CZ209293A3 - Gene of plant chitinase and use thereof - Google Patents

Abstract

A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO.1 or an analogue or subsequence thereof is disclosed. The polypeptide encoded by the DNA sequence, also termed the sugar beet chitinase 4 enzyme, has a high antifungal activity due to a bifunctional catalytic activity (i.e. a chitinase and a lysozyme activity) which makes the enzyme highly effective in inhibiting the growth of chitin-containing fungi. An even improved antifungal effect is obtained when the sugar beet chitinase 4 enzyme is used in combination with other pathogenesis related proteins, especially in combination with a second different chitinase and a beta -1,3-glucanase. A preferred use of the DNA sequence disclosed herein, optionally in combination with DNA sequences encoding other pathogenesis related proteins, is in the construction of genetically transformed plants, especially genetically transfrmed sugar beet plants, having an increased resistance to chitin-containing fungi as compared to untransformed plants.

Description

Field of technology

The invention relates to the DNA sequence of beet referred to in the following sugar beet 4 or to an analogue of this polypeptide having the antifungal effects of sugar beet chitinase, as well as a genetic construct suitable for generating genetically transformed plants which show increased resistance to plant pathogens compared to non-transformed plants. such as phytopathogenic fungi. The genetic construct comprises and is capable of expressing a DNA sequence according to the invention, preferably in combination with a DNA sequence encoding a second chitinase other than sugar beet chitinase 4 and a DNA sequence encoding beta-1,3-glucanase. In another embodiment, the invention relates to a genetically transformed plant, in particular a genetically transformed sugar beet plant, in which a polypeptide exhibiting antifungal effects of sugar beet chitinase 4 is expressed in an increased amount compared to a non-transformed plant, preferably in combination with a polypeptide exhibiting chitinase effects and a polypeptide exhibiting beta-1,3-glucanase effects, resulting in increased resistance to chitin-containing plant pathogens.

Prior art

Most plants are susceptible to infection by pathogens such as microorganisms, and the infection produces various undesirable symptoms of the disease, which cause stunted growth, reduced yields, and consequent economic losses to farmers. The plant responds to infection by various defense mechanisms including phytoalexins, storage of lignin-like substances, accumulation of hydroxyproline-rich cell wall glycoproteins, pathogenesis related proteins (PR-proteins) and increased activity of some lytic enzymes such as chitinases and beta-1. , 3-glucanase. Some of these responses can be induced not only directly by infection, but also by exposure of plants to elicitors isolated from fungal cell walls, and in some cases by exposure to exogenous chemicals such as ethylene. The full capacity of the plant's defense mechanism is normally delayed compared to the onset of infection, and the plant can therefore be severely damaged before the defense mechanism reaches its maximum capacity. The plant's defense mechanism may also not be strong enough on its own to effectively control the pathogenic organism. Therefore, it is a common and necessary measure to treat infected plants or plants susceptible to infection with a chemical, for example a fungicide, either as a prophylactic treatment or shortly after infection.

The use of chemical treatment is neither ecologically nor economically desirable and it would be desirable to increase the defense of the host plant itself by introducing new and / or improved genes through genetic engineering. Another advantageous effect of this strategy is to achieve immediate inhibition of the fungal infestation, which leads to a slowing down of the establishment of an epidemic of the infecting fungus in plant crops and thus to a general reduction of the effects of the infection.

The cell walls of many phytopathogenic fungi contain chitin and glucan, with chitin being a major component of hyphae tips. It has been found that the enzymes chitinase and beta-1,3-glucanase are able to enzymatically cleave fungal cell walls, resulting in particular soluble dimers or oligomers of N-acetyl-D-glucosamine and D-glucose.

Chitinase and beta-1,3-glucanase activity have been observed in plant species such as tobacco, barley, potatoes, rice, corn, beans, tomatoes, cucumbers, wheat germ, rapeseed and peas, and chitinase activity has been found to increase in response on infection by mostly phytopathogenic fungi.

Plant chitinases have been purified and characterized in crops such as tobacco, barley, corn, tomato, beans and peas, and cDNA and genomic clones have been obtained. Plant chitinases have been reviewed by Bol and Linthorst, 1990 and Boiler, 1988.

Several publications have addressed bacterial and plant chitinases and their use in the construction of transgenic plants having increased resistance to various microorganisms, such as fungi.

EP 0 292 435 relates essentially to the regeneration of fertile Zea mays maize plants and states, inter alia, that the tobacco chitinase gene can be introduced into a plant to confer resistance to pathogens. Chitinase genes from sources and plants other than Zea mays are not mentioned.

EP 0 290 123, WO 88/00976 and U.S. Pat. No. 4,940,840 describe the use of chitinases of bacterial origin in the construction of transgenic plants. Chitinase of plant origin is not mentioned or is alternatively only mentioned in general terms.

WO 90/07001 discloses DNA constructs comprising a high throughput promoter operably linked to a DNA sequence encoding a plant chitinase, which constructs are used to transform plants to achieve high chitinase expression in a plant and thereby confer resistance to plant pathogenic fungi. The only plant chitinase mentioned was bean chitinase.

EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304 and EP 0 418 695 describe the construction of transgenic plants containing DNA sequences encoding plant pathogenesis-related proteins (PRPs), for example chitinase and beta -1,3-glucanase. Proteins related to the pathogenesis of sugar beet plants or transgenic sugar beet plants are not mentioned.

EP 0 448 511 also relates to transgenic plants containing recombinant DNA sequences encoding hydrolytic enzymes such as chitinases and glucanases. The reference further relates to compositions containing hydrolytic enzymes such as glucanases and chitinases for use in the control of plant pathogens. Chitinase or glucanase from sugar beet is not mentioned.

WO 91/06312 discloses a crop protection composition comprising endoenzymes such as glucanase or chitinase. No specific source of chitinase or glucanase is mentioned.

Russeau-Limouzin M. and Fritig B. (1991) describe the formation of basic and acidic PR-proteins in sugar beet infected with Cercospora beticola and the serological relationships of these PR-proteins to PR-proteins in tobacco. The PR-proteins described have been found to be serologically related to tobacco PR-proteins, while the sugar beet chitinase 4 of the invention does not show a serological relationship to any known chitinase, see below. No information is given on the amino acid sequence or nucleotide sequence of any of the PR proteins.

Thus, none of the publications cited above discloses any sugar beet chitinase enzyme or its use in the construction of transgenic plants.

At the International Symposium of the Phytochemical Association of Europe, Norwich, UK, April 11 - 13, 1989: Plant Biochemistry and Molecular Biology: Pathogen Interactions, the inventors described the isolation and purification of six chitinase isoenzymes, including chitinase 4, from leaves of Cercospora beticola infected sugar beet plants. , a phytopathogenic fungus containing chitin. Chitinase isoenzymes were characterized by their molecular weight and chitin hydrolysis kinetics. Chitinase preparations have been reported to be able to hydrolyze newly synthesized chitin in the cell walls of growing fungi. No other characteristics were reported and the chitinase enzymes were not analyzed separately.

On the agenda of the APS / CPS (The American Phytopathological Society) joint meeting, which took place on August 4-8, 1990 in Michigan, USA, and at the Fifth International Symposium on Molecular Genetics of Plant-Microbe Interactions, which took place on 9. - 14th September

1990 in Interlaken, Switzerland, the inventors published a paper and a poster showing the amino acid composition of various sugar beet chitinase isoenzymes, including chitinase 4. It was first reported that two serologically distinct groups of chitinase enzymes are present in sugar beet plants. This report has not been discussed in detail.

An author's paper and poster published by the inventor at the Eighth Congress of the Mediterranean Phytopathological Association in Agadir, Morocco, October 28-3, 1990, states that antibodies against wheat germ chitinase, three sugar beet chitinases and two beta 1,3-glucanases and that N-terminal and partial amino acid sequencing of five of the nine purified chitinase isoenzymes was performed. Only the N-terminal sequence of the sugar beet chitinase isoenzyme 2 was detected.

The 1989 Annual Report of the Center for Plant Biotechnology (Denmark), published on 20 August 1990, publishes the above results and states that one of the genes encoding sugar beet chitinase has been isolated and characterized, inter alia by nucleotide sequencing. However, this gene, although not directly stated in this publication, encodes chitinase 1 and not sugar beet chitinase 4. In retrospect and with respect to the description of the invention, it is evident that this chitinase is not chitinase 4, due to the high degree of homology (about 50%) with other known sequences of chitinase genes from tobacco and beans. In contrast, there is a very low degree of homology between chitinase 4 and other known chitinases, as shown in Example 11.

None of the above references mention or mention the amino acid sequence of the sugar beet chitinase 4 enzyme or the DNA sequence encoding sugar beet chitinase 4, nor does it mention or indicate that it would be interesting to look for this sequence. In fact, initial analyzes of sugar beet chitinase 4, which revealed an enzyme with a small functional domain, indicated that the chitinase enzyme has low chitin affinity and thus low enzymatic activity. It did not appear to be an enzyme of particular importance.

Furthermore, none of the references mention or mention a synergistic antifungal effect that can be achieved when the sugar beet chitinase 4 enzyme is combined with acidic chitinase and basic beta-1,3-glucanase. This synergistic antifungal effect is first disclosed in connection with this application.

The invention discloses a novel plant chitinase which, either alone or in combination with other pathogenesis-related proteins, shows promising results in inhibiting chitin-containing fungi.

The essence of the invention

In one embodiment, the invention relates to a DNA sequence comprising said sugar beet chitinase 4 DNA sequence

*. in SEQ ID NO: 1, below, or an analogue thereof, which analog is a DNA sequence encoding a polypeptide having an antifungal

sugar beet chitinase 4 activity as herein described, and i) is a characteristic part of the sequence DNA listed in SEQ ID No. 1, see below, or

ii) hybridizes to the DNA sequence set forth in SEQ ID NO: 1 at 55 ° C under the conditions specified in the Materials and Methods section entitled Identification of DNA Belonging to the Chitinase 4 Gene Family, or iii) encodes a polypeptide having the amino acid sequence of sugar beet chitinase 4. shown in SEQ ID NO: 2, below, or iv) encodes a polypeptide that has been recognized by an antibody raised against sugar beet chitinase 4.

The DNA sequence of chitinase 4, SEQ ID NO: 1, listed below, was determined from a cDNA clone isolated from a sugar beet cDNA library prepared as described in Materials and Methods, see below, by hybridization to a very specific oligonucleotide. probe. An oligonucleotide probe was prepared based on a tryptic peptide made from substantially pure sugar beet chitinase 4 obtained as described in Materials and Methods and in Example 1, see below. The procedure used to isolate the chitinase 4 DNA sequence is described in Example 4, see below.

Prior to the present invention, the amino acid sequence of the sugar beet chitinase 4 enzyme or the DNA sequence encoding sugar beet chitinase 4 was not disclosed, and it was not mentioned that it would be interesting to look for these sequences. In fact, initial analyzes of sugar beet chitinase 4, which revealed an enzyme with a small functional domain, indicated that the chitinase enzyme has low chitin affinity and thus low enzymatic activity. It did not appear to be an enzyme of particular importance.

Elucidation of the amino acid sequence of sugar beet chitinase 4 was an important step in the enzyme analysis. It was clear from the amino acid sequence that sugar beet chitinase 4 belongs to the plant chitinases of the hevein group, in which it contains a leader sequence, a hevein domain and a functional (catalytic) domain. Hevein is a lectin that binds to chitin, and the hevein domain of the enzyme is the part of the enzyme that is thought to bind to chitin and chitin-containing structures, such as in phytopathogenic fungi.

Using hydrophobic clustering analysis using the procedure described by Gaboriaud et al., 1987, the primary structure of chitinase 4 was found to be more compact than the structures of other plant chitinases belonging to the sugar beet chitinase 2 group (as described in more detail below). . The compact structure of chitinase 4 is considered to be an advantage because it allows the enzyme to gain access to chitin structures, for example in the cell walls of phytopathogenic fungi.

Furthermore, chitinase 4 has been found to lack a C-terminal extension, in contrast to other known basic chitinases, meaning that the enzyme is translocated into the intercellular space and thus not into the vacuole. The presence of the enzyme in the intercellular space was experimentally verified.

Sugar beet chitinase 4 has been found to have a surprisingly high antifungal activity and to have a particularly good inhibitory effect on the growth of phytopathogenic fungi. In addition, the use of a combination of the enzyme beet chitinase 4, a second different chitinase and beta-1,3-glucanase to control phytopathogenic fungi has been found to result in even more improved antifungal activity compared to the use of sugar beet chitinase 4 alone. This synergistic antifungal effect was first mentioned in connection with this application.

Accordingly, according to another important embodiment, the invention relates to a genetic construct comprising one or more copies of a DNA sequence comprising the chitinase 4 DNA sequence set forth in SEQ ID NO: 1 or an analogue thereof as defined above, or a subsequence thereof (further defined below). ), one or more copies of a DNA sequence encoding a second chitinase other than sugar beet chitinase 4, and one or more copies of a DNA sequence encoding beta-1,3-glucanase, each of these DNA sequences being operably linked to a promoter and a transcriptional terminator capable of expression DNA sequences into functional polypeptides.

The components of the genetic construct and the synergistic effect are further explained below.

The main use of the genetic construct of the invention is in the production of genetically transformed plants which have increased resistance to chitin-containing plant pathogens compared to plants which do not contain this construct, such as non-transformed or natural plants. Genetically transformed plants are preferably prepared using a plant transformation vector that contains a genetic construct of the invention.

The chitinase 4 DNA sequence or analog thereof, and in particular its specific subsequence (discussed below), can also be used to isolate DNA sequences belonging to the chitinase 4 gene family, as defined above. The DNA sequences of chitinase 4 or an analog or genetic construct thereof of the invention may also be used in a method of preparing polypeptides, for example a recombinant sugar beet chitinase enzyme, or a mixture of polypeptides having strong antifungal activity. The polypeptide or mixture of polypeptides can be prepared using recombinant DNA techniques and can be used in the antifungal treatment of various products, especially food products.

