RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression. Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.

2. Presented By:-
SANDESH V. PAWAR
Department of Plant Pathology
College of Agriculture
Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth,
Dapoli-415 712

3. Contents:-
 Introduction
 History
 Mechanism of RNAi
 Methods to Induce RNAi in Plants
------ Agroinfiltration
------ Micro- Bombardment
------ Virus Inducing Gene Silencing (VIGS)
 RNAi in Plant Disease Management
------ Management of Plant Pathogenic Fungi
------ Management of Plant Pathogenic Bacteria
------ Management of Plant Pathogenic Viruses
 Conclusion and Future Prospects

4.  Plant diseases are a threat to world agriculture. The
losses of the crop due to the attack of pathogen.
 Disease management strategies mainly focus on use of
chemicals or use of disease resistant cultivar which are
developed through conventional breeding techniques.
 During the past few decades, breeding possibilities have
been broadened by G.E and gene transfer technology.
 Transcriptomics, Proteomics, and Metabolomics are
now proved to be in understanding plant metabolic
pathways.

5.  During last decade, the RNA mediated function has been
greatly increased with a discovery of small non-coding
RNAs which play a key role in a process called RNA
Silencing.
 RNAi has revolutionized the possibilities for creating
custom “Konck-downs” of gene activity.
 RNAi operates in both plant and animals.
 Biotechnologist have also used ds RNA’s as a trigger to
targets the homologous mRNA for degradation.
 It has emerged as a method of choice for gene targeting
virulence gene in fungi, viruses, bacteria and plants.

7. RNA interference (RNAi)
dsRNA
siRNA
Dicer
RISC
AAAAcapmRNA
AAAAcapmRNA

8.  In RNAi dsRNA is recognized by an RNase III family
member (i.e. Dicer) and cleaved into siRNAs of 21–
24 nucleotides (Agrawal et al. 2003).
 These siRNAs are incorporated into an RNAi
targeting complex known as RISC, which destroys
mRNAs homologous to the integral siRNA
(Bernstein et al. 2001).
 The target mRNA is cleaved in the center of the
region complementary to the siRNA (Elbashir et al.
2001), cause rapid degradation of the target mRNA
and decreased protein expression.

9. Dicer
Dicer is RNase III family endo-ribonuclease that cleaves
dsRNA into short double-stranded RNA fragments called
siRNA about 21-24 nucleotides long, usually with a two-base
overhang on the 3' end.
Dicer facilitates the formation of the RISC, whose catalytic
component argonaute is an endonuclease capable of degrading
mRNA.
It functions to generate siRNA molecules and loading one of
the two siRNA strands into RISC complex

11. siRNA
 Known as short interfering RNA, they are produced as a
part of RNAi pathway by the enzyme Dicer.
 This is a short double-strand of RNA (dsRNA) with 2-nt
overhangs on either end, including a 5' phosphate group
and a 3' hydroxy (-OH) group.
 They can also be exogenously (artificially) introduced to
bring out knockdown of a particular gene.

13. RISC
 The siRNAs are incorporated into the (RISC) which
consists of an Argonaute (Ago) protein as one of its
main components.
 The Argonaute protein is considered
as the catalytic engine of the RISC
 Ago cleaves and discards the passenger (sense)
strand of the siRNA duplex leading to activation of
the RISC.
 Ago cuts mRNA targets guided by siRNA via its
endonuclease nicknamed “slicer”.

14. The active RISC then targets the homologous transcript by
base pairing interactions and cleaves the mRNA ~12
nucleotides from the 3' terminus of the siRNA and destroys
the cognate RNA.

16. RNA Interference Approaches
• Four types of responses induced by dsRNA

18.  In this system, a number of methods for delivery of dsRNA
or siRNA into different cells and tissues include
transformation with dsRNA forming vectors for selected
gene by an Agrobacterium mediated transformation (Chuang
and Meyerowtiz, 2000; Waterhouse et al., 2001).
 Delivery cognate dsRNA of uidA GUS (β- glucaronidase)
and TaGLP2a:GFP reporter genes into single epidermal
cells of maize, barley and wheat by particle bombardment
(Schweizer et al., 2000)

19.  Introducing a Tobacco rattle virus based vector in tomato
plants by infiltration (Liu et al., 2002).
 Delivery of dsRNA into tobacco suspension cells by
cationic oligopeptide polyarginine- siRNA complex;
infecting plants with viral vector that produce dsRNA
(Tang et al., 2006).
 Among these the commonly used methods are
agroinfiltration, micro-bombardment, and VIGS.

