Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride

Abstract

The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion.

Full text

Plant
Physiol.
(1991)
95,
316-323
0032-0889/91/95/031
6/08/$01
.00/0
Received
for
publication
July
19,
1990
Accepted
October
16,
1990
Ethylene
Biosynthesis-Inducing
Xylanase'
II.
Purification
and
Physical
Characterization
of
the
Enzyme
Produced
by
Trichoderma
viride
Jeffrey
F.
D.
Dean2
and
J.
D.
Anderson*
Plant
Hormone
Laboratory,
Beltsville
Agricultural
Research
Center,
Beltsville,
Maryland
20705
ABSTRACT
The
ethylene
biosynthesis-inducing
endoxylanase
(EIX)
from
xylan-induced
cultures
of
the
fungus,
Trichoderma
viride,
was
purified
to
near
homogeneity
and
compared
with
the
EIX
isolated
from
Cellulysin.
Both
enzymes
migrate
as
9.2
kilodalton
proteins
during
gel
filtration
chromatography
under
nondenaturing
condi-
tions,
but
the
mature
polypeptide
migrates
as
a
22
kilodalton
band
in
sodium
dodecyl
sulfate-polyacrylamide
gel
electropho-
resis.
The
amino
acid
composition
of
the
22
kilodalton
polypeptide
is
enriched
by
Gly,
Ser,
Thr,
Trp,
and
Tyr,
but
depleted
in
Ala,
Glx,
Leu,
and
Lys.
Both
proteins
lack
sulfur-containing
amino
acids.
The
protein
is
glycosylated,
and
inhibition
of
EIX
synthesis
by
tunicamycin
suggests
that
at
least
some
of
the
sugar
moieties
are
linked
to
asparagine
residues.
EIX
appears
to
be
synthesized
initially
as
a
25
kilodalton
precursor
protein
that
is
processed
to
22
kilodalton
during
secretion.
A
factor
limiting
our
understanding
of
plant
defense
re-
sponses
in
plant-pathogen
interactions
has
been
the
paucity
of
elicitors
which
have
been
purified
and
structurally
charac-
terized.
Ethylene
biosynthesis,
one
common
response
of
plant
tissues
to
pathogen
attack
(21),
is
stimulated
by
a
variety
of
plant-
and
pathogen-derived
polysaccharides
and
proteins
(4,
26,
28).
Ethylene
has
been
suggested
to
play
a
role
in
activating
certain
defense
responses
in
some
plant
tissues
(9).
This
laboratory
has
previously
demonstrated
that
an
endo-B-
1,4-
xylanase
(1,4-f3-D-xylan
xylanohydrolase;
EC
3.2.1.8)
purified
from
a
commercial
mixture
of
fungal
enzymes,
Cellulysin3,
is
a
potent
elicitor
of
ethylene
biosynthesis
in
tobacco
(Nicotiana
tabacum
L.
cv
Xanthi)
(
1,
12).
An
EIX4
with
similar
enzy-
'
This
work
was
supported,
in
part,
by
grant
1-1165-86
from
the
U.S.-Israel
Binational
Agricultural
Research
and
Development
Fund
and
by
U.S.
Department
of
Agriculture
Competitive
grant
No.
88-
37261-3680
to
J.
D.
A.
and
J.
F.
D.
D.
2
Current
address:
Center
for
Biological
Resource
Recovery,
De-
partment
of
Biochemistry,
University
of
Georgia,
Athens,
GA
30605.
3
The
mention
of
specific
instruments,
trade
names,
or
manufac-
turers
is
for
the
purpose
of
identification
and
does
not
imply
any
endorsement
by
the
United
States
government.
'
Abbreviations:
EIX,
ethylene
biosynthesis-inducing
endoxylan-
ase;
Tween-20,
polyoxyethylene
(20)
sorbitan
monolaurate;
22
kD-
AB,
antibody
raised
against
the
22
kD
polypeptide
isolated
by
SDS-
PAGE
from
Cellulysin
EIX.
matic,
biological,
and
immunological
characteristics
was
pro-
duced
in
xylan-grown
cultures
of
the
saprophytic
fungus,
Trichoderma
viride,
the
organism
from
which
Cellulysin
is
obtained
(8).
Proteins
with
similar
enzymatic
and
biological
activities
and
immunological
characteristics
are
also
produced
by
at
least
two
pathogenic
fungi,
Fusarium
oxysporum
f.
sp.
pisi
and
Macrophomina
phaseolina
(8).
Subsequent
work
has
shown
that
the
enzyme
from
T.
viride
also
elicits
other
defense
responses
in
tobacco,
including
synthesis
of
pathogenesis-
related
proteins
(18),
tissue
necrosis,
and
electrolyte
leakage
(3).
Other
fungal
endoxylanases
have
been
shown
to
elicit
defense
responses,
such
as
phytoalexin
production
(10),
lipid
peroxidation
(13),
vascular
gelling
(29),
and
cell
death
(5),
in
various
plant
tissues.
However,
with
the
exception
of
the
enzyme
from
Phytophthora
parasitica
f.
sp.
nicotiana
(10),
structural
characterization
of
these
xylanolytic
elicitors
has
been
limited.
