lozano-durán et al, plant cell (supp data) 28-2-11€¦ · lozano-durán et al. (2011). plant cell...

Supplemental Data. Lozano-Durán et al. (2011). Plant Cell 10.1105/tpc.110.08026

SUPPLEMENTAL DATA

Title: Geminiviruses subvert ubiquitination and inhibit jasmonate

signalling by altering CSN-mediated de-rubylation of SCF E3 ligase

complexes

Mock TYLCSV TYLCSV C2T2C

dsDNA

ssDNA

C2T2C WT

dsDNA

ssDNA

TYLCSV

A

B

Supplemental Figure 1. A. Symptoms of N. benthamiana plants mock-inoculated or inoculated with TYLCSV WT or C2T2C. Total DNA was isolated from systemically infected leaves from five plants for each virus (WT or C2T2C) at 21 dpi, and analyzed on DNA gel blots (2 !g/lane). B. Total DNA isolated from N. benthamiana leaf patches agroinfiltrated with TYLCSV WT or TYLCSV C2T2C at 7 days post-infiltration were analyzed in DNA gel blots (2 !g/lane). All membranes were hybridized with a TYLCSV radiolabeled probe. Bands corresponding to double- (dsDNA) and single- (ssDNA) stranded forms of TYLCSV are indicated.


A

B

Supplemental Figure 2. A. Comparison of the amino acid sequence of CSN5 from different plant species. At CSN5A: CSN5A from Arabidopsis thaliana (Q9FVU9). At CSN5B: CSN5B from A. thaliana (Q8LAZ7). Sl JAB-VFNT: CSN5 from S. lycopersicon cultivar VFNT (Q9FR56). Mt CSN5: CSN5 from Medicago truncatula (B7FI77). Pp CSN5: CSN5 from Physcomitrella patens (A9T9Z3). Os CSN5: CSN5 from Oryza sativa (Q8H936). Zm CSN5: CSN5 from Zea mays (B4FUK9). B. CSN5 phylogram tree. Distances are shown inthe branches.

