supplemental carbonell plant cell 2012

ADH …TIIFGAA…IFYRDGVS…IVPPAYYAHLAAF…

DAH …TIIFGAD…IFYRAGVS…IVPPAYYAHLAAF…

DDA …TIIFGAD…IFYRDGVS…IVPPAYYAALAAF…

DDD …TIIFGAD…IFYRDGVS…IVPPAYYADLAAF…

…TIIFGAD…IFYRDGVS…IVPPAYYAHLAAF… DDH (wt)

AG

O1:H

A-A

GO

1

Or

35S

:3xH

A-A

GO

1

C

AAH …TIIFGAA…IFYRAGVS…IVPPAYYAHLAAF…

DAA …TIIFGAD…IFYRAGVS…IVPPAYYAALAAF…

ADA …TIIFGAA…IFYRDGVS…IVPPAYYAALAAF…

AAA …TIIFGAA…IFYRAGVS…IVPPAYYAALAAF… 35S

:HA

-AG

O1

…TIIFGAD…IFYRAGVS…IVPPAYYAHLAAF…

…TIIFGAD…IFYRDGVS…IVPPAYYAHLAAF…

DAH

DDH (wt)

35S

:HA

-A

GO

10- E

D

ADH …VIFMGAA…IFFRDGVS…IVPPAYYAHLAAY…

DAH …VIFMGAD…IFFRAGVS…IVPPAYYAHLAAY…

DDA …VIFMGAD…IFFRDGVS…IVPPAYYAALAAY…

DDD …VIFMGAD…IFFRDGVS…IVPPAYYADLAAY…

…VIFMGAD…IFFRDGVS…IVPPAYYAHLAAY… DDH (wt)

AG

O7:H

A-A

GO

7

…TIIFGAD…IFYRDGVS…IVPPAYYAHLAAF… At AGO1 (DDH)

…VMFIGAD…VIFRDGVS…LVPPVYYADMVAF… At AGO2 (DDD)

A

…TIIFGAD…IFYRDGVS…IVPPAYYAHLAAF… At AGO10 (DDH)

…VIFMGAD…IFFRDGVS…IVPPAYYAHLAAY… At AGO7 (DDH)

ADD …VMFIGAA…VIFRDGVS…LVPPVYYADMVAF…

DAD …VMFIGAD…VIFRAGVS…LVPPVYYADMVAF…

DDA …VMFIGAD…VIFRDGVS…LVPPVYYAAMVAF…

…VMFIGAD…VIFRDGVS…LVPPVYYADMVAF… DDD (wt)

DDH …VMFIGAD…VIFRDGVS…LVPPVYYAHMVAF…

B

AG

O2

:HA

-AG

O2

O

r 3

5S

:HA

-AG

O2

SUPPLEMENTAL FIGURE 1. AGO1, AGO2, AGO7 and

AGO10 wild-type (wt) and mutant constructs.

Partial alignment of the PIWI domains of

(A) A. thaliana AGO1, AGO2, AGO7 and AGO10,

(B) AGO2 wt and active-site substitution forms expressed

through 35S or authentic regulatory sequences,

(C) AGO1 wt and multiple combination active-site

substitution forms expressed through 35S or authentic

regulatory sequences,

(D) AGO7 wt and active-site substitution forms expressed

through authentic regulatory sequences, and

(E) AGO10 wt and active-site substitution forms

expressed through 35S regulatory sequences.

Key residues of the catalytic triad are in bold, those found

in wt sequences are in black, and those mutated are in

red.

