antifungal activity of tobacco osmotin has specificity and

ELSEVIER Plant Science 118 (1996) II-23

CiENCE

Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization

Laura R. Abada, Matilde Paino D’Urzo”, 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- diiethyl-[2-‘*CJoxaxolidine-2,4ione; FCCP, carbonyl cyanide ptrifluoromethoxyphenylhydraxone; FDA, fluorescein diacetate; PDA, potato dextrose agar; PDB, potato dextrose broth; PR, pathogenesis-related. l Corresponding author. Tel.: +I 317 494 7858; fax: +l 317

494 0391; e-mail: [email protected]

1. htroduction

Plants accumulate a number of antifungal proteins. Among these proteins are included the

thionins, the ribosome-inactivating proteins, lipid- transfer proteins, 2S storage albumins, non-

0168~9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII: SO I68-9452(96)04420-2

I2 L.R. Abad et a[. lhn~ Science 118 (1996) II-23

enzymatic chitin-binding proteins, polygalacturo- nase inhibitor proteins, the defensins and pathogenesis-related (PR) proteins 111. PR proteins 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 remain 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], suggesting 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 resistance, the spectrum of their antifungal activities needs to be determined. However, there has been no extensive or systematic survey of the phytopathogenic 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 protein from maize, had no chitinase, glucanase, man- nanase, protease, ribonuclease, phospholipase or

ribosome-inactivating activity i23). Ba& on the ability of zeamatin to cause the leakage of cytoplasmic 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 isolated 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 polyacrylamide 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 f. dianthi oxysporum sp. 2 3 3 Fwarium f. lycopersici oxysponun sp. 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 120°C. 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 germinated. 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 suspension. 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 inhibition 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 Fisher’s 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 medium 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 relative 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 relative 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 inhibition 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 containing 100 pg osmotin. Overall, there was a varia- tion in the degree of sensitivity exhibited by the

fungal isolates tested, ranging from the very sensitive 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. oxysporum f. sp. lycospersici (3); H - F. roseum ‘Sam- bucinum’ (3); I - S. maydis (3); J - T. fongibrachiatum (4).

16 L.R. Abad et al. /Plant Science 118 (19%) 11-23

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, SO and 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 controls 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 germliags were 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; germling 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..

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 regression 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 germinated 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 indicate 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 germination 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 B. cinerea F. graminearum l? moniliforme F. oxysporum f. sp. dianthi F. oxysporum f. sp. lycopersici T. longibrachiarum V. dnhliae

Nonea Nonea Germination reduction Lysis Lysis Lysis Lysis Lysis

-

879.5~~ 5.1C

10.OC 4.6’ 11.Y 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 germination 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.

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 medium. 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

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 membrane 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 uptake 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 uptake by tobacco cells (not shown).

3.3. Osmotin action is affected by salts and sorbitol

The inhibitory effect of osmotin on T. longibrachiatum 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. longibrachiatum hyphae (not shown). These results fur-

18 L.R. Abad et al. /Plant Science 118 (19%) II-23

0 S 10 IS 20 2s Osmotin @g/ml)

S 10 1s 20 2s Osmotin (&ml)

l-

o, . I . I ’ I . 1 0 S 10 1s 20 2s

Osmotin (&ml)

Fig. 3. Effect of osmotin on the ability of cells to maintain a pH gradient. DMO uptake in IO min by (A) T. longibrachiatum hyphae, (B) R so&k hyphae; (C) cultured cells of N. tabacum L. var. Wisconsin 38. The experiments were performed as described in Section 2, Materials and methods.

ther supported the hypothesis that osmotin antifungal action is the result of its ability to dissipate the plasma membrane pH gradient, since both processes are similarly affected by NaCl. Choline Cl was not able to influence osmotin inhibition of T. longibrachiatum spore germination,

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 membranes [23]. However, the effects of these proteins on fungal cells were demonstrated under hypotonic conditions that can cause the growing cells to burst if the treatment resulted in a weakened 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 plasmolyzed cells were able to accumulate FDA equally well in the presence (Fig. 5C) and absence of osmotin (data not shown). The sorbitol had virtually no 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 required or facilitate osmotin action, we can not eliminate some role of turgor in osmotin sensitivity.

