Gene silencing is the process of switching off specific genes through mechanisms like transcriptional gene silencing and post-transcriptional gene silencing, which are regulated by small RNA molecules such as miRNAs and siRNAs. This technology has numerous applications in plant biotechnology, including enhancing nutritional content, extending shelf life, and reducing allergenic properties in crops like tomatoes and apples. RNA interference (RNAi) plays a critical role in these processes, allowing for targeted gene regulation to improve crop traits and mitigate risks associated with food allergies.

2.  Gene silencing is a general term used to describe the "switching off" of a
gene by a mechanism other than genetic modification.
 That is, a gene which would be expressed (turned on) under normal
circumstances is switched off by machinery in the cell.
 Genes are regulated at either the transcriptional or post-transcriptional level.
 Both transcriptional and post-transcriptional gene silencing are used to
regulate endogenous genes.
Meister Gunter; Tuschl Thomas. Mechanisms of gene silencing by double-
stranded RNA. Nature. (2004) 431-343 https://doi.org/10.1038/nature02873
10.1038/nature02873

3. Transcriptional gene silencing (TGS) Post-transcriptional gene silencing (PTGS)
•Transcriptional gene silencing is the
result of histone modifications,
creating an environment
of heterochromatin around a gene that
makes it inaccessible to transcriptional
machinery (RNA polymerase,
transcription factors, etc.).
•Post-transcriptional gene silencing is the
result of mRNA of a particular gene
being destroyed or blocked. The
destruction of the mRNA
prevents translation to form an active
gene product (in most cases, a protein). A
common mechanism of post-
transcriptional gene silencing is RNAi.
• Genomic Imprinting
• Paramutation
• Transposon silencing
• Transgene silencing
• Position effect
• RNA-directed DNA
methylation
• RNA interference
• RNA silencing
• Nonsense mediated
decay

4.  Gene silencing
 RNA silencing
 RNA interference
 In certain fungi: quelling
Most widely held view is that RNAi evolved to protect the genome from
viruses (or other invading DNAs or RNAs)
Gene silencing thus may be part of an ancient immune system protecting
from such infectious DNA elements.

5.  First discovered in plants
(R. Jorgensen, 1990)
 When Jorgensen introduced a re-engineered gene into petunia that had a lot
of homology with an endogenous petunia gene, both genes became
suppressed!
• Also called Co-suppression
• Suppression was mostly due to increased degradation of the mRNAs
(from the endogenous and introduced genes)

6.  In 1990 Rich Jorgensen attempted to alter flower colors in Petunia by
introducing additional copies of a gene encoding chalcone synthase , a key
enzyme for flower pigmentation into petunia.
 The overexpressed gene was expected to result in darker flowers, but
instead produced less pigmented, fully or partially white flowers, indicating
that the activity of chalcone synthase had been substantially decreased; in
fact, both the endogenous genes and the transgenes were downregulated in
the white flowers.
 Further investigation of this phenomenon in plants indicated that the
downregulation was due to post-transcriptional inhibition of gene
expression via an increased rate of mRNA degradation
 This phenomenon was called co-suppression of gene expression, but the
molecular mechanism remained unknown.

7.  RNAi is a process through which the small, double-stranded RNAs
specifically silence the expression of homologous genes, largely through
degradation of their cognate mRNA.
 RNAi discovered in C. elegans (first animal) while attempting to use
antisense RNA in vivo
Craig Mello Andrew Fire (2006 Nobel Prize in
Physiology & Medicine)
RNA interference has an important role in defending cells against parasitic
genes – viruses and transposons – but also in directing development as well as
gene expression in general.

