CiENCE

ELSEVIER Plant Science 118 (1996) II-23

Antifungal activity of tobacco osmotin has specificity and

involves plasma membrane permeabilization

Laura R. Abada, Matilde Paino DUrzo, Dong Liua, Meena L. Narasimhan,

Moshe Reuveni, Jian Kang Zhua, Xiaomu Niua, Narendra K. Singhb,
Paul M. Hasegawaa, Ray A. Bressan*a
Center for Pbmt Environmental Stress Physiology, Purdue University. II65 Horticulture Building, West Lofayette, IN 47907-1165,
USA
bDepartment of Botany and Microbiology, Auburn University, Auburn, AL 36849-5407. USA

Received 12 December 1995; revised 9 April 1995; accepted 9 April 1995

Abstract

Osmotin protein is able to inhibit in vitro the growth of a number of unrelated pathogens. A survey of 31 isolates
representing 18 fungal genera indicated that sensitivity may be determined at the genus level. Hyphal growth of
Aspergillus flavus, Aspergillus parasitica, Rhizoctonia solani and Macrophomina phaseolina was highly resistant to
osmotin whereas the growth of Bipolaris, Fusarium and Phytophthora species was very sensitive. Of all fungi tested
Trichoderma lortgihrachiatum hyphal growth was most inhibited by osmotin treatment. Osmotin either induced spore
lysis, inhibited spore germination or reduced germling viability in seven fungal species that exhibited some degree of
sensitivity in hyphal growth inhibition tests. The species-specific growth inhibition was correlated with the ability of
osmotin to dissipate the fimgal membrane pH gradient. Both growth inhibition and pH gradient dissipation by osmotin
were sensitive to NaCl and other inorganic cations. Cells of T. longibrachiatum were insensitive to osmotin after
plasmolysis, suggesting that the cell wall may be a component of the mechanism by which osmotin permeabilizes the
plasma membrane and kills fungal cells.

Keywords: Antifungal protein; Fusarium sp.; Osmotin; Pathogenesis-related protein; Phytophthora sp.; Trichoderma
longibrachiatum

Abbreviations: BSA, bovine serum albumin; DMO, 5,5- 1. htroduction

diiethyl-[2-*CJoxaxolidine-2,4ione; FCCP, carbonyl
cyanide ptrifluoromethoxyphenylhydraxone; FDA, fluores-
Plants accumulate a number of antifungal pro-
cein diacetate; PDA, potato dextrose agar; PDB, potato dex-
trose broth; PR, pathogenesis-related. teins. Among these proteins are included the
l Corresponding author. Tel.: +I 317 494 7858; fax: +l 317 thionins, the ribosome-inactivating proteins, lipid-
494 0391; e-mail: becky_fagan@hort.purdue.edu transfer proteins, 2S storage albumins, non-

0168~9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved

PII: SOI68-9452(96)04420-2
I2 L.R. Abad et a[. lhn~
enzymatic chitin-binding proteins, polygalacturo-
nase inhibitor proteins, the defensins and
pathogenesis-related (PR) proteins 111. PR pro-
teins were originally observed in tobacco upon
virus infection [2]. The PR proteins comprise a
large super-family of plant defense proteins that
accumulate in response to various biotic and
abiotic stresses (3-S]. They have been classified
into families based on serological relationships,
amino acid sequence similarities and function
14-61. Only members of PR-protein families 1
through 5, in the nomenclature of Van Loon et al.
171,have been shown to possess antifungal activity
in in vitro and in vivo assays [ 1,8- 141. The
mechanisms of action of many of these proteins re-
main obscure. Notable exceptions are members of
the PR-2 and PR-3 families which contain
chitinases and o-1,3-glucanases, respectively.
Chitinases and 8-l ,3-glucanases have been im-
plicated, individually and in combination, in
fungal cell wall degradation.
The PR-5 protein family includes proteins with
molecular weight of about 24 kDa that are
homologous to the sweet tasting protein
thaumatin 115,161. The PR-5 family contains
osmotin, a protein that accumulates in tobacco
cells in response to pathogen attack and osmotic
stress [17-211. Osmotin-like proteins have been
shown to inhibit spore germination in vitro of the
phytopathogenic fungi Phytophthora infestarts and
Fusaritun oxysporum [12,22]. They have also been
shown to inhibit in vitro hyphal growth of P. in-
j&tans, P. nicotianae and Cercospora beticola
(14,201. It has been demonstrated that transgenic
potato plants overexpressing tobacco osmotin
have enhanced resistance to P. infestans [20], sug-
gesting that PR-5 proteins can be used as a novel
source of resistance. In order to evaluate the
potential of these proteins for use as a source of re-
sistance, the spectrum of their antifungal activities
needs to be determined. However, there has been
no extensive or systematic survey of the phyto-
pathogenic fungi which are sensitive to osmotin-
like proteins. There is little information available
concerning the mechanism of inhibition of fungi
by PR-5 proteins. Zeamatin, an osmotin-like pro-
tein from maize, had no chitinase, glucanase, man-
nanase, protease, ribonuclease, phospholipase or
Science 118 (1996) II-23
ribosome-inactivating activity i23). Ba& on the
ability of zeamatin to cause the leakage of cyto-
plasmic contents from fungal hyphae in a rapid
and temperature-independent manner, Roberts
and Selitrennikoff [23] proposed that thaumatin-
like proteins act by permeabilizing fungal cell
membranes and thus termed such proteins as per-
matins.
This paper presents results which demonstrate
that tobacco osmotin inhibits hyphal growth and
spore germination of a large number of
economically important plant pathogens. We also
provide evidence that (a) the ability of osmotin to
inhibit hyphal growth and spore germination is
directly correlated with its ability to increase per-
meability of the fungal plasma membrane to pro-
tons and to the inability of the cells to maintain a
pH gradient in the presence of the protein and (b)
osmotin antifungal activity is affected by a number
of factors such as the growth stage of the fungus,
the proximity of the cell wall and membrane and
by salts.
2. Materials and methods
2.1. Isolation and purification of osmotin
Osmotin was isolated from salt-adapted tobacco
cell suspensions (Nicotiana tabacum L. var.
Wisconsin 38) as described by Singh et al. [18].
Briefly, the basic isoforms of osmotin were isola-
ted after homogenization of tobacco cells in phos-
phate buffer followed by ammonium sulfate
fractionation, CM-25 Sephadex cation-exchange
chromatography and cationic HPLC separation
on S-300 1181. The homogeneity of the protein
preparations was monitored by l- and 2-D poly-
acrylamide gel electrophoresis and on immuno-
blots of these gels using polyclonal antibodies
raised against tobacco osmotin. For antifungal
assays, osmotin was dissolved in sterile distilled
water.
2.2. Fungal strains and media
The pathogens used for in vitro tests of osmotin
antifungal activity are listed in Table 1. Fungi were
maintained on either potato dextrose agar (PDA;
 L.R. Abad et al. /Plant Science 118 (1996) II-23