At CSN5A MEGSSS-----AIARKTWELENNILPVEPTD------SASDSIFHYDDASQAKIQQEKPW 49 At CSN5B MEGSSS-----TIARKTWELENSILTVDSPD------STSDNIFYYDDTSQTRFQQEKPW 49 Sl CSN5 MDALNSYASSAAMAQQTWELENNIVTTDAPSGSAPENSASDAIFHYDDAAQTKFQREKPW 60 Mt CSN5 MEASSS--SSSAIAQQTWELENNIIPMETPS--------DDSIFHYDEAGQAEFQRDKPW 50 Pp CSN5 MEASTS---SAMIARQTWEIENNIVATDSPAAT----SDSDAIFEYDEAAQSSIQQQKPW 53 Os CSN5 MEPTSS----AAMARQTWELENNIPAAASDP------DALDAIYRYDEAAQARVQQEKPW 50 Zm CSN5 MEPTSS----ASIARQTWELENNIPAAASDP------DAMDAIYRYDDASQARAQQEKPW 50 *: .* :*::***:**.* . . * *: **::.*: *::*** At CSN5A ASDPNYFKRVHISALALLKMVVHARSGGTIEIMGLMQGKTEGDTIIVMDAFALPVEGTET 109 At CSN5B ENDPHYFKRVKISALALLKMVVHARSGGTIEIMGLMQGKTDGDTIIVMDAFALPVEGTET 109 Sl CSN5 TSDPHYFKRVKISALALLKMVVHARSGGTIEVMGLMQGKTDGDAIIVMDAFALPVEGTET 120 Mt CSN5 ANDPHYFKRVKISALTLLKMVVHARSGGTIEVMGLMQGKTDADSIIVMDAFALPVEGTET 110 Pp CSN5 TRDPHYFKNVKISALALLKMVVHARSGGTIEVMGLMQGKTDGDTIIIMDAFALPVEGTET 113 Os CSN5 ANDPHPFRRAKISALALLKMVVHARAGGTIEVMGLMQGKCEGDAIVVMDAFALPVEGTET 110 Zm CSN5 ANDPHHFRRTKISALALLKMVVHARAGGTIEVMGLMQGKCEGDAIIVMDAFALPVEGTET 110 **: *:..:****:*********:*****:******* :.*:*::************* At CSN5A RVNAQSDAYEYMVEYSQTSKLAGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQQYQEPF 169 At CSN5B RVNAQDDAYEYMVEYSQTNKLAGRLENVVGWYHSHPGYGCWLSGIDVSTQRLNQQHQEPF 169 Sl CSN5 RVNAQADAYEYMVEYSQTNKQAGRLENVVGWYHSHPGYGCWLSGIDVTTQMLNQQYQEPF 180 Mt CSN5 RVNAQADAYEYMVDYSQTNKQAGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQQFQEPF 170 Pp CSN5 RVNAQADAYEYMVQYSQTNKQVGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQTYQEPF 173 Os CSN5 RVNAQADAYEYMVEYSTINKQAGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQQFQEPF 170 Zm CSN5 RVNAQADAYEYMVDYSTINKQAGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQQFTEPF 170 ***** *******:** .* .*************************:** *** . *** At CSN5A LAVVIDPTRTVSAGKVEIGAFRTYPEGHKISDDHVSEYQTIPLNKIEDFGVHCKQYYSLD 229 At CSN5B LAVVIDPTRTVSAGKVEIGAFRTYSKGYKPPDEPVSEYQTIPLNKIEDFGVHCKQYYSLD 229 Sl CSN5 LAVVIDPTRTVSAGKVEIGAFRTYPEGYKPPDDPISEYQTIPLNKIEDFGVHCKQYYSLD 240 Mt CSN5 LAVVIDPTRTVSAGKVEIGAFRTYPEGYKPADDPISEYQTIPLNKIEDFGVHCKQYYSLD 230 Pp CSN5 LAVVIDPTRTVSAGKVEIGAFRTYPQGYKPPDEPPSEYQTIPLNKIEDFGVHYKQYYSLD 233 Os CSN5 LAVVIDPTRTVSAGKVEIGAFRTYPKDYKPPDEPVSEYQTIPLNKIEDFGVHCKQYYALD 230 Zm CSN5 LAVVIDPTRTVSAGKVEIGAFRTYPKDYKPPDEPVSEYQTIPLNKIEDFGVHCKSYYALD 230 ************************.:.:* .*: ***************** *.**:** At CSN5A ITYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNGDYVAGQISDLAEKLEQAESQLANS-R 288 At CSN5B VTYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNGDYVAGQISDLAEKLEQAESHLVQS-R 288 Sl CSN5 ITYFKSSLDCHLLDLLWNKYWVNTLSSSPLLGNGDYVAGQISDLAEKLEQGENHLSHS-R 299 Mt CSN5 ITYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNGDYVAGQISDLAEKLEQAENQLAHS-R 289 Pp CSN5 ITYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNRDYVAGQVADLAEKLEQAESQLAHSGR 293 Os CSN5 ITYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNRDYVAGQIFDLADKLEQAEGQLAHS-R 289 Zm CSN5 ITYFKSSLDSHLLDLLWNKYWVNTLSSSSLLGNRDYVAGQIFDLADKLEQAEGQLAHS-R 289 :********.******************.** * ******: ***:*:**.*.:* :* * At CSN5A YGGIAPAGHQRRKEDEPQLAKITRDSAKITVEQVHGLMSQVIKDILFNSARQSK------ 342 At CSN5B FGGVVPSSLHKKKEDESQLTKITRDSAKITVEQVHGLMSQVIKDELFNSMRQSNN----- 343 Sl CSN5 FGHLVAAPQ-RKKEEESQLAKITRDSAKITVEQVHGLMSQVIKDILFNSVCKSG------ 352 Mt CSN5 FGPLVAPTP-RKKEEESPLAKITRDSAKITVEQVHGLMSQVIKDILFNSVHQAN------ 342 Pp CSN5 MGGFFMPVQ-KKKEEESQLAKISRDSSKITVEQLHGLMSQVIKDTLFNSVMSSSNAPVAM 352 Os CSN5 YG-MLMPSQRKKEQEESPLAKVTRDSSKITAEQVHGLMSQVIKDILFNSVHPSNK----- 343 Zm CSN5 FGGMIMPSQRKKEQEESPLAKVTRDSSKITAEQVHGLMSQVIKDILFNSVHPSSK----- 344 * . . :::::*. *:*::***:***.**:********** **** : At CSN5A -----KSADDSSDPEPMITS 357 At CSN5B -----KSPTDSSDPDPMITY 358 Sl CSN5 -----KSQTEPSDPEPMVET 367 Mt CSN5 -----KSRTETSDPEPMIES 357 Pp CSN5 SAETSIVATDSSGPEPMVEA 372 Os CSN5 --ASTSAP-DSSGPEPMVEA 360 Zm CSN5 --ASTSAPPDSSGPEPMVEA 362 :.*.*:**:


