060516_UMH_AC

Nuevas herramientas para el control de la
expresión génica en plantas basadas en
pequeños RNAs artificiales
Alberto Carbonell
www.slideshare.net/AlbertoCarbonell1
acarbonell@ibmcp.upv.es
@A_Carbonell_

Gene Silencing
 Eukaryotic evolutionarily conserved, sequence-specific, RNA-based
gene-inactivation system that regulates key biological processes
Development Stress response
Chromatin structure Pathogen defense
Gene Silencing

Gene Silencing
is important for plant growth
Normal
RNA silencing
Defective
RNA silencing

Gene Silencing
is necessary for proper leaf shape
Defective
RNA silencing
Normal
RNA silencing

Gene Silencing
controls developmental timing
Normal
RNA silencing
Defective
RNA silencing

Gene Silencing
defends plants against diseases
Normal
RNA silencing
Defective
RNA silencing
Plant resistant
to virus
Plant susceptible
to virus

Classes of Gene Silencing
Transcriptional
Gene Silencing
(TGS)
An
*
X
Gene
An
mRNA
Protein
No
Gene Silencing
Post-Transcriptional
Gene Silencing
(PTGS)
An*
X

PTGS in Plants
AGO
dsRNA
target RNA
ssRNA
Intramolecular
(folding)
RNA-dependent
RNA polymerase
DCL
RDR
..............
AGO
..............
AGO
.............. An
sRNA
An
An
Translational
repression
.............
RNA-dependent
RNA polymerase
target RNA
RDR

Small RNA (sRNA) Silencing Pathways
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
HYL1
DCL1
An
MIRNA primary transcript
|||||| ||||||
An
miRNA
targetAGO1
AGO1
SE
miRNA
foldback
miRNA pathway
HEN1miRNA
An
tasiRNA
target
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An
AGO1
DRB4
DCL4
DRB4
DCL4
SDE5
RDR6
SGS3
RDR6
TAS primary transcript
AGO1
tasiRNA pathway
HEN1
AGO
dsRNA
tasiRNA
miRNA

Artificial sRNA Silencing Pathways
hpRNA pathway
hpRNA transgene
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
TAS!primary#transcript!
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!
hpRNA primary transcript
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
HEN1#
AGO#
dsRNA!
tasiRNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||DCL4
DRB4
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
Modified#TAS!primary#transcript!
AGO1#
HEN1#
AGO#
dsRNA!
syntasiRNA+1! syntasiRNA+2!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
||||||||||||||| ||||||||||||||||||||||
||||||||||||||| ||||||||||||||||||||||
HYL1%
DCL1%
An%
ed'MIRNA'primary%transcript'
|||||| ||||||
An%
amiRNA%
target%RNA'AGO1%
AGO1%
SE%
miRNA%
oldback'
HEN1%
amiRNA' amiRNA*'
miRNA%
miRNA*%
An
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
hpRNA
siRNA
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
HYL1%
DCL1%
An%
Modified'MIRNA'primary%transcript'
|||||| ||||||
An%
amiRNA%
target%RNA'AGO1%
AGO1%
SE%
amiRNA%
foldback'
HEN1%
amiRNA' amiRNA*'
amiRNA%
amiRNA*%
s
amiRNA pathway
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
HYL1%
DCL1%
An%
|||||| ||||||
An%
amiRNA%
target%RNA'AGO1%
AGO1%
SE%
amiRNA%
foldback'
HEN1%
amiRNA' amiRNA*'
amiRNA%
amiRNA*%
amiRNA transgene
amiRNA amiRNA*
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!
syn-tasiRNA pathway
An#
syntasiRNA+1#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
syntasiR-1
syntasiR-2
syntasiR-1
syntasiR-2
An#
syntasiRNA+2#
target#RNA!
AGO1#
syn-tasiRNA transgene
syntasiR-1
syntasiR-2
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!
amiRNA pathway
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
|||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||
HYL1%
DCL1%
An%
|||||| ||||||
An%
amiRNA%
target%RNA'AGO1%
AGO1%
SE%
amiRNA%
foldback'
HEN1%
amiRNA' amiRNA*'
amiRNA%
amiRNA*%
amiRNA transgene
amiRNA amiRNA*
An#
tasiRNA#
target#RNA!
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
An#
AGO1#
DRB4#
DCL4#
DRB4#
DCL4#
SDE5#
RDR6#
SGS3#
RDR6#
AGO1#
HEN1#
AGO#
dsRNA!
tasiRNA!

