1) A paternally expressed imprinted gene PEG2 acts as a molecular sponge for a transposon-derived small interfering RNA (siRNA854) in Arabidopsis. 2) PEG2 sequesters siRNA854 in triploid seeds, inhibiting the targeting of siRNA854's natural target genes and leading to triploid seed abortion. 3) Mutating the siRNA854 binding site in PEG2 prevents siRNA854 sequestration and allows translation of PEG2 in triploid seeds, restoring their viability. This demonstrates that the balance of siRNA854 levels regulated by PEG2 determines triploid seed survival.

1. Sequestration of a Transposon-
Derived siRNA
by a Target Mimic Imprinted Gene
Induces Post-zygotic Reproductive
Isolation in Arabidopsis
Presented by Nitika ,Kajol B.M Singh , Vijay and Rajan (M.sc.
Botany(F))

2. BACKGROUND
Genomic imprinting is an epigenetic phenomenon occurring in
mammals and flowering plants that leads to parent-of-origin
specific differential expression of maternally and paternally
inherited alleles.
maternally expressed
gene and paternally silent
are referred to as MEGs
(maternally expressed
imprinted genes
paternally expressed
and maternally silent
are referred to as PEGs
(paternally expressed
imprinted genes).
Wolff, P., Jiang, H., Wang, G., Santos-Gonzalez, J., & Köhler, C. (2015). Paternally expressed imprinted genes establish postzygotic hybridization
barriers in Arabidopsis thaliana. Elife, 4, e10074.

3. During reproduction, plant genomes undergo significant
reconfiguration of chromatin structure and DNA methylation
HOW IMPRINTING HAPPEN?
vegetative cell undergoes substantial
DNA demethylation due to reduced
expression of DECREASE in DNA
METHYLATION1 (DDM1) and activity
of the DEMETER (DME) demethylase
that frequently targets TEs
During female gametophyte
development, active
demethylation by DME
occurs throughout the
genome of the central cell
IMPRINTING SETTING
PRIOR TO FERTILIZATION
Gehring, M. (2013). Genomic imprinting: parental lessons from plants. Annu Rev Genet, 47, 187-208.
Lessons From Plants, Review in Advance

4. Imprinting effect is
primarily restricted
to the endosperm
models that integrate
known chromatin
dynamics during
reproduction to explain
how the insertion of a
TE could lead to a MEG
or PEG imprinting
status for a gene.
Ref. Sarah N Anderson and Nathan M Springer, Potential roles for transposable elements in creating
imprinted expression, Current Opinion in Genetics & Development 2018, 49:8–14

5. GENOMIC IMPRINTING AND TRIPLOID BLOCK
fusion of the maternal homodiploid
central cell with a paternal haploid
sperm cell,
the expected ratio of maternal to
paternal transcripts is 2:1
endosperm balance number
changes in the ratio of
maternal and paternal
genomes
Unbalanced expression of
imprinted genes
consequences for
endosperm
development, leading
to seed collapse
Lafon‐Placette, C., & Köhler, C. (2016). Endosperm‐based postzygotic hybridization
barriers: developmental mechanisms and evolutionary drivers. Molecular Ecology,
25(11), 2620-2629.

6. paternally
expressed
imprinted
gene PEG2
transcript
sequesters the
transposable
element (TE)-derived
small interfering RNA
(siRNA) siRNA854 in
the endosperm
reproductive barrier and
prevents successful
hybridizations between
plants differing in
chromosome number
(ploidy)
Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic
imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.

7. Hybridizations of diploid (2x) and tetraploid (4x)
Arabidopsis plants
cause strongly increased expression of paternally
expressed imprinted genes (PEGs)
ADMETOS (ADM), SUVH7, PEG2, and PICKLE RELATED2
share a common mode of action,
acts as a dosage-sensitive
regulator of triploid seed
development and increased ADM
expression in triploid seeds
causes endosperm cellularization
failure leading to seed abortion
functions only
in syncytial
endosperm
Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic
imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.

8. In Arabidopsis, at time of microspore to form
the pollen grain, the vegetative cell undergoes
substantial DNA demethylation
due activity of the DEMETER (DME)
demethylase that frequently targets
Transposable element.
The de-repression of TEs in the vegetative cell
is followed by generation of epigenetically
activated small interfering RNAs (easiRNAs)
increased levels of easiRNAs
negatively correlate with DNA
methylation established by the RNA-
dependent DNA methylation
(RdDM) pathway
suggesting a dose-
dependent role of easiRNAs
in controlling RdDM activity
Furthermore, depletion of easiRNAs
strongly reduces PEG expression and restores viability of
triploid seeds
indicating that easiRNAs act upstream in controlling PEG
expression
Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic
imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.

