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• Classical epigenetic systems

• Gene silencing

• Viral cross-protection

• Epigenetics in plant development

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• Transposons - change of phase

• Paramutation in maize

CLASSICAL EPIGENETIC SYSTEMS

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• Heritable, but reversible

• Epimutants differ in theirdevelopmental expression patterns

• The transition from active to cryptic (and thereverse) takes several plant generations

Changes in Spm activity phase

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• McClintock: an inactive transposon wakes up when anactive transposon is present, but segregates unchanged

• Fedoroff: an active element can heritably wake up aninactive or a cryptic element

• The transition from active to cryptic (and the reverse)takes several plant generations

Genetic analysis of phase change

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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 paramutagenicupon exposure to a paramutagenic allele

Brink, R. A., Styles, E. D. and Axtell, J. D. (1968) Science, 159: 161-170

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R gene paramutation in maize

Walker, E. L. (1998), Genetics, 148: 1973-1981

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Structure of a paramutagenic R allele

Kermicle, J. L., Eggleston, W. B. and Alleman, M. (1995), Genetics, 141: 361-372

• The R-st allele contains several highly homologous repeats

• Paramutagenicity is directly proportional to the number of repeats

• Transcription start sites are methylated

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Structure of the paramutable R-r allele

Walker, E. L. (1998), Genetics, 148: 1973-1981

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Common themes intransposon inactivation and paramutation

• Sequence duplication is central

• Promoter sequences are methylated

• Genes/TEs transcriptionally silenced

• Silencing is heritable, but reversible

• Both involve transposon sequences

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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

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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

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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

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Viral resistance is RNA-mediated

• 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

PVX

W22

PVX. W22

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Gene silencing: a systemic signal

Voinnet, O., and Baulcombe, D. C. (1997). Nature 389: 553

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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

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TGS and PTGS: is there a relationship?

Wassenegger, M., Heimes, S., Reidel, L., and Sanger, H. L. (1994) Cell 76: 567-76.

P35S PSTVd cDNA pAnos P35S PSTVd cDNA pAnos

Replication competentReplication incompetent

Transcription only

No replication

No methylation

Transcription

Replication

Methylation

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microRNAs and silencingRNAs in plants

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

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microRNAs and silencingRNAs in animals

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

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The Arabidops is hy l1 mutation

wildtype

hyl1

0.6 µM ABANo ABA

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The hy l1 mutation affects miRNA levels

wt hyl1 hen1-1 1 3

ARF8

SCL6-III

rRNA

MYB33

35S::HYL1

wt hyl1 hen1-1 1 3

35S::HYL1

miR167

miR171

tRNA +5S rRNA

miR159

UBQ1DCL1

wt hyl1 hen1-1 wt hyl1 hen1-1

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The hyl1 mutation affects mRNA stability

B.

Time (hrs)

% i

n i t i a l v a l u e

10

100

0 4 8 12

MYB33

hyl1

wt

35S::HYL1

50

30

hyl1

wt

35S::HYL1

SCL6-III

0 4 8 12

hyl1

wt

35S::HYL1

0 4 8 12

ANP1

hyl1

wt

35S::HYL1

ARF8

0 4 8 12

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HYL1 is

innuclearbodies

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• TnpA and TnpD are required for transposition

• TnpA is also a weak transcription factor

Spm has one gene,but codes for two proteins

Transposition

active Spm

TnpA

promoter

TnpD

TnpDmRNA

TnpAmRNA

TnpD

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• Promoter methylated, element inactive

• Methylation of GC-rich sequence confers heritability

• Reversed by Spm -encoded TnpA

Changes in Spm activity phase

Methylated site Unmethylated site

cryptic Spm active Spm TnpA

promoter

GC-rich sequence

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• TnpA is a weak transcription factor

• TnpA binds unmethylated and hemimethylated DNA

• TnpA promotes active demethylation

Molecular mechanism of Spm activation

Methyl group promoter

replication

TnpA

TnpA TnpA

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Transposon silencing: the chromatin connection

transposition

silencing

mRNA

siRNAs?

siRNAsDNA methylasehistone deacetylasechromatin remodeling proteins

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The story of papayaringspot virus

http://www.apsnet.org/education

/feature/papaya/Top.htm

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QuickTime™ a nd a

TIFF (Uncompressed) decompressor are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Papaya ringspot virus

http://www apsnet org/education/feature/papaya/Top htm

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http://www.apsnet.org/education/feature/papaya/Top.htm

1940s: PRS virus discovered in Hawaii

1950s: Oahu’s papaya industry wiped out

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

1960s: Papaya industry moves to Puna district


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TGS• No


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1991: First transgenic PRSV-resistant papaya plant

1980s: PRSV-resistance project started under direction of Dennis Gonsalves

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


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http://www.apsnet.org/education/feature/papaya/Top.htm

Papaya

ringspotvirus

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Epigenetic mechanisms: plantevolution, 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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24891