At CSN5A

At CSN5B

Sl CSN5

Mt CSN5

Pp CSN5

Os CSN5

Zm CSN5

0.030

0.015

0.075

0.073

0.066

0.061

0.088

0.091 0.020

0.027

Supplemental Figure 3. Characterization of the transgenic Arabidopsis lines expressing C2/L2. RNA gel blots and segregation data (number of loci) from C2/L2 transgenic plants used in this work. C: negative control (wild-type Col0 plants).

Loci (nº) 1 1 1 1 2 2 1 1 1


A

B

Supplemental Figure 4. CUL1 and CUL3 protein accumulation in transgenic Arabidopsis lines expressing C2/L2. A: Total amount of CUL1 and CUL3 proteins (unrubylated plus rubylated). B: Relative amount of the rubylated CUL1 and CUL3. Protein amount was quantified from !-CUL1 and !-CUL3 inmunoblots using ImageJ software. Equal protein loads were confirmed using !-RPN6 (RPN6 is a non-ATPase regulatory subunit of the 26S proteasome). Error bars represent standard error of four (CUL1) or two (CUL3) independent biological replicates.

0

1

2

3

WT C2TS C2TM L2BC

Tota

l CU

LLIN

acc

umul

atio

n

CUL1

CUL3

0

1

2

3

WT C2TS C2TM L2BC

Rel

ativ

e R

ub/U

nRub

CU

LLIN

acc

umul

atio

n

CUL1

CUL3


0,5

1

1,5

Hyp

ocot

yl le

ngth

(cm

)

WT C2-TS C2-TM L2-BC

* ** *

* *

*

3.3 1.6 1 2.0 1.8 1 3.0 1.7 1

Relative C2 mRNA level

Supplemental Figure 5. Hypocotyl length of nine-day-old dark-grown transgenic C2-TS, C2-TM and L2-BC or wild-type Arabidopsis seedlings. Three independent lines are depicted for each construct. The relative accumulation of C2/L2 mRNA for each line is shown. Bars represent standard deviation. Asterisks indicate a statistically significant difference when compared to the wild-type value according to Mann-Whitney rank sum test. n!30; the experiment was repeated three times.