9. In this work, they show that transposable element (TE)-derived small
interfering RNA (siRNA) siRNA854 present in the vegetative cell of pollen
then transferred to the central cell of the female gametophyte after
fertilization , where it is captured by PEG2
This sequestering inhibits the targeting of siRNA854 natural target
genes like UBP1b
Sequestration of siRNA854 as a consequence of increased PEG2
transcript levels is causally responsible for triploid seed inviability.
Thus, the balance of a male gamete accumulating TE-derived siRNA
and a PEG regulates triploid seed viability,
revealing a trans generational speciation mechanism
.
Wang, G., Jiang, H., de León, G. D. T., Martinez, G., & Köhler, C. (2018). Sequestration of a transposon-derived siRNA by a target mimic
imprinted gene induces postzygotic reproductive isolation in Arabidopsis. Developmental cell, 46(6), 696-705.

10. RESULTS
PEG2 Is Transcribed, but Not Translated, in the Endosperm
PEG2 Translation in the Endosperm Is Impaired by siRNA854
The siRNA854 Binding Site Is Essential to Cause Triploid Seed
Abortion
The PEG2 mRNA Rather Than the PEG2 Protein Is Functionally
Relevant in the Triploid Block
PEG2 Functions as an siRNA Sponge after Fertilization in the
Endosperm
Regulation of UBP1b by siRNA854 Is Causally Connected to Triploid
Seed Viability
siRNA854 Is Transferred to the Central Cell via Sperm Cells

11. Experimental Models
Arabidopsis
thaliana
Columbia
(Col-0)
ubp1b-3
mutant
Peg2
mutant
osd1-1 and
osd1-3
mutants
T-DNA Insertion lines, PCR confirmed
For crossing experiments, designated female plants were emasculated, and the
pistils were manually pollinated 2 days after emasculation.
Transgenic lines Floral Dip method
A. Thaliana Seeds
Stratification for 2 days at 4C
Surface sterilised(5% sodium
hypochlorite and 0.01% Triton
X-100)
Plants transferred into soil
and grown in a growth
chamber
Germination on half strength
MS medium (1% sucrose)
16 h L/8 h D, 21C

12. Vectors
Fusion Vector Over-expression Vector

13. Translational reporter PEG2::PEG2-GFP
Transcriptional reporter PEG2::H2B-GFP
Silent mutant of PEG2 (PEG2::PEG2MUT-GFP)
Frame-shift PEG2 (RPS5A::PEG2FS)
Truncated PEG2 (RPS5A::PEG2TR)
Tissue-specific PEG2 constructs (PHE1::PEG2 &
MGH3::PEG2)
Mutant PEG2 with full sequence
complementarity to siRNA854 (PEG2::PEG2PM-
GFP)
35S::PEG2-GFP
PEG2 GFP
PEG2
H2B GFP
PEG2
PEG2FS GFP
RPS5A
PEG2MUT GFP
PEG2
PEG2
PHE1
PEG2TR GFP
RPS5A
PEG2
MGH3
PEG2PM GFP
PEG2
PEG2 GFP
35S
C
O
N
S
T
R
U
C
T
S

14. Triploid seeds generated from pollinating wild-type plants with diploid pollen of the
omission of second division 1 (osd1) mutant abort at high frequency, while osd1
peg2 double mutant pollen gives rise to viable triploid progeny. .
PEG2 Is Transcribed, but Not Translated, in the Endosperm
PEG2 (AT1G49290) encodes for an unknown protein with no predicted functional
domains
(Pollen 2n) (Pollen 2n)

15. Localized PEG2 using a translational reporter construct of the PEG2 promoter fused to the
PEG2 coding region and the green fluorescent protein GFP; PEG2::PEG2-GFP
Monitored GFP fluorescence and found a strong signal surrounding the sperm nuclei of pollen.
The presence of PEG2::PEG2-GFP in osd1 peg2 restored triploid seed abortion, revealed that this
construct is functional and can complement the peg2 mutant.
Sperm cell
GPF Signal
surrounding
nuclei of
sperm cell

