Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Abstract

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.

Full text

Plant
Physiol.
(1991)
97,
1181-1186
0032-0889/91/97/1181/06/$01
.00/0
Received
for
publication
April
29,
1991
Accepted
June
28,
1991
Ethylene
Biosynthesis-Inducing
Endoxylanase
Is
Translocated
through
the
Xylem
of
Nicotiana
tabacum
cv
Xanthi
Plants1
Bryan
A.
Bailey*,
Rosannah
Taylor,
Jeffrey
F.
D.
Dean2,
and
James
D.
Anderson3
Plant
Hormone
Laboratory,
Beltsville
Agricultural
Research
Center
(West),
Agricultural
Research
Service,
U.S.
Department
of
Agriculture,
Beltsville,
Maryland
20705
ABSTRACT
Ethylene
biosynthesis-inducing
xylanase
(EIX)
from
the
fungus
Trichoderma
viride
elicits
enhanced
ethylene
production
and
tis-
sue
necrosis
in
whole
tobacco
(Nicotiana
tabacum
cv
Xanthi)
plants
at
sites
far
removed
from
the
point
of
EIX
application
when
applied
through
a
cut
petiole.
Symptoms
develop
in
a
specific
pattem,
which
appears
to
be
determined
by
the
interconnections
of
the
tobacco
xylem.
Based
on
results
of
tissue
printing
experi-
ments,
EIX
enters
the
xylem
of
the
stem
from
the
point
of
appli-
cation
and
rapidly
moves
up
and
down
the
stem,
resulting
in
localized
foliar
symptoms
on
the
treated
side
of
the
plant
above
and
below
the
point
of
EIX
application.
The
observation
that
a
fungal
protein
that
elicits
plant
defense
responses
can
be
trans-
located
through
the
xylem
suggests
that
plants
respond
to
path-
ogen-derived
extracellular
proteins
in
tissues
distant
from
the
invading
pathogen.
Some
fungal,
bacterial
(1),
and
viral
(2)
pathogens
are
capable
of
moving
through
plant
vascular
systems
to
induce
disease
symptoms
in
infected
tissues.
Some
of
these
disease
symptoms,
e.g.
hypersensitive
necrosis,
are
believed
to
be
defense
responses
used
by
the
plant
to
limit
progression
of
the
pathogen
into
healthy
tissues
(16).
When
applied
to
the
plant,
numerous
compounds,
some
of
which
are
pathogen
products
and
others
of
which
are
host-derived,
act
to
elicit
responses
resembling
those
in
diseased
tissues
(7,
16).
Recog-
nition
by
the
plant
of
pathogen-generated
elicitors
early
in
the
course
of
infection
could
limit
further
spread
of
the
pathogen
(21),
but
this
requires
movement
of
the
elicitor
or
elicitor-generated
signal
from
diseased
to
healthy
tissue
in
advance
of
the
invading
pathogen.
Unfortunately,
the
move-
ment
of
defense-response
elicitors
through
diseased
plant
tissues
in
vivo
is
very
difficult
to
study
because
of
the
difficulty
in
distinguishing
between
responses
to
pathogen
products
versus
host-derived
materials.
Previous
work
in
this
laboratory
showed
that
an
endo-,B-
1,4-xylanase
(1,4-f3-D-xylan
xylanohydrolase,
EC
3.2.1.8)
pu-
'This
research
was
supported
in
part
by
U.S.
Department
of
Agriculture
Competitive
Research
Grant
Office
grant
No.
88-37261-
3680.
2
Present
address:
Department
of
Biochemistry,
University
of
Geor-
gia,
Athens,
GA
30605.
3
Present
address:
Weed
Science
Laboratory,
Beltsville
Agricultural
Research
Center
(West),
Agricultural
Research
Service,
U.S.
Depart-
ment
of
Agriculture,
Beltsville,
MD
20705.
1181
rified
to
near
homogeneity
from
xylan-grown
cultures
of
the
fungus
Trichoderma
viride,
induces
biosynthesis
of
the
gas-
eous
phytohormone,
ethylene,
in
tobacco
(Nicotiana
tabacum
cv
Xanthi)
leaf
tissue
(1
1,
15).
