RNAi – Mechanism and Its Application In Crop Improvement

Presented by:-

Jadhao Kundansingh R.

09ABT/11

Department of Agril-biotechnology,Orissa University of Agriculture & Technology,BBSR.

Overview

• Small RNA Family• Mechanism of action of RNAi

• Application of RNAi in crop improvement

• Case study I • Conclusions

Molecular Biology’s Central Dogma

DNA

RNA

protein

AAAA

mRNArRNAtRNA

other noncoding RNAs

RNA Family

scnRNA

Constituent RNAs

tRNA

Regulatory RNAs

rRNA

Non-coding RNAs

RNA

mRNA

Coding RNA

miRNA(stRNA)

siRNA snoRNA

Types of small silencing RNAsName Organism Length

(nt)Proteins Source of trigger Function

miRNA Plants,algae,animals,viruses, protists

20-25 Drosha (animals only) + Dicer

Pol II transcription (pri-miRNAs Regulation of mRNA stability,Translation

rasiRNA Plants 24 DCL3 Transposons, repeats Chromatin modification

tasiRNA Plants 21 DCL4 miRNA-cleaved TAS RNAs Post transcriptional regulation

Exo -siRNA Animals, fungi, protists, plants

21-24 Dicer Transgenic, viral or otherexogenous dsRNA

Post transcriptional regulation,antiviral defense

Endosi RNA Plants,algae,animals,fungi, protists

21 Dicer Structured loci, convergent and bidirectional transcription, mRNAs paired to antisense pseudogene transcripts

Post transcriptional regulation oftranscripts and transposonsTranscriptional gene silencing

piRNA germ line Drosophila melanogaster,mammals, zebrafish

24-30 Dicer- independent Long, primary transcripts Transposon regulation, unknownfunctions

piRNA like Drosophila melanogaster

24-30 Dicer- independent In ago2 mutants in Drosophila

Unknown

21U-RNA piRNAs Caenorhabditis elegans

21 Dicer- independent Individual transcription of eachpiRNA

Transposon regulation ,unknownfunctions

26G RNA Caenorhabditis elegans

26 RdRP Enriched in sperm Unknown

What is RNAi?

• RNA interference (RNAi) is an evolutionally highly conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.

• It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegans and later found in a wide variety of organisms, including mammals. 

RNAi like phenomena

•Plants•Petunias

•Fungi•Neurospora

•Animals•Caenorhabditis elegans

Alternate terms to RNAi

• PTGS (Posttranscriptional Gene Silencing)

• Cosuppression

• Quelling

• Virus-induced gene silencing

HISTORY OF RNAi

The discovery of RNA interference

• An Unexpected Result…

• petunias surprisingly developed areas of hypopigmentation when transduced with the gene encoding an enzyme required for pigment synthesis.

1990-Petunias

• Napoli et al. defined an RNAi-like phenomenon and called it “cosupression.”

• chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, the rate-limiting enzyme in anthocyanin biosynthesis, responsible for the purple coloration.

1992-The mold

• Carlo Cogoni and Guiseppe Macino of the Università di Roma La Sapienza in Italy introduced a gene needed for carotenoid synthesis in the mold Neurospora crassa:

• The introduced gene led to inactivation of the mold's own gene in about 30% of the transformed cells. They called this gene inactivation "quelling."

A rosette of the asci

• The answer actually came in the year 1988 from researchers working on C. elegans

Potent and genetic interference by ds RNA in C. elegans

Fire.A,Xus,Montogomery ,Mk Kosta SA Driver SE ,Mello CG 1998 Feb 19.391:806-11.

Double stranded RNA Posses Puzzle

Wagner RW, Sun.L Nature 1988 19,744-5

Unc-22 (Uncoordinated 22)• Codes for a non essential myofilament• It is present several thousand copies/cell

Unc-22 phenotype

• 4-6 hours after injection, eggs collected.

• Screened for phenotypic changes• twiching

Exon Size RNA Phenotype

Exon 21-22 742 SenseAntisenseSense+antisense

WildtypeWildtypeTwicher (100%)

Exon 27 1033 SenseAntisenseSense+antisense

WildtypeWildtypeTwicher (100%)

Timeline for RNAi Dicsoveries

RNA Interference

Attempting to use antisense RNA to knock down gene expression, they found synergistic effects on gene silencing when antisense and sense RNA strands where delivered together,

this phenomenon is later termed as

Nobel Prize in Physiology or Medicine 2006

• Targetted disruption of gene function in Drosophylla by RNA interference : a role for nautilus in embryonic somatic formation. L.Misquitta,B.Paterson,proc.Nat. Acad .Sci,USA,1999.96-1451-1453.