Applications of Plant Artificial sRNAs
 Study gene function
CONCLUSION:
Gene A is necessary for chlorophyll synthesis
Normal function
of plant gene A
Artificial small RNAs shut
down function of plant gene A

Applications of Plant artificial sRNAs
 Induce antiviral resistance
Niu et al. Nature Biotechnology (2006)

Advantages of Plant Artificial sRNAs
 Spacio and temporal regulation of gene expression
-Tissue specific expression of artificial sRNAs
-Inducible expression of artificial sRNAs
 Study of lethal genes
 Simultaneous silencing of multiple:
-Sequence related genes (e.g. gene family)
-Sequence unrelated genes
 Generation of allelic series with different silencing levels
-Transformation process
-Use of expression promoters of distinct strength
-Fine tune regulation of the artificial sRNA efficacy by modifying base-
pairing interactions between the artificial sRNA and target

Limitations of Artificial sRNA Systems
1. Design (WMD3):
-Non-intuitive interface
-Relatively slow
-No syn-tasiRNA design tool
http://wmd3.weigelworld.org/
1st PCR
amiRNA insert
2nd PCR
BamHI cut
EcoRI cut
Gel purification
Gel purification
Entry vector
Cut pBSK vector with EcoRI
Alkaline Phosphatase treatment
Gel purification
BamHI cut
Entry plasmid
Mini-prep
Pick up positive colony
Confirm the sequencing
Ligation
Transform into E. coli
Entry plasmid
Binary vector
Binary plasmid
Cut with restriction enzyme(s)
Gel purification
Mini-prep
Pick up positive colony
Confirm the replacement of the fragment
Transform into Agro
Ligation
Transform into E. coli
Cut with restriction enzyme(s)
Alkaline Phosphatase treatment
Gel purification2. Cloning:
-Long and slow (multi-step)
-Non cost-effective
-Non-high throughput capability
-Lack of convenient syn-tasiRNA
cloning systems
Schwabb et al., Plant Cell (2006)
3. Expression:
-Frequent miss-processing of
amiRNAs (-> off target effects!)
1 2 3 4 5 6 7 8 9
amiRNAs
-21 nt

This platform includes:
a) Web-based tools for the design of artificial sRNAs
b) A new generation of artificial sRNA vectors
GOAL:
To develop a new platform for the:
1. Design
2. Cloning (high-throughput) and
3. Expression
of plant amiRNAs and syn-tasiRNAs in a simple, fast, cost-
effective and effective manner for specific gene silencing in
plants.

P-SAMS
http://p-sams.carringtonlab.org
Fahlgren et al. Bioinformatics (2016)

P-SAMS
Computational Design of Artificial sRNAs
 Step 1: Identification of all possible target sites in target transcript(s) by
cataloguing all possible 21-nucleotide sequences
 Step 2: Remove target sites that contain 15-nt sequence form positions 6-20 (core
target pairing sequence) that perfectly match a non-target transcript
 Step 3: Target sites are grouped by the core target pairing sequence, only target
site groups that contain all input genes are considered further.
 Step 4: Grouped sites are scored and ranked based on group-wise similarity and
the identity of nucleotides at positions 1, 2, 3 and 21.
 Step 5: For each group site, a guide RNA is designed to target all sites with the
additional criteria that position 1 and 19 are a U and a C, and that position
21 is mismatched
 Step 6: P-SAMS uses TargetFinder to predict target RNAs for each guide RNA.
-Optimal Results: include guide RNAs predicted to target exclusively
transcripts from input genes
-Sub-Optimal Results: guide RNAs predicted to target transcripts from
input genes AND from non-input genes

Precursor Selection For AmiRNA Vectors
Ath-MIR390a For Eudicots
A
B
Carbonell et al. Plant Physiology (2014)
Osa-MIR390 For Monocots
A
B
Carbonell et al. Plant Journal (2015)

