supplemental figure s1.€¦ · 2013-06-19 · bbb c d d d d c b bbb aa cell death (+) (+) (+)...
TRANSCRIPT
Supplemental Figure S1.
G GA M K
GGGG LDF I I
Supplemental Figure S2.
MPALGCCVD--ATVSPPLGYAFSSDSSLPTPEFFSSGVPPMTNAAAG-----HSHWSPDLSSALYRVD 61MPALGCCVDA-AVVSPPLSYAFSRDSSLPAPEFFASGVPPTNSAAA-------SHWSPDLSSALYGVD 60MPALACVDTSFVPPAYAFSDTAG--------DVFIPASSPTSAAVVV------DRWSPSLSSSLYRID 54MPALTCFVDGAAALLHPPGYALAGDFTLPLPFTFSAAATITDDADATAVEDSNSIWSPSLSSKLFRID 68
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1 MPALAFVDTP--------IDTFS--------SIFTPSSVSTAVVDGS------CHWSPSLSSSLYRID 46
GWGAPYFSVNSSGDISVRPHGTDTLPHQEIDLLKVAKKAADPKHLGGLGLQMPLVVRFPDILKNRLET 129GWGAPYFSVNSNGDISVRPHGTDTLPHQEIDLLKVVKKASDPKNSGGLGLQLPLVVRFPDVLKNRLES 128GWGAPYFIANSSGNISVRPHGSETLPHQDIDLLKIVKKVTGPKSSGGLGLQLPLIVRFPDVLKNRLEC 122GWGFPYFGVNAAGDISVRPHGSATMSHQEIDLLKVVKKASDPKCCGGLGLQLPLVVRFPDVLKDRLES 136GWGAPYFAANSSGNISVRPHGSNTLPHQDIDLMKVVKKVTDP---SGLGLQLPLIVRFPDVLKNRLEC 111
====================LQSAFDMAVRSRGYEAHYQGVYPVKCNQDRFVVEDIVKFGSPYRFGLEAGSKPELLLAMNCLSKGSAD 197LQSAFDLAVHSQGYGAHYQGVYPVKCNQDRFVVEDIVKFGSPFRFGLEAGSKPELLLAMSCLCKGSAE 196LQSAFDYAIKSQGYDSHYQGVYPVKCNQDRFVVEDIVKFGSSFRFGLEAGSKPEILLAMSCLCKGSPD 190IHAAFDGAIQLQGYESHYQGVYPVKCNQDRYIVEDIVEFGSSFRFGLEAGSKPELLLAMSCLCKGNRE 204LQSAFDYAIQSQGYDSHYQGVYPVKCNQDRFIIEDIVEFGSGFRFGLEAGSKPEILLAMSCLCKGNPE 179
ALLVCNGFKDTEYISLALIARKLLLNTVIVLEQEEELDLVIDISRKMAVRPVIGLRAKLRTKHSGHFG 265GLLVCNGFKDAEYISLALVARKLMLNTVIVLEQEEELDLVIDISHKMAVRPVIGLRAKLRTKHSGHFG 264AFLVCNGFKDAEYISLALLGRKLALNTVIVLEQEEELDLVIELSQKMNVRPVIGLRAKLRTKHSGHFG 258AFLVCNGFKDSEYISLALIARKLALNTVIVLEQEEELDMVVEISNKLCIRPVIGVRAKLKTKHSGHFG 272AFLVCNGFKDSEYISLALFGRKLELNTVIVLEQEEELDLVIDLSQKMNVRPVIGLRAKLRTKHSGHFG 247
--STSGEKGKFGLTTTQILRVVKKLEGSGMLDCLQLLHFHIGSQIPTTELLADGVGEATQIYSELVRLGA 333STSGEKGKFGLTTTQIVRVVKKLEESGMLDCLQLLHFHIGSQIPSTGLLADGVGEAAQIYCELVRLGA 332STSGEKGKFGLTTTQIVRVVRKLRQSGMLDCLQLLHFHIGSQIPSTSLLSDGVAEAAQLYCELVRLGA 326ATSGDKGKFGLTTIQILHVVKKLEQLDMLDCLQLLHFHIGSQIPTTELLADGVREASQIYCELLRLGA 340STSGEKGKFGLTTVQILRVVRKLSQVGMLDCLQLLHFHIGSQIPSTALLSDGVAEAAQLYCELVRLGA 315
