATACTGAAATTGCTCTGAGGGTTGATCATATTATCA GAGGAGATAGTATCAAGGACCCTGCAATTGATCCTAATGCTTTACTAGCCAAACTCCTAATT GACCAGGTTGACAGGAAATTGGTGAAGCAGACAGTAATGACCTCAGTGTATGGTGTTACCTA TGTCGGAGCACGTGAGCAAATCAAAAGAAGATTGGAGGAGAAGGGTCTTATCGATGATGATA GGCTGCTATTTACTGCATCTTGCTATGCTGCTAAAGTGACATTAGCTGCTTTGGGGGAGTTA TTTCAAGCGGCACGTGGCACAATGACTTGGCTTGGTGACTGTGCTAAGGTGATTGCTTTAGA AAATCAGCCAGTGCGATGGACGACACCACTGGGGCTCCCTGTTGTGCAGCCTTACTTTAAAA CTCAGCGGCATGTTATAAGAACTTCTCTTCAAATTTTGGCTTTGCAGCGCGAGGGTGATGCA GTTGAGGTCAGGAAACAGAGAACTGCTTTTCCTCCAAATTTCGTGCACTCACTTGATGGTTC GCATATGATGATGACTGCTGTTGCTTGTAGGGATGCTGGACTACAATTTGCAGGGGTACATG ATTCCTTCTGGACTCATGCATGTGATGTCGACCAGATGAACAGGATACTCCGCGAAAAGTTT GTGGAGCTGTACAGTTTGCCTATTCTTGAAGATTTGCTCGAAAACTTCCAGAAGTCATATCC AGCATTAACATTTCCTCCTCTACCAAAAAGAGGTGATTTCAATTTAAGGGAAGTTCTCGAGT CGCCCTACTTCTTTAACTGA >N.1) ATGCCTATTGGTGTTCCAAAAGTCCCTTTCCGAAGTCCTGGAGAGGAAGATGCATCTTGGGT TGACGTATACAACCGACTTTATCGAGAAAGATTACTTTTTTTAGGCCAAGAGGTTGATAGCG AGATTTCGAATCAACTTATTGGTCTTATGGTATATCTCAGTATCGAGGATGAGACCAAAGAT CTGTATTTGTTTATAAACTCTCCTGGGGGCTGGGTAATACCTGGGGTGGCTATTTATGATAC TATGCAATTTGTGCGACCAGATGTCCATACAATATGCATGGGATTAGCCGCTTCAATGGGAT CTTTTATCCTGGTCGGAGGAGAAATTACCAAACGTCTAGCATTCCCTCACGCTAGGGTAATG ATCCATCAACCTGCTAGTTCTTTTTATGAGGCACAAACAGGCGAATTTGTCCTGGAAGCGGA AGAACTGCTGAAACTGCGTGAAACCCTCACAAGGGTTTATGTACAAAGAACGGGGAAACCCT TATGGGTTGTATCCGAAGATATGGAAAGAGATGTTTTTATGTCAGCAACAGAAGCCCAAGCT TATGGAATTGTTGATCTTGTAGCGGTTGAATGA >N.tabacum_ chloroplast DNA for putative RNA polymerase beta‐subunit (ORF 1070) (GenBank: X12745. chloroplast gene encoding chloroplast protein.tabacum_ ClpP protease (ClpP) mRNA.6.