8.  Homology of the dsRNA and the target gene/mRNA is required. Double-
stranded RNA triggers cleavage of homologous mRNA.
 Targeted mRNA is lost (degraded) after RNAi.
 The effect is non-stoichiometric; small amounts of dsRNA can wipe out an
excess of mRNA (pointing to an enzymatic mechanism).
 Two types of small RNA molecules – microRNA (miRNA) and small
interfering RNA (siRNA) – central to RNA interference.
Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E.
Driver & Craig C. Mello. Potent and specific genetic interference by double-
strandedRNAin Caenorhabditis elegans. Nature. (1998) NATURE. 391: 806-
811.

9. siRNAs
• Small interfering RNAs that have an integral role in the phenomenon of RNA
interference(RNAi).
•They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme
Dicer.
• 21-25 nt fragments, which bind to the complementary portion of the target mRNA and
tag it for degradation.
• A single base pair difference between the siRNA template and the target mRNA is enough
to block the process.
mi RNAs
•A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25
nucleotide long (thought to regulate the expression of other genes).
• Derive from ~70 nt ssRNA (single-stranded RNA), which forms a stemloop; processed to
22nt RNAs
•miRNAs are RNA genes which are transcribed from DNA, but are not translated into
protein.

10. AAAA
RNAi is mediated by small
(~21-25 nucleotide) noncoding RNAs
complementary to the targeted gene
cleavage of
targeted mRNA
(siRNA)
Inhibits protein translation or
causes mRNA degradation
(miRNA)
mRNA:
dsRNA
intermediate

11.  The RNAi pathway initiated by the enzyme Dicer, which cleaves long dsRNA
molecules into short fragments of ~21 nucleotides.
 Thus producing multiple “trigger” molecules from the original single dsRNA.
 The siRNA-Dicer complex recruits additional components to form an RNA-induced
Silencing Complex (RISC) in which the unwound siRNA base pairs with
complementary mRNA, thus guiding the RNAi machinery to the target mRNA
resulting in the effective cleavage and subsequent degradation of the mRNA.
 In this way, the activated RISC could potentially target multiple mRNAs, and thus
function catalytically.
Sayda M. Elbashir, Winfried Lendeckel and Thomas Tuschl. RNA interference is
mediated by 21- and 22-nucleotide RNAs. Genes & Dev. (2001). 15: 188-200

13.  An endonuclease of RNase III family essential for
sequence-specific gene suppression.
 Dicer cleaves dsRNA into small interfering RNA
duplexes (siRNAs) encompassing a length of 21 to
25 nt.
 Dicer facilitates the formation of the RNA-induced
silencing complex (RISC, whose catalytic component
argonoute is an endonuclease capable of degrading
mRNA).
 Argonaute proteins bind different classes of small
non-coding RNAs, including (miRNAs), (siRNAs)
and (piRNAs).
 Small RNAs guide Argonaute proteins to their
specific targets through sequence complementarity,
which typically leads to silencing (endonuclease
activity) of the target.

14.  Class 2 RNaseIII enzyme responsible
for initiating the processing of miRNA.
 pri-miRNA, which is cleaved by Drosha
to produce a characteristic stem loop
structure of about 70 bp long, known as
a pre-miRNA.
 Drosha exists as part of a protein
complex called the Microprocessor
complex, which also contains the
double-stranded RNA binding protein
Pasha , which is essential for Drosha
activity and is capable of binding single-
stranded fragments of the pri-miRNA
that are required for proper processing.
 Drosha was the first human RNase III
enzyme identified and cloned.

15. What is antisense RNA??
 Antisense RNA is a single-stranded RNA that is complementary to mRNA
(sense strand) transcribed within a cell.
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
 Antisense RNA or gene encoding antisense RNA is introduced into target
organism by using a plasmid vector or using a gene gun that shoots
microscopic tungsten pellets coated with the gene .
 This antisense RNA (aRNA) is specific to the gene whose expression is to
be regulated.
 Inhibit the translation machinery by base pairing with the sense RNA and
activating the RNase H.
Andrew J. Hamilton, David C. Baulcombe. A Species of Small Antisense RNA in
Posttranscriptional Gene Silencing in Plants. Science. 286(5441): 950-952

16.  When mRNA forms a duplex with a complementary antisense RNA
sequence, translation is blocked.
 This may occur because-
• The ribosome cannot gain access to the nucleotides in the mRNA or
• Duplex RNA is quickly degraded by ribonucleases in the cell ( RNA
interference ).

