NL9001294A - Fungistatic compsns. contg. plant proteins for storage - comprising osmotic or related proteins, for food preservatives and cosmetics, for house plants agricultural crops and fruit - Google Patents

Abstract

Compsns. contain an osmotin-type protein (I) derived from plant (cells). Pref. (I) is (a) protein AP20 derived from tobacco plants, (b) osmotin I or II derived from tobacco plants, or (c) an osmotin-type protein derived from tomato plants. Proteins are described, e.g. in Plant Physiol., 87, (1987) and 90, 1096 (1989), and their aminoacid sequences are given.

Description

Novel anti-fungal preparations as well as methods for the preparation of such preparations

Field of technology

The invention relates to the inhibition of fungal growth using proteins that can be isolated from plants. The invention provides methods for the preparation of such proteins in an active form, as well as active formulations of such proteins.

Background of the invention

Plant proteins with anti-fungal action are known. A chitinase purified from the bean effects inhibition of the growth of the fungal Trichoderma viride in agar plate assays (Schlumbaum et al. 1986, Nature 324, 365-367). However, said enzyme has only a limited anti-fungal effect on, for example, the ascomycete Cladosporium cucumerinum. and no effect on the growth of, among others, the oomycetes Phvtophthora cactorum, Pvthium aphanidermatum. and Pythium ultimum. An important disadvantage of this enzyme is therefore the limited spectrum of action.

A pea chitinase with a growth inhibitory effect on Trichoderma viride in agar plate assays is described by Mauch et al. (Plant Physiol., 1988, 88 / 936-942. In a similar assay, it was found that β-1,3-glucanase has a growth inhibitory effect. at Fusarium solani f.sp.pisi.

A preparation with a hydrolytic effect on the prepared cell walls of Verticillium alboatrum consisting of a combination of a purified tomato endo-B-1,3 glucanase and an exo-Bl, 3-glucanase of fungal origin is described by Young & Pegg ( 1982, Physiol, Plant, Pathol. 21, 411-423). Neither enzyme had any effect.

A number of thionins, inter alia, from leaves of barley, maize, wheat, rye and various dicotyledonous plants, which show a significant anti-fungal effect in in vitro assays, are further described by Bohlmann, H. et al.

(1988) EMBO J. 7: 1559-1565.

Plant proteins that have an enhancing effect on the anti-fungal effect of antibiotics are further described, inter alia, in patent application PCT / US88 / 03420. These plant proteins are generally referred to by the term synergistic anti-fungal proteins or SAFPs. SAFPs are used, inter alia, in combination with polyoxins and nikkomycins, both of which are already individually active against phytopathogenic fungi; in combination with SAFPs, efficacy improvements can be obtained on the order of a factor of 10 to 100. However, SAFPs do not in themselves have anti-fungal activity.

Due to the above-mentioned drawbacks of already known proteins, there is a need for new proteins with an anti-fungal effect and a preferably broad spectrum of action.

Description of the invention

The invention now provides new proteins which show surprisingly good activity against fungi, new methods for the preparation of such proteins in an active form, and effective formulations of this protein in an anti-fungal preparation. To this end, the preparation contains as active ingredient an osmotin or osmotin-like protein from plants or plant cells.

In general, it has been found that if plants are treated with an incompatible pathogen, a few days after inoculation, protein can be isolated from these plants, from which fractions can be obtained which have a potent fungicidal effect after separation. In tobacco and tomato it was found that the inhibitory effect in some fractions could not be attributed to B-1,3 glucanases or chitinases, since no glucanase or chitinase activity could be detected in the further purified protein preparation with fungicidal activity. . The anti-fungal activity proved to be heat sensitive. The tobacco active ingredient was identified as a hydrophobic protein with a molecular weight of about 24 kD, a basic isoelectric point (pi), and was designated AP20. After purification and determination of the amino acid sequence of the N-terminus of AP20, it was found that this part is identical to the corresponding part of the known protein osmotin from tobacco (Singh, NK et al. Plant Physiol. 1987, 85 / 529-536; Singh, NK et al. Plant Physiol. 1989, 90, 1096-1101) and belongs to the group of plant proteins referred to as osmotins or osmotin-like proteins. Plante osmotins are described, inter alia, by Singh, N.K. et al. supra. Osmotine gets its name from the finding that the protein is synthesized and accumulates during osmotic adaptation of plant cells in a medium containing high concentrations of sodium chloride, potassium chloride or polyethylene glycol; however, accumulation of osmotin is dependent on continued presence of said osmotic agents. Under the influence of (some) non-osmotic agents such as cadmium chloride, no accumulation of osmotin occurs (King et al. 1986, Plant Mol. Biol. 7: 441-449).