4. Dlseusslon

4. I. Species specificity

The results of this study indicate that tobacco osmotin is effective against a range of economically 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 exhibited some sensitivity to osmotin in vitro (Table 1). Although the fungi in this study were selected for their economic importance and not for their taxonomic distinction, representatives of Ascomycetes, Hyphomycetes, Oomycetes and 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?/o reversal (mM)

NaCl 40 KC1 35

MgClz 40 Na*HPO, 40 Na,S04 20 NaHCO, 40

Spore suspensions of T. longibrachiatum (final concentration ca. 5 x IO3 per ml) in PDB were incubated at room temperature with various concentrations of the indicated salts (O-80 mM) in the absence or presence of osmotin (20 &ml). Fungal growth was measured after 48 h as absorbance at 410 nm. The concentration of salt required for 50% reversal of osmotin inhibition of growth was calculated in each instance from data similar to those shown in Fig. 4.

sitive to tobacco osmotin in hyphal growth inhibition assays. This wide-ranging effect suggests that the use of osmotin as a novel defense gene effective against a large number of pathogens is feasible. However, osmotin is not universally effective 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 impervious to the effects of tobacco osmotin. The general trend provided by these data suggests that sensitivity might be determined on the genus level. If this is true, certain genera of pathogens could be targeted for specific crop plants, increasing the usefulness of the conferred resistance.

The antifungal effect of osmotin appears to operate on hyphae and spores. Hyphal growth was inhibited to varying degrees in most of the isolates tested. A similar phenomenon has been observed in fungi treated with chitinases and p-1,3- glucanases [31]. In five of the eight fungi tested, spore lysis occurred in the presence of osmotin. This lytic effect of osmotin has been reported in osmotin-treated sporangia of the Oomycete pathogen P. infestans [12]. Of the fungi whose spores were not lysed by osmotin, treatment with osmotin resulted in a reduction in spore germination in F. graminearum and a reduction of germling viability in B. cinerea. These effects appear to

04 0

r I I

20 40 60 80 Concentration of NaCl (mM)

;‘looo.

b 5 80. ‘\y%

s \‘b- _ - 5 60. -- ----L- _ ___ _;

‘r g 40-

3

p I? 203 : -..&-+---~;

0’ I I I I 0 20 40 60 80

Concentration of CholineCl (pglml)

Fig. 4. Inhibitory effect of ions on osmotin inhibition of spore germination. (A) Effect of NaCl. (B) Effect of choline chloride. T. longibrachiatutn spore suspensions (final concentration ca. 5 x IO3 conidia/ml) in PDB were mixed with osmotin (final concentration 20 &/ml) and the indicated amounts of NaCl or choline chloride and then incubated at room temperature for 48 h. Fungal growth was measured as absorbance at 410 nm. Sym- bols: (0), minus osmotin; (0). plus osmotin.

be the result of different levels of sensitivity to osmotin. The most sensitive spores lysed in the presence of osmotin while less sensitive spores did not lyse, but were killed before germination could occur. Other fungi, like B. cinerea, germinated but were subsequently killed. At the end of the spectrum were fungi such as A. flrtvus, which were largely unaffected by osmotin. It is important to note, however, that there is variability within a spore population to the antifungal effect of osmotin. That is, a proportion of spores, even in 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 Aspergilus flnvus Bipolaris may& Bipolaris zeicola Borryris cinerea Cercospora zeae-maydis Clodoporiton cucumerinum Colktorrichum gleosparioiaks CoNero~richwn grambaicola Colierorrichum laginarium CoNetotrichum sublineolum Fusarium graminearum Fuwrium monirifrme Fusariwn oxysporum f. sp. dianthi Fusarium oxysporum f. sp. lycopersici Fusarium roseum ‘Sambucinum’ Kabar&ila zeae Macrophomina phaseolina Magnaporthe grisea Periconia circina~a Phyrophrhora infesstans Phyrophthora parasirica var. dianthi Phytophlhora parasitica var. nicoknae Rhizocronia salani Stenocarpelka tnaydir Verricillium dohliae

Tomato, potato, others Peanut, maize, others Maize Maize Many hosts Maize Cucurbits Many hosts Maize, Sorghum Cucurbits Sorghum Maize, Sorghum, wheat Maize, Sorghum, others Carnation Tomato Potato Maize Many hosts Rice Sorghum Potato, tomato Carnation Tobacco Many hosts Maize Many hosts

Leaf, stem, fruit blight Seed rot Leaf blight Leaf spot Petal, fruit blights Leaf spot Scab Anthracnose Anthracnose Anthracnose Anthracnose Stalk, root, kernel rot Stalk, root, kernel rot Wilt Wilt Tuber rot Leaf spot Root, stem rot Leaf blight Root rot Leaf blight Root rot Root rot Root, stem rot Bar rot Wilt

L.R. A&d et al. /Plant Science 118 (19%) II-23 2

highest concentrations of osmotin used in the tests. These data are consistent with the observations of Liu et al. [20] who showed that osmotin overexpression 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 gradient 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 inhibited 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

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 resistance 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 nikkomycin (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 combinations 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.

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