• Double stranded RNA induces m-RNA degradation in Trypanosoma bruci. H.Nigo,C.Tschudi,K.Gull.Proc.Nati.Acad.Sci.USA.1998,95,14687-14692

•Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.

R.Water house,M.GrahamM.wang proc.Nat. Acad .sci,USA,1998.95,13959-13964.

It was shown that plants contain an enzyme RNA dependent RNA polymerase .It was responsible for the synthesis of ds RNA in presence of high level of m-RNA

RELOOKING INTO 1990

Dicer

Double-stranded RNA triggers processed into siRNAs

by enzyme RNAseIII family, specifically the Dicer family

Processive enzyme - no larger intermediates.

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.

Contd…..

They can 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

Developmental consequence in Drosophila and

C. elegans

RISC complex

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

triggers mRNA degradation in response to siRNA

some components have been defined by genetics, but function

is unknown, 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

THE SILENCING MECHANISM

• Two-step model to explain RNAi.• I. dsRNA is diced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs (siRNAs).• duplexes of 21 23 nucleotides bearing

two-nucleotide 3' overhanging ends. • II. siRNAs are transferred to a second enzyme complex, designated RISC for RNAi-induced silencing complex.The siRNA guides RISC to the target mRNA, leading to its destruction.• the antisense strand of the siRNA is

perfectly complementary

AAAA

RNAi is mediated by small (~21-25 nucleotide) noncoding RNAs

complementary to the targeted gene

cleavage oftargeted mRNA(siRNA)

inhibitsprotein translation or causes mRNA degradation(miRNA)

mRNA:

dsRNAintermediate

Application of RNAI in crop improvement

Crop quality traits : Sunilkumar et al., 2006. reduced the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinene synthase gene in the gossypol biosynthesis pathway.

Virus resistance : the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinene synthase gene in the gossypol biosynthesis pathway.

Protection from insect pests : Baum et al. 2007. showed that silencing of a

vacuolar ATPase gene (V-type ATPase A gene) in midgut cells of western corn rootworm (WCR) led to larval mortality and stunted growth.

Researchers identified a cytochrome P450 monooxygenase (CYP6AE14) gene important for larval growth expressed in midgut cells with a causal relationship to gossypol tolerance.

Transgenic tobacco and Arabidopsis producing CYP6AE14 dsRNA were fed to larvae, successfully decreasing endogenous CYP6AE14 mRNA in the insect, stunting larval growth and increasing sensitivity to gossypol.

Cont…

Nematode resistance : Yadav et al., 2006. showed transgenic

tobacco having dsRNA targetting two Meloidogyne (root knot) nematode genes had more than 95% resistance to Meloidogyne incognita.

Huang et al., 2006. showed that Arabidopsis plants expressing dsRNA for a gene involved in plant–parasite interaction (16D10) had suppressed formation of root galls by Meloidogyne nematodes and reduced egg production.

Bacterial and fungal risistance : Little progress.Escobar et al. 2001. showed that silencing of two bacterial genes (iaaM and ipt) could decrease the production of crown gall tumors (Agrobacterium tumefaciens) to nearly zero in Arabidopsis, suggesting that resistance to crown gall disease could be engineered in trees and woody ornamental plants.

Application Case study authors

the level of lysine Reduction of lysine catabolism and improving seedgermination generating a dominant high-lysine maizevariant by knocking out the expression of the 22-kDmaize zein storage protein

Zhu et al., Tang et al., Segal et al.

Barley and Rice Resistance of barley to BYDV and producing a ricevariety called LGC-1 (low glutenin content 1) byRNAi technology

Wang et al., Kusaba et al.Williams et al.

Banana Production of banana varieties resistant to the BananaBract Mosaic Virus (BBrMV) by RNAi

Rodoni et al.

Cotton Transgenic cotton plants expressing a RNAi constructof the d-cadinene synthase gene of gossypol synthesisfused to a seed-specific promoter caused seed-specificreduction of Gossypol

Sunilkumar et al.

Jute.

