FEMS Microbiology Letters 114 (1993) 85-92
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
85
FEMSLE 05693
Evidence of two polygalacturonases produced
by a mycorrhizal ericoid fungus during
its saprophytic growth
R e n a t o P e r e t t o a, Vittorio Bettini b and Paola Bonfante .,a
a Dipartimento di Biologia Vegetale, Uni~,ersith di Torino, Torino, Italy and b Centro di Studio sulla Micologia del Terreno, C.N.R.
Viale Mattioli 25, 1-10125 Torino, Italy
(Received 13 June 1993; revision received 30 August 1993; accepted 3 September 1993)
Abstract: A mycorrhizal fungal strain (PS4), forming endomycorrhizae with the fine roots of ericaceous plants, was grown in pure
culture on citrus pectin or sucrose as carbon source. Extracellular polygalacturonase (PG) activity was found only in the
pectin-containing medium. Preparative isoelectric focusing identified two activity peaks (maximal activity at pH 4.2 and 5.7) that
were attributed to two PGs (PG1 and PG2). Viscosimetric analysis revealed that PG1 hydrolyzes the substrate randomly, whereas
PG2 shows an exo-mode of action. The pH optima were 4.6 for PGI and 4.9 for PG2. The optimum temperature was about 55°C
for both the enzymes. Both PG1 and PG2 degraded preferentially polygalacturonic acid and, to a lesser extent, citrus pectin. On
Western blots PGI was specifically labelled by a polyclonal antibody raised against an endopolygalacturonase from Fusarium
moniliforme. The molecular mass of PGI, as revealed by the antibody, was 40 kDa. Labelling with Concanavalin A showed that
PG1 is a glycoprotein.
Key words: Mycorrhiza; Ericoid fungi; Enzyme production; Polygalacturonase
Introduction
Mycorrhizal fungi live in symbiosis with the
roots of most plants, and are classified as endoor ectomycorrhizal, according to their ability or
inability to cross the root host wall [1]. Endomycorrhizal ericoid fungi are Ascomycetes that
* Corresponding author. Tel. (0ID 650 2927. Fax (011) 655
839.
colonise the fine roots of the Ericaceae [2]. Some
ericoid strains possess a battery of extracellular
enzymes, namely proteases, acid phosphatases,
chitinases, cellulases, enabling them to draw N, P
and C from organic compounds [3,4]. It is thus
clear that these fungi possess some saprophytic
capabilities.
Polygalacturonase (PG) is produced by saprophytic microorganisms during their substrate
degradation [5] and by phytopathogenic fungi
during their interaction with their plant host [6].
86
The considerable pathogenicity of PGs is shown
by their fragmentation and solubilization of plant
cell wall homogalacturonans. This both opens the
way to colonization and provides food for the
fungus [6]. No detailed analysis has yet been
made of PG production on the part of mycorrhizal fungi, even if some preliminary data for the
ecto- as well as for both arbuscular and ericoid
mycorrhizal fungi suggest that they produce polygalacturonase in much smaller quantity and at a
later stage than some pathogens [4,7,8].
This paper shows that an ericoid fungal strain
(PS4) produces two acidic polygalacturonases
during its saprophytic phase. A partial definition
of their characteristics is also proposed.
Materials and Methods
Culture conditions
PS4 strain [9] was grown on a liquid mineral
medium supplemented with 1% (w/v) citrus
pectin from Sigma (St. Louis, MO, USA). The
mineral medium contained N a N O 3 (3 g l - l ) ,
K z H P O 4 (1 g l - l ) , M g S O 4 . 7 H 2 0 (0.5 g 1-1),
KCI (0.5 g l - l ) , F e S O 4 . 7 H 2 0 (0.05 g I-1).
Medium p H was adjusted to 5 with 1 N NaOH.
PS4 strain was also grown on a medium containing 1% (w/v) sucrose as the sole carbon source.
100-ml cultures were incubated in the dark at
24°C.