The DNA sequence of chitinase 4, SEQ ID No. 1, encodes the basic enzyme of sugar beet chitinase 4, the amino acid sequence of which is also apparent from SEQ ID No. 2. As used herein, chitinase 4 and sugar beet chitinase 4 are used interchangeably.

One feature of the DNA sequence of chitinase 4 of the invention and its analog is that they encode a polypeptide having the antifungal activity of sugar beet chitinase 4. The antifungal activity of sugar beet chitinase 4 is characterized by the fact that it is a bifunctional activity consisting of chitinase activity and lysozyme activity. To the best of the inventors' knowledge, this bifunctional activity has not yet been detected in any basic plant chitinase from the hevein group.

Accordingly, the term antifungal activity of sugar beet chitinase 4 refers to the characteristic bifunctional activity of this enzyme, i.e. the combination of chitinase activity and lysozyme activity found in sugar beet chitinase 4.

The term chitinase activity refers to the ability of an enzyme to degrade chitin and chitin-containing structures, and this chitinase activity can be determined firstly by biological assays and secondly by chemical assays. In biological assays, the effect of chitinase 4 on the growth of hyphae of pathogenic fungi is directly observed, i.e. the ability of chitinase 4 to destroy the walls of hyphae and thus slow down the growth of hyphae. In chemical tests, the decomposition of 1 H-chitin by chitinase 4 is observed, which mainly produces chitin dimers.

The biological assay can be performed using any of the three methods described herein in the Materials and Methods section under the title Antifungal Activity. If a positive result is obtained according to any of these methods, i.e. destruction of the hyphal walls and retardation of the growth of fungal hyphae is observed, this is considered as evidence of the biological activity of chitinase 4.

The chemical assay can be performed as described in the Materials and Methods section under the name Radiochemical Chitinase

1 test. Chitinase 4 activity is demonstrated in the assay by hydrolysis 3

H-chitin and the resulting formation of mainly chitin dimers.

The term lysozyme activity refers to the ability of an enzyme to lyse fungal cell walls. Lysozyme activity is determined by performing the lysozyme assay described in the Materials and Methods section under the title Lysozyme Assay.

It is understood that the antifungal activity of sugar beet quininase 4 has a qualitative as well as a quantitative component that reflects the ability of the polypeptides to destroy components such as chitin, hyphal wall phytopathogenic fungi and thereby inhibit or retard fungal growth.

An analog of the chitinase 4 DNA sequence is a DNA sequence that has at least one of the properties i) -iv) listed above. The terms used to define the analogs of the invention are explained in more detail below.

The term characteristic moiety, as used in connection with the analogue defined in vi) above, refers to a nucleotide sequence which is obtained from the nucleotide sequence of the DNA chitinase 4 sequence or which has a nucleotide sequence corresponding to part of the DNA chitinase 4 sequence and which encodes a polypeptide having conserved antifungal activity of sugar beet chitinase 4. Typically, a characteristic moiety comprises a chitinase 4 DNA sequence subsequence, which subsequence is either a contiguous portion of the chitinase 4 DNA sequence or is composed of one or more separate nucleotide sequences of chitinase 4 DNA sequence to allow a polypeptide encoded by a characteristic chitinase DNA sequence 4 retains its characteristic antifungal activity, this portion is normally only a small number of nucleotides shorter than the DNA sequence of chitinase 4, for example 1-50, such as 1-25 nucleotides shorter.

Typical examples of a characteristic portion of the chitinase 4 DNA sequence include nucleotides encoding the active site of chitinase 4.

2

The analog defined in ii) above is a DNA sequence that hybridizes to the chitinase 4 DNA sequence under the conditions specified in the Materials and Methods section, see below, entitled Identification of DNA Belonging to the Chitinase 4 Gene Family. The defined conditions under which hybridization is to occur on hybridization experiments performed with many known plant chitinases and sugar beet chitinase 4 and are further described in Example 11, see below.

As used herein, any DNA sequence nybridizing to a chitinase 4 DNA sequence under the hybridization conditions specified in the Materials and Methods section cited above is defined as belonging to the chitinase 4 gene family and is viewed as encoding a polypeptide having structure and antifungal activity. chitinases 4 sugar beets. Furthermore, when polypeptides produced from these DNA sequences react with antibodies raised against sugar beet chitinase 4, this is a strong indication that the DNA sequence of interest belongs to the serological group of sugar beet chitinase 4. Such DNA sequences forming part of the invention may either comprise sequences isolated from natural sources, for example plants, synthetically prepared sequences, or may be synthetically modified DNA sequences, for example as described below. In the following, DNA sequences belonging to the chitinase 4 gene family are also referred to as chitinase 4-related DNA sequences.

An analog as defined in iii) above is a DNA sequence that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, i.e. the amino acid sequence of the mature chitinase 4 enzyme. It is known that the same amino acid can be encoded by different codons. on the preferences of a particular organism expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the chitinase 4 DNA sequence of the invention may be confused with others which, after expression,

3 give the resulting polypeptide identical or substantially identical to the polypeptide of interest encoded by the chitinase 4 DNA sequence.

The analog as defined in iv) above is a DNA sequence encoding a polypeptide that is recognized by an antibody raised against sugar beet chitinase 4. As used herein, the term is recognized to be used interchangeably with the term binds to. As described in Example 3, see below, the sugar beet chitinase 4 enzyme was found to belong to a new serological group of basic chitinases not previously described in the literature. Current serological analyzes of rapeseed chitinase have revealed a close serological similarity between this chitinase and sugar beet chitinase 4, suggesting that this analyzed rapeseed chitinase belongs to the same new group of basic chitinases.

The antibody used to determine the serological affinity between a polypeptide encoded by a chitinase 4 DNA sequence of the invention and a polypeptide encoded by a DNA sequence of another origin may be a monospecific polyclonal antibody or a monoclonal antibody. A particularly suitable antibody is a monoclonal or polyclonal antibody * raised against one or more epitopes encoded by the chitinase 4 DNA sequence. These epitopes are described in more detail below.

The DNA sequences of the invention described herein may comprise natural as well as synthetic DNA sequences, which natural sequences are typically derived directly from cDNA or genomic DNA, usually of plant origin, for example as described below. Synthetic sequences can be prepared by conventional methods for the synthetic preparation of DNA molecules, for example, using the principles of solid or liquid phase DNA synthesis, such as a DNA synthesizer 381 A (Applied Biosystems). The DNA sequence can, of course, also be of mixed cDNA and genomic, mixed cDNA and synthetic and mixed genomic and synthetic origin.

4

In the following, the composition of the chitinase 4 DNA sequence and each of the chitinase 4 enzyme domains encoded by the DNA sequence set forth in SEQ ID No. 1 and the amino acid sequence set forth in SEQ ID No. 2 are further described and compared with other plant chitinases.

The chitinase 4 DNA sequence, SEQ ID NO: 1, contains a leader sequence (nucleotides 2-70) encoding 23 amino acid residues, a portion (nucleotides 71-174) encoding a 35 amino acid residue hevein domain, and a portion (nucleotides 175-793) encoding a functional a domain of 206 amino acid residues. The N-terminal portion of the mature polypeptide chain is blocked and cannot be sequenced by conventional amino acid sequencing methods. However, based on comparisons with wheat germ agglutinin (WGA-A) and potato chitinase DNA sequences and based on analysis by electrospray mass spectrometry (see Example 4), the start codon of the chitinase DNA sequence 4 was derived. Comparison of the chitinase DNA leader sequence 4 (SEQ ID NO: 1) and the chitinase 4 genomic DNA leader sequence (SEQ ID NO: 3, see below) showed that the first two nucleotides in the chitinase 4 DNA leader sequence (SEQ ID NO: 1) are missing. Thus, while the leader sequence of genomic chitinase 4 consists of 24 amino acid residues (SEQ ID NO: 4, see below), the leader sequence of chitinase 4 consists of 24 amino acid residues, although chitinase from almost full length cDNA lacks the first amino acid methionine (SEQ ID NO: 4). 2).

Plant chitinases can be divided into 3 different groups, hevein class, non-hevein class and cucumber class.

Sugar beet chitinase 4 is an alkaline cnitinase belonging to the hevein class, but it is clearly different from other alkaline chitinases of this class. While chitinases from beans, tobacco, tomatoes, potatoes, peas, poplar, barley (T and K) and sugar beets (chitinase 2) have a molecular

5 weight 32 - 38 kDa (see Example 10), chitinase 4 is smaller with a molecular weight of about 26 kDa (as determined for the mature enzyme). Furthermore, since the antibodies raised against chitinase 4 do not recognize the other basic chitinases described above (see Example 10), it is evident that chitinase 4 also belongs to a different serological group than all other basic plant chitinases of the hevein class.

The primary structure of mature chitinase 4, as determined by its amino acid sequence, contains two distinct domains: a hevein domain and a functional domain. In the N-terminal part of the polypeptide chain, 12 of the 35 amino acid residues are conserved with respect to the hevein structure. The functional domain contains 206 amino acid residues. In basic tobacco chitinase Nicotiana tabaccum (cv. Havanna) (Shinshi et al., 1989), the hevein domain consists of 43 amino acid residues and the functional domain contains 263 amino acid residues. Although the hevein domain (i.e., the chitin binding domain) of chitinase 4 is shorter than that of tobacco chitinases, chitinase 4 has a binding affinity of similar magnitude to other basic chitinases belonging to the hevein class. For comparison, when weak chitinases from the non-hevein class are examined, very little or no binding is observed. This class of chitinas does not contain a hevein domain, but only a functional domain. The homology between the functional domains of the hevein class and the non-hevein class is very high. In addition, polyclonal antibodies raised against hevein-class chitinases recognize non-hevein-class chitinases.

In general, the specific activity of the non-hevein class, acidic chitinase from tobacco and basic chitinase C from barley (Kragh Μ. Μ., Thesis, 1,990) is approximately six times lower than that of chitinas of the hevein class.

Because the functional domain of chitinase 4 contains only

206 amino acid residues, compared to 263 amino acid residues in the functional domain of tobacco basic chitinase, were expected to reduce specific activity. However, chitinase 4 works extremely well and has been described by the inventors as better than barley chitinase Τ, K and C (results not shown) when analyzed by the radiochemical enzyme assay described in Materials and Methods, see below.

From the above explanation, it is clear that the most important parts of the chitinase 4 DNA sequence shown in SEQ ID NO: 1 are the part encoding the hevein domain and in particular the part encoding the functional domain of the enzyme. Although in most cases the presence of a leader sequence is a prerequisite that allows a polypeptide expressed from a DNA sequence to be transported out of the cell in which it is produced, the nature and origin of the particular leader sequence used may vary and may not be a leader sequence naturally associated with the chitinase enzyme. 4. In addition, a leader sequence naturally associated with the enzyme chitinase 4 can be used in a heterologous gene construct in the transformation of plants, especially sugar beet plants, as long as the encoded polypeptides are directed to the intercellular space.

Accordingly, a particularly interesting DNA sequence of the invention is a DNA sequence comprising nucleotides 71-793 of the chitinase 4 DNA sequence set forth in SEQ ID NO: 1 and encoding the hevein domain and functional domain of a sugar beet chitinase 4 enzyme, or an analog of the above DNA sequence.

The term analog refers to a DNA sequence that is either

Ai) a characteristic part of the above DNA sequence, or

Aii) hybridizes to a DNA probe prepared from the above DNA sequence, or

Aiii) encodes a polypeptide having the same amino acid

7 a sequence as a polypeptide encoded by the above DNA sequence, or

Aiv) encodes a polypeptide that is recognized by an antibody raised against a polypeptide encoded by the above DNA sequence.

An even more interesting DNA sequence of the invention is a DNA sequence comprising nucleotides 75-793 of the chitinase 4 DNA sequence set forth in SEQ ID NO: 1, encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analog of the above DNA sequence. The term analog refers to a DNA sequence that

Bi) is a characteristic part of the above sequence

DNA, or

Bii) hybridizes to a DNA probe prepared from the above DNA sequence, or

Biii) encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the above DNA sequence, or

Biv) encodes a polypeptide that is recognized by an antibody raised against a polypeptide encoded by the above sequence.

Analogs defined by properties Ai) - Aiv) a

Bi) - Biv), see above, are defined in a similar manner as analogs of the DNA sequence of chitinase 4 defined by properties i) - iv), see above.

In another embodiment, the invention relates to a DNA sequence comprising a sugar beet chitinase 4 gene. As used herein, the term gene is used to refer to a DNA sequence that is involved in the formation of a polypeptide chain and that includes regions preceding and following the coding region (5-upstream and 3'-downstream sequences), as well as middle sequences, so-called introns, which they are located between individual coding segments (so-called exons), or in the 5'-upstream or 3'-downstream region. The 5'-upstream region contains a regulatory sequence that directs gene expression, typically a promoter. The 3-downstream region contains sequences involved in gene transcription termination and, optionally, sequences responsible for transcript polyadenylation and a 3 'untranslated region.

An example of a DNA sequence according to the invention containing the chitinase 4 gene is the genomic DNA sequence of sugar beet contained in the genomic clone of chitinase 4 (chit 4), the isolation of which is described in Example 4. The partial nucleotide sequence of this gene was determined and is given in SEQ ID No. 3. By comparing this partial DNA sequence with the DNA sequence of the chitinase 76 gene shown in SEQ ID NO: 5 and further described below, and the nucleotide sequence of the chitinase 4 cDNA shown in SEQ ID NO: 1 (comparisons are shown in Figure 24), it was determined that nucleotides 356 - 358 of the chitinase 4 gene sequence form the start codon of the chitinase 4 gene.