Table 1
Inhibition of hyphal growth by osmotin

Fungus Degree of hyphal growth inhibition by osmotin

30 ccg 60 cg 1OOCB

Altermaria solani 1 2 2
Aspergillw flaws 0 0 0
Aspergillus parasitica 0 0 0
Bipolaris maydis 1 2 2
Bipolaris reicola 1 2 2
Botrytis cinerea 0 1 1
Cercospora zeae-maydis 2 2 3
Cladosporium cucumerinum 1 1 2
Colletotrichum gleosporioiaks 0 I 1
Colletotrichum graminicola 0 1 1
Colletotrichum laginarium I 2 2
Colletotrichum sublineolum 1 2 2
Fusarium graminearum 1 2 2
Fusariwn moniliforme I 1 2
Fusarium oxysporum f. sp. dianthi 2 3 3
Fwarium oxysponun f. sp. lycopersici 2 3 3
Fusariwn roseurn Sambucinum 2 2 3
Kabatiella zeae 1 1 2
hiacrophomina phaseolina 0 0 0
Magnaporthe grisea 0 0 1
Periconia circinata 1 1 2
Phytophthora infestans 0 1 2
Phytophthora parasitica var. dianthi 1 1 2
Phytophthora parasitica var. nicotianae 0 1 2
Rhizoctonia solani 0 0 0
Sclerotinia sclerotiorum 0 1 2
Stenocarpella maydis 0 1 3
Trichoderma longibrachiatum 2 3 4
Verticillium dahliae (mint) I 1 2
Verticillium akhliae (potato) 2 2 2
Verticillium dahliae (tomato) 1 2 2

Hyphal growth inhibition was measured by placing sterile filter paper disks containing water (control), BSA (100 ag, control) or
osmotin (30, 60 or 100 fig) on the surface of the growth medium adjacent to the colony margin and allowing the fungi to grow for
several days at room temperature. The degree of hyphal growth inhibition around the disks was evaluated using the following visual
rating scale (examples shown in Fig. 1): 0, hyphal growth over disk, no growth inhibition; 1, hyphal growth over disk, slight growth
inhibition at mycelial margin; 2, no hyphal growth on disk, slight inhibition zone surrounding disk; 3, no hyphal growth on disk,
definite inhibition zone surrounding disk; 4, no hyphal growth on disk, wide inhibition zone surrounding disk.

Sigma, St. Louis, MO) or V8 juice agar. Hyphal
assays were performed on a modified PDA
(PDwA) prepared with washed agar. To prepare
PDwA (1 liter), agar (20 g; Difco) was washed by
stirring in a large volume of distilled water for
8-12 h, exchanging water 3-4 times during this
period. The washed agar was collected by tiltra-
tion, added to 36 g potato dextrose broth (PDB;
Sigma), brought up to a volume of 1 liter and
autoclaved for 20 min at 120C. Spore germination
assays were conducted in PDB.
2.3. Measurement of antificngal activity
2.3. I. Hyphal growth inhibition assays
An agar plug containing mycelia of the test fun-
gus was placed at the center of a PDwA plate.
Hyphae were allowed to grow at room tempera-
14 L.R. Abad et al. /Pkmt Science 118 (19%) 11-23
ture until the colony reached a diameter of 5 cm,
at which time sterile filter paper disks were posi-
tioned adjacent to the colony margin. The disks
were saturated with 20 hl of either water, bovine
serum albumin (BSA; 100 pg; Sigma) or tobacco
osmotin (30,60 or 100 rg) and the plates were fur-
ther incubated for several days. Growth inhibition
around each disk was then evaluated visually.
Experiments were conducted twice and the data
pooled.
2.3.2. Spore lysis and germination assays
Fifty microlitres of fungal spores suspended in
2 x PDB (ca. 1 x 10 conidia/ml) were mixed
with 50 11 of water (control), BSA (control) or
tobacco osmotin solution, respectively. The final
treatments contained 5 x lo4 conidia/ml with 0,
1, 10, 50 and 100 &ml osmotin and 100 &ml
BSA (control). Spore suspensions were incubated
at room temperature for 16-22 h or until at least
50% of the spores in the control treatments ger-
minated. After incubation, the total number of
spores and the number of germinated spores were
counted in an aliquot of each sample using a
Spotlite hemacytometer (American Scientific
Products, McGaw Park, IL). The number of viable
spores and germlings was estimated by staining
sample aliquots with fluorescein diacetate (FDA)
and counting the fluorescent spores and/or germ
tubes using the hemacytometer. For staining with
FDA, 5 ~1 of a FDA stock solution (50 mg in 100
ml acetone) were mixed with 100 ~1 of spore sus-
pension. Each treatment contained three replicates
and three samples from each of these replicates
were counted. Experiments were conducted twice
and data from the two experiments were pooled.
The data for each spore germination experiment
were transformed for regression analysis. Treat-
ment concentrations were log transformed and
spore concentration or germination was expressed
first as percent inhibited (or lysed) compared to
the control (water treatment), then this percent in-
hibition was transformed into probits [24]. Sepa-
rate regression analyses were performed on each
experiment and analysis of variance (ANOVA) on
the slopes of each experiment was completed. The
dose at which 5O?h of the spores are affected by
osmotin (ED,) was estimated from the regression
line of those fungal species exhibiting a response to
the treatment. The EDSo values of these species
were in turn subjected to an ANOVA, and a
Fishers LSD comparison made.
To determine the effect of various salts on the
antifungal activity of osmotin, 150 ~1 of a T.
longibrachiatum spore suspension in 2 x PDB (ca.
1 x lo4 conidiaml) were dispensed into each well
of a 96-well microtiter plate. Salt solutions and
osmotin were added to each well as desired and the
volume was adjusted to 300 ~1 with distilled water.
After incubation at room temperature for 48 h,
fungal growth was quantitated by measuring the
absorbance at 410 nm with a microtiter plate
reader (Bio-Tek Instruments, Winooski, VT).
2.4. Determination of membrane leakiness
Plasma membrane integrity was estimated from
the measurement of the capacity of the cells to
maintain a pH gradient between the external medi-
um and the cytosol. This was accomplished by
determining the distribution of the weak acid
5,5dirqethyl-[2-4C]oxazolidine-2,4-dione (DMO;
Amersham, Arlington Heights, IL) between the
cells and the external solution 1251.The weak acid
will accumulae in the alkaline compartment of the
cell, i.e. the cytoplasm [25]. The procedure for
using DMO was described by Reuveni et al.
[26,27] for cytoplasmic pH estimation. The rela-
tive cytoplasmic volume, that is, the volume of the
cells cytoplasm as a percent of the cells volume,
was determined using t3H]H20 (Amersham, Arl-
ington Heights, IL) and [r4C]sorbitol (Amersham,
Arlington Heights, IL) [26]. The amount of DMO
taken up by the cells was corrected using the rela-
tive cytoplasmic volume. For measurement of
DMO uptake, tobacco cells or fungal hyphae were
suspended in their respective growth media. The
suspension cultures of tobacco cells used in these
experiments were maintained as described by
Binzel et al. 1281.Suspension cultures of the fungi
were prepared as follows: 2day-old PDA plate
cultures of the fungus were flooded by swirling
PDB (5 ml) on the surface and the hyphal pieces
so obtained were used to inoculate 200 ml of PDB.
The suspension culture was grown overnight with
shaking at room temperature and aliquots were
 L. R. Abad et al. / Pkznt Science I I8 (19%) 11-23 I5

