• Classical epigenetic systemsClassical epigenetic systems

• Gene silencingGene silencing

• Viral cross-protectionViral cross-protection

• Epigenetics in plant developmentEpigenetics in plant development

• Classical epigenetic systemsClassical epigenetic systems

• Gene silencingGene silencing

• Viral cross-protectionViral cross-protection

• Epigenetics in plant developmentEpigenetics in plant development

• Transposons - change of phase Transposons - change of phase

• Paramutation in maizeParamutation in maize

• Transposons - change of phase Transposons - change of phase

• Paramutation in maizeParamutation in maize

CLASSICAL EPIGENETIC SYSTEMSCLASSICAL EPIGENETIC SYSTEMS CLASSICAL EPIGENETIC SYSTEMSCLASSICAL EPIGENETIC SYSTEMS

• Heritable, but reversibleHeritable, but reversible

• Epimutants differ in theirEpimutants differ in their developmental expression patterns developmental expression patterns

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

• Heritable, but reversibleHeritable, but reversible

• Epimutants differ in theirEpimutants differ in their developmental expression patterns developmental expression patterns

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

Changes in Changes in SpmSpm activity phase activity phase Changes in Changes in SpmSpm activity phase activity phase

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

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

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

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

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

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

Genetic analysis of phase changeGenetic analysis of phase change Genetic analysis of phase changeGenetic analysis of phase change

Paramutation at the Paramutation at the R R locus in maizelocus in maizeParamutation at the Paramutation at the R R locus in maizelocus in maize

• A directed, heritable change in gene expressionA directed, heritable change in gene expression

• r-str-st and and r-mb r-mb termed PARAMUTAGENIC termed PARAMUTAGENIC

• R-r termed PARAMUTABLER-r termed PARAMUTABLE

• Altered expression is heritableAltered expression is heritable

• Partial reversion when homozygousPartial reversion when homozygous

• A paramutable allele can become paramutagenicA paramutable allele can become paramutagenic upon exposure to a paramutagenic allele upon exposure to a paramutagenic allele

• A directed, heritable change in gene expressionA directed, heritable change in gene expression

• r-str-st and and r-mb r-mb termed PARAMUTAGENIC termed PARAMUTAGENIC

• R-r termed PARAMUTABLER-r termed PARAMUTABLE

• Altered expression is heritableAltered expression is heritable

• Partial reversion when homozygousPartial reversion when homozygous

• A paramutable allele can become paramutagenicA paramutable allele can become paramutagenic upon exposure to a paramutagenic allele upon exposure to a paramutagenic allele

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

R R gene paramutation in maizegene paramutation in maize R R gene paramutation in maizegene paramutation in maize

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

Structure of a paramutagenic R alleleStructure of a paramutagenic R alleleStructure of a paramutagenic R alleleStructure of a paramutagenic R allele

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

• The R-st allele contains several highly homologous repeatsThe R-st allele contains several highly homologous repeats

• Paramutagenicity is directly proportional to the number of repeatsParamutagenicity is directly proportional to the number of repeats

• Transcription start sites are methylatedTranscription start sites are methylated

• The R-st allele contains several highly homologous repeatsThe R-st allele contains several highly homologous repeats

• Paramutagenicity is directly proportional to the number of repeatsParamutagenicity is directly proportional to the number of repeats

• Transcription start sites are methylatedTranscription start sites are methylated

Structure of the paramutable R-r alleleStructure of the paramutable R-r allele Structure of the paramutable R-r alleleStructure of the paramutable R-r allele

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

Common themes inCommon themes intransposon inactivation and paramutationtransposon inactivation and paramutation

Common themes inCommon themes intransposon inactivation and paramutationtransposon inactivation and paramutation

• Sequence duplication is centralSequence duplication is central

• Promoter sequences are methylatedPromoter sequences are methylated

• Genes/TEs transcriptionally silencedGenes/TEs transcriptionally silenced

• Silencing is heritable, but reversibleSilencing is heritable, but reversible

• Both involve transposon sequencesBoth involve transposon sequences

• Sequence duplication is centralSequence duplication is central

• Promoter sequences are methylatedPromoter sequences are methylated

• Genes/TEs transcriptionally silencedGenes/TEs transcriptionally silenced

• Silencing is heritable, but reversibleSilencing is heritable, but reversible

• Both involve transposon sequencesBoth involve transposon sequences

Gene silencing (co-suppression) by trangenesGene silencing (co-suppression) by trangenes Gene silencing (co-suppression) by trangenesGene silencing (co-suppression) by trangenes