------------GMKFIDIGGGLGIDYDGSKSSDSDVSVAHGIEEYASAVVQAVQFVCDRKGVKHPVICSESGRAIVSHH 401GMKFIDIGGGLGIDYDGTKSCDSDVSVGYGIQEYASAVVQAVQYVGDRKGVKHPVICSESGRAIVSHH 400HMKVIDIGGGLGIDYDGSKSGESDLSVAYSLEEYAEAVVASVRVVCDRSSVKHPVICSESGRAIVSHH 394QMKVLDIGGGLGIDYDGSKSGDSDESVAYGLEEYAAAVVHAVKYVCDRKNVKHPVICSESGRAIVSHH 408HMKVIDIGGGLGIDYDGSKSGESDLSVAYSLEEYAAAVVASVRFVCDQKSVKHPVICSESGRAIVSHH 383
SIMIFEAVSASTTNVSP-QLSSGGLQSLAETLNEDARADYRNLSAAAVRGEYDTCLLYSDQLKQRCVE 468SILIFEAVSASSTHVSSSHLSSGGLQSMAETLNEDALADYRNLSAAAVRGEYETCVLYSDQLKQRCVE 468SVLIFEAVSADKP--MVHQATPGDIQFLLEGN-EEARANYEDLYAAVMRGDHESCLLYVDQLKQRCVE 459SILIFEASGASTNTAPS--LSSIELQYLGEGLSEEALADYQNISAATLRGEYEACLLYTEQFKKRCVE 474SVLIFEAVSAGQ-----QHETPTDHQFMLEGYSEEVRGDYENLYGAAMRGDRESCLLYVDQLKQRCVE 446
QFNDGSLDIEQLAAVDSICDLVSKAIGVSDPVRAYHVNLSVFTSIPDFWAFGQLFPIVPIHRLDEKPT 536QFKEGSLGIEHLAAVDSICDFVSKAMGAADPVRTYHVNLSIFTSIPDFWAFGQLFPIVPIHRLDEKPA 536GFKEGVLSIEQLASVDGLCEWVLKAIGASDPVHTYNINLSVFTSIPDLWGIDQLFPIVPIHKLDQRPG 527EFKQGTLGIEQLAAVDGLCDLITETIGVKDPVKKYHVNLSVFTSVPDFWGINQLFPIVPIHRLDEKPT 542GFKEGSLGIEQLAGVDGLCEWVIKAIGASDPVLTYHVNLSVFTSIPDFWGIDQLFPIVPIHKLDQRPA 514
MRGILSDLTCDSDGKVDKFIGGESSLPLHDLGSNGGDGGRYYLGMFLGGAYEEALGGLHNLFGGPSVV 604VRGILSDLTCDSDGKVDKFIGGESSLPLHELGSNG-DGGGYYLGMFLGGAYEEALGGLHNLFGGPSVV 603ARGILSDLTCDSDGKINKFIGGESSLPLHELDKNG-SGGRYFLGMFLGGAYEEALGGVHNLFGGPSVV 594ARGILSDLTCDSDGKIDKFIGGESSLPLHEMEGHG---GGYYLGMFLGGSYEEALGGLHNLFGGPSVV 607ARGILSDLTCDSDGKINKFIGGESSLPLHEMDNNGCSGGRYYLGMFLGGAYEEALGGVHNLFGGPSVV 582
RVLQSDSPHSFAVTRSVPGPSCADVLRAMQFEPELMFETLKHRAEESFDQEEEIEDKGLAFE------ 666RVVQSDSAHSFAMTRSVPGPSCADVLRAMQHEPELMFETLKHRAEEFLEQED---DKGLAVE------ 662RVSQSDGPHSFAVTRAVPGQSSADVLRAMQHEPELMFQTLKHRAEEMMNNNNKACGDKGNDK-----L 657RVLQSDGPHGFAVTRAVAGSSCADVLRVMQHEPQLMFETLKHRALEFCGQHD--DDSVVNAG------ 667RVLQSDGPHGFAVTRAVMGQSSADVLRAMQHEPELMFQTLKHRAEEPRHTKGGSEGENEEEEEDDEFN 650