used. For staining with FDA, hyphae were
suspended in PDB containing the desired amount
of sorbitol. The hyphae were stained as described
by Widholm [29]. Photographs were taken with an
Olympus Vanox microscope fitted with an
automatic camera.
3. Results
3.1. Osmotin is specifically active against certain
fungal species
3. I. 1. Hyphal growth inhibition assays
Thirty-one fungal isolates, comprising 18 dif-
ferent genera, were tested for hyphal growth in-
hibition in the presence of tobacco osmotin (Table
1). The hyphal growth of most fimgal species
tested in this manner was inhibited when exposed
to osmotin, with more inhibition evident at higher
concentrations. Zones of growth inhibition were
detectable surrounding filter paper disks saturated
with 30 pg osmotin in 19 of the 31 isolates; 27 of
the same 31 isolates were inhibited by disks con-
taining 100 pg osmotin. Overall, there was a varia-
tion in the degree of sensitivity exhibited by the
fungal isolates tested, ranging from the very sensi-
tive Trichodenna longibrachiatum to the complete-
ly insensitive Rhizoctonia solani, Macrophomina
phaseolina and Aspergillus spp. (Fig. 1; Table 1).
There appeared to be a loose correlation between
members of the same genus and the amount of
hyphal growth inhibition evident in this assay.
Phytophthora and Colletotrichum appeared to be
moderately sensitive as a genus, while Aspergillus
seemed to be insensitive. The five isolates of
Fusarium were all sensitive to some extent. How-
ever, F. graminearum and F. monil$orme exhibited
less inhibition than F. oxysporum and F. roseum,
reflecting the variability within members of a
genus.
3.1.2. Spore iysis and germination inhibition assays
Eight fungi were chosen to examine the effect of
osmotin on spores. In the presence of osmotin,
spore lysis i.e. disappearance of entire spores leav-
ing behind cell wall debris resulting in decrease in
spore concentration in the osmotin treatments
compared with the controls, occurred in five of the
eight fungi tested (Fig. 2A). All five fungi had
fewer than 30% of spores surviving in treatments

