This will provide an overview of how through different mechanisms gene silencing can be done at the transcriptional level.

1. Transcriptional gene
silencing
SHEETAL MEHLA
PhD Molecular Biology Biotechnology and Bioinformatics
Advances in Plant Molecular Biology

2. Gene silencing
 Gene silencing is the regulation of gene expression in a cell to prevent the
expression of a certain gene.
 As the name implies, gene silencing is a technique that aims to reduce or
eliminate the production of a protein from its corresponding gene.Gene
silencing is often considered the same as gene knockdown. When genes are
silenced, their expression is reduced. In contrast, when genes are knocked out,
they are completely erased from the organism's genome and, thus, have no
expression. Gene silencing is considered a gene knockdown mechanism since
the methods used to silence genes, such as RNAi, CRISPR, or siRNA,
generally reduce the expression of a gene by at least 70% but do not
completely eliminate it.

3. Central Dogma:
 Genes are sections of DNA that contain the instructions for making proteins.
 Proteins are essential molecules that perform an array of functions including
signalling between cells, speeding up biochemical reactions, and providing
structural support for the cell.
DNA mRNA Protein
transcription
translation
replication

4.  Instead of directly editing DNA or inhibiting the transcription process, the key
idea behind gene silencing is intervening in gene expression prior
to translation, scientists have been able to effectively decrease levels of
that protein. So by hindering the processes of central dogma it will directly
leads to silencing of gene for exploring various purposes.

6. Types of gene silencing
Transcriptional
• Genomic Imprinting
• Paramutation
• Transposon silencing
• Transgene silencing
• Position effect
• RNA-directed DNA
methylation
Post-
transcriptional
• RNA interference
• RNA silencing
• Nonsense
mediated decay
Meiotic
• Transvection
• Meiotic silencing
of unpaired DNA
Gene
silencing

7. Genomic imprinting
 Genomic imprinting is an inheritance process independent of the classical Mendelian inheritance. It is
an epigenetic process that involves DNA methylation and histone methylation without altering the
genetic sequence.
 The process by which one chromosome of a pair is chemically modified, depending on whether the
chromosome comes from the father or the mother. These modifications lead to differential expression of
a gene or genes on a maternally derived chromosome versus a paternally derived chromosome.
 During fertilisation of the egg cell, a second, separate fertilization event gives rise to the endosperm,
an extra embryonic structure that nourishes the embryo in a manner analogous to the
mammalian placenta. Unlike the embryo, the endosperm is often formed from the fusion of two
maternal cells with a male gamete. This results in a triploid genome. The 2:1 ratio of maternal to
paternal genomes appears to be critical for seed development. Some genes are found to be expressed
from both maternal genomes while others are expressed exclusively from the lone paternal copy. It
has been suggested that these imprinted genes are responsible for the triploid block effect in
flowering plants that prevents hybridization between diploids and auto tetraploids.
 Triploid block is a phenomenon describing the formation of nonviable progeny after hybridization
of flowering plants that differ in ploidy. The barrier is established in the endosperm, a nutritive tissue
supporting embryo growth. This phenomenon usually happens when auto polyploidy occurs
in diploid plants. Triploid blocks lead to reproductive isolation. The triploid block effects have been
explained as possibly due to genomic imprinting in the endosperm.

8.  Genomic imprinting occurs when both maternal and paternal alleles are
present, but one allele will be expressed while the other remains inactive. It
is not completely evident why genes are imprinted. The most prominent
assumption is that this process is necessary for development and may
somehow regulate growth in the embryo and neonate. Evidence for this
suggestion came from experiments with androgenotes (embryos with two
paternal genomes) and gynogenotes (embryos with two maternal genomes),
which were produced by nuclear transplantation. These zygotes were formed,
but neither type was able to undergo further development. From this
situation, it is possible to suggest that the maternal and paternal effects are
complementary here. Each genome contains different viable and necessary
properties.
 Hundreds of imprinted genes are being added to the list through deep
sequencing technologies, but the functional role is yet to be elucidated. DNA
methylation and histone modifications are the two important process involved
in exertion and maintenance of imprinting.