Based on a comparison with the chitinase 76 sequence (containing one intron) and the chitinase 1 DNA sequence shown in SEQ ID NO: 11, see below (containing two introns), the chitinase 4 gene was determined to contain only one intron starting at nucleotide 398 downstream from ATG start codon. The position of the intron corresponds to the position between nucleotides 395 and 396 in the chitinase 4 cDNA sequence set forth in SEQ ID NO: 1.

Possible 5 regulatory sequences of the chitinase 4 gene are listed in Examples 17 and 18 below.

As mentioned above, knowledge of the amino acid sequence of sugar beet chitinase 4 allows the enzyme to be analyzed and important parts of the enzyme to be determined, for example by comparison with the amino acid sequence of other known chitinases. A particularly interesting part of the enzyme is, for example, the part containing the active site of the enzyme, the part containing the epitopes of the enzyme and the part responsible for the substrate specificity of the enzyme and / or the binding properties.

The putative position of the active site of sugar beet chitinase 4 was found by comparison with the active sites of other known enzymes catalyzing the hydrolysis of other oligosaccharides, as explained in Example 16, see below. Thus, the active site of sugar beet chitinase 4 consists of amino acid residues 183 (aspartic acid) and 189 (glutamic acid) in SEQ ID NO: 2.

Based on the spatial structure of the chitinase 4 enzyme, which can be determined using conventional x-ray crystallographic analysis and the amino acid sequence of the enzyme, it is possible to determine the portions of the enzyme that are responsible for specific properties of the enzyme. Thus, in addition to the active site found above, specific amino acids of the enzyme responsible for its substrate specificity and binding to the substrate can also be determined. Amino acid residues forming epitopes of the enzyme can also be found. Based on knowledge of such specific amino acids, it is possible to specifically modify the enzyme to achieve a modified mode of enzyme action, e.g., to increase catalytic activity, improved, i.e., enhanced substrate specificity, improved substrate binding, e.g., chitin, or epitope modification. These modifications can be achieved using known principles of protein engineering, such as site-directed mutagenesis, for example as described in Example 16, see below.

For example, replacing one or more tryptophan residues at positions 169, 204, and 206 with tyrosine residues is expected to alter the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity. Similarly, changes in the amino acid residues forming the active site or amino acid residues that form the structure of the composite enzyme are expected to affect, for example, catalytic activity, substrate specificity and / or substrate binding, and may result in improved properties of the resulting modified enzyme. Of course, the nature of the modifications made will depend on the desired result, i.e., the specific desired function of the resulting modified enzyme.

Correspondingly, as the chitinase 4 enzyme encoded by the DNA sequence of the invention, the DNA sequence encoding the modified chitinase 4 enzyme can be used either alone or in combination with DNA sequences encoding other proteins, for example pathogenesis-related proteins such as thaumatin, osmotin and / or zeamatin ( Viegers, 1991) or thionin (Bohlmann et al., 1988), cercropin (J. Jaynes, 1989) or other enzymes such as chitinases and beta-1,3-glucanases to construct a genetically transformed plant, preferably a sugar beet plant, which has particularly high and advantageous antifungal activity. The modified chitinase 4 enzyme may also present as a particularly interesting component of an antifungal agent, as described below.

In the gene family, a high degree of homology between coding regions of genes is expected, while less homology is expected between non-coding regions. Homology can vary considerably between two different gene families. The term homology is used to denote the presence of some degree of complementarity between the amino acid sequence of a given polypeptide and the amino acid sequence of another polypeptide in an analysis given using the computer program MYERS and MILLER, version 1.05, September 1990, using a comparison matrix: Genetic Code, the Open Gap Cost 6 and the Unit Gap cost 1 (see also Myers and Miller, 1988). The degree of homology between different genes, especially between coding regions, can thus be used to determine the degree of relatedness between different genes. Amino acid sequences may be derived from a DNA sequence or may be obtained by conventional amino acid sequencing methods. The degree of homology is preferably determined based on the mature protein, i.e. without considering any leader sequence.

Accordingly, the invention relates to a DNA sequence encoding a chitinase isoenzyme which is at least 60% homologous to the sugar beet chitinase enzyme encoded by the DNA sequence SEQ ID No. 1a and at most 40% homologous to the sugar beet chitinase 1 encoded by the DNA sequence set forth in SEQ ID NO: 1. ID No. 11. A minimum degree of homology of at least 60% was determined based on analysis of rapeseed chitinase (based on mature protein), which was found to belong to the serological group of sugar beet chitinase 4 (see Example 11). The degree of homology of 40% with chitinase 1 (which does not belong to the chitinase group 4) reflects the minimum degree that is considered acceptable for a polypeptide belonging to the chitinase group 4.

Of course, the higher degree of homology to chitinase 4 and the lower degree of homology to chitinase 1 reflect even greater similarity, and accordingly the DNA sequences described above preferably encode a chitinase isoenzyme that is at least 65%, for example at least 70% homologous, such as at least 75% or preferably 80% homologous to the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID No. 1 and / or at most 38%, such as at most 35% homologous to the sugar beet chitinase 1 enzyme encoded by the DNA sequence of SEQ ID NO: 11.

An example of a DNA sequence encoding a polypeptide that is approximately 75% homologous to the sugar beet chitinase 4 enzyme and at most 40% homologous to the sugar beet chitinase 1 enzyme is a genomic DNA sequence (chitinase 76, the sequence of which is set forth in SEQ ID NO: 5). contained in the chitinase 76 gene clone obtained as described in Example 5.

It is clear from Example 10 that sugar beet chitinase 4 isolated from sugar beet leaves is recognized by an antibody raised against this sugar beet chitinase, but not by an antibody raised against sugar beet chitinase 2. This is a strong confirmation of the fact that sugar beet chitinase 4 belongs to a different group of chitinas than sugar beet chitinase 2, and that therefore there are two different groups of sugar beet chitinases. Other polypeptides belonging to the chitinase 4 family are expected to exhibit similar responses, and accordingly the invention further comprises a DNA sequence that encodes a polypeptide that is recognized by an antibody raised against sugar beet chitinase 4 but not by an antibody raised against chitinase 2. sugar beet.

In another embodiment, the invention relates to a modified DNA sequence comprising a DNA sequence as defined above, comprising a DNA sequence or chitinase 4 gene or analogue thereof, in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted. so as to encode a polypeptide that has retained the antifungal activity of sugar beet chitinase 4 or that has increased antifungal activity compared to sugar beet chitinase 4. The polypeptide encoded by the modified DNA sequence normally has an amino acid sequence that is different from the amino acid sequence of sugar beet chitinase 4. It is understood that the modified DNA sequence of the invention is important in the preparation of novel polypeptides having increased antifungal activity compared to chitinase 4.

If substitution is made, it is; one or more nucleotides in the complete nucleotide sequence are replaced by one or more other nucleotides if addition is made, one or more nucleotides are added to either end of the complete nucleotide sequence if insertion is performed, one or more nucleotides are inserted into the complete nucleotide sequence and if when a deletion is made, one or more nucleotides are removed from the complete nucleotide sequence, either at either end of the sequence or at any suitable site therein.

The modified DNA sequence can be obtained by methods well known in the art, for example, by site-directed mutagenesis.

In another embodiment, the invention relates to a subsequence of the chitinase 4 DNA sequence, SEQ ID NO: 1, encoding a polypeptide that may not, but may have, sugar beet chitinase 4 antifungal activity. Particularly interesting subsequences of the chitinase 4 DNA sequence or genomic DNA sequences are subsequences containing a nucleotide sequence defining the active site of the sugar beet chitinase 4 enzyme. An example of such a subsequence is a DNA sequence containing the active site of a sugar beet chitinase 4 enzyme, for example a DNA sequence encoding the following peptide designated peptide 4-22 (indicated using a common one letter amino acid code) consisting of amino acids 179-200 in SEQ ID NO: 2.

S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R

This sequence is the amino acid sequence of tryptic peptide 4-22 obtained, from purified sugar beet chitinase 4, as described in Example 16, see below. The DNA sequence encoding this polypeptide may be of particular importance for making modifications to the active site to improve the antifungal activity of the resulting polypeptide. Furthermore, this DNA sequence may be linked to a portion of another DNA sequence encoding an enzyme other than sugar beet chitinase 4 or substituted with a portion of such an enzyme encoding its active site to obtain a hybrid enzyme having antifungal activity of sugar beet chitinase 4. Of course, the polypeptide chain of the hybrid enzyme should be able to fold in the correct manner to ensure the correct conformation around the active site.

Another interesting DNA sequence encoding a portion of the chitinase 4 enzyme is a DNA sequence encoding a polypeptide having the following amino acid sequence consisting of amino acids Nos. 183-204 in SEQ ID NO: 6

S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-D-A-V-I-A-F-K

This polypeptide is derived from the DNA sequence of genomic chitinase 76 clone shown in SEQ ID NO: 5 and almost corresponds to the DNA sequence of peptide 4-22 above, except for the most important fact that the highlighted D site is in peptide 4-22 N. It is considered that the chitinase 76-derived polypeptide may have the same or almost the same interesting properties and use as peptide 4-22.

The other two DNA sequences of interest are the sequences encoding the following peptide consisting of amino acids Nos. 163-169 in SEQ ID NO: 2.

G-P-L-Q-I-T-W which is the tryptic peptide 4.19.3 of chitinase 4 and the DNA sequence encoding the tryptic peptide 4-26 consisting of amino acids No. 201-224 in SEQ ID No. 2

T-A-F-W-F-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-S-I which sequences are described in Example 16, see below. The peptides contain one or two tryptophan residues, respectively. Tryptophan residues are thought to be involved in the active site and / or affect the substrate specificity of the chitinase 4 enzyme, for example, as further discussed in Example 16, see below. Of particular interest may be analogs of the aforementioned subsequences in which at least one nucleotide has been deleted, substituted or modified, or in which at least one additional nucleotide has been inserted and which still have catalytic and / or binding activity as the three aforementioned peptides encoded by chitinase 4 DNA subsequences. .

Another example of an interesting subsequence according to the invention is a subsequence of the DNA sequence chitinase 4, SEQ ID No. 1, encoding a polypeptide comprising the hevein domain of the sugar beet chitinase 4 enzyme, or an analogue of said subsequence in which at least one nucleotide has been deleted, substituted or modified or at least one additional nucleotide has been inserted and which subsequence encodes a polypeptide capable of binding to chitin, as determined by affinity column chromatography on regenerated chitin prepared as described in the Materials and Methods section entitled Preparation of Chitin Column.

Because the hevein domain of the chitinase 4 enzyme is compact and is thought to be very efficient, ie capable of forming a tight bond to chitin, this domain may prove very useful in modifying chitinases, such as other plant chitinases, containing either no or a weak hevein domain to impart a stronger chitin-binding ability to such chitinases. Examples of chitinases that can be advantageously modified by inserting a DNA sequence encoding the hevein domain of sugar beet chitinase 4 are non-hevein group or cucumber group chitinases (e.g., sugar beet chitinase SE described herein).

Another interesting subsequence according to the invention is the subsequence of the chitinase 4 DNA sequence, SEQ ID No. 1, encoding the chitinase 4 leader peptide or analogue thereof, in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted, and which is capable of directing a mobile polypeptide to which it is attached out of the cell in which the fused leader and mobile polypeptide are produced to be delivered to the intercellular space.

As explained above, sugar beet chitinase 4 enzyme epitopes can be used to generate monospecific polyclonal and monoclonal antibodies that are useful in identifying chitinase 4 isoenzymes belonging to the serological class of chitinase 4 and prc epitope mapping. The finding of suitable epitopes is expected among the hydrophilic peptides of the amino acid sequence of chitinase 4, SEQ ID NO: 2, because these enzymes appear to differ significantly from the peptide portions of chitinases other than sugar beet chitinase 4. Antibodies (either monoclonal, monospecific, or polyspecific) can be prepared using conventional methods, for example, as described in Materials and Methods, see below, based on synthetically produced peptide portions of the sugar beet chitinase 4 enzyme. Based on routine computer analysis of the DNA sequence and amino acid sequence of chitinase 4, the following possible epitopes of SEQ ID NO: 2 were identified:

Peptide 1: AGCRPHYTHRA (consisting of amino acids No. 87-95)

Peptide 2: CNPSKQYY (consisting of amino acids Nos. 153-160)

Peptide 3: IECNGGNS (consisting of amino acids 230-237)

Peptide 4: TARVGYYTQYCQ (consisting of amino acids Nos. 241-252)

These epitopes are believed to be particularly suitable for the production of monospecific antibodies against sugar beet chitinase 4. Peptide 1 and peptide 4 are considered to be the most suitable peptide sequences for use in the production of monospecific antibodies against chitinase 4.

It is understood that a DNA sequence comprising a subsequence according to the invention in which one or more nucleotides has been modified, for example as mentioned above, and which has substantially retained the function and / or characteristics of the subsequence is within the scope of the invention.

As mentioned above, as well as plant chitinases, there are also bacterial ones. In this context, in which the construction of genetically transformed plants is described as an important use of the DNA sequence according to the invention, plant chitinases are considered to be the most interesting type of chitinase, and the DNA sequence according to the invention or its analogue or subsequence is preferred. which are of plant origin. Particularly interesting DNA sequences of plant chitinas come from species of the family Chenopodiaceae, Solanaceae, Apiaceae, Brassicaceae, Cucurbitaceae or Fabaceae.

Examples of such plants are corn, alfalfa, barley, wheat, rye, rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, tomato, potato, soybean, oilseed rape, cabbage, peppercorn, lettuce, beans and peas.

The terms sequence, subsequence, and analog, as used herein with respect to the sequences, subsequences, and analogs of the invention, are, of course, to be understood not as including these entities in their natural environment, but rather, for example, in isolated, purified, in vitro, or recombinant form.