Two osmotins are known in tobacco, osmotin-I a water-soluble form, and osmotin-II, a detergent-soluble, relatively protease-resistant form. Both tobacco osmotins have a molecular weight of approximately 24 kD, have a very similar amino acid sequence, as well as with a 24 kD osmotin-like protein from tomato (Lycopersicon esculentum), and to a lesser extent with thaumatin-like proteins (including thaumatin from Thaumatococcus daniellii 'pathogenesis-related protein S' (PR-S) from tobacco, and the so-called bifunctional corn trypsin / α-amylase inhibitor Osmotin-like proteins, all serologically related and with a molecular weight corresponding to the osmotins from tobacco and tomato are described inter alia in millet, soy, carrot (Daucus carota), cotton, potato (Solanum tuberosum), (Singh et al. 1987 PNAS USA 84, 739-743), lucerne (Medicago sativa). and bean (Phaseolus) ( King et al. 1986, supra).

An effect of osmotine has not been known so far. It has been generally believed that osmotins have a function in providing osmotic tolerance to the plant when exposed to low water potential (see, for example, Grosset et al. Plant Phys. (1990) 92, 520-527).

Due to the similarity of the physical parameters, namely the substantially identical molecular weight, an amino acid sequence identical to that of tobacco osmotin II and substantially identical to tobacco osmotin I and tomato osmotin (NP24), and corresponding pi of AP20 with known osmotins, it must be concluded that the anti-fungal protein AP20 is an osmotin. Moreover, the osmotin protein NP24 from tomato also appears to have an anti-fungal effect; surprisingly, it has to be concluded that osmotins from tobacco and tomato appear to have an anti-fungal effect and are therefore very suitable for use as an active ingredient in anti-fungal preparations. Given the great homology between osmotins in the plant kingdom, it is obvious that osmotins other than those from tobacco and tomato will also have an anti-fungal effect, provided there is sufficient amino acid sequence homology.

For the sake of clarity, osmotins, or osmotin-like proteins, are understood to mean proteins with an amino acid homology of more than 80% with tobacco osmotin (sequence figure 1) and a basic pi, whose synthesis and accumulation is correlated with osmotic adaptation of plant cells with a high NaCl-containing environment, and which have a growth and / or anti-sporulation inhibiting effect on at least one fungus.

Preferably, for the isolation of osmotins, use will be made of the possibilities to induce the synthesis of the protein, for example by inoculating plants with a pathogen, in particular an incompatible pathogen from the groups of viruses, bacteria or fungi. However, it is not necessary for plants to be inoculated with a pathogen. Other ways of inducing osmotin synthesis are also suitable, such as, for example, exposure of the plants, or plant cells in culture, to NaCl and / or polyethylene glycol. This leads to very high concentrations of osmotin in the cell; accumulation levels of up to 12% of the total cellular protein content have been reported (Singh et al. (1987) Plant physiol., supra). Osmotin synthesis can also be induced by treating plants or plant cells with ABA (Abscisic acid) (Singh et al. (1987) P.N.A.S., supra).

Plants from which osmotins can be isolated include millet, soybean, carrot, cotton, tomato, and potato, described by Singh et al. (1987) P.N.A.S. USA 84: 739-743? and King et al. (1988). Plant Mol. Biol. 10, 401-412, but also osmotins (osmotin-like proteins) occurring in plants other than the above will suffice if they have sufficient homology with osmotin from tobacco plants, or have physical parameters comparable to osmotine from the tobacco plant. In a preferred embodiment of the invention, use is made of osmotin-like proteins isolated from material (including, for example, cell cultures) of tobacco plants exposed to a stress factor, such as, for example, an osmotic agent, drought or a pathogen, preferably the tobacco mosaic virus (TMVj, or tomato plants inoculated with the fungus Phvtophthora infestans.