Rel

ativ

e ro

ot g

row

th

WT C2-TS

*

*

0.2 µM 2,4-D

100

50

0

100

50

0

Rel

ativ

e ro

ot g

row

th

50 µM MeJA

*

Rel

ativ

e ro

ot g

row

th

Mock-treated

Hormone-treated

A

B

Relative C2 mRNA level

1.6 1 2.0 1 1.7 1

WT C2-TS C2-TM L2-BC

Mock-treated

MeJA-treated

Supplemental Figure 6. Reduced response to hormones in transgenic A. thaliana and N. benthamiana plants expressing C2. A. Reduced jasmonate response in transgenic C2/L2 Arabidopsis lines. Hormone sensitivity was measured as root growth inhibition. Two independent lines are depicted for each construct. The relative accumulation of C2/L2 mRNA for each line is shown. Experiments were repeated at least three times independently; results from one of the replicates are represented (n!15). Five-day-old seedlings were grown on exogenous hormone for an additional five days. Bars represent standard error. Asterisks indicate a statistically significant difference when compared to the wild-type value according to Mann-Whitney rank sum test. B. Reduced auxin and jasmonate response in transgenic C2-TS N. benthamiana plants. Hormone sensitivity was measured as root growth inhibition. Experiments were repeated at least three times independently; results from one of the replicates are represented (n!4). Five-day-old seedlings were grown on exogenous hormone for an additional five days. Bars represent standard error. Asterisks indicate a statistically significant difference when compared to the wild-type value according to Mann-Whitney rank sum test.


Fold

exp

ress

ion

AT2G06050 AT3G16470

Control

C2-TS

Supplemental Figure 7. Evaluation of the expression of two differentially expressed genes in transgenic C2-TS Arabidopsis plants for microarray validation. Relative expression of AT2G06050 and AT3G16470 in transgenic C2-TS and control Arabidopsis seedlings determined by quantitative real-time PCR. Actin was used as the internal control. Fold expression values obtained in the microarray are presented in Supplemental table 3. Bars represent standard error.

1.5 -

1.0 -

0.5 -

0 -


Control C2-TS C2-TM L2-BC

Rel

ativ

e C

UL3

mR

NA

leve

l

Supplemental Figure 8. Relative expression of CUL3 in C2-TS, C2-TM, and L2-BC or control Arabidopsis seedlings determined by quantitative real-time PCR. Actin is used as the internal control. Bars represent standard error.

100

50

0


Sup

plem

enta

l Dat

a. L

ozan

o-D

urán

et a

l. (2

011)

. Pla

nt C

ell 1

0.11

05/tp

c.11

0.08

026

TAB

LES

Nam

e (a

,b)