Elevated
ethylene
production,
a
common
symptom
of
plant
infection
(19),
is
also
induced
in
tobacco
by
an
EIX4
produced
by an
important
fungal
pathogen,
Fusarium
oxysporum
(12).
The
T.
viride
EIX
elicits
necrosis
and
other
symptoms
commonly
associated
with
path-
ogenesis
when
introduced
into
detached
tobacco
leaves
and
intact
plants
(3),
even
under
conditions
that
block
ethylene
biosynthesis
and
ethylene
action
(18).
Immunoblots
of
pro-
teins
extracted
from
detached,
EIX-treated
leaves
probed
with
EIX-specific
antibodies
demonstrated
that
EIX
had
moved
into
leaves
from
the
point
of
application
(3).
In
this
report,
the
same
EIX-specific
antibodies
have
been
used
in
conjunc-
tion
with
the
technique
of
tissue
printing
(9)
to
examine
the
means
by
which
EIX
is
transported
in
tobacco
and
how
that
transport
leads
to
particular
patterns
of
symptom
development.
MATERIALS
AND
METHODS
Chemicals
and
Enzymes
The
chemicals
used
were
of
commercial
origin.
The
EIX
was
purified
as
previously
described
(
11)
from
xylan-induced
Trichoderma
viride
cultures.
Treatment
of
Plant
Materials
Tobacco
(Nicotiana
tabacum
L.
Xanthi)
plants
were
grown
under
greenhouse
conditions
until
25
to
30
cm
tall.
Whole
tobacco
plants
were
exposed
in
a
40.6-L
glass
jar
to
120
,uL/
L
ethylene
for
14
h
or
maintained
in
an
atmosphere
depleted
of
ethylene
by
the
use
of
25
g
of
the
organic
absorbent,
Purafill
IIs
for
14
h.
After
the
ethylene pretreatment,
a
leaf
located
midway
up
the
plant
stem
was
removed,
leaving
the
exposed
petiole.
A
4-cm
section
of
Tygon
tubing
was
attached
to
the
petiole,
and
50
Mg
of
EIX
in
100
ML
distilled
water
was
applied
to
the
petiole
through
the
Tygon
tubing.
Control
plants
were
treated
with
50
Mg
of
EIX
boiled
for
10
min.
Some
plants
were
treated
'Abbreviation:
EIX,
ethylene-inducing
xylanase.
I
Mention
of
trademark,
proprietary
product,
or
vendor
does
not
constitute
a
guarantee
of
warranty
by
the
U.S.
Department
of
Agri-
culture
and
does
not
imply
approval
to
the
exclusion
of
other
products
or
vendors
that
may
also
be
suitable.

Plant
Physiol.
Vol.
97,
1991
with
a
1%
solution
of
the
vital
stain
Evans
blue
to
track
the
movement
of
the
applied
solution
in
the
absence
of
EIX.
Symptom
Measurements
The
percentage
of
necrosis
of
leaves
was
monitored
over
48
h
for
five
leaves
above
and
below
the
point
of
EIX
application
and
rated
on
a
scale
of
0
to
100
%
necrosis.
For
determination
of
ethylene
production,
tobacco
plants
were
dissected
20
min
after
EIX
application
by
removing
each
leaf,
leaving
the
petiole
on
the
stem.
Six
1-cm
leaf
discs
were
cut
from
the
left
and
right
sides
of
each
leaf
and
bioassayed
separately
over
a
4-h
period
for
ethylene
production
as
previously
described
(3,
17).
Ethylene
biosynthesis-inducing
activity
is
expressed
as
,L
ethylene
evolved
per
h
per
g
fresh
weight
of
tobacco
tissue.
Immunolocalization
of
EIX
Protein
Tobacco
plants
treated
with
EIX
protein
as
described
above
were
dissected
20
min
after
EIX
application.
Cross-sections
Necrosis
(%
leaf
halfl
of
tobacco
stems
and
petioles
were
printed
onto
nitrocellulose
membranes
(9),
and
EIX
was
immunolocalized
with
poly-
clonal
antibodies
raised
against
the
22-kD
EIX
polypeptide
(12).
