Differential expression of peach ERF transcriptional activators in response to signaling molecules and inoculation with Xanthomonas campestris pv. pruni.

Abstract

Ethylene response factors (ERFs) are a large family of transcription factors (TFs) that have diverse functions in plant development and immunity. However, very little is known about the molecular regulation of these TFs in stone fruits during disease incidence. In the present study, we describe the identification of five peach ERFs (Pp-ERFs), aiming to elucidate their potential roles in defense against Xanthomonas campestris pv. pruni (Xcp), the causal agent of bacterial spot disease. The phylogenetic analysis along with sequence comparisons indicated that all Pp-ERFs are transcriptional activators belonging to groups IX and IIV ERFs. The transactivation capacity of these proteins was verified in vivo where they all induced the expression of the GUS reporter gene and in a GCC-dependent manner. The nuclear localization was also confirmed for two of these proteins, Pp-ERF2.b and Pp-ERF2.c, after their transient expression in onion epidermal cells. The induction kinetics of Pp-ERFs after inoculation with Xcp was determined by qRT-PCR. Except for Pp-ERF2.b, transcript levels of Pp-ERFs increased strongly and rapidly in the resistant 'Venture' compared to the susceptible 'BabyGold 5' cultivar after infection with Xcp. In contrast, the expression of Pp-ERF2.b was several-fold higher in the susceptible cultivar after bacterial infection. The expression of Pp-ERFs was also monitored after treating with signaling compounds; salicylic acid (SA) (1 mM), ethephon (1 mM) and methyl jasmonate (MeJA) (50 μM). Although the results generally emphasize the role of ethylene/jasmonic acid (ET/JA) signaling pathways in regulating the expression of Pp-ERFs, there was a coordination of the timing of ET/JA responses, suggesting compensatory rather than synergistic interactions between these pathways during defense against Xcp.

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Journal
of
Plant
Physiology
169 (2012) 731–
739
Contents
lists
available
at
SciVerse
ScienceDirect
Journal
of
Plant
Physiology
jou
rn al
h o mepage:
www.elsevier.de/jplph
Differential
signaling
expression
of
peach
ERF
transcriptional
activators
in
response
to
molecules
and
inoculation
with
Xanthomonas
campestris
pv.
pruni
S.
Sherifa,b,
I.
El-Sharkawya,c,
G.
Paliyathb,
S.
Jayasankara,∗
aDepartment
bDepartment
cVineland
of
Plant
Agriculture,
University
of
Guelph,
4890
Victoria
Av.
N.,
PO
Box
7000
Vineland
Station,
ON
L0R
2E0,
Canada
of
Plant
Agriculture,
University
of Guelph,
Guelph,
Ontario
N1G
2W1,
Canada
Research
and
Innovation
Centre,
4980
Victoria
Av.
N.,
PO Box
4000
Vineland
Station,
ON,
Canada
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
history:
12
December
2011
in revised
form
12 February
2012
14 February
2012
Keywords:
Biotic
Ethylene-responsive
GCC-box
Plant
Xanthomonas
stress
factor
hormones
campestris
pv.
pruni
a
b
s
t
r
a
c
t
Ethylene
tions
of
of
campestris
sequence
and
the
also
onion
qRT-PCR.
tant
the
The
(SA)
emphasize
Pp-ERFs,
synergistic
response
factors
(ERFs)
are
a
large
family
of
transcription
factors
(TFs)
that
have
diverse
func-
in
plant
development
and
immunity.
However,
very
little
is
known
about
the
molecular
regulation
these
TFs
in
stone
fruits
during
disease
incidence.
In
the
present
study,
we
describe
the
identification
five
peach
ERFs
(Pp-ERFs),
aiming
to
elucidate
their
potential
roles
in
defense
against
Xanthomonas
pv. pruni
(Xcp),
the
causal
agent
of bacterial
spot
disease.
The
phylogenetic
analysis
along
with
comparisons
indicated
that
all
Pp-ERFs
are
transcriptional
activators
belonging
to
groups
IX
IIV
ERFs.
The
transactivation
capacity
of these
proteins
was
verified
in
vivo
where
they
all
induced
expression
of the
GUS
reporter
gene
and
in
a
GCC-dependent
manner.
The
nuclear
localization
was
confirmed
for
two
of
these
proteins,
Pp-ERF2.b
and
Pp-ERF2.c,
after
their
transient
expression
in
epidermal
cells.
The
induction
kinetics
of
Pp-ERFs
after
inoculation
with
Xcp
was
determined
by
Except
for
Pp-ERF2.b,
transcript
levels
of
Pp-ERFs
increased
strongly
and
rapidly
in
the
resis-
‘Venture’
compared
to
the
susceptible
‘BabyGold
5’
cultivar
after
infection
with
Xcp.
In
contrast,
expression
of
Pp-ERF2.b
was
several-fold
higher
in
the
susceptible
cultivar
after
bacterial
infection.
expression
of
Pp-ERFs
was
also
monitored
after
treating
with
signaling
compounds;
salicylic
acid
(1
mM),
ethephon
(1
mM)
and
methyl
jasmonate
(MeJA)
(50
?M).
Although
the
results
generally
the
role
of
ethylene/jasmonic
acid
(ET/JA)
signaling
pathways
in
regulating
the
expression
of
there
was
a
coordination
of
the
timing
of
ET/JA
responses,
suggesting
compensatory
rather
than
interactions
between
these
pathways
during
defense
against
© 2012 Elsevier GmbH. All rights reserved.
Xcp.
Introduction
Transcription
tional
with
factors
transcriptome
(Chen
teins
factors
(TFs)
play
a
central
role
in the
transcrip-
reprogramming
associated
with
the
interaction
of
plants
their
surrounding
environment.
APETALA2/ethylene
response
(AP2/ERFs)
are
a group
of
plant
TFs
known
to modulate
plant
to cope
with
miscellaneous
biotic
and
abiotic
stimuli
et
al.,
2008;
Gao
et
al.,
2008;
Dong
et
al.,
2010).
These
pro-
are
characterized
by
the
existence
of
57–66
conserved
amino
Abbreviations:
APETALA2/ethylene
terns;
ET,
monic
pathogen-associated
triggered
campestris
∗Corresponding
E-mail

