A abidopsis P o ein Phospha ase DBP1 Nuclea es a
P o ein Ne wo k wi h a Role in Regula ing Plan De ense
Jose
´Luis Ca asco
1.
, Ma ı
´a Jose
´Cas ello
´
1.
, Kai Naumann
2
, Ines Lassowska
2
, Ma isa Na a e e-Go
´mez
1
,
Die k Scheel
2
, Pablo Ve a
1,2
*
1Ins i u o de Biologı
´a Molecula y Celula de Plan as, Uni e sidad Poli e
´cnica de Valencia-C.S.I.C, Ciudad Poli e
´cnica de la Inno acio
´n, Valencia, Spain, 2Depa men o
S ess and De elopmen al Biology, Leibniz Ins i u e o Plan Biochemis y, Halle, Ge many
Abs ac
A abidopsis haliana DBP1 belongs o he plan -speci ic amily o DNA-binding p o ein phospha ases. Al hough ecen ly
iden i ied as a no el hos ac o media ing suscep ibili y o po y i us, li le is known abou DBP1 a ge s and pa ne s and
he molecula mechanisms unde lying i s unc ion. Analyzing changes in he phosphop o eome o a loss-o - unc ion dbp1
mu an enabled he iden i ica ion o 14-3-3liso o m (GRF6), a p e iously epo ed DBP1 in e ac o , and MAP kinase (MAPK)
MPK11 as componen s o a small p o ein ne wo k nuclea ed by DBP1, in which GRF6 s abili y is modula ed by MPK11
h ough phospho yla ion, while DBP1 in u n nega i ely egula es MPK11 ac i i y. In e es ingly, g 6 and mpk11 loss-o -
unc ion mu an s showed al e ed esponse o in ec ion by he po y i us Plum pox i us (PPV), and he desc ibed molecula
mechanism con olling GRF6 s abili y was ecapi ula ed upon PPV in ec ion. These esul s no only con ibu e o a be e
knowledge o he biology o DBP ac o s, bu also o MAPK signalling in plan s, wi h he iden i ica ion o GRF6 as a likely
MPK11 subs a e and o DBP1 as a p o ein phospha ase egula ing MPK11 ac i i y, and un eils he implica ion o his
p o ein module in he esponse o PPV in ec ion in A abidopsis.
Ci a ion: Ca asco JL, Cas ello
´MJ, Naumann K, Lassowska I, Na a e e-Go
´mez M, e al. (2014) A abidopsis P o ein Phospha ase DBP1 Nuclea es a P o ein
Ne wo k wi h a Role in Regula ing Plan De ense. PLoS ONE 9(3): e90734. doi:10.1371/jou nal.pone.0090734
Edi o : Abidu Rahman, Iwa e Uni e si y, Japan
Recei ed No embe 26, 2013; Accep ed Feb ua y 3, 2014; Published Ma ch 4, 2014
Copy igh : ß2014 Ca asco e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k was suppo ed by he Spanish MICINN (G an s BFU2009-09771, EUI2009-04009 o PV), Gene ali a Valenciana (P ome eo2010/020 o PV) and
he Ge man DFG (SCHE 235/15-1 o DS). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he
manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
.These au ho s con ibu ed equally o his wo k.
In oduc ion
DNA-binding p o ein phospha ases (DBPs) a e a unique amily
o p o ein phospha ases o he 2C class ha a e dis inc i ely
capable o binding DNA [1]. While p o ein phospha ase ac i i y
lies in he C- e minal domain, he abili y o bind DNA esides in
an N- e minal ex ension, which is ba ely conse ed beyond a mo i
di ec ly in ol ed in binding [1,2]. DBP ac o s, al hough
appa en ly unique o plan s, a e p esen h oughou he plan
kingdom and seem o ha e unc ionally di e si ied du ing
e olu ion [2].
Tobacco DBP1 (N DBP1), he ounding membe o his amily,
was shown o be in ol ed in he ansc ip ional egula ion o a
de ense- ela ed gene in he cou se o compa ible plan - i us
in e ac ions [1]. Mo e ecen ly, in A abidopsis haliana, DBP1 was
ound o media e disease suscep ibili y o po y i uses like Plum pox
i us (PPV) and Tu nip mosaic i us (TuMV) [3]. Mo eo e , DIP2, a
small pep ide o unknown unc ion ha unc ionally modula es
DBP1 in A abidopsis, was iden i ied as an addi ional hos ac o
in luencing PPV in ec ion, he eby emphasizing he signi ican
implica ion o DBP1 in he in e ac ion [4]. DBP1 unc ion is also
modula ed h ough he in e ac ion wi h 14-3-3 p o eins, highly
conse ed ubiqui ous euka yo ic p o eins ecognized as impo an
media o s in he egula ion o di e se biological p ocesses, in
pa icula in signal ansduc ion and ansc ip ion [5]. Ca asco
e al (2006) epo ed ha bo h in obacco and A abidopsis, DBP1
o hologs di ec ly in e ac wi h 14-3-3G and 14-3-3liso o ms,
espec i ely, and his in e ac ion p o okes he nuclea expo o
he complex o he cy oplasm [6]. Re en ion o DBP1 in he
cy osol will in u n p e en binding o DBP1 o he p omo e
egion o de ense- ela ed a ge genes, e en ually elie ing gene
ep ession and concomi an ly p omo ing unc ion o DBP1 in he
cy osol.
Mi ogen-ac i a ed p o ein kinase (MAPK) cascades a e essen ial
componen s o signal ansduc ion mechanisms e olu iona ily
conse ed in all euka yo es. A ypical MAPK module consis s o
h ee p o ein kinases, MAP kinase kinase kinase (MAP3K), MAP
kinase kinase (MAPKK) and MAPK, which sequen ially phos-
pho yla e and ac i a e each o he o media e cell esponses and
de elopmen al pa hways [7]. Once ac i a ed, MAPK phospho -
yla es and modula es speci ic a ge s, such as ansc ip ion ac o s.
MAPK ac i a ion occu s h ough conse ed h eonine and
y osine phospho yla ion by MAPKK, a dual speci ici y kinase
[8]. Dephospho yla ion o ei he esidue by speci ic p o ein
phospha ases inac i a es MAPK [9].
The p esen s udy p o ides u he insigh s in o he molecula
mechanisms unde lying DBP1 unc ion by compa ing he
phosphop o eome o loss-o - unc ion dbp1 mu an plan s o wild
ype. Among he de ec ed p o eins, we iden i ied 14-3-3l(GRF6),
an ea lie desc ibed DBP1 in e ac o , and MPK11, a MAPK
PLOS ONE | www.plosone.o g 1 Ma ch 2014 | Volume 9 | Issue 3 | e90734
ac i a ed du ing he plan immune esponse [10]. Physical
in e ac ions among hese p o eins we e demons a ed in i o by
Bimolecula Fluo escence Complemen a ion analyses and coim-
munop ecipi a ion. We showed ha DBP1 nega i ely egula ed
MPK11 ac i i y, and ha MPK11 media ed phospho yla ion o
GRF6, p omo ing ubiqui ina ion and inc eased GRF6 p o ein
u n-o e . These esul s un eil a phospho yla ion-dependen ,
p o easome-media ed egula o y mechanism o GRF6, and
iden i y a likely subs a e o MPK11, a MAPK wi h unknown
unc ion, whose ac i i y is shown o be, in u n, modula ed by
DBP1. In e es ingly, e e se gene ic s udies e ealed ha bo h
GRF6 and MPK11 a e in ol ed in he esponse o A abidopsis
plan s o in ec ion by he po y i us Plum pox i us, and he desc ibed
egula ion o GRF6 p o ein s abili y by MPK11 was shown o
occu du ing PPV in ec ion, appea ing as a no el signalling
componen in he A abidopsis-PPV in e ac ion.