9. Sexual reproduction in the flowering plant A. thaliana. The gametes are contained
within multicellular haploid structures called gametophytes that are derived by mitosis
from meiotic spores. The fusion of two haploid polar nuclei forms a diploid central cell
in the female gametophyte. At the time of fertilization, the diploid female central cell
and a haploid male sperm cell fuse to give rise to the endosperm, while the haploid
female egg cell and haploid male sperm cell fuse to give rise to the embryo. The
resulting seed is formed of the endosperm, the embryo, and a maternally derived
seed coat.

10. Model for the simultaneous evolution of imprinting and endosperm in the ancestor of angiosperms
(flowering plants). Angiosperms emerged from a gymnosperm (nonflowering seed plant) lineage,
evolving flowers, fruits, and endosperm. Endosperm potentially evolved through the sexualization of
a female gamete companion cell, such as the ventral canal cell (Friedman and Floyd 2001; Rudall
2006), via fusion with one of the two sperm cells of pollen. Activity of a DME-like enzyme in the
sexualized female gamete companion cell would give rise to an endosperm with DNA methylation-
based imprints in the ancestor of modern angiosperms.

11.  a “G” may be found in a specific position in a candidate gene from the mother, and
a “T” in this same position in the gene from the father.
 If the polymorphism is located in an exon of the gene, then the parental origin of
the mRNA transcripts can be distinguished.
 mRNAs prepared from such an informative individual are reverse transcribed (RT)
into matching complementary DNA (cDNA), and are amplified by the polymerase
chain reaction (PCR) to allow analysis of this gene’s mRNAs out of the thousands of
others in the original sample.
 Amplified cDNA products from the pool of mRNA transcripts subjected to this RT-PCR
approach are inspected, usually by direct sequencing or by sequencing of >10
individual clones, to determine whether both, or only one, of the polymorphisms is
present in the sample. Recovery of approximately equivalent levels of each
polymorphism indicates biallelic expression, while the finding of only one of the two
genomic polymorphisms in the cDNA pool suggests monoallelic expression consistent
with imprinting.

12.  Assessment of monoallelic transcription by PCR amplification of expressed
polymorphisms. (a) A simplified pedigree showing a homozygous T/T father, G/G
mother, and heterozygous T/G offspring for a hypothetical candidate gene. (b) RT-
PCR amplification of the candidate gene’s mRNA products. Each parental genomic
DNA structure is shown as three exons, with the embedded T or G polymorphism.
Following reverse transcription of the mRNA, the cDNA is PCR-amplified and
analyzed for the presence of the T or G polymorphism.

13. Paramutation
 Paramutation is a regularly occurring, directed, and heritable alteration of
gene expression resulting from the interaction of two alleles.
 One allele is referred to as paramutable: its expression changes following
paramutation.
 The other allele is paramutagenic: it causes the change in the paramutable
allele.
 It is used to describe both the process and outcome of directed and
meiotically heritable changes in both gene regulation and silencing abilities
that are influenced by trans-homolog interactions (THI). Unlike mutations,
however, paramutations occur in predictable, invariant, and sometimes
reversible manners. Deviations from expected Mendelian ratios of trait
transmission – such as exclusive inheritance of a dominant trait – are one
hallmark of paramutation events.
 Paramutation was first described in 1956 by Brink, but a mechanism to
explain how one allele can heritably affect the expression of another has
remained obscure.

14. In RdDM-type working model for paramutation. Maize components identified by mutations are
presented above the dotted line, and presumed orthologs of select Arabidopsis RdDM proteins are
presented below. In this schematic, Pol IV is transcribing a repetitive enhancer element (arrowed
boxes), the nascent RNA is copied by RDR2, and the resulting double-stranded RNA (red lines) is
cleaved by DCL3 to create 24-nucleotide RNAs. These small RNAs, in complex with argonaute4
(AGO4), facilitate the association with Pol V nascent RNAs and the recruitment of a de novo
methyltransferase (domains-rearranged DNA methyltransferase2 [DRM2]) to accomplish site-
directed cytosine methylation (black lollipops). All proteins are to relative scale based on amino
acid content.