Seq

uenc

e R

estr

ictio

n si

te

Des

tinat

ion

vect

or

Pri

mer

s fo

r cl

onin

g

C2 1

-78 (T

S)-

F G

GG

AC

TCA

TATG

CA

ATC

TTC

GTC

AC

C

Nde

I pG

BK

T7

C2 1

-78 (T

S)-

R

CA

GC

TGC

AG

AA

ATA

TAG

GG

GA

TTTG

T P

stI

pGA

DT7

C

SN

5A44

-357

-F

CG

AG

AC

ATA

TGG

AG

AA

GC

CA

TGG

GC

CTC

CG

N

deI

pGA

DT7

C

SN

5A44

-357

-R

TTTT

TCTC

GA

GC

GC

AA

AA

AC

ATA

ATG

ATC

G

Xho

I pG

AD

T7

C2 1

-78 (

TM)-

F TC

TCA

CA

TATG

CA

AC

CTT

CG

TCA

CC

C

Nde

I pG

BK

T7

C2 1

-78 (

TM)-

R

GTT

ATC

TTC

TGC

AG

AG

AG

GG

GA

TTG

P

stI

pGB

KT7

L2

1-10

8 (B

C)-

F C

TTA

AC

ATA

TGG

AA

AA

CC

AC

GTG

TC

Nde

I pG

BK

T7

L21-

108 (B

C)-

R

GG

AG

CTG

CA

GTT

AC

AG

TGG

ATT

CG

GTA

C

Pst

I pG

BK

T7

CS

N5B

44-3

58-F

TT

CC

ATA

TGG

AG

AA

AC

CG

TGG

GA

G

Nde

I pG

AD

T7

CS

N5B

44-3

58-R

TT

TTTC

TCG

AG

TCA

ATA

TGTA

ATC

ATA

GG

X

hoI

pGA

DT7

JA

B57

-367

-F

TTC

CA

TATG

GA

GA

AG

CC

GTG

GA

CG

N

deI

pGA

DT7

JA

B57

-367

-R

TGA

CTC

GA

GTC

AG

GTT

TCG

AC

CA

TCG

G

Xho

I pG

AD

T7

C2

(TS

) OR

F-F

AA

GC

GG

CC

GC

CA

TGC

AA

TCTT

CC

AG

N

otI

pEN

TR/D

C

2 (T

S) O

RF-

R

AA

GG

CG

CG

CC

CC

TAA

AG

AC

TCTT

AA

A

Asc

I pE

NTR

/D

C2

(TM

) OR

F-F

AA

GC

GG

CC

GC

CA

TGC

AA

CC

TTC

GTC

N

otI

pEN

TR/D

C

2 (T

M) O

RF-

R

AA

GG

CG

CG

CC

CC

TAA

ATA

CTT

TTA

AC

A

scI

pEN

TR/D

L2

(BC

) OR

F-F

AA

GC

GG

CC

GC

CA

TGG

AA

AA

CC

AC

GTG

N

otI

pEN

TR/D

L2

(BC

) OR

F-R

A

AG

GC

GC

GC

CC

TTA

TCC

AA

GTA

TATC

A

scI

pEN

TR/D

C

SN

5A O

RF-

F A

AG

CG

GC

CG

CC

ATG

GA

AG

GTT

CC

TG

Not

I pE

NTR

/D

CS

N5A

OR

F-R

A

AG

GC

GC

CC

TCA

GA

TCTA

ATC

ATG

G

Asc

I pE

NTR

/D

JAB

OR

F-F

AA

GC

GG

CC

GC

CA

TGG

AC

GC

TCTG

AA

TTC

TTA

C

Not

I pE

NTR

/D

JAB

OR

F-R

A

AG

GC

GC

GC

CC

TCA

GG

TTTC

GA

CC

ATC

G

Asc

I pE

NTR

/D

C2

(TS

) OR

F-F

CC

AA

CA

AG

GTA

CC

ATG

CA

ATC

TTC

GTC

AC

CA

TCC

K

pnI

pBIN

X1

C

2 (T

S) O

RF-

R

CC

CC

CC

GA

GC

TCC

TAA

AG

AC

TCTT

AA

AA

AA

TGA

CC

S

acI

pBIN

X1

GFP

-F

CC

GC

GC

GTT

AA

CA

TGA

GTA

AA

GG

AG

AA

GA

AC

H

paI

pBIN

X1

Sup

plem

enta

l Dat

a. L

ozan

o-D

urán

et a

l. (2

011)

. Pla

nt C

ell 1

0.11

05/tp

c.11

0.08

026

GFP

-R

CC

CG

GA

TCC

TGC

TTTG

TATA

GTT

CA

TCC

ATG

C

Bam

HI

pBIN

X1

C2 T

2C-F

G

GG

CC

ATA

AA

AA

AcG

CA

ATC

TTC

GTC

AC

C

N/A

N

/A

C2 T

2C-R

G

GTC

AC

GA

AG

ATT

gCG

TTTT

TTA

TGG

CC

C

N/A

N

/A

Frag

men

t1C

2 T2C

–F

CC

GG

CA

CC

ATA

TGTT

TCTC

C

Nde

I pG

reen

TYA

14

Frag

men

t2C

2 T2C

–R

A

CA

TTTA

CC

CG

GG

ATA

AA

CC

ATG

G

Nco

I pG

reen

TYA

14

qRT-

PC

R P

rim

ers

A

ctin

F C

TAA

GC

TCTC

AA

GA

TCA

AA

GG

CTT

A (B

ertra

nd e

t al.,

200

3)

N/A

N

/A

Act

inR

A

CTA

AA

AC

GC

AA

AA

CG

AA

AG

CG

GTT

(Ber

trand

et a

l., 2

003)