Ma e ials and Me hods
Plan Ma e ial and G ow h Condi ions
A abidopsis plan s we e g own in a g ow h chambe unde a
10/14 h ligh /da k pho ope iod (23 o 19uC, 85% ela i e
humidi y, 100 mEm
22
s
21
luo escen illumina ion). All mu an s
we e in a Col-0 backg ound and we e ob ained om he
Eu opean A abidopsis S ock Cen e a he Uni e si y o
No hingham (h p://a abidopsis.in o/). dbp1 mu an was al eady
desc ibed [3]. mpk11 and g 6 co espond o SALK_049352 and
SALK_075219 lines, espec i ely (Salk Ins i u e Genomic Analysis
Labo a o y, h p://signal.salk.edu/).
Phosphop o ein En ichmen and Analysis
A de ailed desc ip ion o he p o ocol used o phosphop o ein
en ichmen is included in Suppo ing In o ma ion.
T ansien Exp ession in Nico iana ben hamiana Lea es
Tobacco plan s (Nico iana ben hamiana) we e g own in a
phy ochambe unde sho day condi ions a 23uC/19uC. Almos
ully expanded lea es we e in il a ed wi h a suspension o
Ag obac e ium ume aciens C58 bea ing he ele an cons uc in
10 mM MES pH 5.6, 10 mM MgCl
2
, 150 mM ace osy ingone a
an OD
600
= 0.5. A e 3 days, luo escence was analyzed in
in il a ed lea es by con ocal mic oscopy. Fo co-in il a ion,
Ag obac e ium cul u es g own sepa a ely and p ocessed as indica ed
abo e, we e adjus ed o an O.D. = 0.5, and mixed p io o
in il a ion. Ag obac e ium exp essing he i al silencing supp esso
P19 was included in all in il a ions.
Fluo escence Mic oscopy
GFP/YFP luo escence in inocula ed plan s was moni o ed
using Nikon SMZ800, and Leica MZ16F mic oscopes.
Bimolecula Fluo escence Complemen a ion Analysis
The ele an gene coding sequences we e ampli ied by PCR
using speci ic oligonucleo ides bea ing a B si es, cloned in o
pDONR207 (In i ogen), and inally ans e ed o pYFN43 and
pYFC43 plasmids c ea ed by A. Fe ando (IBMCP, Valencia,
Spain; h p://www.ibmcp.up .es), o gene a e C- e minal ans-
la ional usions o he N- e minal (YFP
N
) and C- e minal (YFP
C
)
agmen s o he yellow luo escen p o ein (YFP), espec i ely.
Once ans e ed o Ag obac e ium, cons uc s we e ansien ly
exp essed in N. ben hamiana lea es as desc ibed abo e. Fluo escence
was de ec ed by con ocal mic oscopy h ee days ollowing
in il a ion.
Vi al Inocula ion
PPV was inocula ed by gen ly making a single punc u e on he
dis al pa o a lea wi h a s e ile oo hpick soaked in a suspension
o A. ume aciens bea ing an in ec ious cDNA clone a an O.D. = 1
a 600 nm. The Ag obac e ium suspension was p epa ed as desc ibed
abo e.
RNA Ex ac ion, RT and qPCR
To al RNA was ex ac ed using TRIzolH eagen (In i ogen)
ollowing he manu ac u e ’s ecommenda ions and u he
pu i ied by li hium chlo ide p ecipi a ion. Fo e e se ansc ip-
ion, he Re e Aid
TM
H Minus Fi s S and cDNA Syn hesis Ki
(Fe men as Li e Sciences) was used. Quan i a i e PCR (qPCR)
ampli ica ions and measu emen s we e pe o med using an ABI
PRISM 7000 sequence de ec ion sys em, and SYBR-G een
(Pe kin-Elme Applied Biosys ems). ACTIN2/8 was chosen as he
e e ence gene. The ollowing oligonucleo ides we e u ilized in he
analyses:
PPV-CP-FW: 59-GACTACGGCGTCAATGCTCAAC-39
PPV-CP-RV: 59- GTTTGCAGTTGAGGTCCTGACAC-39
ACTIN2/8-FW: 59-GGTAACATTGTGCTCAGTGGTGG-
39
ACTIN2/8-RV: 59-AACGACCTTATCTTCATGCTGC-39
MPK4-FW: 59-TTCCCAAACATGTCGGCTGGTG-39
MPK4-RV: 59-TGGCACAACGCCTCATCAACTG-39
MPK11-FW: 59-ACCCAAACAGACGCATTACAGTCG-39
MPK11-RV: 59-TCGTGTAGCGGTGCTAAGTAAGG-39
GRF6-FW: 59-TATGCAGGAGCAGATGGACGAG-39
GRF6-RV: 59-GGTGGCAGAAACATCGCGTAAC-39
Wes e n Blo and Immunop ecipi a ion
P o ein c ude ex ac s we e p epa ed by homogenizing g ound
ozen lea ma e ial wi h T is-bu e ed saline (TBS) supplemen ed
wi h 5 mM DTT, p o ease inhibi o cock ail (Sigma-Ald ich), and
p o ein phospha ase inhibi o s (PhosS op, Roche). P o ein con-
cen a ion was measu ed using B ad o d eagen ; 25 mg o o al
p o ein was sepa a ed by SDS-PAGE (12% ac ylamide w/ ) and
ans e ed o ni ocellulose il e s. The il e was s ained wi h
Ponceau-S a e ans e , and used as a loading con ol.
Fo immunop ecipi a ions, ex ac s we e clea ed by wo
successi e cen i uga ion s eps a 160006g and 4uC o 30
minu es, and hen incuba ed a 4uC o 2 h wi h conjuga ed
an ibodies unde gen le ocking. Unbound p o eins we e emo ed
by successi ely washing he esin wi h TBS, TBS 0.5% ( / )
T i on X-100 and TBS 0.3M NaCl, 1% ( / ) NP-40. We used
monoclonal an i-HA clone HA-7 aga ose-conjuga e (Sigma-
Ald ich), and abbi polyclonal GFP-sepha ose conjuga e (Abcam).
Immunop ecipi a ed p o eins we e elu ed wi h Laemmli sample
bu e by hea ing o 5 minu es a 95uC. Fo Wes e n blo s analysis
he ollowing an ibodies we e used: mouse monoclonal an i-GFP
an ibodies (Roche), abbi polyclonal an i-MaBP an ibodies (New
England Biolabs), mouse monoclonal an i-phosphoMyBP clone
P12 an ibodies (Millipo e), abbi polyclonal an i-HA an ibodies
(Sigma), mouse monoclonal an i-phosphose ine Q5 an ibodies
(Qiagen), and mouse monoclonal an i-ubiqui in Ub (P4D1)
an ibodies (San a C uz Bio echnology).