Fig. 1. Hyphal growth inhibition assay with tobacco osmotin. Shown are examples of the hyphal growth inhibition assays rated in
Table 1. The disks contain (in order, clockwise beginning at the top) water; BSA, 100 fig; osmotin 30 pg. 60 rg and 100 pg. The fungi
and their inhibition rating at 100 cg osmotin, in parenthesis, are: A - R. solani [O];B - C. gleosporioiaks (I); C - M. @sea (1);
D - A. so&i (2); E - K &Mae (mint) (2); F - C. zene-maydis (3); G - F. oxysporumf. sp. lycospersici (3); H - F. roseumSam-
bucinum (3); I - S. maydis (3); J - T. fongibrachiatum(4).
16 L.R. Abad et al. /Plant Science 118 (19%)
AF BC FG
Fig. 2. Effect of tobacco osmotin on fungal spores. (A) Spore
lysia. Fungal spores were treated with the indicated coacentra-
tion of tobacco osmotia ia PDB to make final osmotia coacea-
tratioas of 1. 10, SOand 100 &nl. The zero osmotia controls
received water or BSA (100 &ml). Spore suspensions (final
conceatratioa ca. 5 x lo4 ml) were incubated at room temper-
atare for 16-22 h or until at least SC%of the spores in the con-
trols had germinated.The total number of spores were counted
in sample aliquots using a hemacytometer. The spore coacen-
trations were expressed relative to the water aad BSA controls.
Each point represents the average of two experiments, with
three replicates per treameat, sampled three ties each. (B)
Effect of OSmotin on germliag viability. Aliquots of spore
suspeasions prepared as de&bad above were stained with
FDA. The nun&r of germiaated 5p~res and number of viable
germliagswere counted in samples treated with 100 &ti BSA
(control) and 100 &nl osmotin. Germination is expressed as
a percent of spore germination compared to the control; germl-
ing viability is expressed as a percent of the total number of
wings. Symbols: AF, A. @US; BC, B. cinerea; FG, F.
gramineawn; FM, F. monilifonne; FOD, F. oxysporum f. Sp.
dianthi; FOL, F. oxysporum f. sp. lycopersicii; TL, T.
longibrachiatum; VD, V. &hliae..
11-23
containing 100 &ml osmotin. Spore germination
was observed in Aspergillusjlavus, Botrytis cinerea
and Fusarium graminearum, the three species
whose spores were not lysed in the presence of
osmotin. F. graminearum spore germination was
reduced to 43% of control by 100 &ml osmotin
(Fig. 2B). No decrease in spore germination was
observed upon 100 &ml osmotin treatment with
either A. flaws or B. cinerea (Fig. 2B). A regres-
sion analysis revealed that there was a significant
correlation between the dose of osmotin present in
the treatment and the effect on the fungus (either
spore lysis or spore germination reduction) for F.
graminearum, F. moniliforme, F. oxysporum f. sp.
dianthi, F. oxysporum f. sp. lycopersici, T.
longibrachiatum and V. dahliae. There was no such
correlation with A. flaws or B. cinerea (Table 2).
The analysis indicated that there was a significant
difference between the effective dose of osmotin to
obtain spore germination reduction in F.
graminearum and the dose required to cause spore
lysis in F. moniliforme, F. oxysporum f. sp. dianthi,
F. oxysporum f. sp. lycopersici, T. longibrachiatum
and V. aizhliae. There was no significant difference
in the effective dose amongst the five lysis-sensitive
fungi.
3.1.3. Spore viability assays
The spores of the resistant fungal species F.
graminearum, A. jlavus and B. cinerea which ger-
minated in the presence of osmotin (Fig. 2A) were
examined by treatment with FDA, a dye that
causes fluorescence of living cells. Viability of
germlings was reduced to 75% and 87% of un-
treated controls in B. cinerea and F. graminearum,
respectively, in the presence of 100 &ml osmotin
(Fig. 2B). Neither spore germination nor germling
viability was affected in A. flaws at this osmotin
concentration.
The data shown in Tables 1,2 and Figs. 1,2 in-
dicate that osmotin is active against specific fungal
species and can act at different growth stages of
the fungi. A qualitative correlation was observed
between results of the hyphal growth inhibition
tests and spore tests. For example, spore germina-
tion of the fungus A. flaws, which was insensitive
to 100 fig osmotin in the hyphal growth inhibition
tests, was unaffected by 100 &ml osmotin and the
 L.R. Abad et al. /Plant Science 118 (19%) II-23 17

Table 2
Comparison of osmotin ED, on fungal spores

Fungus Effect on spores EDso

(rg/ml)
A. jlavus Nonea
B. cinerea Nonea -
F. graminearum Germination reduction 879.5~~
l? moniliforme Lysis 5.1C
F. oxysporum f. sp. dianthi Lysis 10.OC
F. oxysporum f. sp. lycopersici Lysis 4.6
T. longibrachiarum Lysis 11.Y
V. dnhliae Lysis 24.6

The EDa, i.e. dose at which 50% of the spores were affected by osmotin, was estimated form the regression line of the fungal species
exhibiting a response to the treatment.
*The spores of these fungi were not lysed and spore germination was unaffected by osmotin.
%s ED, value was significantly different from the EDso values marked with superscript c (EDSo values were subjected to an
ANOVA, and a Fishers LSD comparison was made).
%ese ED, values were not significantly different from one another (ED, values were subjected to an ANOVA, and a Fishers LSD
comparison was made).

germlings were all viable. B. cinerea, which was
marginally sensitive to 100 pg osmotin in the
hyphal assay, exhibited no spore lysis at 100 &ml
osmotin. Spore germination was also unaffected at
100 &ml osmotin. However, the germlings were
somewhat sensitive to osmotin and only 75% sur-
vived the treatment. Fungi of the genera Fusarium
and Verticillium were more sensitive than the
Botrytis or Aspergiliw strains in the hyphal growth
inhibition assay and their spores were either lysed
in the presence of osmotin (Fig. 2A) or spore ger-
mination and germling viability were affected (Fig.
2B). These results also suggested that molecules
that are recognized by osmotin and cause osmotin
sensitivity are present in a large number of fungi
and that the accessibility of these molecules to
osmotin is affected at different stages of the
growth cycle, such as hyphae, spores or germlings,
by unknown mechanisms.
of DMO uptake by hyphae of the osmotin-
sensitive fungus T. longibrachiatum in a
concentration-dependent manner (Fig. 3A). The
decrease in DMO uptake was linear for 10 mm
(not shown). At all concentrations tested, osmotin
had no effect on DMO uptake into the osmotin-
insensitive R. solani hyphae (Fig. 3B). Thus, the
ability of osmotin to dissipate the plasma mem-
brane pH gradient correlates with its effects on
hyphal growth.
Certain antifungal proteins such as leaf thionins
are effective in vitro on plant protoplasts as well
[30]. However, osmotin had no effect on DMO up-
take by tobacco cells at concentrations that com-
pletely abolished DMO uptake into T.
Iongibrachiatum (Fig. 3C). The protonophore
FCCP (carbonyl cyanide p-trifluoromethoxy-
phenylhydrazone), however, inhibited DMO up-
take by tobacco cells (not shown).