For the isolation of osmotin-like proteins, use can be made of (combinations of) well-known protein separation techniques, such as, for example, centrifugation, chromatography, electrophoresis and the like. Preferably, techniques will be used which are based on non-denaturing conditions. In a preferred embodiment, use is made of gel chromatography and ion exchange chromatography in combination with hydrophobic interaction chromatography, the elution being monitored by UV spectroscopy. The fractions obtained can be analyzed for the presence of an inhibitory effect on the growth of spores, whether or not pre-germinated, of fungi sensitive to osmotin-like proteins, such as, for example, fungi belonging to the genus

Phvtophthora. Fusarium. Trichoderma. Penicillium and the like, preferably for the growth of spores of P.infestans. The (pre-sprouted) spores (between 1 and 100 spores per μΐ, preferably between 5 and 20 spores per μΐ) can be tested on a suitable medium, such as for example potato dextrose agar (PDA), and the like. The development and growth of the fungus can be easily monitored by staining the mycelium at certain times after fraction administration; by comparing incubations of the fungal spores with a control preparation, the presence of an anti-fungal factor in the fraction can be determined. The fractions exhibiting heat-labile anti-fungal activity can be analyzed for the presence of osmotin-like proteins by molecular weight using electrophoresis and / or immunoblot techniques using antibodies directed against, for example, tobacco osmotin or PR -S. The osmotin-containing fractions can be further fractionated using, for example, Hydrophobic Interaction Chromatography (HIC) or High Performance Liquid Chromatography (HPLC); with this almost complete purity can be obtained. According to the method described here, it is possible to isolate osmotin-like proteins from any plant material. It will be clear that, depending on the starting material, small variations on the described method may be necessary, which are obvious to a person skilled in the art.

For the inhibition of fungal growth, osmotins can be used, among other things, in the preservation of foodstuffs, cosmetics, medicines, the use in spray water for houseplants and agricultural crops, for the preservation of fruit, vegetables and other crops that can be stored temporarily, and furthermore in all those cases where inhibition of fungal growth is desired, provided that osmotins are active under conditions. Hereby, osmotin may be administered per se or in combination with a suitable carrier, optionally in combination with other active compounds, in the form of powders, crystals, suspensions, emulsified suspensions, granules, aerosols, solutions, gels or other types of diluents or carrier materials.

For use in preparations, for example, to broaden the spectrum of action, a combination of osmotin and other anti-fungal agents can also be used, such as classical anti-fungal antibiotics, SAFPs, and chemical fungicides such as polyoxins, nikkomycins, carboxymides, aromatic hydrocarbons, carboxins, morpholines, sterol biosynthetic inhibitors, organophosphines. and the like, enzymes such as glucanases, chitinases and the like. Osmotin can be used alone or in combination with other active ingredients at concentrations effective for its intended purpose; generally between 1 ^ g / ml and 100 mg / ml (0.0001% (w / v) - 10% (w / v), preferably between 5 μg / ml and 5 mg / ml (0.0005% (w / v) ) - 0.5% (w / v), within pH limits between 3.0 and 9.0 In general, it is desirable to use buffered preparations, for example, using a phosphate buffer between 1 mM and 1 M, preferably between 10 mM and 100 mM, in particular between 15 and 50 mM, it being desirable to add a salt at low concentrations of the buffer to increase the ionic strength, preferably NaCl in concentrations between 1 mM and 1M, preferably between 10 and 100 mM.

The following figures illustrate the invention. Figure 1 shows the similarity of the amino acid sequence of AP20 with tobacco osmotins II as published in an article by Singh et al. 1989, supra). tobacco osmotin I (Singh et al., 1987, supra) and a tomato osmotin (King et al. 1988, supra).

The examples given below are illustrative of the invention and are not intended to be limiting in any way.

E. Examples I Isolation of AP20 from TMV-induced tobacco plants

Tobacco leaves (Samsun NN) from 7 to 8 weeks old were inoculated with tobacco mosaic virus (TMV) in a manner known to the investigator in the area. Seven days after inoculation, 400 grams of leaves were harvested and homogenized at 4 ° C in 600 ml 0.5 M NaOAc, 15 mM 2-mercaptoethanol, 4g activated carbon using a Waring blendor. The homogenate was filtered over cheesecloth and the filtrate then centrifuged at 5,000g for 15 minutes. The supernatant was centrifuged at 22,000g for 50 minutes and desalted on a Sephadex G25 (Pharmacia) column, length 60 cm diameter 11.5 cm and equilibrated in 20 mM NaOAc pH 5.2. The protein fraction was left overnight (o / n) and then centrifuged at 22,000g for 45 minutes. The supernatant was passed over a S-sepharose "fast flow" (Pharmacia) column, 5cm in diameter, 5cm in length, equilibrated with 20mM NaOAc pH 5.2, at a flow rate of about 15ml per minute. The unbound protein fraction was collected. The bound protein fraction was eluted with a NaCl gradient up to 500 mM in the above buffer, at a flow rate of 3 ml per minute; fractions of 4.2 ml were collected. Of each second fraction, a portion was electrophoresed in a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) using molecular weight markers (66-20 kD) as a reference. Another part of the same fractions was tested for anti-fungal activity.