Oligonucleotide Design
for Direct AmiRNA Cloning in MIR390-Based Vectors

AmiRNA Cloning in B/c Vectors

Plant expression vectors
AmiRNA B/c Vectors
GATEWAY-compatible
entry vectors

Functionality Of AmiRNA Vectors For Eudicots
G
- U6
- amiRNA
amiR-
Trich
- +
amiR-
Lfy
- +
amiR-
Ch42
- +
amiRNA accumulation
in Arabidopsis transgenic lines
amiR-
Ft
- +
21-
24-
- amiRNA
amiR-CH42
- +
- U6
amiR-
CH42
Severe
Inter-
mediate
Weak
No
phenotype
C
Weak
35S:GUS
Intermediate
Severe
No
phenotype
Silencing of CHLORINA 42 (CH42)
LFY
- +
amiR-Lfy
TargetmRNA
relativeexpression
0
0.2
0.4
0.6
0.8
1
1.2
CH42
- +
amiR-Ch42
RNAaccumulation
amiR-CH42
- +
62/101
25/101
10/101
vector
48/48
35S:GUS
35S:AtMIR390a-Trich
G
- U6
- amiRNA
amiR-
Trich
- +
amiR-
Lfy
- +
amiR-
Ch42
- +
amiRNA accumulation
in Arabidopsis transgenic lines
amiR-
Ft
- +
21-
24-
- amiRNA
- +
- U6
amiR-Trich
amiR-Trichvector
TRY CPC ETC2
- + - + - +
amiR-Trich
RNAaccumulation
LFY
- +
amiR-Lfy
TargetmRNA
relativeexpression
0
0.2
0.4
0.6
0.8
1
1.2
amiR-Trich
- + - + - +
52/5333/33
Silencing of TRICH (TRY, CPC, ETC2)

UC
U AG
C
U
A GACAGGCGUAAGAUUGCG
CGCAAUCUU C GCC UGCUC
A
U
G
C
35S:OsMIR390-AtL-
Bri1
16/20
AC
U UG
C
U
A GACAGGCGUAAGAUUGCG
CGCAAUCUU C GCC UGCUC
A
U
G
C
35S:OsMIR390-
Bri1vector
0/7 7/11
Silencing of BRASSINOSTEROID-
INSENSITIVE 1 (BRI1)
Functionality Of AmiRNA Vectors For Monocots
0
0.2
0.4
0.6
0.8
1
1.2
1.4
amiR-Bri1
- +
BRI1 RNA
-
35S:OsMIR390
35S:OsMIR390-
AtL
+
vector
35S:OsMIR390-
Bri1 AtL-Bri1
21 -
24 -
- amiRNA
- U6
amiR-Spl11
35S:
OsMIR390-
Spl11
35S:
OsMIR390-AtL-
Spl11vector
0/33 8/8 23/23
AC
U UG
C
U
A UGGCUACUGCUCAGAUCG
CGAUCUGAG A GUA GCCCA
U
A
G
A
C
UG
C
U
A UGGCUACUGCUCAGAUCG
CGAUCUGAG A GUA GCCCA
U
A
G
A
U
A
vector
35S:OsMIR390-
Spl11 AtL-Spl11
Silencing of SPOTTED LEAF 11
(SPL11)
- + +
SPL11 RNA
21 -
24 -
- amiRNA
- U6
LFY
- +
amiR-Lfy
TargetmRNA
relativeexpression
0
0.2
0.4
0.6
0.8
1
1.2
vector
35S:OsMIR390
35S:OsMIR390-
AtL

Precursor Selection For Syn-tasiRNA Vectors
Oligonucleotide Design for Cloning in AtTAS1c-
based Vectors

Syn-tasiRNA Cloning in B/c Vectors

pMDC123SB-AtTAS1c-B/c
Gateway-compatible entry clonePlant expression vectors
pMDC32B-AtTAS1c-B/c pENTR-TAS1c-B/c
ccdB
BsaI
BsaIAtTAS1c
5’ 3’
KanRattL2attL
1
ccdB
BsaI
BsaIAtTAS1c
5’ 3’
HygRNosR
B
L
B
2x35S
BsaI
KanR
ccdB
BsaI
BsaIAtTAS1c
5’ 3’
BastaRNosR
B
L
B
2x35S
BsaI
KanR
Syn-tasiRNA B/c Vectors