3.2. Osmotin acts on sensitive fungal species to
dissipate the plasma membrane pH gradient
Fungal and plant cells maintain a proton pH
gradient between the cytosol and the external me-
dium. This gradient provides the driving force for
the uptake of weak acids such as DMO that can be
used as pH probes. Osmotin decreased the amount
3.3. Osmotin action is affected by salts and sorbitol
The inhibitory effect of osmotin on T. longi-
brachiatum spore germination was blocked by salts
(Table 3; Fig. 4). Also, in the presence of 50 mM
NaCl, osmotin (up to 15 rg) was unable to affect
the amount of DMO taken up by T. longi-
brachiatum hyphae (not shown). These results fur-
18 L.R. Abad et al. /Plant Science 118 (19%) II-23

suggesting that the effect was specific for inorganic

cations.

3.4. Osmotin is not active against plasmolyzed cells

It has been postulated that osmotin-like proteins

have a permeabilizing activity on fungal mem-
branes [23]. However, the effects of these proteins
on fungal cells were demonstrated under
0 S 10 IS 20 2s hypotonic conditions that can cause the growing
Osmotin @g/ml)
cells to burst if the treatment resulted in a weaken-
ed cell wall. We therefore treated hyphae of T.
longibrachiatum with osmotin under hypertonic
and hypotonic conditions (Fig. 5). Under
hypotonic conditions (Fig. 5A,B), the uptake of
FDA was greatly reduced in the presence of
osmotin (Fig. 5B). In medium containing 1.5 M
sorbitol, plasmolysis occurred, and the plasmolyz-
ed cells were able to accumulate FDA equally well
in the presence (Fig. 5C) and absence of osmotin
S 10 1s 20 2s (data not shown). The sorbitol had virtually no
Osmotin (&ml) effect on the ability of osmotin to permeabilize the
hyphae until the concentration was high enough to
observe hyphal cell plasmolysis. Deplasmolyzed
cells obtained by transferring the hyphae from 1.5
to 0.15 M sorbitol became sensitive to osmotin and
did not accumulate FDA (Fig. 5D). Although
these experiments demonstrate that close jux-
taposition of the cell wall and membrane are
l- required or facilitate osmotin action, we can not
eliminate some role of turgor in osmotin sensi-
o, . I . I I . 1 tivity.
0 S 10 1s 20 2s
Osmotin (&ml)
4. Dlseusslon
Fig. 3. Effect of osmotin on the ability of cells to maintain a pH
gradient. DMO uptake in IO min by (A) T. longibrachiatum
4. I. Species specificity
hyphae, (B) R so&k hyphae; (C) cultured cells of N. tabacum
L. var. Wisconsin 38. The experiments were performed as The results of this study indicate that tobacco
described in Section 2, Materials and methods. osmotin is effective against a range of economical-
ly important fungal pathogens. The host range and
the diseases caused by some of the test fungi are
listed in Table 4. Fifteen of the 18 genera tested
ther supported the hypothesis that osmotin exhibited some sensitivity to osmotin in vitro
antifungal action is the result of its ability to (Table 1). Although the fungi in this study were
dissipate the plasma membrane pH gradient, since selected for their economic importance and not for
both processes are similarly affected by NaCl. their taxonomic distinction, representatives of
Choline Cl was not able to influence osmotin in- Ascomycetes, Hyphomycetes, Oomycetes and
hibition of T. longibrachiatum spore germination, Basidiomycetes were among those which were sen-
 L.R. Abad et al. /Plant Science 118 (19%) 11-23 19

Table 3
Reversal of osmotin inhibition of T. longibrachiarum spore
germination by salts

Compounds Concentration for 5O?/oreversal

(mM)

NaCl 40
KC1 35
MgClz 40
Na*HPO, 40
Na,S04 20
NaHCO, 40
04 r
I
80
I

Spore suspensions of T. longibrachiatum (final concentration 0 20 40 60

Concentration of NaCl (mM)
ca. 5 x IO3 per ml) in PDB were incubated at room tempera-
ture with various concentrations of the indicated salts (O-80
mM) in the absence or presence of osmotin (20 &ml). Fungal ;looo.
growth was measured after 48 h as absorbance at 410 nm. The
concentration of salt required for 50% reversal of osmotin in- b5 80. \y%
hibition of growth was calculated in each instance from data s \b- _ -
similar to those shown in Fig. 4. 5 -- ----L- _ ____;
60.
r
g 40-
sitive to tobacco osmotin in hyphal growth inhibi-
3
tion assays. This wide-ranging effect suggests that I?
p 203 : -..&-+---~;
the use of osmotin as a novel defense gene effective
against a large number of pathogens is feasible. 0 I I I I
However, osmotin is not universally effective 0 20 40 60 80
Concentration of CholineCl (pglml)
against all fungi. A. flavus, A. parasitica, 44.
phaseolina and R. solani are all pathogens that
occur worldwide on many hosts and appear to be Fig. 4. Inhibitory effect of ions on osmotin inhibition of spore
impervious to the effects of tobacco osmotin. The germination. (A) Effect of NaCl. (B) Effect of choline chloride.
general trend provided by these data suggests that T. longibrachiatutn spore suspensions (final concentration ca.
5 x IO3 conidia/ml) in PDB were mixed with osmotin (final
sensitivity might be determined on the genus level. concentration 20 &/ml) and the indicated amounts of NaCl or
If this is true, certain genera of pathogens could be choline chloride and then incubated at room temperature for 48
targeted for specific crop plants, increasing the h. Fungal growth was measured as absorbance at 410 nm. Sym-
usefulness of the conferred resistance. bols: (0), minus osmotin; (0). plus osmotin.
The antifungal effect of osmotin appears to
operate on hyphae and spores. Hyphal growth was
inhibited to varying degrees in most of the isolates be the result of different levels of sensitivity to
tested. A similar phenomenon has been observed osmotin. The most sensitive spores lysed in the
in fungi treated with chitinases and p-1,3- presence of osmotin while less sensitive spores did
glucanases [31]. In five of the eight fungi tested, not lyse, but were killed before germination could
spore lysis occurred in the presence of osmotin. occur. Other fungi, like B. cinerea, germinated but
This lytic effect of osmotin has been reported in were subsequently killed. At the end of the spec-
osmotin-treated sporangia of the Oomycete trum were fungi such as A. flrtvus, which were
pathogen P. infestans [12]. Of the fungi whose largely unaffected by osmotin. It is important to
spores were not lysed by osmotin, treatment with note, however, that there is variability within a
osmotin resulted in a reduction in spore germina- spore population to the antifungal effect of
tion in F. graminearum and a reduction of germl- osmotin. That is, a proportion of spores, even in
ing viability in B. cinerea. These effects appear to the most sensitive fungi, were not lysed at the
20 L.R. Abader al./PIanr Science 118 (19%) 11-23