Supplemental Data. Carbonell et al. (2012). Plant Cell 10.1105/tpc.111.099945

HA-AGO2

L-Rubisco

HA-AGO2

L-Rubisco

ag

o2

-1 AGO2:HA-AGO2 (ago2-1)

AD

H

DA

H

DD

A

DD

H

Co

l-0

DD

D

(w

t)

- 153 kDa

ag

o2

-1 AGO2:HA-AGO2 (Col-0)

Ve

cto

r (C

ol-0

)

DD

D

(w

t)

AD

H

DA

H

DD

A

DD

H

Co

l-0

- 153 kDa

Ve

cto

r (a

go

2-1

)

A B

SUPPLEMENTAL FIGURE 2. Accumulation of HA-AGO2 forms in Arabidopsis (A) Col-0 and (B) ago2-1 T3 transgenic

lines expressing different AGO2 forms.

L-Rubisco blot is shown as loading control.


No transgene ADH DDA DDH (wt) DDD

AGO1:HA-AGO1

Vector

Col-0

A

DAH

B

-21 nt

-145 kDa

Rela

tive

Accum

ula

tion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Col-0

AD

H

DA

H

DD

A

DD

D

AGO1:HA-AGO1 (Col-0)

Vecto

r (C

ol-0)

DD

H

(w

t)

ago1-2

5

HA-AGO1

L-Rubisco

U6

tasiR255

tasiR255

HA-AGO1

A. thaliana Col-0 transgenic plants

Col-0

No transgene ADH DAH DDA DDH (wt) DDD

AGO7:HA-AGO7

Vector No transgene

No transgene

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8

Col-0

AD

H

DA

H

DD

A

DD

D

tasiR2142

HA-AGO7


Vecto

r (C

ol-0)

DD

H

(w

t)

zip

-1

HA-AGO7

L-Rubisco

U6

tasiR2142

Rela

tive

Accum

ula

tion

-21 nt

-137 kDa

A. thaliana Col-0 transgenic plants

ago1-25

zip-1

SUPPLEMENTAL FIGURE 3. Phenotypic and molecular analyses of Arabidopsis Col-0 transgenic plants expressing

wild-type (wt) or active-site substitution AGO1 and AGO7 forms, respectively.

(A) Pictures of 21 days-old T1 transgenic plants expressing the AGO1 (top panel) or the AGO7 (bottom panel) forms.

(B) Accumulation of TAS-dependent tasiRNAs. Left, accumulation of TAS1c-dependent tasiRNA (tasiR255) and HA-

AGO1 forms in Col-0 T1 transgenic lines. Mean (n=3) relative to tasiR255 (dark blue) and HA-AGO1 (black) levels +

s.d. (Vector and DDH (wt) =1.0 for tasiR255 and HA-AGO1, respectively). Right, accumulation of TAS3a-dependent

tasiRNA (tasiR2142) and HA-AGO7 forms in Col-0 T1 transgenic lines. Mean (n=3) relative to tasiR2142 (green) and

HA-AGO1 (dark orange) levels + s.d. (Col-0 and DDH (wt) =1.0 for tasiR2142 and HA-AGO7, respectively. Other

details are as in Fig. 4B.


B

0

1

2

3

4

5

6

7

8

9

Col‐0

zip‐1

Vector

DDH

ADH

DAH

DDA

DDD

Co

l-0

AD

H

DA

H

DD

A

DD

D

AGO7:HA-AGO7 (zip-1)

Ve

cto

r (z

ip-1

)

DD

H

(w

t)

zip

-1

Le

af n

um

be

r

0

1

2

3

4

5

6

7

8

Col‐0

zip‐1

Vector

DDH

ADH

DAH

DDA

DDD

Co

l-0

AD

H

DA

H

DD

A

DD

D


Ve

cto

r (C

ol-0

)

DD

H

(w

t)

zip

-1

Le

af n

um

be

r

A

Bla

de le

ng

ht/petiole

length

Leaf number

0

1

2

3

4

1 2 3 4 5 6 7

Col-0

zip-1

Col.0"

Zip.1

Vect

Vector (Col-0)

DDH

ADH

DAH

DDA

DDD


7"

DDH"(zip.1)"

ADH"(zip.1)"

DAH"(zip.1)"

