VIGS VECTORS FOR GENE SILENCING: Many Targets, Many Tools

27 Apr 2004 15:11 AR AR213-PP55-19.tex AR213-PP55-19.sgm LaTeX2e(2002/01/18) P1: GDL10.1146/annurev.arplant.55.031903.141803

Annu. Rev. Plant Biol. 2004. 55:495–519doi: 10.1146/annurev.arplant.55.031903.141803

Copyright c© 2004 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on February 25, 2004

VIGS VECTORS FOR GENE SILENCING: ManyTargets, Many Tools

Dominique RobertsonDepartments of Botany and Genetics, North Carolina State University,Raleigh, North Carolina 27695–7612; email: Niki [email protected]

Key Words RNAi, hpRNA, functional genomics, geminiviruses

■ Abstract The discovery that plants recognize and degrade invading viral RNAcaused a paradigm shift in our understanding of viral/host interactions. Combined withthe discovery that plants cosuppress their own genes if they are transformed with ho-mologous transgenes, new models for both plant intercellular communication and viraldefense have emerged. Plant biologists adapted homology-based defense mechanismstriggered by incoming viruses to target individual genes for silencing in a process calledvirus-induced gene silencing (VIGS). Both VIGS- and dsRNA-containing transforma-tion cassettes are increasingly being used for reverse genetics as part of an integratedapproach to determining gene function. Virus-derived vectors silence gene expressionwithout transformation and selection. However, because viruses also alter gene expres-sion in their host, the process of VIGS must be understood. This review examines howDNA and RNA viruses have been modified to silence plant gene expression. I discussadvantages and disadvantages of VIGS in determining gene function and guidelinesfor the safe use of viral vectors.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496Viruses as Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497Biology of VIGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498When the Silencer is the Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501Inoculation of Viral Vectors and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501Optimizing Silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505Pathways for Diffusible Silencing Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506Gene Function Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

FUNCTIONAL GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509

APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

1543-5008/04/0602-0495$14.00 495

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496 ROBERTSON

INTRODUCTION

The first indication that plants can be transformed with engineered genes broke theground for plant biotechnology and promised a new era of crop plant protectionand yield enhancement (8, 32). However, transgene expression is not entirelypredictable. RNA levels from the same gene transformed into different plants canvary and in some cases transgene expression is lost during development or insubsequent generations (4, 7). This was dramatically illustrated by the discoverythat not only transgene but also endogenous plant gene expression is subject tosilencing by ectopic expression of homologous transgenes (104, 145). Despiteactive transcription, mRNA accumulation for both genes decreased, a processcalled post-transcriptional gene silencing (PTGS).

The parallel discovery that transgene silencing can also impact RNA virusinfections if the transgene and virus share significant homology led to a model ofplant-mediated RNA degradation as a defense mechanism (24, 84, 119; reviewedin 83). Subsequent studies demonstrated that the transgene was not required totrigger this plant defense pathway. Nontransgenic plants that displayed recoveryphenotypes from wild-type virus infection also showed characteristics similar togene silencing in that viral RNA was eliminated in tissue where recovery wasevident (2, 20).

The nature of the recovery mechanism was not understood until PTGS1 wasassociated with a diffusible silencing signal. This was first demonstrated by graft-ing transgenic stocks of tobacco showing PTGS with the scions from a plant thatexpressed the same transgene (108). Efficient transmission of a silencing signaloccurred even when the stock and scion were separated by a 30-cm nontransgenicstem (double graft). In a second report, spread of transgene silencing was initi-ated by syringe-mediated agroinfiltration of a reporter gene expression cassetteinto a single leaf ofN. benthamianaactively expressing a chromosomal copy ofthe reporter gene (147). Expression of the reporter gene, encoding jellyfish greenfluorescent protein (GFP) could be visualized using UV illumination. Movementof an unidentified silencing factor into upper leaves and new growth could bechronicled in real time as UV-illuminated silenced tissues lost all green fluores-cence. Remarkably, even promoterless dsDNA with homology to GFP introducedby biolistics triggered systemic GFP silencing (149).

Antisense- and sense-mediated inhibition of gene expression was commonlyused to downregulate gene expression in plants and inC. elegans, but its efficiencyvaried in different transformants. A breakthrough that started the use of RNAias a general silencing tool occurred when it was found that only small amounts

1PTGS is similar to RNAi, which is here referred to as interfering RNA from dsRNA.siRNA refers to 22-nt oligonucleotide dimers used to initiate silencing. hpRNA refers toconstructs for transcribing inverted repeats separated by nonhomologous or intron sequences(sometimes referred to as panhandles). smRNA refers to small species of RNA (less than100 nt) associated with PTGS.

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VIGS VECTORS 497

(a few molecules) of dsRNA injected intoC. eleganswere needed for widespreadsilencing similar to PTGS in plants (19a, 29, 70, 100). The low efficiency of bothsense- and antisense-mediated silencing in plants was then explained by proposingthat dsRNA was produced from complex integration patterns in different transfor-mants. Subsequently, transgene constructs containing an expression cassette ofinverted repeats (IR-PTGS or hpRNA) were found to be much more efficient thaneither antisense- or sense-mediated silencing alone (157). The similarities betweenPTGS-related mechanisms in a variety of different organisms have been reviewed(16, 19).