-FDA

+FDA

Fig. 5. Effect of osmoticum on osmotin action on T. longibrachiatum hyphae as measured by FDA staining. All treatments with
osmotin (final concentration 40 &ml) were for IS min. (A) Hyphae in PDB. (B) Hyphae in PDB containing osmotin. (C) Hyphae
in PDB containing 1.5 M sorbitol and osmotin. (D) Hyphae in PDB containing I .5 M sorbitol were transferred to PDB containing
0.15 M sorbitol and treated with osmotin in the latter medium.

Table 4
Host range of and diseases caused by the pathogens tested for sensitivity to tobacco osmotin

Pathogen Host Typical disease

Altemaria sohmi Tomato, potato, others Leaf, stem, fruit blight

Aspergilus flnvus Peanut, maize, others Seed rot
Bipolaris may& Maize Leaf blight
Bipolaris zeicola Maize Leaf spot
Borryris cinerea Many hosts Petal, fruit blights
Cercospora zeae-maydis Maize Leaf spot
Clodoporiton cucumerinum Cucurbits Scab
Colktorrichum gleosparioiaks Many hosts Anthracnose
CoNero~richwngrambaicola Maize, Sorghum Anthracnose
Colierorrichum laginarium Cucurbits Anthracnose
CoNetotrichum sublineolum Sorghum Anthracnose
Fusarium graminearum Maize, Sorghum, wheat Stalk, root, kernel rot
Fuwrium monirifrme Maize, Sorghum, others Stalk, root, kernel rot
Fusariwn oxysporum f. sp. dianthi Carnation Wilt
Fusarium oxysporum f. sp. lycopersici Tomato Wilt
Fusarium roseum Sambucinum Potato Tuber rot
Kabar&ila zeae Maize Leaf spot
Macrophomina phaseolina Many hosts Root, stem rot
Magnaporthe grisea Rice Leaf blight
Periconia circina~a Sorghum Root rot
Phyrophrhora infesstans Potato, tomato Leaf blight
Phyrophthora parasirica var. dianthi Carnation Root rot
Phytophlhora parasitica var. nicoknae Tobacco Root rot
Rhizocronia salani Many hosts Root, stem rot
Stenocarpelka tnaydir Maize Bar rot
Verricillium dohliae Many hosts Wilt
 L.R. A&d et al. /Plant Science 118 (19%)
highest concentrations of osmotin used in the tests.
These data are consistent with the observations of
Liu et al. [20] who showed that osmotin overex-
pression in transgenic potato delays but does not
prevent development of disease symptoms of black
shank disease.
4.2. Mechanism of Action
Roberts and Selitrennikoff [23] showed that
zeamatin, a corn protein with sequence homology
to osmotin, caused leakage of preloaded
[ C]amino isobutyric acid, a non-metabolizable
amino acid, from Neurospora crassa cells. They
also showed that zeamatin induced hyphal rupture
in N. crassa. Based on these data, they proposed
that zeamatin may be causing lysis by direct inser-
tion into the membrane to form transmembrane
pores.
We have provided evidence that osmotin can
cause membrane leakage and dissipate the pH gra-
dient across the cell wall/membrane of a sensitive
fungal species. The species specificity of osmotin
inhibition of growth and the physiological effect
on the plasma membrane suggests the existence of
membrane receptors. The fact that osmotin can
function on cells only under hypotonic conditions
suggests that cell walls may be required or at least
facilitate osmotin action. Observations consistent
with the idea that cell wall properties affect the
activity of osmotin have been made in the case of
zeamatin. Zeamatin-induced hyphal rupture oc-
curs mostly at the tips or behind the hyphal apical
dome [23] where the cell wall is being deposited
and has unique structural and chemical features
[32]. Also, the activity of osmotin-like proteins is
facilitated by nikkomycin, an inhibitor of chitin
biosynthesis [22,23]. Our data, however, can be
explained without involving the cell wall by invok-
ing the necessity of a particular conformation of
plasma-membrane constituent(s) that is disturbed
by osmotically-induced volume changes in the
fungal cells. They also are consistent with the theo-
ry that osmotin antifungal activity may not be able
to work under high osmoticum.
The action of osmotin on fungal cells was in-
hibited by salts. Such an effect has been observed
for other antifungal proteins [23,33,34,35]. The
significance of the salt effect is not known, but it
II-23 2
may be related to an ionic effect on a receptor or
target since in some cases the effects of salts are
species specific [36]. The lack of effect with choline
chloride might suggest that only inorganic cations
can reverse the action of osmotin.
5. Conclusiolls
We have presented results here that indicate that
osmotin could function as a novel source of resis-
tance against a wide range of fungal pathogens.
Because of its relatively non-selective effect on
fungal pathogens and because some spores of any
given population appear to remain viable even at
high osmotin concentrations, overexpression of
the osmotin gene may boost the level of resistance
in a plant without putting an undue amount of se-
lection pressure on the target pathogens resulting
in a loss of durability. Data presented in this paper
bring forth the possibility that there is a receptor