DD

DDD"(zip.1)"

Col.0"

Zip.1Vect

DDH"(zip.1)"

0

1

2

3

4

1 2 3 4 5 6 7

Bla

de le

ng

ht/petiole

length

Leaf number

DDH"(zip.1)"

ADH"(zip.1)"

7"

DD

DDD"(zip.1)"

Col.0"

Zip.1

zip-1

Vector (zip-1)

ADH

DAH

DDA


Col-0

DDH

DDD


Col.0"

Zip.1

Vect

DDH"(zip.1)"

DDH"(zip.1)"

ADH"(zip.1)"

DAH"(zip.1)"

DD

DDD"(zip.1)"

SUPPLEMENTAL FIGURE 4. Phenotypic analyses of Arabidopsis zip-1 and Col-0 T1 transgenic plants expressing

wild-type (wt) or modified AGO7 forms.

(A) Mean (+ s.d.) leaf position at which first abaxial trichomes were detected in zip-1 (left) or Col-0 (right) T1 transgenic

plants.

(B) Ratio of leaf blade length/petiole length for leaves 1-7 in zip-1 (left) or Col-0 (right) T1 transgenic plants.


CA

-145 kDa

D

TAS1c 3’D2(-)

miR173

in HA in HA in HA in HA

35S:GUS DDH (wt)

35S:TAS1c + 35S:MIR173

-22 nt

HA-AGO1

L-Rubisco

HA in HA in

ADH DAH DDA DDD

35S:HA-AGO1

tasiR255 -21 nt

TAS1c -139 bp

-21 nt

Actin -237 bp

U6

1 2 3 4 5 6 7 8 9 10 11 12

Immunoprecipitation analysis N. benthamiana transient assay

B

in HA in HA in HA in HA

DDH (wt) 35S: GUS

35S:MIR173

DDH (wt) DAH

35S:HA-AGO10

35S: MIR390a

35S:TAS1c

HA-AGO10

L-Rubisco

-139 bp

-237 bp Actin

U6

-22 nt

-137 kDa

miR173

miR390a -21 nt

TAS1c

1 2 3 4 5 6 7 8

Immunoprecipitation analysis N. benthamiana transient assay

0.00

0.50

1.00

1.50

2.00

1 2 3 4 5 6 7 8

- + + + + 35S:TAS1c + + + 35S:MIR173 - - + + + + + +

- - - - - DDA - + -

DDD - - - - - + - -

DAH - - - - - - + - ADH - - - - - - + -

DDH (wt) - - - - - - + -

miR173

tasiR255

-22 nt

-21 nt

Rela

tive

Accum

ula

tion

HA-AGO1

L-Rubisco

-145 kDa

TAS1c transcript

16S/25S rRNA

-1 kb

U6

U6

35S

:HA

-AG

O1

TAS1c transcript

tasiR255

1 2 3 4 5 6 7 8

N. benthamiana transient assay

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1 2 3 4 5 6

miR173

tasiR255

-22 nt

-21 nt

Rela

tive

Accum

ula

tion

HA-AGO10

L-Rubisco

-137 kDa

TAS1c transcript

16S/25S rRNA

-1 kb

U6

U6

35S:HA- AGO10

TAS1c transcript

tasiR255

- + + + 35S:TAS1c +

DDH (wt) - - - - +

DAH - - - - +

miR390a -21 nt

35S:MIR173 - - + + +

35S:MIR390a - - - - -

+

+

-

+

-

1 2 3 4 5 6

N. benthamiana transient assay

SUPPLEMENTAL FIGURE 5. AGO1 and AGO10 interactions with small RNAs and target transcripts in N.

benthamiana transient assays overexpressing wild-type (wt) or modified AGO forms.