The
present
paper
describes
the
purification
to
near
ho-
mogeneity
of
the
EIX
protein
from
the
culture
medium
of
T.
viride.
The
electrophoretic
and
chromatographic
properties
of
the
enzyme
under
denaturing
and
non-denaturing
condi-
tions
are
compared
with
those
of
the
enzyme
isolated
from
the
commercial
T.
viride
enzyme
preparation,
Cellulysin.
The
amino
acid
and
carbohydrate
compositions
of
the
two
EIX
proteins
are
compared,
and
the
effect
of
tunicamycin
on
the
synthesis
of
EIX
by
T.
viride
is
examined.
Data
are
presented
to
suggest
that
the
secreted
EIX
is
initially
synthesized
as
a
larger
precursor
protein.
MATERIALS
AND
METHODS
Biologicals
and
Reagents
Trichoderma
viride
T-
1
(ATCC
52438)
was
originally
ob-
tained
from
G.
C.
Papavizas.
CM-Sepharose
and
mol
wt
markers
for
gel
permeation
chromatography
were
obtained
from
Pharmacia
(Piscataway,
NJ).
Low
mol
wt
protein
mark-
ers
for
SDS-PAGE
were
from
BRL
(Gaithersburg,
MD);
14C-
methylated
SDS-PAGE
markers
were
prepared
by
Amersham
(Arlington
Heights,
IL).
Birchwood
xylan
was
obtained
from
Atomergic
Chemetals
Corp.
(Farmingdale,
NY).
Cellulysin,
Pansorbin,
and
tunicamycin
are
products
of
Calbiochem
(San
Diego,
CA).
All
other
reagents
were
obtained
at
the
highest
purity
available
and
used
without
further
purification.
316

PURIFICATION
OF
AN
ETHYLENE
INDUCING
XYLANASE
Cellulysin
CM-Sepharose
3.000
2.500
12.000
;t-
1.500-
L-0
o
0
X
1.000-
cs
0
>%
0.500
x
Elution
Volume
(mL)
500
1000
Elution
Volume
(mL)
2.500
3,
2.000
>%
5.
;t
0
,.,
1.500
°
U)
1.000
cx
0
>3
0.500
Cellulysin
Phenyl-Superose
0.0008'
i
-
L
-
.
-
---.0o
0
20
40
60
80
100
Elution
Volume
(mL)
3
Trichoderma
Phenyl-Superose
-
20
40
60
Elution
Volume
(mL)
80
IC
30
2.000
-25>
.5
.1.500
20-t
CM
-15C
c
1
.000
._
10-0
-
0.500
5
0
.0
_
-C
I
0.000
25>
'D.
20'w
15c
._
:3
10-0
SC
0
5
0
C
wnIi
Figure
1.
Column
chromatographic
profiles
of
the
purification
of
EIX
from
Cellulysin
and
T.
viride
cultures.
Xylanase
activity
(Amol
min-'
mL-'),
protein
(mg
mL-1),
ethylene
biosynthesis-inducing
activity
(uL
C2H4
h-'
g-'fresh
weight
mL-1),
and
buffer
molarity
(M)
contained
in
each
fraction
are
plotted
against
the
elution
volume
for
Cellulysin
(top
panels)
and
T.
viride
(bottom
panels)
ultrafiltrates.
Fraction
sizes
were
20
mL
for
the
CM-Sepharose
columns
(left)
and
1
mL
for
the
phenyl-Superose
columns
(right).
Protein
was
monitored
at
A280.
Assays
Protein,
xylanase
activity
(8),
and
ethylene
biosynthesis-
inducing
activity
(1
1)
were
assayed
as
described
previously.
Ethylene
biosynthesis-inducing
activity
is
expressed
as
,uL
C2H4
evolved
per
h
per
g
fresh
weight
of
tobacco
tissue.
Units
of
endoxylanase
activity
are
expressed
in
micromoles
of
re-
ducing
sugar
equivalents
released
from
birchwood
xylan
per
min
at
pH
5.0
and
50°C.
Purification
of
the
Ethylene
Biosynthesis-Inducing
Xylanase
T.
viride
was
cultured
for
3
d
in
a
minimal
salts
medium
(8)
containing
0.1%
(w/v)
glucose
prior
to
addition
of
birch-
wood
xylan
to
0.1%
(w/v).
After
2
more
d
of
incubation
with
xylan,
culture
medium
containing
EIX
was
separated
from
bulk
mycelia
by
coarse
filtration
through
a
glass
fiber
filter
(CF
fraction).
When
Cellulysin
was
used
as
source
material
for
EIX
preparation,
a
1%
(w/v)
solution
of
Cellulysin
in
water
was,
likewise,
initially
treated
with
a
coarse
filtration
step.
Tangential-Flow
Ultrafiltration
Material
contained
in
the
CF
fraction
was
subjected
to
two
rounds
of
ultrafiltration
in
a
tangential-flow
apparatus
(Phar-
macia).
Initial
passage
through
a
30,000
mol
wt
cutoff
mem-
brane
cassette
separated
EIX
and
other
low-mol
wt
molecules
from
the
remaining
mycelia
and
larger
extracellular
proteins.