Polygalacturonase (PG) assay
The time course of PG production in the culture media was followed for 40 days. PG activity
was determined by measuring the increase in
reducing end-groups by the copper-arsenomolybdate method of Milner and Avigad [10], using
D-galacturonic acid as the standard. The reaction
mixtures consisted of 0.1 ml of substrate and 0.1
ml of dialyzed culture medium. The substrate was
0.5% ( w / v ) polygalacturonic acid (Fluka, Buchs,
Switzerland) in 50 mM Na-acetate buffer (pH 5).
The reaction mixtures were incubated at 30°C for
an appropriate time. One unit of PG activity
( R G U ) was defined as the amount of enzyme
producing 1 /xmol of reducing end-groups per
min.
Protein recovery from the cultures
Cultures were harvested by filtration through
filter paper. Filtrates were precipitated to 25%
saturation of ammonium sulfate and then centrifuged at 1 6 0 0 0 × g for 30 rain. The supernatant was brought to 85% saturation of ammonium sulfate and then centrifuged as above. Pellets were dissolved in distilled water and then
dialyzed against two changes of distilled water for
about 40 h. Protein content was determined by
the method described by Bradford [11] with
bovine serum albumin (Sigma) as the standard.
Isoelectric focusing (IEF) of proteins
This was performed by using the Rotofor liquid-phase preparative isoelectric focusing column
(BioRad, Richmond, CA, USA). 50 ml of enzyme
sample containing 4% Bio Lyte (Bio-Rad) ampholine (pH 3-10) were applied to the focusing
chamber.
Enzyme characterization
The specific substrate for PG was identified by
incubating the enzyme solution at 30°C with
0.25% P G A or 0.25% citrus pectin. Optimal pH
and temperature of the two PGs were determined by the copper-arsenomolybdate assay on
1EF fractions 5 (PG1) and 10 (PG2). The reaction
mixtures (0.2 ml) contained 0.25% PGA and 5 txl
of fraction 5 or 10/xl of fraction 10. Optimal pH
was estimated in a p H range 3.9-6.7. Reaction
mixtures were buffered with 0.1 M citrate-phosphate buffer and incubated at 30°C for 1 h.
Optimal temperature was determined from 25°C
to 60°C. Reaction mixtures were buffered at pH 5
with Na-acetate buffer and incubated for 45 min.
Viscosimetric assay
Substrate hydrolysis pattern was determined
by measuring the decrease in relative viscosity at
30°C of a 0.5% (w/v) solution of P G A in 100 mM
Na-acetate buffer (pH 5). The reaction mixture (2
ml) contained 0.0139 R G U of PG1 and 0.0112
R G U of PG2. A micro-Ostwald viscosimeter (i.d.
0.70 mm) was used for this purpose. The decrease
in relative viscosity was monitored by an AVS 310
system (Schott Gerate, Germany).
87
Sodium dodecylsulfate-polacrylamide
trophoresis (SDS-PAGE)
gel elec-
SDS-PAGE was performed in a MINI-PROTEAN II Dual Slab Cell (BioRad). Proteins were
precipitated with 20% (v/v) trichloroacetic acid
and redissolved in 5 txl of SDS sample buffer [12].
An amount of about 12 txg protein was applied to
each well. The resolving gel consisted of 10%
acrylamide. The electrophoresis was performed
in electrophoresis buffer (25 mM Tris, 0.19 M
glycine, 0.1% SDS). Gels were stained for protein
with the silver nitrate method. Molecular mass
standards (BRL, Gaithersburg, MD, USA) were
a-chymotrypsinogen (25 kDa), ovalbumin (42
kDa), bovine serum albumin (68 kDa) and phosphorylase B (100 kDa).
Western blotting
Proteins were electrophoretically transferred,
after SDS-PAGE, to nitrocellulose using a MINI
TRANS-BLOT Electrophoretic Transfer Cell
(BioRad). Transfer buffer consisted of 25 mM
Tris, 192 mM glycine, 20% (v/v) methanol. Nitrocellulose was probed with a polyclonal antibody
(1 : 1000) raised against Fusarium moniliforme endopolygalacturonase [13]. Goat-anti-rabbit yglobulin-alkaline phosphatase conjugate (Sigma)
diluted 1:8500 was used as the secondary antibody. The color by alkaline phosphatase was visualized by using the bromochloroindolyl phosp h a t e / n i t r o blue tetrazolium (BCIP/NBT) substrate as described in Harlow and Lane [14].