15.  Inheritance patterns typifying paramutation (gray box). Here, allele A’ is dominant to both A and
recessive a. Regardless of parental origin, only progeny inheriting A’ from parents show trans-
dominant silencing. Alleles of a linked locus (b) are indicated to emphasize that only A forms are
transmitted from A’/A plants yet linkeded b alleles show Mendelian inheritance from A’B/Ab
individuals.

16. Transposon:
 A transposon may be defined as: “a DNA sequence that is able to
move or insert itself at a new location in the genome.” The
phenomenon of movement of a transposon to a new site in the
genome is referred to as transposition.
 Types of Transposons:
 On the basis of mechanism type II (Transposons)
type I (Retrotransposons)
Cut-and-Paste Transposons
Replicative Transposons
Retro Elements auotnomous elements
non- autonomous elements

17. Cut and paste transposon

18. Conservative (Cut and
paste transposons)
 The cut-and-paste transposition
involves two processes excision from
its original position followed by
reinsertion elsewhere.
 This requires two elements :
transposase and the inverted repeats
at the end of the transposon .
 IS elements, P elements in maize,
hobo-elements in drosophila etc,.

19. Replicative transposon :
 They transpose by a mechanism which involves replication of transposable
sequence and this copy of DNA, so formed, is inserted into the target site while
the donor site remains unchanged.
 Thus, in this type of transposition, there is a gain of one copy of transposon and
both-the donor and the recipient DNA molecule are having one-one
transposable sequence each, after transposition.
 Tn3-elements found in bacteria.

20. Retrotransposon:
 The Retro elements may be viral or non-viral. Out of these two, the non-viral retro
elements are important and may further be classified as:
 (A) Retrovirus like elements:
 They carry long terminal repeats (LTR). Examples are copia, gypsy elements in
Drosophila.
 Retroposons:
 LTR are absent. Examples are LINEs and SINEs in humans.

21. Transposase Families
 At least five families have been classified, although this number will
most likely grow as new transposases are characterised. These families
use distinct catalytic mechanisms for break/rejoining of DNA.
 DDE transposases: These transposases carry a triad of conserved amino
acids: aspartate (D), aspartate (D) and glutamate (E), which are required
for the coordination of a metal ion required for catalysis, although the
DDE chemistry can be integrated into the transposition cycle in differing
ways. These employ a cut-and-paste mechanism of the original
transposon. This family includes the maize Ac transposon, as well as the
Drosophila P element, bacteriophage Mu, Tn5 and Tn10, Mariner, IS10,
and IS50.
 Tyrosine (Y) transposases: These also use a cut-and-paste mechanism of
transposition, but employ a site-specific tyrosine residue. The transposon
is excised from its original site (which is repaired); the transposon then
forms a closed circle of DNA, which is integrated into a new site by a
reversal of the original excision step. These transposons are usually
found only in bacteria, and include Kangaroo, Tn916, and DIRS1.

22.  Serine (S) transposase: These transposases use a cut-and-paste (cut-
out/paste-in) mechanism of transposition involving a circular DNA
intermediate, which is similar to that of tyrosine transposases, only
they employ a site-specific serine residue. These transposons are
usually found only in bacteria, and include Tn5397 and IS607.
 Rolling-circle (RC), or Y2 transposase: These employ either a copy-in/
replicative mechanism, where they copy a single strand directly into
the target site by DNA replication, so that the old (template) and new
(copied) transposons both have one newly synthesized strand. These
transposons usually employ host DNA replication enzymes. Examples
include IS91 and helitrons.
 Reverse transcriptases/endonucleaseses (RT/En): Retrotransposons
can vary in their mechanism of transposition. Some use the RT/En
method, employing an endonuclease to nick the target site DNA, the
nick serving as a primer for reverse transcription of an RNA copy by
the reverse transcriptase enzyme. Examples include LINE-1 and TP-
retrotransposons.