MAP Kinase Ac i i y Assay
The assay was pe o med a e MPK11 immunop ecipi a ion,
and p io o elu ion. Resin was esuspended wi h a eac ion mix
consis ing o 50 mM T is-HCl pH 7.5, 10 mM MgCl
2
,1mM
DTT, 0.1 mM ATP and 0.4 mg/ml myelin basic p o ein (MyBP).
Reac ions we e incuba ed o 30 min a 30uC in a shaking
A abidopsis DBP1 Phospha ase
PLOS ONE | www.plosone.o g 2 Ma ch 2014 | Volume 9 | Issue 3 | e90734
incuba o . Following incuba ion Laemmli sample bu e was
added, and samples we e hea ed o 5 minu es a 95uC.
Phospho yla ed MyBP was de ec ed by Wes e n blo using a
speci ic mouse monoclonal an ibody as desc ibed abo e, ollowing
manu ac u e ’s ins uc ions.
Resul s
dbp1 Mu an Phosphop o eome Analysis
DBP1 is a p o ein phospha ase 2C o he DNA-binding DBP
amily. Changes in he phosphop o eome o DBP1-de ec i e plan s
could e eal di ec o indi ec a ge s o DBP1. Consequen ly, he
phosphop o eome o wild- ype Col-0 and dbp1 mu an plan s was
analysed applying an op imized wo k low o highly e icien
phosphop o ein en ichmen om adul plan lea es, including
ammonium sul a e p ecipi a ion and me al-oxide a ini y ch oma-
og aphy [11] (see Me hods S1). Fo a sensi i e and de ailed
sho gun analysis, LTQ O bi ap Velos echnology was used on
h ee biological eplica es pe geno ype and ela i e quan i ica ion
based on spec al coun was pe o med. E iciency and ep oduc-
ibili y o his p ocedu e was analyzed by 1-D-SDS-PAGE and
subsequen P oQ Diamond and CBB s aining. As lis ed in Table
SI, 169 (phospho)p o eins showed di e en ial accumula ion in he
dbp1 mu an ela i e o Col-0 ( - es , p,0.05). In e es ingly, among
he iden i ied p o eins an ea lie desc ibed DBP1 in e ac o was
ound. 14-3-3l(GRF6; A 5g10450) was p e iously shown o
speci ically in e ac wi h DBP1 in i o [6]. In addi ion, iden i ica-
ion o he MAP kinase pai MPK4 (A 4g01370)/MPK11
(A 1g01560) was also pa icula ly signi ican since i p o ides an
insigh in o he signalling p ocesses DBP1 migh be pa o .
Howe e , p o ein iden i ica ion in his case is ambiguous due o
he high sequence simila i y be ween MPK4 and MPK11.
Consis en wi h an 89% iden i y be ween hese wo p o eins, mos
o he yp ic pep ides iden i ied we e p esen in bo h. The e o e,
a his s age we could no asce ain he ac ual ela ionship o hese
wo MAPKs wi h DBP1. MAPKs a e key componen s o signal
ansduc ion pa hways in euka yo es. MPK4 is ac i a ed in
esponse o pa hogen-associa ed molecula pa e ns (PAMPs) and
has been epo ed o be in ol ed in plan immuni y as a nega i e
egula o o he salicylic acid (SA)-media ed de ense pa hway
[12,13]. MPK11 was also ound o be ac i a ed ollowing
applica ion o he PAMP molecule lg22 [10], bu he signalling
pa hways in which i ope a es emain unknown, as uniden i ied
a e i s p o ein a ge s. As p e iously epo ed, he mpk4 mu an
showed se e e dwa ism (Figu e S1B) [12,14]. The e o e, since
DBP1 has no been ela ed so a o any de elopmen al al e a ion
and he dbp1 mu an exhibi s wild- ype mo phology and a chi ec-
u e, we ocused on he mpk11 mu an o u he s udies.
Subcellula Localiza ion and Analysis o P o ein-p o ein
In e ac ions
The di e en ial p o ein accumula ion obse ed in he dbp1
mu an migh e eal a di ec o indi ec e ec o he absence o
DBP1 p o ein phospha ase ac i i y. The e o e, we analysed
whe he he selec ed p o eins in e ac wi h DBP1 in i o using
Bimolecula Fluo escence Complemen a ion (BiFC). The ele an
p o eins mus co-localize o in e ac , and localiza ion migh be
al e ed as a consequence o he in e ac ion. The e o e, we i s
examined he subcellula localiza ion o MPK11 and GRF6 when
ansien ly exp essed as GFP usions in Nico iana ben hamiana lea es
by ag oin il a ion as desc ibed [4]. Bo h obacco and A abidopsis
DBP1 localize in he nucleus and he cy osol [6]. He e, he
selec ed p o eins exhibi ed a simila subcellula dis ibu ion (Fig. 1).
Nuclea localiza ion was e i ied using cen ome ic his one H3
used o Red Fluo escen P o ein (mRFP) as a nuclea ma ke
[15]. The e o e, he espec i e localiza ion pa e ns enabled
in e ac ion wi h DBP1. BiFC sugges ed ha he in e ac ions
ac ually occu in i o a e ansien ly exp essing he ele an
p o eins used o he N- e minal and C- e minal agmen s o he
Yellow Fluo escen P o ein (YFP) in N. ben hamiana by ag oin il a-
ion. The in e ac ion esul s in YFP luo opho e econs i u ion and
a luo escence signal whe e he p o ein complex accumula es.
Fig. 2A depic s con ocal mic oscopy images ob ained ollowing
coexp ession o he indica ed usion p o eins. DBP1-GRF6 and
DBP1-MPK11 complexes we e de ec ed bo h in he cy osol and in
he nucleus. As abo e, mRFP- agged cen ome ic his one H3 was
included in he analysis as indica i e o nuclea localiza ion. Fo
GRF6, his was consis en wi h p e ious obse a ions ha obacco
GRF6 and DBP1 homologues in e ac ed o media e nucleocy o-
plasmic shu ling and DBP1 accumula ion in he cy osol [6].
In e ac ions we e alida ed by coimmunop ecipi a ion o usion
p o eins ansien ly exp essed in lea es o N. ben hamiana (Fig. 2B
and Figu e S2)). DBP1 used o mal ose binding p o ein (MaBP)
was immunop ecipi a ed wi h an i-MaBP an ibodies. When
coexp essed wi h MPK11 used o YFP, o GRF6 agged wi h
he hemagglu inin (HA) epi ope, p esence o he pu a i e pa ne
in he immunop ecipi a e was demons a ed by Wes e n blo using
an i-GFP and an i-HA an ibodies, espec i ely. The e o e, bo h
MPK11 and GRF6 in e ac in i o wi h DBP1.
DBP1 Inac i a es MPK11 in i o
Since MAPK ac i i y is egula ed by phospho yla ion and
DBP1 is a p o ein phospha ase, DBP1 migh inac i a e MPK11.
