1

Jari Valkonen

RNA SILENCINGas a tool in plant research

and plant protection

Jari Valkonen

Department of Applied BiologyUniversity of Helsinki

Faculty of Agriculture and ForestryFaculty of Agriculture and Forestry

University of HelsinkiUniversity of Helsinki

SBLSBL--KPATKPATPlant Pathology GroupPlant Pathology Group

Jari Valkonen

Acknowledgements

Department of Applied Biology, Helsinki

- Wilmer Cuellar- Marjo Ala-Poikela- Tuuli Haikonen

Genetics Center, SLU, Uppsala

- Eugene Savenkov- Xiandong Li- Anna Germundsson- Elin Gammelgård- Andrey Zamyatnin

International Potato Center, Peru

- Jan Kreuze

SBLSBL--KPATKPATPlant Pathology GroupPlant Pathology Group

Faculty of Agriculture and ForestryFaculty of Agriculture and Forestry

University of HelsinkiUniversity of Helsinki

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Jari Valkonen

1- RNA silencing and transgenic plants

2- Using RNA silencing without transgenic plants

• Agro-infiltration• Virus-induced gene silencing (VIGS) as a research tool

3- Viruses are targets of RNA silencing-based host defence

4- Suppression of RNA silencing by viruses

Contents:

Jari Valkonen

Cellular roles of RNA silencing

- Defence against VIRUSES

- Control of transposable elements

- Control of other endogenous non-coding RNAs

- Transcriptional silencing, formation of heterochromatin

miRNAs (micro-RNA)-Controls many biological processes, including development

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Jari Valkonen

RNA silencing

Synonyms:

• co-suppression• antisense-mediated gene silencing• post-transcriptional gene silencing (PTGS)• quelling• RNA interference (RNAi)

The mechanism targets and destroys harmful, unnecessaryor inadvertently expressed RNA molecules in a sequence-specific manner, no matter if they are of cellular or viralorigin. It does not just “interfere” with gene expression or“silence genes”.

Jari Valkonen

1.RNA SILENCING

andTRANSGENIC PLANTS

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”Co-suppression”Beginning of a series of unexpected results on transgenic plants

Petunia plants were transformed with the chalcone synthase geneto enhance pigmentation of flowers.

However, transgenic plants lost expression of the endogenous, homologous geneand developed unpigmented flowers

• Napoli C, Lemieux C, Jorgensen R (1990) Plant Cell 2, 279• Van der Krol A, Mur L, Beld M, Mol J, Stuitje A (1990) Plant Cell, 2, 291

Jari Valkonen

Chalcone

Phenylalanine

Cinnamate

4-Coumarate

4-Coumaroyl-CoA

Naringenin

Dihydrokaempherol

Leucopelargonidin

Pelargonidin

Pelargonidin-3-O-glucoside

Pelargonidin-3-O-rutinoside

Dihydroquercetin

Leucocyanidin

Cyanidin

Cyanidin-O-glucoside

Cyanidin-O-rutinoside

PAL

C4H

4CL

CHS

CHI

F3HF3'H

DFRDFR

ANSANS

3GT 3GT

RT RT

Biosynthesis ofanthocyaninsin Gerbera

(c) Teemu Teeri

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Anthocyanin biosynthesis is blocked in transgenicGerbera which harbors its own CHS gene is in an

antisense orientation

Phenylalanine

Cinnamate

4-Coumarate

4-Coumaroyl-CoA

PAL

C4H

4CL

CHS

(c) Teemu Teeri

35S

CHS

Jari Valkonen

Transformation of Gerbera with antisense forGGLO1 (a B class MADS box gene) converts thebrilliant red petals into greenish sepal like organs.

MADSMADS--box genes and organ identity in Gerberabox genes and organ identity in Gerbera

Yu et al.1999:Plant J. 17, 51-62.

control transformantB

A C

sepa

lpe

talan

ther

carp

el

A C

sepa

lse

pal

carp

elca

rpel

sepal

petal

anthercarpel

(c) Paula Elomaa

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Jari Valkonen

Smith CJS, Watson CF, Morris PC, Bird CR, MorrisPC, Schuch W, Grierson D 1988,

Antisense RNA inhibition of polygalacturonasegene expression in transgenic tomatoes.