23. Transposon silencing
 Transposon silencing is a form of transcriptional gene silencing targeting transposons.
 Transcriptional silencing of transposons is crucial to the maintenance of a genome. The “jumping” of transposons
generates genomic instability and can cause extremely deleterious mutations.
 Transposable element insertions have been linked to many diseases including hemophilia, severe combined
immunodeficiency, and predisposition to cancer. The silencing of transposons is therefore extremely critical in the
germ line in order to stop transposon mutations from developing and being passed on to the next generation.
Additionally, these epigenetic defences against transposons can be heritable.
 Studies in Drosophila, Arabidopsis thaliana, and mice all indicate that small interfering RNAs are responsible for
transposon silencing. In animals, these siRNAS and piRNAs are most active in the gonads.
 Piwi-interacting RNA (piRNA), the largest class of the small RNAs, are between 26 and 31 nucleotides in length and
function through interactions with piwi proteins from the Argonaut protein family (gene silencing proteins). Many
piRNAs are derived from transposons and other repeated elements, and therefore lack specific loci. Other piRNAs
that do map to specific locations are clustered in areas near the centromeres or telomeres of the chromosome.
 It is thought that piRNA-PIWI complexes directly control the activity of transposons. piRNAs bound to PIWI proteins
seem to use post-transcriptional transcript destruction to silence transposons. Transposon insertions in introns can
escape silencing via the piRNA pathway, suggesting that transcript destruction by piRNAs occurs after nuclear export.
piRNAs could, however, act on multiple levels, including guiding heterochromatin assembly and possibly playing a role
in translation as well . The exact biogenesis of piRNAs is still unknown.
 Most piRNAs are antisense to mRNAs transcribed from the silenced transposons, generally associating with Piwi and
Aubergine (Aub) proteins, while sense-strand piRNAs tend to associate with Argonaute 3 (Ago3) instead. A cycle called
“ping pong” amplification proceeds between the sense and anti-sense piRNAs involving extensive trimming and
processing to create mature piRNAs. This process is responsible for the production of most piRNAs in the germline
and could also explain the origin of piRNAs in germ line development.

24.  Potential modes of piRNA-mediated transposon silencing. (1) Transcriptional silencing of target
transposons. piRNAs bound to Piwi, which accumulates in the nucleus, direct heterochromatin
assembly at target elements. (2) Post-transcriptional target destruction. Transposon transcripts
are recognized by Aub–piRNA complexes in the nuage, which catalyse homology-dependent
cleavage. (3) Aub–piRNA complexes bind transposon transcripts and repress translation.

25.  The heterochromatin protein Rhino is a key player in the generation of piRNAs in fly ovaries, which
help to protect the germline genome by silencing transposons. At heterochromatic dual-strand piRNA
clusters, the structure of heterochromatin and transcription are regulated by the competing activities
of Rhino and another heterochromatin protein, HP1. Rhino is preferentially expressed in the germline
and may possibly dislodge other HP1 proteins from the cluster to allow transcription of long precursor
RNAs, which are required for the generation of primary piRNAs and their amplification in the nuage
body.

26. Transgene silencing
 Silencing is triggered by:
 Very high level of gene expression
 dsRNA DERIVED FROM TRANSGENE
 Aberrant RNAs encoded transgene

27.  How these intrusive DNA are detected:
 The ability to recognise self from non-self is a characteristic feature that exist at
cellular level. The observation from genetically engineered plants suggest that an
ability to recognise these exist at nucleic acid level. The possible mechanisms are
as follow:
 Foreign sequence has different base sequence composition from that of
endogenous chromosomal environment.
 Gene transfer via direct DNA has no control over copy number, hence these
introduced genes may form independent domains in the cell and work
independently with the result they get recognized by genome scanning machinery.
 Every cell has its own modification and restriction system that show xenophobic
effect, which does not allow cell to contaminate its own chromosomal
environment by any foreign sequence.
 Genomes are made of isochores that are very long stretches of DNA with high
compositional homogeneity. On the basis of concept of gene space has been given
according to which if a GC rich transgene is integrated in to a GC rich gene
space(isochores) or AT rich transgene in AT isochores, it is normally transcribed.
But if GC rich transgene get integrated into AT rich isochore or vice versa, it is
inactivated as there is no compositional homogeneity with neighbouring sequence.
A powerful initial response by cell for the presence of foreign DNA may be
fragmented of invasive DNA via the action of cytoplasmic nucleases.