Another breakthrough occurred when smRNAs (21–28 nt) homologous togenes silenced by PTGS were found in silenced tissue (43, 44). In mammals andDrosophila, 22-nt siRNA molecules are sufficient to induce silencing spread (12,46). There are reports of 22-nt siRNA-mediated silencing in plants (74, 146), buttransitive RNA, dsRNA that flanks the region of homology, was also found. Transi-tive RNA fractionates with smRNA but lacks homology to the inducing RNA, andis likely produced by the host enzyme, RNA-dependent RNA polymerase (RdRP).In C. elegans, which also shows transitive RNA, siRNAs can initiate silencing butsubstantially less dsRNA is needed if longer RNAs are used (A. Fire, personalcommunication). In other organisms that encode RdRP, such as plants and fungi,dsRNA larger than 22 nt can initiate silencing, but the specific nature of the mobileRNA species in plants and fungi remains to be determined (98, 116).

This review covers the silencing of endogenous plant genes initiated from re-combinant viral vectors, coined virus-induced gene silencing (VIGS) (126). RNAand DNA virus vectors are described and compared with respect to host range,host interactions, and method of use. Examples from the literature are highlightedto show relative effectiveness of silencing in different classes of genes, and the ad-vantages and disadvantages of using viral vectors compared to methods requiringtransformation. An excellent review covering genomic initiatives for using bothVIGS (primarily RNA virus-based VIGS) and hpRNA chromosomal cassettes forsilencing should also be consulted to provide a comprehensive picture of RNAi inplants (158).

Viruses as Vectors

Many different RNA and DNA viruses have been modified to serve as vectors forgene expression (reviewed in 118, 138). Some viruses, such as tobacco mosaicvirus (TMV), potato virus X (PVX), and tobacco rattle virus (TRV), can be usedfor both protein expression and gene silencing (6, 77, 92, 93). Not all RNA virus-derived expression vectors will be useful as silencing vectors because many, suchas TEV, have potent anti-silencing proteins that directly interfere with host silenc-ing machinery (5, 68). DNA viruses have not been used extensively as expressionvectors due to their size constraints for movement (109). However, a nonmobilemaize streak virus-derived vector (MSV, in the Mastrevirus genus ofGeminiviri-dae) has been successfully used for long-term production of protein in maize cellcultures (110).

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498 ROBERTSON

Using viral vectors to silence endogenous plant genes requires cloning homol-ogous gene fragments into the virus without compromising viral replication andmovement. This was first demonstrated in RNA viruses by inserting sequences intoTMV (77), and then for DNA viruses by replacing the coat protein gene with ahomologous sequence (72). These reports used visible markers for gene silencingPDSandChlI, providing a measure of the tissue specificity of silencing.

Table 1 shows some general characteristics for currently available virus-derivedgene silencing vectors. Most viruses are plus-strand RNA viruses or satellites,whereas tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaL-CuV) are DNA viruses. Although RNA viruses replicate in the cytoplasm, DNAviruses replicate in plant nuclei using host DNA replication machinery. Both typesof viruses induce diffusible, homology-dependent systemic silencing of endoge-nous genes. However, the extent of silencing spread and the severity of viral symp-toms can vary significantly in different host plants and host/virus combinations.With the variety of viruses and the diversity of infection patterns, transmissionvectors, and plant defenses it is not surprising that viruses differ with respect tosilencing (137). Because the continuing development of virus-based silencing vec-tors can extend VIGS to economically important plants, it is useful to considersome of the characteristics of successful VIGS vectors.

Biology of VIGS

RNA viruses replicate cytoplasmically using their own polymerase and host cy-toplasmic membranes, ribosomes, and proteins (22, 60, 106). Detailed protocolsfor making and using virus-derived silencing vectors have been published andshould be consulted for more information (23, 89). The RNA viruses shown inTable 1 can be mechanically inoculated using in vitro transcribed RNA copies ofthe plus-strand genome. An easier method uses agroinfection of cloned vectorsfor TRV and PVX (86, 120). Following agroinfiltration of an expression cassettecontaining a cDNA copy of the genome, a single-stranded mRNA resembling thevirus is transcribed by the host RNA polymerase and exported to the cytoplasm.The first sequence on the mRNA is then translated to produce the viral replicase(an RdRP). Replication of similar mRNA molecules containing a 3′-virus-specificrecognition sequence is initiated and the viral vector moves into new cells. RNAviruses with multiple genome components, such as TRV, can be cloned and coinoc-ulated with high efficiency (120). Most RNA virus–derived vectors are insertionvectors (118) and contain a duplicated subgenomic promoter preceding the inser-tion site. There are two versions of the TRV vector, one with an insertion site andone with a duplicate subgenomic promoter and insertion site (86, 89). Becauseeach RNA virus-encoded protein is required for efficient movement and repli-cation, gene replacement vectors have not been successfully used for silencing(118).