Subsequently,
proteins
in
this
filtrate
fraction
were
concen-
trated
about
10-fold
by
ultrafiltration
against
a
1,000
mol
wt
cutoff
membrane
cassette
(UF
fraction).
During
this
concen-
tration
step,
EIX
was
exchanged
into
the
loading
buffer
(20
mM
ammonium
acetate
[pH
5.0])
for
the
next
purification
step.
CM-Sepharose
Column
Chromatography
The
UF
protein
fraction
was
loaded
onto
a
CM-Sepharose
column
(2.6
x
90
cm)
connected
to
an
FPLC
system
and
washed
with
250
mL
of
loading
buffer
at
a
flow
rate
of
2
mL/
min.
Bound
proteins
were
eluted
from
the
column
with
a
linear
gradient
of
20
to
200
mM
ammonium
acetate
(pH
5.0)
(0.18
mM/mL).
Protein
was
monitored
at
A280.
Phenyl-Superose
Column
Chromatography
Fractions
from
the
CM-Sepharose
column
containing
the
major
peak
of
ethylene
biosynthesis-inducing
activity
(Fig.
1)
were
pooled
(CM
fraction)
and
made
to
2.0
M
by
slow
addition
of
finely
ground
(NH4)2SO4.
The
resultant
solution
was
passed
through
a
0.45
Ag
filter
and
loaded
onto
a
phenyl-Superose
FPLC
column
(HR
5/5,
Pharmacia)
equilibrated
with
2.0
M
(NH4)2SO4
in
2
mm
ammonium
acetate
(pH
5.0).
The
column
was
washed
with
equilibration
buffer,
and
bound
proteins
C
0
a.'
:t
x
.2_
5)
CO
0
c
C
0
0
0
>>
A\
c
0
L-
I
T
2.000
1.5001
1.000
.,
L-
0
0.500
>
U.QULU
_
_
_
-L
317
0.000
uV
iO
i

Plant
Physiol.
Vol.
95,
1991
were
eluted
with
a
biphasic
gradient
of
(NH4)2SO4
in
2
mM
ammonium
acetate
that
was
linear
from
2.0
to
1.0
M
(0.2
M/
mL)
and
1.0
to
0.0
M
(0.025
M/mL).
The
flow
rate
was
0.3
mL/min.
Column
fractions
containing
the
majority
of
ethyl-
ene
biosynthesis-inducing
activity
(Fig.
1)
were
pooled
(PS
fraction)
and
stored
at
-20°C
for
more
than
6
months
without
showing
appreciable
loss
of
activity.
SDS-PAGE,
Immunoblotting,
and
Fluorography
Protein
purification
was
monitored
by
SDS-PAGE
using
the
Tris-Tricine
buffer
system
of
Schagger
and
von
Jagow
(22).
Routine
analysis
was
performed
in
gels
with
10%
total
acrylamide
and
bisacrylamide,
3%
cross-linker
(bisacrylam-
ide)
in
relation
to
the
total
concentration
of
acrylamide.
However,
for
molecular
mass
determinations
of
EIX
polypep-
tides,
gels
with
16.5%
total
acrylamide,
6%
cross-linker
were
used.
Silver
staining
and
immunoblotting
of
the
resolved
proteins
were
performed
as
previously
described
(8).
Gels
containing
[3H]leucine-labeled
proteins
were
fixed
by
exten-
sive
washing
wth
50%
methanol
and
impregnated
with
EnHance
(NEN
Research
Products).
The
gels
were
dried
and
exposed
to
X-Omat
x-ray
film
at
-80°C.
Amino
Acid
Analysis,
Tunicamycin
Inhibition,
and
Carbohydrate
Composition
Analysis
Amino
acid
analyses
of
the
purified
EIX
proteins
were
performed
by
Mark
Hermodson
at
the
Purdue
University
Macromolecular
Analysis
Facility.
The
tryptophan
content
of
EIX
was
estimated
from
the
A288/A280
ratio
under
denaturing
and
nondenaturing
conditions
(1).
The
effect
of
tunicamycin
on
EIX
secretion
and
synthesis
was
studied
by
addition
of
the
antibiotic
to
cultures
of
T.
viride
grown
for
3
d
in
glucose
medium
and
induced
by
addition
of
xylan
to
0.1%
(w/v).
Tunicamycin
from
a
stock
solution
(100
mg/mL
in
ethanol)
was
added
to
each
culture
to
a
final
concentration
of
5
Ag/mL
at
2
or
4
h
after
addition
of
xylan.
Secretion
of
mature
EIX
was
monitored
by
SDS-
PAGE
of
proteins
precipitated
with
TCA
from
culture
super-
natants
removed
at
the
indicated
intervals
after
xylan
addi-
tion.
The
EIX
precursor
protein
was
extracted
from
fungal
mycelia
by
grinding
directly
in
SDS-PAGE
sample
buffer
and
subsequently
analyzed
by
SDS-PAGE.
Inhibition
of
EIX
syn-
thesis
was
determined
by
immunoblotting
and
fluorography
of
proteins
in
culture
supernatants
and
mycelial
extracts
after
addition
of
100
,tCi
[3H]leucine
to
tunicamycin-treated,
xy-
lan-induced
cultures.
