TMV Genome Organization
Plant viruses are a highly
diverse group of pathogens.
CAP
Many show a high degree of
similarity with animal
viruses.
SGP
V
183 K
126 K
SGP
V
SGP
V
17 K
30 K
MT
MT
IR
IR
Helicase
Helicase
Polymerase
CAP
Plant viruses have evolved
unique genes/functions to
facilitate plant infection.
Movement
Vector transmission
tRNAhis
17 K
30 K
Subgenomic
RNA
Cell-to-Cell Movement
CAP
17 K
Subgenomic
RNA
Coat Protein
Protein Expression Chart
Synchronous Infections
Magic Box
Virus Life Cycle
Virus Entry
Wound
Replication
5’
Disassembly
Assembly
Progeny Virus
Plant Cell
Plasmodesmata
MP/vRNA/Host Factors
(K. Sutliff)
Cell-to-Cell Movement
Systemic Movement
Samuels, 1935
Single TMV-GFP infection site on an inoculated leaf of tobacco.
(Four days post-inoculation)
1mm
Replication at leading edge
Plasmodesmata gating at leading edge
1
Healthy
Infected
Microarray Data From TMV Infected Whole Leaf Tissues
Transcriptional Alterations During Virus
Replication
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Sheetal Golem, 2003
Andy Maule
John Innes Center, UK
2
Andy Maule
Andy Maule
Virus-Host Interactions
1.1. Essential
Essentialfor
forvirus
virusfunction
function
Replication
Replication
Movement
Movement
2.2. Host
HostResistance
ResistanceInteractions
Interactions
3.3. Inconsequential
Inconsequentialto
tovirus
virusfunction
function
Virus
Virus
Virus - Host Responses
Incompatible
interactions
Compatible
interactions
Sensitive (Disease)
Tolerant
Resistant
Immune
Host
Host
Disturbance
Disturbancein
inthe
thehost
hostphysiology
physiology
Proteome
Proteome
Disease/Resistance
Disease/Resistance
Transcriptome
Transcriptome
Metabolic
MetabolicPathways
Pathwaysetc.
etc.
Chlorosis,
Chlorosis,Necrosis
Necrosis
Growth
Growthabnormalities
abnormalities
Plant
PlantCell
Cell
Immune Response: Essential Role for eIF(iso)4E in
Potyvirus Infection
Disease Resistance / Avoidance
Inhibitor of Protein Kinase R
Double Stranded RNA
Genetic identification of loss-of-susceptibility mutants
Mechanisms for immune response
PKR
PKR
P
Inhibitor of PKR (IPK)
Shutoff of Protein Synthesis
eIF-2α
eIF-2α
P
Protein Synthesis
Lellis et al., 2002 Current Biology
3
Inhibitor of Protein Kinase R
Double Stranded RNA
Adenovirus VAI RNA
bind to dsRNA substrates
and inhibit PKR
Hepatitis C Virus NS5A inhibits
PKR dimerization
PKR
PKR
P58-IPK knockout results in necrosis at the
site of infection.
Interaction w/viral protein important
for pathogenesis.
P
Inhibitor of PKR (IPK)
Shutoff of Protein Synthesis
TMV Replicase
Influenza virus
eIF-2α
eIF-2α
P
Protein Synthesis
Bilgin et al., 2003, Developmental Cell
HELICASE INTERACTION WITH PAP1/IAA26:
A Tolerance Interactions?
MT
IR
HEL
183 K
MT
IR
HEL
MODEL FOR THE FUNCTION OF ARF AND Aux/IAA PROTEINS
Beta Galactosidase assay for interaction between
PAP and Helicase
POL
Helicase domain showed a strong
interaction with Phytochrome
Associated Protein(PAP1) or IAA26.
Beta galactosidase
activity in Miller Units
126 K
- Auxin
+ Auxin
Aux/IAA
120
100
Aux/IAA
80
E2
ARF
60
40
20
0
ETR1
LEX A
SCFTIR1
WT-TMV-HEL
Interactors
ASK
Skp1
Replicase Proteins
Ub
TIR1
Rbx1
Cul1
HEL
IAA26 is an auxin responsive regulatory protein and a member of the
Aux/IAA family of transcription factors
26S
proteasome
ARF
AuxRE
AuxRE
Auxin responsive gene
Auxin responsive gene
Reed J.W, Trends Plant Sci. 2001 6(9)
ARF’s - transcriptional factors that bind to Auxin responsive Elements
(AuxRE) found in the promoters of early auxin responsive genes.
IAA26-DSRED IS HELD OUT OF THE NUCLEUS BY
TMV-126 kDa REPLICASE-GFP
126 Replicase -GFP
IAA26-DsRed
Regulation (+ or -) of gene expression
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’s
TGTCTC element – ARF binding site
• 20 genes contained 2 or more AuxRE’s. ~ 30% of the genes displaying
transcriptional alterations to TMV infection may be linked to auxin response system
N
N
N
N
Fold expression
AFGC Gene
Model ID
Protein name
Number of
AuxRE’s
In TMV
infection
(from microarray)
N
N
N
20 um
?m
m
N
At5g02160
At1g19350
At3g17790
At5g21010
At4g38850
Put protein
Unk protein
Acid Phos’tase type 5
Put stress prot
SAUR-AC1
3
3
2
3
1
-1.9
-2.0
-2.3
-3.5
-1.10
+50µM IAA
(qRT-PCR derived)
-4.09
-1.8
-6.6
-1.5
5.08
Not above 95%confidence interval cutoff
• Auxin induced expression trends similar to that seen in TMV infection
4
TMV V1087I infection results in attenuated
disease symptoms.