aa,
amino
acids;
AD,
acidic
activation
domain;
AP2/ERF,
response
factor;
DAMPs,
damage-associated
molecular
pat-
DPI,
days
post-inoculation;
EAR,
ERF-associated
amphiphilic
repression;
ethylene;
GFP,
green
fluorescent
protein;
HPI,
hours
post-inoculation;
JA,
jas-
acid;
NLS,
nuclear
localization
signal;
ORFs,
open
reading
frames;
PAMPs,
molecular
patterns;
PR,
pathogenesis-related;
PTI,
PAMPs-
immunity;
SA,
salicylic
acid;
TFs,
transcription
factors;
Xcp, Xanthomonas
pv.
pruni.
author.
Tel.:
+1
905
562
4141x134;
fax:
+1 905
562
3413.
address:
jsubrama@uoguelph.ca
(S.
Jayasankar).
acid
et
homology,
lies:
been
genes
and
ization
group
others,
sion
2000;
by
the
(Park
2009).
The
edged
can
two-hybrid
residues
forming
the
DNA
binding
domain,
AP2
(Okamuro
al.,
1997).
Based
on
the
number
of
AP2
domains
and
sequence
the
AP2/ERF
superfamily
is
classified
into
three
fami-
AP2,
ERF
and
RAV
(Nakano
et
al.,
2006).
The
ERF
family
has
identified
in many
plant
species
with
a
total
of
122
and
139
in Arabidopsis
and
the
rice
genome,
respectively
(Gutterson
Reuber,
2004;
Nakano
et
al.,
2006).
The
functional
character-
and
sequence
homology
define
two
groups
of
ERFs.
One
activates
the
transcription
of
downstream
genes
and
the
which
usually
have
the
ERF-associated
amphiphilic
repres-
(EAR)
motif,
work
as
transcriptional
repressors
(Fujimoto
et al.,
McGrath
et al.,
2005).
ERF
proteins
mediate
their
functions
binding
to target
sequences
(e.g.
GCC
and
DRE/CRT
boxes)
in
promoter
of
biotic-
and
abiotic-responsive
genes,
respectively
et al.,
2001;
Hao
et
al.,
2002;
Lee
et al.,
2004;
Zhang
et al.,
role
of
ERFs
in disease
resistance
was
initially
acknowl-
with
the
discovery
that
ERFs,
namely,
Pti4,
Pti5
and
Pti6,
interact
with
the
tomato
disease
resistance
protein
Pto
in yeast
assays
(Zhou
et
al.,
1997).
This
interaction,
along
with
0176-1617/$
doi:10.1016/j.jplph.2012.02.003
– see
front
matter ©

2012 Elsevier GmbH. All rights reserved.

Page 3

Author's personal copy
732
S.
Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
their
recognition
changes
Gu
provides
biotic
the
tance
et
et
2010).
an
in
be
environmental
et
as
et
Champion
In
exert
under
tion
the
growth
otic
Oelmuller,
will
support
and
which
the
2005),
Interestingly,
actions
idea
ity
fine-tuning
plant.
The
characterization
in
compatible
thomonas
spot
ERFs
ing
order
ent
an
ent
stimuli.
phosphorylation
by
Pto,
provides
a direct
link
between
the
of
plant
pathogens
by
R genes
and
the
subsequent
in the
transcriptome
mediated
by ERFs
(Zhou
et al.,
1997;
et
al.,
2002).
The
overexpression
of
ERFs in different
plant
species
further
evidence
for
the
pivotal
role
of
these
proteins
in
stress.
In many
instances
overexpression
of
ERF
genes
under
control
of
constitutive
promoters
has
led
to enhanced
resis-
against
fungal,
bacterial
and
viral
pathogens
(Berrocal-Lobo
al.,
2002;
Gu
et
al.,
2002;
Berrocal-Lobo
and
Molina,
2004;
Chen
al.,
2008;
Liang
et al.,
2008;
Anderson
et al.,
2010;
Dong
et
al.,
Resistance
to these
pathogens
was
mostly
associated
with
increased
transcription
level
of
pathogenesis-related
(PR)
genes
transgenic
plants
(Gu
et
al.,
2002;
Liang
et
al.,
2008).
ERFs can
also
transcriptionally
regulated
by wounding
(Tournier
et
al.,
2003),
stress
(Park
et al.,
2001;
Chen
et
al.,
2002;
Zhang
al.,
2010),
and
after
treatment
with
signaling
molecules
such
salicylic
acid
(SA),
jasmonic
acid
(JA)
and
ethylene
(ET)
(Chen
al.,
2002;
Gu
et
al.,
2002;
Brown
et
al.,
2003;
Lorenzo
et al.,
2003;
et al.,
2009).
contrast
to the
above
general
theme,
some
of
ERFs
can
also
negative
effects
on
the
expression
of
downstream
genes
certain
conditions.
For
instance,
the
insertional
inactiva-
of
Arabidopsis
ERF9
and
ERF14
activates
the
expression
of
pathogenesis-related
PR1
and
PR2
genes
and
attenuates
the
promotion
induced
by the
colonization
of
the
symbi-
fungi,
Piriformospora
indica
in Arabidopsis
roots
(Camehl
and
2010).
Therefore,
it can
be
interpreted
that
these
ERFs
suppress
the
expression
of
PR1
and
PR2
in wild-type
plants
to
their
interactions
with
beneficial
endophytic
fungi
(Camehl
Oelmuller,
2010).
Overexpression
of
AtERF4
in Arabidopsis,
results
in increased
susceptibility
of
transgenic
plants
to
necrotrophic
pathogen
Fusarium
oxysporum
(McGrath
et
al.,
is
another
example
of
the
negative
effects
of
some
ERFs.
AtERF4
is induced
by
JA,
ET,
and
incompatible
inter-
(Brown
et
al.,
2003).
Together
these
examples
support
the
that
the
redundancy
of
ERFs,
combined
with
the
complex-
and
the
specificity
in their
response,
eventually
allows
for
the
of
diverse
biotic
and
abiotic
stresses
perceived
by
the
present
work
describes
the
identification
and
functional
of
five
ERFs
from
peach
(Prunus
persica). Changes
the
transcript
profile
of
these
genes
were
investigated
during
and
incompatible
interactions
of
peach
with
Xan-
campestris
pv.
pruni
(Xcp),
the
causal
agent
of
bacterial
disease
in stone
fruits.
The
induction
kinetics
of
peach
(Pp-ERFs)
was
also
monitored
after
treating
with
signal-
molecules,
i.e.
SA,
methyl
jasmonate
(MeJA)
and
ethephon
in
to investigate
how
these
TFs
might
be
employed
by
differ-
signaling
pathways
during
biotic
stress.
This
study
provides
insight
into
the
selective
nature
of
ERFs
and
how
differ-
classes
of
ERFs
diverge
in their
responses
to surrounding
Materials
and
methods
Plant
material,
bacterial
inoculation
and
hormone
treatments
Branches
(Prunus
ulated
previously
control
stored
with
healthy
leaves
were
collected
from
two
peach
persica
L.
Batsch)
cultivars
‘Venture’
and
‘BabyGold
5?, inoc-
with
Xcp
or
treated
with
signaling
compounds
as
described
(Sherif
et
al.,
2011).
At
designated
times,
treated
and
leaves
were
quickly
frozen
in liquid
nitrogen
and
then
at −80◦C for
RNA
extraction.
Gene
isolation
and
characterization
Full
length
sequences
of
three
ERFs, designated
Pp-ERF1.a,
Pp-ERF2.a
library
The
able
database
two
obtained
genes
Burlington,
tions.
were
HQ825096,
ERF2.a,
The
with
out
(http://www.nrbsc.org/gfx/genedoc/).
constructed
alized
Deduced
protein
(http://expasy.org/prosite/).
and
Pp-ERF2.c,
were
amplified
from
the
peach
cDNA
using
the
Reverse-Transcription
PCR
(RT-PCR)
approach.
primers
to amplify
these
genes
were
designed
from
the
avail-
expressed
sequence
tags
(ESTs)
of
these
genes
at the
ESTree
(http://www.itb.cnr.it/estree/index.php).
For
the
other
genes,
Pp-ERF1.b
and
Pp-ERF2.b,
only
partial
sequences
were
from
the
ESTree
library.
Full
length
sequences
of
these
were
amplified
using
a 3?- and
5?-RACE
kit
(Invitrogen,
ON,
Canada)
according
to the
manufacturer’s
instruc-
The
open
reading
frame
(ORF)
sequences
obtained
herein
deposited
in GenBank
(accession
nos.
HQ825094,
HQ825095,
HQ825097
and
HQ825098
for
Pp-ERF1.a,
Pp-ERF1.b,
Pp-
Pp-ERF2.b
alignment
and
Pp-ERF2.c,
and
respectively).
analysis