For
carbohydrate
composition
analysis,
protein
(1
mg)
in
the
SP
fraction
of
each
EIX
preparation
were
precipitated
with
an
equal
volume
of
ice-cold
20%
TCA
and
subsequently
washed
several
times
with
cold
10%
TCA
followed
by
cold
acetone.
Sugars
were
released
from
the
protein
by
heating
in
a
sealed
vial
with
4.0
N
TFA
at
100°C
for
5
h.
Allose
(20
,ug/
mL)
was
added
to
the
TFA
prior
to
hydrolysis
to
provide
an
internal
standard.
The
released
sugars
were
identified
and
quantified
as
their
alditol
acetates
according
to
Tong
and
Gross
(27).
Analyses
were
performed
in
duplicate
on
EIX
isolated
from
each
source
on
two
separate
occasions
(four
analyses
of
EIX
from
each
source).
In
Vitro
Translation
and
Immunoprecipitation
Eight
hours
after
EIX
induction
by
addition
of
xylan,
total
RNA
was
isolated
from
T.
viride
by
acid
guanidinium
thio-
cyanate-phenol
extraction
(7).
Without
further
purification,
this
RNA
(5
/Lg)
was
translated
in
a
wheat
germ
translation
system
(Promega,
Madison,
WI)
containing
[3H]leucine.
After
90
min,
the
reaction
mixture
containing
T.
viride
RNA
and
a
control
reaction
containing
no
RNA
were
mixed
with
10
,uL
of
Pansorbin
for
1
h
at
room
temperature.
Labeled
proteins
that
bound
to
Pansorbin
were
removed
by
centrifugation.
Polyclonal
antibodies
(10
,ug)
raised
against
the
native
EIX
isolated
from
Cellulysin
(12)
were
added
to
these
superna-
tants,
and
the
mixtures
were
incubated
overnight
at
4°C.
Complexes
of
EIX-specific
antibodies
and
cross-reactive
pro-
teins
were
precipitated
by
a
second
treatment
with
10
,L
Pansorbin
followed
by
a
1-h
incubation
at
room
temperature
Table
I.
Purification
of
the
Ethylene
Biosynthesis-Inducing
Xylanases
Total
Total
Total
Specific
Enrich-
Total
Yield
Enrich-
Fraction
Volume
Protein
Xylanase
Yield
Xylanase
Inducing
Yield
Inducing
ment
Activity
Activity
Activity
mL
mg
units
%
units/mg
-fold
gLC2H4/h/g
%
pLC2H4/h/g/mg
-fold
Cellulysin
(10
mg/
200
276
1970
100
7.1
1.0
12442
100
45
1.0
mL)
Ultrafiltrate
225
29.3
1616
82
55.2
7.8
13104
105
450
10.0
Carboxymethyl
100
10.0
990
50
99.0
13.9
8803
71
880
19.6
sepharose
Phenyl
superose
16
3.4
898
46
267.1
37.6
6584
53
1960
43.6
(9.8)a
(96.7)
(710)
T.
viride
medium
3000
120
2280
100
19.0
1.0
9900
100
82
1.0
Ultrafiltrate
240
125
1452 64
11.6
0.6
7354
74
59
0.7
Carboxymethyl
120
8.4
1390
61
165.4
8.7
7106
72
850
10.2
sepharose
Phenyl
superose
15
3.3
1455
64
440.9
23.2
7651
77
2320
28.0
(20.3)
(71.9)
(378)
a
Parenthetical
values
represent
EIX
protein
as
calculated
from
A280
measurements
of
the
final
purification
pools.
318
DEAN
AND
ANDERSON

PURIFICATION
OF
AN
ETHYLENE
INDUCING
XYLANASE
Ceilulysin
Trichoderma
M,
x10
M
Mr
-
432
-
'
-
25.7
-
_
184A
143
-
25.7
-*-
-184
-
I'
-
~~~~142-
-
62
Figure
2.
SDS-PAGE
and
immunoblots
of
protein
purification
pools.
Aliquots
containing
amounts
of
ethylene
biosynthesis-inducing
activ-
ity
equal
to
that
contained
in
the
PS
sample
were
taken
from
the
crude
filtrate
(CF),
ultrafiltrate
(UF),
CM-Sepharose
(CM),
and
phenyl-
Superose
(PS)
pools
of
Cellulysin
(left)
or
T.
viride
(right).
EIX
and
TCA-precipitable
material
in
each
sample
was
subjected
to
SDS-
PAGE.
Resolved
proteins
were
either
silver
stained
(A)
or
transferred
to
PVDF
membranes
and
probed
with
antibodies
(B).
In
each
case,
the
PS
lane
contained
250
ng
of
protein
as
determined
by
A280-
and
subsequent
centrifugation.
The
precipitates
were
resus-
pended
several
times
in
PBS
containing
0.05%
Tween-20,
and
labeled
proteins
were
released
from
the
Pansorbin
by
boiling
for
5
min
in
10
,uL
of
SDS-PAGE
sample
buffer
(8).
The
immunoprecipitates
were
analyzed
by
fluorography.