Nature 334: 724-726

The Flavr SavrTM tomato of Calgene wasintroduced to the market in USA in 1994.

Commercial applications of RNA silencing - 1

An antisense gene construct for the inhibition of fruit ripening and softening,by preventing production of polygalacturonase, one of the most abundant proteinsin ripe tomato fruit

Calgene was later bought by Monsanto.Flavr Savr tomatoes were withdrawn from the market in 1997.

Jari Valkonen

Commercial utilisation of RNA silencing – 2Transgenic resistance to viruses

Powell-Abel P, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN(1986). Delay of disease development in transgenic plants that express thetobacco mosaic virus coat protein gene. Science 232, 738-743

Sanford & Johnston (1985) The concept of parasite-derived resistance: derivingresistance genes from the parasite´s own genome. J Theor Biol 113: 395-405

”Interfere with the pathogenicity cycleby causing the host to express pathogen gene

at the wrong time, in the wrong amount, or in a counterfunctional form”

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Jari Valkonen

Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, SandersP & Tumer NE (1990) Engineering resistance to mixed virusinfection in a commercial potato cultivar: Resistance to potatovirus X and potato virus Y in transgenic Russet Burbank.Biotechnology 8:127-134

Kaniewski W, Lawson C, Sammons B, Haley L, Hart J, DealnnayX & Tumer NE (1990) Field resistance of transgenic RussetBurbank potato to effects of infection with potato virus X andpotato virus Y. Biotechnology 8: 750-754.

Commercial applications of RNA silencing in plant protection:virus-resistant transgenic potatoes

Reduced growth frominfected seed tuber

PVY-infected Healthy

Bintje

Significant virus disease problemsdue to vegetative propagation(cloning):

• potato• sweetpotato• cassava• garlic, onion• berry plants• fruit trees

Jari Valkonen

Milestones in discovery of RNA silencing

Transgenic plants that express viral sequences and are virus-resistant

Lindbo A, Silva-Rosales L, Proebsting WM, Dougherty WG (1993) Induction of a highly specificantiviral stage in transgenic plants: implications for regulation of gene expressionand virus resistance. Plant Cell 5, 1749-1759 (cited 325 times / Sept. 4, 2005)

Smith HA, Swaney SL, Parks TD, Wernsman EA, Dougherty WG (1994) Transgenic plant virusresistance mediated by untranslatable sense RNAs: expression, regulationand fate of nonessential RNAs. Plant Cell 6, 1441-1453

Goodwin J, Chapman K, Swaney S, Parks TD, Wernsman EA, Dougherty WG (1996)Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.Plant Cell 8, 95-105

• Untranslatable and antisensetranscripts confer resistance

• Transgene is actively transcribed butonly low levels of mRNA accumulate

• Resistance specific to the virus fromwhich the transgene sequence wasderived

à Non protein-mediated

à Post-transcriptionaldegradation of mRNA

à Sequence-specific

Observation: Conclusion on the mechanism:

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Jari Valkonen

Milestones in discovery of RNA silencing

The model by William Dougherty (1995)

“…many of these studies and their unexpected results may be telling us something newabout cellular gene expression. For the sake of argument, it is this latter explanationthat we develop in this article.

If one embraces this concept, then it may be possible to link many seemingly unrelatedbiological phenomena as manifestations of a common regulatory system.

If this turns out to be the case, then an 'underworld' of small RNA moleculeswill negatively regulate many aspects of gene expressionand transgenic organisms will provide a window to view this system.”

Dougherty WG & Parks TD (1995) Transgenes and gene suppression: telling us something new?Current Opinion in Cell Biology 7, 399-405

Jari Valkonen

Milestones in discovery of RNA silencing

Dougherty & Parks (1995) conceptualised the mechanism differently from others,taking the early examples of natural antisense-mediated regulation of endogenous gene expressionto an account (first example from 1984: Izant & Weintraub, Cell, 36, 1007-1015).