(A) Immunoprecipitations with HA-AGO1 proteins. Input and immunoprecipitated fractions from N. benthamiana

following co-expression of 35S:miR173 and 35S:TAS1c with 35S:GUS, 35S:HA-AGO1-DDH (wt), 35S:HA-AGO1-ADH,

35S:HA-AGO1-DAH, 35S:HA-AGO1-DDA and 35S:HA-AGO1-DDD were analyzed. Top, EtBr RT-PCR products

corresponding to a non-cleaved fragment from the TAS1c transcript. Middle, miR173 and tasiR255 blots are shown as


controls for HA-AGO1 miRNA/siRNA binding. The TAS1c3’D2(-) panel shows an HA-AGO1-nonassociated tasiRNA

generated from the TAS1c transcript as an immunoprecipitation control.

(B) Effects of wt and modified AGO1 forms on TAS1c targeting and tasiRNA biogenesis. Accumulation of TAS1c

transcript and TAS1c-dependent tasiRNA (tasiR255) in N. benthamiana leaves from assays testing the AGO1 forms.

Top, mean (n=3) relative TAS1c transcript (light blue) and tasiR255 (dark blue) levels + s.d. (lane 2 and lane 3 = 1.0 for

TAS1c transcript and tasiRNA255, respectively). miR173 and HA-AGO1 blots are shown as controls. Other details are

as in Fig. 2.

(C) Immunoprecipitations with HA-AGO10 proteins. Input and IP fractions from N. benthamiana following co-

expression of 35S:MIR173 and 35S:TAS1c with 35S:GUS, 35S:HA-AGO10-DDH (wt) and 35S:HA-AGO10-DAH, as

well as co-expression of 35S:MiR390a with 35S:TAS1c and 35S:HA-AGO10-DDH, were analyzed. Top, EtBr RT-PCR

products corresponding to a non-cleaved fragment from the TAS1c transcript. EtBr RT-PCR products from N.

benthamiana actin are shown as controls. Middle, miR173 blot is shown as control for HA-AGO10 binding. The

miR390 panel shows a HA-AGO10-nonassociated miRNA and serves as immunoprecipitation control. Bottom, HA-

AGO10 blots. Other details are as in A.

(D) Effects of AGO10 catalytically active and inactive forms on TAS1c targeting and subsequent tasiRNA biogenesis.

Accumulation of TAS1c transcript and tasiR255 in N. benthamiana leaves from assays testing AGO10 forms. miR173,

miR390 and HA-AGO10 blots are shown as controls. Constructs were co-expressed as indicated above the blot

panels. Top, mean (n=3) relative TAS1c transcript (light blue) and tasiR255 (dark blue) levels + s.d. (lane 2 and lane 3

= 1.0 for TAS1c transcript and tasiR255, respectively). Other details are as in B.

SUPPLEMENTAL FIGURE 5 (Cont.)


SUPPLEMENTAL FIGURE 6. AGO1 interactions with small RNA and target transcripts in N.

benthamiana transient assays overexpressing AGO1 wild-type (wt), double or triple active-site

substitution forms together with miR173 and TAS1c.

(A) Immunoprecipitations with HA-AGO1 proteins. Input (in) and immunoprecipitated (HA)

fractions from N. benthamiana following co-expression of 35S:miR173 and 35S:TAS1c with

35S:GUS, 35S:HA-AGO1-DDH (wt), 35S:HA-AGO1-AAH, 35S:HA-AGO1-DAA, 35S:HA-AGO1-

ADA and 35S:HA-AGO1-AAA were analyzed. Other details are as in Fig. 5B and Sup. Fig. 5A.

(B) Effects of AGO1 wt and modified forms on TAS1c targeting and tasiRNA biogenesis. Other

details are as in Sup. Fig. 5B.