RESULTS
Purification
of
EIX
from
Trichoderma
viride
Cultures
and
Cellulyisn
The
ethylene
biosynthesis-inducing
and
endoxylanase
ac-
tivities
were
purified
28.0-
and
23.2-fold,
respectively,
from
xylan-induced
cultures
of
T.
viride
by
a
combination
of
ultrafiltration,
cation-exchange
chromatography,
and
hydro-
phobic
interaction
chromatography
(Table
I).
A
similar
series
of
steps
yielded
a
43.6-
and
37.6-fold
purification
of
the
same
activities,
respectively,
from
the
commercial
T.
viride
enzyme
mixture,
Cellulysin
(Table
I).
Analyses
by
SDS-PAGE
and
immunoblotting
revealed
that
the
final
pool
(PS
fraction)
of
T
viride
EIX
contained
a
predominate
polypeptide
that
cross-
reacted
with
antibodies
originally
raised
against
the
SDS-
PAGE
purified
22
kD
EIX
polypeptide
from
Cellulysin
(22
kD-AB)
as
well
as
a
minor
one
at
14
kD
(Fig.
2).
The
PS
pool
from
the
purification
of
Cellulysin
EIX
contains
three
poly-
peptides
having
electrophoretic
mobilities
and
immunological
characteristics
identical
to
the
three
polypeptides
previously
demonstrated
in
preparations
of
EIX
isolated
from
Cellulysin
by
a
different
protocol
(8,
1
1).
Size
and
Composition
of
EIX
Polypeptides
Samples
of
the
purified
Cellulysin
and
T.
viride
EIXs
were
subjected
to
gel
filtration
chromatography
on
a
calibrated
superose
12
FPLC
column.
The
apparent
molecular
mass
of
both
EIXs
under
nondenaturing
conditions
was
9.2
±
0.4
kD
(Fig.
3A).
The
mobility
of
EIX
on
this
chromatography
column
was
unaffected
by
increasing
the
ionic
strength
from
200
to
500
mm
or
adding
nonionic
detergent
(0.05%
v/v
Tween-20).
Only
a
single
peak
of
protein
was
found
for
each
EIX,
even
though
the
Cellulysin
EIX
peak
was
demonstrated
to
contain
three
polypeptides
by
SDS-PAGE.
Under
the
de-
naturing
conditions
of
SDS-PAGE,
both
preparations
con-
tained
a
polypeptide
with
an
apparent
molecular
mass
of
21.5
kD
(Fig.
3B).
In
addition,
the
T.
viride
EIX
preparation
contained
a
minor
band
of
42
kD,
and
the
Cellulysin
EIX
preparation
contained
two
smaller
polypeptides
with
apparent
molecular
masses
of
14.5
and
5.4
kD,
which
constitute
puta-
tive
degradation
products
of
EIX.
Amino
acid
analyses
of
the
two
EIX
preparations
demon-
strated
the
similarities
in
composition
between
the
two
en-
zymes
and
a
third
endoxylanase
purified
from
Trichoderma
harzianum
(Table
II)
(30).
EIX
lacks
sulfur-containing
amino
acids
(Cys
and
Met)
and
has
reduced
levels
of
Ala,
Glx,
Leu,
and
Lys,
but
elevated
levels
of
Gly,
Ser,
Thr,
Trp,
and
Tyr
in
comparison
with
the
average
amino
acid
composition
of
A
1ooooo-n-
T
o
S
t
(I)
1000
+
tt
5000L
---
13
B
i
-
O
Ov
Ribor
14
,albumin
-0
Chymotrypsinogen
A
uclease
A-O.|
"-EIX
Bovine
Trypsin
Inhibitor
-
_
_
_4
_
1
15
16
17
Elution
Volume
(mL)
-
oa-Chymotrypsinogen
+21.5
kD
*
l-Lactoglobulin
C]
14.5
kD
-
L
Lysozyme
(0
10000i
(I,
k-O_i~
> +
5.4
kD
_°>
s
YPInhib
Insulin-
0
1000
I+
X
0
1
0
20
30
40
Migration
Distance
(mm)
)itor
50
Figure
3.
Molecular
mass
determinations
of
native
and
denatured
EIX
polypeptides.
Samples
of
purified
EIX
(filled
circle)
from
Cellulysin
or
T.
viride
were
applied
to
a
Superose
12
(Pharmacia)
FPLC
column
calibrated
with
the
indicated
standards
(open
circles)
in
200
mm
ammonium
acetate
(pH
5.0)
buffer
at
a
flow
rate
of
0.3
mL/min
(panel
A).
Panel
B
depicts
the
apparent
molecular
masses
of
the
EIX
polypeptides
(filled
circles)
compared
with
the
indicated
standards
(open
circles)
under
the
denaturing
conditions
of
SDS-PAGE.
319
I
I'm

Plant
Physiol.
Vol.
95,
1991
Table
II.
Comparison
of
the
Amino
Acid
Percent
Composition
of
the
Ethylene
Biosynthesis-Inducing
Xylanases
Isolated
from
Cellulysin
and
Trichoderma
viride
with
a
Xylanase
from
T.
harzianum
and
total
proteins
from
E.
coli
Amino
Acid
Cellulysin
T.
viride
T.
harzianuma
E.
