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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/
Searching for miRNAs that target your gene of interest?
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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