N. benthamiana
Tomato
?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.
Developmentally Static:
Less Suitable for Virus Replication
SCFTIR1
ASK
Skp1
- ↓Auxin
- ↑MIR393
Developmentally Active:
More Suitable for Virus Replication
Aux/IAA
TMV-Replicase
Aux/IAA
TIR1
Rbx1
Cul1
Arabidopsis
Mock
WT-TMV
TMV-V1087I
ARF
ARF
AuxRE
AuxRE
Auxin responsive gene
Auxin responsive gene
Regulation (+ or -) of gene expression
Hypersensitive Resistance Against Turnip Crinkle Virus
Resistance to
Cucumber Mosaic Virus
HRT = R gene protein (NBS-LRR)
TIF = NAC family transcription factor
Resistance to Papaya Ringspot Virus
Ren et al., 2005, Virology
RNAi (PTGS, RNA suppression, VIGS)
Resistance to Zucchini Yellow and
Watermelon Mosaic Virus
D. Gonsalves, Cornell University
Vance & Vaucheret Science Vol. 292,2277
Sense RNA
Silencing
siRNA
QDE-3
DNA helicase
MET1
DDM1
DNA methylase
Chromatin remodeling
Anti-Sense RNA
MMMM
aberrant RNA
AGO1
PAZ/Piwi
development
STE3
SGS3
RNA Helicase
Coiled-coil
SGS2
RdRp
Endogenous RNA
Double-Stranded RNA
End-to-End Transgene
Virus RNA
ds RNA
DICER
dsRNase
Micro RNA (miRNA) = Transcribed directly from the genome and used to regulate various genes.
Short Interfering RNA (siRNA) = originate from viruses, mRNAs, transposons, transgenes
Suppressors
rgs-CaM HC-Pro
Sequence Specific RNA degradation
22-28 nucleotide RNAs
Short Interfering RNA (siRNA) = originate from viruses, mRNAs, transposons, heterochromatic DNA,
transgenes
5
Disease responses associated with virus suppression of RNAi
Developmental defects induced by silencing suppressors from five viruses
Viral suppressors of RNAi
Diverse group of viral proteins
sharing no common sequences
Disrupt RNAi pathway at different
points, RISC formation, amplification,
maintenance, systemic signal, etc.
Elisabeth J. Chapman et al. Genes Dev. 2004; 18: 1179-1186
Roth et al., 2004, Virus Research
Scarecrow Transcription Factors
Searching for miRNAs that target your gene of interest?
search the
No cleavage in the
presence of the suppressor
James C. Carrington and Kristin D. Kasschau
http://asrp.cgrb.oregonstate.edu/
Kasschau et al., 2003, Dev. Cell
5 ' CAP
Sp 6 / T7
Pr o m o t e r
Reverse Genetics
ori
p BR3 2 2
Viral genome cloned
into a bacterial plasmid and
used to modify the genome.
1 2 6 / 1 8 3 KDa
Re p lic a s e
Full-length
pTMV cDNA
Am p R
In vitro transcription from
plasmid template produces
genomic viral RNA.
Rub inoculate viral RNA
directly onto leaf surfaces to
initiate infection.
Infectious TMV
RNA Transcript
5 4 KDa
?
3 ' UTR
Kpn I
COATP ROTEI N
( 1 7 . 6 KDa )
EcoR I
(Applications)
3 0 KDa MP
Functional genomics applications - the analysis and commercialization
of gene and protein function.
Nco I
In vitro transcription
Wild-type
1
2
Wi
Virus Vectors:
(Rationale)
High levels of protein expression (dsRNA)
Their genomes can be modified
Travel systemically throughout the plant
Infection occurs rapidly
Manufactured proteins and therapeutics - the manufacture of proteins
and peptides for medicine, chemistry and agriculture.
Mutant
1
2
Linear
PlasmidDNA
RNA
Transcripts
6
Strategy for foreign gene expression from
Tobacco Mosaic Virus Vector
CAP
126 K
SGP
V
17 K
183 K
30 K
CAP
17 K
SGP
V
SGP
V 17 K
Functional Genomic Studies Using Plant Virus Vectors
tRNAhis
Subgenomic
RNA
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
Promoter Duplication
126 K
183 K
tRNAhis
30 K
Polylinker
CAP
TMV U1
TMV U2
CAP
CAP
Your GENE
17 K
Subgenomic
RNA
Subgenomic
RNA
Diverse suppressors of RNA silencing enhance agroinfection by
a viral replicon
Beet yellows virus genome
Leafy gene silenced
[Ruiz, et al, 1998].
Phytoene desaturase Benedito et al., 2003
Diverse suppressors of RNA silencing enhance agroinfection by
a viral replicon
Agro-delivered viral replicons
P21 = Silencing suppressor
Chiba et al., Virology, 2006
Chiba et al., Virology, 2006
7