of

the

Pp-ERF

sequences
their
orthologs
from
other
plant
species
were
carried
using
ClustalX
(Jeanmougin
et
al.,
1998) and
GeneDoc
The
phylogenetic
tree
was
according
to the
neighbor-joining
method
and
visu-
by Phylogeny.fr
web
tool
were
(http://www.phylogeny.fr/).
analyzedusing

sequences

PROSITE
RNA
extraction
and
gene
expression
analyses
Total
RNA
was
extracted
from
treated
and
control
peach
leaves
using
ples
of
cDNA
Premium
ON,
real
Color
Canada).
tion
0.2
program
cycles
further
95◦C.
ized
was
of
sion
treatment
leaves).
listed
RNeasy
Kit
(Qiagen,
Mississauga,
ON,
Canada).
RNA
sam-
were
treated
with
DNase
I (Invitrogen)
prior
to the
synthesis
cDNA
to remove
any
traces
of
genomic
DNA.
The
first-strand
was
synthesized
from
5 ?g of
total
RNA
using
RevertAidTM
First
Strand
cDNA
Synthesis
Kit
(Fermentas,
Burlington,
Canada)
according
to manufacturer’s
instructions.
Quantitative
time-PCR
(qRT-PCR)
was
performed
using
the
MyiQ
Single-
Real-Time
PCR
Detection
System
(Bio-Rad,
Mississauga,
ON,
The
PCR
reaction
was
performed
in 25
?L of
the
reac-
mixture
containing
1×
iQTMSYBR
Green
Supermix
(Bio-Rad),
?M of
each
primer
and
about
10 ng
of
cDNA.
The
amplification
was
performed
as
follows:
3
min
at
94◦C,
followed
by
40
of
30 s at
94◦C and
1 min
at 60◦C.
The
PCR
products
were
analyzed
using
a dissociation
curve
program
from
65◦C to
The
threshold
cycle
(CT)
values
for
each
gene
were
normal-
to those
of
Pp-actin
(BU046508).
The
relative
gene
expression
calculated
as
2−?CT, where
?CT represents
CT of the
gene
interest
minus
the
CT
of
Pp-actin. Fold
change
in
gene
expres-
was
calculated
as
2−??CT, where
??CT
represents
the
?CT of
minus
the
?CT
of
control
(untreated
or mock-inoculated
The
primers
used
to detect
transcript
levels
of
Pp-ERFs are
in Online
Resource
1.
Construction
of
reporter
and
effector
plasmids
Two
reporter
constructs
were
used
in this
study.
One
was
the
provided
was
by
(CATAGCCGCCATTT)
PCR
using
CCGGGAAAT)
to
digested
(Pasquali
promoter
PDF1.2
promoter-GUS
reporter
construct
which
was
kindly
by
Dr.
J.
Memelink
(Pre
et
al.,
2008).
The
other
the
4XGCC-GUS
reporter
plasmid,
which
was
constructed
multimizing
the
14
bp
region
of
Arabidopsis
HLS1
promoter
four
times
in a synthetic
fragment.
The
amplification
of
the
synthetic
fragment
was
performed
primers
(F:
TTCGCGTCTAGAACATAGC,
and
R: AAAAAAAAC-
with
the
recognition
sites
for
XbaI
and
SmaI
added
the
5?and
3?ends,
respectively.
The
amplified
sequences
were
with
XbaI
and
SmaI
and
cloned
into
GUSXX
plasmid
et al.,
1994)
downstream
of
the
CaMV
35S-47
minimal
sequence.
For
the
construction
of
effector
plasmids,
the