28. Mechanism of transgene silencing:
 There are various mechanisms for the silencing of transgene:
 DNA methylation
1. Transcriptional
2. Post transcriptional
 Homology dependent gene silencing
1. Inactivation of homologous transgene
2. Paramutation
3. Co-supression
 Suppression by antisense
 Silencing by RNAi
 Position effect
 High copy number

30.  DNA methylation:
 The inactivation of transgene is often accompanied by an increase in DNA methylation and
inactivation very frequently correlates with the number of copies of integrated transgene. It is
done at two level:
1. Transcriptional; methylation of the promoter and both symmetric and asymmetric methylation
of cytosine is known to occur. The transgene architecture, copy number and genomic position
plays an important role in the determining whether the promoter sequence will be methylated
or repressed.
2. Post transcriptional; methylation of the coding sequence.
 The repression of the methylated promoter probably results from recruitment of chromatin
modifying factor (histone deacetylase) and remodelling factor (SNF2 Helicase) through
methylated DNA binding protein (MeCP2) which prevent access of DNA to the transcription
machinery.
 Nevertheless even when the transgene insert is present in multiple copies many studies have
reported expression. This implies that induction of TGS is a multicomponent process.
 Induction of denovo methylation of transgene involves different modes:
1. DNA methylation via DNA-DNA pairing
2. Multiple transgene- concatamere- DNA coiling- copies comes In front of each other like
homologous sequences-mutual suppression-increase in methylation-inactivation.
3. Transgene recognition: due to older age of transgene or certain stresses transgene get hyper
methylated
4. Insertion into hyper methylated genomic region due to spreading of hyper methylation pattern.

31.  Homology dependent gene silencing:
Homologous sequence not only affects the stability of transgene expression but that
of the endogenous gene. It takes places generally by:
1. Inactivation of homologous transgene
2. Paramutation
3. Co- suppression
 Suppression by antisense gene by disturbing the process of transcription,
mRNA splicing, translation initiation and it’s expression.
 Silencing by RNAi
 Position effect: The integration of transgene at hyper methylated,
heterochromatic, telomeric and compositionally different genomic region, then
due to suppression effect of adjacent region or environment it’s expression is
inactivated.
 Increased copy number: A correlation between the number of integrated
copies and the frequency of inactivation is well documented for copies
arranged in cis position. A reduction of copy number inside a locus was shown
to increase gene expression and decrease suppression effect.

32.  Transcriptional gene silencing can be further divided into two classes:
 Transcriptional cis Inactivation
 In plants, transgenes integrate into the genome at random positions by illegitimate recombination; hence,
copy number, their integration site, and local arrangement differ in each transformation event. Also, an
inverse relation between transgene copy number and gene expression suggests that multicopy integration
can lead to silencing. Integrated foreign genes can undergo TGS in cis when multicopy T-DNA is integrated
at a locus adjacent to hypermethylated regions of the host genome. More rarely, single copy transgene
integration at a hypomethylated locus can lead to cis inactivation. A maize A1 gene involved in floral
pigmentation when overexpressed in Petunia led to silencing of A1; however, it was not silenced
when Gerbera dihydroflavonol-4-reductase was over expressed in Petunia suggesting that the transgene
also influenced the silencing process. Hence, some degree of difference in DNA composition of the
transgene and surrounding host genomic sequences can be recognized by the cellular machinery as foreign
non-compatible DNA, leading to specific methylation and silencing.
 It is believed that cisTGS occurs as a result of pairing between closely associated copies of transgenes or
endogenous genes, which leads to the formation of secondary DNA structures which are sites for DNA
methylation. Cytosine methylation at CpG and CpNpG sites of transgene and the 35S promoter were also
detected in transgenic grapevine transformed with Grapevine fanleaf virus (GFLV) coat protein gene.
 Transcriptional Trans-Inactivation
 Transcriptional gene silencing can result from unidirectional effects of one transgene on another transgene
or homologous endogenous gene. A transgene can be methylated and silenced when it is crossed with a
plant in which the homologous gene is in a silenced state. De novo methylation of one transgene is
mediated by a second transgene under control of the same promoter leading to TGS in trans. Experiments
using dsRNA-containing promoter sequences initiated TGS and subsequently de novo DNA methylation of the
corresponding transgene or endogenous gene, implying a role of an RNA intermediate in TGS. Yamasaki et
al. (2011) reported methylation of asymmetric cytosine in the enhancer region of 35S promoter in
transgenic gentian.