The DNA sequence of chitinase 4 according to the invention or an analogue or subsequence thereof as defined above, and in particular a single-stranded DNA or RNA sequence which is substantially complementary to any strand of this DNA sequence, can be used to isolate corresponding sequences from other plants. , if desired, modified as described herein.

It is clear from the above interpretation that the DNA sequence of chitinase 4 according to the invention, or an analogue or subsequence thereof, may be joined to one or more other nucleotide sequences encoding another polypeptide or part thereof under conditions which ensure that at least part of the DNA sequence according to the invention is expressed in combination with another nucleotide sequence or sequences, for example in the form of a fusion protein. For example, a DNA sequence of the invention encoding a polypeptide having antifungal activity of the sugar beet chitinase 4 enzyme may be advantageously linked to a C-chain sequence encoding a signal peptide that causes transport of the fusion protein expressed therefrom to specific organelles of the organism expressing the polypeptide. The transport-causing signal peptides are discussed in more detail below. Similarly, interesting subsequences of the chitinase 4 DNA sequence, such as those described above, e.g., subsequences encoding a hevein domain and / or epitope, may be joined to DNA sequences encoding other proteins, such as enzymes, e.g., chitinases, to impart desired properties to the polypeptides encoded by the subsequences. chitinase DNA sequence 4.

Also within the scope of the invention is a polypeptide encoded by the chitinase 4 DNA sequence or an analog or subsequence thereof as defined above, preferably in a non-naturally occurring or recombinant form. Compared to the naturally occurring enzyme chitinase 4, the polypeptide of the invention has the advantage that it can be easily produced in large quantities using well known conventional recombinant production techniques, for example as described by Sambrook et al., 1990, and that it can be obtained. in the form of free impurities normally associated with naturally occurring sugar beet chitinase 4. The polypeptide of the invention may be used as a component of an antifungal agent, for example as described below.

As explained above and in the following examples, the sugar beet chitinase 4 enzyme exhibited a number of advantageous properties, including surprisingly high antifungal activity compared to other known chitinases, such as other known sugar beet chitinases, apparently due to its dual chitinase / lysozyme activity and its compact structure. . Advantageous properties of the sugar beet chitinase 4 enzyme also include its strong hevein domain. Thus, it is believed that the use of a DNA sequence encoding sugar beet chitinase 4 or an analogue thereof encoding a polypeptide having antifungal activity as defined above will be of great interest in the construction of genetically modified plants having increased resistance to phytopathogenic fungi compared to non-transformed plants.

Accordingly, according to another important embodiment, the invention relates to a genetic construct comprising

1) a promoter operably linked to

2) a DMA sequence comprising the DNA sequence of chitinase 4 or an analog or subsequence thereof as defined above and

3) a transcription terminator operably linked to the DNA sequence.

The genetic construct can be used in the construction of a genetically modified plant to produce a plant exhibiting increased antifungal activity as determined by the method set forth in Example 2, and thus increased resistance to phytopathogenic fungi. It is further contemplated that the genetic construct may be used to increase the ability of plants to degrade chitin. An example of a genetic construct as defined above is given in Example 18, see below.

Experiments have further shown (see Example 2) that the treatment of phytopathogenic fungi (Cercospora beticola and Trichoderma viride) with a composition containing a polypeptide having antifungal activity of sugar beet chitinase 4, in admixture with acid chitinase and basic beta-1,3-glucanase, results in to drastically reduce the growth of fungal hyphae and reduce the number of germinating spores. In this context, it is expected that a synergistic effect will generally be observed when sugar beet chitinase 4 is used in combination with other chitinases and beta-1,3-glucanases, preferably of plant origin.

In another important embodiment, the invention relates to a genetic construct comprising one or more copies of a DNA sequence as defined above, comprising the chitinase 4 DNA sequence set forth in SEQ ID NO: 1, or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide. which has a second chitinase activity other than sugar beet chitinase 4, and / or one or more copies of a DNA sequence encoding a polypeptide having beta-1,3-glucanase activity, each of these DNA sequences being operably linked to a promoter and a transcription terminator capable of expressing DNA sequences into functional polypeptides.

Polypeptides with chitinase or beta-1,3-glucanase activity are preferably of plant origin. Chitinase and beta-1,3-glucanase activity can be determined as described in Materials and Methods, below.

Of particular interest is a genetic construct comprising one or more copies of a DNA sequence as defined above, comprising the chitinase 4 DNA sequence set forth in SEQ ID NO: 1, or an analog or subsequence thereof, one or more copies of a DNA sequence encoding acidic chitinase having pI equal to or less than 4.0, and one or more copies of a DNA sequence encoding a basic beta-1,3-glucanase having a pi of at least 9.0, each of said DNA sequences being operably linked to a promoter and a transcription terminator capable of expressing the DNA sequences. into functional polypeptides.

As used herein, acid chitinase is defined as a chitinase that has a pi of less than 4.0. Preferably, the acidic chitinase is chitinase, which hydrolyzes chitin to chi31 tooligosaccharides of the hexameric type. Preferably, the acidic chitinase is of plant origin. Examples of such chitinases are cucumber lysozyme / chitinase and Arabidopsis, as well as sugar beet acid SE chitinase having the DNA sequence set forth in SEQ ID NO: 7 below, and the amino acid sequence set forth in SEQ ID NO: 8 below, or an analog the above-mentioned DNA sequence encoding an acidic chitinase having a pI of at most 4.0 and preferably is capable of hydrolyzing ³ -chitinase mainly to hexamers.

As used herein, the term basic beta-1,3-glucanase refers to beta-1,3-glucanase having a pI of greater than 9.0. Preferably, the basic beta-1,3-glucanase is capable of hydrolyzing glucan mainly to dimers, for example as determined by a 3

H-laminarin described in the Materials and Methods section, see below. The basic beta-1,3-glucanase is preferably of plant origin. Examples of suitable basic beta-1,3-glucanases are basic beta-1,3-glucanases obtained from tobacco (Shinshi et al., 1990), barley (Fincher et al., 1986) or sugar beet, for example basic beta-1. Sugar beet 3-glucanase 4, the DNA sequence of which is given in SEQ ID No. 9, see below, or an analogue thereof encoding basic beta-1,3-glucanase, which has a pI of at least 9.0 and is preferably capable of hydrolyzing 1 H- laminarin mainly on beta-1,3-glucan dimers. Alkaline sugar beet beta-1,3-glucanase 4 differs from other plant beta-1,3-glucanases in that it does not contain a C-terminal extension, as seen in the amino acid sequence of SEQ ID NO: 10, see below. The beneficial effect of using basic sugar beet beta-1,3-glucanase 4 may be due in part to the lack of this C-terminal extension.

Another interesting sugar beet chitinase is sugar beet chitinase 1, which shows very low homology to sugar beet chitinase 4 according to the invention, see above. The DNA sequence of sugar beet chitinase 1 is shown in SEQ ID No. 11, see below. The DNA sequence is approximately 6.3 kb in length and encodes a polypeptide having 439 amino acid residues. The poly32 peptide shown in SEQ ID NO: 12, below, contains a 26 amino acid leader leader sequence, a 20 amino acid residue hevein domain, and a 23 amino acid C-terminal extension. In addition, this sequence contains the most interesting domain of 238 amino acids rich in proline, which forms an interesting embodiment of the invention.

The experiments reported in Example 2 below showed that the combination of the sugar beet chitinase 4 enzyme, acid chitinase and basic beta-1,3-glucanase results in increased antifungal activity compared to the antifungal activity of each of the components. The increased antifungal activity observed using this specific combination is probably due in part to the different mode of action of acid chitinase, basic beta-1,3-glucanase, and sugar beet chitinase 4, respectively. Because acid chitinase hydrolyzes chitin primarily to hexamers (compared to chitinase 4, which hydrolyzes chitin primarily to dimers) and basic beta-1,3-glucanase hydrolyzes glucan primarily to dimers, these different methods are thought to cleavage can affect the resulting beneficial overall effect.

It is further believed that the synergistic effect obtained using a combination of sugar beet chitinase 4, a polypeptide having a second chitinase activity other than chitinase 4, e.g. acid chitinase, and a polypeptide having beta-1,3-glucanase activity, e.g. basic beta-1,3- glucanase, is due to the fact that such a combination attacks both the chitin and glucan components of the cell wall of phytopathogenic fungi and also the parts of the cell wall in which the chitin and glucan components are tightly bound to each other. Beta-1,3-glucanase further causes the removal of the outer glucan layer covering the chitin structure of chitin-containing plant pathogens, such as phytopathogenic fungi, which results in exposure of the chitin structure to the enzymatic effects of chitinase.

The DNA sequences encoding the second chitinase mentioned above and beta-1,3-glucanase can be obtained, for example, from already known sources, or can be identified and isolated from natural sources, for example, using the techniques described herein.

Obviously, a large number of different genetic constructs can be designed and prepared as defined above. Without exhaustive list, elements of genetic constructs that may vary the copy number of each of the DNA sequences of the genetic construct, the specific nucleotide sequence of each of the DNA sequences, the type of promoter and terminator attached to each of the DNA sequences, and the type of any accompanying sequences, for example C-terminal or N-terminal sequences (described below). Thus, the genetic constructs of the invention can vary widely. Normally, the combination of each of the aforementioned variable elements of the genetic construct to be selected depends, for example, on the desired strength of antifungal effect to be achieved, which can be determined as a function of gene amount and specific nucleotide sequence of each DNA sequence, and promoter type and strength. and a terminator used for each DNA sequence. With different types of promoter and terminator, expression in specific parts of the plant may also vary in terms of organs and intracellular and extracellular location.

However, in designing a genetic construct of the invention to be expressed in a particular organism, such as a plant, the possible toxic effects of overexpression of one or more proteins encoded by the genetic construct, which may lead to lower yield of the transformed organism, e.g. plants, compared to a non-transformed organism or an organism that does not contain a genetic construct. If the genetic construct of the invention is too large, it may also be difficult to achieve its stable integration into the plant genome, which may lead to excision of part or all of the genetic construct from the plant genome. Thus, the genetic construct should be adapted so that its expression products are generally acceptable to the host organism.

The copy number of the DNA sequences of the genetic construct of the invention, together with the activity of the genes, determines the optimal copy number of the DNA sequences of the genetic construct of the invention. Due to the rapidly growing knowledge of plant genetic engineering, improved transformation and biological control techniques can be developed to allow the introduction of larger foreign genetic fragments into a plant without causing growth retardation, yield reduction or recombination problems, compared to today's options. .

Currently, a construct that contains only a few copies of the DNA sequence of the invention is preferred. Accordingly, it is preferred that each of the DNA sequences of the genetic construct of the invention be present in only one copy. The construction of a genetic construct containing one copy of each of the DNA sequences is shown in the examples below.

As mentioned above, a substantial antifungal effect is obtained with the protein encoded by the chitinase 4 DNA sequence of the invention or an analogue thereof. Accordingly, a genetic construct of the invention in which two copies of the DNA sequence of chitinase 4 of the invention or an analogue thereof, and one copy of each of the DNA sequences encoding acidic chitinase and basic beta-1,3-glucanase are present, is contemplated. may have very strong antifungal effects in the genetically transformed plant according to the invention. It is believed that such a genetic construct will not be too burdensome for the plant in which it is contained. Of course, the choice of, for example, the promoter used for each DNA sequence will also affect the amount of protein expressed therefrom, as will be explained below.

The genetic construct of the invention, as described above, may be present on one or more DNA fragments. Depending on the size of the genetic construct to be introduced into an organism such as a plant, in the case of a plant typically by a plant transformation vector, and a combination of promoters and transcription terminators, it may be advantageous to introduce the construct using two or more plant transformation vectors , and accordingly it may be advantageous for the genetic construct to be present on two or more DNA fragments. If it is desired to use only one vector for plant transformation, it is preferred that the genetic construct be present on a single DNA fragment.

If a polypeptide encoded by a DNA sequence of the invention is to be expressed in an organism, such as a plant, it is desirable that the DNA sequence further comprise a nucleotide sequence encoding a leader sequence. The leader sequence may be a natural leader sequence, or a leader sequence derived from DNA encoding another protein. In each case, the leader sequence should be operably linked to the DNA sequence such that the polypeptide expressed from the resulting nucleotide sequence serves to direct the polypeptide encoded by the DNA sequence out of the cell in which it is produced. Depending on the nature of the leader sequence used, the polypeptide may be directed to specific sites in the organism in which it is produced, for example, to lysosomes or vacuoles, or the motile polypeptide may be secreted into the intracellular space. The leader sequence can be located at either the N-terminus or the C-terminus.

The nature of the N-terminal sequence to be used depends, for example, on the particular organism or part thereof, for example the specific cell or tissue in which the polypeptide encoded by the DNA sequence of the invention is to be produced and the part of the same cell or other site where the organism is to be produced. polypeptide transported. A typical leader peptide has a center of hydrophobic amino acids, and a suitable leader sequence to be used in conjunction with the DNA sequence of the invention is thus a nucleotide sequence comprising a band of codons encoding hydrophobic amino acids.

Examples of leader sequences to be used in this context are the following leader sequences, which are also part of the invention. These leader sequences are the N-terminal leader sequence of the sugar beet chitinase 1 enzyme, the nucleotide and amino acid sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the N-terminal leader sequence from genomic chitinase 76 clone, the nucleotide and amino acid sequence of which are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively, the N-terminal leader sequence of sugar beet SE chitinase, the nucleotide and amino acid sequences of which are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively, and the N-terminal the sequence of beta-1,3-glucanase 4, the nucleotide and amino acid sequences of which are given in SEQ ID No. 9 and SEQ ID No. 10, respectively. Another interesting sequence is the subsequence of sugar beet chitinase 1 encoding the proline-rich domain of the chitinase 1 gene, which contains 132 amino acids and is set forth in SEQ ID NO: 12, which may also be used to direct a polypeptide to a specific site in an organism. The above leader sequences are considered to be non-limiting examples.