A microtiter plate assay was designed to test for anti-fungal activity. Both 24-well and 96-well microtiter plates were used. 250 μΐ (24-well plates) or 50 μΐ (96-well plates) potato dextrose agar (PDA) were pipetted into each well. P. infestans sporangia were suspended in water and added to the wells: 500-700 sporangia (in 50 μΐ) to the 24-well plates, and 100-200 sporangia (in 25 μΐ) to the 96-well plates. The fractions to be tested were dialyzed against 15 mM K2HP04 / KH2P04 pH 6.0, 20 mM NaCl, filter sterilized (0.22 μιη filter) and added to the sporangia suspension (100 and 50 μΐ for the 24-well and the 96, respectively) well plates), in both cases resulting in a final concentration of 10 µg AP20 / ml. Lowering the phosphate concentration proved necessary because P.infestans is sensitive to too high concentrations? 15 mM potassium phosphate buffer was found to have no effect on the growth of P.infestans. Addition of NaCl is desirable for the stabilization of AP20 at low phosphate concentrations. As a control, (parts of) the same fractions were boiled for 10 minutes after dialysis. Plates were then incubated in the dark at 20 ° C for 4 to 5 days. The first signs of the fungus-inhibiting effect can be observed under a microscope after about 20 hours. The fungicidal action manifests itself through lysis of germinating sporangia and of the growing germ tubes or hyphen buds.

Inhibition of the growth of mycelium can also be observed at a later stage.

For further purification of the active component, the active anti-fungal fractions were pooled, dialyzed against 1M (NH4) 2 SO4, 500 mM Κ, ΗΡΟ ^ ΚΙΙ, ΡΟ * pH 7.0 and then filtered through a 0.22 µl filter. The filtrate was then loaded onto a hydrophobic interaction (HIC) column (Phenylsuperose HR 5/5 from Pharmacia) previously equilibrated with dialysis buffer. All previous steps were performed at 4 ° C.

The bound proteins were eluted with a zero (NH4) 2 SO4 gradient in 50 mM K2HPO4 / KH2PO4 at a flow rate of 0.5 ml per minute at room temperature. The elution step was run through as follows: -in 5 minutes from 100 to 30% of the initial concentration (NH4) 2SO4 -eluting for 7.5 minutes with 30% of the initial concentration (NH4) 2SO4 -in 10 minutes from 30% to 0% of the initial concentration (NH4) 2 SO4.

The anti-fungal protein elutes after about 5.5 minutes (about 2.7 ml) at 0% of the initial concentration (NH4) 2 SO4 of the column; the fraction collected is 95% pure. All these steps were performed at room temperature. To get the anti-fungal protein 99% pure, this fraction was dialyzed against 1M (NH4) 2 SO4, 50mM K2HP04 / KH2P04 / pH 7 buffer and after filtration through 0.22 µm filter again separated on the HIC column according to the following elution pattern: -in 7.5 minutes from 100% to 15% - during 12.5 minutes at 15% - in 10 minutes from 15% to 0%.

The anti-fungal protein elutes from the column after 13 minutes (approx. 2.7 ml); this eluate contains 99% pure anti-fungal protein. 400 μg of anti-fungal protein (95% pure) or 200 μg of 99% pure anti-fungal protein can be isolated from 400 g of leaf material.

The anti-fungal protein with an estimated molecular weight of about 24 kD was designated AP20.

AP20 already has a growth-inhibiting effect on P.infestans at concentrations of 1 µg / ml; at 10 μg / ml, the inhibition of P.infestans is complete.

II Identification of AP20 as osmotin

The amino acid sequence of the N-terminal portion of AP20 was determined using techniques known to those skilled in the art (see, for example, Matsudairo, P. et al. (1987) J. Biol. Chem. 262, 10035-10038). The amino acid sequence of AP20 is shown in Figure 1. The Figure shows the complete similarity of the 40 N-terminal amino acids of AP20 with that of the N-terminus of tobacco osmotin I and II and tomato osmotin-like protein NP24.

An accurate determination of the molecular weight of AP20 was performed using 1D-polyacrylamide gel electrophoresis (1D-PAGE) with molecular weight references of 66 to 20 kD. According to this method, the molecular weight was determined to be 24 kD, which is consistent with the electrophoretic mobility of other osmotins.

