TuMV, miRNAs and
Arabidopsis thaliana
Amy Shatswell
HHMI Summer Research 2006
Carrington Lab
Some Background
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Arabidopsis thaliana is a plant used to study genetics because of its
short generation time and sequenced genome.
TuMV stands for Turnip Mosaic Virus, a plant virus that infects
Arabidopsis.
Upon TuMV infection of Arabidopsis, virus-derived siRNAs are
produced from the viral RNA
siRNAs bind complementary viral RNA and inhibit translation
(siRNAs act in cis)
miRNAs (microRNAs) are produced from microRNA genes, bind to
complementary messenger RNAs (targets) and inhibit translation
(miRNAs act in trans)
MicroRNAs regulate many genes involved in important
developmental pathways and cell growth (Sullivan and Ganem,
2006).
siRNAs as an Antiviral Defense Mechanism
RDR6: enzyme responsible for
making RNA double stranded.
TuMV RNA
RDR6
|||||||||||||||||||||||||||||||||||
DCL4
DCL4: enzyme responsible for dicing
RNA into short fragments that can be
recognized by RISC.
|||||||||||
|||||||||||
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v-siRNA duplex
AGO
v-siRNA*
CUAUAACCGCGCCGAGUUAGU
AGO: incorporates siRNA and
degrades viral RNA target.
AGO-siRNA is known as RISC
AGO
RISCv-siRNA
How miRNAs are formed
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miRNAs are derived from single-stranded RNAs
that fold back on themselves to form an
imperfectly matched hairpin structure.
These foldback RNA structures are processed by
enzymes to form the 21 to 24 nucleotide-length
miRNAs.
The newly formed miRNAs can repress gene
expression after transcription (Mallory and
Vaucheret, 2006).
DCL1: a dicer enzyme that
is necessary for the
cleavage of all miRNA
precursors.
MIRNA gene
Pol II
CC UU
C
C
GAUAUUGGC UGGUUCA UCAGAU
|:|||||||| || ||||||||| :||:||| ||||||
UGCUCUC UCA GA
CUAUAACCG GCCGAGU AGU CUA
U CU
C
U
5’ AUGAGAGAGU CU
3’
AAA
44nt loop
DCL1
CC UU
C
C
GAUAUUGGC UGGUUCA UCAGAU
||||||||| :||:||| ||||||
CUAUAACCG GCCGAGU AGU CUA
U CU
C
U
5’ AUGAGAGAGU CU
|:|||||||| ||
44nt loop
3’ UGCUCUC UCA GA
DCL1
C
C
5' UAUUGGC UGGUUCA UCACU 3'
||||||| ||||||| |||
3' CUAUAACCG GCCGAGU AGU 5'
C
U
Nuclear export
RISC: the protein complex
that is responsible for
carrying the miRNA to the
target so it can be silenced.
C
C
5' UAUUGGC UGGUUCA UCACU 3'
||||||| ||||||| |||
3' CUAUAACCG GCCGAGU AGU 5'
C
U
AGO1
RISC
components
UAUUGGCCUGGUUCACUCCACU
miRNA*
CUAUAACCGCGCCGAGUUAGU
AGO1
Target
..... .............
CUAUAACCGCGCCGAGUUAGU
RISCmiRNA
AAA
AGO1
Cleaved target
AAA
More Background
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Some viruses encode
miRNAs that interact with the
host’s mRNAs, resulting in
up-regulation or downregulation of certain genes.
Virus-encoded miRNAs
enhance replication in some
viruses (Sullivan and Ganem,
2006).
www.omafra.gov.on.ca/.../ facts/88091f7.jpg
Hypothesis
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TuMV encodes siRNAs that act like
miRNAs (act in trans) to enhance virus
infectivity through regulating the
expression of host genes.
Predictions based on hypothesis
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siRNAs encoded by TuMV are complementary to
Arabidopsis mRNAs
Those siRNAs arise from a microRNA precursorlike structure
TuMV infection will be compromised in plants
mutant for miRNA processing factors
Expression of targeted host genes is downregulated in infected plants
Targeted host genes are involved in viral defense
Experiment 1- Test the prediction that siRNAs encoded by
TuMV are complementary to Arabidopsis mRNAs
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Sequence through our lab’s strain of TuMV.
