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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
Table 1
Inhibition of hyphal growth 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 autoclaved for 20 min at 120C. Spore germination
assays were performed on a modified PDA assays were conducted in PDB.
(PDwA) prepared with washed agar. To prepare
PDwA (1 liter), agar (20 g; Difco) was washed by 2.3. Measurement of antificngal activity
stirring in a large volume of distilled water for
8-12 h, exchanging water 3-4 times during this 2.3. I. Hyphal growth inhibition assays
period. The washed agar was collected by tiltra- An agar plug containing mycelia of the test fun-
tion, added to 36 g potato dextrose broth (PDB; gus was placed at the center of a PDwA plate.
Sigma), brought up to a volume of 1 liter and 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, line of those fungal species exhibiting a response to
at which time sterile filter paper disks were posi- the treatment. The EDSo values of these species
tioned adjacent to the colony margin. The disks were in turn subjected to an ANOVA, and a
were saturated with 20 hl of either water, bovine Fishers LSD comparison made.
serum albumin (BSA; 100 pg; Sigma) or tobacco To determine the effect of various salts on the
osmotin (30,60 or 100 rg) and the plates were fur- antifungal activity of osmotin, 150 ~1 of a T.
ther incubated for several days. Growth inhibition longibrachiatum spore suspension in 2 x PDB (ca.
around each disk was then evaluated visually. 1 x lo4 conidiaml) were dispensed into each well
Experiments were conducted twice and the data of a 96-well microtiter plate. Salt solutions and
pooled. osmotin were added to each well as desired and the
volume was adjusted to 300 ~1 with distilled water.
2.3.2. Spore lysis and germination assays After incubation at room temperature for 48 h,
Fifty microlitres of fungal spores suspended in fungal growth was quantitated by measuring the
2 x PDB (ca. 1 x 10 conidia/ml) were mixed absorbance at 410 nm with a microtiter plate
with 50 11 of water (control), BSA (control) or reader (Bio-Tek Instruments, Winooski, VT).
tobacco osmotin solution, respectively. The final
treatments contained 5 x lo4 conidia/ml with 0, 2.4. Determination of membrane leakiness
1, 10, 50 and 100 &ml osmotin and 100 &ml
BSA (control). Spore suspensions were incubated Plasma membrane integrity was estimated from
at room temperature for 16-22 h or until at least the measurement of the capacity of the cells to
50% of the spores in the control treatments ger- maintain a pH gradient between the external medi-
minated. After incubation, the total number of um and the cytosol. This was accomplished by
spores and the number of germinated spores were determining the distribution of the weak acid
counted in an aliquot of each sample using a 5,5dirqethyl-[2-4C]oxazolidine-2,4-dione (DMO;
Spotlite hemacytometer (American Scientific Amersham, Arlington Heights, IL) between the
Products, McGaw Park, IL). The number of viable cells and the external solution 1251.The weak acid
spores and germlings was estimated by staining will accumulae in the alkaline compartment of the
sample aliquots with fluorescein diacetate (FDA) cell, i.e. the cytoplasm [25]. The procedure for
and counting the fluorescent spores and/or germ using DMO was described by Reuveni et al.
tubes using the hemacytometer. For staining with [26,27] for cytoplasmic pH estimation. The rela-
FDA, 5 ~1 of a FDA stock solution (50 mg in 100 tive cytoplasmic volume, that is, the volume of the
ml acetone) were mixed with 100 ~1 of spore sus- cells cytoplasm as a percent of the cells volume,
pension. Each treatment contained three replicates was determined using t3H]H20 (Amersham, Arl-
and three samples from each of these replicates ington Heights, IL) and [r4C]sorbitol (Amersham,
were counted. Experiments were conducted twice Arlington Heights, IL) [26]. The amount of DMO
and data from the two experiments were pooled. taken up by the cells was corrected using the rela-
The data for each spore germination experiment tive cytoplasmic volume. For measurement of
were transformed for regression analysis. Treat- DMO uptake, tobacco cells or fungal hyphae were
ment concentrations were log transformed and suspended in their respective growth media. The
spore concentration or germination was expressed suspension cultures of tobacco cells used in these
first as percent inhibited (or lysed) compared to experiments were maintained as described by
the control (water treatment), then this percent in- Binzel et al. 1281.Suspension cultures of the fungi
hibition was transformed into probits [24]. Sepa- were prepared as follows: 2day-old PDA plate
rate regression analyses were performed on each cultures of the fungus were flooded by swirling
experiment and analysis of variance (ANOVA) on PDB (5 ml) on the surface and the hyphal pieces
the slopes of each experiment was completed. The so obtained were used to inoculate 200 ml of PDB.
dose at which 5O?h of the spores are affected by The suspension culture was grown overnight with
osmotin (ED,) was estimated from the regression 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 fungal isolates tested, ranging from the very sensi-
suspended in PDB containing the desired amount tive Trichodenna longibrachiatum to the complete-
of sorbitol. The hyphae were stained as described ly insensitive Rhizoctonia solani, Macrophomina
by Widholm [29]. Photographs were taken with an phaseolina and Aspergillus spp. (Fig. 1; Table 1).