To es his hypo hesis, we ansien ly exp essed MPK11 in N.
ben hamiana as a YFP N- e minal usion, alone o in combina ion
wi h DBP1 used o MaBP. MPK11 was hen immunop ecipi a ed
wi h an i-GFP an ibodies, and MAPK ac i i y was measu ed in
he immunop ecipi a e using myelin basic p o ein (MyBP) as a
subs a e and an i-phospho-MyBP an ibodies o p oduc de ec-
ion. The expe imen was epea ed h ee imes. Albei he le el o
ac i i y o MPK11 was a iable, likely because ac i a ion o
exp essed MPK11 elies on endogenous kinases om N.
ben hamiana, he de ec ed ac i i y was ma kedly educed in e e y
case when DBP1 was coexp essed wi h MPK11. As shown in
Fig. 3A o a ep esen a i e expe imen , simila MPK11 p o ein
amoun s we e immunop ecipi a ed in bo h cases (uppe panel), bu
when coexp essed wi h DBP1 (middle panel), MPK11 ac i i y was
signi ican ly educed (lowe panel). The e o e, DBP1 is a p o ein
phospha ase di ec ly egula ing MPK11 ac i i y.
Since DBP1 inhibi ed MPK11, di e en ial (phospho)p o ein
accumula ion in Col-0 and he dbp1 mu an migh no exclusi ely
e lec di ec DBP1-media ed dephospho yla ion, bu also indi ec
e ec s o inc eased MPK11 ac i i y in he mu an . Fo ha eason,
and o help cla i y how his eme ging p o ein ne wo k ope a es, we
analysed i GRF6 migh be a MPK11 subs a e. Using BiFC, we
i s showed ha GRF6 in e ac ed wi h MPK11 in i o (Fig. 3B).
The complex la gely accumula ed in he nucleus, indica ing ha
he in e ac ion likely occu ed in he nuclea compa men . The
in e ac ion was con i med by coimmunop ecipi a ion (Fig. 3C).
When coexp essed by ag oin il a ion in N. ben hamiana oge he
wi h GRF6-HA, YFP-MPK11 was de ec ed by Wes e n-blo in he
an i-HA immunop ecipi a e.
MPK11 Phospho yla es GRF6 in i o
The MPK11-GRF6 in e ac ion sugges ed ha GRF6 migh be
a MPK11 subs a e. To e i y his hypo hesis, GRF6 phospho -
yla ion was analyzed by Wes e n blo a e immunop ecipi a ion,
using wo di e en monoclonal an ibodies ecognizing phospho -
A abidopsis DBP1 Phospha ase
PLOS ONE | www.plosone.o g 3 Ma ch 2014 | Volume 9 | Issue 3 | e90734
yla ed se ine and h eonine esidues. The an i-phosphose ine and
an i-phospho h eonine an ibodies a e comme cial an ibodies
which ecognize he espec i e phospho yla ed esidues ega dless
o su ounding amino acids. Di e ences we e only ound in se ine
phospho yla ion. GRF6 was phospho yla ed a se ine esidues
when exp essed in N. ben hamiana by an endogenous kinase ac i i y
(Fig. 4A, le lane, bo om panel). Se ine phospho yla ion
signi ican ly inc eased when GRF6was coexp essed wi h YFP-
MPK11 (Fig. 4A, middle lane), sugges ing addi ional phospho -
yla ion as media ed by MPK11. Whe he phospho yla ion may be
occu ing di ec ly o indi ec ly ia ano he kinase ac i i y ha is
modi ied by MPK11 emains s ill unknown. In he p esence o
DBP1, he MPK11-dependen inc ease in GRF6 se ine phos-
pho yla ion was p e en ed (Fig. 4A, igh lane), u he suppo ing
MPK11 inac i a ion by DBP1.
MPK11 P omo es Deg ada ion o GRF6 in i o
When ansien ly coexp essed wi h MPK11 in N. ben hamiana,
GRF6 ep oducibly accumula ed a lowe le els han when
exp essed alone (Fig. 4B). This sugges ed lowe GRF6 s abili y
in he p esence o MPK11. The ubiqui in-p o easome sys em
(UPS) is a majo componen o he cell machine y engaged in he
con ol o p o ein abundance, and plays an impo an ole in he
egula ion o mul iple biological p ocesses [16]. UPS a ge
p o eins a e ubiqui ina ed by E3 ubiqui in ligases, ecognized by
he 26S p o easome complex and deg aded. The e o e, we
analyzed GRF6 ubiqui ina ion ollowing ansien exp ession in
N. ben hamiana and immunop ecipi a ion wi h an i-HA an ibodies.
Wes e n blo analysis using an i-ubiqui in monoclonal an ibodies
e ealed ha a ac ion o immunop ecipi a ed GRF6 was
ubiqui ina ed (Fig. 4C, le panel), accumula ing p edominan ly
as a likely e aubiqui ina ed o m acco ding o i s es ima ed
molecula mass. To con i m he iden i y o his ubiqui ina ed
p o ein as GRF6, a Wes e n blo was immunodeco a ed wi h an i-
HA an ibodies, e ealing de ec ion o he same p o ein along wi h
he as mig a ing and mo e abundan unmodi ied GRF6 p o ein
(Fig. 4C, igh panel). In e es ingly, abundance o he ubiqui i-
na ed ac ion inc eased when GRF6 was coexp essed wi h
MPK11, sugges ing a highe ubiqui ina ion a e upon MPK11-
media ed GRF6 phospho yla ion. This co ela ed wi h he
inc eased GRF6 ins abili y we obse ed in he p esence o
MPK11, as shown in Fig. 4B. Rein o cing he unc ional
in e ela ionship exis ing among hese h ee p o eins, DBP1
p e en ed ubiqui ina ion o GRF6. When DBP1 and MPK11
we e simul aneously exp essed along wi h GRF6 in N. ben hamiana
lea es, he p esence o DBP1 es ic ed MPK11-p omo ed GRF6
ubiqui ina ion (Fig. 4C). This was likely due o he inhibi o y e ec
o DBP1 on MPK11 ac i i y which, as shown abo e, also
p e en ed GRF6 phospho yla ion media ed by MPK11 (Fig. 4A).
Candida e DBP1 Ta ge s had Di e en Roles in PPV
In ec ion
DBP1 unc ions as a hos suscep ibili y ac o du ing PPV
in ec ion [3], whe eas DIP2, a small polypep ide unc ionally
modula ing DBP1, con ibu es o esis ance [4]. The implica ion
o he selec ed p o eins in he A abidopsis-PPV in e ac ion was
examined by selec ing homozygous mu an plan s bea ing a T-
DNA inse ion in he ele an gene ha ma kedly educes gene
exp ession (Fig. S1A). Plan s o hese lines we e inocula ed wi h a
PPV-GFP i al s ain, and in ec ion p og ession was moni o ed by
luo escence mic oscopy and by RT-qPCR using speci ic p ime s
o he i al coa p o ein gene. In e es ingly, mpk11 showed
inc eased suscep ibili y o PPV, whe eas g 6 exhibi ed enhanced
esis ance (Fig. 5A, B). Complemen a ion o he analyzed mu an s
wi h genomic agmen s bea ing he wild- ype gene sequence
showed ha he obse ed pheno ypes we e indeed due o loss o
unc ion o he ele an genes (Figu e S1C).
