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 LabHHMI 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 virus-

derived 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

dsRNA

Dicer

RISC forms

Targeted RNA cleaved by DCLs

RNA Silencing-hpRNA

-viral RNA

-Effector complex-Contains AGO protein-Incorporates one strand

siRNAs 21-26 nt

RDR forms dsRNA

- Signal Amplification

-mRNA-viral ssRNA

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

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SPECIFIC AIMS

• Purification of TuMV-GFP-AS9

• To determine the requirement of A. thaliana genes in antiviral silencing

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Purification of TuMV-GFP-AS9

Injection with Agrobacterium

• 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:

Purify virus

Titrate on dcl2/3/4 mutants

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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?

Titration of Wild Type TuMV-GFP and Mutant AS9

0

5

1015

20

25

30

1x Mutant 1X Wt X/5 Wt X/10 Wt

Virus

Ave

rag

e G

FP

Fo

ci p

er

Lea

f

Conclusions:

-The AS9 viral prep is infective

-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 rdr1/2/6Collect 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

Partial DCL1

activity

InfectionInfectionNo infection

Lacks RDR1,

RDR2, & RDR6

activity

Lacks DCL2, DCL3, & DCL4 dicing activity

Infection

Wild type

rdr1/2/6dcl2/3/4dcl1-7Col-0A. thaliana Genotype

Mutation

Predictions

AS9 Mutant

Wild type Infection InfectionInfectionInfection

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

Lack activity of a single RDR protein

InfectionInfectionNo

infection

Lacks RDR1,

RDR2, & RDR6

activity

Infection

Wild type

A. thaliana

Genotype

Mutation

Predictions

dcl2/3/4

rdr1/2/6

rdr6-15rdr2-1rdr1-1Col-0

AS 9Mutant

Wild type InfectionInfection InfectionInfection

Lacks DCL2,

DCL3, & DCL4 dicing activity

RESULTS

Wt

Mutant

*Plants with systemic GFP

*

*

7 dpi

• AS9 infects all single RDR mutants

Conclusions:

•rdr1, rdr2, and rdr6 are all required in antiviral silencing

•rdr1 seems to have the largest effect in local leaves

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