Role of RNAi (RNA interference) Mediated Gene

Silencing Technology in Plant Disease Management

ANAM MOOSA
PH.D PLANT PATHOLOGY

DEPT OF PLANT PATHOLOGY

UNIVERSITY OF
AGRICULTURE
FAISALABAD

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 Introduction
Introduction

• Plant diseases (A challenge to world food

production)
• Chemical management strategies extensively
used in developing countries
• Hazardous effects on environment and human
health
• Breeding for resistance - environment friendly
approach
• Transgenic plants are gaining importance
worldwide (eco-friendly, suppression of specific
gene)
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 GENE SILENCING

“Switching off” the expression of a gene

Genes are silenced at either the transcriptional or post-
transcriptional level.
Transcriptional gene silencing - Result of modifications of
either the histone or DNA.
Post-transcriptional gene silencing (PTGS )-Result of the
mRNA of a particular gene being destroyed or blocked.
A common mechanism of PTGS is RNAi.

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RNAi RNA INTERFERENCE

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RNA FAMILY

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 RNAI (RNA
INTERFERENCE)

• A biological process in which dsRNA molecules

inhibit gene expression by causing the destruction of
specific mRNA molecules

• Evolutionary conserved process of Post

Transcriptional Gene Silencing

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Alternate terms to RNAi are…
Quelling
PTGS (POST
TRANSCRIPTIONAL
Co-
(In Fungi)
GENE SILENCING)

suppression
RNAi

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 Historical Overview
• Discovery of RNAi
• In 1990 Napoli and Jorgensen
• Attempted to alter flower colors in petunias,
• Additional copies of a gene encoding chalcone synthase were introduced
• Chalone Synthase (A key enzyme for flower pigmentation into petunia plants of normally
pink or violet flower color).
• Overexpressed gene was expected to result in darker flowers
• Instead, less pigmented, fully or partially white flowers
• Indicating that the activity of chalcone synthase had been substantially decreased
• This phenomenon was called co-suppression of gene expression
• Molecular mechanism remained unknown.

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RNAi induced silencing in Petunia Plants

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 Quelling (IN FUNGI)

Carlo Cogoni and Guiseppe Macino

in 1992
Introduced a gene needed for
carotenoid synthesis in the mold
Neurospora crassa
It led to the inactivation of mold’s
own genes in about 30% in the
transformed cells
Expected :Orange pigment
Result: Albino
A rosette of Neurospora crassa
This gene inactivation was called as asci
“Quelling”
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 C. elegans
The actual reason came into light in 1998…
Craig C. Mello and Andrew Fire’s injected double
stranded RNA into C. elegans.
Investigated the regulation of muscle protein
production
Observation: “Neither mRNA nor antisense RNA
injections had an effect on protein production”
“Instead double-stranded RNA successfully
silenced the targeted gene”
As a result of this work, they coined the term RNAi.
Reported in “NATURE” a potent gene silencing
effect

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RNAi won Noble prize

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 Biological Functions
Immunity

Down regulation of genes

Up-regulation of genes

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 Applications
• Gene Knockdown
• Functional Genomics
• Medicine
[Link]
[Link]
• Biotechnology
[Link]
[Link]
[Link] plants
• Genome scale screening
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• Two types of small RNA molecules are central to RNA
interference (RNAi)

miRNA
microRNA

siRNA
Small Interfering RNA

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 siRNA (small interfering RNA’s)

siRNA (small interfering RNA’s)

Also known as “short interfering RNA’s” or
“silencing RNA’s”
siRNA is a synthetic duplex specifically designed to
target a particular mRNA for degradation
Gene knockdown in a variety of cell lines

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 Micro RNA’s (miRNA)

• miRNAs are non-coding RNAs

• Found only in eukaryotic cells.
• Small in size with an average length of 22 nucleotides.
• Transcribed by RNA polymerase II from independent
genes or introns of protein-coding genes
• Gene-regulatory roles in both plants and animals.
• The first miRNA (lin-4) was discovered in [Link] in
the year 1993

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MECHANISM OF RNAi
INTEREFERENCE

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 Mechanism of SIRNA

dsRNA enter into RNAi pathway

Dicer proteins convert these dsRNA into small
RNA molecules (21-25nt) known as siRNAs
small interfering RNA’s
These siRNA then assemble into a complex
called RNA induced silencing complex known as
(RISC), It starts the unwinding
The siRNA strands then recognize the
complementary sequences on a particular mRNA
and bind to them
Cleavage of mRNA at the points where siRNA
binds.
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 Dicer
DICERProtein
PROTEINS