The
polyclonal
antibodies
were
exposed
to
tobacco
protein
extracts
localized
on
nitrocellulose
membrane
prior
to
use
to
reduce
background
levels
of
cross-reactivity.
Tissues
from
plants
treated
with
Evans
blue
were
similarly
printed
onto
nitrocellulose.
RESULTS
Pattern
of
Necrosis
Development
When
EIX
(50
,ug)
was
introduced
into
the
intact
tobacco
vascular
system
by
application
to
a
cut
petiole
(leafO),
necrotic
lesions
developed
in
specific
leaves
up
and
down
the
stem
from
the
point
of
application
(3).
When
symptom
develop-
ment
in
a
treated
tobacco
plant
was
charted
on
a
radial
map
by
leaf
position
around
the
central
axis
(Fig.
1),
a
pattern
became
apparent.
The
arrangement
of
leaves
on
Xanthi
to-
Ethylene
(uL/gm/H)
-5
_-
Above
Application
Petiole
Right
side
of
leaf
blade
Left
side
of
leaf
blade
Below
Applicaton
Petiole
URtWAM/01/:/.A
Rlght
sle
of
leaf
blade
Left
side
of
leaf
blade
Figure
1.
Radial
diagrams
depicting
necrosis
development
and
ethylene
biosynthesis
by
tobacco
plants
in
response
to
EIX.
Whole
tobacco
plants
were
placed
under
an
ethylene
atmosphere
of
120
,/L/L
for
14
h
prior
to
EIX
treatment.
A
4-cm
section
of
Tygon
tubing
was
attached
to
an
exposed
petiole
(leaf
0)
midway
up
the
plant
and
50
,ug
of
purified
EIX
protein
in
distilled
water
was
applied
to
the
petiole
through
the
Tygon
tubing.
Necrosis
development
and
ethylene
production
were
determined
on
separate
plants
for
each
leaf
half
for
five
leaves
above
(positive
numbers
outside
circle)
and
below
(negative
numbers
outside
circle)
the
point
of
EIX
application.
A
visual
estimate
of
necrosis
was
made
after
24
h
using
a
scale
from
0
to
100%
necrotic.
Control
plants
treated
with
boiled
EIX
or
distilled
water
failed
to
produce
necrosis.
Ethylene
production
was
measured
by
dissecting
tobacco
plants
20
min
after
EIX
application
and
cutting
6
discs
from
each
leaf
half.
Ethylene
production
by
the
leaf
discs
was
quantitated
by
GC
after
4
h
of
incubation.
Leaves
from
control
plants
failed
to
produce
ethylene
in
excess
of
0.02
AL/g/h
and
averaged
0.009
AL/g/h.
-2
2
2
0
1182
BAILEY
ET
AL.
vmr...

TRANSLOCATION
OF
AN
ENDOXYLANASE
IN
TOBACCO
XYLEM
Table
I.
Development
of
Necrosis
in
Air-
and
Ethylene-Pretreated
Tobacco
Plants
Treated
with
EIX
through
Petiole
Application
Plants
were
pretreated
for
14
h
in
an
ethylene
purged
atmosphere
(air)
or
an
atmosphere
of
120
,L/
L
ethylene.
Necrosis
development
was
visually
estimated
at
4,
8,
24,
and
48
h
after
EIX
application
(50
Ag)
to
the
cut
petiole
of
leaf
0.
Values
are
the
averages
from
three
plants
each
for
air
and
ethylene
pretreatments.
Leaf
Necrosis
after
EIX
Application
Leaf
Ethylene
Air
Number
4
h
8
h
24
h
48
h
4
h
8
h
24
h
48
h
+5
0
45
45
45
0
0
6
33
+4
0
0
0
0
0
0
0
0
+3
13
78 79
80
0
0
30
53
+2
2
58
67
67
0
0
10
45
+1
0
2
5
5
0 0
0
22
0
-1
2
11
13
13
0
0
0
7
-2
32
65
67
67
0 0
5
33
-3
58
72
72
72
0
0
1
7
-4
0
0
0
0
0
0
0
1
-5
63
73
75
75
0
0 2
8
bacco
plants
approaches
a
2/5
phyllotaxy;
that
is,
the
leaves
rotate
around
the
central
axis
of
the
plant
in
a
steep
helix
with
approximately
1400
between
leaves.