4-chloro-l-naphthol. 50 /xl of 6% I-t20 2 were
added just before use.
Results
In vitro production of polygalacturonase (PG)
The time course of PG activity in liquid media
containing different carbon sources is represented in Fig. 1. PG secretion was delayed in time
on citrus pectin and reached a plateau about 40
days after inoculation. No PG production was
recorded when the fungus was grown on sucrose.
lsoelectric focusing (IEF)
Extracellular proteins secreted by PS4 strain in
the pectin-containing medium were separated by
preparative IEF. The distribution of PG activity
in the IEF fractions was detected by using polygalacturonic acid as the substrate. Two acidic
polygalacturonases (PG1 and PG2) were revealed. The pI of PG1 and PG2 were 4.2 and 5.7,
respectively, as shown in Fig. 2.
Enzyme characteristics
Viscosimetric analysis revealed that the two
enzymes possess different modes of action. Determinations of reducing end-groups at a time
corresponding to a 50% loss in relative viscosity
(Ts~~) showed that PG1 cleaved 2.3% of the glyco-
.=
5O
Con A affinoblotting
The glycoprotein nature of PG was revealed by
affinoblotting with Concanavalin A (Con A), a
lectin which recognizes glucose and mannose.
Proteins were transferred to nitrocellulose as described in the previous section. The sheet was
incubated overnight with 10 txg/ml Con A in
buffer A (50 mM TBS, 0.05% Tween 20, 1 mM
CaC12, 1 mM MnCI 2, pH 7.4). After washing with
buffer A, nitrocellulose was treated for 1 h with
10 # g / m l horseradish peroxidase (HRP) in buffer
A. The labeling was visualized by using a substrate solution for HRP. The solution was prepared by mixing 25 ml of 50 mM TBS (pH 7.4)
and 5 ml of cold methanol containing 15 mg of
4O
~
=
~
a5
~
2s
~
20
~,
is,
~
10
~
5
30
0
'
0
=
,
10
-
¶
" ,
20
"
.-
"1
3O
"
,"
,=
40
Days
Fig. 1. Production of PG by PS4 strain in culture. The fungus
was grown for 40 days on 1% citrus pectin (o) or 1% sucrose
( • ) . Each point is the average of the values for three flasks.
88
sidic bonds of PGA, whereas PG2 cleaved 17.7%
of the bonds. According to Bateman and Basham
[15], PG1 hydrolyzed the substrate in an 'endo'
fashion, while PG2 showed a prevalent 'exo' mode
of action.
Both PG1 and PG2 degraded PGA to a higher
extent (about 3-fold) than they degraded citrus
pectin. This indicates that these enzymes are
polygalacturonases rather than polymethylgalacturonases.
The optimum pH were 4.6 for PG1 and 4.9 for
PG2 (Fig. 3A). PG1 showed a narrower pH activity curve in respect to PG2. At pH 6 PG1 activity
was completely inhibited, whereas PG2 maintained 30% of its maximal activity.
The optimum temperature for activity of PG1
was between 50 and 55°C (Fig. 3B). PG2 showed
a maximum at 55°C. At 60°C PG1 exhibited only
40% of its maximal activity, while PG2 was still
strongly active (about 78% of its maximum).
100-
80
60
_=
==
40E
20-
"s
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
pH
--
ca
E
100 -
80
60
SDS-PAGE and irnmunoblot
Proteins in the IEF fractions with PG activity
were separated by SDS-PAGE. In the fractions
with PG1 activity, two main bands corresponding
to an apparent molecular mass of 105 kDa and 40
kDa were detected (Fig. 4). Their distribution in
each fraction mirrored its PG activity as shown in
"~
40
E
-~
20
~
o
0
t
i
~
i
i
30
40
50
60
70
Temperature
~H
'12
3,0 -
11
2,5
"7.