As bo h genes appea ed o be implica ed in he A abidopsis-
PPV in e ac ion, we analyzed hei exp ession dynamics ela i e o
i al accumula ion in Col-0. PPV-inocula ed plan s we e inspec ed
unde he luo escence mic oscope and in ec ed lea es we e
g ouped in o ou di e en ca ego ies based on he deg ee o i al
coloniza ion. Lea ma e ial exhibi ing no (1), low (2), medium (3),
Figu e 1. Subcellula dis ibu ion o he iden i ied DBP1 pu a i e a ge s. Full-leng h coding sequences we e used o he N- e minus o
g een luo escen p o ein (GFP) o o he C- e minus o yellow luo escen p o ein (YFP), and ansien ly exp essed in Nico iana ben hamiana lea es
by ag oin il a ion. Lea sec ions we e inspec ed by con ocal mic oscopy 3 d a e in il a ion. Fusion p o eins we e exp essed in epide mal cells.A
la ge cen al acuole occupies mos o he cellula space, cons aining cy osol o a na ow pe iphe al a ea. Co-exp ession wi h mRFP- agged
cen ome ic his one H3 e eals nuclei localiza ion.
doi:10.1371/jou nal.pone.0090734.g001
A abidopsis DBP1 Phospha ase
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and high (4) i al accumula ion we e ha es ed, and gene
exp ession was analyzed by RT-qPCR, e e ed o ACT2/8 and
no malized using simila samples om unin ec ed plan s (Fig. 5C).
MPK11 ansc ip accumula ion inc eased du ing in ec ion, while
GRF6 exp ession was ep essed ollowing MPK11 induc ion.
Fig. 5D shows inc easing i al accumula ion h ough he selec ed
ca ego ies as quan i ied by RT-qPCR.
To demons a e he biological ele ance o hese indings, we
analysed GRF6 accumula ion du ing he cou se o PPV in ec ion
in N. ben hamiana. P o ein le el was measu ed a e 3 d o
exp ession a di e en ime poin s o in ec ion (Fig. 6A, B). As
desc ibed abo e, GRF6 accumula ion was ep oducibly dimin-
ished when coexp essed wi h MPK11. He e, he magni ude o he
dec ease obse ed in non-inocula ed issue (0 dpi) is lowe han
ha shown ea lie . This is no su p ising, since de-s abiliza ion o
GRF6 media ed by MPK11 depends on he exp ession le el o
MPK11 and also on he deg ee o ac i a ion o he kinase, which
elies on endogenous MAPKKs. A signi ican u he educ ion in
GRF6 p o ein accumula ion was obse ed 4 d pos -inocula ion
wi h PPV. This educ ion was speci ic and no obse ed o
cy osolic asco ba e pe oxidase (APX), he e used as a nega i e
con ol, nei he by Ponceau-S s aining o he p o ein ex ac s.
GRF6 p o ein decline was ma kedly accele a ed in he p esence o
Figu e 2. Iden i ied p o eins in e ac
in i o
wi h DBP1. A, BiFC
assay wi h YFP usions ansien ly exp essed in N. ben hamiana lea es
by ag oin il a ion. Bo h ecip ocal combina ions a e shown o each
in e ac ion. As a nega i e con ol, DBP1 and he un ela ed ansc ip ion
ac o BREVIS RADIX (BRX) YFP usions we e used. Cen ome ic his one
H3 used o mRFP was co-exp essed as a ma ke o nuclea localiza ion.
B, Coimmunop ecipi a ion alida ed he epo ed in e ac ions. Rele an
usion p o eins we e ansien ly exp essed in N. ben hamiana lea es by
ag oin il a ion as indica ed, and immunop ecipi a ed wi h an i-MaBP
an ibodies. Wes e n blo s o he immunop ecipi a ed ac ions deco-
a ed wi h he indica ed an ibodies a e shown. An i-GFP an ibodies
allow o de ec ion o YFP.
doi:10.1371/jou nal.pone.0090734.g002
Figu e 3. MPK11 in e ac s wi h GRF6 and is nega i ely
modula ed by DBP1. A, DBP1 inhibi ed MPK11 kinase ac i i y.
MPK11 and DBP1 we e ansien ly exp essed as ansla ional usions o
YFP and MaBP, espec i ely, in N. ben hamiana lea es by ag oin il a-
ion. YFP-MPK11 was immunop ecipi a ed 3 d a e in il a ion wi h
an i-GFP an ibodies and MAPK ac i i y was measu ed in he immuno-
p ecipi a e by Wes e n blo wi h an ibodies speci ically ecognizing
phospho yla ed MyBP (lowe panel). A ac ion o he immunop ecip-
i a ed p o ein was analyzed using an i-GFP (uppe panel) and an i-MaBP
(middle panel) an ibodies. B, MPK11 in e ac ed wi h GRF6 in i o. BiFC
analysis in N. ben hamiana lea es showing speci ic MPK11-GRF6
in e ac ion. The MPK11-GRF6 complex was de ec ed p edominan ly in
he nucleus. C, GRF6 and MPK11 co-immunop ecipi a ion. GRF6-HA was
exp essed in N. ben hamiana lea es and immunop ecipi a ed wi h an i-
HA an ibodies. YFP-MPK11 was de ec ed by Wes e n blo wi h an i-GFP
an ibodies in he an i-HA immunop ecipi a e when coexp essed wi h
GRF6 (uppe panel), con i ming in e ac ion be ween he wo p o eins.
Lowe panel shows GRF6-HA immunode ec ion in he immunop ecip-
i a e by Wes e n blo .
doi:10.1371/jou nal.pone.0090734.g003
A abidopsis DBP1 Phospha ase
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MPK11, co obo a ing he inc eased GRF6 u n-o e caused by
MPK11. To asce ain he implica ion o he p o easome in his
p ocess, GRF6 and MPK11 we e ansien ly exp essed in lea es o
N. ben hamiana which had been p e iously inocula ed wi h PPV in
o de o p omo e ac i a ion o MPK11 and GRF6 deg ada ion. As
shown in Fig. 6C, ea men wi h he p o easome inhibi o MG-132
24 h p io o collec ing he issue e e ed he educ ion in GRF6
s abili y obse ed when coexp essed wi h MPK11, indica ing ha he
p o easome is media ing GRF6 deg ada ion p omo ed by MPK11.
In con as , in he absence o MPK11, addi ion o MG-132 did no
ha e any signi ican e ec on GRF6 p o ein accumula ion, as
expec ed i phospho yla ion o GRF6 by MPK11 is he molecula
e en ha a ge s GRF6 o deg ada ion ia he p o easome.
Discussion
DBPs a e plan -speci ic DNA-binding p o ein phospha ases
[1,2], and ha e been ound o play a ole in plan - i us
in e ac ions [1,3,4]. Howe e , li le is known abou he molecula
mechanisms in which DBP ac o s a e in ol ed. The e o e, we
sea ched o pu a i e DBP1 a ge s by analysing phosphop o eome
changes esul ing om DBP1 loss-o - unc ion. F om he iden i ied
p o eins we selec ed 14-3-3l(GRF6) and MPK11 o u he
analyses.