Thus,
the
fifth
leaf
above
(leaf
+5)
and
the
fifth
below
(leaf
-5)
the
point
of
EIX
application
were
attached
to
the
stem
almost
directly
above
and
below
leaf
0,
respectively,
and
consequently
displayed
severe
necrosis.
Symptomatic
leaves
-3,
-2,
and
+3
were
also
attached
to
the
main
stalk
on
the
same
side
of
the
plant
as
leaf
0,
and
symptoms
in
leaves
-3
and
-2
were,
in
fact,
limited
to
the
half
of
the
leaf
blade
closest
to
the
EIX-treated
side
of
the
plant.
The
remaining
leaves,
attached
to
the
stalk
opposite
the
site
of
application,
generally
failed
to
develop
necrotic
lesions.
The
development
of
necrosis
was
greatly
enhanced
by
ex-
posure
of
whole
plants
to
ethylene
prior
to
EIX
application
(Table
I).
Although
variation
existed
between
plants,
leaves
+2,
+3,
+5,
-2, -3,
and
-5
normally
developed
necrotic
areas,
whereas
the
remaining
leaves
evaluated
showed
little
symptom
development.
The
timing
of
necrosis
development
in
air-pretreated
plants
was
delayed
in
comparison
with
eth-
ylene-pretreated
plants.
Pattem
of
Enhanced
Ethylene
Production
Enhanced
ethylene
production
occurred
in
leaf
discs
cut
from
leaves
20
min
after
EIX
application,
suggesting
that
movement
of
EIX
or
a
product
of
EIX
action
is
rapid
(Fig.
1).
The
pattern
of
enhanced
ethylene
production
closely
fol-
Table
II.
Ethylene
Production
by
Leaf
Discs
Cut
from
Fresh
and
Ethylene-Pretreated
Tobacco
Plants
Treated
with
EIX
through
Petiole
Application
Air
Pretreated8
Ethylene
Pretreated
Ler
Control
(Rb
Plant
1
(R)
Plant
2
(L)
Plant
3
(L)
Control
(R)
Plant
1
(R)
Plant
2
(R)
Plant
3
(L)
Left
Right
Left
Right
Left
Right
Left
Right
Left
Right
Left
Right
Left
Right
Left
Right
nL/g
tissue/h
+5
7
8
2
3
24
20
10
15
4
8
734 779
48
104
194
241
+4
5
12
2
1
6 8
1
3
5
8
2
1
5 5
1
3
+3
2
3
2
3
40
17
11
11
8
8
324
3
259
48
77
436
+2
2
3
11
11
62
40 20
6
5
6
157
170
5
48
26
145
+1
5
5
1
1
28
17
4
7
9
6 2
1
6
6
0
2
0
-1
3
5
1
1
11
17
5
6
6
20
2 2
2 2
90 42
-2
7
6
62
2
52
80
65
117
12
9
29
2
64
11
12
106
-3
3
3
23
85
147
119
134
52
6
15
23
27
84
247
406
240
-4
5 5
3
3
11
23
8
31
10
12
1
2
0 0
3 3
-5
5
8
79
68
36
52
158
188
10
8
18
9
123
236
204
16
a
Plants
were
pretreated
for
14
h
in
an
ethylene
purged
atmosphere
(air)
or
an
atmosphere
of
120
AL/L
ethylene.
Twenty
minutes
after
EIX
(50
gg)
application
to
the
cut
petiole
of
leaf
0,
the
leaves
(+5
to
-5)
were
cut
from
the
plant,
six
discs
removed
from
both
the
left
and
right
sides
of
each
leaf
and
assayed
separately
for
ethylene
production.
Control
plants
were
treated
with
50
Mg
boiled
EIX.
b
R
and
L
indicate
location
of
leaf
+1
in
relation
to
leaf
0.
L
=
left
1400;
R
=
right
1400.
1183

Plant
Physiol.
Vol.
97,
1991
lowed
the
pattern
previously
described
for
necrosis.
The
leaves
located
on
the
stem
opposite
the
point
of
enzyme
application
showed
little
enhanced
ethylene
biosynthesis.