Satellite viruses are small RNAs (800–1300 nt) that usually encode their owncoat protein but rely on a second virus for replication (22). A silencing system

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500 ROBERTSON

[a satellite virus–induced silencing system (SVISS)] was developed that uses satel-lite TMV (STMV) as a vector for spreading silencing fragments and TMV as ahelper virus in tobacco (38). In this system, 100–300-nt fragments are inserted intothe satellite genome and the resulting vector is coinoculated with a helper virus.PVX, TMV, and the TMV satellite virus do not have strong anti-silencing proteins,whereas the cucumber mosaic virus, tobacco etch virus, and cymbidium ringspotvirus have proteins that can reverse transgene silencing (10, 40, 49, 91; reviewedin 144).

Two geminiviruses, TGMV and CaLCuV, have been used to generate silenc-ing vectors. As with other members of the Begomovirus genus ofGeminiviridae,the TGMV and CaLCuV genomes consist of two single-stranded circular DNAmolecules called the A and B components, each is about 2.5 kb in size (45, 53).DNA viruses replicate through dsDNA intermediates in plant nuclei. They dependon host transcription and translation to produce proteins that interact with cellcycle regulators to induce host DNA replication machinery (25, 76). Infectiousvectors have been cloned that contain direct repeats of the viral-replication origin–containing common region flanking the viral genome (124). Replicating unit lengthvectors depends on the AC1 gene and results in infectious DNA genomes (26).DNA replication occurs using host enzymes and double-stranded viral DNA in-termediates (42, 47, 47a).

Geminivirus-derived vectors move systemically without the 800-bp coat pro-tein gene and can carry foreign sequence instead (14, 50, 133), but only in certainhost/virus combinations. Although the A component can replicate in the absenceof the B component (124), movement proteins encoded in the B component arerequired for systemic infection (81). The TGMV genome is stably propagated withinserts ranging from 100–800 bp (112), but African cassava mosaic virus (ACMV)vectors acquire additional sequence from other parts of its genome by recombiningto restore its original size (75). Results for both TGMV and ACMV vectors usedN. benthamiana, suggesting that the viruses themselves exhibited different proper-ties with respect to movement. The molecular size of geminivirus-derived vectorsis easily determined by DNA gel blot hybridizations of restriction enzyme digestedviral DNA because the virus replicates to high numbers in plant nuclei (138). Thus,DNA gel blots can be used to determine the stability of the inserted DNA silencingfragment.

Because geminiviruses induce host DNA replication and alter plant gene ex-pression (9, 103; reviewed in 47), their vectors may be considered unsuitable fortesting gene function. However, two results argue against this conclusion: Bothviral DNA replication and induction of PCNA are cell autonomous (103), and thereplication of geminivirus-derived silencing vectors is greatly reduced comparedto replication of the wild-type virus (73, 112). Both in situ hybridization of viralDNA in silenced tissues and DNA gel blots show that viral DNA levels are re-duced in silenced tissue. SilencingChlI from a mutant, phloem-limited TGMVfurther demonstrates that extensive silencing can occur in uninfected cells (112).Vectors carrying similarly sized unrelated DNA can be used to control the impact

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VIGS VECTORS 501

of virus-associated changes in gene expression. Similar controls have been used toseparate target gene silencing phenotypes and virus effects in RNA vectors (159).

When the Silencer is the Target

RNA viruses are targets for silencing because their genome is directly affectedby host-mediated dsRNA degradation. Inoculation of movement-competent PVX-GFP into GFP-expressing plants results in eliminating the inducing vector (126).However, the same vector carrying sequence homologous to the endogenousgeneRbcS(small subunit of ribulose-bisphosphate carboxylase/oxygenase) wasnot eliminated from PVX:RbcS-silenced plants. DNA methylation is associatedwith PVX-GFP-silenced GFP transgenes, but the PVX:RbcS-silenced endogenousgenes lacked extensive methylation (64). RNA-directed DNA methylation waspreviously reported (114, 155) and may be a general epigenetic mechanism (65,154, 156). It is not understood why transgenes are methylated by VIGS whereasendogenous genes are not.

Levels of recombinant TMV vectors with homology to an endogenous genefluctuate, andChlH silencing also fades and reappears (55). Table 2 shows thatsome RNA vectors produce strong silencing but then it decreases. Although elim-inating a recombinant PVX silencing vector did not occur when an endogenousgene was targeted, it was not clear how long silencing was maintained in newgrowth (126). In contrast to RNA viruses, there is no evidence for eliminationof recombinant geminivirus genomes, even when they contain sequences withhomology to a transgene. For example, systemic spread of GFP silencing is notfollowed by recovery from the virus when a CaLCuV vector is used to infectArabidopsiscontaining a 35S-GFP transgene (140). In contrast to ssDNA virusesstudied so far, the wild-type retrovirus-like CaMV can be eliminated by silencing-related recovery (2), perhaps because the RNA form of its genome is a target(112).

Wild-type geminivirus infections can show attenuation of symptoms similar toRNA viruses, but viral DNA accumulation is reduced, not eliminated (59, 123).Replicating DNA viruses do not tolerate methylation (28) and it is enigmatic thatDNA viruses can elicit silencing of endogenous plant genes while maintainingenough expression of their own genes to sustain infection. The requirement ofRdRP for DNA VIGS, but not RNA VIGS, inArabidopsis(160) provides furtherevidence that silencing does not eliminate DNA viruses. Note that even thoughwild-type tomato yellow leaf curl geminivirus infections are associated with siR-NAs, the symptoms of this pathogen are severe (90).