CO/ib
Alanine
3.8
3.9
4.6
13.0
Arginine
3.6
3.7
3.5
5.3
Asp/Asn
12.1
14.3
11.6
9.9
Cysteine
0
0
0
1.8
Glu/Gln
4.9
5.0
5.2
10.8
Glycine
14.7
14.6
13.9
7.8
Histidine
1.3
1.8
2.3
0.7
Isoleucine
5.7
4.2
5.2
4.4
Leucine
4.7 3.8
2.9
7.8
Lysine
1.4
1.6
2.3
7.0
Methionine
0
0
0.6
3.8
Phenylalanine
4.0
4.3
4.0
3.3
Proline
3.4
3.3
3.5
4.6
Serine
10.6
11.8
11.6
6.0
Threonine
6.5
7.7
9.2
4.6
Tryptophan
3.5c
3.9c
3.5
1.0
Tyrosine
7.8
8.7
9.2
2.2
Valine
6.2
7.2
6.9
6.0
a
From
ref.
30.
b
From
ref.
17.
c
Tryptophan
determined
from
the
A288/A280
ratio
(1).
Escherichia
coli
total
protein
hydrolysates
(
17).
Quantitation
of
amino
acids
in
the
protein
hydrolysates
yielded
Em.1
[A280]
values
of
1.0
and
1.2
x
105
L
mol'
cm-'
for
the
Cellulysin
and
T.
viride
EIXs,
respectively.
Neither
EIX
preparation
contained
polypeptides
whose
amino
termini
were
available
for
sequencing
by
automated
Edman
degradation.
Carbohydrates
Associated
with
EIX
When
xylan-induced
cultures
of
T.
viride
were
treated
with
tunicamycin
(5
Ag/mL),
synthesis
of
EIX
in
mycelia
and
secretion
of
EIX
into
the
culture
medium
immediately
ceased
(Fig.
4,
panels
A,
B).
Fungal
growth,
as
measured
by
increased
mycelial
weight,
did
not
slow
significantly
until
8
to
10
h
after
addition
of
tunicamycin.
Extracts
of
xylan-induced
T.
viride
mycelia
contained,
in
addition
to
the
22
kD
EIX
protein,
a
25
kD
polypeptide
that
cross-reacted
on
immunoblots
with
the
22
kD-AB
(Fig.
4C).
The
synthesis
of
this
25
kD
cross-
reactive
polypeptide
also
was
abruptly
inhibited
by
the
addi-
tion
of
tunicamycin
to
the
culture
medium.
Xylan-induced
fungal
cultures
treated
with
tunicamycin,
and
subsequently
labeled
with
[3H]leucine,
had
a
slight
reduction
of
label
incor-
poration
into
total
proteins
secreted
into
the
medium
or
extracted
from
mycelia
when
compared
to
xylan-induced
controls
(Fig.
5),
but
incorporation
specifically
into
EIX
was
drastically
curtailed.
These
results
suggested
that
EIX
contains
Asn-linked
sugar
moieties.
GC-MS
analyses
of
TFA-hydrol-
ysates
of
the
two
EIXs
revealed
that
both
enzymes
contain
small
(<1%
by
weight)
amounts
of
Gal,
GlcN,
Glc,
and
Man
(Table
III).
EIX
prepared
from
Cellulysin
consistently
dem-
onstrated
significantly
higher
levels
of
GlcN
than
EIX
isolated
from
the
fungal
cultures.
In
Vitro
Translation
of
RNA
Isolated
from
Xylan-lnduced
T.
viride
Total
RNA
isolated
8
h
after
addition
of
xylan
to
T.
viride
cultures
was
translated
in
a
wheat
germ
lysate
system,
and
translation
products
labeled
with
[3H]leucine
were
immuno-
precipitated
with
antibodies
raised
against
the
native
EIX
enzyme
purified
from
Cellulysin.
Fluorography
of
the
im-
munoprecipitated
polypeptides
resolved
by
SDS-PAGE
and
transferred
to
a
PVDF
blotting
membrane
showed
one
major
band
at
about
38
kD
and
a
strong
doublet
at
22
and
21
kD
(Fig.
6).
Faint
bands
are
also
apparent
at
25
and
18
kD.
DISCUSSION
Endoxylanases
have
previously
been
isolated
from
com-
mercial
preparations
of
T.
viride
enzymes
(12,
15,
23)
as
well
as
the
culture
medium
of
several
other
Trichoderma
species
(2,
25,
32).
The
overall
yields
and
specific
activities
provided
by
our
protocol
compare
favorably
with
those
previous
meth-
ods.
The
amino
acid
composition
and
other
physical
charac-
teristics
of
the
two
EIX
proteins
are
very
similar
to
those
of
some
xylanases
previously
isolated
from
bacteria,
yeast,
and
other
fungi
(16,
31).
The
apparent
size
of
EIX
(9.2
kD)
fits
with
the
ability
of
a
similar
xylanase
from
T.
harzianum
to
pass
through
a
10,000
mol-wt-cutoff
ultrafiltration
membrane
(30).
Although
EIX
and
similar
xylanases
have
a
demonstrated
propensity
to
bind
to
dextran-based,
gel
filtration
media
at
salt
concentrations
as
high
as
150
mm
(2,
11,
23,
32),
EIX
mobility
remained
maximal
on
Superose
12
at
salt
concentrations
above
200
mm.