The
total
eth-
ylene
produced
by
tobacco
leaves
was
enhanced
by
ethylene
pretreatment
(Table
II).
In
addition
to
the
previously
de-
scribed
phyllotaxy,
tobacco
plants
also
display
handedness.
Some
plants
rotate
upward
clockwise
from
leaf
0,
1400,
whereas
others
rotate
counterclockwise
140°
to
reach
leaf
+1.
When
this
was
considered,
an
even
more
subtle
pattern
of
symptom
development
was
recognized.
For
example,
leaf
-2
tended
to
produced
more
ethylene
on
the
left
side
of
the
midrib
than
on
the
right
side
in
right-handed
plants,
whereas
the
opposite
was
true
for
left-handed
plants.
Immunolocalization
of
EIX
by
Tissue
Printing
Techniques
Cross-reactive
proteins,
indicating
the
presence
of
EIX,
were
present
within
the
stem
vascular
system
of
plants
treated
with
EIX
and
were
restricted
to
the
EIX-treated
side
of
the
plant
(Fig.
2).
Cross-reactive
proteins
were
also
detected
in
petiole
cross-sections
of
leaves
-5,
-3,
-2,
+2,
+3,
and
+5
(not
all
shown),
but
were
not
detected
in
the
petioles
of
leaves
taken
from
the
opposite
side
of
the
plant.
Leaves
displaying
enhanced
ethylene
production
on
one
side
of
the
leaf
blade
(leaf
+3)
generally
contained
EIX
only
in
the
xylem
on
that
same
side
of
the
petiole
as
shown
in
petiole
+3.
On
occasion,
limited
amounts
of
necrosis
and
enhanced
ethylene
produc-
STEM
PETIOLE
+4
.~
.-
....
Ol~~~~
Figure
2.
Immunolocalization
of
EIX
protein
by
tissue
printing
techniques.
Tobacco
plants
were
dissected
20
min
after
EIX
application.
Cross-
sections
of
tobacco
stems
and
petioles
were
printed
onto
nitrocellulose
membranes
(12),
and
EIX
was
immunolocalized
with
polyclonal
anti-
bodies
raised
against
the
22-kD
EIX
polypeptide
(9).
Negative
and
positive
numbers
represent
leaves
below
and
above
the
point
of
EIX
appli-
cation,
respectively.
Stem
cross-sections
were
made
at
the
juncture
of
each
leaf
with
the
stem.
Experiments
were
repeated
four
times
and
con-
sistently
showed
EIX
to
be
spread
up
and
down
the
stem
more
than
five
leaves
and
in
the
petioles
of
leaves
-2,
-3,
-5,
+2,
+3,
and
+5
(not
all
shown).
Areas
of
cross-reactivity
with
the
EIX
antibodies
are
indicated
by
arrows.
Control
plants
treated
with
distilled
water
showed
no
cross-reactivity
under
these
conditions.
+3
0
-3
-4
1184
BAILEY
ET
AL.
1*
*
d*

TRANSLOCATION
OF
AN
ENDOXYLANASE
IN
TOBACCO
XYLEM
tion
occurred
in
leaves
-1
and
+
1
(Tables
I
and
II),
suggesting
that
limited
amounts
of
EIX
not
detectable
by
our
techniques
or
some
product
of
EIX
action
may
move
into
these
leaves.
The
pattern
demonstrated
for
EIX
movement
was
mimicked
when
Evans
blue,
a
vital
stain,
was
applied
to
whole
tobacco
plants
(Fig.
3).
These
experiments
were
all
performed
with
tobacco
plants
preincubated
in
an
atmosphere
purged
of
ethylene
or
in
an
ethylene
atmosphere
(14
h,
120
,gL/L).
As
shown
previously
(3),
ethylene
pretreatment
led
to
more
rapid
and
severe
symp-
tom
development
but
had
little
effect
upon
the
overall
pattern
or
speed
of
EIX
protein
movement
(data
not
shown).
STEM
DISCUSSION
This
work
provides
evidence
that
EIX,
a
22-kD
fungal
protein
capable
of
eliciting
plant
defense
responses,
can
be
transported
both
upward
and
downward
through
the
tobacco
xylem.