The model (1995) included: Experimental approval:

-dsRNA The sole inducer (Caenorhabditis elegans);Fire et al, 1998: Nature 391:806-810 (cited 2026 times)

- Endogenous RNase III ‘Dicer’ (Drosophila); Bernstein et al, 2001: Nature 409, 363-366

- Hairpin structures of Micro-RNA (miRNA) in plants; Llave et al, 2002: Science 297, 2053-2056endogenous RNA

- Small endogenous antisense A small temporal RNA, let-7, processed by ’Dicer’ and theRNAs (21 nt or 69 nt) RNA silencing machinery;(e.g., lin-4 of C. elegans; Hutvágner et al, 2001: Science 293, 834-838Lee et al, 1993: Cell, 75, 843-854)

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Milestones in discovery of RNA silencing

Dougherty & Parks 1995 (continues...)

The model included: Experimental approval:

- Helicase Fungal QDE-3 (Neurospora crassa);Cogoni & Macino 1999: Science 286, 2342-2344

- Endogenous RNA-dependent Fungal QDE-1 (N. crassa); Cogoni & Macino 1999: Nature 399, 166-169RNA polymerase (RdRp)

-“Interfering small RNA” siRNA (in plants); Hamilton & Baulcombe 1999: Science 286, 950-952

Nuclease fraction from Drosophila contains siRNA specific tothe inducing dsRNA: RNA-induced silencing complex, RISC(Hammond et al, 2000: Nature 404, 293-296)

Jari Valkonen

RNA silencing

siRNA - small interferring dsRNA (effector), 21-25 nt.Contain 2-nt 3’ overhangs and a 3’ hydroxyl group.Prime sequence-specific RNA degradation.

dsRNA: inducer

RNase III DICER (DCL; multidomain protein):RNase III domains bind and cleave dsRNAto produce siRNA

Scans the cell for homologousRNA molecules for degradation

RISC RISC incorporates Dicer and the (-)strandof siRNA

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2.USING RNA SILENCING

WITHOUT TRANSGENIC PLANTS

Jari Valkonen

Instead of ”antisense RNA silencing”, endogenous genes could be silenced byadministrating homologous, short dsRNA to the nematode

Fire et al. 1998, Nature 391: 806-811“Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans”

Administration of dsRNA (or any RNA) to plants is difficult technically.However, endogenous production of dsRNA from a ”hairpin” construct results inefficient silencing of homologous transcripts.

Waterhouse et al. 2000, Nature 407: 319-320

Technical steps forward in using RNA silencing

11

Jari ValkonenWaterhouse et al. 2000, Nature 407: 319-320

”Hairpin-RNA” for efficient induction of RNA silencing

Jari Valkonen

dsRNA-activatedproteinkinase(PKR)

Binding todsRNA PKR-

PKR(dimer)

Autophos-phorylation

ActivatedPKR

Replicatingviral RNA

eIF2

Phosphorylation

Shut-down ofprotein synthesisCell deathReplicating

viral RNA

Interferon-inducedviral defence inmammals

Operates also in plantsBilgin et al. 2003. Devel. Cell 4:651-661

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USING RNA SILENCING WITHOUT TRANSGENIC PLANTScontinues....

Local and systemic silencing

Techniques:

Agro-infiltration

Virus-induced gene silencing (VIGS)

à How does silencing spread in plants?

Jari Valkonen

Systemic movementof TMV-GFP

Site of inoculation

Kreuze et al. 2005,J Virol 79:7227-7238

(Tobacco mosaic virus, a gene vector)

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Jari Valkonen Plant Cell 14, Suppl. 2002, S303

Vascular tissueand plasmodesmalconnectionsbetween cellsin plants

Jari Valkonen

AGROINFILTRATIONto induce silencing in leaf of an

endogenous gene (in this experimentalcase transgenic plant that expresses

green fluorescent protein, GFP)

GFP silencing

hpGFP

HairpinRNA,

inducer

hpGFPAgrobacterium

+GFP

Fluorescence disappears

Signaling for RNA silencing in plants

Agroinfiltration method:

Kapila et al. 1997, Plant Sci. 122:101-108.

