Welcome

Biotechnology and Plant Disease control – Role of

RNA InterferencePresented By:- SANDESH V. PAWAR

Department of Plant Pathology

College of AgricultureDr. Balasaheb Sawant Konkan Krishi

Vidyapeeth, Dapoli-415 712

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

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.

INTRODUCTION

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.

Mechanism0f RNAi

RNA interference (RNAi)dsRNA

siRNA

Dicer

RISC

AAAAcapmRNA

AAAAcapmRNA

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.

DicerDicer 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

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.

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”.

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.

RNA Interference Approaches

• Four types of responses induced by dsRNA

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)

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.

Agroinfiltration

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.

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.

Micro Bombardment

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.

Fig: Construction of Micro-Bombardment

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)

Virus Induced Gene Silencing

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)

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)

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.

RNAi in Plant Disease

Management

Management of Plant Pathogenic

Fungi

• 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)

• 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

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.

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.

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.

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.

Management of Plant Pathogenic Bacteria

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.

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)

Management of Plant Pathogenic Virus

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

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.

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

• 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.

REFERENCESH. 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.

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