Since all of the above leader sequences of the invention are specific for sugar beet plants, these leader sequences according to another embodiment of the invention may be operably linked to a DNA sequence different from the DNA sequences of the invention, which DNA sequence is to be used to transform a sugar beet plant. Such a DNA sequence may in particular be a DNA sequence which does not occur naturally in the sugar beet plant. The use of a leader sequence normally present in sugar beet may be an advantage in the transformation of a sugar beet plant, as such a leader sequence is known to function in sugar beet. The leader sequence of the invention may thus serve to direct the poly37 peptides expressed from the nucleotide sequence to specific sites in the cell or organism where it is produced.

Another interesting subsequence according to this embodiment of the invention is the proline-rich domain of chitinase 1, set forth in SEQ ID NO: 12, which consists of 132 amino acids. It is thought that the proline-rich domain may be involved in the attachment of the chitinase 1 protein to the cell wall after modification of prolines to glycosylated hydroxyprolines. Thus, a subsequence containing a proline-rich domain can be used to control and maintain the polypeptide at a desired site in the cell and / or organism where the polypeptide is produced.

In addition, it may be advantageous if at least one of the DNA sequences of the genetic construct of the invention further comprises a C-terminal sequence encoding a signal peptide capable of directing the polypeptide encoded by the DNA sequence to the part of the organism in which it is to be deposited. Thus, the same DNA sequences may be present in the same genetic construct with or without a C-terminal sequence. The C-terminal sequence may be a C-terminal extension normally associated with the DNA sequence, or may be obtained from the host in which the genetic construct is to be expressed, or may be of another origin. This is particularly important in the context of the DNA sequence of chitinase 4 and the DNA sequence of sugar beet basic beta-1,3-glucanase and acid chitinase SE, all of which lack a C-terminal extension. In DNA sequences that normally contain a C-terminal extension, the native C-terminal sequence may be replaced by another sequence.

Non-limiting examples of C-terminal sequences that may be included in the genetic construct of the invention are C-terminal sequences selected from the group consisting of the following sequences:

The C-terminal sequence of sugar beet chitinase 1, the amino acids of which are set forth in SEQ ID NO: 12, encoding the following polypeptide, which consists of amino acids Nos. 413-439

NLDGYRQTPFDWGLKKLQGARESWSSS *

C-terminus of sugar beet chitinase 4, encoding the following polypeptide, which consists of amino acids No. 26 1-264 in SE

ID No. 2

N L R C *

The C-terminal sequence of bean chitinase (PHA), encoding the following polypeptide set forth in SEQ ID NO: 13

NLDCYSQTPFGNSLLLSDLVTSQ *

The C-terminal sequence of basic tobacco chitinase encoding the following polypeptide set forth in SEQ ID NO: 14

NLDCGNQRSFGNGLLVDTM *

The C-terminal sequence of tobacco acid chitinase encoding the following polypeptide set forth in SEQ ID NO: 15

NLDCYNQRNCFAG *

The C-terminal sequence of barley chitinase CH26 encoding the following polypeptide set forth in SEQ ID NO: 16

NLDCYSQRPFA *, a

The C-terminal sequence of the basic beta-1,3-glucanase of tobacco encoding the following polypeptide set forth in SEQ ID NO: 17

GVSGGVWDSSVETNATASLVSEM

The decision whether to attach the C-terminal sequence to one or more DNA sequences of the genetic construct is made, for example, based on which part of the plant the polypeptide expressed from the sequence is to be directed to. Thus, if it is desired to control a phytopathogenic fungus present mainly in the intercellular space of a plant, it may be desirable not to use a C-terminal sequence. If a phytopathogenic fungus present mainly intracellularly is to be controlled, it may be desirable for most or all of the DNA sequences of the genetic construct to be provided with a C-terminal sequence capable of transporting polypeptides expressed from the DNA sequences into the vacuole.

As is apparent from the above explanation, it is important to achieve sufficient expression of the polypeptides encoded by the genetic construct in plants containing the construct to allow the polypeptides to exert their intended function, i.e., to exert their antifungal activity. One of the necessary elements to achieve sufficient expression is to ensure satisfactory regulation of the transcription and expression of the DNA sequence or gene from which the polypeptide is expressed.

Expression of each of the DNA sequences of a genetic construct of the invention or a gene containing such DNA sequences is achieved by a regulatory sequence operably linked to the DNA or gene sequence so as to achieve expression of that sequence or gene under the control of an inserted regulatory sequence. Typically, this regulatory sequence is a promoter, which may be constitutive or regulatable.

The term promoter refers to a short DNA sequence to which RNA polymerase and / or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is operably linked, thereby allowing transcription. The promoter is usually located upstream (5) from the coding sequence. In a broader sense, the term promoter includes an RNA polymerase binding site as well as regulatory sequence elements located several hundred base pairs apart, sometimes further, from the transcription start site. Such regulatory sequences include, for example, sequences that are involved in the binding of protein factors that control the efficiency of transcription initiation in response to physiological conditions.

A constitutive promoter is a promoter that is not subject to essentially any regulation such as induction or repression, but that allows constant and substantially unchanged transcription of the DNA sequence to which it is operably linked in all active cells of the organism, provided other requirements are met for the course of transcription. The constitutive promoter may be affected by the enhancer.

A regulatable promoter is a promoter whose function is regulated by one or more factors. The presence of these factors can either ensure the expression of the corresponding DNA sequence, or alternatively it can suppress the expression of the DNA sequence, so that their absence causes the expression of the DNA sequence. Thus, the promoter and, optionally, its concomitant regulatory sequences may be activated by the presence or absence of one or more factors that affect the transcription of each of the DNA sequences of the genetic construct of the invention.

Other types of regulatory sequences are upstream and downstream sequences involved in the control of transcription termination (transcription terminators) and intron deletion, as well as sequences responsible for polyadenylation and translation initiation. If the regulatory sequence is to function in a plant, it is preferably of plant origin.

Factors regulating promoter activity may vary depending on, inter alia, the type of promoter used, as well as the organism in which they are to function. Tissue-specific regulation can be achieved by certain intrinsic factors that ensure that genes encoding tissue-specific proteins are expressed. Examples of tissue-specific promoters are leaf-specific promoters, such as the chlorophyll a / b promoter and the sugar beet acetic acid synthase (AHAS) promoter, as well as root-specific, stem-specific promoters, seed-specific and flower-specific promoters. Promoters have also been found to regulate factors such as pathogenic infestation or certain biological factors. Also of interest may be promoters of the heat irritation response and promoters involved in plant developmental regulation.

In this context, a suitable constitutive promoter is selected from the group consisting of plant promoters, fungal promoters, bacterial promoters and plant virus promoters.

A preferred group of plant virus promoters are promoters derived from cauliflower mosaic virus (CaMV). These promoters are normally strong constitutive promoters. Examples of preferred CaMV promoters are the CaMV 19S promoter and the CaMV 35S promoter (Odeli et al., 1985).

Additional promoters can be obtained from a Ti-plasmid, such as the octopine synthase promoter, the nopaline synthase promoter (Herrera-Estrella et al., 1983), the mannopine synthase promoter, and promoters from other open reading frames in T-DNA, such as · Is 0RF7. Other examples of suitable promoters are MAS / 35S (Janssen and Gardner, 1989), MAS dual Tr 1,2 (Velten et al., 1984) and the T-2 DNA gene promoter (T-2 DNA gene 5 promoter, Konz and Schell , 1986).

The regulatory sequence may be a chitinase promoter, i.e. a promoter that is naturally associated with and involved in the transcription of chitinase genes. The chitinase promoter can be obtained from an isolated chitinase gene, for example, from a known chitinase gene or a gene that can be identified and isolated, for example, by the methods described herein. Typically, the chitinase promoter should be obtained from a plant that has shown a rapid response to pathogen infestation. In this context, rapid responses have been observed in peas and barley, and it is believed that chitinase promoters from these plants may be suitable for this purpose. An example of such a promoter is the pea chitinase promoter (K. Vad, 1991). Examples of another promoter that is expected to be useful in this regard are the sugar beet chitinase 1 promoter (SEQ ID NO: 11) and the sugar beet acetic acid synthase (AHAS) promoter (P. Stougard and K. Bojsen, Danisco A / S, Denmark, personal communication). In addition, sugar beet promoters from acid chitinase SE, chitinase 1, chitinase 76 and chitinase 4 or beta-1,3-glucanase 4 may also be suitable.

If desired, the native promoter may optionally be modified for the purpose, for example by modifying the nucleotide sequence of the promoter to obtain a promoter that functions differently from the native promoter, preferably to activate gene transcription earlier after pathogen challenge or to make the promoter stronger. .

As noted above, each of the DNA coding sequences of the genetic construct of the invention is operably linked to a transcription terminator. The transcription terminator terminates the transcription of DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences, and plant virus terminator sequences.

Specific examples of suitable transcriptional terminators are the transcriptional terminator sequences of the NOS and OCS genes of the opin synthase genes from the genus Agrobacterium (Herrera-Estrella et al., 1983), the transcriptional terminator sequences 35S from cauliflower mosaic virus (Paszkowski et al., 1984) and the transcriptional terminator PADG4. gene 4 DNA (Wing et al., 1989) and the trans43 crip terminator PADG7 gene 7 T-DNA.

One or more DNA sequences of the genetic construct of the invention may advantageously be operably linked to an enhancer sequence, resulting in increased transcription and expression of the DNA sequence or sequences. Suitable enhancer sequences and methods for achieving increased transcription and expression are known in the art.

The particular promoters or specific terminators associated with each of the DNA sequences of the genetic construct may be the same or different. It may be advantageous to use different promoters or terminators, as this avoids the risk of recombination problems that can lead to the exudation of part or all of the genetic construct.

In another embodiment, the invention relates to a vector capable of replication in a host organism and which carries a DNA sequence of the invention comprising the DNA sequence of chitinase 4, substantially as set forth in SEQ ID NO: 1, or an analogue or subsequence thereof, or a genetic construct according to of the invention. The vector may be either capable of autonomous replication, such as a plasmid, or is replicated with a host chromosome, such as a bacteriophage, or is integrated into the plant genome via the flanking sequences of Ti vectors. For production purposes, a vector is an expression vector capable of expressing DNA sequences in an organism selected for production. Thus, an expression vector is a vector that carries the regulatory sequences necessary for expression, such as a promoter, an initiation signal, and a termination signal, etc. These regulatory sequences may be sequences that carry a genetic construct of the invention. The vector can also be used to identify and optionally isolate chitinase genes or mediators from other organisms, such as other plants, for which expression is not required. This can be done, for example, as described below.

In yet another embodiment, the invention relates to an organism that carries and is capable of replicating or expressing an inserted DNA sequence as defined above, i.e. a chitinase 4 DNA sequence comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 1 or an analogue thereof, or a chitinase gene or pseudogen containing the above-mentioned DNA sequence.

The term inserted indicates that a DNA sequence (or subsequence or analogue, or gene or pseudogen) has been inserted into an organism or its ancestor using genetic manipulation, in other words, an organism that does not naturally or inheritably contain such a DNA sequence in its genome, or it may be an organism that naturally or inheritably contains such a DNA sequence, but in a lower amount, so that organisms with an inserted DNA sequence or genetic construct contain a higher number of such sequences than naturally occurring analogous organisms.

The DNA sequence carried by the organism may be part of the genome of the organism, or may be carried by a vector as defined above which is contained in the organism. The DNA sequence may be present in the genome or expression vector as defined above in frame with one or more other DNA sequences encoding the second polypeptide or part thereof, and thus encode a fusion protein, for example as defined above.

The organism may be a higher organism, such as a plant, or a lower organism, such as a microorganism. For the production of recombinant polypeptides as defined above, a lower organism such as a bacterium, for example a gram-negative bacterium such as a bacterium of the genus Escherichia, for example Ξ, is suitable. coli, or of the genus Pseudomonas, for example P. putida and P. fluorescens, or gram-positive bacteria, such as bacteria of the genus Bacillus, for example B. subtilis, or yeast, such as yeast of the genus Saccharomyces or a fungus, for example of the genus Aspergillus. Although many organisms inherit chitinase production, insertion of the DNA sequence or genetic construct of the invention can lead to a significantly increased ex45 presi of chitinase and possibly beta-1,3-glucanase and a corresponding increase in antifungal activity. Recombinant production can be performed using conventional techniques, for example, as described by Sambrook et al., 1990.

As will be discussed further below, the chitinase-producing microorganism can be used to combat soil plant pathogens, i.e., pathogens present in the soil and responsible for growth retardation or plant death. Examples of such plant pathogens are soil fungi present, for example, in the rhizosphere.

The organism may also be a cell line, for example a plant cell line. Most preferably, the organism is a plant, i.e. a genetically modified plant, as will be discussed in more detail below.

As mentioned above, the genetic construct is preferably used in plant modification. Accordingly, the invention also relates to a genetically transformed plant which comprises in its genome a genetic construct as defined above. A genetically transformed plant has increased antifungal activity compared to a plant that does not contain a genetic construct of the invention, for example an untransformed or natural plant or a plant that has been genetically transformed but not using a genetic construct of the invention. Normally, constitutive expression of polypeptides encoded by a genetic construct is desired, but in some cases it may be of interest for the expression of polypeptides encoded by the genetic construct to be regulated by various factors, such as temperature, pathogens, and biological factors.

Chitinase genes have been found in monocotyledonous as well as dicotyledonous plants and have been found to be expressed in chitinases active in killing cell walls of phytopathogenic fungi.

