Role of Arabidopsis RNADependent RNA Polymerase Genes
3, 4 and 5 in Antiviral Defense
Nick Lowery
Dr. James C. Carrington
Dr. Hernan Garcia-Ruiz
Oregon State University
HHMI 2010
RNAi
• Major post-transcriptional regulatory pathway
– Regulates expression of endogenous genes
– Regulates exogenous genetic elements
• e.g. Transposons, viruses
• Areas for research / commercial potential :
– Cancer
– Selective gene silencing
– Antiviral silencing
P0
HC-Pro
Virus mRNA secondary
structure?
P38
P19
AGO
Dicing
An
An
DRB4
Endogenous
foldbacks?
DRB
AGO
HEN1
Primary
targeting of
virus mRNA
DCL4
DCL2
DRB
DCL3
Viral
replication
intermediates
?
2b
SGS3
RDR6
SDE5
An
RDR1
RDR2
Amplification
RDR
DRB
DRB4
DRB
DRB
DCL
HEN1
AGO
Secondary
targeting of
virus
mRNA
Systemi
c
spread
An
HC-Pro
AGO
P19
RNA-dependent RNA Polymerases
• In Arabidopsis: 6 RDR genes.
– Functions for RDR1, RDR2 and RDR6 have been
characterized for TuMV infections
• RDR1 and RDR6 are the primary effectors, acting
cooperatively or redundantly
• RDR2 plays a minor role in local infection
– RDR3, RDR4 and RDR5 currently have no biological
function assigned to them
RDR3, RDR4, RDR5
Basal siRNA Biogenesis
TuMV
siRNAs
U6
Col-0
Col-0
rdr1
rdr2
rdr6
dcl2
dcl3
dcl4
Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96
Hypothesis
• RDR3, RDR4 and RDR5 play a role in antiviral
RNA silencing during TuMV infection of
Arabidopsis
– May be primary or secondary
– One, two or all three genes may be involved
Experimental Design
• Knock out genes and infect with virus
– Compare severity of infection to wild-type plants
and dcl triple mutants as controls
• Points that must be considered
– Viral silencing suppressor, HC-Pro
– Masking of effects by other RDR genes
– Generation of mutants
Viral Infection
• TuMV-GFP
– Allows for visualization of infection under UV
– Monitor for ability to establish infection foci and to move
systemically throughout the plant
Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96
Silencing Suppressor
• TuMV encodes a silencing suppressor
protein, HC-Pro
– Argonaute imposter; sequesters siRNA,
so no antiviral response can be mounted
• Solution: TuMV-AS9
Wt
– Mutant virus lacking functional copy of
HC-Pro
– Unable to infect wild-type Arabidopsis
– Wt infection serves as positive control
AS9
Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96
Hierarchical Action of RDRs
• RDR1,2,6 pathway primary for TuMV infection
– Will likely see effects only in absence of primary
pathway, either in whole or in part
– Strategy: gradually remove RDR1,2,6 pathway in
rdr3 rdr4 rdr5 background
1.
2.
3.
4.
5.
rdr3 rdr4 rdr5 in Col-0
rdr3 rdr4 rdr5 in rdr1,2,6 single mutants
rdr3 rdr4 rdr5 in rdr1,2,6 double mutants
rdr null plant
rdr3,4,5 single mutants in rdr1,2,6 mutants
–
used to determine which genes are active
Generation of Mutants
• Single mutants: Salk Institute
• Combination Mutants
– Crosses
• rdr1,2,6 single and combination mutants onto
rdr3,4,5 single mutants
– Transformation
• Required to overcome linkage
• Express artificial miRNA to silence genes
• rdr3 rdr4 rdr5 triple mutants
Progress
• All mutant lines generated
– Both crosses and dipping result in introduction of
one copy of mutant allele
– Need to bring to homozygous condition
Screen for rdr5-3; rdr5-3 X rdr1-1 rdr2-1 rdr6-15, F2 generation
rdr5-3
Wt
RDR5
rdr5-3
heterozygote
amiRrdr345 Expression
Future Projects
• Infect with different viruses
– Different viruses affect different steps in RNAi
pathway during infection
• Mapping experiments
– RDR null plant incapable of producing secondary
siRNA?
• Sequence and map primary siRNA back to TuMV
genome to determine primary target sites.
Acknowledgements
•
•
•
•
Dr. Hernan Garcia-Ruiz
Dr. James C. Carrington
Steen Hoyer, Noah Fahlgren and everyone in the lab
HHMI, the Cripps scholarship & Dr. Kevin Ahern