17.  The tomato was the first whole food created by antisense RNA technology
that was evaluated by the FDA.
• One by Calgene reduces polygalacturonase activity to retard softening
in 1992. Licensed in May 17, 1994.
• While the other blocks ethylene synthesis to retard overall ripening.
• These products were not successful due to limitations in the quality of the
base germplasm, the development of competitive non transgenic products
and the difficulty of obtaining premium prices when shelf life is not a
primary consumer concern.
• The increased popularity of ready-to-eat and convenience foods will drive
the need for products with improved shelf life. For example, sales of
prepackaged lettuce have increased over the past 5 years.

24.  Epidemiologic studies have suggested a potential benefit of
the carotenoid lycopene in reducing the risk of prostate
cancer, as a nonpolar carotenoid, lycopene is more soluble
in a lipid base; in addition, carotenoid-binding proteins
are broken down during processing, leading to greater
bioavailability.
 While modifying polyamines to retard tomato ripening,
an unanticipated enrichment in lycopene was discovered,
with levels up by 2- to 3.5-fold compared with the
conventional tomatoes. This is a substantial enrichment,
exceeding that so far achieved by conventional means. This
novel approach may work in other fruits and vegetables.

25.  (National institute of Plant Genome Research) in Feb,2010 has developed a
tomato by antisense technology which can last long upto 45 days.
 NIPGR scientist had silenced the expression of two important gene which
are responsible for loss in firmness and textures during ripening (Meli et al.,
2010).
 The two gene silenced are alpha-man and beta-hex of Glycosyl hydrolase,
enzyme that breaks the chemical bond holding a sugar to either another
sugar or some other molecule, like a protein.

27.  Abiotic stress tolerance
 Biotic stress tolerance
 Nutritional Improvement
 Deletion of Allergens
 Removal of toxic compounds
 Prolongation of shelf life
 Modulation of flower colour
Daniel H. Kim & John J. Rossi. RNAi mechanisms and applicatons.
Biotechniques. (2018) 5(4): https://doi.org/10.2144/000112792

28.  In recent years, RNAi technology has been used in metabolic engineering of
plants with respect to different traits and targets.
 RNAi has shown promise in development of tomato (Lycopersicon
esculentum) fruit with enhanced carotenoid and flavonoid content, both of
which are highly beneficial for human health.
 Davuluri et al. (2005) used fruit specific promoter combined with RNAi to
suppress an endogenous photomorphogenesis regulatory gene Det1 in
tomato, which represses several signaling pathways controlled by light. In
contrast to control wild type (non transgenic) tomato, the transgenic
tomatoes so obtained showed specific degradation of Det1, along with an
increase in the carotenoid and flavonoid content.

29.  Similarly, the carotenoid content of rapeseed (Brassica napus) were also
enhanced by utilizing RNAi to downregulate the expression of lycopene
epsilon cyclase (ε-CYC). The transgenic Brassica seeds thus obtained
showed increased levels of β-carotene, zeaxanthin, violaxanthin and lutein
(Yu et al., 2007).
 RNAi was successfully used to silence the N-demethylase gene, designated
as CYP82E4 for suppressing nicotine to nornicotine conversion in tobacco
(Nicotiana tabacum). Nor-nicotine is the precursor of N'-nitrosonornicotine
(NNN), which is a tobacco specific nitrosamine (TSNA) having
carcinogenic properties (Gavilano et al., 2006).