Accordingly, the plant to be transformed using the genetic construct of the invention may be monocotyledonous as well as dicotyledonous, as the genetic construct is expected to be active in these groups of plants. Non-limiting examples of monocotyledonous plants that can be transformed are corn, oats, wheat, rye, rice, barley and sorghum.

Non-limiting examples of dicotyledonous plants that can be genetically transformed are alfalfa, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, tomato, potato, soybean, oilseed rape, cabbage, peppercorn, lettuce, beans, and peas.

From the above description, it is clear that the genetically transformed plant of the invention has increased resistance to plant pathogens containing chitin, such as phytopathogenic fungi and nematodes, compared to plants that have not been genetically transformed according to the invention or compared to plants that do not contain the genetic construct. as defined above.

The most important chitin-containing plant pathogens to be controlled according to the invention are phytopathogenic fungi. Phytopathogenic fungi differ in the way they interact with their host plant during infection. Some species attack the plant through natural openings or injured tissues and grow between plant cells, in the intercellular space, throughout the infection cycle. Fungal hyphae secrete toxins or enzymes that weaken or destroy plant cells, thereby supplying the fungus with cellular components that escape from plant cells. Other fungal pathogens immediately destroy host cells by penetrating the cell walls of healthy host cells and disintegrating their protoplasts.

Below are some examples of phytopathogenic fungi containing chitin and glucan with different host interaction strategies, all of which are expected to be susceptible to the transgenic plants of the invention.

Cercospora spp. is a fungus whose growth is restricted to the intercellular space. Conidia (i.e. spores) of the fungus germinate on the leaf surface and penetrate through the leaf vents. Inside the leaf, plant cells near hyphae growing in the intercellular space are strongly affected by toxins secreted by the fungus. Toxins cause degradation of the plasma membrane and thus the contents of the cell are poured into the intercellular space. Later in the infection cycle, plant cells collapse and necrotic areas containing dead plant cells and fungal mycelium appear.

Verticilium alboatrum is a root pathogen that spreads in the intercellular space, but which penetrates holes that appear when lateral roots grow, through mechanically damaged areas, or by direct penetration of hyphae through fine root tissue in the area of cell elongation or meristematic activity. The fungus destroys parenchymal cells and the conductive elements are mechanically clogged.

Other fungi pathogenic to plants with an intercellular infection cycle include Sclerotinia sclerotiorum, Rhizoctonia solani, Phytophtora megasperma and Helmintosporium spp.

Colletotricum lindemuthianum causes anthracnose of beans. The conidia of this fungus germinate in a layer of water at the site of infection and the germ formed penetrates the cuticle and grows into the epidermal cells of the leaves and beans of the beans. During the ensuing infection, the fungus acts as a parasitic pathogen, penetrating living cells and causing protoplast disintegration.

Fusarium spp. is typical of soil-surviving fungus infecting plants through roots, where hyphae penetrate the epidermal cells of young roots and attack the xylem of roots and stems. The blood vessels become clogged with granular material and the cells surrounding the outer phloem and cortex are destroyed.

Puccinia graminis causes stalks in wheat. Sporidia germinate in a layer of water on the surface of the plant and the germs penetrate the cuticle. The growing mycelium forms haustoria that penetrate the walls of host cells and invagine their protoplasts.

Ustilago maydis is a fungus that grows mainly intercellularly, but occasionally penetrates the cell walls of host cells.

In another embodiment, the invention relates to seeds, seedlings and plant parts obtained by growing a genetically transformed plant as described above. It is understood that any plant part or cell derived from a genetically transformed plant of the invention is within the scope of the invention.

At present, great efforts are being made to develop suitable methods for constructing new plants or plant cells having specific and desired properties by transferring new genetic information encoding the desired properties to the plant, and many methods based on recombinant DNA and suitable plant systems are now available. Typically, genetic information is introduced into a plant using a vector system or direct introduction, for example, using the procedures described by Herrera-Extrella et al., 1988; Rogers et al., 1988; Saul et al., 1988; An et al. 1988, Hooykaas, 1988; Horsch et al., 1988; Reynaerts et al., 1988, and Tomes et al., 1990.

on transformational technology

Thus, in another embodiment, the invention relates to a transformation system comprising at least one vector which carries a genetic construct as defined above and which is capable of introducing this genetic construct into the genome of a plant such as Chenopodiaceae, in particular beet (Chenopodiaceae). Beta), especially beet species (3eta vulgaris).

Normally, transformation systems are based on the use of plasmids or plasmid derivatives from Agrobacterium. The best known species of the genus Agrobacterium are Agrobacterium tumefaciens and Agrobacterium rhizogenes (their plasmids are hereinafter referred to as pTi and pRi, respectively). The use of these plant transformation systems is based on the ability of Agrobacterium to transfer a specific portion of DNA (T-DNA) to a plant cell in an injured area. In nature, T-DNA is located between specific DNA border sequences on pTi and pRi, which in turn carry the virulence genes necessary for T-DNA transfer into the plant. The Agrobacteria transformation system mediates the transfer of any DNA sequence located between the borders, and thus it is possible to replace the standard type of agrobacterial T-DNA with any desired part of the DNA to be introduced into the plant.

Preferably, the plant transformation system according to the invention is based on disarmed, Agrobacterium-derived derivatives of pTi or pRi, from which the standard type T-DNA has been removed.

Normally, the vector system by which the plant is transformed contains one or two plasmids. In a single plasmid system (also referred to as a cointegrated vector system), pTi or pRi T-DNA was removed and replaced with DNA to be transferred into a plant cell using homologous recombination. In a two-plasmid system (also called a binary vector system), both T-DNA and border were removed from pTi or pRi. Introduction of a small plasmid containing the DNA to be transferred between identical pTi or pRi harvests and suitable origins of replication into disarmed Agrobacteria results in a vector system where virulence functions are located on disarmed pRi or pTi and T-DNA and boundaries are located on another plasmid.

An example of a suitable plant transformation vector is pBI121 and its derivatives, for example as described by Jefferson, 1987.

Preferably, the vector to be used is provided with suitable markers, eukaryotic as well as prokaryotic, e.g. genes encoding antibiotic resistance or herbicide or glucuronidase resistance (GUS), e.g. hygromycin or other known markers, e.g. markers described by Lindsey, 1990 and Reynaerts et al., 1938. The marker should be present to determine if the DNA insert has been inserted at the desired site on the plasmid and to select for plant cells transformed with the vector.

The use of more than one vector in transformation, in accordance with currently known plant transformation techniques, normally requires the presence of different selective genes on each of the vectors in order to determine the success of the plant transformation.

When constructing a transgenic plant using plasmids such as pTi or pRi or derivatives thereof, it is preferred that the genetic construct to be introduced into the plant be first constructed in a microorganism in which the plasmid can replicate and be easily manipulated. An example of a suitable microorganism is Escherichia coli, but other microorganisms having the above properties may be used. If a plasmid or vector system as defined above has been constructed in Escherichia coli, it is transferred, if necessary, to a suitable strain of Agrobacteria, for example Agrobacterium tumefaciens.

Thus, a plasmid containing a genetic construct of the invention is preferably transferred to a suitable Agrobacteria strain, for example Agrobacterium tumefaciens, to obtain an Agrobacteria cell containing a genetic construct of the invention, the DNA of which is subsequently transferred to the plant cell to be modified. This transformation can be performed in a number of ways, for example as described by An et al. (1988).

Direct infection of plant tissues with Agrobacterium is a simple technique that is widely used and described by Butcher et al. (1980). Typically, the plant to be infected is injured, for example by cutting the plant with a razor blade or piercing the plant with a needle or rubbing the plant with an abrasive or scratching the plant with a steel brush (for example as described in Example 15). The wound is then inoculated with Agrobacterium, for example in suspension. Alternatively, the infection of the plant may be performed on a certain part or tissue of the plant, i.e. on a part of the leaf, root, stem or other part of the plant. The inoculated plant or part of the plant is then subjected to selection and regeneration and allowed to grow on a suitable culture medium and allowed to develop mature plants. This is done using methods known per se.

Other very suitable methods for plant transformation are the use of sonication, electroporation (Joersbo, 1990) or particle gun methods, for example as described by Klein et al., 1989.

If genetically transformed plant cells are produced, these cells can be grown and maintained in accordance with well-known tissue culture procedures, such as culturing the cells in a suitable culture medium supplemented, if necessary, with growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of transformed cells into genetically modified plants can be performed using known methods for regenerating plants from cell or tissue cultures, for example, by selecting transformed parts using antibiotics and subculturing these parts on medium containing necessary nutrients, plant horoms, etc.

In accordance with well-known plant propagation techniques, it will be appreciated that the production of a genetically transformed plant may be performed as a double transformation (introducing a genetic construct in two transformation cycles) or may be combined using conventional propagation techniques. Thus, two genetically modified plants of the invention can be crossed to obtain a plant that contains a genetic construct from each of the parent plants.

As will be apparent from the introductory part of the description, the DNA sequence of chitinase 4 according to the invention or an analogue thereof can be used for diagnostic purposes, which will be explained in more detail below.

Various types of diagnoses can be made using the chitinase 4 DNA sequence of the invention. For example, chitinase mediator RNAs transcribed from genes belonging to the chitinase 4 gene family can be quantitatively determined by hybridization to a DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analog or subsequence thereof under conditions suitable for such hybridization. Furthermore, genes belonging to the chitinase 4 gene family present in an organism, such as a plant, can be identified and isolated using the DNA sequence of the invention, for example by screening a gene library of such an organism.

If a DNA sequence comprising a chitinase 4 DNA sequence or an analog or subsequence thereof is to be used for diagnostic purposes, it is often convenient to label it with a label that can be used for detection. Suitable designations are known per se and include, for example, a fluorophore, a radioactive isotope, or a complexing agent such as biotin.

The DNA sequence according to the invention comprising the DNA sequence of chitinase 4 or an analogue or subsequence thereof can also be used to isolate a gene or mediator belonging to or derived from the chitinase 4 gene family from an organism, for example a plant, especially a dicotyledonous. This method involves hybridizing a nucleic acid-containing sample obtained from a gene library or cDNA library of an organism to a DNA sequence of the invention comprising a chi53 tinase 4 DNA sequence or an analog or subsequence thereof, optionally in labeled form, denatured form, or with its RNA- copies, under conditions suitable for hybridization between the DNA or RNA-copy sequence and the nucleic acid in the sample, and further comprising regenerating the hybridized clone to obtain a gene or cDNA of an organism belonging to the chitinase 4 gene family.

Identification and isolation of a gene clone or cDNA clone in a sample belonging to the chitinase 4 gene family using the chitinase 4 DNA sequence of the invention or an analog thereof, especially a subsequence thereof, can be based on standard procedures, e.g., as described by Sambrook et al., 1990. For example, to characterize chitinase 4-related genes in other plants, it is advantageous to use the standard Southern technique.

The DNA sequence of chitinase 4 of the invention or an analog or subsequence thereof can also be used to quantify the amount of chitinase 4-related mediator present in various tissues of an organism, such as a plant. This method comprises hybridizing a nucleic acid-containing sample obtained from an organism to a DNA chitinase 4 sequence of the invention comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 1, or an analogue thereof, especially a subsequence thereof, optionally in labeled form, in denatured form, or with its RNA-copy, under conditions suitable for hybridization between the denatured DNA sequence or RNA-copy and RNA from the sample, and further comprising determining the amount of hybridized nucleic acid (Barkardottir et al., 1987).

Hybridization should be performed in accordance with conventional hybridization procedures under appropriate conditions with respect to, for example, hybridization stringency, incubation time, temperature, ratio of DNA sequence of the invention containing chitinase 4 DNA sequence or analog or subsequence thereof to be used for identification, and the sample to be analyzed, the buffer and salt concentrations and other conditions relevant to hybridization. The choice of conditions depends, inter alia, on the degree of complementarity between the fragments to be hybridized, i.e. a high degree of complementarity requires more stringent conditions such as low salt concentration, low ionic strength of buffer and higher temperatures, while a low degree of complementarity requires less stringent conditions. for example, higher salt concentration, higher ionic strength of the buffer, and lower temperature, for hybridization to occur.

The support to which the DNA or RNA fragments from the sample to be analyzed are bound in denatured form is preferably a solid support and may be any of the support materials commonly used in DNA and RNA analyzes.

The DNA sequence used to detect the presence of chitinase 4-related genes is preferably labeled, for example, as described above, and the nature of the hybridized DNA is determined by autoradiography, scintillation counting, luminescence, or chemical reactions.

Another approach for detecting the presence of a specific chitinase 4-related gene, e.g. introduced by genetic methods described previously, or part thereof in an organism, e.g. a plant, especially a dicotyledonous, is to use the principles of well known polymerase chain reaction, e.g. and methods, see below.

The sample to be analyzed for the presence of the chitinase 4-related gene or a portion thereof, according to the procedures described above, may be taken from a group of plant parts consisting of leaves, stems, tubers, flowers, roots, sprouts, shoots and seeds.

The same principles as described above can be used to isolate DNA sequences to be used in the preparation of a genetic construct of the invention, for example DNA sequences encoding a polypeptide having chitinase or beta-1,3-glucanase activity.

Restriction fragment length polymorphisms (RFLPs) are increasingly being used to monitor specific gene alleles in various organisms. Alleles are either observed on their own or are used as markers (unlinked or connected) in crosses affecting other characteristics, such as pathogen resistance and morphological characteristics such as tuber color. Until now, this procedure has been used mainly in humans, but it has also been used in plants. It is contemplated that the DNA sequence of chitinase 4 according to the invention or an analogue thereof may be useful in the RFLP analysis of chitinase 4-related genes, in particular in sugar beet.