for osmotin which exists in a large variety of
fungal species and that its accessibility is affected
by the growth stage of the fungus and other factors
such as salt, sorbitol (high osmoticurn?) or nik-
komycin (inhibitor of cell wall chitin biosynthesis)
and cell wall properties. It is therefore predicted
that the efficacy and spectrum of osmotin action in
transgenic plants overexpressing the osmotin gene
can be enhanced by (1) the co-expression of
glucanases or chitinases which would result in a
weakened cell wall of the pathogen, (2) the co-
application of small amounts of fungicides or
chemicals which would weaken the fungus to im-
prove accessibility to osmotin. In this category of
chemicals could be inhibitors of some step of cell
wall biosynthesis, or even general inhibitors of
protein/RNA synthesis. There are, in fact, reports
of synergism between chitinaseiglucanase com-
binations and chitinase/antibiotic combinations
[31,37-391.
Acknowledgements
This work was supported by grants from the
National Peanut Foundation, Pioneer Hi-Bred In-
ternational, The Consortium for Plant
Biotechnology Inc. and BARD No. US-2233-92.
Dong Liu was supported by a fellowship from The
Rockefeller Foundation. This is journal paper
22 L.R. Abad et al. /Plant Science 118 (1996) II-23
number 14887 of the Purdue University Agricul-
tural Experiment Station. Gifts of fungal stocks
from Dr R. Hammerschmit, University of
Michigan; Dr A. Barta, Mycogen Plant Sciences;
Dr Y. Elad, Volcani Center, Israel; Dr R. Hanau,
Purdue University; Dr L. Dunkle, Purdue Univer-
sity; Dr T. Kommedahl, University of Minnesota;
Dr J. Hamer, Purdue University; Dr W. Fry,
Cornell University; Dr J. Chappell, University of
Kentucky and Dr R. Green, Purdue University are
gratefully acknowledged. We thank MS Jean
Clithero for excellent technical assistance.
References
Ill D.-J. Yun, R.A. Bressan and P.M. Hasegawa, Plant anti-
fungal proteins, Plant Breed. Rev., (1996) in press.
121L.C. Van Loon and A. Van Kammen, Polyacrylamide
disc electrophoresis of the soluble leaf proteins from
Nicotia?ta tabacwn var. Samsun and Samsun NN II.
Changes in protein constitution after infection with
tobacco mosaic virus. Virology, 40 (1970) 199-211.
[31 J.F. Bol, H.J.M. Linthorst and B.J.C. Cornelissen, Plant
pathogenesis-related proteins induced by virus infection.
Annu. Rev. Phytopathol., 28 (1990) 113-138.
[41 H.J.M. Linthotst, Pathogenesis-related proteins of
plants. 0%. Rev. Plant Sci., 10 (1991) 123-150.
151 A. Stintzi, T. Heitz, V. Prasad, S. Wiedemann-
Merdinoglu, S. Kaufmann, P. Geoffroy, M. Legrand and
B. Fritig, Plant pathogenesis-related proteins and their
role in defense against pathogens. Biocbimie, 75 (1993)
687-706.
I61 L.C. Van Loon, W.S. Pierpoint, T. Boller and V. Cone-
jero, Recommendations for naming pathogenesis-related
proteins. Plant Mol. Biol. Rep., 12 (1994) 413-437.
[71 L.C. Van Loon, Y.A.M. Gerritaen and C.E. Ritter, Iden-
tification, purification, and characterization of
pathogenesis-related proteins from virus-infected Sam-
sun NN tobacco leaves. Plant. Mol. Biol., 9 (1987)
593-609.
Bl D. Alexander, R.M. Goodman, M. Gut-Rella, C.
Glascock, K. Weymann, L. Friedrich, D. Maddox, P.
Ahl-Goy, T. Luntz, E. Ward and J. Ryals, Increased tol-
erance to two oomycete pathogens in transgenic tobacco
expressing pathogenesis-related protein la. Proc. Natl.
Acad. Sci. USA, 90 (1993) 7327-7331.
[91 K. Broglie, I. Chet, M. Holliday, R. Cressman, P. Biddle,
S. Knowlton, C.J. Mauvalis and R. Broglie, Transgenic
plants with enhanced resistance to the fungal pathogen
Rhizoctonia solani. Science, 254 (199 1) 1194- 1197.
WI A.S. Ponstein, S.A. Bres-Vloemans, M.B. Sela-Buurlage,
P.J.M. van den Elzen, L.S. Melchers and B.J.C. Cor-
nelissen, A novel pathogen- and wound-inducible tobac-
co (Nicotiana &&mm) protein with antifungal activity.
Plant Physiol., 104 (1994) 109-118.
Ill1 M.B. Sela-Buurlage, A.S. Ponstein, S.A. Bres-Vloemans,
L.S. Melchers, P.J.M. van den Elzen and B.J.C. Cor-
nelissen, Only specific tobacco (Nicotiam tabacxm)
chitinases and &1,3glucanases exhibit antifungal activ-
ity. Plant Physiol., 101 (1993) 857-863.
I121 C.P. Woloshuk, J.S. Meulenhoff, M. Sela-Buurlage,
P.J.M. van den Elzen and B.J.C. Comelissen, Pathogen-
induced proteins with inhibitory activity toward Phyto-
phthora infestam. Plant Cell, 3 (1991) 619-628.
El31 A.J. Vigers, W.K. Roberts and C.P. Selitrennikoff, A
new family of plant antifungal proteins. Mol. Plant-
Microbe Interact., 4 (1991) 315-323.
v41 A.J. Vigers, S. Wiedemann, W.K. Roberts, M. Legrand,
C.P. Selitrennikoff and B. Fritig, Thaumatin-like
pathogenesis-related proteins are antifungal. Plant Sci.,
83 (1992) 155-161.
1151 H. Van der We1 and K. Loeve, Isolation and
characterization of thaumatin I and II, the sweet-tasting
proteins from Thaumatococcus aianiellii Benth. J.
B&hem., 31 (1972) 221-225.
WI L. Edens, L. Heslinga, R. Klok, A.M. Ledeboer, J. Maat,