AtTAS1c-D3&D4-Trich
target mRNA
5’ TCCCATTCGATACTGCTCGCC
||||||||||||||||||:|
3’ AGGGTAAGCTATGACGAGTGA
TRY
TCCCATTCGATACTGCTCGCC 3’
||||||||||||||||||:|
AGGGTAAGCTATGACGAGTGA 5’
AtTAS1c-D3&D4-Ft
target mRNA
5’ TTGGTTATAAAGGAAGAGGCC
|||||||||||||||||:|||
3’ AACCAATATTTCCTTCTTCGG
TTGGTTATAAAGGAAGAGGCC 3’
|||||||||||||||||:|||
AACCAATATTTCCTTCTTCGG 5’
AtTAS1c-D3Trich-D4Ft
target mRNA
||||||||||||||||||:|
|||||||||||||||||:|||
FT
TRY TRY
FT FT
CPC
||||||||||||||:|||:||
3’ AGGGTAAGCTATGATGAGTGG
ETC2
||||||||||| ||:|||:||
3’ AGGGTAAGCTACGATGAGTGA
CPC
||||||||||||||:|||:||
AGGGTAAGCTATGATGAGTGG 5’
ETC2
||||||||||| ||:|||:||
AGGGTAAGCTACGATGAGTGA 5’
CPC
5’ TCCCATTCGATACTGCTCGCC 3’
||||||||||||||:|||:||
3’ AGGGTAAGCTATGATGAGTGG 5’
2x35S:AtTAS1c
miR173
target site
miR173
5’ GUGAUUUUUCUCUACAAGCGAA 3’
|||||||:||||| ||||||||
3’ CACUAAAGAGAGACGUUCGCUU 5’
syn-tasiRNAs
syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+])
AtTAS1c-D3&D4-Trich
target mRNA
||||||||||||||||||:|
TRY
||||||||||||||||||:|
AtTAS1c-D3&D4-Ft
target mRNA
|||||||||||||||||:|||
|||||||||||||||||:|||
AtTAS1c-D3Trich-D4Ft
target mRNA
||||||||||||||||||:|
|||||||||||||||||:|||
FT
AtTAS1c-D3Ft-D4Trich
target mRNA
|||||||||||||||||:|||
FT
||||||||||||||||||:|
TRY
TRY TRY
FT FT
CPC
||||||||||||||:|||:||
3’ AGGGTAAGCTATGATGAGTGG
ETC2
||||||||||| ||:|||:||
3’ AGGGTAAGCTACGATGAGTGA
CPC
||||||||||||||:|||:||
AGGGTAAGCTATGATGAGTGG 5’
ETC2
||||||||||| ||:|||:||
AGGGTAAGCTACGATGAGTGA 5’
CPC
||||||||||||||:|||:||
ETC2
||||||||||| ||:|||:||
3’ AGGGTAAGCTACGATGAGTGA 5’
CPC
||||||||||||||:|||:||
ETC2
||||||||||| ||:|||:||
3’ AGGGTAAGCTACGATGAGTGA 5’
miR173
5’ GUGAUUUUUCUCUACAAGCGAA 3’
|||||||:||||| ||||||||
3’ CACUAAAGAGAGACGUUCGCUU 5’
syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+])
Functionality of Syn-tasiRNA Vectors
35S:GUS Trich Ft
35S:AtMIR390-
35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D
35S:AtMIR390- 35S:AtTAS1c-
vector syntasiRNA-Trich
syntasiRNA-Ft
35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D4Trich
35S:AtMIR390- 35S:AtTAS1c-