SLISGLAQSFHNMPYLVAPSSCCFTAATAKGGGY-YYYNEDNAAD------CATAEDEIWSYCTA 724SLASSVAQSFHNMPYLVAPSSCRFTAATDNNGGYNYYYSDENAAD------SATGEDEIWSYCTA 721 (86%)VVASCLAKSFNNMPYLSMETS--TNALTAAVNNLGVYYCDEDVYDY-----ISA----------- 711 (71%)VLANSLAQSFDNMPYLVSSTTCCLNALTNNNGFY-YCSGDDFSADTVSVATSVAGEDENWSY--- 728 (69%)NVAASLDRSFHNMPYLATEQASPSNSLSAAISNLGFYYCDEAAAGG-----GGKGKDENWSYFG- 702 (68%)
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
CaADC1 Tobacco ADC
Arabidopsis ADC2 Garden pea ADC
Arabidopsis ADC1
A
Leaf
Stem
Roo
t
Flow
er
Gre
en fr
uit
Red
frui
t
CaADC1
rRNA
CaADC1
rRNA
0 1 5 10 15 20 25h
Salicylic acid
B
CaADC1
rRNA
0 1 5 10 15 20 25 hMethyl jasmonate
CaADC1
rRNA
0 1 5 10 15 20 25h
EthyleneC
Supplemental Figure S3.
D
35S:EV(OD600=0.2)
35S:CaADC1 K154A
(OD600=0.2)35S:CaADC1(OD600=0.2)
35S:CaADC1K154A /
35S:avrBsT(OD600=0.05)
35S:avrBsT(OD600=0.2)
35S:avrBsT(OD600=0.05)
35S:CaADC1 / 35S:avrBsT
(OD600=0.05)
35S:CaADC1K154A / 35S:BAX
(OD600=0.05)35S:BAX
(OD600=0.2)35S:BAX
(OD600=0.05)
35S:CaADC1 / 35S:BAX
(OD600=0.05)
AB
25
20
15
10
5
0
Con
duct
ivity
(μS
cm-1
)
Supplemental Figure S4.
0 24 48Hours after infiltration
35S:EV (OD600=0.2)
35S:CaADC1 (OD600=0.2)
35S:CaADC1 K154A (OD600=0.2)
35S:avrBsT (OD600=0.05)
35S:CaADC1 / 35S:avrBsT (OD600=0.05)
35S:CaADC1 K154A / 35S:avrBsT (OD600=0.05)
35S:avrBsT (OD600=0.2)
35S:CaADC1 / 35S:BAX (OD600=0.05)
35S:BAX (OD600=0.05)
35S:CaADC1 K154A / 35S:BAX (OD600=0.05)
35S:BAX (OD600=0.2)
aa a a a a a a aa a a aa a a bb bc
dd
d d
c
b b b b baaa
Cell death (+) (+) (+) (+++)
Cell death (+) (++) (+) (+++)
Cell death (-) (-) (-)
35S:EV(OD600=0.2)
35S:CaADC1 K154A
(OD600=0.2)35S:CaADC1(OD600=0.2)
35S:CaADC1K154A /
35S:avrBsTC222A
(OD600=0.05)
35S:avrBsTC222A
(OD600=0.2)
35S:avrBsTC222A
(OD600=0.05)
35S:CaADC1 / 35S:avrBsT
C222A(OD600=0.05)
35S:avrBsT(OD600=0.2)
B
35S:avrBsT C222A (OD600=0.05)
35S:CaADC1 / 35S:avrBsT C222A (OD600=0.05)
35S:CaADC1 K154A / 35S:avrBsT C222A (OD600=0.05)
35S:avrBsT C222A (OD600=0.2)
C
25
20
15
10
5
0
Con
duct
ivity
(μS
cm-1
)
Supplemental Figure S5.