30.  Amylose content in wheat has been markedly increased with RNAi
approach, by suppressing simultaneously the expression of SBEIIa and
SBEIIb. The suppression yielded >70% amylose in wheat (Regina et al.,
2006).
 In 2003, Kusaba et al. reported hpRNA mediated silencing of Low Glutelin
Content 1 (Lgc1) gene in rice. Glutelin is a major seed storage protein,
which accounts for about 60% of total endosperm protein in rice In mutant
line LGC1, the glutelin content is reduced so that kidney patients, who need
to restrict their protein intake might benefit from this.
 RNAi was applied to engineer decaffeinated coffee (Coffea canephora)
plants by using constructs containing CaMXMT1 sequence that encodes the
theobromine synthase gene involved in the caffeine biosynthetic pathway.
The RNAi mediated suppression of theobromine synthase thus led to
reduction of caffeine content by up to 70% in comparison to controls (Ogita
et al., 2003).

31.  RNAi technology has also been used successfully in genetically modifying
the fatty acid composition of cotton seed oil. Liu et al. (2002) utilized a
hairpin RNA (hpRNA) mediated RNAi method to downregulate two key
fatty acid desaturase genes encoding stearoyl-acyl-carrier protein Δ9-
desaturase and oleoyl-phosphatidylcholine ω6-desaturase. Downregulation
of these two genes in cotton resulted in nutritionally improved high stearic
and high oleic cotton seed oils which are essential fatty acids for better
health of the human heart.
 RNAi has even been used to increase the shelf life of tomato by delaying its
ripening. Tomato being a climacteric fruit, has a burst of autocatalytic
ethylene during ripening process. Xiong et al. (2005) introduced a unit of
ACC oxidase dsRNA in tomato and thus blocked the expression of its gene
which result in delayed ripening. In fruits of transgenic plants, the ethylene
production rate of ripened fruits and leaves was significantly inhibited.

32.  lysine is one of the most important essential amino acids and due to its
presence in limiting levels in major food crops. In corn (Zea mays),
production of high lysine was reported by RNAi mediated suppression of
the lysine catabolic enzyme lysine-ketoglutarate reductase/saccharopine
dehydrogenase (ZLKR/SDH) in endosperm (Houmard et al., 2007). Lysine
ketoglutarate reductase is the first enzyme in the α-amino adipic acid
pathway, which catabolizes lysine in glutamate, α-amino adipic acid and
acetyl CoA. Since lysine is an important essential amino acid, increasing its
content in major cereal crops like rice and wheat through such RNAi
approaches will prove to be fruitful.
 RNAi technology has also been used in soybean (Glycine max) in order to
silence the myo-inositol-1-phosphate (GmMIPS1) gene, which resulted in a
drastic reduction (up to 94.5%) of phytate content in the developed
transgenic lines (Nunes et al., 2006).

33.  Food allergies, though a rare phenomenon, are a cause of concern, in today’s
world. People allergic to a particular food or food items often avoid that which
causes the allergy, which results in deprivation of the diet of a wide range of
common plant foods that have important nutritional value.
 RNAi being sequence-specific is highly efficient in silencing specific allergens
and toxic metabolites, to the extent that it limits only their allergic potential
without hampering the essential cellular functions, which these allergens and
metabolites may perform.
 Le et al. (2006) have produced tomato fruits with reduced allergenicity. They
reported efficient downregulation of of Lyc e3, a tomato allergen in transgenic
tomato plants. The Lyc e 3 encodes a non-specific lipid transfer protein (ns-
LTP), which is hydrophilic and enhances specific intermembrane lipid transfer.
Specific dsRNAi constructs of LTPG1 and LTPG2 were utilized for the
suppression of Lyc e 3 accumulations. Tests were conducted for the allergenic
potential of transgenic tomato plants by measuring the histamine release from
sensitized human basophils as against parental lines.