• Transgenes can silence endogenous genesTransgenes can silence endogenous genes

• More transgenes, more gene silencingMore transgenes, more gene silencing

• Inverted repeats are especially effectiveInverted repeats are especially effective

• Silenced genes are often methylatedSilenced genes are often methylated

• Silencing can be heritableSilencing can be heritable

• Silenced genes can be “paramutagenic”Silenced genes can be “paramutagenic”

• Transgenes can silence endogenous genesTransgenes can silence endogenous genes

• More transgenes, more gene silencingMore transgenes, more gene silencing

• Inverted repeats are especially effectiveInverted repeats are especially effective

• Silenced genes are often methylatedSilenced genes are often methylated

• Silencing can be heritableSilencing can be heritable

• Silenced genes can be “paramutagenic”Silenced genes can be “paramutagenic”

Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9 Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9

Transcriptional and post-transcription silencingTranscriptional and post-transcription silencing (TGS and PTGS)(TGS and PTGS) Transcriptional and post-transcription silencingTranscriptional and post-transcription silencing (TGS and PTGS)(TGS and PTGS)

• Silencing can be transcriptional, post-transcriptional or bothSilencing can be transcriptional, post-transcriptional or both

• TGS is associated with promoter methylationTGS is associated with promoter methylation

• PTGS is associated with coding sequence methylationPTGS is associated with coding sequence methylation

• Promotor methylation is not required for initiation of silencingPromotor methylation is not required for initiation of silencing

• Methylation is required for the maintenance of silencingMethylation is required for the maintenance of silencing

• Silencing can be transcriptional, post-transcriptional or bothSilencing can be transcriptional, post-transcriptional or both

• TGS is associated with promoter methylationTGS is associated with promoter methylation

• PTGS is associated with coding sequence methylationPTGS is associated with coding sequence methylation

• Promotor methylation is not required for initiation of silencingPromotor methylation is not required for initiation of silencing

• Methylation is required for the maintenance of silencingMethylation is required for the maintenance of silencing

Gene silencing and viral resistanceGene silencing and viral resistance Gene silencing and viral resistanceGene silencing and viral resistance

• Viral infection confers immunity to further infectionViral infection confers immunity to further infection

• Transgenic plants expressing coat protein are resistantTransgenic plants expressing coat protein are resistant

• Viral infection confers immunity to further infectionViral infection confers immunity to further infection

• Transgenic plants expressing coat protein are resistantTransgenic plants expressing coat protein are resistant

Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560

Viral resistance is RNA-mediatedViral resistance is RNA-mediated Viral resistance is RNA-mediatedViral resistance is RNA-mediated

• Transgene-induced resistance resembles PTGSTransgene-induced resistance resembles PTGS

• Resistance is mediated by RNAResistance is mediated by RNA

• Virus infection can result in co-suppression Virus infection can result in co-suppression

• Transgene-induced resistance resembles PTGSTransgene-induced resistance resembles PTGS

• Resistance is mediated by RNAResistance is mediated by RNA

• Virus infection can result in co-suppression Virus infection can result in co-suppression

Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276: 1558-1560

PVXPVX

W22W22

PVX. PVX. W22W22

Gene silencing: a systemic signalGene silencing: a systemic signal Gene silencing: a systemic signalGene silencing: a systemic signal

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

The systemic gene silencing signal is RNAThe systemic gene silencing signal is RNA The systemic gene silencing signal is RNAThe systemic gene silencing signal is RNA

• Non-overlapping gene fragments cross-silenceNon-overlapping gene fragments cross-silence

• RNA moves between cells in plantsRNA moves between cells in plants

• Plants encode RNA-dependent RNA polymerasesPlants encode RNA-dependent RNA polymerases

• Non-overlapping gene fragments cross-silenceNon-overlapping gene fragments cross-silence

• RNA moves between cells in plantsRNA moves between cells in plants

• Plants encode RNA-dependent RNA polymerasesPlants encode RNA-dependent RNA polymerases

TGS and PTGS: is there a relationship?TGS and PTGS: is there a relationship? TGS and PTGS: is there a relationship?TGS and PTGS: is there a relationship?

