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.