33. Position effect
 Position-effect variegation (PEV) is a variegation caused by the silencing of
a gene in some cells through its abnormal juxtaposition with heterochromatin via
rearrangement or transposition. It is also associated with changes in chromatin
conformation.
 PEV is a position effect because the change in position of a gene from its original
position to somewhere near a heterochromatic region has an effect on
its expression. The effect is the variegation in a particular phenotype i.e., the
appearance of irregular patches of different colour(s), due to the expression of
the original wild-type gene in some cells of the tissue but not in others as seen in
the eye of mutated Drosophila melanogaster.
 Among a number of models, two epigenetic models are popular. One is the cis-
spreading of the heterochromatin past the rearrangement breakpoint. The trans-
interactions come in when the cis-spreading model is unable to explain certain
phenomena.
 In plants, PEV has been observed in Oenothera blandina. The silencing of
euchromatic genes occurs when the genes get placed into a new heterochromatic
neighborhood.

34.  CIS SPREADING
 According to this model, the
heterochromatin forces an altered
chromatin conformation on the
euchromatic region. Due to this, the
transcriptional machinery cannot access
the gene which leads to the inhibition of
transcription.
 In other words, the heterochromatin
spreads and causes gene silencing by
packaging the normally euchromatic
region. But this model fails to explain
some aspects of PEV.
 For example, variegation can be induced
in a gene located several megabases
from the heterochromatin-euchromatin
breakpoint due to rearrangements in
that breakpoint. Also, the austerity of
the variegated phenotype can be altered
by the distance of the heterochromatic
region from the breakpoint.
 TRANS- INTERACTIONS
 These are interactions between the different
heterochromatic regions and the global
chromosomal organisation in the interphase
nucleus. The rearrangements due to PEV places
the reporter gene in a new compartment of the
nucleus where the transcriptional machinery
required is not available, thus silencing the
gene and modifying the chromatin structure.

35. RNA directed DNA methylation
 RNA-directed DNA methylation (RdDM) is an epigenetic process first discovered in
plants. During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24 nucleotide
small interfering RNAs (siRNAs) and guide methylation of homologous DNA. In plants
dsRNAs may be generated from four sources:
 Viral replication intermediates
 Products of the endogenous RNA-directed RNA polymerase
 Transcribed inverted repeats
 Transposable elements
 Besides RNA molecules, a plethora of proteins are involved in the establishment of RdDM,
like Argonautes, DNA methyltransferases, chromatin remodelling complexes. and the plant-
specific Polymerase IVand Polymerase V. All these act in concert to add a methyl-group at
the 5' position of cytosines. In contrast to animals, cytosines at all sequence context (CG,
CHG, CHH) may get de novo methylated in plants.

36.  Proposed model for RdDM in A.thaliana. One form of RNA polymerase IV, POLIVa, gets recruited to a target
genomic site (for example a transposon or DNA tandem repeats), through an unknown mechanism. Once
recruited, POLIVa synthesizes a single-stranded RNA (ssRNA). RDR2 uses this ssRNA as a template to synthesize
a dsRNA that is then processed by DCL3 into siRNAs that bind AGO4 proteins. SiRNAs-bound to AGO4 proteins
together with POLIVb, the second form of POLIV that interacts with AGO4, initiate in a sequence-specific
manner de novo DNA methylation, histone methylation as well as a probable ATP-dependent chromatin
remodeling. Given that at some RdDM targeted loci, siRNAs are not detected in absence of the de novo
cytosine methyltransferase DRM2, it is possible that a positive feedback loop (like in fission yeast) is required
for efficient siRNA-dependent de novo methylation and gene silencing.