Page 4

Author's personal copy
S. Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
733
Fig.
proteins:
and
alignment
black
1.
Structure
of
Pp-ERF
proteins
and
comparison
of
AP2/ERF
domain
sequences.
(A)
Schematic
representation
of
major
domains
and
signature
sequences
in Pp-ERF
AP2
is the
DNA-binding
domain,
AD is the
putative
acidic
activation
domain,
NLS
indicates
the
amino
acid
residues
that
could
serve
as
a nuclear
localization
signal,
MCGG
refers
to the
MCGGII/L,
the
motif
of
unknown
function.
The
table
on the
right
shows
the
length
(aa)
and
molecular
weight
(kDa)
of
each
protein.
(B)
Amino
acid
of
the
ERF/AP2
domain
among
Pp-ERFs
and
other
orthologs
from
different
plant
species.
The
intensity
of
shading
shows
the
similarity
among
amino
acids,
with
shading
representing
100%
similarity.
Amino
acid
residues
that
bind
to the
GCC-box
are
indicted
by
arrows.
ORFs
were
listed
and
2005)
of
Pp-ERF1.a,
Pp-ERF1.b,
Pp-ERF2.a,
Pp-ERF2.b
and
Pp-ERF2.c
amplified
from
the
peach
cDNA
library
using
the
primers
in Online
Resource
1.
PCR
fragments
were
digested
with
SmaI
BglII
and
cloned
into
the
pSAT1A-cEYFP-N1
vector
(Chung
et
al.,
upstream
of
the
CaMV
dual
35S
promoter.
Protoplast
transient
expression
and
GUS
quantification
The
isolation
of
Arabidopsis
protoplasts
and
the
PEG-mediated
transfection
(Yoo
toplasts
each
10
solution
experiments
were
performed
as
previously
described
et
al.,
2007)
with
minor
modifications.
The
number
of
pro-
in MMG
solution
was
adjusted
to 4–4.5
× 105per
mL.
For
transfection
assay,
100
?L of
protoplasts
was
transfected
with
?L of
plasmid
DNA
(2 ?g/?L) in the
presence
of
110
?L PEG
(0.2
M mannitol,
100
mM
CaCl2, 36%
PEG
4000).
For
each
assay,
together
along
protoplasts
then
supernatant
frozen
?-Glucuronidase
(Cervera,
extraction
Na2ethylenediaminetetraacetic
and
1
1.5
d-glucuronide
4 ?L of
reporter
and
6 ?L of
effector
plasmids
were
mixed
and
introduced
into
protoplasts.
Empty
effector
plasmids
with
reporter
plasmids
were
used
as
controls.
The
transfected
were
kept
in 2 mL
W5
solution
for
18–20
h at 22◦C and
collected
by
centrifugation
at 8000
rpm
for
3 min
at
4◦C.
The
was
carefully
discarded
and
the
pellets
were
directly
in liquid
nitrogen
and
stored
at -80◦C until
further
use.
(GUS)
activity
was
measured
fluorometrically
2005).
Frozen
protoplasts
were
homogenized
in 200
?L
buffer
(50
mM
sodium
phosphate
buffer
(pH
7.0),
10 mM
acid
(pH
8.0),
0.1%
Triton
X-100
10
mM
?-mercaptoethanol)
and
centrifuged
at 13,000
rpm
for
min
at
4◦C.
100
?L of
the
supernatant
was
transferred
to a
brown
mL
tube
containing
100
?L of
2 mM
4-methyl-umbelliferyl-?-
(MUG)
solution,
mixed
gently
and
then
incubated
in

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Author's personal copy
734
S.
Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
Fig.
plete amino
constructed
tree
Pp-ERF2.a
ina
(FJ026006)];
lycopersicum
tiana
(AAS20427)],
(ABO40237)]
2.
A rooted
phylogenetic
tree
of
ERFs
from
different
plant
species.
The
com-
acid
sequences
were
aligned
by
Clustal
X,
and
the
phylogenetic
tree
was
according
to the
neighbor-joining
method.
The
ERFs
used
to build
this
are
the
following:
P. persica
[Pp-ERF1.a
(HQ825094),
Pp-ERF1.b
(HQ825095),
(HQ825096),
Pp-ERF2.b
(HQ825097)
and
Pp-ERF2.c
(HQ825098)];
P. salic-
[Ps-ERF1a
(FJ026009),
Ps-ERF1b
(FJ026008),
Ps-ERF2a
(FJ026007),
Ps-ERF2b
Arabidopsis
thaliana
[AtERF1
(BAA32418),
AtEBP
(CAA05084)];
Solanum
[SlERF2
(AAO34704),
Pti4
(AAC50047),
JERF1
(AAK95687)];
Nico-
tabacum
[NtERF2
(Q40479);
Capsicum
annuum
[CaPF1
(AAP72289),
CaERELP1
Cucumis
melo
[CmERELP
(BAD01556)],
Medicago
truncatula
[MtERF1a
and
Gossypium
hirsutum
[GhERFEBP
(AAV51937)].
a water
of
vortexing.
tilabel
4-methylumbelliferone
quantified
GUS
protein.
similar
bath
at 37◦C for
1,
2 or
3 h.
To stop
the
reaction,
900
?L
Na2CO3(200
mM)
was
added
and
the
contents
were
mixed
by
Color
development
was
quantified
in a Victor
1420
Mul-
Fluorescence
Plate
Reader
(Wallac
Oy,
Turku,
Finland),
using
(MU)
as
standards.
The
total
protein
was
in each
sample
using
the
Bradford
assay
and
the
total
activity
was
calculated
as
pmol
of
MU
min−1?g−1of
total
Each
transfection
assay
was
repeated
three
times,
with
a
number
of
technical
replicates
each
time.
Gene
proteins
bombardment
and
subcellular
localization
of
Pp-ERF
The
coding
sequence
of
Pp-ERF2.b
and
2.c
was
cloned
into
the
pGreen
and
fied
1 and
site.
visualization
vector
(Hellens
et
al.,
2000)
downstream
the
GFP
protein
upstream
the
CaMV
dual
35S
promoter.
The
ORFs
were
ampli-
from
peach
cDNA
using
the
primers
listed
in Online
Resource
cloned
into
the
pGreen
vector
using
the
BamHI
restriction
The
transient
expression
in onion
epidermal
cells
and
GFP
were
done
as
described
previously
(Sherif
et
al.,
2011).
Statistical
analyses
All
parameters
variance,
Analysis
MIXED
tute,
to compare
presented
were
tested
for
normality
prior
to analysis
of
and
a log
transformation
was
performed
when
required.
of
variance
(ANOVA)
was
performed
using
the
GLM
or
procedures
of
SAS
statistical
software
(release
9;
SAS
Insti-
Cary,
NC).
The
Tukey–Kramer
HSD
(P < 0.05)
test
was
used
among
means.
Treatment
means
and
standard
errors
in figures
were
calculated
from
non-transformed
data.
Fig.
Scheme
reporter
of
mal
into
The
Fluorometric
simultaneously
the
protoplasts.
independent
3. Assay
on
transcription
activity
of
Pp-ERFs
in
Arabidopsis
protoplasts.
(A)
of
the
reporter
and
effector
constructs
used
in the
GUS-reporter
assay.
For
constructs,
the
expression
of
the
gus
A gene
is driven
by either
1047
bp
the
Arabidopsis
PDF1.2
promoter
or 4 copies
of
the
GCC-box
fused
to the
mini-
sequence
of
the
CaMV
35S-promoter.
For
effector
constructs,
ERFs
were
cloned
the
pSAT1A-cEYFP-N1
plasmid
upstream
of
the
CaMV
dual
35S
promoter.
transcription
of
effector
genes
is driven
by the
CaMV
dual
35S-promoter.
(B)
quantification
of
GUS
activity
in Arabidopsis
protoplasts
transfected
with
4 ?L reporter
and
6 ?L effector
plasmids.
In control
treatments,
pSAT1A-cEYFP-N1
empty
vector
was
introduced
with
reporter
plasmids
into
Values
are
presented
as the
mean
(±SE)
of
GUS
activity
from
three
experiments.
Results
Cloning
and
molecular
characterization
of Pp-ERFs
Full
length
sequences
of
five
ERFs, designated
Pp-ERF1.a,
Pp-
ERF1.b,
peach
with
amino
acid
ERFs
Pp-ERF1.b
Pp-ERF2.c
Sequence
cated
domain,
tively
domain
among
signature
and
fied
(MCGGII/L)
The
ncbi.nlm.gov/blast)
plant
anism,
AtERF1
PP-ERF2.a,
Pp-ERF2.b
and
Pp-ERF2.c
were
isolated
from
cDNA
library.
These
genes
are
predicted
to encode
proteins
calculated
molecular
masses
of
287,
260,
382,
321
and
289
acids
(aa),
respectively
(Fig.
1a).
Comparison
of
the
amino
sequences
along
with
phylogenetic
analysis
indicated
that
Pp-
belong
to two
evolutionarily
divergent
groups.
Pp-ERF1.a
and
are
located
in group
IX,
while
Pp-ERF2.a,
Pp-ERF2.b
and
are
located
in group
VII
(Nakano
et al.,
2006)
(Fig.
2).
analysis
using
PROSITE
(http://expasy.org/prosite/) indi-
that
all
Pp-ERFs
have
a single
DNA
binding
domain,
the
AP2
comprising
58–59
aa between
groups
VII
and
IX,
respec-
(Fig.
1a).
The
seven
amino
acid
residues
within
the
AP2
that
bind
the
GCC-box
(Allen
et
al.,
1998)
are
conserved
all
Pp-ERFs
(Fig.
1b).
In addition
to the
AP2
domain,
other
sequences
including
the
acidic
activation
domain
(AD)
the
nuclear
localization
signal
(NLS)
have
also
been
identi-
in all
Pp-ERFs.
The
N-terminal
of
group
VII
has
another
motif
with
results
an
unknown
of
function
BLASTP
(Fig.
1a).
online

the

(http://www.
indicated
that
Pp-ERFs
are
homologues
to
proteins
with
demonstrated
functions
in defense
mech-
including
tomato
Pti4
(He
et al.,
2001),
and
Arabidopsis
(Berrocal-Lobo
and
Molina,
2004)
within
group
IX;
hot

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Author's personal copy
S. Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
735
pepper
2009)
the
orthologs
CaPF1
(Yi
et al.,
2004)
and
soybean
GmERF3
(Zhang
et
al.,
within
group
VII
(Fig.
1a).
As
expected,
Pp-ERFs
showed
highest
percentage
of
sequence
similarity
(93–96%)
with
their
in plums
(Prunus
salicina) (Online
Resources
2 and
3).
Pp-ERFs
activate
the
transcription
of
GCC
box-containing
genes
The
GCC-box,
with
a
core
sequence
(AGCCGCC),
is a cis-
regulatory
(Broglie
et
et
the
assay,
were
of
4X
moter)
into
activate
in
sequences.
(P
promoter
of
exhibited
total
teins.
obtained
by
element
found
in the
promoter
of
many
PR genes
et al.,
1989;
Ohme-Takagi
and
Shinshi,
1995;
Penninckx
al.,
1996;
Chakravarthy
et
al.,
2003;
Lorenzo
et al.,
2003;
Sherif
al.,
2011).
To
examine
the
transactivation
capacity
of
peach
ERFs,
GUS
reporter
assay
in Arabidopsis
protoplasts
was
used.
In this
ERF
ORFs
under
the
control
of
the
CaMV
dual
35S
promoter
used
as
effector
plasmids,
and
the
gus
A gene
under
the
control
either
the
Arabidopsis
PDF1.2
promoter
(PDF1.2
promoter)
or
the
GCC-box
fused
to the
minimal
CaMV
35S
promoter
(4X
GCC
pro-
were
used
as
reporter
plasmids
(Fig.
3a).
When
introduced
Arabidopsis
protoplasts,
all
Pp-ERF
effectors
were
able
to trans-
gus
A expression
over
the
control
(the
empty
vector),
and
a manner
dependent
on
the
number
of
GCC-boxes
in promoter
In all
assays,
the
GUS
activity
was
significantly
higher
< 0.001)
when
the
transcription
of
gus
A was
driven
by
the
4X GCC
compared
to the
PDF1.2
promoter,
which
has
two
copies
the
GCC-box
(Zarei
et
al.,
2011) (Fig.
3b).
For
group
IX,
Pp-ERF1.b
more
GUS
activity
(nearly
4000
pmol
MU
min−1mg−1
proteins)
than
Pp-ERF1.a
(∼3000)
when
used
as
effector
pro-
Among
group
VII,
the
maximum
GUS
activity
(5400)
was
when
Pp-ERF2.a
was
used
as
an
effector
protein,
followed
Pp-ERF2.c
(4300)
(Fig.
3b).
Pp-ERF2.b
and
Pp-ERF2.c
are
localized
to the
cell
nucleus
Potential
NLS
sequences
were
predicted
for
all
peach
ERFs
based
on
Sharkawy
and
with
siently
To
the
or
mal
the
cipally
was
the
sequence
homology
with
their
orthologs
in plums
(El-
et
al.,
2009) (Online
Resources
2 and
3).
Plum
Ps-ERF1a
Ps-ERF1b,
which
share
a high
percentage
of
sequence
similarity
group
IX Pp-ERFs,
showed
nuclear
localization
when
tran-
expressed
in
tobacco
protoplasts
(El-Sharkawy
et
al.,
2009).
examine
the
subcellular
localization
of
group
VII
Pp-ERFs
in vivo,
expression
vectors
p35S::Pp-ERF2.b-GFP,
p35S::Pp-ERF2.c-GFP
the
control
vector
p35S::GFP
were
introduced
into
onion
epider-
cells
by the
gene
bombardment
procedure.
As
shown
in Fig.
4,
chimeric
Pp-ERF2.c-GFP
and
Pp-ERF2.b-GFP
proteins
were
prin-
expressed
in the
nucleus
of
the
cell,
whereas
the
GFP
protein
distributed
evenly
throughout
the
cell.
Expression
of
Pp-ERF
genes
after
inoculation
with
Xcp
To
investigate
responses
lyzed
and
fold
HPI,
treatment)
tivar,
post-inoculation.
sion
increasing
and
significant
levels
Group
inoculation
chiefly
whether
Pp-ERFs
are
involved
in
peach
defense
to Xcp, the
expression
patterns
of
Pp-ERFs
were
ana-
following
compatible
and
incompatible
interactions
of
peach
Xcp. For
group
IX ERFs, transcripts
of
Pp-ERF1.a
increased
(∼5-
over
mock
treatment)
in the
resistant
‘Venture’
cultivar
at 1
declined
at 4 and
8 HPI
and
peaked
again
(∼4-fold
over
mock
at
24
and
48
HPI.
In the
susceptible
‘BabyGold
5’
cul-
the
expression
of
Pp-ERF1.a
was
negligible
after
all
times
Pp-ERF1.b
transcripts
exhibited
a
similar
expres-
pattern
in both
compatible
and
incompatible
interactions;
at
1 HPI,
peaking
at
4 HPI
(∼5-fold
over
mock
treatment)
then
decreasing
at 8,
24
and
4 h HPI
(Fig.
5).
There
were
no
differences
(P =
0.221)
between
cultivars
in expression
of
Pp-ERF1.b
after
bacterial
infection.
VII
ERFs exhibited
different
expression
patterns
after
with
Xcp. While
Pp-ERF2.a
and
Pp-ERF2.c
were
induced
in the
resistant
cultivar,
Pp-ERF2.b
transcript
levels
were
Fig.
The
were
sues
microscopy.
4.
Nuclear
localization
of
Pp-ERF2.b-
and
Pp-ERF2.c-GFP
chimeric
proteins.
constructs
of
p35S::GFP
(A),
p35S::Pp-ERF2.b-GFP
(B),
p35S::Pp-ERF2.c-GFP
(C)
introduced
into
onion
epidermal
cells
by gene
bombardment.
Transfected
tis-
were
kept
at room
temperature
for
18
h, and
then
visualized
by
fluorescent
The
scale
= 10
?m.
higher
Pp-ERF2.c
‘Venture’
at
and
than
were
rial
the
however;
‘BabyGold
transcript
transcripts
in
can
IX and
in
in the
susceptible
cultivar.
Transcripts
of
Pp-ERF2.a
and
reached
high
levels
(∼5-fold
over
mock
treatment)
in
at 1 HPI.
While
the
expression
of both
genes
declined
4 and
8 HPI,
induction
peaks
were
observed
for
Pp-ERF2.c
at 24
48
HPI
with
approximately
8-fold
higher
transcript
abundance
mock
treatment.
Levels
of
Pp-ERF2.a
and
Pp-ERF2.c
transcripts
significantly
lower
(P < 0.001)
in ‘BabyGold
5’
after
bacte-
infection.
For
Pp-ERF2.b,
transcript
levels
increased
in both
susceptible
and
the
resistant
cultivar
after
bacterial
infection,
transcript
levels
were
significantly
higher
(P <
0.001)
in
5’
than
in ‘Venture’.
The
largest
difference
in
Pp-ERF2.b
levels
between
cultivars
was
recorded
at 4
HPI,
when
reached
a maximum
of
>30-fold
over
mock
treatment
‘BabyGold
5’
compared
to only
7-fold
in ‘Venture’.
Therefore,
it
be
concluded
that,
except
for
Pp-ERF2.b,
transcription
of groups
VII
ERFs was
induced
at
higher
levels
in the
resistant
cultivar
at least
one
time-point
after
inoculation
with
Xcp.

Page 7

Author's personal copy
736
S.
Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
Fig.
ratio
replicates.
5.
Expression
patterns
of
Pp-ERFs in peach
cultivars,
‘Venture’
and
‘BabyGold
5’,
after
1,
4, 8,
24
and
48 HPI
by
Xcp. The
fold
change
in gene
expression
is shown
as the
of
the
relative
gene
expression
of
each
sample
to that
in mock-inoculated
leaves
of
the
same
cultivar,
and
values
are
the
mean
and
standard
error
of
three
biological
Effect
of
signaling
molecules
and
wounding
To

elucidate

the

molecular
defense

regulation
signaling,

of

Pp-ERFs
the

by
phytohormones-dependent
pattern
the
MeJA
mainly
treatment
induced
respectively,
to
Pp-ERF1.a
a peak
mechanical
which
(Fig.
Transcription
MeJA
55-fold
was
hon
control)
transcripts.
group

transcript
of
the
Pp-ERFs
in ‘Venture’
leaves
was
investigated
after
exogenous
application
of
three
signaling
molecules;
ethephon,
and
SA.
Transcription
of
group
IX ERFs was
upregulated
by
ethephon
and
MeJA
and
to a lesser
extent
by SA
(Fig.
6).
Expression
of
Pp-ERF1.a
and
Pp-ERF1.b
was
to a maximum
of
20-
and
15-fold
over
untreated
controls,
after
4 h of
ethephon
treatment
and
then
declined
the
basal
level
at
24 h post-treatment
(HPT).
Expression
of
was
also
upregulated
by
MeJA
treatment
and
reached
of
about
13-fold
over
control
at 24
HPT.
The
effect
of
wounding
was
significant
on
Pp-ERF1.b
transcription,
increased
by 6.6-fold
over
control
within
1 h of
treatment
6).
of
group
VII
ERFs was
upregulated
primarily
by
treatment
with
Pp-ERF2.c
showing
a maximum
induction
of
over
control
(Fig.
6).
The
induction
of
these
ERFs by
MeJA
characterized
by
a sustained
expression
up to 24
HPT.
Ethep-
treatment
showed
a
relatively
weak
(about
3-
and
5-fold
over
but
rapid
(4 HPT)
induction
for
Pp-ERF2.a
and
Pp-ERF2.c
SA exhibited
only
small
effect
on
the
transcription
of
VII
ERFs. Mechanical
wounding
induced
the
expression
of
group
observed
Pp-ERFs
ing
both
effect
effect
exert
VII
ERFs with
a maximum
induction
(>13-fold
over
control)
for
Pp-ERF2.c
at
8 HPT.
Analysis
of
these
data
implies
that
transcription
is synchronized
in a timely
manner
depend-
on
the
signaling
pathway.
While
group
IX ERFs are
upregulated
by
ET and
JA,
ethylene
seems
to have
a more
rapid
and
stronger
than
does
JA.
On
the
other
hand,
JA seems
to have
a dominant
on
the
expression
of
group
VII
ERFs, but
takes
longer
time
to
its
maximum
effects.
Combined
analysis
of
peach
PR and
ERF
transcripts
To get
inducible
the
Pp-TLP3) (Sherif
present
genes
in
to
24
To
are
for
with
80%
a better
idea
about
transcript
kinetics
of
defense-
genes
after
inoculation
with
Xcp, a
combined
analysis
of
five
peach
PR
genes
(Pp-PR1a, Pp-PR1b, Pp-TLP1, Pp-TLP2
and
et
al.,
2011) and
the
five
Pp-ERFs
described
in the
study
was
performed
(Online
Resource
4).
Among
the
10
studied,
6 genes
(60%)
showed
higher
expression
(≥2-fold)
‘Venture’
than
‘BabyGold
5’
at
1 HPI.
This
percentage
decreased
only
10%
and
20%
at
4 and
8 HPI,
respectively,
increased
again
at
HPI
and
reached
50%
at
48 HPI.
further
examine
whether
defense-inducible
genes
in peach
regulated
by
the
same
signaling
pathways,
a combined
analysis
peach
PR and
ERF
transcripts
was
performed
after
treatments
SA,
MeJA
or ethephon.
Ethephon
treatment
accounted
for
of
the
gene
induction
at 1 HPI,
followed
by
MeJA
and
SA

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S. Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
737
Fig.
change
three
6.
Gene
expression
analyses
of
ERFs
in ‘Venture’
leaves
after
1,
4, 8 and
24 h of
treatments
with
MeJA
(50
?M),
SA (1 mM),
ethephon
(1
mM)
or wounding
(WN).
The
fold
in gene
expression
is shown
as the
ratio
of
the
relative
gene
expression
of
each
sample
to that
in untreated
leaves,
and
values
are
the
mean
and
standard
error
of
biological
replicates.
Treatments
marked
by an
asterisk
(*)
are
significantly
greater
than
untreated
controls
(P < 0.05).
(Online
lation
In
amplified
treatment
inducible
Resource
5).
The
effect
of
ethephon
on transcript
accumu-
declined
at 4 HPT
and
reached
its
lowest
levels
at
24
HPT.
contrast,
the
effect
of
MeJA
treatment
on
gene
expression
was
over
time
and
reached
its
highest
level
at
24
HPT.
SA
showed
minimal
effects
of
the
transcription
of
defense-
genes
in peach.
Discussion
In
the
present
study,
five
ERFs were
isolated
from
peach
cDNA
library.
typical
sor
(Fujimoto
five
as
tion
and
analysis
rich
(Fujimoto
transactivation
in
gene
The
the
have
a
The
deduced
proteins
from
these
five
ERFs
contain
the
ERF/AP2
domain
of
the
ERF
family
and
lack
the
EAR
repres-
domain,
and
hence
are
considered
to be
activator-type
ERFs
et
al.,
2000).
Consistent
with
their
role
as
TFs,
all
the
Pp-ERFs
have
basic
amino
acid
regions
that
potentially
serve
NLS
to target
the
proteins
to the
nucleus.
The
nuclear
localiza-
was
further
confirmed
by transient
expression
of
Pp-ERF2.b
Pp-ERF2.c
in onion
epidermal
cells.
In addition,
the
sequence
of
Pp-ERF
proteins
indicated
stretches
of
24–25
aa that
are
in aspartate
and
glutamate
residues
and
which
could
act
as
ADs
et
al.,
2000;
Tournier
et
al.,
2003;
Zhang
et
al.,
2004).
The
capacities
of
the
Pp-ERFs
were
further
confirmed
vivo
where
they
all
triggered
the
expression
of
the
GUS
reporter
higher
than
the
empty
effector
construct.
induction
of
GUS
expression
was
linearly
dependent
on
number
of
GCC-boxes
in the
promoter
sequence.
NMR
studies
revealed
that
the
AP2/ERF
domain
binds
the
GCC-box
through
motif
formed
by seven
amino
acid
residues,
i.e.,
Arg29,
Arg31,
Trp33,
all
in
for
hand,
ity
the
Glu39
among
residues
ences
Possibly
their
Based
(2006), Pp-ERFs
groups
Berrocal-Lobo
tomato
CaPF1
all
pathogen,
functions
the
defense
needed
whether
Temporal
showed
Glu39,
Arg41,
Arg49
and
Trp51
(Allen
et al.,
1998).
Although
these
residues
are
conserved
among
Pp-ERFs,
the
divergence
other
regions
within
the
AP2/ERF
domain
might
be
the
reason
different
transactivation
capabilities
of
Pp-ERFs.
On
the
other
molecular
dynamic
simulations
have
predicted
some
dispar-
among
Arabidopsis
ERFs
in terms
of
their
binding
specificity
to
GCC-box
(Wang
et al.,
2009).
While
the
interactions
of Arg29,
and
Arg41
to G7,
C6
and
G4,
respectively,
were
conserved
the
four
studied
AP2/ERF
domain-GCC
complexes,
three
Arg31,
Arg49
and
Trp51
exhibited
different
binding
prefer-
to DNA
bases
or the
phosphate
backbone
(Wang
et
al.,
2009).
such
differences
exist
among
Pp-ERFs
as
well,
explaining
different
capacities
in triggering
GUS
expression
(Fig.
3).
on
the
former
classification
of
ERFs
by
Nakano
et
al.
fall
in two
groups;
XI
and
VII.
Genes
within
these
include
Arabidopsis
AtERF1
(Berrocal-Lobo
et al.,
2002;
and
Molina,
2004),
At
ERF2
(McGrath
et al.,
2005),
Pti4
and
Pti5
(He
et
al.,
2001;
Gu
et
al.,
2000),
pepper
(Yi
et al.,
2004)
and
soybean
GmERF3
(Zhang
et al.,
2009),
of
which
provide
enhanced
disease
resistance
against
microbial
when
overexpressed
in transgenic
plants.
The
conserved
of
these
ERF
classes
in activating
plant
defenses
lead
to
prediction
that
Pp-ERFs
could
play
a
regulatory
role
in peach
mechanisms
against
Xcp. However,
further
studies
are
to determine
which
of these
play
an
important
role
and
they
can
act
alone.
and
quantitative
analysis
of Pp-ERF
transcripts
more
rapid
and
vigorous
gene
expression
of all
the
Pp-

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738
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Sherif
et al.
/ Journal
of
Plant
Physiology
169 (2012) 731–
739
ERFs, except
with
suggested
two
intermediate
of
and
guard
(PAMPs)
et al.,
immunity
related
ERFs were
flg22,
bacterial
PR
entry
3 HPI
(Melotto
through
PTI
6
expression
response
stage
of
Polysaccharides
dle
(DAMPs)
and
On
susceptible
to different
activator-type
with
biotrophic
times
when
Fusarium
sion
(Zhang
lowing
been
pv.
in
expression
Signaling
elicitors
sion
Lorenzo
tion
ethephon
These
pathways
such
2011).
highlighted
(Menke
was
hon
(Online
earum
signaling
vated
of
(Champion
Pp-ERF2.b,
in the
resistant
cultivar
after
inoculation
Xcp. The
combined
analysis
of
peach
PR and
ERF
transcripts
a pattern
similar
to a double-sigmoid
curve,
in which
phases
of
gene
induction
occurred
at
1 and
24
HPI
and
an
stationary
phase
observed
at
4–8
HPI.
The
first
phase
gene
induction
at 1 HPI
was
accompanied
by stomatal
closure
probably
illustrates
the
immunity
responses
of
epidermal
and
cells
triggered
by pathogen-associated
molecular
patterns
such
as
bacterial
flagellin
(Asai
et
al.,
2002;
Nurnberger
2004).
This
form
of
immunity
referred
to as
PAMPs-triggered
(PTI)
is associated
with
the
induction
of
many
defense-
genes.
For
instance,
more
than
1000
genes
including
several
induced
in Arabidopsis
within
30
min
of
treatment
with
a short
peptide
representing
the
elicitor-active
epitope
of
flagellin
(Zipfel
et al.,
2004).
The
dramatic
reduction
of
and
ERF
transcripts
at
4–8
HPI
is
possibly
due
to the
excessive
of
bacteria
via
stomata,
which
reopened
in both
cultivars
at
(Sherif
et al.,
2011) and
which
probably
stay
open
at 4 HPI
et
al.,
2006).
At
this
stage,
bacterial
effectors
delivered
type
three
secretion
system
(TTSS)
might
suppress
early
responses,
as
only
7% of flg22-responsive
genes
were
induced
at
HPI
with
Pseudomonas
syringae
(Navarro
et al.,
2004).
The
second
peak
at
24 and
48
HPI
probably
represents
the
immune
of
mesophyll
cells.
The
increased
bacterial
growth
at
this
(Sherif
et
al.,
2011)
is likely
coupled
with
the
degradation
cell
wall
components
and
cell
collapse
(Aarrouf
et
al.,
2008).
resulting
from
the
degradation
of
the
cell
wall
mid-
lamella
might
serve
as
damage-associated
molecular
patterns
to elicit
another
phase
of
PTI
responses
against
Xcp
at
24
48 HPI.
the
other
hand,
higher
expression
levels
of Pp-ERF2.b
in the
cultivar
may
be due
to the
specificity
of
different
ERFs
types
of
pathogens.
For
instance,
the
wheat
TaERF3, an
ERF,
is
expressed
differently
following
inoculation
various
pathogens.
After
infection
with
Blumeria
graminis, a
pathogen,
the
transcript
level
of
TaERF3
was
about
six
higher
in resistant
than
in susceptible
wheat
lines.
However,
wheat
lines
were
infected
with
the
necrotrophic
pathogens,
graminearum
and
Rhizoctonia
cerealis, the
TaERF3
expres-
was
about
three
to six
times
higher
in the
susceptible
lines
et
al.,
2007).
The
induction
of
ERF
gene
expression
fol-
both
compatible
and
incompatible
interactions
has
also
reported
in interactions
involving
Arabidopsis
and
P. syringae
tomato
DC3000,
suggesting
altogether
a role
of
certain
ERFs
orchestrating
the
proper
temporal
responses
in defense
gene
(Onate-Sanchez
and
Singh,
2002).
molecules
such
as
ethephon,
MeJA
and
SA are
known
of
plant
immune
responses
and
also
regulate
the
expres-
of
several
ERF
genes
(Park
et
al.,
2001;
Brown
et
al.,
2003;
et
al.,
2003;
Zhang
et
al.,
2004).
The
exogenous
applica-
of
these
compounds
altered
the
expression
of
Pp-ERFs, with
and
MeJA
exhibiting
more
impact
than
SA treatment.
results
are
consistent
with
the
role
of
JA-
and
ET-signaling
in enhancing
resistance
to hemibiotrophic
pathogens
as
Xcp
(Li
and
Yen,
2008;
Ding
et
al.,
2011;
Sherif
et
al.,
The
significance
of
ET/JA
cross-talk
in disease
resistance
is
by
their
induction
of
the
same
defense-related
genes
et
al.,
1999;
van
der
Fits
and
Memelink,
2001).
However,
it
clear
from
the
temporal
analysis
of
gene
expression
that
ethep-
triggers
the
expression
of
peach
PRs
and
ERFs earlier
than
MeJA
Resource
5).
Similarly,
in interactions
involving
F.
gramin-
(a hemibiotrophic
fungus)
and
wheat,
JA-mediated
defense
was
activated
at 12
HPI,
while
ET
signaling
was
acti-
at 6–12
HPI
(Ding
et al.,
2011).
Given
the
cumulative
effects
ET and
JA signaling
pathways
on
transcript
induction
of
ERFs
et
al.,
2009;
Memelink,
2009),
plants
might
modulate
the
synergistic
action
robust
sive
addition,
susceptible
activation
pathways
tially
an
Taken
play
in peach.
cates
in
tion
early
emphasizes
the
ular
should
peach
timing
of
ET/JA
responses
to allow
for
compensatory
rather
than
interactions
between
these
pathways
during
the
inter-
with
Xcp. These
compensatory
interactions
would
afford
responses
while
minimizing
the
negative
impacts
of
exces-
immune
responses
on
plant
fitness
(Tsuda
et al.,
2009).
In
Pp-ERF2.b,
which
showed
higher
transcript
levels
in the
cultivar,
was
also
induced
by
MeJA.
This
coordinated
of
negative
and
positive
regulators
by
the
same
signaling
could
also
be
a strategy
that
plants
use
to avoid
poten-
self-inflicted
damage
while
triggering
a defense
response
to
invading
pathogen
(Kazan,
2006).
together,
these
results
strongly
indicate
that
Pp-ERFs
an essential
role
in resistance
against
bacterial
spot
disease
The
combined
analysis
of
peach
PR
and
ERF
genes
indi-
that
induction
of
these
genes
upon
bacterial
infection
occurs
two
phases.
These
two
phases
probably
illustrate
the
recogni-
of
PAMPs
and
DAMPs
by
epidermal
and
mesophyll
cells,
at
and
late
hours
post-inoculation,
respectively.
This
study
also
the
role
of
the
ET/JA
signaling
pathways
in regulating
expression
of
Pp-ERFs
during
defense
against
Xcp. The
molec-
characterization
of
other
components
within
these
pathways
enhance
our
understanding
of
the
defense
mechanisms
in
and
other
stone
fruits
against
pathogen
attack.
Acknowledgments
We
would
like
to thank
Dr.
Johan
Memelink
(Leiden
University,
the
GUS
of
grant
(Canada;
Netherlands)
for
providing
us
with
the
GUSXX
and
PDF1.2-
vectors.
This
Wok
was
supported
by
funds
from
the
Ministry
Higher
Education
(Egypt;
SS)
and
a
Sustainable
Production
from
Ontario
Ministry
of Agriculture,
Food
and
Rural
Affairs
SJ).
Appendix
A.
Supplementary
data
Supplementary
the
data
associated
with
this
article
can
be
found,
in
online
version,
at doi:10.1016/j.jplph.2012.02.003.
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