GRF6 is liso o m wi hin he 14-3-3 amily, highly conse ed
p o eins in ol ed in egula ing mul iple biological p ocesses and
signalling pa hways in euka yo es. 14-3-3 p o eins usually bind
phosphopep ide mo i s and modi y unc ion o hei a ge s a
di e en le els [17]. GRF6 was p e iously shown o in e ac wi h
DBP1 using he yeas wo-hyb id sys em [6], an in e ac ion
conse ed in obacco and A abidopsis. MPK11 is one o he abou
20 MAPKs p esen in A abidopsis. Toge he wi h MAPKKs and
MAP3Ks, MAPKs o m signalling cascades media ing de elop-
men al p ocesses and esponses o bo h bio ic and abio ic s esses
in all euka yo ic o ganisms. Iden i ica ion o subs a es and ac o s
egula ing kinase ac i i y is essen ial o a be e unde s anding o
Figu e 4. MPK11 phospho yla ed GRF6 p omo ing ubiqui ina ion and inc eased u no e . A, De ec ion o GRF6 phospho yla ion. GRF6-
HA was ansien ly exp essed, alone o oge he wi h YFP-MPK11 and MaBP-DBP1 as indica ed in N. ben hamiana lea es by ag oin il a ion, and
immunop ecipi a ed using an i-HA an ibodies. The h ee uppe panels show de ec ion by Wes e n blo o he di e en exp essed p o eins wi h he
indica ed an ibodies. The wo lowe panels show immunop ecipi a ion o GRF6-HA using an i-HA an ibodies: an aliquo o he immunop ecipi a ed
ac ion was loaded abo e as a loading con ol, whe eas in he bo om panel se ine phospho yla ion de ec ed using an i-phosphose ine monoclonal
an ibody is shown. B, MPK11 media ed inc eased GRF6 u no e . GRF6-HA and YFP-MPK11 we e exp essed in N. ben hamiana lea es by
ag oin il a ion. Wes e n blo o p o ein ex ac s om in il a ed lea es 3 d pos -in il a ion immunodeco a ed wi h he indica ed an ibodies o
speci ic exp essed p o ein de ec ion. Ponceau-S s aining o he ni ocellulose memb ane is shown a he bo om. C, GRF6 was ubiqui ina ed in i o
and MPK11 inc eased ubiqui ina ion a e. The indica ed p o eins we e ansien ly exp essed in N. ben hamiana lea es by ag oin il a ion, and GRF6-
HA was immunop ecipi a ed wi h an i-HA an ibodies 3 d pos -in il a ion. Le panel, Wes e n blo o he an i-HA immunop ecipi a e
immunodeco a ed wi h an i-ubiqui in an ibodies. Righ panel, he same blo shown in he le panel was s ipped, and immunodeco a ed wi h
an i-HA an ibodies, indica ing ha he de ec ed ubiqui ina ed p o ein co esponds o GRF6-HA and e ealing unmodi ied GRF6.
doi:10.1371/jou nal.pone.0090734.g004
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cell esponses o de elopmen al, ho mone and en i onmen al
signals. MAPKs a e ac i a ed by phospho yla ion, whe eas
dephospho yla ion leads o inac i a ion. In yeas and mammals,
y osine and dual speci ici y phospha ases a e conside ed he
majo MAPK phospha ases [18]. In plan s, se ine/ h eonine
p o ein phospha ases 2C ha e also been shown o dephospho yla e
and modula e MAPK ac i i y [19–23]. When ac i a ed, MAPKs
phospho yla e a a ie y o subs a es, he eby modula ing hei
unc ion. He e we show ha DBP1 nega i ely egula ed MPK11
ac i i y and ha GRF6 seems o be a MPK11 subs a e. In
A abidopsis, only a ew MAPK a ge s ha e been iden i ied in i o
so a . Popescu e al. (2009) unde ook an ex ensi e sea ch o
subs a es o 10 MAPKs using p o ein mic oa ays, bu MPK11
was no included in he s udy [24]. We demons a ed ha GRF6
in e ac s wi h MPK11, and ha MPK11 p omo es phospho yla-
ion o GRF6 in i o. Mo eo e , DBP1 p e en ed MPK11-
media ed GRF6 phospho yla ion. In e es ingly, GRF6 accumu-
la ed a lowe le els when coexp essed wi h MPK11, sugges ing a
phospho yla ion-dependen egula ion o GRF6 p o ein abun-
dance. Indeed, GRF6 was ubiqui ina ed in i o, and ubiqui ina ion
was enhanced in he p esence o MPK11, leading o highe
deg ada ion a es. In con as , he p esence o DBP1 p o ec ed
GRF6 om he inc eased ubiqui ina ion p omo ed by MPK11.
This is likely a consequence o he inhibi ion o MPK11 ac i i y
exe ed by DBP1. Mo eo e , GRF6 deg ada ion was a enua ed in
he p esence o he p o easome inhibi o MG-132, indica ing ha
GRF6 is deg aded ia he p o easome. These esul s p o ide
compelling e idence o a egula o y p o ein ne wo k nuclea ed by
DBP1 which modula es s abili y o GRF6, wi h GRF6 in u n
media ing nucleo-cy oplasmic shu ling o DBP1. A ubiqui in
ligase was ecen ly epo ed o a ge 14-3-3 p o eins in a s imulus-
speci ic manne in he cellula C/N balance esponse [25].
The e o e, o he 14-3-3 p o eins a e subjec ed o a simila
egula ion h ough induced deg ada ion ia UPS.
Figu e 5. Pu a i e DBP1 a ge s played a ole in PPV in ec ion, posi i ely o nega i ely a ec ing PPV mul iplica ion. A, P og ession o
PPV in ec ion in loss-o - unc ion mu an s. Plan s o he indica ed geno ypes we e inocula ed wi h PPV-GFP and in ec ion ex en was analyzed by
luo escence mic oscopy o moni o GFP dis ibu ion. B, PPV accumula ion in loss-o - unc ion mu an s. Vi al accumula ion was measu ed by RT-qPCR
using p ime s speci ically ampli ying he i al coa p o ein gene. Da a we e no malized using ACT2/8 as a e e ence gene. Rela i e accumula ion wi h
espec o Col-0 is shown as he mean o h ee independen expe imen s 6SD. C, Gene exp ession analysis du ing he cou se o PPV-GFP in ec ion in
Col-0 plan s. Inocula ed lea issue showing p oduc i e in ec ion was pooled in o ou di e en ca ego ies acco ding o he deg ee o i al
coloniza ion de e mined unde he luo escence mic oscope. Lea ma e ial wi h no isible (1), low (2), medium (3), and high (4) i al accumula ion was
ha es ed, and gene exp ession was analyzed by RT-qPCR using ACT2/8 as a e e ence gene. Exp ession da a we e no malized o hose ob ained o
simila samples collec ed om unin ec ed plan s. The mean o h ee independen expe imen s 6SD is shown. D, PPV accumula ion in he selec ed
in ec ion ca ego ies. Vi al accumula ion was quan i ied by RT-qPCR as abo e, and e e ed o he alue in (1). Al hough no isible signal was obse ed
in hese lea es when assessed mic oscopically, he e was an incipien in ec ion de ec able by RT-qPCR.
doi:10.1371/jou nal.pone.0090734.g005
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MPK11 p omo ed enhanced GRF6 deg ada ion, nega i ely
egula ing GRF6 unc ion. BiFC analysis p o ides a cellula basis
o be e in e p e hese biochemical da a. As DBP1, GRF6 also
localized bo h o he nucleus and he cy osol (Fig. 1), and, in u n,
in e ac ion be ween hese wo ac o s, as de ec ed by BiFC, was
also obse ed in he wo cellula compa men s (Fig. 2A).