21.  The injection of the Agrobacterium carrying similar DNA
construct into the intracellular spaces of leaves for
triggering RNA silencing is called Agroinoculation or
Agroinfiltration. (Hily and Liu, 2007).
 It is use to initiate systemic silencing.
 In plants cytoplasmic RNAi they can acts as similar to that
of T-DNA vector.

22.  In this method dissecting the mechanism of gene silencing
especially concerned with is suppressors, systemic
silencing signal and also for simple protein purification.
(L. K. Johansen and F. Tenllado, 2003)
 They provide a rapid, versatile and convenient way for
achieving a very high level of gene expression in a distinct
and defined zone.

23. Micro
Bombardment

24.  Linear or circular template is transferred into nucleus.
 Synthetic siRNA are delivered into plants by biolistic
pressure.
 Bombarding particle are coated with dsRNA, siRNA or
DNA.
 The silencing effect of RNAi is detected as early as a day
after bombardment and it continues up to 3 to4 days of post
bombardment.

25. Fig: Construction of Micro-Bombardment

26.  Silencing occurs 2 weeks later and it is manifested by the
vascular tissues of the non targeted leaves.
 After one month the loss of GFP expression seen in non
vascular tissues.
 RNA blot hybridization with systemic leaves indicated that
the biolistically delivered siRNAs induce de novo formation
of siRNAs, which accumulated to cause systemic silencing.
(U. Klahre, nad P. Crete, 2002)

28. Modified viruses as a RNA silencing triggers are used as a
mean for inducing RNA in plants.
Different types of RNA and DNA viruses are modified for
this system, such as TMV, PVX, TRV. (M. H. Kumagai and
J. Donson. 1995)

29. All RNA virus derived expression vectors will not be useful
as silencing vectors.
Similarly, DNA viruses have not been used extensively as
expression vectors.
 Non mobile Maize streak virus (MSV) have been used
successfully production of protein. (M. H. Kumagai and J.
Donson. 1995)

30.  Using virus vectors as a gene silencing in plants require
cloning homologous gene fragments. This was
demonstrated in RNA virus by inserting sequence in TMV.
(M. J. Dallwitz and E. J. Zurcher. 1996)
 Phytoene desaturase (PDS) and Chalcone synthase
(CHS) were used as markers for gene silencing.

33. • It is used as reverse tool.
• Homology based gene silencing is used in many plant
pathogenic fungi.
• Hypermorphic mechanism- used in polyploid and
polykaryotic fungi.
• Simultaneous silencing of several unrelated genes by
introducing a single chimeric construct has been
demonstrated in case of Venturia inaequalis. (A. Fitzgerald
and J. A. Van Kha. 2004)

34. • Table 1. RNAi effects on targeted region
in some fungal plant pathogen
Pathogen Targeted
region
Result References
Magnaporthae
oryzae
eGFP Sequence specific
degradation of
mRNA
N. Kadotani,
H.
Nakayashiki
Cladosporium
fulvum
Cgl 1 and
Cgl 2
Blocking disease
infection spread
G. C. Segers,
W. Hamada
Venturia inaequalis Multiple
inverted
repeats
- A. Fitzgerald,
J. A. Van Kha
Fusarium
graminearum
- - H.
Nagayashiki
Blumeria graminis Mlo Immunity P. Schweizer,
J. Pokorny

35. Silencing in Cladosporium fulvum:
HCf-1 gene that codes for hydrophobin of C.fulvum,
which is co-suppressed by ectophic integration. (P.
Spanu, 1997)
The transcription rate of the HCf-1 in the co-suppressed
isolates are higher in the untransformed strain.
This is due to ectopic integration of transgene next to
promoters which initiate transcription.

36. Silencing in Venturia inaequalis:
Hairpin vector technology – Silencing GFP and THN
gene. (A. Fitzgerald 2004)
THN silenced transformants exhibited a distinctive light
brown phenotype and maintained the ability to infect
apple.
Frequency of silencing of both gene is 51% of all the
transformants.