A

B

TAS1c 3’D2(-)

miR173

in HA in HA in HA

35S: GUS DDH (wt)

35S:TAS1c + 35S:MIR173

-22 nt

HA-AGO1

L-Rubisco

HA in

AAH DAA

35S:HA-AGO1

tasiR255 -21 nt

-21 nt

HA in HA in

ADA AAA

U6

-145 kDa

TAS1c

Actin

-139 bp

-237 bp

1 2 3 4 5 6 7 8 9 10 11 12

0.00

0.50

1.00

1.50

2.00

1 2 3 4 5 6 9 10

miR173

tasiR255

-22 nt

-21 nt

Rela

tive

Accum

ula

tion

HA-AGO1

L-Rubisco

-145 kDa

U6

U6

- + + + + 35S:TAS1c + + +

35S:MIR173 -

- + + + + + +

TAS1c transcript

25S/16 rRNA

- - - - - + - - DAA

- - - - - + - - AAH

- - - - - + - - DDH (wt)

- - - - - - + - AAA

- - - - - - + - ADA

TAS1c transcript

tasiR255

35S

:HA

-AG

O1

1 2 3 4 5 6 7 8

-1 kb


% transformants/seeds plated

Days to bolting (mean, days)

Flowering time (mean, days)

Seeds/plant (mean)

Supplemental Table 1. Phenotypic analyses of Arabidopsis ago1-25 and Col-0 transgenic plants expressing wild-type and modified AGO1 constructs

Plant Genotype

b

a At least 15000 seeds for each transformed construct were screened (n>15000).

b

c a

Sterile plants/ Total plants

Plant height (mean, cm)

d Analyses were done in 40 days-old plants

n.d.: not determined e

not determined as a consequence of strong developmental defects in inflorescences that impede to score accurately the exact day when the first flower opens f

Plants bolting/ Total plants

23.56 +/- 2.79 0.57

0.65

0.55

0.41

0.38

0.61

23.27 +/- 1.92

22.75 +/- 1.39

22.52 +/- 1.03

23.24 +/- 1.66

22.52 +/- 0.77

25.06 +/- 2.69

25.36 +/- 2.06

24.06 +/- 1.39

24.32 +/- 1.35

25.14 +/- 1.66

24.19 +/- 1.11

Vector (Col-0)

DDH (wt)

ADH

DAH

DDA

DDD


28.42+/- 4.66

25.87 +/- 4.90

26.67 +/- 5.00

28.82 +/- 4.61

21.77 +/- 3.22

32.88 +/- 4.41

18/18

33/33

32/32

31/31

23/23

31/31

0/18

0/33

0/32

0/31

0/23

0/31

Col-0 23.41 +/- 1.45 n.d. 24.85 +/- 1.63 e 29.70 +/- 4.50 27/27 0/27

A plant is considered to have bolted when its main bolt reaches 1 cm in height. c Flowering time is measured as the age of the plant (in days) when the first flower is opened

27.85+/- 2.19 0.57

0.65

0.55

0.41

0.38

0.61

23.06 +/- 1.22

28.11 +/- 2.31

28.71 +/- 2.34

26.72 +/- 1.66

22.31 +/- 1.39

31.30 +/- 2.15

24.88 +/- 1.26

n.d.

n.d.

n.d.

25.31 +/- 1.93

Vector (ago1-25)

DDH (wt)

ADH

DAH

DDA

DDD

AGO1:HA-AGO1 (ago1-25)

15.15 +/- 3.33

25.36 +/- 2.06

2.41 +/- 2.86

4.57 +/- 4.11

7.56 +/- 4.83

33.70 +/- 4.43

e, f

26/26

32/32

19/33

28/32

29/32

29/29

0/26

0/32

32/33

31/32

31/32

0/29

e, f

e, f

ago1-25 26.81 +/- 1.90 n.d. 31.10 +/- 2.23 16.50 +/- 3.05 29/29 0/29 e

d

n.d. e

n.d. e

n.d. e

8561.16 +/- 3907.86

9660.41 +/- 3254.39

5565.12. +/- 3310.77

8550.55 +/- 4269.54

7860.63 +/- 4623.67

6490.45 +/- 3285.34

6154.78+/- 2687.63

8762.97+/- 3142.89

7796.71 +/- 3328.3

9881.48 +/- 8612.21

8083.55 +/- 2552.95

Supplem

ental Data. C

arbonell et al. (2012). Plant C

ell 10.1105/tpc.111.099945

Supplemental Table 2. Oligonucleotides useda,b,c.