N

/A

N/A

E

RF1

-F

TTC

CG

ATC

AA

ATC

CG

TAA

GC

N

/A

N/A

E

RF1

-S

AC

CC

TCTC

ATC

GA

GA

AA

GC

A

N/A

N

/A

ER

S1-

F C

TCTC

TGG

CA

AA

GG

TTA

CG

C

N/A

N

/A

ER

S1-

R

GG

TGTC

CTC

ATC

TCG

TGG

TT

N/A

N

/A

PIN

1-F

TTTG

TGTG

GA

GC

TCA

AG

TGC

N

/A

N/A

P

IN1-

R

TTG

TTG

GTG

GC

ATC

AC

CTT

A

N/A

N

/A

IAA

19-F

G

TGTG

GC

CTT

GA

AA

GA

TGG

T N

/A

N/A

IA

A19

-R

TGA

AC

CA

GC

TCC

TTG

CTT

CT

N/A

N

/A

MFC

19.1

3-F

CA

GA

GG

ATG

CA

GA

AC

GTG

AA

N

/A

N/A

M

FC19

.13-

R

GA

TCTT

CC

CA

ATT

CC

AG

CA

A

N/A

N

/A

MH

J24.

10-F

TC

TCC

AA

TCC

GA

TCA

AG

TCC

N

/A

N/A

M

HJ2

4.10

-R

TTA

GC

TCC

GG

TAA

CC

AC

AC

C

N/A

N

/A

OP

R3-

F C

GG

CG

TTG

GC

AG

AG

TATT

AT

N/A

N

/A

OP

R3-

R

TCC

CTT

AG

CG

TGA

AC

TGC

TT

N/A

N

/A

JR1-

F G

AG

TTG

CTT

GG

GA

TGA

TGG

T N

/A

N/A

JR

1-R

C

CTC

GA

GTC

CA

TAC

GG

GA

TA

N/A

N

/A

CU

L3-F

C

GTA

CA

AC

ATG

GTC

CTG

CA

C

N/A

N

/A

CU

L3-R

TC

CA

CA

AG

TTA

AG

CC

CC

ATC

N

/A

N/A

a F

indi

cate

s fo

rwar

d. b R

indi

cate

s re

vers

e.

Sup

plem

enta

l Tab

le 1

. Olig

onuc

leot

ides

use

d in

this

stu

dy. T

he n

ucle

otid

e th

at in

trodu

ces

the

T2C

mut

atio

n is

sho

wed

in lo

wer

case

.

Sup

plem

enta

l Dat

a. L

ozan

o-D

urán

et a

l. (2

011)

. Pla

nt C

ell 1

0.11

05/tp

c.11

0.08

026

Pla

smid

V

ecto

r ba

ckbo

ne

Frag

men

t P

rim

ers

/frag

men

t C

loni

ng s

ite

Use

d fo

r

pGre

enTY

A14

pG

reen

0029

(a)

Not

I/Xho

I fra

gmen

t fro

m

pTY

A14

(RE

F)

Not

I/Xho

I

fragm

ent

from

pTY

A14

(RE

F)

Not

I/Xho

I TY

LCS

V in

fect

ion

pBS

SK

-TY

A14

Nde

I/Nco

I pB

lues

crip

t SK

II+ (b

) N

deI/N

coI

fragm

ent

of

TYLC

SV

com

pris

ing

the

T1

630C

poi

nt m

utat

ion

Frag

men

t1C

2 T2C

–F

/ Fr

agm

ent2

C2 T

2C -R

N

deI/N

coI

Clo

ning

inte

rmed

iate

st

ep

pGre

enTY

A14

C2 T

2C

pGre

enTY

A14

(th

is

wor

k)

Nde

I/Nco

I

fragm

ent

from

pB

SS

K-

TYA

14N

deI/N

coI

- -

TYLC

SV

C2 T

2C in

fect

ion

pGB

KT7

-C2 1

-78

(TS

) (H

éric

ourt

et

al,

in

prep

arat

ion)

pGB

KT7

(c)

C2-

TS1-

78

- -

Yea

st tw

o hy

brid

pGA

DT7

-CS

N5A

44-3

57

pGA

DT7

(c)

Ara

bido

psis

C

SN

5A44

-

357

CS

N5A

44-3

57-F

/ C

SN

5A44

-357

-R

Nde

I/Xho

I Y

east

two

hybr

id

pGB

KT7

-C2 1

-78 (

TM)

pGB

KT7

(c)