34.  An allergen Mal d 1 in apple (Malus domestica), leads to cross-reactive IgE
antibody responses, which causes adverse reactions in allergic patients. In order to
inhibit the expression of this allergen RNA interference approach was used
(Gilissen et al., 2005). An intron containing Mal d 1 gene was isolated from the
cultivar Gala, to build the RNAi construct for successful gene silencing of Mal d 1.
The results suggested about 10-fold reduction in the expression of Mal d 1.
 High quality protein content in cottonseed makes it a nutrient rich resource for food
but cannot be utilized due to the presence of toxic gossypol within the seed tissue.
Inspite of being toxic to humans, gossypol is required by the plant to protect itself
against insects and pathogens. In 2006, Sunil Kumar et al. reported successful use
of RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with
the expression of the δ-cadinene synthase gene during seed development. A tissue
specific promoter for this RNAi approach was used, so that the gossypol content
was reduced only in the seeds of cotton. The transgenic cottonseeds obtained
showed 99% reduction of gossypol as compared to non transgenic wild types.
However, no such reduction was observed in the other parts of transgenic plants.

35. •A serious shortcoming of many insecticides is that they can kill non-target
species.
•Sequence specificity of RNA interference (RNAi) used to design orally-
delivered ds RNAs that selectively killed target species.
•Fruit flies (Drosophila melanogaster), flour beetles (Tribolium castaneum), pea
aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta) were
selectively killed when fed species-specific dsRNA targeting vATPase transcripts.
•They demonstrated that even closely related species can be selectively killed by
feeding on dsRNAs that target the more variable regions of genes, such as the 3’
untranslated regions (UTRs)
•Four species of the genus Drosophila were selectively killed by feeding on short
(<40 nt) dsRNAs that targeted the 3’UTR of the ɤ-tubulin gene.

36.  The ɤTub gene is highly conserved in four species of Drosophila. Four
drosophilid species were selected D. melanogaster, Drosophila pseudoobscura,
Drosophila sechellia , and Drosophila yakuba for study.
 To deliver ɤ-tub-dsRNA (long dsRNAs, enzymatically diced dsRNAs, or
synthesized siRNAs) to D. melanogaster larvae, neonates were soaked in
solutions of dsRNA with encapsulation within cationic liposomes. The insects
were soaked for a period of 1–2 h and then transferred to normal diet and all
species suffered high mortalities following ingestion of conspecific dsRNA.
 Quantitative RT-PCR confirmed that the reduction in GUS enzyme activity
correlated quite closely with the extent of gus transcript knockdown.
 The larvae were also offered the dsRNA by droplet feeding, similar levels of
RNAi were observed.

38. Insecticidal dsRNAs in non-drosophilids
•To demonstrate that dsRNA targeting a single gene could be used selectively to target
different pest insects, they designed species-specific dsRNAs to silence the gene
encoding the E-subunit of V-ATPase in four insect species: the fruit fly (Drosophila
melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum),
and tobacco hornworms (Manduca sexta). V-ATPase is a membrane-bound protein
that acts as a proton pump to establish the pH gradient within the gut lumen of
many insects.
•To examine the specificity of the dsRNA to selectively kill the target species, the four
species of insects were fed each dsRNA for the targeted species. When insects were fed
on a diet laced with D. melanogaster-specific vATPase dsRNA , only D. melanogaster
showed reduced growth and development, while the other three species were unaffected
by the Drosophila dsRNA. Similarly, by feeding each of the other three species
conspecific dsRNA, they selectively killed each species, without adversely affecting any
of the others.
•Ingested dsRNA results in systemic RNAi :To determine whether the ingested dsRNA
induced RNAi in tissues other than gut cells, dsRNA fed larvae were dissected and the
level of GUS activity and gus transcripts were assessed in isolated guts and other tissues.
No significant knockdown of GUS activity or gus transcripts was detected in other
tissues even when using the highest concentration of dsRNA, which suggests that the
dsRNA did not move from the gut cells.