These
observations
may
help
explain
other
instances
in
which
necrosis
is
observed
in
areas
distant
from
the
point
of
elicitor
application
(20).
Although
specific
plant
proteins
have
been
shown
to
be
localized
to
the
plant
vascular
system
(6),
transport
of
these
proteins
within
the
vascular
system
has
not
been
demonstrated.
Given
the
demonstrated
ability
of
fungal
and
plant
cell
wall
polysaccharides
to
elicit
plant
de-
PETIOLE
.4
.3
0
-3
-4
Figure
3.
Movement
of
Evans
blue
in
tobacco
plants.
A
1%
solution
of
Evans
blue
(100
ML)
was
applied
through
Tygon
tubing
to
exposed
petioles
on
whole
tobacco
plants.
The
plants
were
dissected
20
min
after
application
of
Evans
blue
and
cross-sections
of
stems
and
petioles
were
printed
onto
nitrocellulose
membranes.
Negative
and
positive
numbers
represent
leaves
below
and
above
the
point
of
Evans
blue
appli-
cation,
respectively.
Stem
cross-sections
were
made
at
the
juncture
of
each
leaf
with
the
stem.
The
presence
of
Evans
blue
on
tissue
prints
is
indicated
by
arrows.
Fin
...e,-.
.....~n
a..
.g~~a
'a
*{
~
-
1185
I
.zj

Plant
Physiol.
Vol.
97,
1991
fense
responses
(4,
10,
13),
it
is
expected
that
oligosaccharides
released
from
plant
cell
walls
by
EIX
activity
would
be
the
actual
elicitor
molecules
traveling
through
the
tobacco
xylem.
However,
previous
efforts
to
demonstrate
the
release
of
such
heat-stable
signals
have
been
unsuccessful
(14).
The
extremely
localized
induction
of
pathogenesis-related
protein
synthesis
in
tobacco
mesophyll
tissue
injected
with
minute
amounts
of
EIX
also
suggests
that
EIX
must
be
present
locally
to
induce
defense
responses
(18).
Thus,
even
if
EIX
generates
polysac-
charide
fragment
elicitors,
these
molecules
must
be
labile
or
have
very
limited
mobility
in
the
tobacco
system.
At
this
point,
it
is
of
interest
to
note
that
the
reported
size
of
the
nondenatured
EIX
protein
is
equivalent
to
a
9200
D
globu-
lar
protein
(11).
Such
a
small
size
would
allow
EIX
to
pene-
trate
even
the
smallest
cell
wall
pores
(8)
and
reach
the
plasmalemma.
The
movement
of
EIX
both
upward
and
downward
through
the
xylem
appears
to
contradict
the
generally
ac-
cepted
model
of
water
flow
in
response
to
transpirational
forces.
However,
Evans
blue
dye,
introduced
into
the
vascular
system
via
the
same
route
used
for
EIX,
moved
in
exactly
the
same
pattern.
If
the
xylem
elements
leading
into
any
given
leaf
actually
intersect
the
primary
stem
xylem
at
points
below
the
site
of
leaf
attachment,
then
a
change
in
the
water
source-
sink
relationships
between
the
leaves
and
stalk
will
suffice
to
explain
these
observations.
The
water
(+EIX)
that
remains
in
the
cut
petiole
and
xylem
elements
connecting
it
with
the
stem
xylem
becomes
a
passive
water
source
after
removal
of
the
leaf.
This
water
(+EIX)
is
then
drawn
downward
into
the
stem
by
the
transpirational
pull
of
the
remaining
leaves
and
then
travels
upward
once
it
reaches
vessels
leading
to
actively
transpiring
leaves.
Because
xylem
elements
have
limited
lat-
eral
connections,
the
radial
diffusion
of
EIX
around
the
tobacco
stalk
is
limited
to
only
one
side
of
the
plant.
The
ability
of
fungus-produced
enzymes
to
travel
through
the
plant
vascular
system
raises
new
questions
about
the
mechanisms
involved
in
pathogenesis.
Many
types
of
patho-
gens
are
known
to
progress
through
the
plant
via
the
xylem
(1).
One
of
these,
the
fungus
F.
oxysporum,
is
responsible
for
numerous
vascular
wilt
diseases
and,
as
previously
noted,
is
capable
of
synthesizing
an
EIX
protein
when
cultured
on
xylan.