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Jari ValkonenVoinnet 2005, Nature Rev. Genet 6:206-220

Agro-infiltration

Jari Valkonen

Local and systemic signaling for RNA silencingstudied in GFP-expressing transgenic plant by agroinfiltration

Himber et al. 2003, EMBO J. 22: 4523-4533 • Palaqui et al. 1997, EMBO J 16: 4738-4745(systemic signal exists)

• Yoo et al. 2004, Plant Cell 16: 1979-2000(transport of siRNA in phloem sap)

• Huang et al. 2005, Science 309: 1694-1696(mRNA is transported in phloem to actin other parts of the plant)

Kreuze et al. 2005, J. Virol. 79:7227-7238

Local spread of silencing signal(red ”borders”)

Anna Germundsson

Systemic GFP silencing

Infiltration

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Jari Valkonen

VIRUS-INDUCED GENE SILENCING (VIGS)

Commonly used (in plant research) for silencing cellular genes

Potato virus X vector, (+)ssRNA virus or any host geneor fragment thereof

Kumagai et al. 1995, PNAS 92:1679-1683

Jari Valkonen

How to obtain silencing-inducing siRNA from thevector virus?

Cellular RNA-dependent RNA polymerase (RdRp)

•During (tombus)virus infection in plants, siRNA is generated from many but not all partsof the viral RNA genome; mostly from positions that form imperfect hairpinsSzittya et al. 2002, Plant Cell 14:369-372

•QDE-1 of Neurospora crassa synthesises template-long copies of the RNA moleculeor 9-21 nt long oligomers from any position of the templateMakeyev & Bamford 2002, Mol. Cell 10, 1417-1427

• Arabidopsis RDR2 is needed for synthesis of siRNA (Xie et al. 2004, PLoS Biol. 2, E104)

•Amplification of siRNA using the (-)strand as a primer à spread of silencing(plants and nematodes)

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Jari Valkonen

VIRUS-INDUCED GENE SILENCING (VIGS) continues...

Potato virus X vector, (+)ssRNA virus or any host geneor fragment thereof

1. Post-transcriptional silencing:

• Virus replicates to high titres in cytoplasm• Virus-derived siRNAs accumulate (including the added sequences), which targets the virusand the homologous host mRNA to degradation in cytoplasm

• The encoding region of the gene may ultimately become methylated (in the nucleus)

2. Transcriptional silencing:

If the gene promoter sequence was expressed from the vector virus (in cytoplasm),promoter becomes methylated (in nucleus)

Jari Valkonen

Detection of mRNA by RT-PCR:

Lanes 1- 5 = PCR cycle numbers21, 24, 27, 30 and 35.

Virus-induced gene silencingin tomato fruit

Fu et al. 2005, Plant Journal 43: 299-308

Tobacco rattle virus was usedas a vector to silence LeEILs,a gene of the ethylene synthesispathway (ethylene is involved infruit ripening)

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Jari Valkonen

Park JA, Kim TW, Kim SK, et al.Silencing of NbECR encoding a putative enoyl-CoA reductase results indisorganized membrane structures and epidermal cell ablation in Nicotiana benthamianaFEBS LETTERS 579 (20): 4459-4464 AUG 15 2005

Hein I, Pacak MB, Hrubikova K, et al.Virus-induced gene silencing-based functional characterization of genesassociated with powdery mildew resistance in barley.PLANT PHYSIOLOGY 138 (4): 2155-2164 AUG 2005

Scofield SR, Huang L, Brandt AS, et al.Development of a virus-induced gene-silencing system for hexaploid wheatand its use in functional analysis of the Lr21-mediated leaf rust resistance pathwayPLANT PHYSIOLOGY 138 (4): 2165-2173 AUG 2005

Nasir KHB, Takahashi Y, Ito A, et al.High-throughput in planta expression screening identifies a class II ethylene-responsiveelement binding factor-like protein that regulates plant cell death and non-host resistancePLANT JOURNAL 43 (4): 491-505 AUG 2005

Park JA, Ahn JW, Kim YK, et al.Retinoblastoma protein regulates cell proliferation, differentiation, and endoreduplication in plantsPLANT JOURNAL 42 (2): 153-163 APR 2005

Valentine T, Shaw J, Blok VC, et al.Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vectorPLANT PHYSIOLOGY 136 (4): 3999-4009 DEC 2004

Jari Valkonen

OPEN QUESTIONS CONCERNING RNA SILENCING

How does RdRp recognise the ssRNA that should be degraded,so that it won’t synthesise dsRNA from RNAs that need tosurvive?