Olympus Vanox microscope fitted with an There appeared to be a loose correlation between
automatic camera. members of the same genus and the amount of
hyphal growth inhibition evident in this assay.
3. Results Phytophthora and Colletotrichum appeared to be
moderately sensitive as a genus, while Aspergillus
3.1. Osmotin is specifically active against certain seemed to be insensitive. The five isolates of
fungal species Fusarium were all sensitive to some extent. How-
ever, F. graminearum and F. monil$orme exhibited
3. I. 1. Hyphal growth inhibition assays less inhibition than F. oxysporum and F. roseum,
Thirty-one fungal isolates, comprising 18 dif- reflecting the variability within members of a
ferent genera, were tested for hyphal growth in- genus.
hibition in the presence of tobacco osmotin (Table
1). The hyphal growth of most fimgal species 3.1.2. Spore iysis and germination inhibition assays
tested in this manner was inhibited when exposed Eight fungi were chosen to examine the effect of
to osmotin, with more inhibition evident at higher osmotin on spores. In the presence of osmotin,
concentrations. Zones of growth inhibition were spore lysis i.e. disappearance of entire spores leav-
detectable surrounding filter paper disks saturated ing behind cell wall debris resulting in decrease in
with 30 pg osmotin in 19 of the 31 isolates; 27 of spore concentration in the osmotin treatments
the same 31 isolates were inhibited by disks con- compared with the controls, occurred in five of the
taining 100 pg osmotin. Overall, there was a varia- eight fungi tested (Fig. 2A). All five fungi had
tion in the degree of sensitivity exhibited by the 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%) 11-23
Table 2
Comparison of osmotin ED, on fungal spores
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 of DMO uptake by hyphae of the osmotin-
marginally sensitive to 100 pg osmotin in the sensitive fungus T. longibrachiatum in a
hyphal assay, exhibited no spore lysis at 100 &ml concentration-dependent manner (Fig. 3A). The
osmotin. Spore germination was also unaffected at decrease in DMO uptake was linear for 10 mm
100 &ml osmotin. However, the germlings were (not shown). At all concentrations tested, osmotin
somewhat sensitive to osmotin and only 75% sur- had no effect on DMO uptake into the osmotin-
vived the treatment. Fungi of the genera Fusarium insensitive R. solani hyphae (Fig. 3B). Thus, the
and Verticillium were more sensitive than the ability of osmotin to dissipate the plasma mem-
Botrytis or Aspergiliw strains in the hyphal growth brane pH gradient correlates with its effects on
inhibition assay and their spores were either lysed hyphal growth.
in the presence of osmotin (Fig. 2A) or spore ger- Certain antifungal proteins such as leaf thionins
mination and germling viability were affected (Fig. are effective in vitro on plant protoplasts as well
2B). These results also suggested that molecules [30]. However, osmotin had no effect on DMO up-
that are recognized by osmotin and cause osmotin take by tobacco cells at concentrations that com-
sensitivity are present in a large number of fungi pletely abolished DMO uptake into T.
and that the accessibility of these molecules to Iongibrachiatum (Fig. 3C). The protonophore
osmotin is affected at different stages of the FCCP (carbonyl cyanide p-trifluoromethoxy-
growth cycle, such as hyphae, spores or germlings, phenylhydrazone), however, inhibited DMO up-
by unknown mechanisms. take by tobacco cells (not shown).
3.2. Osmotin acts on sensitive fungal species to 3.3. Osmotin action is affected by salts and sorbitol
dissipate the plasma membrane pH gradient
The inhibitory effect of osmotin on T. longi-
Fungal and plant cells maintain a proton pH brachiatum spore germination was blocked by salts
gradient between the cytosol and the external me- (Table 3; Fig. 4). Also, in the presence of 50 mM
dium. This gradient provides the driving force for NaCl, osmotin (up to 15 rg) was unable to affect
the uptake of weak acids such as DMO that can be the amount of DMO taken up by T. longi-
used as pH probes. Osmotin decreased the amount brachiatum hyphae (not shown). These results fur-
18 L.R. Abad et al. /Plant Science 118 (19%) II-23
Table 3
Reversal of osmotin inhibition of T. longibrachiarum spore
germination by salts
NaCl 40
KC1 35
MgClz 40
Na*HPO, 40
Na,S04 20
NaHCO, 40
04 r
I
80
I
-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
highest concentrations of osmotin used in the tests. may be related to an ionic effect on a receptor or
These data are consistent with the observations of target since in some cases the effects of salts are
Liu et al. [20] who showed that osmotin overex- species specific [36]. The lack of effect with choline
pression in transgenic potato delays but does not chloride might suggest that only inorganic cations
prevent development of disease symptoms of black can reverse the action of osmotin.
shank disease.