 syn-tasiRNA vectors for targeting single or multiple (sequence
unrelated) genes
-Arabidopsis (and close species) vectors: AtTAS1c-based
-In other species if MIR173 is co-expressed
 amiRNA vectors for targeting single or multiple (sequence related)
genes:
-Eudicot vectors: AtMIR390a-based
-Monocot vectors: OsMIR390-AtL-based
Development of a new platform to design, clone and express plant
artificial small RNAs in a simple, fast, cost-effective and effective
manner to silence single or multiple genes in plants
Summary
 P-SAMS webtool with two apps (P-SAMS amiRNA Designer and
P-SAMS syn-tasiRNA Designer) for the automated design of
amiRNAs and syn-tasiRNAs, respectively AtMIR390a-B/c
vector
amiRNA
insert
Oligo annealing
BsaI digestion
+ ligation
E. coli transformation
Plasmid purification
Sequencing
amiRNA
construct
1 h
5 min
Plant
transformation
Day
6
Agrobacterium
liquid culture
Day
7
Day
2
amiRNA
oligos
Agrobacterium
transformation
Day
4
E. coli
liquid culture
Day
1
P-SAMS
amiRNA Design
Oligo ordering
Day
3
1-10 min
amiRNA
sequence

World-Wide Usage Of P-SAMS
Number of Sessions

B/c Vectors are Available @ www.addgene.org

Some Institutions Having Requested B/c Vectors

Viroids
 Single-stranded circular RNA (246-401 nt)
 High secondary structure content
 Do not code for proteins
 Need host factors for replication
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 Plant pathogens
Trifoliate orange
Semancik and Weathers Virology (1972)
Gross et al. Europ. J. Biochem. (1982)
Tomato
Chrysanthemum

Potato Tuber Spindle Viroid
(PSTVd)
Potato Tomato
1
C
G
GA CUAA
A
UU
C
ACACCU
GA
CCUC
CU
GAGCAG
AA
AAGA
A
AA
A
AGAAGGCGG CUCGG
A
GG
A
G
C
UCCCGAG
AA
CCGCUUUUU
C
U
C
U
A
UCUU
AC
UGCUU
C
GGGG
C
G
A
GGGUGU
UU
AGCC
C
U
U
GGAACCGCAGUUGGUUC
C
U
GCUUCAG
G
G
A
UCC C
G
U
GGA
A
A
C
A
A
CUGAAGC
CGGG
G
A
A
A
C
CUGGAGCGA
A
C
U
GGC
A
A
A
GC
GCUGUCGCUUCGG
C
U
AC
U
ACCCG
AAAGG
AC
C
CCUUU
GGUGGGGAGUG
CACCCCUCGCC
C
AC
CCAGCGGCCG
CGCCCGCAGG
AC
CG
AGGAG
UUCCU
UA
CC
AUUCCCG
CGGGUGU
CC
UU
GAAA
C
AGGGU
U
U
U
C
ACCCU
U
C
C
UUUC
20 40 60 80
100
120 140 160
180200220240
260
280300320340
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A C
C
GUGGUUCC
U
G
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1
C
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A
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C
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AA
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A
AA
A
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A
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A
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C
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AA
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C
U
C
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A
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AC
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C
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C
G
A
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C
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C
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G
G
A
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G
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A
A
C
A
A
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CGGG
G
A
A
A
C
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A
C
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A
A
A
GC
GCUGUCGCUUCGG
C
U
AC
U
ACCCG
AAAGG
AC
C
CCUUU
GGUGGGGAGUG
CACCCCUCGCC
C
AC
CCAGCGGCCG
CGCCCGCAGG
AC
CG
AGGAG
UUCCU
UA
CC
AUUCCCG
CGGGUGU
CC
UU
GAAA
C
AGGGU
U
U
U
C
ACCCU
U
C
C
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20 40 60 80
100
120 140 160
180200220240
260
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N. benthamiana
Gross et al. Nature (1978)
Diener Virology (1971)

Goal:
To induce PSTVd resistance in tomato plants using efficient
plant artificial sRNAs:
-amiRNAs
-syn-tasiRNAs

