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

• 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
•
Heritable, but reversible
• Epimutants differ in their
developmental expression patterns
• The transition from active to cryptic (and the
reverse) takes several plant generations
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
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
R gene paramutation in maize
Walker, E. L. (1998), Genetics, 148: 1973-1981
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
Structure of the paramutable R-r allele
Walker, E. L. (1998), Genetics, 148: 1973-1981
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
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
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
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
Viral resistance is RNA-mediated
PVX
W22
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
Gene silencing: a systemic signal
Voinnet, O., and Baulcombe, D. C. (1997). Nature 389: 553
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
TGS and PTGS: is there a relationship?
Replication incompetent
P35S
PSTVd cDNA
pAnos
Replication competent
P35S
PSTVd cDNA
pAnos
Transcription only
Transcription
No replication
Replication
No methylation
Methylation
Wassenegger, M., Heimes, S., Reidel, L., and Sanger, H. L. (1994) Cell 76: 567-76.
microRNAs and silencingRNAs in plants
Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:120-125.
microRNAs and silencingRNAs in animals
Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:120-125.
The Arabidopsis hyl1 mutation
No ABA
wildtype
hyl1
0.6 µM ABA
The hyl1 mutation affects miRNA levels
35S::HYL1
wt
hyl1 hen1-1 1
35S::HYL1
3
wt
hyl1 hen1-1 1
miR159
wt
3
MYB33
miR167
ARF8
miR171
SCL6-III
tRNA +
5S rRNA
rRNA
wt
hyl1 hen1-1
DCL1
hyl1 hen1-1
UBQ1
The hyl1 mutation affects mRNA stability
B.
hyl1
100
hyl1
hyl1
wt
hyl1
wt
wt
wt
35S::HYL1
% initial value
50
35S::HYL1
35S::HYL1
35S::HYL1
30
MYB33
SCL6-III
ANP1
ARF8
10
0
4
8
12 0
4
8
12 0
Time (hrs)
4
8
12 0
4
8
12
HYL1 is
in
nuclear
bodies
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
mRNA
TnpD
active Spm
Transposition
TnpD
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
Molecular mechanism of Spm activation
• TnpA is a weak transcription factor
• TnpA binds unmethylated and hemimethylated DNA
• TnpA promotes active demethylation
Methyl group
promoter
TnpA
replication
TnpA
TnpA
Transposon silencing: the chromatin connection
silencing
mRNA
siRNAs?
transposition
siRNAs
DNA methylase
histone deacetylase
chromatin remodeling proteins
The story of papaya
ringspot virus
http://www.apsnet.org/education
/feature/papaya/Top.htm
Papaya ringspot virus
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http://www.apsnet.org/education/feature/papaya/Top.htm
Papaya ringspot virus
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1940s: PRS virus discovered in Hawaii
1950s: Oahu’s papaya industry wiped out
1960s: Papaya industry moves to Puna district
Papaya ringspot virus
TGS
• No
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
Papaya
ringspot
virus
http://www.apsnet.org/education/feature/papaya/Top.htm
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)