Name Sequence (5'->3')

3xHA-AGO1-F caccATGGCCTATCCTTATGATGTACCTGATTATGCCTACCCATACGACGTTCCAGACTACGCTTACCCATACGACGTTCCA

GACTACGCTGTGAGAAAGAGAAGAACGGATGC

3xHA-AGO10-F caccATGGCCTATCCTTATGATGTACCTGATTATGCCTACCCATACGACGTTCCAGACTACGCTTACCCATACGACGTTCCA

GACTACGCTCCGATTAGGCAAATGAAAGATAG

ACT-Benth 123-F CAGCCACACTGTCCCAATTTATGAG

ACT-Benth 480-R TGGATTCCGGCAGCTTCCATTC

ACT2-F GCC ATC CAA GCT GTT CTC TC

ACT2-R GAA CCA CCG ATC CAG ACA CT

Adaptor 1 ACACTCTTTCCCTACACGACGCTCTTCCGATC*T

Adaptor 2 /5Phos/G*ATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

AGO1-3'UTR-AscI-F caccGGCGCGCCGTTGATTCACCCTCTATCTATC

AGO1-3'UTR-R TTTAGGCATTTTCCACGCAAACC

AGO1-ADH-F TATATTTGGTGCTGCTGTTACCCACCCTCACCC

AGO1-ADH-R TGAGGGTGGGTAACAGCAGCACCAAATATAATGG

AGO1-DAH-F CATCTTCTACAGGGCTGGAGTCAGTGAGGG

AGO1-DAH-R CCTCACTGACTCCAGCCCTGTAGAAGATGA

AGO1-DDA-F CCCTGCATATTATGCAGCTCTAGCAGCTTTTAG GGC

AGO1-DDA-F CCCTAAAAGCTGCTAGAGCTGCATAATATGCAGGGG

AGO1-DDD-F CCTGCATATTATGCAGATCTAGCAGCTTTTAGGGC

AGO1-DDD-F CCTAAAAGCTGCTAGATCTGCATAATATGCAGGGG

AGO1-Prom-F caccCCATGGGAATTCGCGGCCGCCGCTTGTTAAAACTCATAATC

AGO1-Prom-NotI-R CCATGGGAATTCGCGGCCGCGATGATTCCTGTGAAAATAAC

AGO1-R TCAGCAGTAGAACATGACAC

AGO10-3pUTR-F2 caccGTGCACTCGGTCGGTCTCTATAGTTCC

AGO10-3pUTR-R2 TATTCTTTTTCCAAATATGGCCGAG

AGO10-D795A-F CGAATTATCTTTTATCGTGCTGGAGTAAGCGAAGGGC

AGO10-D795A-R GCCCTTCGCTTACTCCAGCACGATAAAAGATAATTCG

AGO10-R TTAGCAGTAGAACATTACTCTC

AGO2-ADD-F TGTTCATTGGTGCTGCTGTCAATCATCCCGC

AGO2-ADD-R GCGGGATGATTGACAGCAGCACCAATGAACA

AGO2-DAD-F TGTGATATTCCGTGCTGGTGTCAGCGATGC

AGO2-DAD-R GCATCGCTGACACCAGCACGGAATATCACA

AGO2-DDA-F GCCGGTGTATTATGCTGCCATGGTTGCTTTTAGAGG

AGO2-DDA-R CCTCTAAAAGCAACCATGGCAGCATAATACACCGGC

AGO2-DDH-F CGCCGGTGTATTATGCTCACATGGTTGCTTTTAGAGG

AGO2-DDH-R CCTCTAAAAGCAACCATGTGAGCATAATACACCGGCG

amiR173-5'A-F TGTAATCGCTTGCAGAGAGAAATCACATGATGATCACATTCGTTATCTATTTTTTGTGATTCTCTGTGTAAGCGAT

amiR173-5'A-R AATGATCGCTTACACAGAGAATCACAAAAAATAGATAACGAATGTGATCATCATGTGATTTCTCTCTGCAAGCGAT

AP2-CS-F CTGAGAACCACCGGTTTGAT

AP2-CS-R TGTGATGATGAGGAGAGAATCC