Generate all possible siRNAs from TuMV
sequence using bioinformatics.
Generate list of host mRNAs complementary to
TuMV siRNAs using bioinformatics.
Results
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TuMV cDNA clone sequenced with four-fold
redundancy (contig submitted to Genbank)
Bioinformatics generated 10594 possible TuMV
siRNAs and a list of Arabidopsis transcripts
putatively targeted by TuMV siRNAs.
TuMV contig Genbank entry:
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
REFERENCE
AUTHORS
TITLE
JOURNAL
REFERENCE
AUTHORS
TITLE
JOURNAL
COMMENT
bankit843647
10644 bp
mRNA
circular VRL 27-SEP-2006
Turnip mosaic virus-GFP mRNA, complete cds.
843647
.
Turnip mosaic virus
Turnip mosaic virus
Viruses; ssRNA positive-strand viruses, no DNA stage; Potyviridae;
Potyvirus.
1 (bases 1 to 10644)
Lellis,A.D., Kasschau,K.D., Whitham,S.A. and Carrington,J.C.
Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an
essential role for eIF(iso)4E during potyvirus infection
Current Biology 12, 1046-1051 (2002)
2 (bases 1 to 10644)
Chapman,E.J., Shatswell,A.L., Lellis,A.D. and Carrington,J.C.
Direct Submission
Submitted (27-SEP-2006) Center for Genome Research and Biocomputing
and Department of Botany and Plant Pathology, Oregon State
University, 3021 ALS, Corvallis, OR 97331, USA
Vector Explanation: Turnip mosaic virus cDNA was engineered to
contain GFP reporter gene
Bankit Comment: Sequence of mRNA encoded by binary plasmid
pCBTuMV-GFP; transcript represents Turnip mosaic virus (strain UK1)
carrying a soluble modified red-shifted GFP reporter.
Experiment 2- Test the prediction that siRNAs siRNAs arise
from a microRNA precursor-like structure
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Predict secondary structure of TuMV RNA using
mFOLD
Compare TuMV to TEV, a related virus known to
form foldback structure, using ClustalW
Results
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2 foldback structures were identified in TuMV
TuMV and TEV are 58% identical in these
regions
Stem-loop
structures
identified in TuMV.
They are very
similar to those
found in TEV
(Haldeman-Cahill et al., J. Virol.
.
1998)
dG = -58.8
dG = -117.1
Comparison of TEV Sequence and
TuMV sequence
Experiment 3: test the prediction that TuMV infection will be
compromised in plants mutant for miRNA processing factors
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Infect RNA pathway mutants
with TuMV that has been labeled
with a GFP marker.
Infect wildtype control plants.
Examine plants under UV light
for several weeks and record
infection progress.
Compare infection in mutant
plants to wildtype plants.
Determine whether infection is
compromised in miRNA
processing mutants.
Photo courtesy of Andrew Lellis
Results of Experiment 3
5 dpi
n = 23
n = 24
n = 24
n = 33
n = 34
50
n = 23
75
n = 30
75
n = 31
100
100
n = 30
% infected
TuMV-GFP Infection 4 dpi
25
25
0
50
Col-0
rdr6-15/dcl4-2
dcl1-7
0
Col-0
rdr6-15/dcl4-2
dcl1-7
inoculum dilution
none
1:10
1:100
% infected
6 dpi
9 dpi
100
100
75
75
50
50
25
25
0
Col-0
rdr6-15/dcl4-2
dcl1-7
0
Col-0
rdr6-15/dcl4-2
dcl1-7
Summary
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TuMV-GFP sequence was determined and
submitted to Genbank
Bioinformatics generated 10594 possible TuMV
siRNAs and a list of putative targets in
Arabidopsis.
Secondary structures of TuMV RNA were
predicted.
Virus infectivity did not appear to depend upon
Arabidopsis genotype.
Future Directions
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Test prediction that expression of predicted
targets is down-regulated in infected plants
(analyze microarray data)
Test prediction that targeted host genes are
involved in viral defense
Acknowledgements
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The Howard Hughes Medical Institute
Elisabeth Chapman
Kristin Kasschau
Jim Carrington