Classical epigenetic systems

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
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 competent
Replication incompetent
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, 7120-125.
18
microRNAs and silencingRNAs in animals
Mallory, A. C., and Vaucheret, H. (2004) Current
Opinion in Plant Biology, 7120-125.
19
The Arabidopsis hyl1 mutation
0.6 µM ABA
No ABA
wildtype
hyl1
20
The hyl1 mutation affects miRNA levels
21
The hyl1 mutation affects mRNA stability
B.

hyl1
100
hyl1
hyl1
wt
hyl1
wt
wt
wt
35SHYL1
50
35SHYL1
35SHYL1
35SHYL1
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
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
http//www.apsnet.org/education/feature/papaya/Top
.htm
Papaya ringspot virus
1940s PRS virus discovered in Hawaii
1950s Oahus 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)