• RNAseIII like dsRNA

• Specific ribonuclease
• Involved in initiation of RNAi
• Digests dsRNA’s into uniformly sized small RNA’s
(siRNA’s)
• Dicer family proteins are ATP-dependent nucleases.
• These proteins contain an amino-terminal helicase
domain, dual RNAseIII domains in the carboxy-
terminal segment, and dsRNA-binding motifs

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• They also contain a PAZ
domain, which is thought to be
important for protein-protein
interaction.

• Dicer homologs exist in many

organisms including C. elegans,
Drosphila, yeast and humans

• Loss of dicer: loss of silencing,

processing in vitro

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 Risc complex

• RISC is a large (~500-kDa) RNA-multiprotein complex,

• Agronautt protein is the key player which triggers mRNA degradation
• e.g.
• – unwinding of double-stranded siRNA (Helicase !?)
• – ribonuclease component cleaves mRNA (Nuclease !?)
• – amplification of silencing signal (RNA-dependent RNA polymerase !?)
• cleaved mRNA is degraded by cellular exonucleases

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 RNAi in Plant Disease Management

• RNAi has rapidly gained favor as a “reverse

genetics”
• Tool to knock down the expression of targeted
genes in plants
• siRNAs-mediated RNA silencing (RNAi)
mechanism was used to trigger RNA silencing
by targeting pathogen genomes
• Target: multiple gene family members with a
single RNAi-inducing transgene

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• Transgenic plants (Resistant to pathogens)
• The application of RNAi to plant disease resistance focuses on
three approaches
Approaches to induce RNAi

Selection of RNAi targets

Pathogens targeted by RNAi

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 Methods to induce RNAi in
Plants

Micro-bombardment Agroinfilteration
• Microprojectile bombardment • In the method a suspension of
involves the coating of gold or Agrobacterium tumefaciens is
tungsten particles with DNA and injected into a plant leaf, where it
accelerating them at high velocity transfers the desired gene to
into target plant tissue plant cells
• Some particles will penetrate into • Speed and convenience
the nuclei of some of the cells,
where the DNA may integrate into
the plant's genome.

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 Virus induced gene silencing
(VIGS) • There are a number of viruses which
have been modified to silence the gene
of interest in a sequence-specific
• Virus-induced gene silencing (VIGS) manner.
is one of the reverse genetics tools • Advancement in methodologies
• Viral vectors carrying a target gene • Virus-derived inoculations are
fragment to produce dsRNA which performed on host plants using
trigger RNA-mediated gene silencing different methods such as agro-
are employed  infiltration and in vitro transcriptions.

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 Agroinfiltration

• 1st step: Introduction of a gene of interest to

a strain of Agrobacterium
• 2nd step: The strain is grown in a liquid culture 
• 3rd step: The liquid culture is suspended into a
suitable buffer solution.
• 4th step: This solution is then placed in a syringe
• 5th step: Syringe tip is pressed against the
underside of a leaf
Injected into the airspaces inside the leaf
through stomata

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 RNAi mediated resistance against
Viruses

• Remarkable contributions of Plant Virologists

• Transgenic Plants (Resistant against Viruses)
• The RNAi mediated gene silencing technology has been widely used in
producing virus resistant plants
• Many viruses have evolved to express VSR ( Viral silencing repressors)
proteins to counter host antiviral RNA silencing
• It prompted the virologists to use VSR as a target of engineering
resistance in plants

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The first attempt, by Niu et al. (2006)
• They expressed amiRNAs (based on an A. thaliana miR159 precursor) targeting
the sequence of two VSRs
1. P69 of the turnip yellow mosaic virus (TYMV)
2. HC-Pro of the turnip mosaic virus (TuMV)
• Transgenic plants expressing resistance to TYMV and TuMV
PVY and SCMV resistant potato and sugarcane plants were developed
respectively through siRNA technology by targeting capsid protein gene of
respective virus
Tabassum et al. 2011

Golden mosaic virus resistant bean

Ring spot virus resistant papaya

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 VIRUS TARGET REGION HOST
Poplar mosaic virus Gfp N . benthamiana