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
A
Modified TA
primary tran
syn-tasiRNAs
TAS1c-
tasiRNAs
(…)
miR173
target site
miR173
dsR
DCL4
Virus A
An
RDR6
AGO1
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
DCL4
AGO1
AGO1 AGO1
AGO1 AGO1
TAS1c-bas
syn-tasiRN
B.
B.
P-SAMS design of multiple amiRNAs
against a given plant virus
(repeat this process for each target virus)
amiRNA
constructs
A.
syn-tasiRNA
construct
Cloning in B/c
amiRNA vector
Plant transformation
Rapid amiRNA screening
(e.g. agroinfiltration
in N. benthamiana)
Selected
sRNAs
Cloning in B/c
syn-tasiRNA vector
Transgenic plant expressing
antiviral syn-tasiRNAs
Analysis of the plant
multiviral resistance
Transgenic plant long lasting
resistant to multiple viruses
sRNA
sequences
Carbonell et al. RNA&Disease (2016)
Methodology

P-SAMS-Based amiRNA Design
164 optimal results
PSTVd(+)-amiRNAs
6 amiRNAs selected*
PSTVd(-)-amiRNAs GUS-amiRNAs
2 amiRNAs selected
148 optimal results 3 optimal results
6 amiRNAs selected*
*Criteria:
-Low score
-Target different genomic locations
-With non-overlapping target sites
1
C
G
G A A C U
A
AAC U
C
G U G G U U C C
U
G
U
G G U U
C
A C A C C U G A C
C U C C
U
G A C A A G
A A
A A G A
A
A A
A
A G A A G G C G G C U C G G
A
G G
A
G
C
UCCCGAG
AA
CCGCUUUUU
C
U
C
U
A
UCUU
A
CUG
C
UC
C
GGGG
C
G
A
GGGUGU
UU
AGCC
C
U
U
GGAACCGCAGUUGGUUC
C
U
G C U U C A G
G
G
A
U C C C
G
U
GGA
A
A
C
A
A
CUGAAGC
C G G G
G
A
A
A
C
C U G G A G C G A
A
C
U
G G C
A
A
A
GC
GCUGUCGCUUCGG
C
U
AC
U
A
CCCG
A A A G G
A C
C
CCUUU
G G U G G G G A G U G
CACCCCUCGCC
C
AC
C C A G C G G C C G
CGCCCGCAGG
A C
CG
A G G A G
UUCCU
U A
CC
A U U C C C G
CGGGUGU
C C
UU
G A A A
C
A G G G U
U
U
U
C
ACCCU
U
C
C
UUUC
20 40 60 80
100
120
140 160
180200220
260
280300320
340
U
G U
U
C
U
C
A
A
AA
AU
U
A
U
U
TCR (11-26)
CCR (81-108;258-283)
(+)-4
(+)-1
(+)-2(+)-3(-)-1 (-)-3
(-)-6
(-)-4
(-)-5 (+)-5
(+)-6
(-)-2
Location of PSTVd-amiRNA Target Sites

0
0.5
1
1.5
2
1 2 3 4 5 6 7 8 9 10
amiR-PSTVd(+)
1 2 3 4 5 6- 1
GUS
2
amiR-GUS
PSTVd
*
* *
- PSTVd
0
0.5
1
1.5
1 2 3 4 5 6 7 8 9 10
amiR-PSTVd(-)
1 2 3 4 5 6- 1
GUS
2
amiR-GUS
PSTVd
*
*
*
- PSTVd
Screening of AmiRNA Anti-PSTVd Activity
Nicotiana benthamiana transient assays
amiRNA
2x35S T
PSTVd
2x35S T
-Collect leaves 2 dpi
-RNA extraction
-Northern-blot analysis
From Wikipedia.org
amiRNAs against PSTVd(-)amiRNAs against PSTVd(+)

Syn-tasiRNAs Against PSTVd
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|||||||||||||||||||
An
PSTVd
syn-tasiRNAs
miR173
target site
miR173
DCL4
RDR6
AGO1
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6
|||||||||||||||||||
|||||||||||||||||||
|||||||||||||||||||
DCL4
AGO1
AGO1 AGO1
AGO1
AGO1
syn-tasiRNA
construct
PSTVd

Acknowledgements
Mockler lab
Todd Mockler
Skyler Mitchell
Kevin Cox
Kevin Reilly
DDPSC
Bioinformatics
Noah Fahlgren
Steven Hill
Carrington lab
Jim Carrington
Atsushi Takeda
Josh T. Cuperus
Daròs lab
José Antonio Daròs
Teresa Cordero
Verónica Aragonés

060516_UMH_AC

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