Accumula ion o GRF6-DBP1 complex in he cy osol is cong uen
wi h DBP1 elocaliza ion om he nucleus o he cy osol, as
p omo ed by 14-3-3 G in obacco [6]. In con as , he in e ac ion
be ween GRF6 and MPK11 was de ec ed p edominan ly in he
nucleus. MPK11 could be p e en ing GRF6-DBP1 in e ac ion in
he nucleus by p omo ing GRF6 deg ada ion. Phospho yla ion is
well known o in luence ubiqui ina ion ei he posi i ely o
nega i ely [26]. Thus, he ac i i y o E3 ubiqui in ligases may be
egula ed by phospho yla ion. Al e na i ely, phospho yla ion o
he a ge p o ein can enable o p e en E3 ubiqui in ligase
ecogni ion, o egula e E3 ubiqui in ligase access o i s a ge by
modi ying i s subcellula localiza ion. In he case o GRF6, bo h
mechanisms a e possible. Phospho yla ion by MPK11 migh
c ea e a binding si e o E3 ubiqui in ligase, o could ec ui GRF6
o he nucleus, whe e he E3 ubiqui in ligase migh be loca ed.
Al hough he MPK11-GRF6 complex accumula es in he nucleus,
we canno exclude ha he in e ac ion akes place in he cy osol,
and p omo es apid nuclea a ge ing o GRF6 o ubiqui ina ion,
he eby p e en ing in e ac ion wi h DBP1.
Li le is known abou he biology o DBP ac o s. DBP1 was
ecen ly epo ed o ac as a po y i us suscep ibili y ac o , since
he absence o DBP1 unc ion hinde ed in ec ion by he
po y i uses Plum pox i us (PPV) and Tu nip mosaic i us (TuMV)
[3]. Complex se s o hos -pa hogen in e ac ions de e mine he
ou come o plan i us in ec ion. These in e ac ions, he unde -
lying mechanisms and he in ol ed signalling pa hways a e la gely
unknown. We showed ha bo h MPK11 and GRF6 also play a ole
in he A abidopsis-PPV in e ac ion, since he espec i e loss-o -
unc ion mu an s exhibi ed an al e ed esponse o PPV in ec ion.
MPK11 eme ges as a signalling ac o likely media ing he de ense
esponse o PPV in ec ion, since loss o unc ion leads o inc eased
suscep ibili y. Th ee weeks a e inocula ion, PPV accumula ion
Figu e 6. MPK11 p omo es inc eased GRF6 p o ein u n-o e du ing PPV in ec ion. A, Analysis o GRF6 accumula ion in in ec ion by PPV.
GRF6-HA was ansien ly exp essed in N. ben hamiana lea es alone o oge he wi h MPK11 as indica ed. P o ein accumula ion was analyzed 3 days
pos -in il a ion in lea es ha we e no inocula ed wi h PPV (0 dpi), lea es ha we e inocula ed wi h PPV one day a e GRF6-HA ag oin il a ion
(2 dpi; in hese lea es, PPV in ec ion is a day 2 when GRF6 p o ein accumula ion was analyzed), and lea es ha we e inocula ed wi h PPV one day
be o e GRF6-HA ag oin il a ion (4 dpi; in hese lea es, PPV in ec ion is a day 4 when GRF6-HA accumula ion was eco ded). As an in e nal con ol,
accumula ion o asco ba e pe oxidase (APX) is shown. Ponceau-S s aining o memb ane indica es equal p o ein loading. PPV and ACTIN2/8 RNA
accumula ion was de e mined by RT-PCR. B, Quan i ica ion o GRF6 and APX bands in Wes e n blo using ImageJ so wa e. Values we e e e ed o
hose in he absence o PPV and MPK11. The expe imen was epea ed h ee imes wi h simila esul s. C, P o easome media es GRF6 deg ada ion
p omo ed by MPK11. GRF6-HA and YFP-MPK we e ansien ly exp essed in N. ben hamiana lea es 4 days a e inocula ion o he same lea es wi h
PPV. Two days la e , lea es we e in il a ed ei he wi h he p o easome inhibi o MG-132 o wi h he same concen a ion o he sol en DMSO, and
a e 24 h, lea ma e ial was collec ed. P o ein accumula ion was analyzed by Wes e n blo using speci ic an ibodies as indica ed.
doi:10.1371/jou nal.pone.0090734.g006
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was app oxima ely 10- old highe in he mpk11 mu an han in
Col-0 plan s, as de e mined by RT-qPCR (Fig. 5A). The ac ha
MPK11 exp ession was mode a ely and ansien ly induced du ing
in ec ion suppo s his hypo hesis (Fig. 5C). This assigns a
biological ole o MPK11, and sheds ligh on he signalling
p ocesses igge ed in esponse o PPV in ec ion. The e o e,
MPK11 shows p omise o enginee ing imp o ed esis ance
agains PPV. In con as , GRF6-de icien plan s displayed
enhanced esis ance o PPV in ec ion, a pheno ype simila o ha
o dbp1 mu an s, sugges ing ha DBP1 and GRF6 likely coope a e
o con e suscep ibili y, whe eas MPK11 would be an agonizing
his e ec . Since MPK11 p omo es GRF6 deg ada ion, o es
whe he his egula ion plays a ole in he A abidopsis-PPV
in e ac ion, we analyzed GRF6 accumula ion du ing he cou se o
PPV in ec ion in N. ben hamiana lea es (Fig. 6A). In PPV-inocula ed
lea es, GRF6 p o ein le el was signi ican ly educed as he i us
accumula ed in he locally in ec ed issue. Howe e , when co-
exp essed wi h MPK11, GRF6 u no e was speci ically accele -
a ed. This u he sugges s ha MPK11 would be in ol ed in a
plan local de ense esponse aimed a educing GRF6 p o ein le el
in ea ly s ages o PPV in ec ion inc easing i s p o easome-
media ed deg ada ion.
The e o e, ou esul s ha e a signi ican impac no only in he
biology o DBP ac o s, bu also con ibu e o a be e knowledge
o MAPK signalling pa hways in A abidopsis h ough he
iden i ica ion o GRF6 as a likely MPK11 subs a e and o
DBP1 as a p o ein phospha ase egula ing MPK11 ac i i y, and
un eils he implica ion o his p o ein module in he esponse o
PPV in ec ion in A abidopsis.
Suppo ing In o ma ion
Figu e S1 T-DNA inse ion mu an s used in his s udy.
A, RT-qPCR analysis o gene exp ession. Da a we e no malized
using ACT2/8 as a e e ence gene and a e exp essed ela i e o he
espec i e exp ession le el in wild- ype Col-0 plan s. B, Images o
4 week-old ep esen a i e plan s o each mu an line compa ed o
he wild- ype Col-0 eco ype. No isible al e a ion was obse ed in
plan mo phology and a chi ec u e a any de elopmen al s age,
excep o mpk4 mu an which showed se e e de elopmen al
de ec s. C, mpk11 and g 6 mu an lines we e ans o med wi h
genomic agmen s encompassing he co esponding wild- ype
s uc u al genes and mo e han 1500 bp o p omo e sequence.