How is dsRNA recognised initially?

(After generation of siRNA, RISC finds new homologous RNA with help of thesiRNA (-)strand that incoporates in RISC)

What is the systemic signal?

How does siRNA mediate its effects on DNA methylation in thenucleus?

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3.VIRUSES AS TARGETS

OF RNA SILENCING

Jari Valkonen

Viruses as targetsof RNA silencing

Voinnet 2005 6: 206-220

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Recovery from virus infection

Ratcliff et al. 1997. Science 276:1558-1560.

Jari Valkonen

Resistance to poliovirus infection in human cellsGitlin, Karelsky &Andino 2002. Nature 418:430-434

• Poliovirus-derived dsRNA introducedà virus accumulation decreased, viruscleared from most cells

• Sequence-specific: a single nt-change within the target sequence enables the virusto escape the resistance mechanism

(Not attributable to “classical antisense mechanisms” or interferon-mediatedresponse)

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A cellular miRNA mediates antiviral defense in human cells

Lecellier et al. 2005, Science 308:557-560

Primate foamy virus type 1 (PFV-1), a HIV-like retrovirus

• Human miRNA-32 and PFV-1 have significant sequence identity

• Preventing miRNA pairing to PVF-1 (mutated) caused a 3-fold increasein PFV-1 accumulation

• Expressing plant tombusvirus P19 silencing suppressor proteinin the mammalian cells greatly increased PFV-1 accumulation(P19 sequesters siRNA)

• PFV-1 encodes Tas protein that suppresses RNA silencingin mammalian and plant cells

Jari Valkonen

Ebstein-Barr virus (herpesvirus) and other herpesviruses encode miRNAsthat regulate viral gene expression

However, based on lack of sequence identity,the viral miRNA presumably do not regulate host gene expression

Pfeffer et al. 2005, Nature Methods 2: 269-276(opposite to the conclusion of Pfeffer et al. 2004, Science 304:734-736)

Virus-derived siRNA is rare in infected cells of vertebrates.

How important is RNA silencing as a virus defence system in vertebrates?

Masked by other defence systems (immuno-response and interferon-mediateddsRNA-induced defence)?

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4.VIRUSES SUPPRESS

RNA SILENCING

Jari Valkonen

SUPPRESSION OF TRANSGENIC RESISTANCE byINFECTION WITH A HETEROLOGOUS VIRUSA: N. benthamiana plants transformed with the PVA coat protein (CP) geneare resistant (immune) to PVA; no CP transgene mRNA is detected bynorthern analysis

B: Following infection with PVY, the CP transgene mRNA accumulates todetectable levels; plants are susceptible to PVA

A: healthy B: PVY-infected

Resistant Susceptible

PVA 5’-UTR+CP

1 2 3 4 5 6 1’ 2’ 3’ 4’ 5’ 6’c

Savenkov & Valkonen 2001,J. Gen. Virol. 82: 2275-2278

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P19, the best-characterisedviral RNA silencing suppressor

Tombusviruses, (+)ssRNA genome

Tomato bushy stunt virus (TBSV)Carnation Italian ringspot virus (CIRV)

Silhavy et al. 2002, EMBO J 21:3070-3080Vargason et al. 2003, Cell 115:799-811

Vargason et al. 2003, Cell 115:799-811

P19 sequesters siRNA:

(A) It binds siRNA duplexes (dsRNA) as a tail-to-tail dimer.Contacts are sequence-independent (via phosphate groups and end-capping)

(B) The N- and C-terminus interact in the homodimer(green, C-terminus; blue, N-terminus; violet, siRNA)

Jari Valkonen

P19, the best-characterisedviral RNA silencing suppressor (continues...)