The isoelectric point was determined by isoelectric focusing chromatography between pi 4.3 and 10.6. The mobility of AP20 in the gel / buffer system used did not allow an exact determination of the pi, since AP20 was found to have a higher pl value than the most basic marker (pH = 10.6); it must be concluded that the pi of AP20 is greater than 10.6. A comparison of the physical parameters of AP20 with that of tobacco and tomato osmotins (identical molecular weight, very basic pI, and identical amino acid sequence of the 40 most N-terminal residues) shows that AP20 is an osmotin. From the determination of the anti-fungal effect of the osmotin (AP20) isolated by us and the high degree of conservation of AP20 with other osmotins, as well as between the other osmotins, it is obvious that the whole group of osmotin-like proteins in the plant kingdom have a corresponding anti-fungal effect.

III Inoculation of tomato with P.infestans and isolation of osmotin from tomato

The method followed for the inoculation of tomato plants is described by Heller & Gessler (J.

Pathology, 1986, 116, 323-328).

Tomato plants (Lvcopersicon esculentum cv. Money maker) were inoculated with zoospores of P.infestans approximately 2 months after sowing. The zoospores were formed in a suspension of 1 x 105 sporangia per ml of incubation medium. 6 drops of 10 μΐ of this suspension were pipetted onto the leaves per leaf. The plants were incubated at 15 ° C, at a humidity of 95-100%, and low light intensity, until the lesion development became visible (after about 3 days). Then the temperature was increased to 22eC, the humidity level decreased to 75% and the light intensity brought to normal. After an additional three days of incubation, the leaves were harvested with dry necrotic lesions and stored at -80eC.

The isolation procedure of osmotin from tomato leaves (including the test for P, infestans) is the same as for the isolation of AP20 from tobacco, described in Example I.

In the following Examples, the effect of AP20 on other related and less related fungi is tested.

IV The effect of AP20 on Phytophthora nicotianae

The anti-fungal effect of AP20 was tested on spores of P.nicotianae as described for the test on P.infestans. with the proviso that AP20 is dissolved in 50 mM potassium phosphate buffer pH 6.0, without NaCl. To this end, AP20 in the elution buffer containing residues (NH4) 2 SO4 was dialyzed against 50 mM K2HPO4 / KH2PO4 pH 6.0 for one hour. To control side effects caused by the dialysis buffer, the effect of AP20 in elution buffer was compared to the effect of an amount of elution buffer without protein, and elution buffer with AP20 heated at 100 ° C for 10 minutes. The test was performed on 25-50 spores pre-seeded in microtiter plates (on 50 μΐ potato dextrose agar (PDA) per well) for 5-20 hours. After 1-2 days, growth inhibition can be seen in the wells to which AP20 was added, relative to the control.

V The anti-fungal effect of AP20 on Trichoderma viride

The test was performed as described for P.nicotianae. After about an hour, lysis of the hyphen buds or germ tubes can be seen in the wells to which AP20 was added. After approximately 5 hours, lysis was observed in 72% of the hyphen buds (50 hyphen buds counted). After about a day, growth inhibition was noticeable compared to the control.

Control incubations with buffer or AP20 heated at 100 ° C for 10 minutes showed hardly any lysis of hyphen buds or germ tubes.

VI The anti-fungal effect of AP20 on Fusarium oxysporum

The test was performed as described for P.nicotianae (Example IV). 76% of the hyphen buds counted were lysed. Growth inhibition was seen after about a day. No lysis or growth inhibition was observed in the control.

VII The anti-fungal effect of AP20 on Penicillium digitalis

The test was performed as described for P.nicotianae (Example IV). 2% of the hyphen buds counted were lysed. However, growth inhibition was noticeable after about a day. No lysis or growth inhibition was observed in the control.

Claims (4)

Anti-fungal preparation comprising an active ingredient and a suitable carrier, characterized in that the active ingredient contains an osmotin-like protein from plants or plant cells in the composition. Anti-fungal preparation according to claim 1, characterized in that the osmotin-like protein comes from plants or plant cells selected from one of the groups; -Solanaceae. Lecmminosae. Gramineae. Umbelliferae. or cotton. Anti-fungal preparation according to claim 1, characterized in that said plants or plant cells are selected from the varieties of tobacco (Nicotiana tabacum) or tomato (Flexopersicon esculentum). Method for the preparation of an anti-fungal preparation according to claim 1, characterized in that for the preparation of osmotin protein, use is made of material from plants which have been previously exposed to a form of stress such as osmotic stress, drought - stress, and / or pathogen stress - to isolate and fractionate total soluble protein from this material using a combination of protein separation techniques under non-denaturing conditions - test fractions obtained for anti-fungal activity using a test on a osmotin-sensitive fungus, such as P.infestans.