16. No GFP fluorescence in diploid or triploid seeds generated from pollinations with haploid or diploid PEG2::
PEG2-GFP pollen. This was in sharp contrast to the strong increase of the PEG2 transcript levels in triploid
seeds, indicating that these transcripts accumulate but fail to be translated. Transcripts derived from
PEG2::PEG2-GFP accumulated to similar levels as those of the endogenous PEG2 gene
A transcriptional reporter for PEG2 containing the PEG2 promoter sequence driving a nuclear-localized histone
H2B-GFP fusion was highly active in sperm cells and in the endosperm
Seed without
GFP Signal at
4 days after
polliantion

17. PEG2 Translation in the Endosperm Is Impaired by siRNA854
Queried PEG2
sequence for possible
microRNA (miRNA)
target sites
5 members of the
miRNA854/siRNA854 family
predicted to target the PEG2
cDNA sequence at
nucleotides 614 to 634.
These mismatches indicate
a potential destabilization of
the siRNA854 cleavage at its
central position that leads
to translational repression.
Target site contained a
mismatch at position 10 and
two non-Watson-Crick base
interactions at positions 9
and 11 of the siRNA/PEG2
mRNA hybrid.
Introduced three
silent mutations into
the siRNA854 binding
site, to test whether
it is responsible for
PEG2 translational
inhibition
Mismatch and
non watson-
crick base
interaction

18. Showed GFP fluorescence detected
surrounding sperm nuclei when
expressed mutated version of PEG2
(PEG2MUT), similar to the signal
detected when expressing the non-
mutated version of PEG2.
In contrast to the non-mutated
version, this construct was
translated in the endosperm, as
evidenced by a detectable GFP
signal surrounding the endosperm
nuclei

19. Consistently, the PEG2MUT-GFP protein was detected by immunoblot
analysis, while no signal was detected in seeds expressing PEG2-GFP .
Jointly, these results strongly support that PEG2 translation in the
endosperm is impaired by siRNA854.

20. • Introduced the two versions of PEG2 with the natural and mutated siRNA854 binding sites into the
osd1 peg2 double mutant and tested viability of the progeny obtained from crossing these plants to
a diploid maternal plant.
• In contrast to the complete complementation of the abortion phenotype when expressing
PEG2::PEG2-GFP, PEG2::PEG2MUT-GFP was not able to confer full complementation .
• While the decrease in viability of triploid peg2; PEG2::PEG2MUT-GFP seeds was significant
compared to triploid peg2 seeds, the difference was rather minor and differed drastically from the
complete suppression of viability conferred by expressing PEG2::PEG2-GFP.
The siRNA854 Binding Site Is Essential to Cause Triploid
Seed Abortion

22. So, this study says that PEG serves as
sponge for epigenetically controlled siRNA
(produced in the pollen grain).Dosage of
siRNA854 in endosperm is responsible for
triploid seed survival.
Its already know ,Coding and non coding
RNAs can act as molecular sponge to
regulate abundance of miRNAs (Franco-
Zorilla et al. 2007)
According to Author, No one before this
paper reported about molecular sponge
for TE-Derived siRNA. But this paper does!
So know we know that dosage of TE-
derived siRNA854 work as a reproductive
barrier b/w plants with different ploidy.
As per Borges et al., It is know that
easiRNA is actually responsible for triploid
block by affecting the imprinted genes, and
offcourse siRNA is a member of easiRNA
(LTR retrotransposons).These LTR
retrotransposons activity depends on
various DCL 2,4 etc.
From above points we can say that,
dependence of PEG2 to deplete siRNA854
depends on easiRNA.

23. The possible explanation for the paradox
is that reduced dosage of easiRNA in
triploid seeds restores wild type levels of
PEG2 and other PEG such as ADM in the
intro portion . They are responsible for
seed abortion ,but mutation in ADM
leads to viable seed formation.

24. Model Explaining the Role of PEG2 in Establishing
Interploidy Seed Abortion
-In pollen , PEG2 is expressed in sperm cells.
-The siRNA854 is present in the vegetative cell, and transported to sperm cells.
-In sperm,siRNA854 doesn’t efficiently inhibit translation of PEG2, and PEG2 surrounds sperm
nuclei .

25. AFTER FERTILIZATION

26. INFERENCES
Normal fertilisation.
PEG2 highly regulated
,cause depletion of
siRNA854.
Hence , reduction in
PEGs expression
,bypasses the need of
siRNA854 and restores
triploid seed viability.