Inoculation of Viral Vectors and Safety

Recombinant viruses require special treatment and approval from plant healthregulatory authorities must be obtained before the viruses are received and used.Because RNA viruses, especially barley stripe mosaic virus (BSMV), are usedin their entirety for silencing vectors, unintentional inoculation by mechanical

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loss

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l

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nth

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ian

aP

hoto

blea

chin

gS

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ing

fade

san

dre

turn

sM

echa

nica

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assa

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er(5

5,56

)ov

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inoc

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ls

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VIGS VECTORS 503Ta

rget

gene

Viru

sH

ost

Sile

ncin

gph

enot

ype

Com

men

tsIn

ocul

atio

nP

heno

typi

cas

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men

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efer

ence

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BS

MV

Bar

ley

Pho

tobl

each

ing

Qua

ntita

tive

PC

Ras

says

show

Mec

hani

cal,

thre

eH

PLC

used

to(5

8)si

lenc

ing

incr

ease

sth

enin

vitr

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ribed

dete

ctph

ytoe

nede

crea

ses

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een

5–25

dpi

RN

As

AtT

auA

TR

V35

SA

tTau

A-G

FP

Loss

of35

S-T

uA-G

FP

Sho

wed

that

TM

V-d

sRed

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hani

cali

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latio

nC

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icro

scop

y,(3

6)tr

ansg

enic

expr

essi

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mov

esin

35S

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ofin

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sgen

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pres

sion

N.b

en

tha

mia

na

sile

nced

tissu

e,bu

tsile

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gin

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mai

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otyp

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brid

izat

ion

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en

tha

mia

na

syst

emic

necr

osis

TM

V::G

FP

;or

TR

V::E

DS

then

TR

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toor

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300

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rious

phen

otyp

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otyp

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tro

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en

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en

tha

mia

na

infe

ctio

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and

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ctor

sus

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rex

pres

sion

ofF

LAG

-or

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ract

ion

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ntin

ue

d)

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504 ROBERTSON

TAB

LE2

(Co

ntin

ue

d)

Targ

etge

neV

irus

Hos

tS

ilenc

ing

phen

otyp

eC

omm

ents

Inoc

ulat

ion

Phe

noty

pic

asse

ssm

ent

Ref

eren

ce

NbF

tsH

PV

XN

.be

nth

am

ian

aP

hoto

blea

chin

gP

heno

copi

esAra

bid

op

sisv

ar1

Mec

hani

cali

nocu

latio

nR

T-P

CR

,im

mun

oblo

t(1

29)

mut

atio

n(F

tsH

hom

olog

)of

invi

tro

tran

scrip

ts

NbP

AF,

NbR

PN

9T

RV

N.b

en

tha

mia

na

Ass

ayed

20dp

iE

xcel

lent

cont

rols

,N,C

,and

Agr

o-in

ocul

atio

nS

emi-q

uant

itativ

e(7

1)fo

rpr

ogra

mm

edfu

ll-le

ngth

PA

Fsi

lenc

ing

RT

PC

Rof

mul

tiple

cell

deat

hco

ntru

cts

assa

yed

gene

ste

sted

NbP

DS

,HvP

DS

TM

VN

.be

nth

am

ian

a,P

hoto

blea

chin

gIR

inse

rten

hanc

edsp

read

ofM

echa

nica

lV

isua

l,Q

PC

R(7

9)B

SM

Vba

rley

begi

ns12

dpiT

MV,

sile

ncin

g,40

–60

ntIR

used

,14

dpib

arle

y20

0nt

IRno

teffe

ctiv

e

NtC

DP

K1,

NtR

PN

3P

VX

N.b

en

tha

mia

na

Cel

ldea

th,a

bnor

mal

RT

PC

Rof

mul

tiple

gene

sA

gro-

inoc

ulat

ion

RT-

PC

R(8

2)st

omat

alde

velo

pmen

tto

test

com

plex

resp

onse

,as

says

at20

dpi

NbW

IPK

,NbS

IPK

PV

XN

.be

nth

am

ian

aH

yper

sens

itive

Tim

eco

urse

RT-

PC

Rov

er4

hM

echa

nica

lR

T-P

CR

(131

)re

spon

sefo

llow

ing

chal

leng

ew

ithin

ocul

atio

nof

Imm

unob

lots

,kin

ase

fung

alel

icito

rin

vitr

o-m

ade

assa

ysfo

llow

ing

tran

scrip

tsw

ound

ing

orel

icito

r

WIP

K,S

IPK

PV

X35

S-N

gene

Loss

ofT

MV

-indu

ced

Bot

hW

IPK

and

SIP

KA

gro-

inoc

ulat

ion

Imm

unob

lots

(85)

N.b

en

tha

mia

na

kina

seex

pres

sion

prot

eins

asse

ssed

inre

cipr

ocal

sile

ncin

gex

pts

P58

(IP

K)

PV

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.be

nth

am

ian

aP

lant

deat

hfo

llow

ing

VIG

Sre

sults

confi

rmed

inA

gro-

inoc

ulat

ion

Vis

ual,

RT-

PC

R(1

3)vi

rus

infe

ctio

nA

rab

ido

psi

skno

ckou

tmut

ant

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VIGS VECTORS 505

transmission must be considered and appropriate measures must be taken toprevent it.