"~
" 10
2,0
.
Fig. 3. pH (A) and temperature (B) optima of PGI (e) and
PG2 ( • ) activity, expressed as % of the maximum enzyme
activity (copper-arsenomolybdate assay). In A the maximum
values (expressed as R G U x 10 3)were 2.25 for PG1 and 1.3
for PG2. In B the maximum values were 2.55 for PGI and 3.7
for PG2. Each point is the average of two determinations.
8
1,5'
"7
1,0
5
-4
0,5
i3
i2
0,0
0
(°C)
2
4
6
8
10
12
14
16
Fraction
18
2
number
Fig. 2. PG activity (OD6o0) detected in fractions following
preparative IEF of proteins collected from cultures of PS4
strain grown on 1% citrus pectin. - - o - - , PG activity; - - o - - ,
pH.
Fig. 2. When the proteins were electroblotted on
nitrocellulose and probed with a polyclonal antibody against F. moniliforme endopolygalacturonase, only the 40 kDa protein showed a strong
reactivity with the antibody (Fig. 5). The 105 kDa
band was not labelled. We can conclude that PG1
has a molecular mass of 40 kDa. In the fractions
with PG2 activity some weak bands were detected
(Fig. 4). None of them reacted with the antibody
(Fig. 5).
89
MW
kDa
p
3
4
5
6
8
9
10
11
206
100
68
42
Fig. 4. SDS-PAGE of proteins contained in selected IEF fractions. Lane numbers correspond to the fraction numbers in Fig. 2.
Arrows point to the 105 kDa and 40 kDa bands, p, Total proteins recovered from pectin-containing cultures of PS4 strain. MW,
Molecular masses.
Con A affinoblotting
L a b e l l i n g of t h e e x t r a c e l l u l a r p r o t e i n s with C o n
A r e v e a l e d t h a t PG1 is a g l y c o p r o t e i n w h o s e
glycidic p a r t c o n t a i n s m a n n o s e a n d / o r glucose
(Fig. 6).
Discussion
M a n y p h y t o p a t h o g e n i c a n d s a p r o p h y t i c fungi
p r o d u c e p e c t i n o l y t i c enzymes. A m o n g these, P G s
11
10 9
8
6
5
4
3
p MW kDa
play a key role in p l a n t - f u n g a l i n t e r a c t i o n s since
t h e y not only allow p l a n t tissue c o l o n i z a t i o n , b u t
also r e l e a s e o l i g o m e r i c f r a g m e n t s that r e g u l a t e
v a r i o u s physiological events i n c l u d i n g elicitation
o f the p l a n t d e f e n s e r e s p o n s e s [6]. P G s from
s a p r o p h y t i c fungi, such as Aspergillus niger a n d
A. tubigensis, a r e of c o n s i d e r a b l e e c o n o m i c imp o r t a n c e since t h e y a r e e m p l o y e d in t h e p r o c e s s ing of a g r i c u l t u r a l p r o d u c t s in the food industry
[5]. By contrast, m y c o r r h i z a l fungi s e e m to prod u c e P G in s m a l l e r quantities, since they obviously d e p e n d on the c o n t i n u i n g viability of the
host cells.
11 10
9
8
6 4+5 3
MW
kDa
,100
68
,42
25
Fig. 5. Western blots of some selected IEF fractions. Lane
numbers correspond to the fraction numbers in Fig. 2. p,
Total proteins recovered from pectin-containing cultures of
PS4 strain. MW, Molecular masses. A polyclonal antibody
raised against Fusarium moniliforme endoPG was used for
immunodetection. PG1 is specifically immunodetected.
-100
_68
_42
-25
Fig. 6. Con A affinoblot of some selected IEF fractions. Lane
numbers correspond to the fraction numbers as reported in
Fig. 2. Fractions 4 and 5 have been joined together. PGI
(arrow) is visualized as a glycoprotein. MW, Molecular masses.