35S:EV (OD600=0.2) 35S:CaADC1 (OD600=0.2)
35S:avrBsT (OD600=0.2)35S:CaADC1 K154A (OD600=0.2)
0 24 48Hours after infiltration
a
b
b
aa a a a a a a aa aa aa a a a aa a a
Cell death (-) (-) (-) (+++)
Cell death (-) (-) (-) (-)
AvrBsT:cMyc
AvrBsT C222A:cMyc
CaADC1:HA
+ +
+ +--
- -
IP: cMyc IP: HA
α-cMyc
α-HA
CBB
CaADC1:HA
-
-
AvrBsT:cMyc /AvrBsT C222A:cMyc
+- - +-
- +
- + +
+ +--
- - -
-+- - +-
- +
-
A
0 1 2Asp Glu Asn Ser Gln Gly His Arg Thr Ala GABA Pro Tyr Val Ile Leu Phe Trp Lys
35S:avrBsT (OD600=0.2)35S:CaADC1 (OD600=0.2)35S:EV (OD600=0.2)
6
5
4
3
2
1
0 0 1 2 0 1 2 0 1 2 0 1 2 0 1 20 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 dai
Supplemental Figure S6.
Amin
o ac
id (
μm
ol g
-1FW
)
aaaa
aa
a
a
a
a
a
a
a
aa
a
a
a
aaa aa
b
b
aa
aaa
a
aa
aa
a
aa
bb
b
bb
c
c
aaa
a
a
aaaa aa aa
aaaa
b bb
b
b
bb
bc
a
aaa aabb
b
c
caaa
a a
a
aa
a
a
a
a
a
a
aaa
bb
b
a
aa
b
b
c
aaa aaaaaa aaa aa
aaa
a
aaa aaa
aa aaa aaa
b
b
b
bb
c
a aaa aaa aa
a
aaaaaaaaaaaaaaa
Visible UV
TRV:
00
cfu mL-1
107
108
Xcv Ds1(EV)
Xcv Ds1(avrBsT)
TRV:
CaA
DC
1
Visible UV
Xcv Ds1(EV)
Xcv Ds1(avrBsT)
Xcv Ds1(EV)
Xcv Ds1(avrBsT)
Xcv Ds1(EV)
Xcv Ds1(avrBsT)
Supplemental Figure S7.
3
2
1
0
3
2
1
0
Asp Glu Asn Ser Gln Gly His Arg Thr Ala GABA Pro Tyr Val Ile Leu Phe Lys0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 dai
TRV:00 TRV:CaADC1
Am
ino
acid
(μ
mol
g-1
FW)
Xcv Ds1 (EV)
Xcv Ds1 (avrBsT)
Supplemental Figure S8.
*****
*
*
*
*
*
*
*
*
*
* * * ** * *
8
6
4
2
0
Log
cfu
cm-2
0 8 16 24 0 8 16 24
Hours after incubation
Xcv Ds1 (EV) Xcv Ds1 (avrBsT)
Mock 1mM Alanine 10mM Alanine 1mM GABA 10mM GABA
Supplemental Figure S9.
aaa a a
a a a a a
a a a a a
a a a a a
a a a a a
a a a a a
a a a a a
a a a a a
GABA
H2O2
Cell death & defense response
Arginine
Agmatine
Putrescine
Spermidine
Spermine
CaADC1
AvrBsT
Supplemental Figure S10.
NO
SA
Pyrroline