Viruses from DNA vectors such as CaLCuV lack a coat protein and are notinfectious, but the same precautions should be taken, especially when the coatprotein is present. Geminiviruses are transmitted by whiteflies (Begomoviruses)or leaf hoppers (Mastreviruses and Curtoviruses). The TGMV vector comes froma virus that can no longer be transmitted by whiteflies and is no longer infectious intomato. Other isolates of TGMV were recently reported in Brazil and are infectiousin tomato (35). CaLCuV was identified in Florida as a pathogen of cabbage (53).The CaLCuV silencing vector developed forArabidopsislacks the coat proteinand therefore cannot be transmitted by whiteflies (139). Without the coat protein,CaLCuV is not infectious in cabbage, broccoli, and cauliflower by microprojectilebombardment (M. Flores & D. Robertson, unpublished data). Geminiviruses canevolve rapidly under field conditions (117), and even noninfectious vectors derivedfrom viral pathogens should be contained at all times (135, 136). The presenceof whiteflies should be monitored carefully and, in certain agricultural regions,geminivirus vectors should be used only if they lack a coat protein.

Several methods are commonly employed to deliver viral silencing vectorsto plants, including: mechanical inoculation using in vitro transcribed RNA orextracts from infected leaves, agroinoculation, and microprojectile bombardment.Mechanical inoculation is time consuming but can increase the efficiency of silenc-ing in certain hosts such asArabidopsis(120). Agroinoculation or agroinfiltrationhave been developed for both DNA and RNA viruses, as well as for transient si-lencing in the absence of virus (27, 130, 141). Tobacco, tomato, and barley vectorshave been developed that show extensive silencing with attenuated symptoms, andagroinfiltration of TRV vectors is becoming the vector of choice for many investiga-tors (Table 2). A web-based movie demonstrating syringe inoculation of Agrobac-terium into leaves can be found at http://www.sainsbury-laboratory.ac.uk/david-baulcombe/Services/agroInfil1.mpg, courtesy of D. Baulcombe. Agroinfiltrationof N. benthamianaleaves is routine and a similarly robust procedure is being devel-oped forArabidopsis(S. Kjemtrup, personal communication). The use of syringeswith needles can be used reliably (J. Ascensio & L. Hanley-Bowdoin, unpublisheddata), but the procedure is not suitable for high throughput.

Microprojectile bombardment of plasmid DNA-coated tungsten or gold micron-sized particles have been extremely useful for DNA viruses (102). Alternativemethods for virus inoculation are also available (34, 122).

Optimizing Silencing

The effectiveness of VIGS depends on environmental variables. For example,plants grown at higher temperatures show a stronger silencing response (97, 134).For TGMV, N. benthamianagrown in a 4′′ pot shows better silencing than whengrown in a 2′′ pot (C. Jordan & D. Robertson, unpublished work). Silencing inArabidopsisis more effective under short days, and inoculation at the 4–6-leaf

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506 ROBERTSON

stage produces silencing in rosette leaves whereas inoculation at 10–12 leavestargets inflorescence tissue (140). These variables are easier to sort out when usinga target gene with a predicted visual or quantifiable phenotype.

Having a single gene as a silencing reference can help to sort out these variables.Choosing a target gene for a positive silencing control should be done carefullybecause different targets may impact VIGS vector replication, movement, andextent or timing of silencing, especially if unknown targets are characterized.Because transgene silencing can be propagated independently of the vector, its useas a silencing control for endogenous gene silencing is limited (54, 64, 72, 112,126).

GSA, PDS, ChlI, ChlH, andPAIgenes have visual phenotypes following silenc-ing (11, 57, 61, 77). The sequence from the tobaccoChlI ortholog (also calledsu)was originally identified by transposon tagging thesulfur locus of tobacco (31, 48,105).Chl1-silencedN. benthamianashow a uniform progression of silencing andflower normally and set seed, but they have reduced leaf size, stature, and numberof flowers (112). Silencing ofChlI from TGMV has been maintained for morethan 52 days by pruning plants to prevent them from producing seed (112).

Choosing only one gene for a silencing control is important for standardiz-ing epigenitic variation due to environmental conditions. For example, silencingdifferent subunits (ChlH versus ChlI) of the magnesium chelatase complex is ex-pected to produce different results. Expression levels of the nuclear-encodedLHCPmRNA in ChlH andChlI Arabidopsismutants showed that the nuclear-encodedLHCP mRNA decreased inchlH but not inchlI. TheChlH subunit participatesin a plastid-to-nucleus signaling pathway that regulates photosynthesis-associatedgene expression (99). Photobleaching could also impact silencing results; however,when chloramphenicol was added tochlH mutants to inhibit chloroplast transla-tion, LHCP expression was restored. Therefore, repression depended on activechloroplast translation in the albino plants. Although silencingChlH may causeunintended downregulation of other photosynthesis-related genes, bothChlH andChlI are reliable markers and are useful for studying the silencing dynamics ofendogenous genes (55, 56).