40.  They observed that dsRNA targeting the E-subunit of vATPase can be
selectively designed and fed to a broader range of insects, including larvae
of another species of beetle (T. castaneum), moth larvae (M. sexta), aphid
nymphs (A. pisum), and dipteran larvae (D. melanogaster).
 They also demonstrated that this method could also be used to target closely
related species, for this they selected a gene that was even more highly
conserved, the ɤTub23C gene, and by targeting the more variable 3’ UTR
sequences, it is possible to design dsRNAs that are species-specific.
 They described that feeding of dsRNA to a range of different insect species,
showing that even highly conserved genes can be exploited to induce
species-limited RNAi, without affecting non-target species.
 This was the first demonstration of RNAi following ingestion of dsRNA in
all of the species tested, and the method offers promise of both higher
throughput RNAi screens and the development of a new generation of
species-specific insecticides.

42.  In this study, three potential candidate genes shown to be involved in
abiotic stress response pathways in Arabidopsis thaliana were selected for
VIGS experiments in wheat.
 Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8’-hydroxylase),
and Sal1 (inositol polyphosphate 1-phosphatase).
 Gene homologues for these three genes were identified in wheat and
cloned in the viral vector barley stripe mosaic virus (BSMV) in the
antisense direction, followed by rub inoculation of BSMV viral RNA
transcripts onto wheat plants.
 Quantitative real-time PCR showed that VIGS-treated wheat plants had
significant reductions in target gene transcripts. When VIGS-treated
plants generated for Era1 and Sal1 were subjected to limiting water
conditions, they showed increased relative water content, improved water
use efficiency, reduced gas exchange, and better vigour compared to water-
stressed control plants inoculated with RNA from the empty viral vector
(BSMV0).
 In comparison, the Cyp707a-silenced plants showed no improvement over
BSMV0-inoculated plants under limited water condition.
 These results indicate that Era1 and Sal1 play important roles in conferring
drought tolerance in wheat.

43. Phenotypes of wheat plants at 24 dpi with BSMV RNA transcripts containing one of
the following wheat genes: Sal1, Cyp707A,or Era1. Water stress was imposed on these
plants by withholding water until 50% of field capacity. Water-stressed BSMV0
inoculated plants served as control. Non-silenced well-watered (100%), non-silenced
water-stressed (50%) plants, and water-stressed (50%) plants were included for
comparison of phenotypes.

44. Effect of Era1 silencing on wheat seed germination and response to pathogens. (A)
Number of germinated seeds in Era1-silenced and non-silenced plants, determined
according to the 2-mm radicle extrusion criterion. (B) Cumulative lesion area
observed after 7 days post infection with Xanthomonas translucens B74 and B75
on Era1silenced or non-silenced plants under drought or non-drought conditions.
Era1, plants silenced in Era1; BSMV0, plants inoculated with empty BSMV viral
vector; non-silenced, plants not inoculated with BSMV.

45. Bayer Crop Science has acquired an exclusive worldwide license to
develop, market, and sell some plant varieties in which the RNAi
technology has been successfully applied by the CSIRO scientists. Using
this technique this group has developed varieties of barley that are resistant
to BYDV (barley yellow dwarf virus) (Wang et al. 2000). Their results
showed that the barely plants developed through RNAi technology are
resistant to viral infection while the control plants became infected with the
yellow dwarf virus.

46.  RNAi has been recognized as an attractive tool for plant gene function
analysis and also for manipulation of both desirable and undesirable genes
to generate plants with improved quality traits and having better potentiality
of protection against abiotic and biotic stresses. In recent years, the studies
focused on RNAi have revealed a much clearer picture about its
mechanisms and applications, but still there is more, which needs to be
known and understood.
 The Antisense RNA technology shows the potential for diverse applications
to basic research and therapy. Antisense technology offers almost unlimited
scope for the development of new methods of drug design and one of the
most approved approaches among several others, for inactivating a single
chosen gene. However, the full commitment of this promise is yet to be
established.