complete cds. partial cds.1) ATGTCACCACAAACAGAGACTAAAGCAAGTGTTGGATTCAAAGCTGGTGTTAAAGAGTACAA ATTGACTTATTATACTCCTGAGTACCAAACCAAGGATACTGATATATTGGCAGCATTCCGAG TAACTCCTCAACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCT ACTGGTACATGGACAACTGTATGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCG ATGCTACCGCATCGAGCGTGTTGTTGGAGAAAAAGATCAATATATTGCTTATGTAGCTTACC CTTTAGACCTTTTTGAAGAAGGTTCTGTTACCAACATGTTTACTTCCATTGTAGGTAACGTA TTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTCCTGCTTATGT TAAAACTTTCCAAGGTCCGCCTCATGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTATG GTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAAACTACGGT AGAGCCGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACTAAAGATGATGAGAACGTGAA CTCACAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAG CACAGGCTGAAACAGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGTACATGCGAA GAAATGATCAAAAGAGCTGTATTTGCTAGAGAATTGGGCGTTCCGATCGTAATGCATGACTA CTTAACGGGGGGATTCACCGCAAATACTAGCTTGGCTCATTATTGCCGAGATAATGGTCTAC TTCTTCACATCCACCGTGCAATGCATGCGGTTATTGATAGACAGAAGAATCATGGTATCCAC TTCCGGGTATTAGCAAAAGCGTTACGTATGTCTGGTGGAGATCATATTCACTCTGGTACCGT 194 .6. tabacum_ coupling factor beta‐subunit gene.5‐ biphosphate carboxylase/ oxygenase large subunit gene. and ribulose‐1. Supplements ATCCTAGTCACTATGGACGTATTTGCCCAATTGACACATCTGAAGGAATCAATGTTGGACTT ATTGGATCCTTAGCAATTCATGCGAGGATTGGTCATTGGGGATCTCTAGAAAGCCCTTTTTA TGAAATTTCTGAGAGGTCAACCGGGGTACGGATGCTTTATTTATCACCAGGTAGAGATGAAT ACTATATGGTAGCGGCAGGAAATTCTTTAGCCTTAAATCAGGATATTCAGGAAGAACAGGTT GTTCCAGCTCGATACCGTCAAGAATTCTTGACTATTGCATGGGAACAGGTTCATCTTCGAAG TATTTTTCCTTTTCAATATTTTTCTATTGGAGCTTCCCTCATTCCTTTTATCGAACATAATG ATGCGAATCGAGCTTTAATGAGTTCTAATATGCAACGTCAAGCAGTTCCTCTTTCTCGCTCC GAGAAATGCATTGTTGGAACTGGGTTGGAACGACAAGCAGCTCTAGATTCGGGGGCTCTTGC TATAGCCGAACGCGAGGGAAGGGTCGTTTATACCAATACTGACAAGATTCTTTTAGCAGGTA ATGGAGATATTCTAAGCATTCCATTAGTTATATATCAACGTTCCAATAAAAATACTTGTATG CATCAAAAACTCCAGGTTCCTCGGGGTAAATGCATTAAAAAGGGACAAATTTTAGCGGATGG TGCTGCTACGGTTGGTGGCGAACTTGCTTTGGGGAAAAACGTATTAGTAGCTTATATGCCGT GGGAGGGTTACAATTCTGAAGATGCAGTACTTATTAGCGAGCGTTTGGTATATGAAGATATT TATACTTCTTTTCACATACGGAAATATGAAATTCAGACTCATGTGACAAGCCAAGGCCCTGA AAAAGTAACTAATGAAATACCGCATTTAGAAGCCCATTTACTCCGCAATTTAGATAAAAATG GAATTGTGATGCTGGGATCTTGGGTAGAGACAGGTGATATTTTAGTAGGTAAATTAACACCC CAGGTCGTGAAAGAATCGTCGTATGCCCCGGAAGATAGATTGTTACGAGCTATACTTGGTAT TCAGGTATCTACTTCAAAAGAAACTTGTCTAAAACTACCTATAGGTGGCAGGGGTCGGGTTA TTGATGTGAGGTGGATCCAGAAGAGGGGTGGTTCTAGTTATAATCCCGAAACGATTCGTGTA TATATTTTACAGAAACGTGAAATCAAAGTAGGCGATAAAGTAGCTGGAAGACACGGAAATAA AGGTATCATTTCCAAAATTTTGCCTAGACAAGATATGCCTTATTTACAAGATGGAAGATCCG TTGATATGGTCTTTAACCCATTAGGAGTACCTTCACGAATGAATGTAGGACAGATATTTGAA TGTTCACTAGGGTTAGCAGGGAGTCTGCTAGACAGACATTATCGAATAGCACCTTTTGATGA GAGATATGAACAAGAAGCTTCGAGAAAACTTGTGTTTTCTGAATTATATGAAGCCAGTAAGC AAACAGCGAATCCATGGGTATTTGAACCCGAATATCCAGGAAAAAGCAGAATATTTGATGGA AGGACGGGGAATCCTTTTGAACAACCCGTTATAATAGGAAAGCCTTATATCTTGAAATTAAT TCATCAAGTTGATGATAAAATCCATGGGCGCTCCAGTGGACATTATGCGCTTGTTACACAAC AACCCCTTAGAGGAAGAGCCAAACAGGGGGGACAGCGGGTAGGAGAAATGGAGGTTTGGGCT CTAGAAGGGTTTGGGGTTGCTCATATTTTACAAGAGATGCTTACTTATAAATCGGATCATAT TAGAGCTCGCCAGGAAGTACTTGGTACTACGATCATTGGGGGAACAATACCTAATCCCGAAG ATGCTCCAGAATCTTTTCGATTGCTCGTTCGAGAACTACGATCTTTAGCTCTGGAACTGAAT CATTTCCTTGTATCTGAGAAGAACTTCCAGATTAATAGGAAGGAAGCT >N. chloroplast genes for chloroplast products (GenBank: J01450.