Carnation Italian ringspot virus (CIRV)

Vargason et al. 2003, Cell 115:799-811

Trp39 and Trp42 (N-terminus)form stacking interactions with

the exposed basepairsat each end of the siRNA duplex

End-capping at each endof siRNA duplex,a ”pocket” fitting inthe dsRNA

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Jari Valkonen

Revealing viral RNA silencing suppressors by AGRO-INFILTRATION

Co-infiltration

GFP silencing

hpGFP

A RSS?

Test protein,suppressor (?)

gene AAgrobacterium

GFP

Silencinginducer,hairpin

hpGFP

Reporter,GFP

Agrobacterium

Agrobacterium

GFP expression

+GFP

GFP Fluorescenceindicates suppression of silencing

Infiltration

Jari Valkonen

Agro-infiltration for co-expression ofGFP + hpGFP and one or two viral proteins

The leaf was photographed from undersideunder UV light at 10 days post-infiltration.

Green fluorescence observed in the spotsinfiltrated with p22 or HC-Pro indicated thatthese proteins suppressed RNA silencing.

Kreuze et al. 2005, J. Virol 79, 7227-7238

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Jari Valkonen

RNA silencing suppressors affect different stepsof the silencing process

Kreuze et al. 2005, J. Virol. 79:7227-7238

Local spread of silencing signal(red ”borders”) inhibited

Himber et al. 2003, EMBO J. 22: 4523-4533

Potyvirus-encoded HCpro suppressorprevents:- initiation of silencing- maintenance of silencing- local movement of thesilencing signal

but not systemic signaling for silencing

Some suppressors inhibitonly the systemic signaling step

HCpro suppresses RNA silencingalso in animal cells

Jari Valkonen

Viral RNA silencing suppressors influence plant developmentvia interference with miRNA biogenesis

and, hence, induce symptoms!

Naturally silenced chalcone synthase genebecomes active in virus-infected soybeansdue to viral RNA silencing suppressor

Senda et al. 2004, Plant Cell 16:807-818

Naturally silenced chalcone synthase gene in the white sectorsof petunia flowers become active in virus-infected flowers

Teycheney & Tepfer 2001, J. Gen. Virol. 82: 1239-1243

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Voinnet 2005, Nature Reviews Genetics 6:206-220

Viral RNA silencing suppressors influence plant developmentvia interference with miRNA biogenesis (continues...)

Influence of different viral viral RNA silencing suppressors on development of Arabidopsis

Jari Valkonen

Voinnet 2005,Nature Reviews Genetics6:206-220

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Lu et al. 2005, Science 309: 1567-1569; viewpoint pp. 1525-1526

75,000 different (non-redundant)small RNA sequencesexpressed in Arabidopsis(especially large diversityof sequences is found in flowers)

Physical distribution of originin the genome reveals sparse todense clusters (no. of sRNAderived per 500 nt)

[Arabidopsis has 26178 genes]

The small-RNA world, much to explore

Jari Valkonen

RNase III

- Endo-ribonucleases

- dsRNA substrate

- Prokaryotes and eukaryotes

DICER, DCL (siRNAs)

DROSHA (miRNAs)

E. coli, SPCSV(homodimer)

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Sweet potato chlorotic stunt virus (SPCSV)Single-stranded, positive-sense, bi-partite RNA genome

The sub-genomic RNAs (sgRNA) corresponding toRnase3 and p22 are expressed early in infection

sgRNAs

Jari Valkonen

Viral Rnase3 enhances suppression ofp22-mediated RNA silencing

in agroinfiltrated leaves

RNA analysisAgroinfiltrated leaf

R3-Western Blot

Kreuze et al. 2005, J. Virol 79, 7227-7238

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Jari Valkonen

1- RNA silencing and transgenic plants

2- Using RNA silencing without transgenic plants

• Agro-infiltration• Virus-induced gene silencing (VIGS) as a research tool

3- Viruses are targets of RNA silencing-based host defence

4- Suppression of RNA silencing by viruses

Summary