5. Conclusiolls
4.2. Mechanism of Action
We have presented results here that indicate that
Roberts and Selitrennikoff [23] showed that osmotin could function as a novel source of resis-
zeamatin, a corn protein with sequence homology tance against a wide range of fungal pathogens.
to osmotin, caused leakage of preloaded Because of its relatively non-selective effect on
[ C]amino isobutyric acid, a non-metabolizable fungal pathogens and because some spores of any
amino acid, from Neurospora crassa cells. They given population appear to remain viable even at
also showed that zeamatin induced hyphal rupture high osmotin concentrations, overexpression of
in N. crassa. Based on these data, they proposed the osmotin gene may boost the level of resistance
that zeamatin may be causing lysis by direct inser- in a plant without putting an undue amount of se-
tion into the membrane to form transmembrane lection pressure on the target pathogens resulting
pores. in a loss of durability. Data presented in this paper
We have provided evidence that osmotin can bring forth the possibility that there is a receptor
cause membrane leakage and dissipate the pH gra- for osmotin which exists in a large variety of
dient across the cell wall/membrane of a sensitive fungal species and that its accessibility is affected
fungal species. The species specificity of osmotin by the growth stage of the fungus and other factors
inhibition of growth and the physiological effect such as salt, sorbitol (high osmoticurn?) or nik-
on the plasma membrane suggests the existence of komycin (inhibitor of cell wall chitin biosynthesis)
membrane receptors. The fact that osmotin can and cell wall properties. It is therefore predicted
function on cells only under hypotonic conditions that the efficacy and spectrum of osmotin action in
suggests that cell walls may be required or at least transgenic plants overexpressing the osmotin gene
facilitate osmotin action. Observations consistent can be enhanced by (1) the co-expression of
with the idea that cell wall properties affect the glucanases or chitinases which would result in a
activity of osmotin have been made in the case of weakened cell wall of the pathogen, (2) the co-
zeamatin. Zeamatin-induced hyphal rupture oc- application of small amounts of fungicides or
curs mostly at the tips or behind the hyphal apical chemicals which would weaken the fungus to im-
dome [23] where the cell wall is being deposited prove accessibility to osmotin. In this category of
and has unique structural and chemical features chemicals could be inhibitors of some step of cell
[32]. Also, the activity of osmotin-like proteins is wall biosynthesis, or even general inhibitors of
facilitated by nikkomycin, an inhibitor of chitin protein/RNA synthesis. There are, in fact, reports
biosynthesis [22,23]. Our data, however, can be of synergism between chitinaseiglucanase com-
explained without involving the cell wall by invok- binations and chitinase/antibiotic combinations
ing the necessity of a particular conformation of [31,37-391.
plasma-membrane constituent(s) that is disturbed
by osmotically-induced volume changes in the Acknowledgements
fungal cells. They also are consistent with the theo-
ry that osmotin antifungal activity may not be able This work was supported by grants from the
to work under high osmoticum. National Peanut Foundation, Pioneer Hi-Bred In-
The action of osmotin on fungal cells was in- ternational, The Consortium for Plant
hibited by salts. Such an effect has been observed Biotechnology Inc. and BARD No. US-2233-92.
for other antifungal proteins [23,33,34,35]. The Dong Liu was supported by a fellowship from The
significance of the salt effect is not known, but it 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- Ill1 M.B. Sela-Buurlage, A.S. Ponstein, S.A. Bres-Vloemans,
tural Experiment Station. Gifts of fungal stocks L.S. Melchers, P.J.M. van den Elzen and B.J.C. Cor-
nelissen, Only specific tobacco (Nicotiam tabacxm)
from Dr R. Hammerschmit, University of
chitinases and &1,3glucanases exhibit antifungal activ-
Michigan; Dr A. Barta, Mycogen Plant Sciences; ity. Plant Physiol., 101 (1993) 857-863.
Dr Y. Elad, Volcani Center, Israel; Dr R. Hanau, I121 C.P. Woloshuk, J.S. Meulenhoff, M. Sela-Buurlage,
Purdue University; Dr L. Dunkle, Purdue Univer- P.J.M. van den Elzen and B.J.C. Comelissen, Pathogen-
sity; Dr T. Kommedahl, University of Minnesota; induced proteins with inhibitory activity toward Phyto-
phthora infestam. Plant Cell, 3 (1991) 619-628.
Dr J. Hamer, Purdue University; Dr W. Fry,
El31 A.J. Vigers, W.K. Roberts and C.P. Selitrennikoff, A
Cornell University; Dr J. Chappell, University of new family of plant antifungal proteins. Mol. Plant-
Kentucky and Dr R. Green, Purdue University are Microbe Interact., 4 (1991) 315-323.
gratefully acknowledged. We thank MS Jean v41 A.J. Vigers, S. Wiedemann, W.K. Roberts, M. Legrand,
Clithero for excellent technical assistance. C.P. Selitrennikoff and B. Fritig, Thaumatin-like
pathogenesis-related proteins are antifungal. Plant Sci.,
83 (1992) 155-161.
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