37. RNA-directed DNA methylation. This model is based on genetic evidence from Arabidopsis thaliana. In the nucleus, dsRNA can be
produced by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) activity on ssRNA templates or by transcription of inverted DNA repeats by
a DNA-dependent RNA polymerase such as RNA polymerase II (not shown). RNA signals that direct DNA methylation are produced
through processing of dsRNA by enzymes of the DICER-LIKE family (in some cases known to be DCL3). These RNA signals (wavy red
lines) target site-specific methyltransferases — MET1 for CGs and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) primarily for
nonCGs — to catalyse de novo methylation of DNA. RNA-directed DNA methylation (RdDM) also requires the activity of the SNF2-like
protein DEFECTIVE IN RNA- DIRECTED DNA METHYLATION 1 (DRD1), perhaps to make DNA accessible to RNA signals. Methylation of
CGs and CNGs can be maintained to some extent in the absence of the RNA trigger through the activity of MET1 and
CHROMOMETHYLASE 3 (CMT3), respectively. Maintenance of CG methylation requires the activity of the histone deacetylase HDA6
and the SNF2-like protein DECREASE IN DNA METHYLATION 1 (DDM1). CNG methylation is accompanied by H3K9 methylation (not
shown), catalysed by SUVH4 (also known as KRYPTONITE). The Argonaute proteins AGO4 and AGO1 are involved in de novo
methylation or maintenance of methylation of some loci, respectively. The DNA glycosylase domain-containing proteins DEMETER
(DME) and REPRESSOR OF SILENCING 1 (ROS1) are needed for loss of methylation — and therefore reactivation — of selected genes,
which might also involve small RNAs (see also BOX 2). m, methyl group

38. DNA methylation
 In eukaryotes DNA methylation occurs predominantly at CpG dinucleotide
sequence. The enzyme DNA methyl transferase mediates the transfer of a
methyl group to cytosine, generating 5-methylcytosine.
 The region of the genome with high number of methylated cytosine are
usually transcriptionally inactive, silencing is either due to direct inhibition of
transcription factors binding as a result of methylated cytosine or binding of
other proteins with methyl binding domain to the DNA.
 In eukaryotes there are two types of DNA methylation:
 Maintainence methylation
 De novo methylation

41. Advantages
 This method could be effective against a broad spectrum of a pathogen’s
species and have implication for the control of other plant parasites and
pathogens.
 It is reliable, reduce labor, lower expenses, easy, increase cropping choices and
eliminate the need for chemicals that may be harmful to the environment.
 It could be used for functional analysis of plant genes through loss-of-function
of genes.
 It generates rapid phenotype that plant transformation is not needed.
 There is no need to screen large populations to detect the function of a specific
gene, only a single plant is enough to follow phenotype with targeted silencing.
Therefore, repeating the experiment is easy and time effective.
 VIGS system is particularly useful on plants which are difficult or impossible to
transform.
 It has the ability to target multiple gene family members with a single RNAi-
inducing transgene.

42. Disadvantages
 Since, the system relies on sequence information, it can only be used for
specific gene silencing if only the sequence information is known.
 The approach also depends on pathogen-host interaction, so pathogen
infection may manipulate host function and alter development and morphology.
There should be positive control in all VIGS assays to mark the effect of viral
inoculation on silenced plant.
 VIGS might suppress non-targeted gene in silenced plant cell or tissue.
 Gene of interest that we want to be expressed in an organism generally get
silenced so interfering in the expression of gene.

43.  At present, there are no reports of promoter-silencing strategies in GM crops
in the literature. Promoters are sequences that control the expression of
genes, thus silencing promoter inactivates transcription from the gene it
controls. The resulting transcriptional silencing may be advantageous because
it might be more stable than post-transcriptional silencing.