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

P35S PSTVd cDNA pAnos P35S PSTVd cDNA pAnos P35S PSTVd cDNA pAnos P35S PSTVd cDNA pAnos

Replication competent Replication competent Replication competent Replication competent Replication incompetent Replication incompetent Replication incompetent Replication incompetent

Transcription onlyTranscription only

No replicationNo replication

No methylationNo methylation

Transcription onlyTranscription only

No replicationNo replication

No methylationNo methylation

TranscriptionTranscription

ReplicationReplication

MethylationMethylation

TranscriptionTranscription

ReplicationReplication

MethylationMethylation

microRNAs and silencingRNAs in plantsmicroRNAs and silencingRNAs in plants microRNAs and silencingRNAs in plantsmicroRNAs and silencingRNAs in plants

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

microRNAs and silencingRNAs in animalsmicroRNAs and silencingRNAs in animals microRNAs and silencingRNAs in animalsmicroRNAs and silencingRNAs in animals

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

The Arabidopsis hyl1 mutation The Arabidopsis hyl1 mutation

wildtypewildtype

hyl1hyl1

0.6 µM ABA0.6 µM ABANo ABANo ABA

The hyl1 mutation affects miRNA levelsThe hyl1 mutation affects miRNA levels

wtwt hyl1hyl1hen1-1hen1-1 11 33

ARF8ARF8

SCL6-IIISCL6-III

rRNArRNA

MYB33MYB33

35S::HYL135S::HYL1

wtwt hyl1hyl1hen1-1hen1-1 11 33

35S::HYL135S::HYL1

miR167miR167

miR171miR171

tRNA +5S rRNAtRNA +5S rRNA

miR159miR159

UBQ1UBQ1DCL1DCL1

wtwt hyl1hyl1 hen1-1hen1-1 wtwt hyl1hyl1 hen1-1hen1-1

The hyl1 mutation affects mRNA stabilityThe hyl1 mutation affects mRNA stability

B.

Time (hrs)Time (hrs)

% in

itial

val

ue%

initi

al v

alue

10

100

00 44 88 1212

MYB33MYB33

hyl1hyl1

wtwt

35S::HYL135S::HYL1

50

30

hyl1hyl1

wtwt

35S::HYL135S::HYL1

SCL6-III SCL6-III

00 44 88 1212

hyl1hyl1

wtwt

35S::HYL135S::HYL1

00 44 88 1212

ANP1ANP1

hyl1hyl1

wtwt

35S::HYL135S::HYL1

ARF8 ARF8

00 44 88 1212

HYL1 is in

nuclear bodies

HYL1 is in

nuclear bodies

• TnpA and TnpD are required for transpositionTnpA and TnpD are required for transposition

• TnpA is also a weak transcription factorTnpA is also a weak transcription factor

• TnpA and TnpD are required for transpositionTnpA and TnpD are required for transposition

• TnpA is also a weak transcription factorTnpA is also a weak transcription factor

SpmSpm has one gene, has one gene, but codes for two proteinsbut codes for two proteins

SpmSpm has one gene, has one gene, but codes for two proteinsbut codes for two proteins

TranspositionTransposition

active active SpmSpmTnpATnpA

promoterpromoter

TnpD

TnpDmRNA

TnpAmRNA

TnpD

• Promoter methylated, element inactivePromoter methylated, element inactive

• Methylation of GC-rich sequence confers heritabilityMethylation of GC-rich sequence confers heritability

• Reversed by Reversed by SpmSpm-encoded TnpA-encoded TnpA

• Promoter methylated, element inactivePromoter methylated, element inactive

• Methylation of GC-rich sequence confers heritabilityMethylation of GC-rich sequence confers heritability

• Reversed by Reversed by SpmSpm-encoded TnpA-encoded TnpA

Changes in Changes in SpmSpm activity phase activity phase Changes in Changes in SpmSpm activity phase activity phase

Methylated siteMethylated siteUnmethylated siteUnmethylated site

cryptic cryptic SpmSpm active active SpmSpmTnpATnpA

promoterpromoter

GC-rich sequenceGC-rich sequence

• TnpA is a weak transcription factor TnpA is a weak transcription factor

• TnpA binds unmethylated and hemimethylated DNATnpA binds unmethylated and hemimethylated DNA