In another embodiment, the invention relates to an antifungal composition comprising a polypeptide encoded by a DNA sequence comprising the chitinase 4 DNA sequence set forth in SEQ ID NO: 1 or an analog or subsequence thereof as defined above or a genetic construct of the invention as defined above and a suitable carrier medium. . In another embodiment, the invention relates to an antifungal composition comprising a microorganism capable of expressing a polypeptide encoded by a DNA sequence comprising the chitinase 4 DNA sequence set forth in SEQ ID NO: 1 or an analog or subsequence thereof as defined above or a genetic construct of the invention as defined above. environment. Microorganisms suitable as components of an antifungal agent are listed above.

The antifungal composition of the invention can be prepared by a method comprising culturing a microorganism comprising and capable of expressing a DNA sequence of the invention comprising the DNA sequence of chitinase 4 set forth in SEQ ID NO: 1 or an analogue or subsequence thereof, or a genetic construct of the invention. under conditions that cause expression of one or more antifungal polypeptides encoded by DNA sequences, further optionally disrupting the microorganisms to release their contents with the ex56 primed polypeptide or polypeptides into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide or polypeptides to drying by freezing or spray drying, thereby obtaining an antifungal composition comprising the antifungal polypeptide or polypeptides. Alternatively, the antifungal proteins may be secreted into the medium, and optionally after removal of the microorganisms by conventional methods or after purification of the proteins by conventional methods or after purification of proline by conventional methods, the medium may be used directly or after freeze-drying.

The antifungal composition of the invention can be used to control or inhibit the germination and / or growth of phytopathogenic fungi in or on a plant or any other material in which the presence of fungi is undesirable, as will be discussed below.

The antifungal composition of the invention may, of course, be adapted to its intended purpose, having regard both to the carrier medium to be used and to the form in which the antifungal agent is present. The term antifungal agent refers to the active ingredient of an antifungal agent that is responsible for or involved in providing antifungal activity. The term antifungal polypeptide refers to a polypeptide encoded by the chitinase 4 DNA sequence of the invention or an analog or genetic construct thereof of the invention that exhibits antifungal activity, i.e. chitinase activity and optionally beta-1,3-glucanase activity as defined above.

The antifungal composition may contain, in addition to the polypeptide encoded by the chitinase 4 DNA sequence of the invention or an analogue or genetic construct thereof of the invention having antifungal activity, i.e. chitinase activity and optionally beta-1,3-glucanase activity as defined above, one or more several chemicals, such as fungicides, conventionally used to control fungi either therapeutically or prophylactically.

Normally, the antifungal agent is itself a microorganism, or is prepared with the aid of a microorganism. In most cases, the simplest and cheapest way to prepare an antifungal composition is to use the microorganism as such or the medium in which it has been grown as an antifungal agent. The antifungal polypeptide or polypeptides expressed in the microorganism may be secreted into the medium, for example due to the action of a suitable signal polypeptide capable of directing the polypeptide out into the medium, or may be released from the microorganism by well known mechanical or chemical methods. Prior to use, it may be advantageous to remove microorganisms or any cell debris from the medium.

The medium can in principle serve as a carrier medium for the antifungal agent, but it is advantageous to add another carrier medium corresponding to the particular intended use.

A culture of microorganisms expressing the antifungal polypeptide or polypeptides can be obtained as described above using methods known per se. As mentioned above, it may be necessary or advantageous to subject the culture of microorganisms to further treatment to release the contents of the antifungal polypeptide or polypeptide into the medium, or to increase the amount released by secretion.

The medium containing a substantial amount of antifungal polypeptide or polypeptide can be applied directly to the soil in which the plants are present or on which the plants are to be grown, or to the plants or plant parts or to the irrigation water. Alternatively, they may be treated with a seed medium, optionally in combination with a conventional seed coating composition.

Microorganisms expressing the antifungal polypeptide or polypeptides can be applied in a variety of formulations containing agronomically acceptable carriers, i.e., excipients and carriers, at dosages and concentrations selected to maximize the beneficial effect of the microorganism. However, the microorganisms can also be distributed as such in circumstances that allow the microorganisms to settle in the material being treated. If the microorganism is a microorganism commonly found in the soil, for example a root bacterium, it will generally be desirable for the transformed microorganism to settle in the soil so that it can continuously secrete the antifungal polypeptide or polypeptides into the soil surrounding the plant.

It may be advantageous to add microorganisms or medium containing the antifungal polypeptide or polypeptides to premixed mixtures, for example artificial growth medium or other soil mixtures used in culturing a particular plant. For these purposes, it is preferred that the microorganisms or medium be in solid form, for example in powder form or in the form of granules. The powder form can be obtained by conventional methods, for example, by applying the microorganism to a particulate carrier, spray-drying or the equivalent method.

If the microorganism expressing the antifungal polypeptide or polypeptides is to be used in a wet state, it may be in the form of a suspension or dispersion, for example an aqueous suspension.

In order to induce chitinase activity in the transformed microorganism, it may be advantageous to add a small amount of chitin to the medium in which the transformed microorganism is present.

In accordance with the foregoing, the invention also relates to a method of inhibiting the germination and / or growth of a chitin-containing plant pathogen, such as a phytopathogenic fungus, in or on a plant, the method comprising

1) transforming the plant or part thereof with a genetic construct as defined above and regenerating the resulting transformed plant or plant part into a genetically transformed plant, and / or

2) treating the plant or part thereof, the seedling or seed from which the plant is to grow, or the medium on which it is grown, with an antifungal agent as defined above.

Although genetic transformation of plants is advantageous for most purposes, it may be advantageous to combine transformation with treatment of a plant with an antifungal agent of the invention. Since genetic transformation is a time-consuming and in some respects complex process, it may be advantageous to use a biologically based agent instead of or in addition to conventionally used and environmentally undesirable chemical fungicides.

In most cases, the material to be treated with the antifungal composition of the invention is a plant. However, there are many chitin-containing fungi that infect non-plant materials, such as food products such as bread or bread products, dairy products, cheese, meat, vegetables, cereals, in which the presence and growth of fungi is undesirable. It is contemplated that the antifungal composition of the invention may be used to control or control such fungi. In this regard, it is contemplated that beverages or containers (any portion thereof) used for food products or beverages may also be treated with the antifungal composition of the invention, either as a prophylactic treatment or as a fungicide treatment.

The invention is further illustrated by the following sequences, examples and additional figures, which, however, do not limit the scope of the invention in any way.

Description of the picture:

Giant. 1 describes the purification of sugar beet chitinases 2, 3 and 4 by cation exchange chromatography (Mono-S) at pH 4.5. Protein elution was performed with a linear NaCl gradient. Absorbance was measured at 280 [mu] m. The symbol t indicates the time in minutes.

Giant. 2 shows a polypeptide pattern of sugar beet chitinase 2, 3 and 4 after purification by liquid column chromatography (Mono-S FPLC). Lanes contain 50 .mu.g of the following proteins: pathways a and b: chitinase 4, d and e: chitinase 3, 'f and g: chitinase 2, c and h: molecular weight markers. Proteins were stained with silver. MW is the molecular weight

Giant. 3 shows the analysis of water-soluble products

- 3 3 released from H-chitin by chitinase 4. H-chitin was incubated with 4 μg of chitinase 4 at 37 ° C for 0.25, 0.5, 3 'and 24 hours. As a control, ^J H-chitin was incubated without enzyme at 37 ° C for 24 hours. The released chitooligosaccharides were separated by thin layer chromatography and identified by comparing their migration with the migration of N-acetylglucosamine (monomer) (Fig. 3A), chitobiose (dimer) (Fig. 3B), chitotriose (trimer) (Fig. 30 and chitotetraose) standards. (tetramer) (Fig. 3D) Radioactivity representing chitooligosaccharides was determined by scintillation analysis after cutting the chromatographic plate into sections, symbol t denotes time in hours, symbol p number of pulses per minute, symbol 0 denotes incubation without enzyme Fig. 3A refers to monomers , Fig. 3B dimers, Fig. 3C trimers and Fig. 3D tetramers.

Giant. 4 shows the lysozyme activity of chitinase 4.

/ μg of enzyme was incubated with Micrococcus lysodeikticus cell walls and the decrease in absorbance at 450 nm was determined at the indicated time intervals. 1 .mu.g SE (Sure Ellen) was used as a control, and lysozyme (lys) (50 ng and 5 .mu.g) was used as a standard. The symbol t indicates the time in minutes.

Giant. 5 shows the inhibition of Cercospora growth by a combination of chitinase 4, acid chitinase SE and beta-1,3-glucanase 4 using a microscopic bioassay. After 48 hours of incubation, the culture was stained with Calcofluor White and examined under fluorescent light.

Giant. 5A shows fungal growth when 20 [mu] g of each of the enzymes chitinase 4, acid chitinase SE and beta-1,3-glucanase 4 were added to the culture at time 0.

Giant. 5B shows the growth of a control culture to which no antifungal proteins were added.

Figure 6 shows inhibition of Cercospora growth by chitinases using a microtiter bioassay. Time curves (absorbance at 620 nm) describe fungal growth during the first 92 hours of incubation. Absorbance (growth indicator) was measured at eight to sixteen hour intervals and each measurement is the average of five replicates. Curve A is a control curve showing the growth of Cercospora if no growth inhibitors were added to the culture. Curve B shows the growth of the fungus after the addition of 20 .mu.l of chitinase-containing fraction from the chitin column at time 0. In the case of curve C, 20 .mu.g of purified chitinase 4 was added to the culture at time 0. Symbol t means time in hours, symbol and absorbance at 620. nm, A: KP buffer, B: chitin column eluate, C: chitinase isozyme 4.

Giant. 7 is an autoradiography showing the effect of chitinase 4 on chitin at the tips of Cercospora fungi. Incorporation of H-labeled N-acetylglucosamine into Cercospora beticola hyphae was performed by growing the fungus for 20 minutes on growth medium containing radioactive chitin monomer. The incorporation of N-acetylglucosamine into the cell wall at the tip of the hyphae is visible as black dots.

Giant. 7A shows hyphae before treatment with purified chitinase 4.

Giant. 7B shows hyphae for radioactive incorporation followed by treatment with purified chitinase 4 for 24 hours.

Giant. 8 shows the separation of tryptic peptides of chitinase 4 by reverse phase high performance liquid chromatography on a Vydac RP-18 column. Peptides were eluted with a linear gradient from 10% to 45% acetonitrile over 25 to 75 minutes. Buffer A was water, while B was acetonitrile. Both solvents contained 0.1% trifluoroacetic acid. The flow rate was 0.7 ml per minute. The symbol t denotes time in minutes, the symbol and the absorbance at 214 nm.

Giant. 9 shows the separation of three acidic SE chitinase isozymes on an anion exchange column (Mono P) using a liquid chromatography system (FPLC). Proteins were eluted with a linear gradient of sodium chloride in 25 mM Bis-Tris buffer at pH 7.0. The symbol t represents the time in minutes.

Figure 10 describes two different serological classes of sugar beet, class chitinase 2 and chitinase 4. 5 μg of chitinase 2 (32 kD) and no (26 kD) were blotted onto the nitrocellulose membrane before reaction with sugar beet chitinase 2 antibody (labeled A2 ) or with an antibody against sugar beet chitinase 4 (labeled A4). MW is the molecular weight

Giant. 11 shows the hybridization of different chitinase genes with the chitinase 4 cDNA probe under specific hybridization conditions. Spots of various chitinase genes were applied to Hybond N-nylon membranes as 1 μl of a probe of plasmid preparations containing chitinase sequences.

clone chitinases 1 from sugar beet clone chitinases 4 from sugar beet clone chitinases 76 from sugar beet clone chitinases from peas clone sour chitinases SE from sugar beet clone 1 chitinases from tobacco clone 2 chitinases from tobacco clone 3 chitinases from tobacco clone fulo chitinase chitinase 4 from rapeseed.

Hybridization was performed overnight at 55 ° C in the following hybridization buffer: 2 x sodium chloride citrate buffer (SSC), 0.1% sodium dodecyl sulfate (SDS), 10 x Denhardt s, 50 μg / ml salmon sperm DNA (Salmon sperm DNA) and as a probe for chitinase 4 cDNA sequence. Washing conditions were 55 ° C twice for fifteen minutes, 2 x SSC, 0.1%

SDS followed by washing twice after fifteen minutes with 1 x SSC, 0.1% SDS at 55 ° C.

Giant. 12 shows the induction of chitinase and beta-1,3-glucanase in sugar beet leaves after Cercospora beticola infection. The plants were inoculated with a suspension of fungal spores. The leaves were harvested at the indicated time intervals and crude extracts were prepared. The activity of the enzymes chitinase (Fig. 12A) and beta-1,3-glucanase (Fig. 12B) was measured using radioactive assays with 1 H-chitin and 1 H-laminarin as substrate, respectively. The symbol t indicates the time after inoculation in days, I means infected, K means control. Giant. 12A refers to chitinase and W indicates ng chitinase per 1.25 mg of leaf tissue, FIG. 12B refers to beta-1,3-glucanase, and A indicates enzyme activity in pulses per minute.

Giant. 13 shows the immunodetection of sugar beet chitinase 2 and 4 and beta-1,3-glucanase 3 in protein extracts of sugar beet leaves infected with Cercospora. Lanes I and C contain protein extracts from infected and control plants, respectively. Antibodies generated for 64 chitinase 2 (left), chitinase 4 (middle) and beta-1,3-glucanase (right) were used. C2 is chitinase 2, C4 is chitinase 4 and G3 is glucanase 3, I is infected, C is control. MW is the molecular weight.