45. Aim of this study
 In this study, we aimed to induce RdDM‐mediated TGS of endogenous genes
with different expression profiles (constitutive expression, endoplasmic
reticulum [ER] stress‐inducible expression and seed‐specific expression) in
rice. Several TGS rice lines were obtained and TGS was inherited until at least
the third generation after the removal of the TGS trigger gene by segregation in
some of these lines. These TGS lines lacking the trigger gene are epimutants,
and our strategy to produce such a TGS rice line can be called epigenome
editing. Our results show that RdDM‐mediated TGS may be useful as an NPBT
in rice breeding, although some problems remain to be solved before its reliable
application.

46. Experimental procedures
 Plant material: Rice (Oryza sativa L. cv. Kitaake) was used for transformation.
 Vector construction and generation of transgenic rice: Approximately 1‐kb promoter regions of
genes encoding calnexin (CNX, −1003 to 3 bp), OsbZIP50 (−965 to −1 bp), globulin (Glb‐1, −858 to
−3 bp), glutelin B4 (GluB4, −997 to −1 bp), protein disulphide isomerase like 1‐1 (PDIL1‐1) and luminal
binding protein 1 (OsBiP1, −1017 to −5 bp) were cloned into pCR‐Blunt II‐TOPO vector using a Zero
Blunt TOPO cloning kit (Invitrogen)
 Total protein extraction and immunoblot analysis: Mature seed, leaves and roots were ground into
a fine powder with a Multi‐Beads Shocker (Yasui Kikai). For protein extraction, 300 μL (10 mg of leaves
and roots) or 500 μL (one seed) of extraction buffer [50 mM Tris‐HCl, 8 M urea, 4% SDS, 20% glycerol,
5% 2‐mercaptoethanol, 0.01% Bromophenol Blue] was added, and the samples were vortexed for 1 h
at room temperature. The mixture was centrifuged at 12,000 × g for 10 min at room temperature, and
the supernatant (protein extract) was decanted into a new tube. Total protein (2 μL of seed extract,
5 μL of leaf and root extracts) was electrophoresed in 12% SDS‐PAGE gels and subjected to
immunoblot analysis. Antibodies against calnexin, globulin, OsbZIP50, PDIL1‐1, GluA, GluB, GluC and
GluD were used.
 Reverse transcription (RT)‐PC: Total RNA was extracted using an RNeasy Plant Mini kit (Qiagen).
An RT reaction was performed with ReverTra Ace qPCR RT Master Mix with gDNA Remover
(Toyobo).
 Quantitative PCR (qPCR: Genomic DNA (200 ng) was digested overnight with the
methylation‐sensitive restriction enzyme HapII in a 20‐μL reaction mixture.
 MethylC‐seq: DNA was isolated from rice leaves using the CTAB method. Genomic DNA (1 μg) was
used for MethylC‐seq library preparation.

47. MethylC‐seq

48.  Induction efficiency of
transcriptional gene silencing in six
endogenous genes
 Plants with severely inhibited
growth and withered plants were
counted as TGS lines.

49. Summary
 To induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.),
we expressed double‐strand RNA of each promoter region and thus induced RNA‐directed
DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin
(CNX), protein disulphide isomerase (PDIL1‐1) and luminal binding protein (BiP1); an
endoplasmic reticulum stress‐inducible gene (OsbZIP50); and genes with seed‐specific
expression encoding α‐globulin (Glb‐1) and glutelin‐B4 (GluB4). TGS of four genes was
obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50,
63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1‐1 (33.3%) and Glb‐1 TGS
lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of
silencing of the target gene were investigated in some of the TGS lines. In progeny
of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except
in the endosperm) even after the removal of trigger genes (T‐DNA) by segregation. TGS
of CNXwas reverted by demethylation treatment, and a significant difference in CG and CHG
methylation levels in the −1 to −250 bp region of the CNX promoter was detected between the
TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of
promoter. TGS exhibited specific suppression towards the target gene compared with
post‐transcriptional gene silencing when GluB4gene from glutelin multigene family was
targeted. Based on these results, future perspectives and problems to be solved in the
application of RdDM to new plant breeding techniques in rice are discussed