Homozygous plan s bea ing single inse ions we e inocula ed wi h
PPV and p og ession o in ec ion was analyzed by RT-qPCR
using p ime s o he i al coa p o ein gene. Exp ession alues
we e no malized using ACT2/8 and e e ed o Col-0.
(TIF)
Figu e S2 Speci ici y o he an ibodies used in immu-
nop ecipi a ion assays. The indica ed p o ein combina ions
we e ansien ly exp essed in N. ben hamiana lea es by ag oin il a-
ion, and immunop ecipi a ed wi h he an ibodies indica ed on
op. The co esponding immunop ecipi a ed ac ions we e
analyzed by Wes e n blo using he an ibodies e e ed on he le .
(TIF)
Me hods S1
(DOCX)
Table S1
(XLS)
Acknowledgmen s
Au ho s wish o hank V. Rami ez, J. Ga cı
´a-And ade and A. Lo´pez om
P. Ve a’s lab o help ul discussions.
Au ho Con ibu ions
Concei ed and designed he expe imen s: JLC MJC KN IL MNG.
Pe o med he expe imen s: JLC MJC KN IL MNG. Analyzed he da a:
JLC MJC KN IL MNG DS PV. W o e he pape : JLC PV.
Re e ences
1. Ca asco JL, Ancillo G, Mayda E, Ve a P (2003) A no el ansc ip ion ac o
in ol ed in plan de ense endowed wi h p o ein phospha ase ac i i y. EMBO J
22: 3376–3384.
2. Ca asco JL, Ancillo G, Cas ello´ MJ, Ve a P (2005) A no el DNA-binding mo i ,
hallma k o a new amily o plan ansc ip ion ac o s. Plan Physiol 137: 602–
606.
3. Cas ello´ MJ, Ca asco JL, Ve a P (2010) DNA-binding p o ein phospha ase
A DBP1 Media es Suscep ibili y o wo Po y i uses in A abidopsis. Plan Physiol
153: 1521–1525.
4. Cas ello´ MJ, Ca asco JL, Na a e e-Go´mez M, Daniel J, G ano D e al. (2011)
A plan small polypep ide is a no el componen o DNA-binding p o ein
phospha ase 1-media ed esis ance o Plum pox i us in A abidopsis. Plan
Physiol 157: 2206–2215.
5. Denison FC, Paul AL, Zupanska AK, Fe l RJ (2011) 14-3-3 p o eins in plan
physiology. Sem Cell De Biol 22: 720–727.
6. Ca asco JL, Cas ello´ MJ, Ve a P (2006) 14-3-3 media es ansc ip ional
egula ion by modula ing nucleocy oplasmic shu ling o obacco DNA-binding
p o ein phospha ase-1. J Biol Chem 281: 22875–22881.
7. Colcombe J, Hi H (2008) A abidopsis MAPKs: a complex signalling ne wo k
in ol ed in mul iple biological p ocesses. Biochem J 413: 217–226.
8. Kiege l S, Ca dinale F, Siligan C, G oss A, Baudouin E e al. (2000) SIMKK, a
mi ogen-ac i a ed p o ein kinase (MAPK) kinase, is a speci ic ac i a o o he sal
s ess-induced MAPK, SIMK. Plan Cell 12: 2247–2258.
9. Camps M, Nichols A, A kins all S (2000) Dual speci ici y phospha ases: a gene
amily o con ol o MAP kinase unc ion. FASEB J 14: 6–16.
10. Be hke G, Peche P, Eschen-Lippold L, Tsuda K, Ka agi i F e al. (2012)
Ac i a ion o he A abidopsis haliana mi ogen-ac i a ed p o ein kinase MPK11 by
he lagellin-de i ed elici o pep ide, lg22. Mol Plan -Mic obe In e ac 25: 471–
480.
11. Wolschin F, Wienkoop S, Weckwe h W (2005) En ichmen o phospho yla ed
p o eins and pep ides om complex mix u es using me al oxide/hyd oxide
a ini y ch oma og aphy (MOAC). P o eomics 5: 4389–4397.
12. Pe e sen M, B ode sen P, Naes ed H, And easson E, Lindha U e al. (2000)
A abidopsis MAP kinase 4 nega i ely egula es sys emic acqui ed esis ance. Cell
103: 1111–1120.
13. Asai T, Tena G, Plo niko a J, Willmann MR, Chiu WL e al. (2002) MAP kinase
signalling cascade in A abidopsis inna e immuni y. Na u e 415: 977–983.
14. Kose su K, Ma sunaga S, Nakagami H, Colcombe J, Sasabe M e al. (2010)
The MAP kinase MPK4 is equi ed o cy okinesis in A abidopsis haliana. Plan
Cell 22: 3778–3790.
15. Ko ole a OA, Tomlinson ML, Leade D, Shaw P, Doonan JH (2005) High-
h oughpu p o ein localiza ion in A abidopsis using Ag obac e ium-media ed
ansien exp ession o GFP-ORF usions. Plan J 41: 162–174.
16. Vie s a RD (2009) The ubiqui in-26S p o easome sys em a he nexus o plan
biology. Na Re Mol Cell Biol 10: 385–397.
17. Go¨ki mak T, Paul AL, Fe l RJ (2010) Plan phosphopep ide-binding p o eins as
signalling media o s. Cu Opin Plan Biol 13: 527–532.
18. Keyse SM (2000) P o ein phospha ases and he egula ion o mi ogen-ac i a ed
p o ein kinase signaling. Cu Opin Cell Biol 12: 186–192.
19. Gup a R, Luan S (2003) Redox con ol o p o ein y osine phospha ases and
mi ogen-ac i a ed p o ein kinases in plan s. Plan Physiol 132: 1149–1152.
20. Ka ou S, Ka i a E, Yamakawa H, Seo S, Mi suha a I e al. (2005) Ca aly ic
ac i a ion o he plan MAPK phospha ase N MKP1 by i s physiological
subs a e salicylic acid-induced p o ein kinase bu no by calmodulins. J Biol
Chem 280: 39569–39581.
21. Schweigho e A, Kazana iciu e V, Scheikl E, Teige M, Doczi R e al. (2007)
The PP2C- ype phospha ase AP2C1, which nega i ely egula es MPK4 and
MPK6, modula es inna e immuni y, jasmonic acid, and e hylene le els in
A abidopsis. Plan Cell 19: 2213–2224.
22. Ulm R, Re enko a E, di Sansebas iano GP, Bech old N, Paszkowski J (2001)
Mi ogen-ac i a ed p o ein kinase phospha ase is equi ed o geno oxic s ess
elie in A abidopsis. Genes De 15: 699–709.
23. Yamakawa H, Ka ou S, Seo S, Mi suha a I, Kamada H e al. (2004) Plan
MAPK phospha ase in e ac s wi h calmodulins. J Biol Chem 279: 928–936.
A abidopsis DBP1 Phospha ase
PLOS ONE | www.plosone.o g 9 Ma ch 2014 | Volume 9 | Issue 3 | e90734