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Plant viruses are a highly diverse group of pathogens.

Many show a high degree of similarity with animal viruses.

Plant viruses have evolved unique genes/functions to facilitate plant infection.

MovementVector transmission

126 K 183 K30 K

17 K

MT IR Helicase

30 K17 K

17 K

VSGP

VSGP

CAP

CAP

CAP

Cell-to-Cell Movement

Coat Protein

SubgenomicRNA

SubgenomicRNA

tRNAhis

TMV Genome Organization

VSGP

MT IR Helicase Polymerase

Protein Expression ChartSynchronous InfectionsMagic Box

5’

Disassembly

Wound

Progeny Virus

Plant Cell

Virus Entry

Plasmodesmata

Cell-to-Cell Movement Systemic Movement

MP/vRNA/Host Factors

(K. Sutliff)

Virus Life Cycle

Replication

Assembly

Samuels, 1935

Single TMV-GFP infection site on an inoculated leaf of tobacco.

1mm

(Four days post-inoculation)

Replication at leading edgePlasmodesmata gating at leading edge

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HealthyHealthy InfectedInfected

Microarray Data From TMV Infected Whole Leaf Tissues

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

Sheetal Golem, 2003

Transcriptional Alterations During VirusReplication

Andy MauleJohn Innes Center, UK

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Andy Maule Andy Maule

Virus Host

1. Essential for virus functionReplicationMovement

2. Host Resistance Interactions3. Inconsequential to virus function

Disturbance in the host physiologyProteomeTranscriptomeMetabolic Pathways etc.

Virus-Host Interactions

Disease/ResistanceChlorosis, Necrosis

Growth abnormalities

Plant Cell

Virus Host

1. Essential for virus functionReplicationMovement

2. Host Resistance Interactions3. Inconsequential to virus function

Disturbance in the host physiologyProteomeTranscriptomeMetabolic Pathways etc.

Disease/ResistanceChlorosis, Necrosis

Growth abnormalities

Plant Cell

Virus - Host Responses

Compatibleinteractions

Incompatibleinteractions

Sensitive (Disease) Tolerant Resistant Immune

Lellis et al., 2002 Current Biology

Immune Response: Essential Role for eIF(iso)4E in Potyvirus Infection

Genetic identification of loss-of-susceptibility mutants Mechanisms for immune response

eIF-2αP

eIF-2α

Shutoff of Protein Synthesis

Protein Synthesis

PKR PKR P

Double Stranded RNA

Inhibitor of PKR (IPK)

Inhibitor of Protein Kinase RDisease Resistance / Avoidance

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eIF-2αP

eIF-2α

Shutoff of Protein Synthesis

Protein Synthesis

PKR PKR P

Double Stranded RNA

Inhibitor of PKR (IPK)

Inhibitor of Protein Kinase R

TMV Replicase

Influenza virus

Hepatitis C Virus NS5A inhibitsPKR dimerization

Adenovirus VAI RNA bind to dsRNA substratesand inhibit PKR

Bilgin et al., 2003, Developmental Cell

P58-IPK knockout results in necrosis at the site of infection. Interaction w/viral protein importantfor pathogenesis.

HEL

Beta Galactosidase assay for interaction between PAP and Helicase

020406080

100120

ETR1 LEX A WT-TMV-HEL

Interactors

Bet

a ga

lact

osid

ase

acti

vity

in

Mil

ler

Uni

ts

HELICASE INTERACTION WITH PAP1/IAA26:A Tolerance Interactions?

Helicase domain showed a strong interaction with Phytochrome Associated Protein(PAP1) or IAA26.

ARF’s - transcriptional factors that bind to Auxin responsive Elements (AuxRE) found in the promoters of early auxin responsive genes.

Reed J.W, Trends Plant Sci. 2001 6(9)

IAA26 is an auxin responsive regulatory protein and a member of the Aux/IAA family of transcription factors

126 K

183 K

HELMT IR

HELMT IR POL

Replicase Proteins

Regulation (+ or -) of gene expression

Aux/IAAARF

MODEL FOR THE FUNCTION OF ARF AND Aux/IAA PROTEINS

- Auxin

ARF

26Sproteasome

+ Auxin

SCFTIR1

Skp

1

Rbx1Cul1

TIR1

AS

K

Aux/IAAE2

Ub

AuxRE Auxin responsive gene AuxRE Auxin responsive gene

N

126 Replicase -GFP

20 ?m

-IAA26-DsRed

N N

126 Replicase -GFP

20 ?m20 um20 m

-

N

IAA26-DSRED IS HELD OUT OF THE NUCLEUS BY TMV-126 kDa REPLICASE-GFP

NN

NN

TRANSCRIPTIONALLY ALTERED ARABIDOPSIS GENES CONTAIN AUXIN RESPONSIVE ELEMENTS WITHIN THEIR PROMOTERS• Microarray data identified 68 Arabidopsis genes altered in infected tissue

(Golem et al. 2003)

• Promoter analysis of 2 kb region upstream of each gene to identify Aux RE’sTGTCTC element – ARF binding site

• 20 genes contained 2 or more AuxRE’s. ~ 30% of the genes displayingtranscriptional alterations to TMV infection may be linked to auxin response system

AFGC Gene Model ID

Protein name Number of AuxRE’s

Fold expressionIn TMV infection

+50µM IAA

At5g02160At1g19350At3g17790At5g21010At4g38850

Put proteinUnk proteinAcid Phos’tase type 5Put stress protSAUR-AC1

33231

-1.9-2.0-2.3-3.5-1.10

-4.09-1.8-6.6-1.55.08

(from microarray) (qRT-PCR derived)

Not above 95%confidence interval cutoff

• Auxin induced expression trends similar to that seen in TMV infection

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TMV V1087I infection results in attenuated disease symptoms.