Although
we
have
not
established
whether
EIX
is
produced
during
host-pathogen
interactions,
the
possibility
exists,
and
we
are
vigorously
pursuing
this
avenue
of
research.
The
potential
for
inducing
systemic
resistance
to
plant
path-
ogens
(5,
20,
2
1)
by
exogenous
application
of
EIX
remains
to
be
explored.
ACKNOWLEDGMENTS
We
would
like
to
thank
H.
David
Clark
for
his
excellent
help
in
photographing
the
figures
presented
in
this
article.
LITERATURE
CITED
1.
Agrios
CN
(1978)
Plant
Pathology.
Academic
Press,
New
York,
pp
66-67,
324-340,
464-467
2.
Atabekov
JG,
Dorokhov
YL
(1984)
Plant
virus-specific
transport
function
and
resistance
of
plants
to
viruses.
Adv
Virus
Res
29:
3
13-364
3.
Bailey
BA,
Dean
JFD,
Anderson
JD
(1990)
An
ethylene
biosyn-
thesis-inducing
endoxylanase
elicits
electrolyte
leakage
and
necrosis
in
Nicotiana
tabacum
cv
Xanthi
leaves.
Plant
Physiol
94:
1849-1854
4.
Baldwin
EA,
Biggs
KH
(1988)
Cell-wall
lysing
enzymes
and
products
of
cell-wall
digestion
elicit
ethylene
in
citrus.
Physiol
Plant
73:
58-64
5.
Biles
CL,
Martyn
RD
(1989)
Local
and
systemic
resistance
induced
in
watermelons
by
Formae
Speciales
of
Fusarium
oxysporum.
Phytopathology
79:
856-860
6.
Biles
CL,
Martyn
RD,
Wilson
HD
(1989)
Isozymes
and
general
proteins
from
various
watermelon
cultivars
and
tissue
types.
Hortic
Sci
24:
810-812
7.
Bowles
DJ
(1990)
Defense-related
proteins
in
higher
plants.
Annu
Rev
Biochem
59:
873-908
8.
Carpita
NC
(1982)
Limiting
diameters
of
pores
and
the
surface
structure
of
plant
cell
walls.
Science
218:
813-814
9.
Cassab
GI,
Varner
JE
(1989)
Immunocytolocalization
of
exten-
sin
in
developing
soybean
seed
coats
by
immunogold-silver
staining
by
tissue
printing
on
nitrocellulose
paper.
J
Cell
Biol
105:
2581-2588
10.
Chappell
J,
Hahlbrock
K,
Boller
T
(1984)
Rapid
induction
of
ethylene
biosynthesis
in
cultured
parsley
cells
by
fungal
elicitor
and
its
relationship
to
the
induction
of
phenylalanine
ammo-
nia-lyase.
Planta
161:
475-480
11.
Dean
JFD,
Anderson
JD
(1991)
The
ethylene
biosynthesis-in-
ducing
xylanase.
II.
Purification
and
physical
characterization
of
the
enzyme
produced
by
Trichoderma
viride.
Plant
Physiol
95:
316-323
12.
Dean
JFD,
Gamble
HR,
Anderson
JD
(1989)
The
ethylene
biosynthesis-inducing
xylanase:
its
induction
in
Trichoderma
viride
and
certain
plant
pathogens.
Phytopathology
79:
1071-1078
13.
Doares
SH,
Bucheli
P,
Albersheim
P,
Darvill
AG
(1989)
Host-
pathogen
interactions
XXXIV.
A
heat-labile
activity
secreted
by
a
fungal
phytopathogen
releases
fragments
of
plant
cell
wall
that
kill
plant
cells.
Mol
Plant-Microbe
Interact
2:
346-353
14.
Fuchs
Y,
Anderson
JD
(1987)
Purification
and
characterization
of
ethylene
inducing
proteins
from
Cellulysin.
Plant
Physiol
84:
732-736
15.
Fuchs
Y,
Saxena
A,
Gamble
HR,
Anderson
JD
(1989)
Ethylene
biosynthesis-inducing
protein
from
Cellulysin
is
an
endoxylan-
ase.