C2-

TM1-

78

C2 1

-78

(TM

)-F/

C

2 1-7

8 (T

M)-

R

Nde

I/Pst

I Y

east

two

hybr

id

pGB

KT7

-L2 1

-108

(BC

) pG

BK

T7 (c

) L2

-BC

1-10

8 L2

1-10

8 (B

C)-

F/

L21-

108

(BC

)-R

N

deI/P

stI

Yea

st tw

o hy

brid

pGA

DT7

-CS

N5B

44-3

58

pGA

DT7

(c)

Ara

bido

psis

C

SN

5B44

-

358

CS

N5B

44-3

58-F

/ C

SN

5B44

-358

-R

Nde

I/Xho

I Y

east

two

hybr

id

pGA

DT7

-JA

B57

-367

pG

AD

T7 (c

) To

mat

o JA

B (

CS

N5)

57-

357

JAB

57-3

67-F

/ JA

B57

-367

-R

Nde

I/Xho

I Y

east

two

hybr

id

pEN

TR/D

-C2

(TS

) pE

NTR

/D (d

) C

2-TS

OR

F C

2 (T

S)

OR

F-F/

C

2 (T

S) O

RF-

R

Not

I/Asc

I G

atew

ay c

loni

ng

pEN

TR/D

-C2

(TM

) pE

NTR

/D (d

) C

2-TM

OR

F C

2 (T

M)

OR

F-F/

C

2 (T

M) O

RF-

R

Not

I/Asc

I G

atew

ay c

loni

ng

pEN

TR/D

-L2

(BC

) pE

NTR

/D (d

) L2

-BC

OR

F L2

(B

C)

OR

F-F/

L2

(B

C) O

RF-

R

Not

I/Asc

I G

atew

ay c

loni

ng

pEN

TR/D

-CS

N5A

pE

NTR

/D (d

) A

rabi

dops

is

CS

N5A

O

RF

CS

N5A

OR

F-F/

CS

N5A

O

RF-

R

Not

I/Asc

I G

atew

ay c

loni

ng

pEN

TR/D

-JA

B

pEN

TR/D

(d)

Tom

ato

JAB

(C

SN

5)

OR

F JA

B O

RF-

F/ J

AB

OR

F-R

N

otI/A

scI

Gat

eway

clo

ning

pBiF

P2-

C2

(TS

) pB

iFP

1 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

2-TS

G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

2 (T

S)

Gat

eway

LR

site

s B

iFC

pBiF

P3-

C2

(TS

pB

iFP

2 (e

) G

atew

ay f

ragm

ent

from

G

atew

ay f

ragm

ent

from

G

atew

ay L

R s

ites

BiF

C

Sup

plem

enta

l Dat

a. L

ozan

o-D

urán

et a

l. (2

011)

. Pla

nt C

ell 1

0.11

05/tp

c.11

0.08

026

pEN

TR/D

-C2-

TS

pEN

TR/D

-C2

(TS

) pB

iFP

2-C

2 (T

M)

pBiF

P1

(e)

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-C2-

TM

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-C2

(TM

) G

atew

ay L

R s

ites

BiF

C

pBiF

P3-

C2

(TM

) pB

iFP

2 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

2-TM

G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

2 (T

M)

Gat

eway

LR

site

s B

iFC

pBiF

P2-

L2 (B

C)

pBiF

P1

(e)

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-L2-

BC

G

atew

ay f

ragm

ent

from

pE

NTR

/D-L

2 (B

C)

Gat

eway

LR

site

s B

iFC

pBiF

P3-

L2 (B

C)

pBiF

P2

(e)

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-L2-

BC

G

atew

ay f

ragm

ent

from

pE

NTR

/D-L

2 (B

C)

