RNA GENE SILENCING AND SUPRESSORS IN PLANTS

RNA silencing/RNAi involves the knock-down of genes through two types of small RNA molecules, miRNAs and siRNAs, that are involved in post-transcriptional and transcriptional gene silencing as an antiviral mechanism; short double-stranded RNAs are processed by the Dicer enzyme into siRNAs which are incorporated into the RISC complex to guide degradation of homologous mRNA targets; RNAi is an important endogenous gene regulatory mechanism and has applications in gene function analysis, gene therapy, and cancer treatment.

1. RNA GENE SILENCING
AND SUPRESSORS

2. CONTENTS
Introduction
Components of the system
Mechanism of RNAi
RNAi-Mediated resistance
Application of exogenous dsRNA
RNA Silencing suppresors
Functional assays
Mechanismofsuppression
Futuredirections

3. INTRODUCTION
•Regulatory mechanism of gene expression in eukaryotic
organisms
•Post-transcriptional gene silencing (PTGS)
•Transcriptional gene silencing (TGS)
•RNA silencing
•RNA interference (RNAi)
•Co-suppression
•Gene silencing
•Quelling

4. HISTORY
1998-Andrew Fire and Craig. C. Mello
discovered a mechanism that can degrade mRNA
from a specific gene.
Noble Prize in 2006

5. CENTRAL DOGMA

6. COMPONENTS OF THE SYSTEM
ds RNA
Dicer like Protein (DCL)
Argonaute (AGO) protein
RISC - RNA induced silencing
complex

7. ds RNA
siRNA: dsRNA
21-22 nt
miRNA: ssRNA
19-25nt
shRNA: ssRNA
20-25 nt

8. •RNAse III-like dsRNA-specific ribonuclease
• Enzyme involved in the initiation of RNAΙ.
•It is able to digest dsRNA into uniformly sized small RNAs
(siRNA)
Dicer Product
DCL1 miRNA
DCL2 22nt siRNA
DCL3 24nt siRNA
DCL4 21nt siRNA
DICER

9. •RISC is a large (~500-kDa) RNA-multiprotein complex, which
triggers mRNA degradation in response to siRNA/miRNA/shRNA.

•The active components of an RISC are endonucleases called
argonaute proteins which cleave the target mRNA strand.
RISC - RNA induced silencing complex

10. Cont..
To produce RISC certain enzymes are needed
S.NOProtein NatureFound in
1 Slicer RNA se HC. elegans
2 Arganout RNA IIIPlants and Animals
3 Dicer
(Endo nuclease)
(Exo nuclease)
RNA IIIAll Eukaryotes

11. RNA
siRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt.
shRNA: ssRNA 20-25 nt. Encoded by non protein coding
genome
RISC
RNA induced Silencing Complex, that cleaves mRNA
Enzymes
Dicer : produces 20-21 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin and to form pre miRNA;
which is later processed by Dicer
The players in the interference

12. TheprocessisinitiallytriggeredbydsRNA,whichcanbe
introducedexperimentally orarise from
STEP:1

13. ThedsRNAtriggeriscleavedbyaribonucleaseIII(RNAseIII)-
likeenzymetermedDicerinto21–24nucleotideduplexestermed
short-interferingRNAs(siRNAs).
Italsoinvolvesinteractionwith
otherproteins,including
i.Anargounautelikeprotein
ii.AdsRNAbindingproteinand
(Voinnetetal.,2014)
iii.RNAhelicase
STEP: 2

15. STEP:3
The antisense siRNA/miRNA/shRNA act as guides for RISC to
associate with complimentary single-stranded mRNAs.

16. STEP:4
WithintheactivatedRISC,single-strandedsiRNAsactas
guide to bring the complex into contact with complemantary
mRNAs and there by cause their degradation
(Bolognaetal.,2014)

17. RNA I Overview
•During RNAi Double-stranded RNAs cut into short fragments of
RNAs, siRNA/miRNA/shRNA by an enzyme called Dicer.
•These then base pair to an mRNA through a dsRNA-enzyme
complex. This will either lead to degradation of the mRNA strand
si RNA
sh RNA
mi RNA
dS RNA
These products lead to
“RNA mediated Gene
silencing”

18. M
E
C
H
A
N
I
S
M

19. Case study
The major objective of this article is the identification of the GLS
gene and downregulation of that gene by using siRNA to
decrease the production of ß 1, 3 -glucan, a major fungal cell
wall component.

