rna silencing in plant

1. RNA SILENCING IN
PLANTS
BY
SHRUSHTI JOSHI
1850302

2. INTRODUCTION
• RNA silencing or RNA interference refers
to a family of gene silencing effects by
which gene expression is negatively
regulated by non-coding RNAs such as
microRNAs.
• RNA silencing may also be defined as
sequence-specific regulation of gene
expression triggered by double-stranded
RNA (dsRNA).
• They are associated with the regulatory
activity of small non-coding RNAs that
function as factors involved in inactivating
homologous sequences, promoting

3. BACKGROUND
Model proposed by Lindbo
et al. (1993) to explain the
RNA degradation and
antiviral state observed in
their study.
Matzke et al. (1989) Nicotiana
tabacum
Napoli et al. (1990) Petunia
hybrida
Lindbo et al. (1993) Nicotiana
tabacum
Ratcliff et al. (1997) Nicotiana
clevelandii

4. MECHANISM
• DsRNA or hpRNA is processed by a
Dicer or DCL protein into 20-24
nucleotide (nt) small RNA (sRNA)
duplex with 2-nt 3’ overhangs at
both ends.
• One strand of the sRNA duplex is
incorporated into an AGO forming
an RNA-induced silencing complex
(RISC).
• The sRNA molecule guides the RISC
to the complementary region of
single-stranded RNA, and the AGO
protein then cleave the RNA at the
nucleotides corresponding to the
central region (usually nt. 10-11) of

5. TYPES
• According to the source of dsRNA or hpRNA precursor and the
functional target of sRNAs, RNA silencing in plants can be
classified into 4 overlapping but functionally distinct pathways:
• microRNA (miRNA) pathway
• Trans-acting small interfering RNA (tasiRNA) pathway
• RNA-directed DNA methylation pathway
• Exogenic RNA silencing pathway
• Associated with the diversification of RNA silencing pathways,
plants have evolved multiple RNA silencing factors.

6. miRNA PATHWAY
• Transcription by RNA polymerase II to generate
primary miRNA transcript (pri-miRNA).
• Formation of “fold-back” stem-loop or hairpin
structure due to the existence of intra-molecular
sequence complementarity.
• Processed by DCL1 in the nucleus with the assistance
of the dsRNA-binding protein DRB1 or HYL1 generate
a 21-nt imperfect RNA duplex comprised of mature
miRNA and miRNA*.
• The 3’ terminal nucleotides are methylated at the 2’-
O-hydroxyl group by the RNA methylase HUA
ENHANCER1 (HEN1).
• miRNA : miRNA* duplexes are exported into the

7. TRANS-ACTING SMALL INTERFERING RNA
(tasiRNA) PATHWAY
• tasiRNA biogenesis is initiated by
specific miRNAs that direct the
cleavage of TAS precursor RNA.
• The miRNA cleavage fragments of TAS
transcript are converted to long dsRNA
by RDR6, which is then processed by
DCL4 into 21-nt siRNAs with 21-nt
phasing starting from the miRNA
cleavage site.
• Like miRNAs, tasiRNAs are methylated
by HEN1 and interact with either AGO1
or AGO7 to direct the degradation of

8. RNA-DIRECTED DNA METHYLATION PATHWAY
• RdDM is directed by 24-nt siRNAs, which is
generated by a combined function of the
plant-specific RNA polymerase IV (PolIV),
RDR2, and DCL3.
• PolIV transcribes methylated and highly
repetitive DNA to generate aberrant RNA and
RDR2 this ssRNA into dsRNA, which is
processed by DCL3 into 24-siRNAs that are
methylated by HEN1.
• The 24-nt siRNAs are loaded onto AGO4 to
form RISC . This AGO4-siRNA complex then
interacts with long non-coding RNA
transcribed from target DNA by another plant-
specific RNA Polymerase V (PolV) to recruit
other factors including Domains Rearranged

9. EXOGENIC RNA SILENCING PATHWAY
• The term “exogenic RNA
silencing” refers to RNA silencing
induced by sense transgenes and
viruses.
• Exogenic RNA silencing overlaps
with the endogenous siRNA and
RdDM pathways.
• It is divided into - Sense
Transgene-induced RNA Silencing
and Anti-viral RNA Silencing

11. RNA SILENCING
TECHNOLOGIES IN PLANTS
• The Basic RNA Silencing Pathway - Hairpin RNA
(hpRNA) Transgene
• The miRNA Pathway - Artificial miRNA (amiRNA)
• The tasiRNA and phsiRNA Pathways – Artificial
tasiRNAs
• Intrinsic Direct-repeat Transgene
• 3’ UTR Inverted-repeat (IR) Transgene
• Terminator-less Transgenes
• The RdDM Pathway – Promoter-targeting Transgenes

12. APPLICATIONS
BIOLOGICAL FUNCTIONS
• Immunity against viruses or
transposons
• Down-regulation of genes
• Up-regulation of genes
• RNA silencing also gets
regulated
PLANT BIOTECHNOLOGY
• Enhancement of Resistance to
Biotic Stresses
• Alteration of Plant
Architecture and Flowering
Time
• Development of Seedless
Fruits
• Secondary Metabolites for
Neutraceutical and
Pharmaceutical Applications

13. ADVANTAGES
• They are now well established technologies and easy to use.
• Complete knock-out of essential genes is lethal to plants and
therefore such mutants cannot be recovered by CRISPR/Cas9-
like mutagenesis technologies.
• RNA silencing technologies allow for tissue-specific silencing of
a gene using a tissue-specifically expressed transgene,
whereas genetic mutation result in gene knock-out in all
tissues.
• RNA silencing technologies can be used to simultaneously
silence multiple genes using transgenes containing either a
conserved sequence or a composite sequence from multiple

14. DISADVANTAGES
• Post-transcriptional RNA silencing usually does not result in
complete gene silencing.
• In addition, it remains unclear if RNA silencing technologies can
be used to consistently switch off endogenous gene promoters
in plants, which, appear to be resistant to siRNA-directed
transcriptional silencing.
• complete or stable gene knock out is hard and almost
impossible to achieve.

15. REFERENCES
• RNA Silencing in Plants: Mechanisms, Technologies and
Applications in Horticultural Crops by Qigao Guo, Qing Liu ,
Neil A. Smith, Guolu Liang, and Ming-Bo Wang.
• RNA Silencing in Plants: Yesterday, Today, and Tomorrow by
Andrew Eamens,* Ming-Bo Wang, Neil A. Smith, and Peter M.
Waterhouse
• A biochemical framework for RNA silencing in plants by
Guiliang Tang, Brenda J. Reinhart, David P. Bartel, and Phillip D.
Zamore

16. THANK YOU