Mock TMV-V1087IWT-TMV

Tomato

Arabidopsis

N. benthamiana

?REPLICASE - AUX/IAA INTERACTION ALTERS CELL ACTIVITY?Older/Stressed Tissue-Lower Concentration of Auxin-Lower Levels of TIR1 due to Increased Accumulations

of the Stress-Inducible MicroRNA MIR393 (Sunkar and Zhu, 2004)-Increased Levels of AUX/IAA Proteins.

ARF

SCFTIR1 Skp

1

Rbx1Cul1

TIR1

AS

K

AuxRE Auxin responsive gene

Aux/IAA- ↓Auxin- ↑MIR393

Developmentally Static:Less Suitable for Virus Replication

Regulation (+ or -) of gene expression

TMV-Replicase

ARF

AuxRE Auxin responsive gene

Aux/IAA

Developmentally Active:More Suitable for Virus Replication

Hypersensitive Resistance Against Turnip Crinkle Virus

HRT = R gene protein (NBS-LRR)TIF = NAC family transcription factor

Ren et al., 2005, Virology

Resistance to Papaya Ringspot Virus

Resistance to Zucchini Yellow and Watermelon Mosaic Virus

Resistance to Cucumber Mosaic Virus

D. Gonsalves, Cornell University

Micro RNA (miRNA) = Transcribed directly from the genome and used to regulate various genes.Short Interfering RNA (siRNA) = originate from viruses, mRNAs, transposons, transgenes

RNAi (PTGS, RNA suppression, VIGS)

SilencingsiRNA

Sense RNA

Anti-Sense RNA

Double-Stranded RNA

Virus RNA

MMM M

MET1DNA methylase

DDM1Chromatin remodeling

QDE-3DNA helicase

aberrant RNA

STE3RNA Helicase

AGO1PAZ/Piwidevelopment

SGS2RdRp

SGS3Coiled-coil

ds RNADICERdsRNase rgs-CaM HC-Pro

Suppressors

Sequence Specific RNA degradation22-28 nucleotide RNAs

Endogenous RNA

End-to-End Transgene

Vance & Vaucheret Science Vol. 292,2277

Short Interfering RNA (siRNA) = originate from viruses, mRNAs, transposons, heterochromatic DNA,transgenes

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Viral suppressors of RNAi

Diverse group of viral proteinssharing no common sequences

Disrupt RNAi pathway at differentpoints, RISC formation, amplification,maintenance, systemic signal, etc.

Roth et al., 2004, Virus ResearchElisabeth J. Chapman et al. Genes Dev. 2004; 18: 1179-1186

Developmental defects induced by silencing suppressors from five viruses

Disease responses associated with virus suppression of RNAi

No cleavage in the presence of the suppressor

Scarecrow Transcription Factors

Kasschau et al., 2003, Dev. Cell

James C. Carrington and Kristin D. Kasschau

http://asrp.cgrb.oregonstate.edu/

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Reverse Genetics

Viral genome cloned into a bacterial plasmid andused to modify the genome.

In vitro transcription from plasmid template producesgenomic viral RNA.

Rub inoculate viral RNAdirectly onto leaf surfaces toinitiate infection.

orip BR3 2 2

Am p R

Sp 6 / T7Pr o m o t e r

1 2 6 / 1 8 3 KDaRe p lic a s e

5 4 KDa?

3 0 KDa MP

5 ' CAP

3 ' UTR

Infectious TMV RNA Transcript

COAT P ROTEI N( 1 7 . 6 KDa )

Full-lengthpTMV cDNA

Kpn I

Nco I

EcoR I

In vitro transcriptionWild-type Mutant

Wi1 2 1 2

LinearPlasmidDNA

RNATranscripts

Virus Vectors: (Rationale)

High levels of protein expression (dsRNA)Their genomes can be modifiedTravel systemically throughout the plantInfection occurs rapidly

(Applications)Functional genomics applications - the analysis and commercializationof gene and protein function.

Manufactured proteins and therapeutics - the manufacture of proteins and peptides for medicine, chemistry and agriculture.

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126 K 183 K30 K

17 KVCAP tRNAhis

SGP

CAP

Strategy for foreign gene expression from Tobacco Mosaic Virus Vector

126 K 183 K30 K

17 KVtRNAhis

SGPV

SGP

Polylinker

TMV U1 TMV U2

Promoter Duplication

SubgenomicRNACAP Your GENE

17 KCAPSubgenomic

RNA

17 KCAPSubgenomic

RNA[Ruiz, et al, 1998].

Silencing of host genes to study function. Termed: Virus Induced Gene Silencing (VIGS).Advantages: Rapid, No plant transformation, overcomes functional redundancy, works in different genetic backgrounds

Leafy gene silenced

Functional Genomic Studies Using Plant Virus Vectors

Phytoene desaturase Benedito et al., 2003

Chiba et al., Virology, 2006

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon

Agro-delivered viral replicons

P21 = Silencing suppressor

Beet yellows virus genome

Chiba et al., Virology, 2006

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon