Pepper

pectin

methylesterase

inhibitor

protein

is

required

for

antifungal

activity

basal

disease

resistance

and

abiotic

stress

tolerance

Pectin

is

one

of

the

main

components

of

the

plant

cell

wall

that

functions

as

the

primary

barrier

against

pathogens

Among

the

extracellular

pectinolytic

enzymes

pectin

methylesterase

-LRB-

PME

-RRB-

demethylesterifies

pectin

which

is

secreted

into

the

cell

wall

in

a

highly

methylesterified

form

Here

we

isolated

and

functionally

characterized

the

pepper

-LRB-

Capsicum

annuum

L.

-RRB-

gene

which

encodes

a

pectin

methylesterase

inhibitor

protein

-LRB-

PMEI

-RRB-

in

pepper

leaves

infected

by

Xanthomonascampestris

pv

vesicatoria

-LRB-

Xcv

-RRB-

transcripts

are

localized

in

the

xylem

of

vascular

bundles

in

leaf

tissues

and

pathogens

and

abiotic

stresses

can

induce

differential

expression

of

this

gene

Purified

recombinant

protein

not

only

inhibits

PME

but

also

exhibits

antifungal

activity

against

some

plant

pathogenic

fungi

Virus-induced

gene

silencing

of

in

pepper

confers

enhanced

susceptibility

to

Xcv

accompanied

by

suppressed

expression

of

some

defense-related

genes

Transgenic

lines

exhibit

enhanced

resistance

to

Pseudomonas

syringae

pv

tomato

mannitol

and

methyl

viologen

but

not

to

the

biotrophic

pathogen

Hyaloperonospora

parasitica

Together

these

results

suggest

that

an

antifungal

protein

may

be

involved

in

basal

disease

resistance

as

well

as

in

drought

and

oxidative

stress

tolerance

in

plants

Introduction

Plant

cell

wall

the

first

barrier

of

defense

against

invading

pathogens

is

composed

of

cellulose

microfibrils

cross-linked

by

hemicellulose

pectin

and

extensin

Pectin

comprises

a

highly

heterogeneous

group

of

polymers

that

includes

homogalacturonans

and

rhamnogalacturonans

I

and

II

-LRB-

Willats

et

al.

b

-RRB-

In

pectin

polymers

the

galacturonic

acid

carboxyl

groups

can

be

methylesterified

by

a

group

of

pectinases

These

galacturonic

acid

methylesters

are

hydrolyzed

by

pectin

methylesterase

-LRB-

PME

-RRB-

-LRB-

Hagerman

and

Austin

Pelloux

et

al.

-RRB-

Many

physiological

processes

such

as

fruit

maturation

microsporangenesis

seed

germination

and

pollen

growth

are

affected

by

the

degree

to

which

PME

changes

the

methlyesterification

of

galacturonic

acid

-LRB-

Tieman

and

Handa

Kagan-Zur

et

al.

Ren

and

Kermode

Bosch

et

al.

-RRB-

The

number

and

distribution

of

free

and

unesterified

galacturonate

carboxyl

groups

along

the

homogalacturonan

chain

has

a

great

influence

on

the

pectin

properties

and

cell

wall

firmness

-LRB-

Willats

et

al.

b

-RRB-

Plant

pathogens

hydrolyze

the

cell

wall

components

of

plants

using

extracellular

pectinolytic

enzymes

-LRB-

Collmer

and

Keen

-RRB-

and

PME

is

found

in

many

plant

pathogenic

bacteria

and

fungi

-LRB-

Asoufi

et

al.

-RRB-

The

black

filamentous

fungus

Aspergillus

niger

secretes

a

set

of

pectin-degrading

enzymes

that

include

PME

polygalacturonase

and

pectin

lyase

and

these

decompose

the

plant

cell

wall

to

establish

infection

and

absorb

nutrients

from

the

host

-LRB-

de

Vries

and

Visser

-RRB-

In

soft

rot

disease

caused

by

Erwinia

chrysanthemi

bacterial

PME

activity

is

induced

during

infection

of

Saintpaulia

plants

however

PME-deficient

mutants

are

noninvasive

to

the

host

cells

-LRB-

Boccara

and

Chatain

-RRB-

PMEs

have

also

been

found

in

higher

plants

they

play

significant

roles

in

physiological

processes

and

interactions

with

pathogens

-LRB-

Micheli

-RRB-

In

tobacco

plants

host

cell

pectin

methylesterases

are

required

for

the

tobacco

mosaic

virus

movement

protein

to

transfer

the

viruses

between

host

cells

-LRB-

Chen

et

al.

-RRB-

Furthermore

PME-degraded

polygalacturonans

are

associated

with

recognition

of

fungal

pathogens

-LRB-

Wietholter

et

al.

-RRB-

PME

is

also

involved

in

symbiosis-specific

functions

-LRB-

Lievens

et

al.

-RRB-

For

example

plant

PME

isoenzymes

may

undergo

organism-specific

post-translational

processing

for

structural

and

functional

integrity

during

interactions

with

various

microorganisms

-LRB-

Micheli

-RRB-

The

PME

enzyme

activity

is

modulated

specifically

by

inhibitor

proteins

such

as

the

pectin

methylesterase

inhibitor

-LRB-

PMEI

Micheli

-RRB-

Moreover

the

PMEIs

that

inhibit

demethylesterification

of

highly

heterogeneous

polymers

-LRB-

pectins

-RRB-

are

the

plant

invertase

inhibitor-related

proteins

which

are

inhibitors

of

important

metabolic

enzymes

-LRB-

Koch

-RRB-

Plant

invertase

inhibitor-related

proteins

play

key

roles

in

wounding

the

plant

defense

reaction

and

developmental

transitions

-LRB-

Raush

and

Greiner

-RRB-

as

well

as

during

osmotic

stress

senescence

and

seed

development

-LRB-

Greiner

et

al.

-RRB-

Investigation

of

gain

and

loss-of-function

mutants

of

tobacco

cell

wall

invertase

inhibitor

-LRB-

NtCIF

-RRB-

protein

demonstrated

that

these

inhibitor

proteins

play

a

role

in

seed

development

-LRB-

Raush

et

al.

-RRB-

Overexpression

of

the

tobacco

vacuolar

invertase

inhibitor

protein

-LRB-

NtVIF

-RRB-

gene

in

transgenic

potatoes

is

of

potential

use

in

the

field

of

food

technology

-LRB-

Greiner

et

al.

-RRB-

However

there

is

little

known

about

the

in

vivo

functions

of

PMEI

protein

Proteinaceous

inhibitors

have

been

purified

from

kiwi

-LRB-

Actinidia

deliciosa

-RRB-

-LRB-

Giovane

et

al.

-RRB-

Arabidopsis

-LRB-

Wolf

et

al.

Raiola

et

al.

-RRB-

rice

-LRB-

Han

et

al.

-RRB-

and

the

jelly

fig

-LRB-

Ficus

awkeotsang

cv

Makino

-RRB-

-LRB-

Jiang

et

al.

-RRB-

The

kiwi

PMEI

is

specific

for

PME

-LRB-

Balestrieri

et

al.

-RRB-

and

is

active

against

PMEs

from

several

plants

including

kiwi

orange

apple

tomato

apricot

carrot

potato

and

banana

-LRB-

Ly-Nguyen

et

al.

-RRB-

Four

Cys

residues

conserved

in

several

isoforms

of

PMEI

are

involved

in

the

formation

of

disulfide

bridges

-LRB-

Camardella

et

al.

-RRB-

PME

and

PMEI

form

a

stoichiometric

complex

in

which

the

interaction

between

the

PME

and

the

inhibitor

occurs

in

close

proximity

to

the

putative

active

site

-LRB-

Di

Matteo

et

al.

-RRB-

Since

PME

activity

can

be

modulated

by

pH

the

stability

of

the

PME

PMEI

complex

is

also

affected

by

pH

-LRB-

Denès

et

al.

-RRB-

Crystallographic

work

has

revealed

that

an

α-helical

hairpin

motif

plays

a

structurally

important

role

in

PMEI

activation

-LRB-

Hothorn

et

al.

-RRB-

Many

cDNAs

encoding

PMEIs

have

been

isolated

and

functionally

characterized

from

plants

-LRB-

Rausch

and

Greiner

-RRB-

however

their

role

in

plant

defense

remains

relatively

unknown

To

date

the

functional

analyses

of

genes

associated

with

defense

responses

in

plants

have

utilized

reverse-genetics

approaches

based

on

loss-of-function

via

double-stranded

RNA

interference

-LRB-

Robertson

-RRB-

or

gain-of-function

via

transgenic

gene

expression

-LRB-

Clough

and

Bent

-RRB-

Virus-induced

gene

silencing

-LRB-

VIGS

-RRB-

has

been

proven

to

be

a

useful

method

for

assessing

the

function

of

target

genes

in

Solanum

species

-LRB-

Brigneti

et

al.

-RRB-

In

particular

VIGS

studies

have

been

used

to

investigate

disease

resistance

signaling

and

defense-related

genes

such

as

-LRB-

Liu

et

al.

Peart

et

al.

-RRB-

-LRB-

Liu

et

al.

Peart

et

al.

-RRB-

and

-LRB-

Liu

et

al.

-RRB-

in

Nicotiana

benthamiana

is

among

the

genes

encoding

pectic

enzyme-related

proteins

it

encodes

polygalacturonase

-LRB-

PG

-RRB-

inhibiting

protein

-LRB-

PGIP

-RRB-

and

an

antisense

gene

was

used

to

silence

its

expression

in

transgenic

Arabidopsis

plants

-LRB-

Ferrari

et

al.

-RRB-

Previous

studies

have

revealed

that

overexpression

of

two

closely

related

genes

and

conferred

resistance

against

Botrytis

cinerea

infection

-LRB-

Ferrari

et

al.

-RRB-

Silencing

of

resulted

in

enhanced

susceptibility

to

infection

as

well

as

reduced

activity

of

PGIP

-LRB-

Ferrari

-RRB-

However

gene-silencing

techniques

such

as

VIGS

and

antisense

RNA

have

not

yet

been

used

to

investigate

the

role

played

in

plant

defense

by

other

PMEI-encoding

genes

Here

we

used

a

macroarray

technique

to

isolate

and

functionally

characterize

a

pectin

methylesterase

inhibitor

gene

from

a

cDNA

library

of

pepper

-LRB-

Capsicum

annuum

L.

-RRB-

leaves

infected

with

Xanthomonas

campestris

pv

vesicatoria

-LRB-

Xcv

Jung

and

Hwang

-RRB-

Local

and

systemic

induction

of

was

investigated

in

pepper

plants

following

inoculation

with

pathogenic

and

non-pathogenic

bacteria

We

also

examined

the

involvement

of

in

defense-related

signal

transduction

cascades

via

exogenous

application

of

abiotic

elicitors

to

pepper

plants

Recombinant

proteins

were

expressed

in

E.

coli

and

exhibited

antifungal

activity

against

plant

pathogenic

fungi

Since

it

is

difficult

to

transform

pepper

plants

we

performed

gene

silencing

and

overexpression

in

pepper

and

Arabidopsis

respectively

to

identify

the

cellular

functions

of

the

gene

The

functional

data

obtained

by

VIGS

and

transgenic

ectopic

expression

of

suggest

that

this

pepper

pectin

methylesterase

inhibitor

is

involved

in

plant

defense

and

abiotic

stress

responses

Materials

and

methods

Plant

materials

and

growth

conditions

Pepper

-LRB-

Capsicum

annuum

L.

cv

Nockwang

-RRB-

plants

were

grown

at

°C

under

a

h

day

at

μmol

photons

m

s

Plants

were

seeded

into

a

plastic

tray

-LRB-

-RRB-

containing

steam-sterilized

soil

mix

-LRB-

peat

moss

perlite

and

vermiculite

v/v/v

-RRB-

and

loam

soil

-LRB-

v/v

-RRB-

At

the

two-leaf

stage

seedlings

were

transplanted

into

plastic

pots

-LRB-

-RRB-

containing

the

soil

mix

previously

described

Arabidopsisthaliana

-LRB-

ecotype

Columbia

-RRB-

plants

were

grown

in

pots

containing

vermiculite

peat

moss

and

perlite

-LRB-

v/v/v

-RRB-

in

a

growth

chamber

under

a

h

h

dark

photoperiod

-LRB-

μmol

photons

m

s

-RRB-

at

°C

and

relative

humidity

Prior

to

sowing

the

seeds

were

surface-sterilized

using

sodium

hypochlorite

and

vernalized

at

°C

for

days

to

break

dormancy

Pathogens

inoculation

procedures

disease

rating

and

tissue

staining

Xanthomonas

campestris

pv

vesicatoria

-LRB-

Xcv

-RRB-

strains

and

were

used

in

this

study

Bacteria

were

cultured

overnight

in

yeast-nutrient

-LRB-

YN

-RRB-

broth

-LRB-

g

L

yeast

extract

g

L

nutrient

broth

-RRB-

at

°C

Prior

to

inoculation

bacteria

were

harvested

by

centrifugation

and

resuspended

in

sterile

tap

water

-LRB-

cfu

mL

-RRB-

Pepper

plants

were

inoculated

at

the

six-leaf

stage

by

infiltrating

the

bacterial

suspension

into

the

abaxial

side

of

fully

expanded

leaves

using

an

atomizer

Infected

plants

were

then

incubated

for

h

at

°C

in

a

moist

chamber

with

relative

humidity

Bacteria-infected

leaves

were

sampled

at

various

time

intervals

after

inoculation

To

evaluate

systemic

induction

in

the

upper

leaves

bacterial

suspensions

-LRB-

cfu

ml

-RRB-

were

infiltrated

into

the

lower

leaves

of

pepper

plants

at

the

two-leaf

stage

using

a

needless

syringe

The

bacterial

strains

used

for

this

study

included

virulent

and

avirulent

strains

of

X.

campestris

pv

vesicatoria

-LRB-

and

-RRB-

a

non-pathogenic

strain

-LRB-

Pseudomonasfluorescens

-RRB-

and

Escherichiacoli

Leaves

of

Arabidopsis

plants

were

infiltrated

with

a

suspension

-LRB-

-RRB-

of

virulent

Pseudomonas

syringae

pv

tomato

strain

-LRB-

Pst

-RRB-

The

bacteria

were

cultured

overnight

at

°C

and

suspended

in

mM

To

determine

bacterial

growth

leaf

discs

were

cut

from

infected

leaves

at

different

time

intervals

after

inoculation

Bacterial

growth

was

monitored

by

performing

serial

dilutions

onto

KB

agar

containing

μg

mL

rifampicin

Each

experiment

was

replicated

three

times

Hyaloperonospora

parasitica

isolate

was

propagated

by

weekly

subculturing

on

to

Arabidopsis

seedlings

The

seedlings

were

inoculated

with

an

H.

parasitica

asexual

inoculum

-LRB-

conidiosporangia

mL

-RRB-

The

seedlings

inoculated

with

H.

parasitica

were

covered

with

a

plastic

dome

to

maintain

a

high

relative

humidity

-LRB-

-RRB-

and

grown

in

a

growth

chamber

at

°C

Seven

days

after

inoculation

disease

rating

was

scored

for

more

than

plants

per

treatment

A

visual

disease

rating

was

expressed

as

the

number

of

sporangiophores

on

each

cotyledon

and

was

divided

into

five

classes

and

sporangiophores

per

cotyledon

The

cotyledons

from

inoculated

plants

were

stained

with

lactophenol-trypan

blue

-LRB-

mL

lactic

acid

mL

glycerol

g

phenol

and

mg

trypan

blue

dissolved

in

mL

of

distilled

water

-RRB-

to

assess

H.

parasitica

growth

At

days

after

inoculation

the

infected

cotyledons

were

boiled

for

min

in

the

staining

solution

and

de-stained

overnight

in

chloral

hydrate

-LRB-

g

chloral

hydrate

dissolved

in

mL

distilled

water

-RRB-

The

destained

cotyledons

were

subsequently

mounted

in

glycerol

for

microscopic

observation

Isolation

and

sequence

analysis

of

pathogen-induced

cDNAs

To

construct

a

pathogen-induced

cDNA

library

pepper

leaves

were

inoculated

with

the

avirulent

strain

X.

campestris

pv

vesicatoria

The

pathogen-induced

cDNA

library

was

constructed

using

μg

poly

-LRB-

A

-RRB-

mRNA

extracted

from

inoculated

pepper

leaves

-LRB-

Kim

and

Hwang

-RRB-

To

isolate

pathogen-inducible

cDNAs

from

the

pepper

cDNA

library

we

performed

differential

hybridization

as

described

previously

by

Jung

and

Hwang

-LRB-

-RRB-

Digoxigenin

-LRB-

DIG

-RRB-

labeled

single-stranded

cDNA

probes

were

generated

from

total

RNA

of

healthy

and

leaves

using

RT-PCR

Nylon

membranes

were

pre-hybridized

at

°C

for

h

in

SSC

sodium

lauroylsarcosine

SDS

and

blocking

reagent

-LRB-

Boehringer

Mannheim

Mannheim

Germany

-RRB-

Hybridization

was

then

performed

overnight

at

°C

in

the

same

buffer

with

single-stranded

cDNA

probes

Hybridized

membranes

were

rinsed

twice

for

min

with

SSC

and

SDS

at

room

temperature

and

twice

for

min

with

SSC

and

SDS

at

°C

The

hybridization

signals

were

detected

according

to

the

manufacturer

's

protocol

-LRB-

Boehringer

Mannheim

-RRB-

We

selected

cDNA

clones

that

were

expressed

strongly

in

pathogen-infected

leaves

compared

with

those

of

healthy

leaves

Clones

were

sequenced

with

an

ABI

DNA

sequencer

-LRB-

Applied

Biosystems

Foster

City

CA

USA

-RRB-

using

the

PRISM

Big

Dye

Terminator

Cycle

Sequencing

Ready

Reaction

Kit

-LRB-

PE

Biosystems

Foster

City

CA

USA

-RRB-

Sequencing

results

were

analyzed

using

BLAST

-LRB-

National

Center

for

Biotechnology

Information

Altschul

et

al.

-RRB-

Treatment

with

abiotic

elicitors

and

environmental

stresses

The

leaves

of

pepper

plants

at

the

six-leaf

stage

were

sprayed

with

mM

salicylic

acid

-LRB-

SA

-RRB-

μM

methyl

jasmonate

-LRB-

MeJA

-RRB-

or

μM

absicisic

acid

-LRB-

ABA

-RRB-

Pepper

plants

treated

with

methyl

jasmonate

were

sealed

tightly

in

plastic

bags

For

ethylene

treatment

whole

plants

were

removed

from

soil

and

then

placed

in

a

water-containing

glass

chamber

followed

by

injection

of

ethylene

gas

-LRB-

μL

L

-RRB-

For

cold

stress

treatment

plants

were

placed

at

°C

in

a

cold

room

For

wounding

stress

the

leaves

were

pricked

with

needles

To

impose

drought

stress

the

plants

were

removed

from

the

soil

and

then

incubated

at

room

temperature

without

water

treatment

was

performed

by

spraying

leaves

with

mM

solution

Leaves

treated

with

various

elicitors

and

abiotic

stresses

were

removed

from

the

plants

frozen

in

liquid

nitrogen

and

stored

at

°C

until

used

for

RNA

isolation

RNA

isolation

and

RNA

gel

blot

analysis

Total

RNA

was

extracted

from

pepper

leaves

stems

roots

flowers

and

fruits

using

the

guanidine

isothiocyanate

method

-LRB-

Chomczynski

and

Sacchi

-RRB-

Frozen

tissues

-LRB-

g

-RRB-

were

ground

to

a

powder

and

homogenized

in

mL

extraction

buffer

-LRB-

M

guanidine

isothiocyanate

mM

sodium

citrate

-LSB-

pH

-RSB-

-LSB-

w/v

-RSB-

N-laurylsarcosine

and

M

-RRB-

A

mixture

of

M

sodium

acetate

-LRB-

pH

-RRB-

water-saturated

phenol

and

chloroform

isoamylalcohol

-LRB-

-RRB-

was

added

to

the

homogenate

followed

by

precipitation

Total

RNA

-LRB-

μg

-RRB-

was

separated

by

formaldehyde-agarose

gel

electrophoresis

and

then

blotted

onto

Hybond-N

membranes

-LRB-

Amersham

Buckinghamshire

UK

-RRB-

Transferred

RNA

was

fixed

to

the

membrane

using

UV

cross-linking

The

UTR

region

of

was

amplified

for

use

in

the

generation

of

a

gene-specific

probe

The

primers

used

for

amplification

of

the

gene-specific

region

were

and

GTTAACAAATGCATA

The

coding

region

was

amplified

with

the

primers

and

The

coding

region

was

amplified

using

the

primers

ATGGTTTCCAAAAGT

and

TATGCTTAACAATTATTACTGAATA

PCR-amplified

products

were

using

a

random

priming

kit

-LRB-

Boehringer

Mannheim

-RRB-

Hybridization

was

performed

overnight

at

°C

in

dextran

sulfate

M

disodium

phosphate

-LRB-

pH

-RRB-

-LRB-

w/v

-RRB-

sodium

dodecyl

sulfate

-LRB-

SDS

-RRB-

and

mM

EDTA

Following

hybridization

the

membranes

were

washed

twice

for

min

with

SSC

and

SDS

at

room

temperature

and

then

twice

for

min

with

SSC

and

SDS

at

°C

Equal

loading

of

RNA

was

confirmed

by

ethidium

bromide-staining

of

ribosomal

RNA

In

situ

RNA

localization

In

situ

RNA

localization

was

performed

as

described

previously

-LRB-

Lee

et

al.

-RRB-

Leaf

tissue

was

fixed

for

h

in

a

solution

of

phosphate-buffered

saline

-LRB-

PBS

-RRB-

paraformaldehyde

and

μL

mL

Triton

by

vacuum

infiltration

for

min

and

shaking

for

h

at

room

temperature

The

fixed

samples

were

washed

with

PBS

dehydrated

through

a

graded

ethanol

and

xylol

series

and

then

embedded

in

liquid

paraplast

at

°C

-LRB-

Sherwood

Medical

St.

Louis

MO

USA

-RRB-

Paraplast-embedded

sections

-LRB-

μm

in

thickness

-RRB-

were

placed

on

glass

slides

coated

with

poly-l-lysine

-LRB-

Sigma

St.

Louis

MO

USA

-RRB-

and

incubated

at

°C

Sections

were

deparafinated

using

xylene

and

followed

by

rehydration

with

serial

dilutions

of

ethanol

Tissue

samples

were

treated

with

M

Tris

HCl

-LRB-

pH

-RRB-

and

bovine

serum

albumin

-LRB-

BSA

-RRB-

for

min

followed

by

incubation

in

mM

Tris

HCl

solution

-LRB-

pH

-RRB-

containing

proteinase

K

-LRB-

mg

mL

-RRB-

and

mM

EDTA

for

min

at

°C

Sections

were

treated

with

acetic

anhydride

in

mM

triethanolamine

-LRB-

pH

-RRB-

for

min

at

room

temperature

to

inhibit

non-specific

signals

Digoxigenin

-LRB-

DIG

-RRB-

labeled

probes

were

prepared

using

the

Dig

High

Prime

DNA

Labeling

and

Detection

kit

according

to

the

manufacturer

's

instructions

-LRB-

Boehringer

Mannheim

-RRB-

Sections

were

prehybridized

and

hybridized

at

°C

in

formamide

SSC

μg

mL

tRNA

and

blocking

reagent

-LRB-

Boehringer

Mannheim

-RRB-

After

hybridization

the

sections

were

washed

twice

with

formamide

and

SSC

at

°C

twice

with

SSC

and

then

once

with

diethyl

pyrocarbonate

-LRB-

DEPC

-RRB-

treated

water

The

DIG

signal

was

detected

according

to

the

manufacturer

's

instructions

-LRB-

Boehringer

Mannheim

-RRB-

Color

reactions

were

developed

overnight

at

°C

with

nitro-blue

tetrazolium

chloride

-LRB-

NBT

-RRB-

and

phosphate

-LRB-

BCIP

-RRB-

and

reactions

were

stopped

with

TE

buffer

-LRB-

mM

Tris

HCl

mM

EDTA

pH

-RRB-

Sections

were

photographed

with

Kodak

ISO

film

under

a

bright

field

Olympus

microscope

-LRB-

Olympus

Tokyo

Japan

-RRB-

To

demonstrate

the

specificity

of

in

situ

hybridization

control

hybridizations

were

performed

without

DIG-labeled

probes

Purification

of

recombinant

protein

The

coding

region

including

the

stop

codon

was

amplified

by

PCR

using

the

forward

and

reverse

primers

and

respectively

Amplification

products

were

cloned

into

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

which

was

digested

with

EcoRI

and

XhoI

and

ligated

into

the

similarly

digested

-LRB-

Novagen

Madison

WI

USA

-RRB-

Escherichia

coli

-LRB-

-RRB-

pLysS

-LRB-

Novagen

-RRB-

which

is

defective

for

thioredoxin

reductase

was

used

as

a

host

for

recombinant

protein

expression

Cultures

were

started

from

single

colonies

grown

in

LB

broth

at

°C

and

then

diluted

at

After

dilution

bacteria

were

grown

to

a

density

of

induced

with

mM

IPTG

and

grown

for

a

further

h

at

°C

Cells

were

harvested

by

centrifugation

for

min

at

g

extracted

with

denaturation

buffer

-LRB-

M

urea

mM

sodium

phosphate

buffer

-LSB-

pH

-RSB-

mM

NaCl

-RRB-

and

disrupted

by

sonication

Following

centrifugation

at

g

for

min

at

°C

the

supernatant

was

loaded

onto

a

mL

column

of

Ni-NTA

resin

-LRB-

Qiagen

Hilden

Germany

-RRB-

which

was

washed

with

an

initial

denaturing

wash

buffer

-LRB-

M

urea

mM

sodium

phosphate

buffer

-LSB-

pH

-RSB-

mM

NaCl

-RRB-

followed

by

a

second

denaturing

wash

buffer

-LRB-

M

urea

mM

sodium

phosphate

buffer

-LSB-

pH

-RSB-

mM

NaCl

-RRB-

Bound

fusion

protein

was

eluted

with

a

final

denaturing

elution

buffer

-LRB-

M

urea

mM

sodium

phosphate

buffer

-LSB-

pH

-RSB-

mM

NaCl

-RRB-

Recombinant

proteins

were

dialyzed

against

a

buffer

containing

mM

Tris

-LRB-

pH

-RRB-

and

Triton

according

to

the

manufacturer

's

protocol

As

a

control

thioredoxin

was

purified

using

a

native

buffer

that

did

not

contain

urea

The

purified

recombinant

protein

was

digested

for

h

at

room

temperature

with

recombinant

enterokinase

-LRB-

μg

recombinant

protein

Novagen

-RRB-

SDS

polyacrylamide

gel

electrophoresis

was

dissolved

in

SDS

sample

buffer

-LRB-

g

glycerol

SDS

bromophenol

blue

mL

mercaptoethanol

and

L

-RRB-

separated

by

SDS-PAGE

as

described

previously

-LRB-

Laemmli

-RRB-

and

stained

with

Coomassie

Brilliant

Blue

Molecular

weights

were

estimated

using

to

kDa

marker

proteins

-LRB-

Bio-Rad

Hercules

CA

USA

-RRB-

Pectin

methylesterase

enzyme

inhibitor

assay

The

inhibitory

effect

of

on

the

enzymatic

activity

of

pectin

methylesterase

-LRB-

PME

-RRB-

was

assayed

under

standard

conditions

-LRB-

Grsic-Rausch

and

Rausch

-RRB-

The

reaction

mixture

comprised

μL

mM

NAD

in

mM

phosphate

buffer

-LRB-

pH

-RRB-

μL

-LRB-

w/v

-RRB-

pectin

-LRB-

Sigma

-RRB-

in

μL

formaldehyde

dehydrogenase

-LRB-

U

Sigma

-RRB-

and

μL

alcohol

oxidase

-LRB-

U

Sigma

-RRB-

After

mixing

the

reaction

was

started

with

the

addition

of

μL

-LRB-

mU

-RRB-

PME

from

orange

peel

-LRB-

Sigma

-RRB-

To

analyze

inhibition

of

PME

μL

inhibitor

solution

-LRB-

mg

fusion

protein

mM

Tris

buffer

-LSB-

pH

-RSB-

M

NaCl

-RRB-

was

mixed

with

μL

PME

-LRB-

mU

-RRB-

and

μL

M

K-acetate

buffer

-LRB-

pH

-RRB-

followed

by

incubation

for

min

at

room

temperature

All

reaction

temperatures

were

maintained

at

°C

Reaction

rates

were

recorded

continuously

at

nm

using

a

UV-visible

spectrophotometer

-LRB-

Beckman

Fullerton

CA

USA

-RRB-

PME

PMEI

interactions

were

determined

by

measuring

the

rate

of

NADH

formation

per

minute

at

pH

and

°C

In

vitro

antifungal

activity

To

determine

the

antifungal

activity

of

we

examined

its

effect

on

plant

pathogenic

fungi

Fusarium

oxysporum

f.sp

matthiole

and

Alternaria

brassicicola

were

incubated

on

potato

dextrose

agar

-LRB-

PDA

-RRB-

at

°C

for

weeks

B.

cinerea

was

incubated

at

°C

for

weeks

Fungi

were

grown

in

plates

-LRB-

Cell

Wells

Corning

Glass

Works

Corning

NY

USA

-RRB-

containing

sterile

potato

dextrose

broth

-LRB-

PDB

μL

-RRB-

and

a

range

of

concentrations

of

purified

protein

-LRB-

μg

mL

-RRB-

Spore

suspensions

-LRB-

spores

mL

-RRB-

were

prepared

and

μL

of

inoculum

was

added

to

each

microwell

Plates

containing

B.

cinerea

were

incubated

at

°C

the

other

fungi

were

incubated

at

°C

for

days

To

determine

the

inhibitory

effect

of

on

spore

germination

and

hyphal

growth

-LRB-

μg

mL

-RRB-

or

the

thioredoxin

control

were

added

to

F.

oxysporum

f.sp

matthiolae

spore

suspensions

-LRB-

spores

mL

-RRB-

placed

on

glass

slides

and

incubated

for

h

at

°C

After

h

incubation

germinated

spores

were

examined

using

a

haemocytometer

and

the

lengths

of

individual

hyphae

were

determined

The

experiment

was

repeated

three

times

Plasmid

construction

and

plant

transformation

The

coding

region

was

PCR-amplified

without

stop

codon

using

the

following

primers

to

generate

XbaI

and

BamHI

sites

TCTAGAATGGAAGGTGGCA

and

GGATCCGCCGTGAAGGGCAGCCAGACGGT

The

fragment

was

inserted

into

-LRB-

Invitrogen

-RRB-

and

digested

with

XbaI

and

BamHI

The

construct

was

confirmed

by

sequencing

To

generate

the

reporter

construct

the

fragment

was

inserted

into

the

XbaI

BamHI

sites

of

the

binary

vector

was

generated

by

fusing

the

gene

encoding

smGFP

from

the

vector

into

digested

with

HindIII

and

EcoRI

The

construct

was

introduced

into

the

Agrobacterium

tumefaciens

strain

using

electroporation

Transgenic

Arabidopsis

plants

were

generated

by

floral-dipping

wild-type

-LRB-

-RRB-

plants

into

an

A.

tumefaciens

culture

containing

the

appropriate

construct

-LRB-

Clough

and

Bent

-RRB-

Agrobacterium

was

grown

at

°C

and

rpm

in

YEP

medium

-LRB-

g

Bacto

peptone

g

yeast

extract

g

NaCl

-RRB-

supplemented

with

kanamycin

-LRB-

μg

ml

-RRB-

Cells

were

harvested

by

centrifugation

for

min

at

g

and

resuspended

in

inoculation

media

containing

sucrose

and

Silwet

-LRB-

OSi

Specialties

Inc.

Danbury

CT

USA

-RRB-

to

Arabidopsis

plants

with

cm

long

bolts

were

inoculated

by

dipping

into

an

Agrobacterium

suspension

-LRB-

-RRB-

and

then

left

in

the

dark

overnight

prior

to

return

to

a

growth

chamber

Seeds

from

the

transformed

Arabidopsis

plants

were

collected

and

screened

for

kanamycin

resistance

Virus-induced

gene

silencing

-LRB-

VIGS

-RRB-

The

TRV-based

VIGS

system

was

used

for

silencing

of

in

pepper

plants

as

described

previously

-LRB-

Liu

et

al.

Chung

et

al.

-RRB-

The

pepper

coding

region

was

cloned

into

to

generate

the

construct

The

fully

extended

cotyledons

of

pepper

seedlings

were

co-infiltrated

with

A.

tumefaciens

carrying

or

-LRB-

for

each

construct

-RRB-

Plants

were

placed

in

a

growth

room

at

°C

under

a

h

h

dark

photoperiod

to

allow

for

growth

and

viral

spread

Experiments

were

performed

weeks

after

the

induction

of

silencing

RT-PCR

analysis

The

RT

reactions

-LRB-

μL

-RRB-

were

performed

at

°C

with

total

RNA

-LRB-

μg

-RRB-

oligo

p

-LRB-

dT

-RRB-

primer

-LRB-

Roche

Mannheim

Germany

-RRB-

and

AMV

reverse

transcriptase

-LRB-

Roche

-RRB-

Aliquots

-LRB-

μL

-RRB-

of

the

RT

reaction

products

were

used

for

RT-PCR

analysis

with

the

following

gene-specific

primers

and

ATCAAAGGC

for

-LRB-

accession

no

-RRB-

TGTCGAAGGTGGTCC

and

for

-LRB-

accession

no

-RRB-

and

CCCTCACTGTGGCCT

for

-LRB-

accession

no

-RRB-

and

CAGGGAGATGAATTCTGA

and

for

-LRB-

accession

no

-RRB-

RT-PCR

conditions

were

°C

for

min

and

cycles

of

°C

for

s

°C

for

s

and

°C

for

min

and

s.

Single

bands

for

PCR

products

were

confirmed

on

an

agarose

gel

Drought

stress

treatment

and

evaluation

For

germination

tests

seeds

from

Arabidopsis

wild-type

and

transgenic

lines

were

surface-sterilized

and

placed

on

MS

media

-LRB-

Murashige

and

Skoog

-RRB-

containing

or

mM

mannitol

The

seeds

were

maintained

at

°C

for

h

under

dark

conditions

to

synchronize

germination

and

then

transferred

to

a

growth

chamber

Experiments

were

repeated

at

least

three

times

using

approximately

seeds

Surface-sterilized

seeds

were

also

used

for

examination

of

relative

root

length

Seedlings

were

grown

on

plates

placed

vertically

in

growth

chambers

and

root

length

was

estimated

for

days

To

assess

mannitol

tolerance

seedlings

were

grown

for

days

in

MS

agar

media

supplemented

with

sucrose

and

then

transferred

to

wells

containing

MS

liquid

media

supplemented

with

or

mM

mannitol

For

drought

treatment

soil-grown

plants

were

deprived

of

water

for

days

and

then

re-watered

on

day

To

minimize

experimental

variation

both

the

transgenic

and

control

plants

were

grown

in

the

same

tray

Experiments

were

repeated

at

least

three

times

For

transpiration

rate

measurements

leaves

of

plants

were

detached

and

maintained

at

room

temperature

Leaf

weight

was

determined

every

min

for

h

and

then

every

h

thereafter

Each

measurement

was

performed

using

eight

leaves

Experiments

were

repeated

at

least

three

times

with

similar

results

Oxidative

stress

treatment

and

evaluation

For

analysis

of

oxidative

stress

tolerance

surface-sterilized

seeds

from

Arabidopsis

wild-type

vector

control

and

transgenic

lines

were

placed

on

MS

medium

containing

different

concentrations

of

methyl

viologen

-LRB-

MV

Sigma

-RRB-

After

synchronized

germination

seeds

were

maintained

in

a

growth

chamber

To

investigate

whether

or

not

MV

treatment

caused

retardation

of

seedling

development

seedlings

were

randomly

transferred

into

well-plates

containing

MS

liquid

medium

supplemented

with

or

μM

MV

and

then

grown

for

a

further

weeks

To

determine

leaf

senescence

during

MV

treatment

fully

expanded

leaves

were

detached

from

plants

and

rinsed

briefly

with

ethanol

The

detached

leaves

were

floated

on

MS

medium

containing

MV

for

h

following

extraction

with

liquid

nitrogen

Their

chlorophyll

content

was

measured

spectrophotometrically

according

to

the

formula

-LRB-

Ca

b

-RRB-

where

C

is

the

chlorophyll

concentration

-LRB-

Ca

b

-RRB-

in

micrograms

per

mL

and

A

is

absorption

-LRB-

Lichtenthaler

-RRB-

Results

Isolation

and

sequence

analysis

of

cDNA

in

pepper

To

identify

the

molecular

mechanisms

involved

in

plant

defense

against

the

invasion

of

microbial

pathogens

we

performed

a

differential

hybridization

screening

of

a

cDNA

library

constructed

from

pepper

leaves

undergoing

an

incompatible

interaction

with

Xcv

-LRB-

Jung

and

Hwang

-RRB-

and

Myb

transcription

factor

were

among

the

pathogen-induced

genes

isolated

by

this

screen

In

addition

we

isolated

a

full-length

cDNA

-LRB-

bp

-RRB-

designated

-LRB-

Capsicum

annuumpectin

methylesterase

inhibitor

protein

accession

no

-RRB-

which

contains

a

bp

untranslated

sequence

and

a

bp

untranslated

region

The

predicted

open

reading

frame

encodes

a

full-length

protein

of

amino

acid

residues

with

an

estimated

molecular

mass

of

kDa

and

an

isoelectric

point

-LRB-

pI

-RRB-

of

BLAST

searches

were

performed

between

the

predicted

amino

acid

sequence

of

-LRB-

accession

no

-RRB-

from

C.

annuum

and

the

EMBL/GenBank

database

These

sequence

analyses

revealed

that

contains

a

conserved

pectin

methylesterase

inhibitor

-LRB-

PMEI

-RRB-

domain

of

amino

acids

-LRB-

Fig.

-RRB-

Four

cysteine

residues

were

conserved

in

all

the

aligned

proteins

-LRB-

Fig.

-RRB-

and

these

are

predicted

to

be

involved

in

the

formation

of

disulfide

bridges

-LRB-

Camardella

et

al.

-RRB-

The

predicted

amino

acid

sequence

of

is

identical

to

that

of

Nicotiana

tabacum

protein

-LRB-

accession

no

-RRB-

and

has

identity

with

Arabidopsis

thaliana

ripening-related

protein

-LRB-

accession

no

-RRB-

with

Pinus

radiata

pectinesterase

homolog

-LRB-

accession

no

-RRB-

and

with

A.

thaliana

invertase

homolog

-LRB-

accession

no

-RRB-

Fig.

acid

sequence

alignments

of

pepper

with

Nicotiana

tabacum

protein

-LRB-

accession

no

-RRB-

A.

thaliana

ripening-related

protein

-LRB-

accession

no

-RRB-

A.

thaliana

invertase

homolog

-LRB-

accession

no

-RRB-

and

Pinus

radiata

pectinesterase

homolog

-LRB-

accession

no

-RRB-

The

boxed

amino

acid

sequences

represent

the

pectin

methylesterase

inhibitor

-LRB-

PMEI

-RRB-

domain

The

conserved

cysteine

residues

are

marked

by

asterisks

and

the

shaded

regions

represent

conserved

amino

acid

residues

Organ-specific

expression

of

in

pepper

tissues

RNA

gel

blot

analysis

was

performed

to

examine

the

organ-specific

expression

of

in

pepper

plants

Although

high

levels

of

transcription

were

observed

in

stem

tissues

only

relatively

low

levels

of

expression

were

found

in

leaf

root

flower

and

green

fruit

-LRB-

Fig.

-RRB-

transcripts

were

not

detected

in

red

fruit

Fig.

gel

blot

analysis

of

expression

of

and

in

pepper

plants

Membranes

were

hybridized

with

probes

from

the

UTR

region

of

pepper

cDNA

or

full-length

cDNA

Equal

loading

-LRB-

μg

-RRB-

was

verified

by

visualizing

RNA

on

a

gel

stained

with

ethidium

bromide

H

healthy

M

mock-inoculated

or

mock-treated

expression

in

various

organs

of

pepper

plants

b

Expression

of

and

in

pepper

leaves

at

various

time

intervals

after

inoculation

with

C.

coccodes

or

virulent

strain

-LRB-

compatible

interactions

with

pepper

susceptible

response

-RRB-

and

avirulent

strain

-LRB-

incompatible

interactions

with

pepper

resistant

response

-RRB-

of

expression

in

lower

-LRB-

local

-RRB-

infected

and

upper

-LRB-

systemic

-RRB-

uninfected

leaves

at

various

time

intervals

after

inoculation

with

the

virulent

and

avirulent

strains

and

of

Xcv

P.fluorescence

and

E.coli

The

lower

leaves

of

pepper

plants

were

inoculated

at

the

stage

For

the

mock-inoculation

the

lower

leaves

were

infiltrated

with

mM

d

Expression

of

and

in

pepper

leaves

at

various

time

intervals

after

treatment

with

salicylic

acid

-LRB-

SA

mM

-RRB-

methyl

jasmonate

-LRB-

MeJA

μM

-RRB-

ethylene

-LRB-

μl

L

-RRB-

and

abscisic

acid

-LRB-

μM

-RRB-

e

Expression

of

and

in

pepper

leaves

at

various

time

intervals

after

treatment

with

drought

wounding

cold

and

induction

by

pathogen

infection

To

assess

the

accumulation

of

transcripts

in

pepper

leaves

during

the

compatible

and

incompatible

interactions

with

pathogens

leaves

were

inoculated

with

C.

coccodes

and

Xcv

-LRB-

virulent

compatible

-RRB-

as

well

as

Xcv

-LRB-

avirulent

incompatible

-RRB-

transcripts

were

not

detected

in

leaves

mock-inoculated

with

sterilized

water

-LRB-

Fig.

c

-RRB-

During

fungal

infection

at

the

four

and

eight-leaf

stages

expression

was

induced

at

and

h

after

inoculation

respectively

suggesting

that

the

adult

plant

stage

is

more

resistant

to

C.

coccodes

-LRB-

Fig.

upper

panel

-RRB-

transcripts

increased

rapidly

within

min

after

infection

by

both

the

virulent

-LRB-

-RRB-

and

avirulent

-LRB-

-RRB-

strains

of

Xcv

-LRB-

Fig.

lower

panel

-RRB-

High

transcript

levels

accumulated

by

h

after

infection

with

the

virulent

strain

-LRB-

-RRB-

that

causes

a

susceptible

response

however

they

started

to

decline

rapidly

by

this

time

following

infection

with

the

avirulent

strain

-LRB-

-RRB-

that

causes

a

resistant

response

-LRB-

Capsicum

annuum

basic

-RRB-

expression

was

observed

to

confirm

the

success

of

individual

inoculations

transcripts

were

detected

at

h

after

inoculation

with

the

virulent

and

avirulent

strains

of

Xcv

and

increased

gradually

during

the

h

following

inoculation

-LRB-

Fig.

-RRB-

Local

and

systemic

expression

of

in

infected

pepper

plants

The

local

and

systemic

expression

was

analyzed

in

pepper

leaves

inoculated

with

Xcv

and

the

non-pathogenic

bacteria

Pseudomonas

fluorescens

and

E.

coli

-LRB-

Fig.

-RRB-

In

leaves

infected

with

the

virulent

-LRB-

-RRB-

and

avirulent

-LRB-

-RRB-

strains

of

Xcv

transcripts

were

detected

h

after

inoculation

and

levels

decreased

thereafter

Infection

of

lower

-LRB-

local

-RRB-

leaves

by

either

Xcv

strain

induced

systemic

accumulation

of

transcripts

in

the

upper

leaves

The

systemic

induction

by

the

avirulent

strain

-LRB-

-RRB-

infection

was

stronger

than

that

by

the

virulent

strain

-LRB-

-RRB-

infection

Local

or

systemic

induction

of

transcripts

was

not

observed

h

after

infection

In

response

to

the

non-pathogenic

bacteria

P.

fluorescens

and

E.

coli

transcripts

were

detected

h

after

inoculation

and

decreased

subsequently

in

both

local

and

systemic

leaves

induction

following

treatment

with

plant

hormones

Plant

hormones

are

involved

in

defense-related

signal

transduction

pathways

-LRB-

Hammond-Kosack

and

Jones

Chung

et

al.

-RRB-

We

examined

whether

or

not

expression

is

induced

by

plant

hormones

using

pepper

leaves

treated

with

salicylic

acid

-LRB-

SA

-RRB-

ethylene

methyl

jasmonate

-LRB-

MeJA

-RRB-

or

abscisic

acid

-LRB-

ABA

-RRB-

-LRB-

Fig.

-RRB-

Salicylic

acid

strongly

induced

expression

of

at

and

h

as

well

as

between

and

h

after

treatment

Following

treatment

with

ethylene

and

MeJA

expression

was

strongly

induced

for

h

In

addition

strong

induction

was

observed

h

after

treatment

with

ABA

In

comparison

SA

only

induced

transcription

h

after

treatment

-LRB-

Fig.

-RRB-

although

transcripts

were

detected

h

after

treatment

with

ethylene

and

MeJA

In

response

to

ethylene

treatment

transcripts

increased

rapidly

between

and

h

after

treatment

but

increased

gradually

between

and

h

after

MeJA

treatment

expression

was

induced

between

and

h

after

ABA

treatment

induction

by

abiotic

stresses

Abiotic

stresses

resulted

in

differential

induction

of

in

pepper

leaves

-LRB-

Fig.

-RRB-

The

gene

was

responsive

to

mechanical

injury

and

expression

was

observed

between

min

and

h

after

wounding

although

no

transcription

was

detected

thereafter

transcription

also

increased

between

and

h

after

cold

treatment

transcription

was

induced

at

h

of

drought

stress

but

was

undetectable

thereafter

In

response

to

treatment

transcript

levels

increased

for

the

first

h

and

declined

thereafter

The

marker

genes

and

were

used

for

comparison

as

they

also

exhibit

differential

induction

by

these

abiotic

stresses

In

situ

localization

of

transcripts

We

performed

an

in

situ

hybridization

using

a

probe

to

examine

spatial

expression

of

in

pepper

leaf

tissues

-LRB-

Fig.

-RRB-

No

hybridization

signals

were

observed

in

non-treated

leaves

-LRB-

Fig.

b

-RRB-

Intense

localization

of

transcripts

was

observed

in

the

xylem

area

of

the

vascular

bundle

in

non-treated

stems

-LRB-

Fig.

-RRB-

transcripts

were

detected

in

the

vascular

bundle

of

leaves

inoculated

with

C.

coccodes

-LRB-

Fig.

-RRB-

and

in

the

vascular

bundles

and

upper

epidermis

of

leaves

treated

with

ethylene

-LRB-

Fig.

-RRB-

No

transcript

signals

were

observed

in

any

pepper

tissues

hybridized

with

the

sense

DIG-labeled

RNA

probe

-LRB-

Fig.

d

f

-RRB-

These

in

situ

hybridization

results

are

supported

by

the

temporal

expression

patterns

observed

in

the

RNA

gel

blot

analysis

of

expression

-LRB-

Fig.

-RRB-

Fig.

situ

localization

of

transcripts

in

pepper

leaf

and

stem

tissues

Cross

sections

of

leaf

tissues

were

hybridized

with

antisense

-LRB-

a

c

e

g

-RRB-

and

sense

-LRB-

b

d

f

h

-RRB-

DIG-labeled

RNA

probes

and

then

photographed

under

bright-field

conditions

The

transcript

signal

is

purple

a

b

Untreated

leaf

tissues

c

d

Untreated

stems

e

f

Leaf

tissues

at

h

after

inoculation

with

C.

coccodes

g

h

Leaf

tissues

treated

with

μl

L

ethylene

P

phloem

X

xylem

UE

upper

epidermis

LE

lower

epidermis

Vs

vascular

bundle

C

cortical

cell

Inhibition

of

pectin

methylesterase

-LRB-

PME

-RRB-

activity

by

protein

To

prepare

recombinant

protein

the

coding

region

was

PCR-amplified

and

cloned

into

E.

coli

strain

-LRB-

-RRB-

pLysS

was

used

as

a

host

for

the

recombinant

expression

of

the

construct

and

an

empty

vector

control

yielding

recombinant

and

thioredoxin

respectively

The

purified

-LRB-

Fig.

lane

-RRB-

and

thioredoxin

-LRB-

Fig.

lane

-RRB-

were

examined

by

SDS-PAGE

analysis

The

purified

thioredoxin

fusion

protein

formed

a

single

band

with

an

apparent

molecular

mass

of

kDa

-LRB-

Fig.

lane

-RRB-

Following

cleavage

of

thioredoxin

by

enterokinase

digestion

the

purified

protein

-LRB-

Fig.

lane

-RRB-

was

used

for

biological

function

determination

The

cleaved

recombinant

protein

was

found

exclusively

in

the

insoluble

fraction

as

inclusion

bodies

-LRB-

data

not

shown

-RRB-

After

attempting

unsuccessfully

to

renature

the

cleaved

protein

using

Triton

as

a

detergent

-LRB-

Kim

and

Hwang

-RRB-

we

performed

the

PME

inhibition

assay

using

crude

and

purified

fusion

proteins

Fig.

of

pectin

methylesterase

-LRB-

PME

-RRB-

activity

by

the

pepper

pectin

methylesterase

inhibitor

-LRB-

CaPMEI

-RRB-

a

Recombinant

expression

in

E.

coli

-LRB-

-RRB-

pLysS

Cells

were

grown

in

LB

media

and

recombinant

protein

expression

was

induced

with

mM

IPTG

M

molecular

marker

-LRB-

kDa

-RRB-

Lane

Uninduced

E.

coli

cell

extracts

Lane

Soluble

fraction

of

E.

coli

cell

extracts

encoding

thioredoxin

protein

after

IPTG

induction

Lane

Purified

thioredoxin

Lane

Crude

protein

extracts

of

E.

coli

cells

producing

the

thioredoxin

fusion

protein

after

IPTG

induction

Lane

Purified

thioredoxin

fusion

protein

Lane

Cleaved

and

thioredoxin

proteins

following

enterokinase

digestion

Protein

staining

was

performed

using

Coomassie

brilliant

blue

b

Inhibition

of

PME

activity

following

treatment

with

For

the

inhibition

assay

crude

-LRB-

μg

-RRB-

was

mixed

with

mU

of

orange

peel

PME

in

a

total

volume

of

μL

and

then

preincubated

at

°C

for

min

followed

by

addition

to

the

reaction

solution

contains

a

pectin

methylesterase

inhibitor

-LRB-

PMEI

-RRB-

domain

-LRB-

Fig.

-RRB-

and

shares

similarity

with

a

plant

pectin

methylesterase

inhibitor

related

to

regulation

of

pectin

degradation

-LRB-

Raiola

et

al.

-RRB-

In

addition

PMEIs

have

been

found

to

play

crucial

roles

in

plant

and

microbial

enzyme

regulation

of

pectin

degradation

by

de-esterification

-LRB-

Raiola

et

al.

-RRB-

To

determine

whether

or

not

orange

peel

PME

activity

is

inhibited

in

the

presence

of

crude

fusion

protein

we

used

the

PME

enzyme

assay

to

measure

the

effect

of

PME

inhibitor

on

the

formation

of

NADH

-LRB-

Fig.

-RRB-

Both

crude

and

purified

-LRB-

ca.

μg

-RRB-

strongly

inhibited

PME

enzyme

activity

and

a

higher

concentration

of

purified

-LRB-

ca.

μg

-RRB-

exhibited

the

strongest

inhibition

However

thioredoxin

alone

did

not

exhibit

any

inhibitory

effect

These

results

suggest

that

functions

as

an

inhibitor

of

PME

activity

Antimicrobial

activity

of

exhibited

antifungal

activity

against

the

three

plant

pathogenic

fungi

examined

F.

oxysporum

f.sp

matthiole

A.

brassicicola

and

B.

cinerea

-LRB-

Fig.

-RRB-

The

thioredoxin

fusion

protein

-LRB-

μg

mL

-RRB-

suppressed

mycelial

growth

of

the

three

plant

pathogenic

fungi

whereas

thioredoxin

alone

did

not

Furthermore

the

purified

thioredoxin

-LRB-

μg

mL

-RRB-

fusion

protein

inhibited

spore

germination

and

hyphal

growth

of

F.oxysporum

f.sp

matthiole

whereas

thioredoxin

alone

did

not

-LRB-

Fig.

-RRB-

Increasing

concentrations

of

recombinant

thioredoxin

fusion

protein

increasingly

inhibited

both

spore

germination

and

hyphal

growth

-LRB-

Fig.

-RRB-

Fig.

of

thioredoxin

fusion

protein

antimicrobial

activity

a

Inhibitory

effects

of

the

thioredoxin

fusion

protein

on

mycelial

growth

of

the

plant

pathogenic

fungi

F.

oxysporum

f.sp

matthiolae

A.

brassicicola

and

B.

cinerea

In

each

plate

the

upper

wells

-LRB-

a

-RRB-

were

treated

with

purified

thioredoxin

and

the

lower

wells

-LRB-

b

-RRB-

were

treated

with

the

purified

fusion

protein

b

Inhibition

of

germination

and

hyphal

growth

of

F.oxysporum

f.sp

matthiolae

Fungal

spores

were

allowed

to

germinate

and

grow

in

μL

potato

dextrose

broth

medium

alone

-LRB-

top

-RRB-

or

with

μg

mL

thioredoxin

-LRB-

middle

-RRB-

or

μg

mL

thioredoxin

fusion

protein

-LRB-

bottom

-RRB-

Photographs

were

taken

after

incubation

for

h

at

°C

Bars

μm

c

Inhibition

of

spore

germination

and

hyphal

growth

of

F.oxysporum

f.sp

matthiolae

by

The

percentage

of

germinated

spores

and

the

length

of

fungal

hyphae

were

determined

by

light

microscopy

Data

represent

means

SD

from

three

independent

experiments

Enhanced

susceptibility

of

pepper

plants

to

Xcv

infection

Since

inoculation

with

Xcv

strongly

induced

expression

in

pepper

plants

we

performed

the

virus-induced

gene

silencing

-LRB-

VIGS

-RRB-

technique

-LRB-

Liu

et

al.

Chung

et

al.

-RRB-

to

examine

its

cellular

function

during

pathogen

infection

The

full-length

ORF

of

was

used

to

construct

Five

to

six

weeks

after

induction

of

silencing

empty

vector

control

-LRB-

TRV

-RRB-

and

-LRB-

TRV

-RRB-

pepper

plants

were

inoculated

with

virulent

and

avirulent

strains

and

of

Xcv

respectively

To

assess

the

efficiency

of

VIGS

transcript

levels

were

examined

by

RT-PCR

-LRB-

Fig.

-RRB-

transcripts

were

nearly

undetectable

in

both

non-inoculated

empty

vector

control

and

plants

However

at

h

after

inoculation

with

virulent

and

avirulent

Xcv

we

observed

strong

induction

of

in

the

empty

vector

control

plants

whereas

only

weak

or

undetectable

transcript

levels

were

found

in

plants

These

results

confirm

the

effective

silencing

of

the

target

gene

in

pepper

plants

Fig.

analysis

of

expression

of

and

several

defense-related

genes

in

empty

vector

control

-LRB-

TRV

-RRB-

and

gene-silenced

-LRB-

TRV

-RRB-

pepper

plants

h

after

inoculation

with

the

virulent

-LRB-

C

compatible

-RRB-

and

avirulent

-LRB-

I

incompatible

-RRB-

strains

of

Xcv

-LRB-

cfu

mL

-RRB-

rRNA

levels

were

visualized

as

a

loading

control

This

experiment

was

repeated

three

times

with

similar

results

H

healthy

leaves

basic

pathogenesis-related

protein

putative

ribonuclease-like

protein

ascorbate

peroxidase

and

To

determine

whether

or

not

the

expression

of

defense-related

genes

is

affected

by

bacterial

infection

in

the

silenced

plants

we

used

RT-PCR

to

analyze

transcript

levels

of

some

defense-related

genes

-LRB-

Fig.

-RRB-

In

the

empty

vector

control

plants

both

-LRB-

basic

pathogenesis-related

protein

-RRB-

and

-LRB-

putative

ribonuclease-like

protein

-RRB-

were

strongly

induced

by

virulent

and

avirulent

Xcv

infection

However

induction

of

these

genes

was

slightly

reduced

in

plants

infected

with

virulent

Xcv

but

was

unaffected

in

plants

infected

with

avirulent

Xcv

Thus

gene

silencing

did

not

alter

the

induction

of

-LRB-

ascorbate

peroxidase

-RRB-

or

-LRB-

SAR

-RRB-

in

response

to

Xcv

infection

To

examine

the

function

of

the

gene

in

basal

defense

or

gene-for-gene

resistance

empty

vector

control

and

pepper

plants

were

infected

with

virulent

or

avirulent

Xcv

-LRB-

Fig.

-RRB-

gene

silencing

significantly

increased

susceptibility

to

virulent

Xcv

infection

but

not

to

avirulent

Xcv

infection

The

pepper

leaves

exhibited

more

severe

disease

symptoms

days

after

virulent

Xcv

inoculation

than

did

empty

vector

control

plants

and

these

symptoms

were

accompanied

by

severe

chlorosis

and

enlarged

water-soaked

lesions

-LRB-

Fig.

-RRB-

However

we

did

not

observe

any

phenotypical

changes

in

the

cell

death

response

of

plants

following

avirulent

Xcv

inoculation

-LRB-

Fig.

-RRB-

At

days

after

inoculation

with

virulent

Xcv

bacterial

growth

was

tenfold

greater

in

plants

than

in

the

empty

vector

controls

-LRB-

Fig.

-RRB-

However

silencing

of

conferred

a

slightly

enhanced

susceptibility

to

avirulent

Xcv

These

findings

suggest

that

may

function

in

basal

resistance

of

pepper

plants

against

Xcv

infection

rather

than

gene-for-gene

resistance

Fig.

disease

susceptibility

of

pepper

plants

to

infection

by

the

virulent

Xcv

strain

but

not

the

avirulent

Xcv

strain

a

Disease

symptoms

developed

on

the

leaves

at

different

time

points

after

inoculation

with

the

virulent

Xcv

strain

-LRB-

cfu

mL

-RRB-

and

the

avirulent

Xcv

strain

-LRB-

various

bacterial

concentrations

-RRB-

b

Bacterial

growth

in

leaves

of

empty

vector

control

-LRB-

TRV

-RRB-

or

-LRB-

TRV

-RRB-

pepper

plants

at

different

time

points

after

inoculation

with

the

virulent

Xcv

strain

or

the

avirulent

Xcv

strain

-LRB-

cfu

mL

-RRB-

Data

represent

the

mean

SD

from

three

independent

experiments

Enhanced

resistance

of

plants

to

Pst

The

and

overexpression

-LRB-

OX

-RRB-

plants

were

generated

by

transformation

of

A.

thaliana

using

the

floral

dipping

method

-LRB-

Clough

and

Bent

-RRB-

Plants

transformed

with

the

empty

vector

were

used

as

a

control

Seeds

-LRB-

-RRB-

were

collected

from

each

transformed

plant

and

screened

for

resistance

to

kanamycin

Northern

blot

analysis

was

performed

on

plants

exhibiting

kanamycin

resistance

to

determine

the

integrity

of

the

inserted

transgene

Three

plants

showing

strong

expression

were

selected

for

analyses

in

planta

-LRB-

Fig.

-RRB-

Fig.

of

wild-type

-LRB-

-RRB-

Arabidopsis

and

transgenic

plants

to

infection

with

P.

syringae

pv

tomato

a

RNA

gel

blot

analysis

confirming

overexpression

-LRB-

OX

-RRB-

in

the

transgenic

Arabidopsis

lines

Total

RNA

-LRB-

μg

-RRB-

was

loaded

into

each

lane

The

UTR

region

of

pepper

cDNA

was

used

as

a

probe

b

Growth

of

Pst

in

the

leaves

of

wild-type

and

transgenic

plants

The

mature

leaves

of

the

plants

were

infiltrated

with

a

Pst

suspension

-LRB-

cfu

mL

-RRB-

and

the

degree

of

bacterial

growth

was

rated

at

and

days

after

inoculation

c

Disease

symptoms

on

leaves

of

plants

infiltrated

with

virulent

Pst

-LRB-

cfu

mL

-RRB-

d

Expression

of

pathogen-related

-LRB-

PR

-RRB-

genes

in

transgenic

plants

Northern

blot

analyses

were

performed

with

μg

total

RNA

prepared

from

leaves

of

the

wild-type

-LRB-

WT

-RRB-

vector

control

-LRB-

smGFP

-RRB-

and

transgenic

-LRB-

smGFP

-RRB-

plants

The

samples

were

collected

at

and

h

following

pathogen

infiltration

with

a

suspension

of

the

virulent

strain

Pst

-LRB-

cfu

mL

-RRB-

To

determine

the

contribution

of

to

Arabidopsis

resistance

wild-type

and

plants

were

inoculated

with

virulent

P.

syringae

pv

tomato

-LRB-

Pst

cfu

mL

-RRB-

Bacterial

titers

were

determined

days

after

inoculation

plants

exhibited

much

less

growth

of

Pst

bacteria

than

wild-type

or

empty

vector

control

plants

-LRB-

Fig.

-RRB-

In

the

transgenic

plants

this

reduced

bacterial

multiplication

was

most

pronounced

at

days

after

inoculation

At

days

after

inoculation

wild-type

-LRB-

-RRB-

and

empty

vector

control

plants

developed

typical

chlorotic

symptoms

in

the

infected

leaves

whereas

the

transgenic

plants

displayed

few

disease

symptoms

-LRB-

Fig.

-RRB-

Expression

of

defense-related

genes

in

plants

To

gain

insight

into

the

role

played

by

in

PR

gene

induction

we

examined

the

expression

patterns

of

the

well-established

marker

genes

and

in

the

defense

responses

of

wild-type

vector

control

and

the

Arabidopsis

plants

infected

with

virulent

Pst

-LRB-

Fig.

-RRB-

The

expression

of

and

was

very

similar

in

the

wild-type

and

vector

control

plants

expression

was

not

induced

in

either

un-inoculated

or

inoculated

wild-type

or

vector

control

plants

However

there

was

significant

expression

of

in

the

transgenic

plants

at

and

h

after

Pst

infection

and

expression

is

known

to

be

regulated

via

the

SA-dependent

pathway

in

Arabidopsis

-LRB-

Uknes

et

al.

-RRB-

and

in

nahG

plants

lack

of

SA

reduces

expression

of

these

three

PR

genes

during

pathogenesis

-LRB-

Delany

et

al.

Nawrath

and

Metraux

-RRB-

In

our

experiments

-LRB-

defensin

-RRB-

transcripts

were

not

detected

in

wild-type

vector

control

or

plants

-LRB-

data

not

shown

-RRB-

Responses

of

wild-type

and

plants

to

Hyaloperonospora

parasitica

We

examined

whether

or

not

ectopic

expression

in

Arabidopsis

plants

affected

resistance

to

the

virulent

biotrophic

oomycte

pathogen

H.

parasitica

isolate

-LRB-

Fig.

-RRB-

Over

seedlings

of

both

wild-type

-LRB-

-RRB-

and

transgenic

lines

were

inoculated

with

spores

of

H.

parasitica

isolate

-LRB-

conidiosporangia

mL

-RRB-

At

days

after

inoculation

over

plants

in

each

line

were

sampled

to

estimate

the

disease

and

assess

the

degree

of

asexual

sporulation

which

was

quantified

and

expressed

as

the

mean

number

of

sporangiophores

per

cotyledon

Fig.

of

wild-type

-LRB-

-RRB-

Arabidopsis

and

transgenic

plants

to

infection

with

H.

parasitica

isolate

a

Disease

symptoms

and

trypan

blue-stained

pathogen

structures

on

cotyledons

of

wild-type

and

transgenic

plants

days

after

inoculation

dpi

days

post-inoculation

Bars

mm

b

Quantification

of

asexual

sporangiophores

per

cotyledon

for

at

least

cotyledons

of

wild-type

and

transgenic

plants

days

after

inoculation

The

average

number

of

sporangiophores

produced

on

the

cotyledons

of

wild-type

and

transgenic

lines

are

shown

below

each

of

the

lines

tested

As

shown

in

Fig.

the

cotyledons

of

wild-type

and

transgenic

seedlings

responded

to

H.

parasitica

infection

by

stimulating

similar

levels

of

asexual

parasite

sporulation

Their

trypan

blue-stained

cotyledons

also

exhibited

abundant

hyphae

conidiospores

and

oospores

-LRB-

Fig.

-RRB-

Quantitative

disease

ratings

are

shown

in

Fig.

Both

the

transgenic

and

the

wild-type

plants

showed

a

high

level

of

susceptibility

to

H.

parasitica

isolate

and

similar

levels

of

heavy

asexual

sporulation

-LRB-

sporangiophores

per

cotyledon

-RRB-

were

observed

on

both

lines

Drought

tolerance

of

plants

To

investigate

a

possible

role

for

in

the

dehydration

response

we

tested

seed

germination

and

seedling

growth

under

osmotic

stress

The

seeds

of

wild-type

vector

control

and

plants

were

placed

on

MS

media

supplemented

with

various

concentrations

of

mannitol

We

observed

no

significant

differences

in

seed

germination

between

these

lines

-LRB-

Fig.

-RRB-

However

treatment

with

and

mM

mannitol

strongly

inhibited

germination

in

the

wild-type

and

vector

control

plants

compared

with

the

plants

Fig.

lines

exhibit

enhanced

tolerance

to

drought

stress

a

Seed

germination

in

wild-type

smGFP

and

transgenic

plants

on

the

MS

media

containing

and

mM

mannitol

The

data

represent

the

mean

SD

of

seeds

for

each

line

tested

b

Relative

root

length

of

wild-type

smGFP

and

transgenic

lines

in

MS

agar

medium

containing

different

concentrations

of

mannitol

Three

independent

experiments

were

performed

with

seedlings

of

both

wild-type

and

transgenic

lines

c

Drought

tolerance

test

of

transgenic

seedlings

Wild-type

smGFP

and

transgenic

lines

were

germinated

and

grown

in

MS

agar

medium

Each

seedling

was

transferred

to

liquid

medium

containing

mM

mannitol

d

Wild-type

smGFP

and

the

transgenic

Arabidopsis

plants

after

days

without

water

e

Water

loss

from

the

excised

leaves

of

wild-type

smGFP

and

transgenic

plants

Data

represent

the

mean

SD

from

three

independent

experiments

We

also

tested

the

sensitivity

of

root

growth

to

osmotic

stress

-LRB-

Fig.

-RRB-

Drought

tolerance

was

observed

during

post-germination

growth

The

root

growth

of

wild-type

and

vector

control

seedlings

was

inhibited

in

the

presence

of

and

mM

mannitol

However

root

elongation

of

plants

was

less

sensitive

to

mannitol-induced

osmotic

stress

and

in

comparison

with

wild-type

and

vector

control

plants

mutant

seedlings

grew

well

in

liquid

medium

supplemented

with

mM

mannitol

-LRB-

Fig.

-RRB-

In

addition

we

noticed

that

adult

plants

exhibited

enhanced

drought

tolerance

Following

days

without

water

wild-type

and

vector

plants

had

withered

severely

whereas

the

plants

remained

healthy

To

determine

the

effect

of

on

survival

these

plants

were

rewatered

on

day

wild-type

and

vector

control

plants

died

whereas

the

transgenic

lines

survived

-LRB-

Fig.

-RRB-

Next

we

examined

transpiration

rates

by

measuring

fresh

weight

loss

in

detached

leaves

The

leaves

of

lines

exhibited

slightly

slower

water

loss

than

those

of

wild-type

or

vector

control

plants

-LRB-

Fig.

-RRB-

Together

these

results

indicate

that

overexpression

enhanced

water

stress

resistance

Oxidative

tolerance

of

plants

To

investigate

the

response

of

plants

to

oxidative

stresses

seeds

of

wild-type

vector

control

and

plants

were

exposed

to

MS

medium

containing

methyl

viologen

-LRB-

MV

-RRB-

-LRB-

Fig.

-RRB-

Treatment

with

or

μM

MV

significantly

inhibited

germination

of

wild-type

and

vector

control

seeds

compared

with

seeds

-LRB-

Fig.

-RRB-

Seven-day-old

seedlings

were

transferred

to

a

medium

containing

different

MV

concentrations

-LRB-

μM

-RRB-

and

grown

for

weeks

Wild-type

and

vector

control

seedlings

turned

white

and

started

to

die

after

days

of

MV

treatment

but

lines

were

much

less

affected

by

the

treatment

-LRB-

Fig.

-RRB-

Furthermore

these

differences

in

plant

phenotype

were

also

observed

with

respect

to

the

higher

fresh

weight

of

transgenic

seedlings

compared

to

those

of

wild-type

and

vector

control

plants

-LRB-

Fig.

-RRB-

Detached

leaves

of

plants

were

treated

with

μM

MV

for

h

after

which

chlorophyll

content

was

measured

-LRB-

Fig.

-RRB-

The

leaves

of

transgenic

lines

treated

with

MV

retained

more

chlorophyll

than

those

of

wild-type

or

vector

control

plants

Fig.

lines

exhibit

tolerance

to

oxidative

stress

a

Effects

of

methyl

viologen

on

the

seed

germination

of

transgenic

lines

Seeds

from

wild-type

vector

control

and

transgenic

lines

were

plated

on

media

with

or

without

methyl

viologen

-LRB-

MV

and

μM

-RRB-

and

incubated

for

days

The

data

represent

mean

SD

of

seeds

for

each

line

tested

b

Phenotypes

of

wild-type

vector

control

and

transgenic

line

seedlings

treated

with

different

concentrations

of

MV

c

Fresh

weights

of

seedlings

grown

in

the

indicated

concentrations

of

MV

for

weeks

The

results

are

presented

as

the

average

fresh

weight

per

seedling

Data

represent

mean

SD

from

three

independent

experiments

d

Chlorophyll

content

of

MV-treated

leaves

of

wild-type

vector

control

and

transgenic

plants

which

were

floated

on

and

μM

MV

in

MS

medium

and

then

incubated

for

h

in

a

growth

chamber

Discussion

Plants

possess

a

diverse

range

of

cell

wall-modified

enzymes

which

are

post-transcriptionally

regulated

by

numerous

inhibitor-related

proteins

-LRB-

Tymowska-Lalanne

and

Kreis

Raush

and

Greiner

-RRB-

Several

invertase

inhibitor-related

proteins

have

been

isolated

from

higher

plants

such

as

kiwi

tobacco

and

Arabidopsis

-LRB-

Greiner

et

al.

Wolf

et

al.

Giovane

et

al.

Raiola

et

al.

-RRB-

Attempts

to

characterize

the

activity

of

invertase

inhibitor

proteins

from

plant

species

other

than

kiwi

or

Arabidopsis

have

been

either

unsuccessful

or

resulted

in

the

isolation

of

invertase

inhibitors

that

share

structural

similarities

with

pectin

methylesterase

inhibitor

proteins

-LRB-

PMEI

-RRB-

but

which

represent

completely

different

target

enzymes

-LRB-

Greiner

et

al.

Scognamiglio

et

al.

-RRB-

In

this

study

we

identified

and

functionally

characterized

a

novel

pepper

gene

encoding

a

PMEI

This

protein

contains

the

four

cysteine

residues

that

are

conserved

among

other

PMEI

proteins

-LRB-

Camardella

et

al.

Sato

et

al.

-RRB-

These

residues

are

expected

to

be

engaged

in

two

disulfide

bridges

which

constitute

a

common

structural

motif

within

the

PMEI

domain

-LRB-

Camardella

et

al.

-RRB-

At

the

molecular

level

expression

was

induced

in

pepper

leaves

by

infection

with

bacterial

pathogens

and

treatment

with

plant

hormones

such

as

SA

ethylene

MeJA

and

ABA

In

particular

these

hormone

treatments

strongly

induced

transcription

suggesting

that

this

gene

may

be

involved

in

the

early

stages

of

the

active

defense

responses

to

bacterial

pathogen

infection

and

exogenous

treatment

with

plant

hormones

In

enzymatic

assays

purified

proteins

significantly

inhibited

activity

of

plant

pectin

methylesterase

-LRB-

PME

-RRB-

In

addition

exhibited

antifungal

activity

against

a

broad

range

of

plant

pathogenic

fungi

including

F.

oxysporum

f.sp

matthiolae

A.

brassicicola

and

B.

cinerea

To

penetrate

the

cuticular

layer

plant

fungal

pathogens

produce

plant

cell

wall-degrading

enzymes

such

as

polygalacturonase

pectin

lyase

and

cellulase

-LRB-

Collmer

and

Keen

-RRB-

An

aggressive

Phaeosphaeria

nodorum

isolate

was

shown

to

produce

high

amounts

of

xylanase

cellulase

polygalacturonase

and

butyrate

esterase

in

vitro

-LRB-

Lalaoui

et

al.

-RRB-

Recently

it

was

found

that

plant

PMEIs

do

not

inhibit

the

PMEs

produced

by

plant

pathogens

-LRB-

Giovane

et

al.

Di

Matteo

et

al.

-RRB-

However

Arabidopsis

plants

expressing

either

or

showed

reduced

infection

by

B.

cinerea

-LRB-

Lionetti

et

al.

-RRB-

This

finding

suggests

that

the

increased

level

of

pectin

methylesterification

caused

by

overexpression

of

results

in

the

inhibition

of

fungal

endopolygalacturonase

activity

Thus

the

increase

in

PMEI

activity

resulted

in

reduced

accessibility

for

fungal

pectin

degrading-enzymes

and

hence

provided

increased

resistance

to

pathogens

-LRB-

Boudart

et

al.

Lionetti

et

al.

-RRB-

Consistent

with

these

findings

our

results

suggest

that

may

function

as

part

of

a

new

group

of

plant

pectin

methylesterase

inhibitors

which

restrict

fungal

pathogen

infection

in

plants

We

used

virus-induced

gene

silencing

-LRB-

VIGS

-RRB-

to

investigate

the

effect

of

loss-of-function

in

pepper

plants

during

Xcv

infection

The

plants

were

susceptible

to

Xcv

infection

and

in

particular

to

infection

with

the

virulent

strain

which

resulted

in

enhanced

bacterial

growth

and

reduced

and

gene

expression

Basal

resistance

is

activated

during

the

compatible

bacterial

interaction

which

restricts

the

spread

of

pathogens

in

the

host

plants

to

a

certain

extent

-LRB-

Glazebrook

-RRB-

In

addition

basal

resistance

is

also

effective

in

retarding

proliferation

of

a

wide

range

of

microbial

pathogens

-LRB-

Chisholm

et

al.

-RRB-

but

it

is

dependent

upon

SA

accumulation

-LRB-

Cao

et

al.

Kinkema

et

al.

-RRB-

Therefore

we

conclude

that

expression

may

be

involved

in

basal

resistance

by

triggering

downstream

PR

gene

induction

in

pepper

plants

To

determine

the

effect

of

gain-of-function

inplanta

we

generated

the

Arabidopsis

transgenic

lines

and

investigated

their

response

to

P.

syringae

and

H.

parasitica

infection

because

these

well-known

model

pathogens

have

been

used

extensively

for

the

study

of

disease

resistance

mechanisms

in

Arabidopsis

-LRB-

Quirino

and

Bent

Slusarenko

and

Schlaich

-RRB-

The

lines

were

resistant

to

Pst

infection

but

not

to

infection

by

the

biotrophic

oomycete

H.

parasitica

which

uses

living

cells

as

a

nutrient

source

during

the

infection

cycle

-LRB-

Alfano

and

Collmer

Heath

-RRB-

Moreover

as

transcripts

localize

intensively

in

the

xylem

of

vascular

bundles

in

leaf

tissues

they

may

not

affect

the

accessibility

of

host

plant

cells

to

H.parasitica

In

contrast

we

hypothesize

that

intercellular

growth

of

P.

syringae

may

be

restricted

by

the

extracellular

secretion

of

into

host

cells

resulting

in

triggering

of

the

basal

resistance

response

We

found

that

fungal

growth

was

inhibited

by

treatment

with

the

recombinant

protein

in

vitro

suggesting

that

may

interfere

directly

with

pathogen

infection

of

host

plants

Plant

pathogenic

microorganisms

have

been

shown

to

produce

a

variety

of

pectinolytic

enzymes

that

macerate

and

kill

plant

tissues

-LRB-

Collmer

and

Keen

-RRB-

Cell

wall

fragments

released

by

these

pectinolytic

enzymes

may

elicit

the

plant

defense

response

-LRB-

D'Ovidio

et

al.

-RRB-

The

polygalacturonase-inhibiting

protein

-LRB-

PGIP

-RRB-

plays

an

important

role

in

the

recognition

and

inhibition

of

fungal

polygalacturonase

-LRB-

PG

-RRB-

Overexpression

of

PGIPs

in

Arabidopsis

not

only

significantly

reduces

disease

symptoms

but

also

enhances

defense

gene

activation

during

pathogen

infection

-LRB-

Ferrari

et

al.

-RRB-

Thus

we

suggest

that

can

disrupt

invading

pathogenic

microorganisms

by

inhibiting

pectin

methylesterases

produced

by

these

pathogens

The

reduced

bacterial

growth

observed

in

Arabidopsis

lines

may

result

from

the

expression

of

SA-inducible

genes

and

overexpression

The

SA-inducible

genes

such

as

and

are

activated

in

the

SA

defense

pathway

-LRB-

Uknes

et

al.

-RRB-

proteins

are

similar

to

thaumatin

which

is

a

sweet-tasting

protein

from

Thaumatococcus

daniellii

-LRB-

Hu

and

Reddy

-RRB-

and

several

pathogens

can

induce

these

proteins

in

a

wide

range

of

plant

species

-LRB-

Ward

et

al.

Hu

and

Reddy

Reuber

et

al.

-RRB-

Members

of

the

group

have

been

shown

to

exhibit

antifungal

activity

against

a

broad

spectrum

of

fungal

pathogens

-LRB-

Coca

et

al.

-RRB-

and

to

participate

in

the

coordinated

induction

of

systemic

acquired

resistance

-LRB-

SAR

-RRB-

against

TMV

-LRB-

Ward

et

al.

-RRB-

Therefore

the

concomitant

induction

of

PR

genes

may

contribute

to

the

enhanced

resistance

of

Arabidopsis

plants

to

bacterial

pathogens

The

Arabidopsis

lines

showed

a

strong

tolerance

to

drought

stress

and

overexpression

resulted

in

reduced

transpiration

and

enhanced

root

elongation

In

contrast

transgene

overexpression

did

not

cause

any

obvious

phenotypic

differences

under

optimal

growing

conditions

Dehydration

factors

such

as

a

mannitol

and

polyethyleneglycol

-LRB-

PEG

-RRB-

have

been

used

to

evaluate

the

effects

of

decreased

water

availability

and

simulate

drought

conditions

in

wild-type

and

Arabidopsis

plants

-LRB-

Gupta

and

Kaur

Verslues

et

al.

-RRB-

Other

plant

invertase

inhibitor-related

protein

genes

such

as

NtCIF

and

NtVIF

are

also

strongly

induced

by

treatment

with

PEG

or

ABA

-LRB-

Rausch

and

Greiner

-RRB-

However

the

molecular

and

genetic

roles

played

by

PMEIs

in

drought

and

osmotic

stress

tolerance

remain

poorly

understood

Since

plant

responses

to

different

abiotic

stresses

may

be

related

to

the

accumulation

of

ROS

and

the

mechanisms

for

their

detoxification

the

role

played

by

ROS

in

stress

signaling

has

been

studied

extensively

-LRB-

Apel

and

Hirt

-RRB-

Methyl

viologen

binds

to

the

thylakoid

membranes

of

chloroplasts

and

in

the

presence

of

light

transfers

electrons

to

in

a

chain

reaction

causing

continuous

formation

of

superoxide

radicals

and

oxidative

stress

-LRB-

Asada

-RRB-

The

lines

exhibited

tolerance

to

oxidative

stress

both

during

seed

germination

and

seedling

growth

This

tolerance

to

oxidative

stress

may

reduce

the

damage

caused

by

other

stresses

via

the

antioxidizing

system

which

suggests

that

overexpression

results

in

detoxification

of

endogenous

superoxide

Here

we

have

determined

that

from

pepper

plays

a

role

as

an

antifungal

protein

and

has

an

inhibitory

effect

on

PME

Furthermore

we

have

shown

that

Arabidopsis

lines

are

resistant

to

bacterial

pathogens

In

addition

they

exhibit

tolerance

to

drought

and

oxidative

stress

In

conclusion

these

multivariate

functions

of

provide

valuable

insights

into

understanding

the

physiological

significance

of

PMEIs

in

plant

disease

resistance

and

abiotic

stress

tolerance

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

-LRB-

DOC

kb

-RRB-

pectin methylesterase inhibitor protein
capsicum annuum
antifungal activity
disease resistance
drought tolerance
oxidative stress tolerance