Transcript Establishment of a System to Replicate, Purify, and Use

Establishment of a System to Replicate,
Purify, and Use a Mutant RNA Virus to
Study the Antiviral Defense Response
in Plants
Katie Brempelis
Mentors: Dr. James C. Carrington, Dr. Kristin Kasschau,
Dr. Hernan Garcia-Ruiz
Dr. James C. Carrington Lab
HHMI Program, Summer 2008
BACKGROUND
• RNA Silencing
• Used in antiviral defense and gene regulation
• mRNA and viral ssRNA degradation, modification of DNA and
histones, and translational repression
• What happens when a plant is infected by a virus?
• The plant enacts antiviral RNA silencing, producing virusderived small RNAs (siRNAs)
• The virus produces suppressors proteins that counteract the
plant’s RNA silencing response
• Experiment Model
• Arabidopsis thaliana and Nicotiana benthamiana
• TuMV-GFP (Turnip mosaic virus with green fluorescent
protein)wild type virus
• TuMV-GFP-AS9 (silencing suppression deficient)mutant virus
Components of RNA Silencing Pathways
• Proteins:
• DCLs- dsRNA-specific ribonucleases
• RDRs- RNA-dependent RNA polymerases
• AGOs- RNAse
• RISC- RNA Induced Silencing Complex
• RNA Components:
• miRNA- gene regulation
• tasiRNA- gene regulation
• siRNA- antiviral defense
RNA Silencing
-hpRNA
dsRNA
-viral RNA
Dicer
siRNAs
21-26 nt
RISC
forms
-Effector complex
-Contains AGO protein
-Incorporates one strand
Targeted RNA
cleaved by DCLs
-mRNA
-viral ssRNA
RDR forms
dsRNA
- Signal Amplification
Proposed Model
• The Proposed Three-Phase Model for
Antiviral Silencing
•DCLs- recognition
and cleavage
•AGO- part of RISC
•RDRs- amplification
MOTIVATION
• Effect of mutating A. thaliana genes
is masked by the virus-encoded
silencing suppressor
• A suppressor-deficient virus is
needed to provide a clear distinction
between plant genes required and
dispensable for antiviral RNA
silencing
upload.wikimedia.org
SPECIFIC AIMS
• Purification of TuMV-GFP-AS9
• To determine the requirement of A.
thaliana genes in antiviral silencing
people.whitman.edu
Purification of TuMV-GFP-AS9
• Established protocol for wild type virus, using
N. benthamiana as a host
• Hypothesis: A highly concentrated inoculum
of the mutant virus can be prepared using a
similar approach
• Prediction: A semi-pure mutant virus preparation
will be highly infective
• Method:
Injection with
Agrobacterium
www.sgn.cornell.edu
Purify
virus
www.focuscience.org
Titrate on
dcl2/3/4
mutants
Maximizing Efficiency of AS9 Harvest
• What is the best day to harvest AS9-infected
N. Benthamiana leaves?
Conclusions:
•GFP does not necessarily indicate virus
accumulation
•Highest viral accumulation at 5 and 6 dpi
•Leaves were senescing
•Harvest leaves at 4 dpi
Measuring Infectivity
• What is the infectivity of the AS9 prep?
Average GFP Foci per
Leaf
Titration of Wild Type TuMV-GFP and
Mutant AS9
Conclusions:
-The AS9 viral prep
is infective
30
25
20
15
10
5
0
1x Mutant
1X Wt
X/5 Wt
Virus
X/10 Wt
-Use a 20-fold
dilution of the wild
type prep for similar
infectivity with the
mutant AS9 prep
Proposed Model
• The Proposed Three-Phase Model for
Antiviral Silencing
•DCLs- recognition
and cleavage
•AGO- part of RISC
•RDRs- amplification
Determining A. thaliana genes required
in antiviral silencing
• Hypothesis: Both DCL1 and RDR6 proteins are
required for antiviral RNA-silencing
• Prediction: AS9 mutant virus accumulation in dcll-7 and
rdr6-15 mutants will be higher than in Col-0 wild type plants
• Method
Inoculate A.
thaliana Col-0,
dcl1-7, dcl2/3/4,
and rdr1/2/6
Collect
infected
tissue at 7,
10, 15 dpi
Western blot
detection of
TuMV-CP
Inoculate A. thaliana
Col-0, rdr1-1, rdr2-1,
rdr6-15, rdr1/2/6,
dcl2/3/4
Method for DCL1 Experiment
A. thaliana
Genotype
Mutation
Predictions
AS9 Mutant
Wild type
dcl2/3/4
rdr1/2/6
Wild type
Partial
DCL1
activity
Lacks DCL2,
DCL3, & DCL4
dicing activity
Lacks
RDR1,
RDR2, &
RDR6
activity
No
infection
Infection
Infection
Infection
Infection
Infection
Infection
Col-0
Infection
dcl1-7
RESULTS
• AS9 did not infect dcl1-7 mutants
Probed for TuMV-CP, 7 dpi inflorescence clusters
Wt
AS9 Wt
AS9 Wt
AS9 Wt
AS9
Method for RDR Experiment
A. thaliana
Col-0
rdr1-1
rdr2-1
rdr6-15
rdr1/2/6
dcl2/3/4
Lacks
RDR1,
RDR2, &
RDR6
activity
Lacks
DCL2,
DCL3, &
DCL4
dicing
activity
Infection
Infection
Infection
Infection
Infection
Infection
Genotype
Mutation
Wild
type
Lack activity of a single RDR
protein
Predictions
AS 9
Mutant
Wild type
No
infection
Infection
RESULTS
• AS9 infects all single RDR mutants
7 dpi
Conclusions:
Wt
*
•rdr1, rdr2, and rdr6 are all
required in antiviral silencing
Mutant
•rdr1 seems to have the largest
effect in local leaves
*
*Plants with systemic GFP
ACKNOWLEDGEMENTS
• Howard Hughes Medical Institute
• Cripps Scholarship Fund, College of
Science
• Mentors:
• Dr. James C. Carrington
• Dr. Kristin Kasschau
• Dr. Hernan Garcia-Ruiz
• Dr. Kevin Ahern