basta resistance: 3195 to 3746 (AY836546.6. Supplements AGTAGGTAAACTTGAAGGTGAAAGAGACATAACTTTGGGCTTTGTTGATTTACTGCGTGATG ATTTTGTTGAACAAGATCGAAGTCGCGGTATTTATTTCACTCAAGATTGGGTCTCTTTACCA GGTGTTCTACCCGAGGCTTCAGGAGGTATTCACGTTTGGCATATGCCTGCTCTGACCGAGAT CTTTGGGGATGATTCCGTACTACAGTTCGGTGGAGGAACTTTAGGACATCCTTGGGGTAATG CGCCAGGTGCCGTAGCTAATCGAGTAGCTCTAGAAGCATGTGTAAAAGCTCGTAATGAAGGA CGTGATCTTGCTCAGGAAGGTAATGAAATTATTCGCGAGGCTTGCAAATGGAGCCCGGAACT AGCTGCTGCTTGTGAAGTATGGAAAGAGATCGTATTTAATTTTGCAGCAGTGGACGTTTTGG ATAAGTAA >N.1) ATGACTGCAATTTTAGAGAGACGCGAAAGCGAAAGCCTATGGGGTCGCTTCTGTAACTGGAT AACTAGCACTGAAAACCGTCTTTACATTGGATGGTTTGGTGTTTTGATGATCCCTACCTTAT TGACGGCAACTTCTGTATTTATTATTGCCTTCATTGCTGCTCCTCCAGTAGACATTGATGGT ATTCGTGAACCTGTTTCAGGGTCTCTACTTTACGGAAACAATATTATTTCCGGTGCCATTAT TCCTACTTCTGCAGCTATAGGTTTACATTTTTACCCAATCTGGGAAGCGGCATCCGTTGATG AATGGTTATACAACGGTGGTCCTTATGAACTAATTGTTCTAGACTTCTTACTTGGCGTAGCT TGTTACATGGGTCGTGAGTGGGAGCTTAGTTTCCGTCTGGGTATGCGACCTTGGATTGCTGT TGCATATTCAGCTCCTGTTGCAGCTGCTACCGCAGTTTTCTTGATCTACCCAATTGGTCAAG GAAGTTTTTCTGATGGTATGCCTCTAGGAATCTCTGGTACTTTCAATTTCATGATTGTATTC CAGGCTGAGCACAACATCCTTATGCACCCATTTCACATGTTAGGCGTAGCTGGTGTATTCGG CGGCTCCCTATTCAGTGCTATGCATGGTTCCTTGGTAACTTCTAGTTTGATCAGGGAAACCA CAGAAAATGAATCTGCTAATGAAGGTTACAGATTCGGTCAAGAGGAAGAAACTTATAACATC GTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCAATATGCTAGTTTCAACAACTCTCG TTCGTTACACTTCTTCCTAGCTGCTTGGCCTGTAGTAGGTATCTGGTTTACCGCTTTAGGTA TCAGCACTATGGCTTTCAACCTAAATGGTTTCAATTTCAACCAATCTGTAGTTGACAGTCAA GGCCGTGTAATTAATACTTGGGCTGATATCATTAACCGTGCTAACCTTGGTATGGAAGTTAT GCATGAACGTAATGCTCACAACTTCCCTCTAGACCTAGCTGCTATCGAAGCTCCATCTACAA ATGGATAA >T‐DNA vector pDs‐Lox.1) ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGT CTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGG AACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCC GAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTA CGACTGGACGGCCGAGTCAACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCT CCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCT GTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCG CGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGC TGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATTTGA 195 . complete sequence.tabacum_ chloroplast gene P32 for thylakoid membrane protein (GenBank: X00616.