Pathways for Diffusible Silencing Signals

To use VIGS to target specific genes, it is useful to understand the dynamic natureof silencing. Homology-dependent gene silencing was previously classified asTGS or PTGS depending on whether transcripts for the silenced gene were foundin the nucleus (94). Silencing machinery is not necessarily active in TGS, whereasPTGS degrades incoming viral RNA and causes a dominant silencing phenotype(37, 80). There is now abundant evidence that RNA causes changes in chromatinstructure that prevent transcription leading to TGS (63, 113, 114, 150, 151, 155).In plants, a picture is emerging where transgenes are methylated in regions thatcorrespond to RNA homology, whereas endogenous genes appear to escape suchmethylation (95, 96).

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VIGS VECTORS 507

The idea that there are similar pathways for virus movement and silencingsignals is supported by the identification of nontoxic levels of cadmium as aninhibitor of PTGS spread and tobamovirus movement in plants (142) and thecorrelation of anti-silencing and viral RNA accumulation in different tissues andplants (69, 128, 148). Other pathways for silencing signals cannot be ruled out andevidence for two kinds of movement has been presented (54). The dynamic natureof plasmodesmatal trafficking of nucleic acids was shown using a TMV vectorengineered to carry an additional copy of its movement protein translationallyfused to GFP (107). Epidermal spread of GFP from an inoculated area was transientand occurred only in cells at the leading edge of the TMV infection.

The dynamic nature of early events in endogenous gene silencing by TGMV::ChlI in Nicotiana is unusual in that discrete yellow spots 0.5–3 mm form ap-proximately five days post-bombardment. Spots are also seen whenN. tabacum,a TGMV host that requires the coat protein gene for movement, is bombarded(C. Peele & D. Robertson, unpublished work). Similar spots are not seen inCaLCuV::ChlI-infectedArabidopsisand it takes 12–14 days to see evidence of si-lencing in new growth (140). The rapid appearance and uniformity of TGMV::ChlI-induced silencing spots inN. benthamianacould be evidence for an apoplasticcomponent of spread (112). Noncell-autonomous movement of nucleic acids andprotein is an exciting area of research (51, 127, 159).

VIGS of transcribed sequences is confined to the inoculated plant and is lostalong with the viral inducer in subsequent generations. VIGS of pretranscribedsequences, such as promoters, can cause methylation, presumably due to RNA-DNA interactions (64). Methylated promoters are not transcribed efficiently, andshow reduced expression of their genes. Methylation is associated with chromatinchanges and can be inherited independently from the VIGS vector. Paradoxically,TRV carrying 35S sequence can cause methylation of 35S promoter sequencesthat extend into subsequent generations, but the TRV vector itself is not seedtransmitted, although the wild-type TRV is (65; Table 1).

Gene Function Studies

VIGS has been used to silence a wide variety of genes in plants (Table 2). Therehave been elegant studies combining VIGS with biochemical and genetic methodsto determine gene function, and they are producing a coherent picture of genefunction (13, 15, 62, 71, 82). Loss of function mutations of some of these geneswould be embryo lethals (such as PCNA, which is required for DNA replication,and mutations affecting chlorophyll stability). VIGS has been especially power-ful for dissecting signaling components involved in disease resistance (87, 111,125). Dramatic phenotypic effects are seen in pathogen-infected host plants si-lenced for the corresponding defense genes. Note that most of these studies usedN. benthamianaas a host (Table 2).

N. benthamianahas been extensively used for silencing studies because it is asuitable host for a wide range of viruses (Table 2). It can be readily transformed by

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508 ROBERTSON

Agrobacterium cocultivation of leaf discs, self-pollinates, flowers rapidly, and is ofsmaller stature than tobacco.N. benthamianahas advantages over Arabidopsis thatinclude limited symptoms and abundant leaf material for biochemical analysis.N.tabacum, tomato, and barley also have well-developed VIGS vectors. VIGS vectorsfor Arabidopsis are currently limited by the need to passage vectors throughN.benthamianato get virions (TRV) or severe symptoms (CaLCuV). Modificationsin these vectors, or host plant mutations, may increase the effectiveness of TRVand CaLCuV in Arabidopsis.

The power of VIGS is its rapid initiation of silencing in intact wild-type ortransgenic plants. The ability to reliably silence 1–2 genes can provide materialfor biochemical analysis, metabolic profiling, and transcript profiling, if suitablecontrols are included. The use of RNA-based silencing to modulate mRNA lev-els can be an advantage and some RNA vectors show predictable increases anddecreases of silencing over time (58). Networks of genetic and protein interac-tions change when mRNA levels for individual genes are altered and informationcan be obtained for both medium and high levels of silencing, as demonstrated inArabidopsis (18). Keep in mind that silencing is a method for modulating geneexpression, not eliminating it. Quantitative measures of target mRNA levels shouldbe measured for each silencing event to be useful for genomic studies.