Generating jute varieties with low lignin content byRNAi technology

Williams et al.

Lathyrus sativus RNAi construct designed to silence the genesencoding the two starch-branching isozymes ofamylopectin synthesisRNAi technology can be used tosilence the gene(s) responsible for production ofBOAA

Regina et al

Tomato RNAi-mediated suppression of DET1 expressionunder fruit-specific promoters has recently shown toimprove carotenoid and flavonoid levels in tomatofruits with minimal effects on plant growth

Williams et al

Coffee RNAi technology has enabled the creation of varietiesof Coffee that produces natural coffee with low orvery low caffeine content

Davuluri et al.

TraitTarget Gene Host Application

Enhancednutrient content

Lyc Tomato Increased concentration of lycopene (carotenoid antioxidant)

DET1 Tomato Higher flavonoid and b-carotene contents

SBEII Wheat, Sweet potato, Maize Increased levels of amylose for glycemic management and digestive health

FAD2 Canola, Peanut, Cotton Increased oleic acid content

SAD1 Cotton Increased stearic acid content

ZLKR/SDH Maize Lysine-fortified maize

Reduced alkaloid production

CaMXMT1 Coffee Decaffeinated coffee

COR Opium poppy Production of non-narcotic alkaloid, instead of morphine

CYP82E4 Tobacco Reduced levels of the carcinogen nornicotine in cured leaves

Heavy metalaccumulation

ACR2 Arabidopsis Arsenic hyperaccumulation for phytoremediation

Reduced polyphenolproduction

s-cadinene synthase gene

Cotton Lower gossypol levels in cottonseeds, for safe consumption

Ethylenesensitivity

LeETR4 Tomato Early ripening tomatoes

ACC oxidase gene Tomato Longer shelf life because of slow ripening

Reducedallergenicity

Arah2 Peanut Allergen-free peanuts

Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass

Reduced production of lachrymatory factor synthase

lachrymatory factor synthase gene

Onion "Tearless" onion

CASE STUDY

RNAi-mediated disruption of squalene synthase improves drought tolerance and yield in riceLakshmi P. Manavalan et.al.2012

A. Construct containing PMI marker gene under the control of Act1 promoter and SQS gene with maize UBI1-13 promoter. (LB: left border; RB: right border)B. PCR amplification of SQS in transgenic RNAi lines (lane 1 and 2) with WT in well 1. Lane 3 and 4 represent the PCR amplification of genomic DNA for internal control gene CBR (cytochrome b5 reductase) C. RT-PCR analysis of abundance of endogenous SQS transcript in WT and three independent RNAi lines. Equal amounts of RNA was extracted from WT and RNAi lines and were used to synthesize 1st strand cDNA. 3ll of this cDNA was amplified using gene-specific primers (see materials and methods for details) for SQS D. RT-PCR analysis of rice ubiquitin5 gene with 3ll template was used as the housekeeping control

Seedling development of SQSRNAi lines under ABA treatment.A. Event D2 (top) and WT grown in different concentrations of ABA (0, 3, 5,7, 10 lmol ABA) B. Lateral root growth in 3 events three days after sowing (DAS) C&D.WT and transgenic line D2-2 grown in agar plates infused with 0 and 5 lmol ABA 7DAS E, F, G. growth, root length and lateral root number of 3 events in 0 and 3lmol ABA H. Reduced SQS gene expression on root tissues of transgenic event D2 in 0 lmol ABA Lanes 2,3,6,7: internal controls Act, ElFa, for WT and D2 Lanes 5 and 9 SQS in WT and D2 4 and 8 no DNA* significantly different (p<0.05 level) from WT and bars followed by different letters differ significantly at p<0.05 level.

Improved drought resistance of SQS RNAi rice at vegetative stage.A. Transgenic plants showing less drying at 15 days after drought. B. Recovery after 32 days drought and 2 days after rewatering C. Reduced water loss of transgenic plants depicted by weightof soil+plant D. Survival rate and number of green leaves retained by transgenics in comparison with WT and negative controls.

Response of RNAi lines to reproductive stage drought. A. morphology at 23 days after withholding water B. Recovery 5 days after rewatering (arrows indicate flowering) C. grain yield (filled grain weight) per plant of different treatments/events D. Percentage reduction of grain yield over control (well-watered) * indicates significantly higher yield than WT under WS at p<0.05 level.