2005 10/2003 – 02/2004 10/1999 – 10/2005 22th of June. Foundation of Re‐ search and Technology Hellas (FoRTH‐IMBB) in Heraklion. Austria Mobile: +43 699 10402842 E‐Mail: Jutta. Greece Study of Food Science and Biotechnology with emphasis on biochemistry and cell biology at the University of Natural Resources and Applied Life Sciences in Vienna. funded by a Marie Curie Fellowship at the Institute of Molecular Biology and Biotechnology. unmarried.6.6 Curriculum vitae (March. Univ. Austria. Austria Brücklweg 20. Kriton Kalantidis Graduation to “Diplomingenieurin” in Food Science and Biotechnology (comparable to MSc) with excellent success Master thesis: Characterization of the myeloperoxidase mutant Met243Thr produced in CHO cells. Greece. Austria 09/2006 – current 14th of October. Dr.at Austrian citizenship. Austria GCSE A‐level with good success at the BG/BRG Gmunden. 1981 in Gmunden. DI Dr. 1999 196 . Mag.Helm@gmx. Prof. Marie‐Theres Hauser expected end: 2011 PhD thesis: The putative RNA silencing protein ERL‐1 is involved in chloroplast ribosomal RNA processing in plants. supervised by Ao. no children Education Doctorate Studies in Agricultural Sciences at the University of Natural Resources and Applied Life Sciences in Vienna. Prof. Christian Obinger Grade: excellent Exchange semester at the Aristoteleio Panepistimio Thessa‐ lonikis in Thessaloniki. supervised by Ao. supervised by Ass. Univ. 2011) Personal Information DI Jutta Maria Helm Born the 11th of July. 4663 Laakirchen. Prof. Dr.
validation of cali‐ bration functions and assistance in the microbiological labo‐ ratory. Germany Reflectometric. SCA Laakirchen (Quality Assurance) in Laakirchen. Austria Sewage and physical paper analysis. Austria Assistant within the GENAU‐Project: Heritable stress ef‐ fects in plants SCA Graphic Sundsvall AB (Ortviken Paper Mill – Devel‐ opment Department) in Sundsvall. Austria Chemical analysis of food raw material. Supplements Work experience Ebewe Pharma GesmbH Nfg KG (Sandoz Business Unit Oncology Injectables) in Unterach. Merck Life Science & Analytics (Research & Development Department) in Darmstadt. Sweden Evaluation and validation of a protocol for the determina‐ tion of COD release during the thermo‐mechanical pulping process and its application at different refiner loads. Austria Frost Nahrungsmittel GmbH (Quality Assurance) in Groß‐Enzersdorf. Austria Laboratory support in analytic chemistry for undergradu‐ ate students. regulatory maintenance University of Natural Resources and Applied Life Sciences in Vienna.6. Austria Teaching assignment: Exercises in Molecular Biology (Eng‐ lish) University of Natural Resources and Applied Life Sciences in Vienna. 04/2010 – current 01/2010 04/2006 – 08/2006 06/2005 – 07/2005 07/2004 – 09/2004 08/2003 07/2002 – 08/2002 197 . photometric and potentiometric determina‐ tion of wine quality parameters and optimization of the probe preparation for the Reflectoquant® determination of yeast‐usable nitrogen. regional ex‐ pert CIS/SEE. Austria Regulatory Affairs Manager: product expert. 03/2005 – 05/2005 University of Natural Resources and Applied Life Sciences in Vienna.
Stopped‐Flow Spectroscopy. Greek: very good. (2011) Local RNA silencing mediated by agroin‐ filtration. Confocal‐/Electron‐/ Lightmicro‐ scopy MS Office 2003 & 2007. (2006). Schumacher HT. play‐ ing guitar Software Driving licenses Interests Publications Helm JM. Southern/Northern/Western Blotting. Methods in Molecular Biology 744 – in press Kalantidis K. Vamvaka E.6. Supplements Additional skills Languages Methods German: mother tongue. Helm JM. competent with internet applications. 100(1):13‐26 Furtmüller PG. 3’‐ RACE. 445(2):199‐213 198 . Zederbauer M. Generation of transgenic plants. English: excellent. animal cell culture (CHO cells). RNA silencing move‐ ment in plants. French. Obin‐ ger C. Jakopitsch C. Biol Cell. Kalantidis K. A role for plant ERL1 in rRNA biogenesis – in preparation Helm JM. reading. swimming. RNAi & Plant Gene Function Analysis. Schumacher HT. working with radioactivity. liquid chromatography. ELISA. Arch Biochem Biophys. Isola‐ tion/Transformation of protoplasts. ba‐ sics in SPSS A. to experience foreign cultures and habits. sports especially bicycling. COI. my dog Mavrodafni. Bogner M. SDS‐PAGE. Flow Cytometry. Helm J. Kalantidis K. SAP. Dadami E. Cloning. B (own car available) Studying languages and travelling. DNA‐/RNA‐/Protein‐Extraction. Alexiadis T. Jantschko W. real‐time PCR. hiking and volleyball. Spanish: good PCR. Active site structure and catalytic mechanisms of human peroxidases. Adobe Photoshop CS3. FPLC. Agroinfiltration. (2008). eCTD‐Manager.
Goethe 200 . ich armer Tor! Und bin so klug als wie zuvor… Faust. Da steh ich nun.
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