The
anomalous
binding
of
EIX
and
other
xylanases
at
lower
salt
concentrations
is
similar
to
that
described
for
the
interaction
between
Sephadex
and
peptides
containing
high
proportions
of
tyrosine
(
14);
thus,
the
elevated
levels
of
tyro-
320
DEAN
AND
ANDERSON

PURIFICATION
OF
AN
ETHYLENE
INDUCING
XYLANASE
Cly
+TunicamyCin
-Tunicamycln
EIX
0h
2h
4h
8h
14h
24h
4h
8h
14h
24h
Mr)10
to
43
2
-_
~215
-14A
I
I
I
I
I
I
I
I
I
,
-t
4
.
-'
-
I
-43.0
-
312
_________
215
*
.
;;
..ff-
144
I1
I
I
I
I
I
Figure
4.
Inhibition
of
EIX
secretion
by
tunicamycin.
Cultures
of
T.
viride
induced
with
xylan
for
2
h
were
incubated
with
(+)
or
without
(-)
tunicamycin.
Aliquots
(25
ML)
of
culture
medium
were
removed
at
various
times
(0,
2,
4,
8,
14,
and
24
h),
and
mycelia
were
removed
by
centrifugation.
TCA-precipitable
material
was
resolved
by
SDS-
PAGE
and
analyzed
by
silver
staining
(A)
or
immunoblotting
(B).
Proteins
in
mycelial
extracts
were
also
analyzed
by
immunoblotting
(C).
Purified
Cellylysin
EIX,
Cly
EIX,
was
used
as
a
marker.
SUPE
+
MYCL
MrX1O
SLPE
MYOL
+
-
+
-
20-
-
97A
-
-
fee
-
430-
-
26.7
-
j_
18A
143
-
Figure
5.
Tunicamycin
inhibition
of
[3H]leucine
incorporation
into
EIX.
Cultures
of
T.
viride
induced
with
xylan
for
4
h
were
incubated
with
(+)
or
without
(-)
tunicamycin
for
a
further
2
h
before
addition
of
[3H]
leucine.
Cultures
were
harvested
4
h
after
addition
of
[3H]leucine.
Proteins
(5
,g)
precipitated
with
TCA
from
the
culture
medium
(supe)
or
mycelial
extracts
(mycl)
were
resolved
by
SDS-PAGE.
The
resolved
proteins
were
visualized
by
fluorography
(A)
or
immunoblotting
(B).
Table
l1l.
Comparison
of
the
Carbohydrate
(CHO)
Composition
of
the
Ethylene
Biosynthesis-Inducing
Xylanases
(EIX)
Isolated
from
Cellulysin
and
T.
viride
Cellulysin
T.
viride
ug
CHOI
mol
%
gg
CHO/
mol
%
mg
EIX
CHO
mg
EIX
CHO
Galactose
1.0
±
0.1
13
1.5
±
0.2
26
Glucosamine
3.8
±
0.3
41
0.6
±
0.2
8
Glucose
2.2
±
0.9
29
2.0
±
0.1
35
Mannose
1.2
±
0.5
16
1.7
±
0.7
30
Total
EIX
Carbohydrate
0.8%
CHO
0.6%
CHO
Content
(w/w)
sine
in
EIX
(Table
II)
may
be
responsible
for
the
affinity
of
the
enzyme
for
dextran-based
matrices
under
these
conditions.
The
size
of
EIX
we
report
in
this
study
is
quite
different
from
that
reported
previously
(1
1).
In
the
earlier
study,
the
purified
protein
was
concentrated
by
lyophylization,
a
process
which
we
have
subsequently
found
to
cause
solubility
and
aggregation
problems.
The
apparent
size
of
native
EIX
may
prove
very
important
to
the
mechanism
whereby
the
enzyme
induces
ethylene
biosynthesis
in
plant
tissues.
A
9.2
kD
EIX
protein
would
be
capable
of
penetrating
through
even
the
smallest
plant
cell
wall
pores
(6)
to
interact
directly
with
the
plasmalemma.
We
have
been
unable
to
demonstrate
that
the
xylanase
component
of
EIX
releases
any
oligosaccharide
frag-
ments
harboring
heat-stable,
ethylene
biosynthesis-inducing
activity
from
purified
xylans,
or
from
isolated
or
in
situ
plant
cell
walls.
The
localization
of
plant
responses
only
to
those
areas
actually
containing
the
EIX
protein
(3,
18)
suggests
that
the
biological
activity
of
EIX
is
either
carried
by
a
labile
cell
wall
fragment of
limited
mobility
or
is
derived
from
direct
recognition
of
the
EIX
protein
by
the
plant
cell.
Glycosylation
is
thought
to
be
responsible,
in
part,
for
the
multiplicity
of
xylanases
produced
by
a
variety
of
microor-
ganisms
(31).
In
contrast
to
a
Cryptococcus
albidus
xylanase
whose
secretion
is
speeded
by
addition
of
tunicamycin
(19),
EIX
secretion
and
synthesis
are
rapidly
and
specifically
shut
off
when
glycosylation
in
T.
viride
is
blocked
with
tunicamy-
cin.