90
Our results offer a partial characterization of
two PGs produced by an ericoid mycorrhizal
strain, PS4. This strain easily forms coils inside
the epidermal cells of Calluna roots [9] and releases PG activity in the culture medium. The
activity peaks after 40 days, compared with the
few days reported for necrotrophic or saprophytic
fungi [13,16,17]. This difference in timing may be
ascribable to the different nutritional strategies.
PG secretion from PS4 strain depends on the
carbon sources, since it is revealed in the presence of citrus pectin, but not of sucrose. Two
regulation mechanisms are thought to occur during PG secretion by pathogenic fungi [6]: (i) the
enzyme is specifically induced by the substrate
(i.e. pectin) or (ii) the enzyme is constitutive, but
its expression is restricted by the presence of a
simple sugar such as sucrose (catabolite repression). Both possibilities may apply to PS4 strain.
Viscosimetric experiments reveal that PS4
strain produces an e n d o P G (PG1) and an exoPG
(PG2), like some pathogenic fungal strains. Sclerotinia sclerotiorum and Bottytis cinerea produce
both exo- and endoPGs, which vary in molecular
mass and p l [18,19]. The ability to produce different PGs may allow a fungus to fully degrade
the substrate thanks to a multiple activity [7].
This hypothesis could be of particular interest in
the case of a mycorrhizal fungus, which has to
survive in both the soil and the specialized niche
provided by a plant cell.
PG1 shows an acidic p l (4.2) whereas many
PGs from pathogenic fungi are basic or only
slightly acidic. It has been suggested that the
basic p l allows physical interaction between the
enzyme and its substrate (cell wall pectins) at the
cell pH [7]. However, presence of PGs with rather
acidic p l has been demonstrated in some
pathogenic strains such as B. cinerea and S. sclerotiorum [18,19], as well as in saprophytic fungi
such as A. niger and A. tubigensis [5]. ExoPGs, in
pathogenic fungi, have received less attention
than endoPGs. Those purified from B. cinerea
[18] and S. sclerotiorum [19] possess an acidic p l
like that shown by the exoPG (PG2) produced by
PS4 strain.
The molecular mass of PG1 (40 kDa) is in the
range for most homogeneous fungal PGs (25-40
kDa; according to Cervone et al. [20]). The
molecular mass of PG2 was not determined, since
there was no cross-reaction with the antibody
raised against an e n d o P G from F. moniliforme.
This is in agreement with the observations that
there are no cross-reactions between antibodies
raised against endoPGs and the exoPG forms [21]
and vice versa [22].
PG1 possesses a glycidic component, as revealed by its binding with Con A, like other PGs
produced by plant pathogens [16,19]. It has already been suggested that this component may
play a role in the molecular basis of the plantfungal interaction [20].
In conclusion, the occurrence and expression
in the culture medium of two PGs by a mycorrhizal fungus raises the question of the significance of their activity during the fungus saprophytic growth as well as in the establishment of
the symbiosis.
Acknowledgements
The Authors are grateful to Prof. F. Cervone
(Universit?a 'La Sapienza', Roma) for providing
the anti-PG antibody; to Dr. F. Favaron (Universith di Padova, Padova) for the viscosimetric
analysis; to Dr. G. Papa (CMST, CNR, Torino)
for introducing us to the use of the Rotofor
apparatus. This research was supported by the
Italian National Council for Research, Special
project RAISA, Subproject no. 2, no. 000.
References
1 Harley, J.L. (1989) The significance of mycorrhiza. Mycol.
Res. 92, 92-129.
2 Read, D.J. (1983) The biology of mycorrhiza in the Ericales. Can. J. Bot. 61,985-1004.
3 Read, D.J. (1991) Mycorrhizas in ecosystems. Experientia
47, 376-391.
4 Peretto, R., Benini, V., Didoli, P., Faccio, A. and Bonfante, P. (1993) Cell wall-degrading enzymes in cricoid
mycorrhizal fungi. Giorn. Bot. Ital. 127, 339-341.