22. RNAi-MEDIATED RESISTANCE AGAINST PHYTOPATHOGENIC BACTERIA
Target
organism
Disease
RNAi
approach
Target
sequence
Experimental
plants
References
Rhizobium
radiobacter
Crown gallTransgenic
plants
(hairpin-loop
structure)
iaaM and ipt
oncogene
mRNAs
Arabidopsis thaliana
Lycopersicon
esculentum (tomato)
Juglans regia L.
(walnut)
Escobar et al.
(2002)
RNAi-MEDIATED RESISTANCE AGAINST PHYTOPATHOGENIC NEMATODES
Target
organism
Disease
RNAi
approach
Target
sequence
Experimental
plants
References
Meloidogyne
incognita
Root knot Transgenic
plants
(hairpin-loop
structure)
M. incognita
16D10
peptide
mRNA
A. thalianaHuang et al.
(2006)

26. RNAi-MEDIATED RESISTANCE AGAINST PLANT VIRUS AND VIRIODS
Target
organism
Disease
RNAi
approach
Target
sequence
Experimental
plants
References
CVYV,
MNSV,
MWMV,
and
ZYMV
Different
virus
diseases
Transgenic plant
(hairpin-loop
structure)
Cm-eIF4E
mRNA
Cucumis melo
(muskmelon)
Rodriguez et al.
(2012)
PNRSV Ringspot
diseases of
prunus
Transgenic plants
(hairpin-loop
structure)
CP coding
sequence
Prunus avium
Prunus cerasus
Song G et al.
(2013)
TYLCV Tomato
yellow
leaf curl
Transgenic plants
(hairpin-loop
structure)
CP coding
sequence
Solanum
lycopersicum
Fuentes A et al.
(2016)
CBSV and
UCBSV
Cassava
brown
streak
Transgenic plants
(hairpin-loop
structure)
CP coding
sequence
Manihot
esculenta
Beyene G et al.
(2016)
PSTVd Potato
spindle
tuber
Transient leaf
assays
(amiRNAs)
PSTVd positive
and
negative strands
N. benthamianaCarbonell A et
al. (2017)

27. What is
suppressors in
RNAi
Role of
suppressors in
pathogen virulence

28. In virus
➢Coat proteins,
➢Replicases,
➢Movement proteins,
➢Helper components for viral transmission,
➢Proteases or
➢Transcriptional regulators.

29. Why are only viral
suppressors are
predominantly involved,
and not fungal or
bacterial suppressors?

30. Roger Hull, 1937

31. Pathogen Host Suppressor Suppression mechanism Reference
Puccinia
graminis
Nicotiana
benthamiana
PgtSR1 Prevents R gene-triggered
hypersensitive response
Yin et al.
(2019)
Verticillim
dahliae
A. thaliana,
N. benthamiana
VdSSR1 Interferes with the nuclear export of
AGO1–miRNA complexes reducing
plant- to-fungi trafficking of trans-
kingdom siRNA
Zhu et al.
(2022)
Phytophthora
sojae
N. benthamianaPSR1 Impairs assembly of microRNA-
processing complexes and causes
reduced abundance of siRNAs
Qiao et al.
(2015)
P. sojae N. benthamianaPSR2 Interferes with biogenesis of
secondary siRNAs and inhibit
salicylic acid defence pathway
Qiao et
al.(2013)
Candidatus
Phytoplasma
tritici
N. benthamianaSWP16
Inhibits biogenesis of some
miRNA
Wang et al.
(2021)

32. Do all plant viruses have suppressors of silencing?
•Althoughmanydifferentviralsuppressorshave
identified,whichsuppressthe RNAsilencing.
been
•Somevirusesmayhaveevolvedotherwaystotrytoavoid
silencing, suchasbyreplicatingwithinspherules in theER
•wherethedsRNAishidden,
•orbyreplicatingand
•movingrapidlyenoughto outrunthemobile
silencingsignal.
(Schwartzetal.,2002)

33. APPLICATION OF EXOGENOUS dsRNA
Das and Sherif, (2020)

34. ADVANTAGES
•Targeted Defense Mechanism
•Broad Spectrum of Action
•Heritable Resistance
•No Need for Chemical Pesticides
•Efficient Response to Viral Infections
•Minimal Risk of Resistance Development
•Potential for Transgenic Crops
•Regulation of Plant-Pathogen Interactions
•Enhancement of Plant Immunity in Combination with Other
Mechanisms
•Adaptability

35. FUTUREDIRECTIONS
•ViralsuppressorsoftentargetRNA-silencingpathwayssuchassiRNAsor
effectorssuchasAGOandDCLproteins,insomecasesasingleVSRcan
targetmorethanoneelementinthesilencingpathway
•ItispossiblethatmanyotherVSRsinteractinmultiplewayswith
RNA-silencingpathways,whichremaintobediscovered.
•There are still several gaps in our knowledge regarding the effectors of
plant silencing machinery
•The replication, subcellular localization and regulation of the
expressions of viral genes including VSRs, which are still not known for
many plant viruses.

36. Conclusion