Barley stripe mosaic Virus Pds Barley , wheat

Bean pod mottle virus Pds Actin Soybean

Brome mosaic  Pds , actin 1 rubisco activase Barley , rice, maize

Pea early browning virus Pspds, uni, kor Pea

Potato virus X Pds, gfp Potato, N. benthamiana

Satellite tobacco mosaic virus Several genes Tobacco

Tobacco mosaic virus Pds, psy Tobacco, N. benthamiana

Tobacco rattle virus Rarl , EDS1, NPR1/NIM1, pds , N . benthamiana , Arabidopsis ,

rbcS , gfp tomato, Solanum species, Chilli
pepper , opium, poppy

Tomato bushy stunt virus Gfp N. benthamiana

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 Fungal Resistance

• Little progress.
• Unlike plant viral pathogens, which replicate and propagate inside of
the infected plant cells
• Fungal interactions require a highly specialized cell called a haustorium
• Act as a interface for signal exchange as well as nutrient uptake
• This close contact of the interaction partners might also facilitate the…
• Uptake of dsRNA or siRNA by fungi from the host plant cells
• RNA silencing-mediated resistance via degradation of mRNA
sequences of fungal pathogen

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• The Host-induced gene silencing (HIGS) of fungal genes was
recently obtained for the barley powdery mildew Blumeria
graminis, a biotrophic fungal pathogen
• Via transgenic expression of the dsRNA directed against B.
graminis target transcripts in barley, a significant reduction of
disease symptoms of a B. graminis infection was observed

PATHOGEN TARGET OUTCOME

Magnaporthae oryzae eGFP Sequence specific

degradation of mRNA
Cladosporium Falvum cgl 1 and cgl 2 Blocking disease infection
spread
Blumeria graminis Mlo   Immunity

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 Bacterial Resistance

• Silencing of two bacterial genes (iaaM and ipt)

• Result: Decreased production of crown gall tumors
(Agrobacterium tumefaciens) to nearly zero in Arabidopsis
Escobar et al. 2001.

Resistance to crown gall disease could be engineered

in trees and woody ornamental plants.

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 Nematode Resistance

• Transgenic tobacco having dsRNA targeting two

Meloidogyne (root knot) nematode genes provided more than
95% resistance to Meloidogyne incognita.
(Yadav et al., 2006)
• Arabidopsis plants expressing dsRNA for a gene involved in
plant–parasite interaction (16D10)
• Suppressed formation of root galls by Meloidogyne
nematodes and reduced egg production.
(Huang et al., 2006)

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Nematode Target Region RNAi effect
M. incognita Cysteine proteinase Delayed development, Decrease in established
nematodes population
Dual oxidase Decrease in established nematodes population and
fecundity 
Splicing factor Reduction in gall formation and Female nematode
integrase Population 
Secreted Peptide Reduction in gall formation and established
16D10 nematode Population
H. glycines Cysteine proteinase Increased male: female ratio
C-type lectin Reduction in established nematode population
Major sperm protein Reduction in mRNA level

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 Advantages of RNAi

• High degree of specificity- It is highly specific to the mRNA

• Highly potent and effective- Only a few dsRNA molecules per

cell are required for effective interference

• Systemic silencing- The interfering activity can cause

interference in cells and tissues far from the site of introduction

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 Limitations of RNAi

• i. Non specific effect –

• Nonspecific effects can also occur.
• These effects are concentration dependent
• siRNA used at lower concentrations, can reduce the concentration-dependent
nonspecific side effects.
• ii. Off target effect –
• siRNA have a similar cellular machinery with miRNA
• For the loose homology requirements for activation of miRNA function
• siRNA can works as a miRNA even in a low concentration.

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 Future prospects

• A lot of research is to be conducted by use of RNAi due to the specificity of

RNAi in silencing target genes.
• Research should be conducted to design specific siRNA against specific
targets
• An additional future application of RNAi includes inhibition of viral
infections.
• New generation RNAi and miRNA should be developed.

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 Conclusion

• A powerful tool in antiviral research

• RNA silencing efficacy are the results of interaction between many
factors, including sequence similarity, target selection and
environmental temperature
• Difficult to accurately predict the resistance efficacy.
• Therefore, further scientific research is required to uncover the
factors affecting RNA silencing-mediated resistance in specific cases
• Test of resistance efficacy in field
• Eco friendly, bio safe and evergreen technology

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