5 Bussink, H.J.D., Buxton, F.P. and Visser, J. (1991) Expression and sequence comparison of the Aspergillus niger and
Aspergillus tubigensis genes encoding polygalacturonase II.
Curr. Genet. 19, 467-474.
91
6 Hahn, M.G., Bucheli, P., Cervone, F., Doares, S.H.,
O'Neill, R.A., Darvill, A. and Albersheim, P. (1989) The
role of cell wall constituents in plant-pathogen interactions. In: Plant-Microbe Interactions (Nester, E. and Kosuge, T., Eds.), pp. 131-181. McGraw Hill, New York.
7 Keon, J.P.R., Byrde, R.J.W. and Cooper, R.M. (1987)
Some aspects of fungal enzymes that degrade plant cell
walls. In: Fungal Infection of Plants (Pegg, G.F. and
Ayres, P.G., Eds.), pp. 133-157. Cambridge University
Press, Cambridge, UK.
8 Garcia-Romera, 1., Garcia-Garrido, J.M. and Ocampo,
J.A. (1991) Pectinase activity in vesicular-arbuscular mycorrhiza during colonization of lettuce. Symbiosis 12, 189198.
9 Perotto, S., Peretto, R., Mor6, D. and Bonfante, P. (1990)
Ericoid fungal strains from an alpine zone: their cytological and cell surface characteristics. Symbiosis 9, 167-172.
10 Milner, Y. and Avigad, G. (1967) Colorimetric assay of
hexuronic acids and some keto sugars. Carbohydr. Res. 4,
359.
11 Bradford, M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 72,
248-254.
12 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature
222, 680-685.
13 De Lorenzo, G., Salvi, G., Degr~, R., D'Ovidio, R. and
Cervone, F. (1987) Induction of extracellular polygalacturonase and its mRNA in the phytopathogenic fungus
Fusarium moniliforme. J. Gen. Microbiol. 133, 3365-3373.
14 Harlow, E. and Lane, D. (1988) Antibodies. A laboratory
15
16
17
18
19
20
21
22
Manual, p. 505. Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
Bateman, D.F. and Basham, H.G. (1976) Degradation of
plant cell walls and membranes by microbial enzymes. In:
Encyclopedia of Plant Physiology, vol. IV, Physiological
Plant Pathology (Heitefuss, R. and Williams, P.H., Eds.),
pp. 316-355. Spriger-Verlag, Berlin.
Walton, J.D. and Cervone, F. (1990) Endopolygalacturonase from the maize pathogen Cochliobolus carbonum.
Physiol. Mol. Plant Pathol. 36, 351-359.
Blais, P., Rogers, P.A. and Charest, P.M. (1992) Kinetic of
the production of polygalacturonase and pectin lyase by
two closely related forrnae speciales of Fusarium oxysporum. Exp. Mycol. 16, 1-7.
Riou, C., Freyssinet, G. and Fevre, M. (1992) Purification
and characterization of extracellular pectinolytic enzymes
produced by ScIerotinia sclerotiorum. Appl. Environm. Microbiol. 58, 578-583.
Johnston, D.J. and Williamson, B. (1992) Purification and
characterization of four polygalacturonases from Botrytis
cinerea. Mycol. Res. 96, 343-349.
Cervone, F., De Lorenzo, G., Salvi, G. and Camardella, L.
(1986) Molecular evolution of fungal polygalacturonase.
In: Biology and Molecular Biology of Plant Pathogen
Interactions (Bailey, J.A., Ed.), NATO ASI Series, Vol.
HI, pp. 386-392. Springer-Verlag, Berlin.
Johnston, D.J. and Williamson, B. (1992) An immunological study of the induction of polygalacturonases in Botrytis
cinema. FEMS Microbiol. Lett. 97, 19-24.
Riou, C., Fraissinet-Tachet, L., Freyssinet, G. and Fevre,
M. (1992) Secretion of pectic isoenzymes by Sclerotinia
sclerotiorum. FEMS Microbiol. Lett. 91,231-238.