In-depth characterization of gene function must be accompanied by other meth-ods to verify VIGS-related analyses. Reverse transcriptase PCR (RT-PCR) of genenetworks, testing overexpression lines, and using transgenic lines carrying hairpinsilencing cassettes can help to insure that effects are gene specific rather than VIGSspecific. For plants with sequenced genomes, homology searches can be done toavoid unintended silencing. Potential effects of transitive silencing, which refer tosilencing initiated from mRNA sequences adjacent to the area of target homology,should also be considered. In plants, transitive RNAi has only been demonstratedfor transgenes (121, 143, 146).

FUNCTIONAL GENOMICS

Integration of RNAi data in combination with transcript profiling and protein-protein interaction studies is yielding computer-intensive ways to explore develop-ment and gene regulation (39). RNAi inC. eleganshas been used for genome-widefunctional genomics (33, 66, 67). Because different kinds of information can begained from large-scale genomics approaches, it would be useful to develop similarsystems in plants. hpRNA has been used on plants in functional genomics (152).VIGS can supplant hpRNA studies by targeting genes that are hard to silence byhpRNA and by providing a bridge for testing conservation of gene function fromArabidopsisto crop plants.

Large-scale silencing projects using transformation with constructs that con-tain hpRNA are in progress and high-throughput vectors are available (41, 52).

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VIGS VECTORS 509

There is an extensive collection of RNA silencing–based transformants, PCRprimers, and vectors forArabidopsischromatin-associated genes (for informa-tion, see http://www.chromdb.org/). The project also shows quantitative data foreach transformant using RNA gel blots or RT-PCR. This is especially importantbecause RNA-based silencing does not eliminate gene expression and silencinglevels vary in different transformants (18).

There are caveats to using hpRNA to determine gene function. Although thehpRNA-induced silencing is heritable, epigenetic effects may result in unexpectedsilencing in subsequent generations, and transcriptional silencing of the inducinghpRNA locus may restore target gene expression (7, 78). Archiving and maintain-ing seed stocks are now being done for T-DNA insertion lines, but they requirean enormous investment. Archiving vectors is easier, and the Chromatin databaseproject has made such constructs available through TheArabidopsisInforma-tion Resource (TAIR). Additionally, unless transformation technologies improve,it will be hard to generate similar resources for plants that are recalcitrant totransformation.

Summaries of high-throughput screens using VIGS were reported inNicotianausing TMV and in barley using BSMV (30). In a second report, a screen of 5000genes for disease resistance resulted in approximately 100 clones whose silencinginterfered with a cell death assay inN. benthamiana(89). Of these, about 10 geneswere directly involved in disease resistance and 90 had unrelated loss of cell deathphenotypes. Although only a small number of genes were identified, this studydemonstrates that VIGS can be used to rapidly identify candidates for furthertesting.

Although there has been a lack of a suitable high-throughput VIGS vectorfor Arabidopsis, there are many public resources available for determining genefunction (one is TAIR at http://arabidopsis.org/). VIGS could be a useful additionfor verifying mutant phenotypes from plants that may have more than one mutationand for silencing combination of genes. The geminivirus vector CaLCuV causesextensive silencing inArabidopsisbut the associated symptoms limit the usefulnessof this vector, especially for developmental studies (140). Because geminivirussymptoms and silencing can be uncoupled (101a), refinements to the CaLCuVVIGS system will improve its usefulness in the near future.

Future Directions

Libraries of VIGS vectors with sequenced inserts would be useful resources forfunctional genomics studies. Unlike transgenes, which are subject to epigeneticmodifications, VIGS vectors can be used for reliable silencing and can be used indifferent genetic backgrounds and for various screens. This versatility is especiallyuseful because many phenotypes are not evident until proper environmental con-ditions are reached. Environment by genotype variations (for example, in stresstolerance assays) will likely require large numbers of plants and it is difficult topredict what genes will be useful for further testing before these kinds of studies are

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performed. Because seed storage and archiving can be labor-intensive processes,developing community resources for RNA silencing–based vectors would be use-ful. Known combinations of PCR primers for silencing sequences could be archivedalong with descriptions of genotypes used and resulting phenotypes. For example,the internationalC. elegansdatabase incorporates a searchable index for RNAiphenotypes at http://www.wormbase.org/db/searches/rnaisearch. The databasearchives information for PCR primers and includes time-lapse studies of develop-ment (115).

An advantage of using viruses for reverse genetics is that most hard-to-transformcrop plants are susceptible to viruses, which could in turn be potential silencingvectors. Viruses with broad host range, such as BSMV and TRV, will be usefulfor extending functional data from model systems to crop plants. Geminivirusesare also attractive for gene silencing vectors because their genome structure isconserved and they infect a wide range of crop plants including soybean, cotton,and vegetable crops. Once a VIGS system is identified and optimized, one can testorthologous genes from model systems for conservation of function because it ispossible that similar genes will produce proteins that acquire different functionsin different plants (21, 101).

Identifying a plant that is supersensitive to silencing may be possible whenthe mechanism of silencing in plants is better understood. Such a mutant inC. eleganshas been identified and maps (counterintuitively) to a RdRP-like gene(132). However, until a method for inducing PTGS of endogenous genes in plantsis developed that is comparable to RNAi inC. elegans, combinations of VIGSand hpRNA stable transformation methods will likely be the method of choice forfunctional genomics in most plant species.