Plant
Physiol
89:
138-143
16.
Hahn
GH,
Bucheli
P,
Cervone
F,
Doares
SH,
O'Neill
RA,
Darvill
A,
Albersheim
P
(1989)
Role
of
cell
wall
constituents
in
plant-
pathogen
interactions.
In
T
Kosuge,
EW
Nester,
eds,
Plant-
Microbe
Interactions,
Vol
3.
McGraw
Hill,
New
York,
pp
131-181
17.
Lieberman
M,
Kunishi
AT,
Mapson
LW,
Wardale
DA
(1966)
Stimulation
of
ethylene
production
in
apple
tissue
slices
by
methionine.
Plant
Physiol
41:
376-382
18.
Lotan
T,
Fluhr
R
(1990)
Xylanase,
a
novel
elicitor
of
pathogen-
esis-related
proteins
in
tobacco,
uses
a
nonethylene
pathway
for
induction.
Plant
Physiol
93:
811-817
19.
Pegg
GF
(1976)
The
involvement
of
ethylene
in
plant
pathogen-
esis.
In
R
Heitfuss,
PH
Williams,
eds,
Encyclopedia
of
Plant
Physiology,
Vol
4.
Springer-Verlag,
New
York,
pp
582-591
20.
Ricci
P,
Bonnet
P,
Huet
J-C,
Sallantin
M,
Beauvais-Cante
F,
Bruneteau
M,
Billard
V,
Michel
G,
Pernollet
J-C
(1989)
Structure
and
activity
of
proteins
from
pathogenic
fungi
Phy-
tophthora
eliciting
necrosis
and
acquired
resistance
in
tobacco.
Eur
J
Biochem
183:
555-563
21.
van
Loon
LC
(1989)
Stress
proteins
in
infected
plant.
In
T
Kosuge,
EW
Nester,
eds,
Plant-Microbe
Interactions,
Vol
3.
McGraw
Hill,
New
York,
pp
198-239
1186
BAILEY
ET
AL.

Plant
Physiol.
(1991)
97,
1608
0032-0889/91/97/1
608/01/$01
.00/0
CORRECTION
Vol.
97,
1181-1186,
1991
Bryan
A.
Bailey,
Rosannah
Taylor,
Jeffrey
F.
D.
Dean,
and
James
D.
Anderson.
Ethylene
Biosynthesis-Inducing
En-
doxylanase
Is
Translocated
through
the
Xylem
of
Nicotiana
tabacum
cv
Xanthi
Plants.
An
error
occurred
in
the
printing
of
color
Figure
2
on
page
11
84.
The
correct
figure
and
its
legend
are
shown
below.
STEM
.4
PETIOLE
4.
*
.W
Figure
2.
Immunolocalization
of
EIX
protein
by
tissue
printing
techniques.
Tobacco
plants
were
dissected
20
min
after
EIX
application.
Cross-
sections
of
tobacco
stems
and
petioles
were
printed
onto
nitrocellulose
membranes
(12),
and
EIX
was
immunolocalized
with
polyclonal
anti-
bodies
raised
against
the
22-kD
EIX
polypeptide
(9).
Negative
and
positive
numbers
represent
leaves
below
and
above
the
point
of
EIX
appli-
cation,
respectively.
Stem
cross-sections
were
made
at
the
juncture
of
each
leaf
with
the
stem.
Experiments
were
repeated
four
times
and
con-
sistently
showed
EIX
to
be
spread
up
and
down
the
stem
more
than
five
leaves
and
in
the
petioles
of
leaves
-2,
-3,
-5,
+2,
+3,
and
+5
(not
all
shown).
Areas
of
cross-reactivity
with
the
EIX
antibodies
are
indicated
by
arrows.
Control
plants
treated
with
distilled
water
showed
no
cross-reactivity
under
these
conditions.
+3
0
7.
:5
*
S
N
I.~~~~~~~~~~~~%
-..
.W4
.1
-0
%-.
%1
%e
I4
It:
.4
SW'
'a.
1608
if-,
'v.
-..a
/,
I
,
6f
up
/
0
-4_e
-4
A".,
.
I
.
.
-*,
.4
1
41
.101
,I
#
.
V--,.
"0
-*.
....