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Moss Systems Biology for Translational Research

Moss Systems Biology for Translational Research. Ralf Reski. Moss Physcomitrella patens : A New Model Plant. Small Few cell types Cell lineage Tightly controlled growth conditions Predominant generation haploid Fully sequenced genome Efficient homologous recombination

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Moss Systems Biology for Translational Research

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  1. Moss Systems Biology for Translational Research Ralf Reski

  2. Moss Physcomitrella patens: A New Model Plant • Small • Few cell types • Cell lineage • Tightly controlled growth conditions • Predominant generation haploid • Fully sequenced genome • Efficient homologous recombination  targeted gene knockout / replacement unique to higher eukaryotes

  3. Modelling Plant Development 1: Light Microscope

  4. Modelling Plant Development 2: Pattern Recognition

  5. no branching yes type of branching F-type branching, left F-type branching, right Y-type of branching Growth! Modelling Plant Development 3: Simulation

  6. miRNAs Regulate Gene Expression Qickly • micro RNAs: • not translated • more efficient than synthesis of regulatory proteins (e. g. repressors) • faster regulatory circuits

  7. Dicer RISC RISC HASTY AGO AGO HEN HYL CAP AAAAA Biogenesis and Function of micro RNAs pri-miRNA pre-miRNA Nucleus Cytosol Post-transcriptional regulation of gene expression

  8. ANIMALS PLANTS Animals and Plants: Different Sets of DICER

  9. Targeted knockout of DCL1a: Drastic Effects WT KO1 KO2 WT miRNA156 miRNA160 miRNA166 miRNA390 7 WT 6 5 KO1 4 Rel. Transcript Level KO2 3 2 1 0 PpSBP3 PpARF PpHDZIP PpHB10 PpTAS1 RT-PCR analysis of miRNAs No DCL1a: No miRNAs, enhanced TF levels, serverely affected growth DCL1a KO1 RT-PCR analysis of target transcripts DCL1a KO2

  10. Targeted knockout of DCL1b: Less Drastic Effects miRNA Expression PpDCL1b KO miRNA156 WT 1 2 3 4 miRNA160 miRNA166 miRNA390 miRNA535 miRNA538 U6 snRNA Wild Type DCL1b miRNAs still present BUT developmental arrest

  11. KO 1 KO 2 KO 3 KO 4 WT DCL1b: miRNAs Cannot Cleave Target mRNAs PpSBP3 (Squamosa Promoter Binding Protein) PpSBP3 5’ U 3’ GUGCUC CUCUCUUCUGUCA CACGAG GAGAGAAGACAGU miR156 3’ U 5’ PpC3HDZIP (HD leucine zipper) PpC3HDZIP1 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCU miR165 3’CC C 5’ PpHB10 (HD leucine zipper) PpHB10 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCU miR165 3’CC C 5’ Control PpGNT1 (no miRNA target)

  12. miRNA Target Genes Silenced in DCL1b Mutant PpDCL1b-Mutants WT 1 2 3 4 PpARF PpHDZIP1 miRNA target transcripts PpHB10 PpSBP3 PpGNT1 Controls (no miRNA targets) EF1 miRNAs cannot cleave target mRNAs in the mutant, nevertheless transcript levels of target genes reduced. Novel feed-back loop: Transcriptional control by miRNAs

  13. Group 3 PpARF3-1 PpARF3-2 Tandem duplication miRNA160 regulates ARF Transcription Factors PpARF1-1 PpARF1-2 PpARF1-3 PpARF1-4 PpARF1-5 PpARF1-6 PpARF1-7 PpARF1-8 • Arabidopsis: 21 ARF Genes • Rice: 24 ARF-Genes • Physcomitrella: 15 ARF Genes Group 1 PpARF1-9 PpARF1-10 PpARF1-11 PpARF1-12 Group 2 Group 3 PpARF3-1 PpARF3-2 Phypa_61245 Tandem duplication

  14. miRNA160: Part of Multilevel Regulatory Network Wild type ARFm Mutant Auxin Auxin miRNA 160 miRNA 160 ARF ARF GH3-2 GH3-2

  15. t ggc atg cag ggg gcc aggca G M Q G A R Pau I t ggc atg caa ggc gcgcga ca G M Q G A R Precise Manipulation of miRNA160 Network Abolish miRNA160 binding site without changing amino acids in the transcription factor Targeted base-specific mutation of endogenous gene!

  16. 3=0 in the ARFm mutant Dynamic Modelling of 4-Level Regulatory Network Impact of GH3-2 on [Auxin] Endogenous auxin synthesis Auxin application Auxin: Activation of miR160 by auxin Activation of miR160 by ARF miR160: Activation of ARF by auxin Inhibition of ARF by miR160 ARF: Activation of GH3-2 by ARF GH3-2:

  17. Expectations from Dynamic Modelling Auxin application 2.0 Steady- State GH3-2 ARF Mutant miRNA 1.5 ARF GH3-2 miRNA [Expression] 1.0 Wild type 0.5 ARF 0 10 20 30 40 50 [Time]

  18. WT ARFm Mutant Mutant: Elevated ARF mRNA levels ARF transcript levels respond to auxin 1,6 1,4 1,2 1 Relative Expression 0,8 0,6 0,4 0,2 0 Control 1h 4h 8h 12h 24h Auxin treatment

  19. WT ARFm Mutant Elevated Transcript Levels of Downstream Gene GH3 conjugates (= inactivates) auxin GH3-2 mRNA levels enhanced by auxin 2,5 2 1,5 Relative Expression 1 0,5 0 control 1h 4h 8h 12h 24h Auxin treatment

  20. Auxin Elevated Levels of Conjugated (= Inactive) Auxin Free & conjugated Auxin Free Conjugated GH3-2: Auxin conjugating enzyme NAA + IAA [ng/g Fresh Weight] WT ARF Mutant

  21. Auxin Mutant is Hyposensitive to Auxin ARFm mutant confirms prediction. WT ARFm Mutant 0 0.1 µM 0.5 µM 1 µM [Auxin]

  22. Extended Model to Include Diurnal Changes

  23. Current Modelling Approaches

  24. The Players Gotelinde Seumel Basel Kraiwesh Asif Arif Erika Lang Philipp Rödel Marta Tomek Jutta Ludwig-Müller Olaf Rönneberger Stefan Jansen Andreas Schlosser Daniel Lang Marco Vervliet-Scheebaum Wolfgang Frank Jens Timmer

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