We
have
found
that
native
EIX
purified
from
T.
viride
cultures
by
the
current
protocol
may
retain
up
to
50%
by
weight
carbohydrate
(data
not
shown),
but
precipitation
and
extensive
washing
of
EIX
with
TCA
prior
to
carbohydrate
composition
analysis
appears
sufficient
to
remove
noncova-
lently
bound
carbohydrate.
Xylanases
have
previously
been
shown
capable
of
retaining
substantial
amounts
of
polysac-
charide
during
nondenaturing
purification
procedures
(20).
Stuttgen
and
Sahm
(24)
demonstrated
such
carbohydrate
binding
by
an
endoxylanase
from
Trichosporon
cutaneum
but
subsequently
declared
that
the
enzyme
was
not
glycosylated,
since
the
total
carbohydrate
content
after
denaturation
and
gel
filtration
in
SDS
was
only
about
1%
by
weight.
Our
data
show
a
carbohydrate
content
of
<1%
for
denatured
EIX,
but
this
level
of
sugar
is
very
reproducible,
and
it
is
difflcult
to
claim
that
the
protein
is
not
glycosylated
given
that
tunica-
mycin
specifically
inhibits
EIX
synthesis
by
the
fungus.
B
C
321
"I-,
---
-4.
"r
-
-
21,5

Plant
Physiol.
Vol.
95,
1991
a-
Wl
Li
'1.2
M
rX
9-
460
-143_
Figure
6.
Immunoprecipitation
of
in
vitro
translation
products
of
RNA
isolated
from
xylan-induced
T.
viride.
Total
RNA
isolated
from
mycelia
of
T.
viride
8
h
after
addition
of
xylan
was
translated
in
a
wheat
germ
lysate
containing
[3H]leucine.
The
immunoprecipitated,
labeled
pro-
teins
(PPT)
resolved
by
SDS-PAGE
were
visualized
by
fluorography
and
compared
with
an
EIX
standard
labeled
with
[3H]tyrosine
(EIX)
and
[14C]methylated-protein
molecular
weight
markers
(MW).
The
25
kD
cross-reactive
polypeptide
detected
on
immu-
noblots
of
mycelial
extracts
from
xylan-induced
T.
viride
(Fig.
4C)
most
likely
represents
a
precursor
form
of
EIX
containing
an
N-terminal
transit
peptide
sequence
that
is
removed
during
secretion.
Such
signal
peptides
have
been
identified
in
xylan-
ases
from
bacteria
and
yeast
(3
1).
Such
a
precursor
form
of
EIX
would
be
expected
as
a
primary
reaction
product
of
the
in
vitro
translation
of
RNA
from
the
xylan-induced
fungus.
The
fluorograph
of
radiolabeled
polypeptides
immunoprecip-
itated
from
the
in
vitro
translation
mixtures
shows
only
a
very
faint
band
at
25
kD
but
a
strong
doublet
at
about
22
If
the
25
kD
polypeptide
detected
in
mycelial
extracts
is
actually
the
EIX
precursor,
then
this
precursor
either
must
be
effi-
ciently
processed
to
the
mature
22
kD
form
in
the
wheat
germ
lysate
system
or
it
is
not
well
recognized
by
the
antibodies
raised
against
the
native
form
of
the
Cellulysin
BIX
that
were
used
for
immunoprecipitation.
The
identity
of
the
38
kD
radiolabeled
peptide
immunoprecipitated
from
the
transla-
tion
reactions
is
unknown.
It
may
be
another
T.
viride
protein
which
resembles
EIX
in
its
native
structure,
but,
as
it
could
not
be
detected
on
immunoblots
of
mycelial
extracts,
it
must
not
resemble
EIX
in
its
denatured
state.
Alternatively,
it
may
be
an
unrelated
fungal
protein
that
becomes
tightly
associated
with
EIX
and
is
consequently
coprecipitated
under
the
non-
denaturing
conditions
used
for
immunoprecipitation.
Previous
work
in
this
laboratory
demonstrated
that
EIX
acts
to
induce
the
ethylene
biosynthetic
pathway
through
the
regulatory
enzyme,
-aminocyclopropane-
-carboxylic
acid
synthase
(3).
Other
work
with
purified
EIX
has
demonstrated
its
effectiveness
in
stimulating
the
synthesis
of
pathogenesis-
related
proteins
(
18)
and
causing
tissue
necrosis
(3).
We
hope
that
the
combination
of
a
well-characterized
host
plant
and
an
easily
purified
protein
elicitor
will
facilitate
study
of
not
only
ethylene
biosynthesis,
but
also
other
responses
associated
with
pathologic
states
in
plants.
ACKNOWLEDGMENTS
The
investigators
wish
to
thank
Dr.
Mark
Hermodson,
Biochem-
istry
Dept.,
Purdue
University,
Dr.
Kenneth
Gross,
Horticultural
Crops
Quality
Laboratory,
USDA/ARS,
and
Mr.
Dave
Clark
for
their
help
in
analyzing
amino
acid
and
glycosyl
compositions,
and
photo-
graphic
reproduction,
respectively.
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