M.Y. Toonen, C. Visser and C.T. Vet-rips, Cloning of
cDNA encoding the sweet-tasting plant protein
thaumatin and its expression in Escherichia coli. Gene, 18
(1982) 1-12.
u71 N.K. Singh, A.K. Handa, P.M. Hasegawa and R.A.
Bressan, Protein associated with adaptation of cultured
tobacco cells to NaCl. Plant Physiol., 79 (1985) 126- 137.
WI N.K. Singh, C.A. Bracker, P.M. Hasegawa, A.K. Handa,
S. Buckel, M.A. Hermodson, E. Pfankoch, F.E. Regnier,
R.A. Bressan, Characterization of osmotin. A
thaumatin-like protein associated with osmotic adapt-
ation in plant cells. Plant Physiol., 85 (1987) 529-536.
1191 N.K. Singh, D.E. Nelson, D. Kuhn, P.M. Hasegawa and
R.A. Bressan, Molecular cloning of osmotin and regula-
tion of its expression by ABA and adaptation to low
water potential. Plant Physiol., 90 (1989) 1096-l 101.
PI D. Liu, K.G. Raghothama, P.M. Hasegawa and R.A.
Bressan, Ckmotin overexpression in potato delays
development of disease symptoms. Proc. Natl. Acad. Sci.
USA, 91 (1994) 1888-1892.
WI A.K. Kononowicz, K.G. Raghothama, A.N. Casas, D.E.
Nelson, D. Liu, M.L. Naraaimhan, P.C. LaRosa, N.K.
Singh, R.A. Bressan, P.M. Hasegawa, Structure, regula-
tion and function of the osmotin gene, in: J.H. Cherry
(Ed.), Biochemical and Cellular Mechanisms of Stress
Tolerance in Plants, NATO-AS1 Series, Vol. H86,
Springer-Verlag, 1994, pp. 381-414.
P21 J. Hejgaard, S. Jacobsen and I. Svendsen, Two antifungal
thaumatin-like proteins from barley grain. FEBS L&t.,
291 (1991) 127-131.
v31 W.K. Roberts and C.P. Selitrennikoff, Zeamatin, an
antifungal protein from maize with membrane-
permeabilizing activity. J. Gen. Microbial., 136 (1990)
1771-1778.
1241 D.J. Finney, Probit Analysis. A Statistical Treatment of
the Sigmoid Response Curve. Cambridge University
Press, 1952.
 L.R. Abad et al./Plant
WI H. Rottenbcrg, The measurement of membrane potential
and pH in cells, organelles, and vesicles. Methods
Enzymol., 55 (1979) 547-567.
1261M.M. Reuveni, H.R. Lemer and A. Poljakoff-Mayber,
Changes in membrane potential as a demonstration of
selective pore formation in the plasmalemma by poly-L-
lysine treatment. Plant Physiol., 79 (1985) 406-410.
1271 M.M.Reuveni, H.R. Lemer and A. Poljakoff-Mayber,
Osmotic adjustment and dynamic changes in ion distri-
bution of low molecular weight solutes between compart-
ments of carrot and beet root cells exposed tJ salinity.
Am. J. Bot., 76 (1991) 601-609.
WI M.L. Bit&, P.M. Hasegawa, A.K. Handa and R.A.
Bressan, Adaptation of tobacco cells to NaCI. Plant
Physiol., 79 (1985) 118-125.
1291 J.M. Widholm, The use of fluorescein diacetate and
phenosafranine for determining viability of cultured
plant cells. Stain Technol., 47 (1972) 189-194.
[301 H. Bohlmann and K. Apel, Thionins. Annu. Rev. Plant
Physiol. Plant Mol. Biol., 42 (1991) 227-240.
1311 F. Mauch, B. Mauch-Mani and T. Boiler, Antifungal hy-
drolases in pea tissue. II. Inhibition of fungal growth by
combinations of chitinase and &1,3glucanase. Plant
Physiol., 88 (1988) 936-942.
1321 J.G.H. We&s, Developmental regulation of fungal cell
wall formation. Annu. Rev. Phytopathol., 32 (1994)
413-437.
1331 FRG Terras, LJ. Goderis, F. Van Leuven, J.
Vanderleyden, B.P.A. Cammue and W.F. Broekaert. In
vitro antifungal activity of a radish (RaphanuF sativus L.)
seed protein homologous to nonspecific lipid transfer
proteins. Plant Physiol., 100 (1992) 1055-1058.
Science 118 (19%) 11-23 23
[34] FRG Terras, H.M.E. Schoofs, M.F.C. De Bolle, F. Van
Leuven, S.B. Recs, J. Vanderleyden, B.P.A. Cammue
and W.F. Broekaert, Analysis of two novel classes of an-
tifungal proteins from radish (Raphanus sativw L.) seeds.
J. Biol. Chem., 267 (1992) 15301-15309.
[35] B.P.A. Cammue, M.F.C. De Belle, FRG Terras, P. Pro-
ost, J. Van Damme, S.B. Rees, J. Vanderleyden and W.F.
Broekaert, Isolation and characterization of a novel class
of plant anti-microbial peptides from Mirabilis jalapa L.
seeds. J. Biol. Chem., 267 (1992) 2228-2233.
[36] W.F. Broekaert, J. Van Parijs, A.K. Allen and W.J.
Peumans, Comparison of some molecular, enzymatic and
antifungal properties of chitinases from thorn-apple,
tobacco and wheat. Physiol. Mol. Plant Pathol., 33
(1988) 319-331.
1371 M. Lorito, C.K. Hayes, A. Di Pietro, S.L. Woo and E.
Hatman, Purification, characterization and synergistic
activity of a glucan 1,3-&glucosidase and an N-acetyl-j3-
glucosaminidase from Trichodermn harzianum.
Phytopathology, 84 (1994) 398-405.
[38] M. Lo&o, C. Peterbauer, C.K. Hayes and G.E. Hannan,
Synergistic interaction of fungal cell wall degrading en-
zymes and different compounds enhances inhibition of
spore get&ration. Microbiology, I40 (1994) 623-629.
1391 L.S. Melchers, M.B. Sela-Buurlage, S.A. Vloemans, C.P.
Woloshuk, J.S.C. Van Roekel, J. Pen, P.J.M. Van den
Elzen and B.J.C. Comelissen, Extracellular targeting of
the vacuolar tobacco proteins AP 24, chitinasc and o-1,3-
glucanase in transgenic plants. Plant Mol. Biol., 21
(1993) 583-593.