ARF16-CS-F TCCCAAGCAATCCCCTTATT

ARF16-CS-R TGTAAACCCACGGGAACATT

Cloning Linker 1 /5rApp/CTGTAGGCACCATCAAT/3ddC/

P5-Primer AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

P7-modban AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

PE-F AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

PE-R CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

RNA 5' adapter GUUCAGAGUUCUACAGUCCGACGAUC

RT-Primer ATTGATGGTGCCTACAG

SCL6-CS-F AGCTGGTTAAGGCAGCAGAG

SCL6-CS-R ACGGGAGAAGAGAGCTGTTG

SPL2-CS-F CACACATGGGTGCTTCTCAA

SPL2-CS-R AAGGGTAAAACGCCTTGGTT

TAS1c-573-5’RACE AGCAACTGTTCTTTAGACGACTTGAAAATCTCAT

TAS1c-707-5’RACE GATGATGCTTCTTCGCTACACCTCGGAG

TAS1c-A388T-F GTGATTTTTCTCTACAAGCGATTAGACCATTTATCGGTGG

TAS1c-A388T-R CCACCGATAAATGGTCTAATCGCTTGTAGAGAAAAATCAC

TAS1c-CS-F CAATTTTCACCAGCCATGTG

TAS1c-CS-F CCACCGATAAATGGTCTATTCG

TAS3a-CS-F TTCGTTCGAGTCATTTTCTCC

TAS3a-CS-R AGAAAAACGTCAACTTCTTTATTGA


Name Sequence (5'->3')

TUB8-F GCGAGAGATTCTTCACATACAAGGT

TUB8-R CGTTGTAGTAGACATTGACTCGTTC

anucleotides differing from the wt sequences are highlighted in bold.bextra nucleotides added for pENTR D-TOPO cloning are in small letters. c * - Phosphorothioate bond

/5Phos/ - 5' phosphorylation

/5rApp/ - 5' adenylated

/3ddC/ - 3'-end blocked with a dideoxy-C base


Supplemental Table 3. Probes for small RNA Northern blot.

Oligonucleotides Sequence (5'->3')aHybridization temperature (°C)

(-)miR156_DNA G+TGC+TCA+CTC+TCT+TCT+GTC+A 56

(-)miR160_LNA T+GGC+ATA+CAG+GGA+GCC+AGG+CA 62

(-)miR171_DNA GATATTGGCGCGGCTCAATCA 38

(-)miR172_LNA A+TGC+AGC+ATC+ATC+AAG+ATT+CT 50

(-)miR173_DNA GTGATTTCTCTCTGCAAGCGAA 38

(-)miR173-5'A_DNA GTGATTTCTCTCTGCAAGCGAT 38

(-)miR390_DNA GGCGCTATCCCTCCTGAGCTT 38

(-)tasiR255_LNA T+ACG+CTA+TGT+TGG+ACT+TAG+AA 40

(-)tasiR2142_LNA G+GGG+TCT+TAC+AAG+GTC+AAG+AA 45

(-)TAS1c3'D2(-)_LNA T+ATT+CTA+AGT+CCA+ACA+TAG+CG 55

(-)U6_DNA AGGGGCCATGCTAATCTTCTC 38

aLNA nucleotides are followed by a '+' sign.


supplemental carbonell plant cell 2012

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