ACKNOWLEDGMENTS

I am indebted to George Allen, Chad Jordan, and Miguel Flores for many stimu-lating discussions, and to Susanne Kjemtrup, Anton Calloway, and Linda Hanley-Bowdoin for suggestions and help.

APPENDIX

PDS, phytoene desaturase;ChsA, chalcone synthase;RbcS, small subunit ofribulose-bisphosphate carboxylase/oxygenase;TK, plastid transketolase;CesA1,cellulose synthase subunit A;AlS, acetolactate synthase.Ppx, protoporphyrin IXoxidase;Gln1, glutamine synthetase;RPII, RNA polymerase II;Cat1, catalase1 PK1, kinasePARP, poly(ADP-ribose) polymerase;N gene, Toll Interleukin1/nucleotide-binding site/leucine-rich repeat resistance gene; TuA, alpha tubu-lin, WIPK, wound-induced protein kinase,SIPK, salicylic acid–induced proteinkinase;ChlH (magnesium chelatase subunit H),p58(IPK) ortholog of dsRNA-dependent PKR inhibitor;PAF, alpha subunit of 26S proteosome,RPN1, secondcomponent of 26S proteosome.

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The Annual Review of Plant Biologyis online at http://plant.annualreviews.org

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P1: FRK

March 24, 2004 1:1 Annual Reviews AR213-FM

Annual Review of Plant BiologyVolume 55, 2004

CONTENTS

AN UNFORESEEN VOYAGE TO THE WORLD OF PHYTOCHROMES,Masaki Furuya 1

ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANTMITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,and Thomas E. Elthon 23

DNA METHYLATION AND EPIGENETICS, Judith Bender 41

PHOSPHOENOLPYRUVATE CARBOXYLASE: A NEW ERA OFSTRUCTURAL BIOLOGY, Katsura Izui, Hiroyoshi Matsumura,Tsuyoshi Furumoto, and Yasushi Kai 69

METABOLIC CHANNELING IN PLANTS, Brenda S.J. Winkel 85

RHAMNOGALACTURONAN II: STRUCTURE AND FUNCTION OF ABORATE CROSS-LINKED CELL WALL PECTIC POLYSACCHARIDE,Malcolm A. O’Neill, Tadashi Ishii, Peter Albersheim, and Alan G. Darvill 109

NATURALLY OCCURRING GENETIC VARIATION IN ARABIDOPSISTHALIANA, Maarten Koornneef, Carlos Alonso-Blanco, andDick Vreugdenhil 141

SINGLE-CELL C4 PHOTOSYNTHESIS VERSUS THE DUAL-CELL (KRANZ)PARADIGM, Gerald E. Edwards, Vincent R. Franceschi,and Elena V. Voznesenskaya 173

MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,Tai-ping Sun and Frank Gubler 197

PHYTOESTROGENS, Richard A. Dixon 225

DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES,Jeffrey F. Harper, Ghislain Breton, and Alice Harmon 263

PLASTID TRANSFORMATION IN HIGHER PLANTS, Pal Maliga 289

SYMBIOSES OF GRASSES WITH SEEDBORNE FUNGAL ENDOPHYTES,Christopher L. Schardl, Adrian Leuchtmann, Martin J. Spiering 315

TRANSPORT MECHANISMS FOR ORGANIC FORMS OF CARBON ANDNITROGEN BETWEEN SOURCE AND SINK, Sylvie Lalonde,Daniel Wipf, and Wolf B. Frommer 341

vii

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March 24, 2004 1:1 Annual Reviews AR213-FM

viii CONTENTS

REACTIVE OXYGEN SPECIES: METABOLISM, OXIDATIVE STRESS,AND SIGNAL TRANSDUCTION, Klaus Apel and Heribert Hirt 373

THE GENERATION OF Ca2+ SIGNALS IN PLANTS,Alistair M. Hetherington and Colin Brownlee 401

BIOSYNTHESIS AND ACCUMULATION OF STEROLS, Pierre Benveniste 429

HOW DO CROP PLANTS TOLERATE ACID SOILS? MECHANISMS OFALUMINUM TOLERANCE AND PHOSPHOROUS EFFICIENCY,Leon V. Kochian, Owen A. Hoekenga, and Miguel A. Pineros 459

VIGS VECTORS FOR GENE SLIENCING: MANY TARGETS,MANY TOOLS, Dominique Robertson 495

GENETIC REGULATION OF TIME TO FLOWER IN ARABIDOPSIS THALIANA,Yoshibumi Komeda 521

VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,Eric Lam, Naohiro Kato, and Koichi Watanabe 537

THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,Jan Smalle and Richard D. Vierstra 555

RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,Stephen P. Long, Elizabeth A. Ainsworth, Alistair Rogers,and Donald R. Ort 591

INDEXESSubject Index 629Cumulative Index of Contributing Authors, Volumes 45–55 661Cumulative Index of Chapter Titles, Volumes 45–55 666

ERRATAAn online log of corrections to Annual Review of Plant Biologychapters may be found at http://plant.annualreviews.org/

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VIGS VECTORS FOR GENE SILENCING: Many Targets, Many Tools

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