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Classical epigenetic systems Gene silencing Viral cross-protection Epigenetics in plant development

PLANT EPIGENETIC MECHANISMS. Classical epigenetic systems Gene silencing Viral cross-protection Epigenetics in plant development. CLASSICAL EPIGENETIC SYSTEMS. Transposons - change of phase Paramutation in maize. Changes in Spm activity phase.

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Classical epigenetic systems Gene silencing Viral cross-protection Epigenetics in plant development

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  1. PLANT EPIGENETIC MECHANISMS • Classical epigenetic systems • Gene silencing • Viral cross-protection • Epigenetics in plant development

  2. CLASSICAL EPIGENETIC SYSTEMS • Transposons - change of phase • Paramutation in maize

  3. Changes in Spm activity phase • Heritable, but reversible • Epimutants differ in their • developmental expression patterns • The transition from active to cryptic (and the • reverse) takes several plant generations

  4. Genetic analysis of phase change • McClintock: an inactive transposon wakes up when an • active transposon is present, but segregates unchanged • Fedoroff: an active element can heritably wake up an • inactive or a cryptic element • The transition from active to cryptic (and the reverse) • takes several plant generations

  5. Paramutation at the R locus in maize • A directed, heritable change in gene expression • r-st and r-mb termed PARAMUTAGENIC • R-r termed PARAMUTABLE • Altered expression is heritable • Partial reversion when homozygous • A paramutable allele can become paramutagenic • upon exposure to a paramutagenic allele Brink, R. A., Styles, E. D. and Axtell, J. D. (1968) Science, 159: 161-170

  6. R gene paramutation in maize Walker, E. L. (1998), Genetics, 148: 1973-1981

  7. Structure of a paramutagenic R allele • The R-st allele contains several highly homologous repeats • Paramutagenicity is directly proportional to the number of repeats • Transcription start sites are methylated Kermicle, J. L., Eggleston, W. B. and Alleman, M. (1995), Genetics, 141: 361-372

  8. Structure of the paramutable R-r allele Walker, E. L. (1998), Genetics, 148: 1973-1981

  9. Common themes in transposon inactivation and paramutation • Sequence duplication is central • Promoter sequences are methylated • Genes/TEs transcriptionally silenced • Silencing is heritable, but reversible • Both involve transposon sequences

  10. Gene silencing (co-suppression) by trangenes • Transgenes can silence endogenous genes • More transgenes, more gene silencing • Inverted repeats are especially effective • Silenced genes are often methylated • Silencing can be heritable • Silenced genes can be “paramutagenic” Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9

  11. Transcriptional and post-transcription silencing (TGS and PTGS) • Silencing can be transcriptional, post-transcriptional or both • TGS is associated with promoter methylation • PTGS is associated with coding sequence methylation • Promotor methylation is not required for initiation of silencing • Methylation is required for the maintenance of silencing

  12. Gene silencing and viral resistance • Viral infection confers immunity to further infection • Transgenic plants expressing coat protein are resistant Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560

  13. W22 PVX Viral resistance is RNA-mediated PVX. W22 • Transgene-induced resistance resembles PTGS • Resistance is mediated by RNA • Virus infection can result in co-suppression Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560

  14. Gene silencing: a systemic signal Voinnet, O., and Baulcombe, D. C. (1997). Nature 389: 553

  15. The systemic gene silencing signal is RNA • Non-overlapping gene fragments cross-silence • RNA moves between cells in plants • Plants encode RNA-dependent RNA polymerases

  16. TGS and PTGS: is there a relationship? Replication incompetent Replication competent P35S PSTVd cDNA pAnos P35S PSTVd cDNA pAnos Transcription only No replication No methylation Transcription Replication Methylation Wassenegger, M., Heimes, S., Reidel, L., and Sanger, H. L. (1994) Cell 76: 567-76.

  17. microRNAs and silencingRNAs in plants Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:120-125.

  18. microRNAs and silencingRNAs in animals Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:120-125.

  19. The Arabidopsishyl1 mutation No ABA 0.6 µM ABA wildtype hyl1

  20. 35S::HYL1 35S::HYL1 wt hyl1 hen1-1 1 3 wt hyl1 hen1-1 1 3 miR159 MYB33 miR167 ARF8 miR171 SCL6-III tRNA + 5S rRNA rRNA wt hyl1 hen1-1 wt hyl1 hen1-1 UBQ1 DCL1 The hyl1 mutation affects miRNA levels

  21. The hyl1 mutation affects mRNA stability B. hyl1 100 hyl1 hyl1 wt hyl1 wt wt wt 35S::HYL1 50 35S::HYL1 35S::HYL1 35S::HYL1 30 % initial value MYB33 ANP1 SCL6-III ARF8 10 0 4 8 12 0 4 8 12 0 4 8 12 0 4 8 12 Time (hrs)

  22. HYL1 is in nuclear bodies

  23. Spm has one gene, but codes for two proteins • TnpA and TnpD are required for transposition • TnpA is also a weak transcription factor promoter TnpA mRNA TnpA TnpD TnpD mRNA active Spm Transposition TnpD

  24. Changes in Spm activity phase • Promoter methylated, element inactive • Methylation of GC-rich sequence confers heritability • Reversed by Spm-encoded TnpA promoter GC-rich sequence TnpA cryptic Spm active Spm Methylated site Unmethylated site

  25. Molecular mechanism of Spm activation • TnpA is a weak transcription factor • TnpA binds unmethylated and hemimethylated DNA • TnpA promotes active demethylation Methyl group promoter replication TnpA TnpA TnpA

  26. Transposon silencing: the chromatin connection silencing mRNA siRNAs? transposition siRNAs DNA methylase histone deacetylase chromatin remodeling proteins

  27. The story of papaya ringspot virus http://www.apsnet.org/education/feature/papaya/Top.htm

  28. Papaya ringspot virus

  29. 1960s: Papaya industry moves to Puna district http://www.apsnet.org/education/feature/papaya/Top.htm Papaya ringspot virus 1940s: PRS virus discovered in Hawaii 1950s: Oahu’s papaya industry wiped out

  30. Papaya ringspot virus TGS • No

  31. Papaya ringspot virus 1980s: PRSV-resistance project started under direction of Dennis Gonsalves 1991: First transgenic PRSV-resistant papaya plant 1992: PRSV discovered in Puna district 1992: First field trials PRSV-resistant papaya plants 1994: USDA granted permission for large scale field trials 1995-97: Approvals for release from USDA, EPA, FDA 1992-1977: PRVS spread; many farmers went out of business 1998: Seeds released, free of charge, to growers 2000: Papaya industry bounced back; crop back to pre-1995 levels

  32. Papaya ringspot virus http://www.apsnet.org/education/feature/papaya/Top.htm

  33. Epigenetic mechanisms: plant evolution, defense and development • Gene silencing is a response to gene duplication • (evolution of duplicated genes; transposon control) • Gene silencing is a response to gene overexpression • (dosage compensation) • Gene silencing is a defense response • (viral cross protection; rapid environmental responses) • Epigenetic mechanisms are used in plant development • (JAW miRNA in leaf morphogenesis)

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