Gat

eway

LR

site

s B

iFC

pBiF

P2-

CS

N5A

pB

iFP

1 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

SN

5A

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-CS

N5A

G

atew

ay L

R s

ites

BiF

C

pBiF

P3-

CS

N5A

pB

iFP

2 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-C

SN

5A

Gat

eway

fra

gmen

t fro

m

pEN

TR/D

-CS

N5A

G

atew

ay L

R s

ites

BiF

C

pBiF

P2-

JAB

pB

iFP

1 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-J

AB

G

atew

ay f

ragm

ent

from

pE

NTR

/D-J

AB

G

atew

ay L

R s

ites

BiF

C

pBiF

P3-

JAB

pB

iFP

2 (e

) G

atew

ay f

ragm

ent

from

pE

NTR

/D-J

AB

G

atew

ay f

ragm

ent

from

pE

NTR

/D-J

AB

G

atew

ay L

R s

ites

BiF

C

pBIN

X1-

GFP

pB

INX

1 (f)

G

FP

GFP

-F/ G

FP-R

H

paI/B

amH

I S

ub-c

ellu

lar l

ocal

izat

ion

pBIN

X1-

GFP

-C2

pBIN

X1-

GFP

(th

is

wor

k)

C2-

TS O

RF

C2

(TS

) O

RF-

F/

C2

(TS

) OR

F-R

K

pnI/S

acI

Sub

-cel

lula

r loc

aliz

atio

n

pBIN

-C2-

TS

pBIN

+ (g

) X

baI

fragm

ent

from

pS

XC

2TS

X

baI

fragm

ent

from

pS

XC

2TS

X

baI

Exp

ress

ion

in p

lant

s

pBIN

X-C

2-TM

pB

INX

1 (f)

S

maI

/Kpn

I fra

gmen

t fro

m p

C2T

M

Sm

aI/K

pnI

fragm

ent

from

pC

2TM

H

paI/K

pnI

Exp

ress

ion

in p

lant

s

pBIN

X-L

2-B

C

pBIN

X1

(f)

Sm

aI/K

pnI

fragm

ent

from

pL2

BC

S

maI

/Kpn

I fra

gmen

t fro

m p

L2B

C

Hpa

I/Kpn

I E

xpre

ssio

n in

pla

nts

pSX

C2T

S

pSX

SN

1 (f)

C

2-TS

OR

F C

2 (T

S)

pSX

SN

1-F

/ C

2 (T

S) p

SX

SN

1-R

H

paI

Clo

ning

inte

rmed

iate

st

ep

pC2T

M

pBlu

escr

ipt S

KII+

(b)

C2-

TM O

RF

C2

(TM

) pB

INX

-F /

C

2 (T

M) p

BIN

X-R

E

coR

V

Clo

ning

inte

rmed

iate

st

ep

pL2B

C

pBlu

escr

ipt S

KII+

(b)

L2-B

C O

RF

L2 (

BC

) pB

INX

-F /

L2

(B

C) p

BIN

X-R

E

coR

V

Clo

ning

iint

erm

edia

te

step

S

uppl

emen

tal

Tabl

e 2.

Pla

smid

s ge

nera

ted

in t

his

wor

k. T

he b

ackb

one,

fra

gmen

t, pr

imer

use

d, c

loni

ng s

ite a

nd u

se a

re s

how

n. B

ackb

one

orig

in is

: (a)

(H

elle

ns e

t al.,

200

0); (

b) S

trata

gene

; (c)

Clo

ntec

h; (

d) In

vitro

gen;

(e)

(de

Luc

as e

t al.,

200

8); (

f) S

ánch

ez-D

urán

et

al.,

subm

itted

; (g)

(va

n

Eng

elen

et a

l., 1

995)


Supplemental Table 3. Hallmark genes of the jasmonate response down-regulated in

the microarray of C2-TS transgenic plants.

AGI code Gene product Molecular function Fold P-value

AT3G45140 LOX2 Lipoxygenase activity -6.4 0,00E+0

AT3G28930 AIG2 Molecular function unknown -3.1 0,00E+0

AT2G06050 OPR3 12-oxophytodienoate reductase activity -2.1 0,00E+0

AT5G42650 AOS Allene oxide synthase activity -2.1 0,00E+0

AT3G16470 JR1 Lectin -1.7 5,84E-7

lozano-durán et al, plant cell (supp data) 28-2-11€¦ · lozano-durán et al. (2011). plant cell...

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