37.  Silencing in Magnaporthae grisea:
 Using green florescent protein gene as a model. (N. Kadotani
and H. Nakayashuki, 1998)
In M.grisea plasmid construct expressing sense, antisense, and
hairpin RNA are introduced into an eGFP expressing
transformants.
The fluorescence of eGFP in the transformants are silenced and
the accumulation of eGFP mRNA are drastically reduced.
Later on a protocol for silencing the mpg1 and polyketide
synthase like gene.

38. Developing RNA silencing vector, pSilent-Dual1 (pSD1) that carries
two convergent promoters, the Aspergillus nidulans tryptophane
promotor (PtrpC) and the A. nidulans glyceraldehyde -3- phosphate
dehydrogenase promoters (pSD1).
This promoters are used for gene expressing in large number of
filamentous fungi.
The greatest merit of pSD1 system over others, such as hpRNA or
ihpRNA silencing system is that it allows a single step cloning for
generation of an RNAi construct.
The main bottleneck of this system is its lower silencing efficiency
compared with hpRNA or ihpRNA expressing RNA silencing vectors.

39. Management of Plant
Pathogenic Bacteria

40. One of the striking example of bacterial disease management with the
help of RNAi. Remarkable type of gene regulation was documented
by Escobar et al (2001)
Developed crown gall disease management with iaaM and ipt
oncogenes.
Transgenic plants Arabidopsis thaliana and Lycopersicon esculentum
showed resistance to crown gall disease.

41.  The incoming bacteria could not make the hormones needed to
cause tumour. (P. Dunoyer, and C. Himber, 2007)
 The natsiRNA (nat-siRNAATGB2) are strongly induced in
Arabidopsis upon infection by Pseudomonas syringae pv tomato
and down regulates a PPRL gene that encodes a negative regulator
of the RPS2 disease resistance pathway. (S. Katiyar- Agarwal, R.
Morgan, 2006)

43. Host system Virus Targeted
Region
References
N. benthamiana African
cassava mosaic
virus
Pds, su,
cyp79d2
I.B. Fofana,
A. Sangara
Barley and
Wheat
Barley stripe
mosaic virus
pds S. Holzberg,
C. Cakir
Barley, Rice,
Maize
Brome mosaic
virus
Pds, actin
1,rubisco
activase
X. S. Ding,
W. L. Schneider
Arabidopsis Cabbage leaf
curl virus
Gfp,
CH42, pds
M. A. Turnage,
N. Muangsan
Table No. 2: Effect of targeted region of RNAi in
various plant virus system

44. Salient features of RNAi
 Double stranded RNA rather than single-stranded
antisense RNA is the interfering agent.
 High degree of specific gene silencing with less effort.
 Highly potent and effective (only a few double stranded
RNA molecules per cell are required for effective
interference).
 Silencing can be introduced in different developmental
stages.
 Systemic silencing.
 Avoids problems with abnormalities caused by a knocked
out gene in early stages (which could mask desired
observations).
 Silencing effects passed through generations.

45. Conclusion and Future prospectus…
• RNAi and miRNA technologies of gene silencing are newly
developed genomics tools that have great advantages
techniques.
• RNAi technology can be considered an eco-friendly, biosafe
and ever green technology as it eliminates even certain risks
associated with development of transgenic
• Since RNAi triggers the formation of dsRNA molecules that
target and facilitate the degradation of the gene of interest as
well as the transgene itself to avoid problems arising from the
synthesis of gene sequences as well as non coding regions of
gene, thus limiting undesirable recombination events

46. • Future directions will focus on developing finely RNAi-
based gene silencing vectors that are able to operate in
a temporally and spatially controlled manner.
• However, a better and comprehensive understanding
of RNAi would allow the researchers to work effectively
and efficiently in order to improve crop plants
nutritionally and manage various crop plants diseases and
other maladies.

47. REFERENCES
H. Nakayashiki, “RNA Silencing in Fungi: Mechanisms and
Applications,” Federation of European Biochemical
Societies Letters, Vol. 579, 2005, pp. 5950-5970.
W. Hamada and P. D. Spanu, “Co-Suppression of the Hydrophobin
Gene Hcf-1 is Correlated with Antisense RNA Biosynthesis
in Cladosporium fulvum,” Molecular and General Genetics,
Vol. 259, 1998, pp. 630-638.
P. Spanu, “HCf-1, a Hydrophobin from the Tomato PathoGen
Cladosporium fulvum,” Gene, Vol. 93, 1997, pp.89-96.