• TnpA promotes active demethylation TnpA promotes active demethylation

• TnpA is a weak transcription factor TnpA is a weak transcription factor

• TnpA binds unmethylated and hemimethylated DNATnpA binds unmethylated and hemimethylated DNA

• TnpA promotes active demethylation TnpA promotes active demethylation

Molecular mechanism of Molecular mechanism of Spm Spm activationactivation Molecular mechanism of Molecular mechanism of Spm Spm activationactivation

Methyl groupMethyl grouppromoterpromoter

replicationreplication

TnpATnpA

TnpATnpATnpATnpA

Transposon silencing: the chromatin connection Transposon silencing: the chromatin connection Transposon silencing: the chromatin connection Transposon silencing: the chromatin connection

transpositiontranspositiontranspositiontransposition

silencingsilencingsilencingsilencing

mRNAmRNA

siRNAs?siRNAs?siRNAs?siRNAs?

siRNAssiRNAsDNA methylaseDNA methylasehistone deacetylasehistone deacetylasechromatin remodeling proteins chromatin remodeling proteins

siRNAssiRNAsDNA methylaseDNA methylasehistone deacetylasehistone deacetylasechromatin remodeling proteins chromatin remodeling proteins

The story of papaya ringspot virus

The story of papaya ringspot virus

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

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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

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Papaya ringspot virusPapaya ringspot virus

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

1940s: PRS virus discovered in Hawaii1940s: PRS virus discovered in Hawaii

1950s: Oahu’s papaya industry wiped out1950s: Oahu’s papaya industry wiped out

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1960s: Papaya industry moves to Puna district1960s: Papaya industry moves to Puna district

Papaya ringspot virusPapaya ringspot virus

TGSTGSTGSTGS

• NoNo• NoNo

Papaya ringspot virusPapaya ringspot virus

1991: First transgenic PRSV-resistant papaya plant1991: First transgenic PRSV-resistant papaya plant

1980s: PRSV-resistance project started under direction of Dennis Gonsalves1980s: PRSV-resistance project started under direction of Dennis Gonsalves

1992: PRSV discovered in Puna district1992: PRSV discovered in Puna district

1992: First field trials PRSV-resistant papaya plants1992: First field trials PRSV-resistant papaya plants

1994: USDA granted permission for large scale field trials1994: USDA granted permission for large scale field trials

1995-97: Approvals for release from USDA, EPA, FDA1995-97: Approvals for release from USDA, EPA, FDA

1992-1977: PRVS spread; many farmers went out of business1992-1977: PRVS spread; many farmers went out of business

1998: Seeds released, free of charge, to growers1998: Seeds released, free of charge, to growers

2000: Papaya industry bounced back; crop back to pre-1995 levels2000: Papaya industry bounced back; crop back to pre-1995 levels

Papaya ringspot virusPapaya ringspot virus

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

Papaya ringspot

virus

Papaya ringspot

virus

Epigenetic mechanisms: plantEpigenetic mechanisms: plantevolution, defense and development evolution, defense and development

Epigenetic mechanisms: plantEpigenetic mechanisms: plantevolution, defense and development evolution, defense and development

• Gene silencing is a response to gene duplication Gene silencing is a response to gene duplication (evolution of duplicated genes; transposon control)(evolution of duplicated genes; transposon control)

• Gene silencing is a response to gene overexpression Gene silencing is a response to gene overexpression (dosage compensation)(dosage compensation)

• Gene silencing is a defense response Gene silencing is a defense response (viral cross protection; rapid environmental responses)(viral cross protection; rapid environmental responses)

• Epigenetic mechanisms are used in plant development Epigenetic mechanisms are used in plant development (JAW miRNA in leaf morphogenesis)(JAW miRNA in leaf morphogenesis)

• Gene silencing is a response to gene duplication Gene silencing is a response to gene duplication (evolution of duplicated genes; transposon control)(evolution of duplicated genes; transposon control)

• Gene silencing is a response to gene overexpression Gene silencing is a response to gene overexpression (dosage compensation)(dosage compensation)

• Gene silencing is a defense response Gene silencing is a defense response (viral cross protection; rapid environmental responses)(viral cross protection; rapid environmental responses)

• Epigenetic mechanisms are used in plant development Epigenetic mechanisms are used in plant development (JAW miRNA in leaf morphogenesis)(JAW miRNA in leaf morphogenesis)