Giant. 14: Site-directed mutagenesis of amino acids thought to form part of the active site of the chitinase 4 enzyme using the polymerase chain reaction (PCR) technique described in Materials and Methods. SDO is used as the 5 'primer for all of the polymerase chain reactions mentioned. The sequence is indicated by an arrow and is selected 5 ”from the unique BamHI site. The sequences for primers SD1, SD2, SD3, SD4 and SD5 are indicated by arrows. Complementary sequences with the indicated substitutions are used for these 3-primers. The primers can be used for the following substitutions with respect to the genomic sequence of DNA chitinase (SEQ ID NO: 3) encoding the amino acid sequence set forth in SEQ ID NO: 4. The numbers in parentheses indicate the number of corresponding amino acids encoded by cDNA chitinase 4 (SEQ ID NO: 2). )

SD1: He suffers from 70— * Tyr (169) TGG ~ * TAC SD2: Glu190— ♦ Gin (189) GAA- »CAA SD3: Asp1 84— »Asn (183) GAT— »AAT SD4: Trp207— * Tyr (206) TGG— »TAC SD5: Trp205— »Tyr (204) TGG— * TAC PCR products are splits appropriate restriction enzymes and exchanged with the corresponding sequences of the chitinase 4 gene. Giant. 14A and 14B are considered to be one figure.

Giant. 15: Construction of a hybrid gene construct of beta-1,3-glucanase with a C-terminal extension from tobacco

Giant. 15A: sugar beet beta-1,3-glucanase cDNA clone with underlined tobacco C-terminal extension.

Giant. 153 and FIG. 15C: PCR primers that can be used to change the termination codon and to introduce parts of the C-terminal extension, the Dral site is formed at the 3-terminus. Arrows indicate PCR primers; for the 5-primer the sequence under the arrow is used, for the 3-primer the complementation of the sequence with the indicated substitutions is used. Giant. 15B and 15C are considered to be one figure.

Giant. 15D: Four thermally hybridized synthetic oligonucleotides containing the last part of the C-terminal extension, a termination codon, a SmaI site and a BglII site.

The fusion gene product can be generated by digesting the glucanase gene with XbaI and EcoRI and ligating it with the PCR product digested with XbaI and Dral and thermally hybridized synthetic oligonucleotides digested with SmaI and BglII.

Giant. 16: Construction of a hybrid gene construct of chitinase 4 with a C-terminal extension

Giant. 16A: Chitinase 4 with underlined tobacco C-terminal extension.

Giant. 16B and 16C: PCR primers that can be used to introduce the SmaI site near the termination codon of the chitinase 4 gene. Arrows indicate PCR primers; for the 5-primer the sequence under the arrow is used, for the 3-primer the complementary sequence with the indicated substitutions is used. Giant. 16B and 16C are considered to be one figure.

Giant. 16D: Four thermally hybridized synthetic oligonucleotides containing a C-terminal extension sequence, an altered termination codon, a SmaI site, and an EcoRI site.

The fusion gene product can be created by digesting the chitinase 4 gene with BamHI and EcoRI and ligating it with a PCR product digested with BamHI and SmaI and thermally hybridized synthetic oligonucleotides digested with SmaI and EcoRI.

Giant. 17: Construction of a transformation vector for plants pBKL4K4 containing the DNA sequence of chitinase 4 shown in SEQ ID No. 1. The framed sequences refer to the cDNA sequences of the B15 chitinase 4 cDNA, the enhanced 35S promoter and the 35S terminator used for the construct. pB15K4.1 is a pBluescript carrying a 966 bp ExoRI fragment encoding chitinase 4. The shaded boxes indicate the coding regions contained in the final product. Kb3 (= KB3) and Kb4 (= K34) are synthetic oligonucleotides acting as primers in the polymerase chain reaction (PCR) using pB15K4.1 DNA as template. The DNA sequences of KB3 and KB4, respectively, are shown in Example 18 and shown in SEQ ID NO: 49 and SEQ ID NO: 50. Plasmid pPS48 carries a conventional amplified 35S promoter and a conventional 35S terminator separated by a polylinker containing unique cloning sites. The transformation vector for plants pBKL4 (modification pBin 19 Bevon, 1989) carries the right and left border sequences of the Agrobacterium Ti plasmid pTiT37, the GUS gene with the 35S promoter and the conventional NOS terminator, the conventional NPTII gene with the 35S promoter and the conventional OCS terminator. A polylinker containing several unique cloning sites is located between the GUS and NPTII genes. Giant. 17A and 17B are considered to be one figure.

Giant. 18: Construction of a transformation vector for plants pBKL4K4KSE1 containing DNA sequences encoding chitinase 4 and acidic chitinase SE, respectively, set forth in SEQ ID NO: 1 and SEQ ID NO: 8. with the construct shown in Figure 17. pSurl is a pBluescript carrying the 5-terminus of the SE gene, pSE22 is similarly a pBluescript carrying almost the entire SE cDNA. The shaded boxes indicate the coding regions contained in the final product. AGCTGTAC1 is the adapter used to ligate KpnI-HindIII, pPS48 is mentioned in connection with Fig. 17. The construction of a plant transformation vector containing the chitinase 4 sequence (pBKL4K4) is described in Fig. 17. 18A and 18B are considered to be one figure.

Giant. 19: Construction of a transformation vector for plants pBKL4K76 containing the genomic chitinase 76 gene, of which

The boxed sequences of the enhanced pUC19 promoter carrying the sequence fragment are set forth in SEQ ID NO: 5 and the sequences of the chitinase 76 gene,

35S and terminator 35S. pK76.1 is

HindIII-EcoRI, encoding chitinase 76, at the HindIII / EcoRI site of the pUC19 polylinker. The shaded boxes indicate the coding regions contained in the final product. KB3 and 340 are synthetic oligonucleotides acting as primers in the polymerase chain reaction (PCR) using pK76.1 as a template. The DNA sequences of KB3 and 340, respectively, are shown in Example 18 and shown in SEQ ID NO: 49 and SEQ ID NO: 51. Plasmid pPS48 was used in connection with Fig. 17. The transformation vector for plants pBKL4 is described in Fig. 17. 19A and 19B are considered to be one figure.

Giant. 20: PCR amplification of a part of the cDNA of acid chitinase SE using mRNA as template. mRNA was reverse transcribed using a primer consisting of oligo-dT linked to two restriction sites (270) (see Example 7). Amplification was performed using a gene specific mixed oligonucleotide linked to a restriction site (XbaI-KB7) as the 5'-primer and 270 as the 3-primer. The second round of amplification was then performed using another gene-specific mixed oligonucleotide linked to a restriction site (BamHI-KB9) as the 5'-primer and 270 as the 3'-primer. The DNA sequence 270 is given in Example 7 and in SEQ ID NO: 30. The symbol RT denotes reverse transcription, the symbol A1 the first amplification reaction and the symbol A2 the second amplification reaction, S1 denotes the first strand of the cDNA.

Giant. 21 describes the separation of sugar beet beta-1,3-glucanases 1, 2, 3 and 4 by cation exchange chromatography (Mono-S) at pH 4.5. Elution was performed with a linear NaCl gradient. Absorbance was measured at 280 nm. The symbol t indicates the time in minutes.

Giant. 22 shows the construction of a plant transformation vector pBKL4K4KSE1G1 containing DNA sequences encoding chitinase 4, acidic chitinase SE, and beta-1,3-glucanase, respectively, as set forth in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 9. The boxed sequences refer to beta-1,3-glucanase cDNA, an enhanced 35S promoter, and a 35S terminator. pGluc 1 is a pBluescript carrying a 1249 bp EcoRI fragment encoding beta-1,3-glucanase. The shaded box indicates the coding area. Plasmid pPS48M is the same as pPS48 described in connection with the construct shown in Figure 17, except that this plasmid is supplemented with two additional restriction sites (EcoRI and KpnI) on each side of the E35S-35St frame. The construction of a plant transformation vector containing the chitinase 4 and SE sequences is described in Fig. 17 and Fig. 18. 22A and 22B are considered to be one figure. The symbol G means glucanase.

Giant. 23 shows the immunodetection of chitinase 4 and sugar beet chitinase in protein extracts from the transgenic plant Nicotiana benthaminana using antibodies raised against sugar beet chitinase 4.

C = control plants containing the GUS gene construct a

NPT,

SE = acid chitinase,

K76 = genomic chitinase (see Fig. 19)

K4 = chitinase 4 (see Fig. 17)

K4 + SE = chitinase 4 and acid chitinase (SE) (see Fig. 18), Std. = 10 pg of purified chitinase 4 of sugar beet,

MW is the molecular weight.

Giant. 24: DNA sequence comparison of the chitinase 4 cDNA sequence set forth in SEQ ID NO: 1 and the chitinase 76 genomic clone set forth in SEQ ID NO: 5. The position of the chitinase 76 intron is readily seen at positions 875-1262. The sequence homology is about 73%. Giant. 24A, Fig. 24B, Fig. 24C, Fig. 24D, Fig. 24E and Fig. 24F are considered as one figure.

The two sequences to be compared are: B15CHIT4, total base 966 and CHIT76, total base 1838.

Open gap cost: 25

Unit gap cost: 2

A character indicating that the two residues are identical is

Identity: 707 (73.2%)

Number of spaces inserted in B15CHIT4: 4

Number of spaces inserted in CHIT76: 1

Giant. 25: Comparison of the amino acid sequence of the cDNA sequence of chitinase 4 shown in SEQ ID No. 2 and chitinase 76 shown in SEQ ID No. 6. Homology of about 80% was found.

The three additional amino acids in chitinase 76 are amino acids (Ser, Thr, Pro) at position 62-64. FIG. 25A and FIG. 253 are considered to be one figure.

The two sequences to be compared are: B15CHIT4, total number of residues 264 and CHIT76, total number of residues 268. Comparison matrix: matrix of the genetic code

Open gap cost: 6

Unit gap cost: 1

A character indicating that the two residues are identical is

Identity: 214 (81%)

Number of spaces inserted in B15CHIT4: 1

Number of spaces inserted in CHIT76: 0

Giant. 26: Comparison of the non-coding 5-sequences of the genomic sequences of chitinase 4 and chitinase 76 shown in SEQ ID No. 3 and SEQ ID No. 5, respectively. There are 8 boxes with strong homology in the non-coding 5-region. It is anticipated that some of these boxes may have regulatory significance. Giant. 26A and 26B are considered to be one figure.

The two sequences to be compared are: CHIT4GENOM, total base 691 and CHIT76, total base 1838.

Open gap cost: 25

Unit gap cost: 2

The character indicating that the two residues are identical is

List

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID

SEQ ID sequences:

No. 1: cDNA sequence of chitinase (contained in cDNA-clone B15 of chitinase 4 sugar beet)

No. 2: amino acid sequence of chitinase 4 (contained in cDNA clone B15 of chitinase 4 sugar beet)

No. 3: partial DNA sequence of the genomic clone of chitinase 4

No. 4: partial amino acid sequence of the genomic clone of chitinase 4

No. 5: DNA sequence of the genomic clone of chitinase 76

No. 6: deduced amino acid sequence of the genomic clone of chitinase 76

No. 7: cDNA sequence of sugar beet SE chitinase SE

No. 8: deduced amino acid sequence of sugar beet SE chitinase SE

No. 9: sugar beet basic beta-1,3-glucanase cDNA sequence

No. 10: deduced amino acid sequence of sugar beet basic beta-1,3-glucanase

No. 11: DNA sequence of the whole sugar beet chitinase 1 gene including introns, promoter and leader sequence, and amino acid sequence derived from the coding region of the chitinase 1 gene

SEQ ID NO: 12: amino acid sequence derived from the coding region of the chitinase 1 gene

SEQ ID NO: 13: C-terminal amino acid sequence of bean chitinase (PHA)

SEQ ID NO: 14: C-terminal amino acid sequence of basic tobacco chitinase

SEQ ID NO: 15: C-terminal amino acid sequence of tobacco acid chitinase

SEQ ID NO: 16: C-terminal amino acid sequence of barley chitinase CH26

SEQ ID NO: 17: C-terminal amino acid sequence of basic beta-1,3-glucanase from tobacco

SEQ ID NO: 18: amino acid sequence of a tryptic peptide from sugar beet chitinase 3

SEQ ID NO: 19: amino acid sequence of a tryptic peptide from sugar beet chitinase 3

SEQ ID NO: 20-: amino acid sequence of a tryptic peptide from sugar beet chitinase 3

SEQ ID NO: 21: amino acid sequence of a tryptic peptide from sugar beet chitinase 3

SEQ ID NO: 22: amino acid sequence of a tryptic peptide from sugar beet chitinase 3

SEQ ID NO: 23: N-terminal amino acid sequence of the amino acid sequence of the chitin binding protein from WGA-A (Triticum aestivum)

SEQ ID NO: 24: N-terminal amino acid sequence of the amino acid sequence of the chevin binding protein from Hevea brasiliensis

SEQ ID NO: 25: N-terminal amino acid sequence of the amino acid sequence of bean chitinase (Phaseolus vulgaris)

SEQ ID NO: 26: N-terminal amino acid sequence of the amino acid sequence of tobacco chitinase (Nicotiana tabacum)

SEQ ID NO: 27: N-terminal amino acid sequence of the amino acid sequence of sugar beet chitinase 2

SEQ ID NO: 28: DNA primer named KB-7 constructed in part from the polypeptide sequence of sugar beet acid chitinase (SEQ ID NO: 8)

SEQ ID NO: 29: a complementary DNA primer named KB-9 constructed in part from the polypeptide sequence of sugar beet acid chitinase (SEQ ID NO: 8)

SEQ ID NO: 30: a complementary DNA primer named Oligo-dT constructed on the basis of a broad knowledge of polyA mRNA acids

SEQ ID NO: 31: amino acid sequence of cucumber (Cucumis sativus) lysozyme / chitinase

SEQ ID NO: 32: amino acid sequence of Arabidopsis thaliana lysozyme / chitinase

4

SEQ ID NO. 33: amino acid subsequence of the 3-15 amino acid sequence of sugar beta-1,3-glucanase beets SEQ ID NO. 34: amino acid subsequence of the 3-17 amino acid sequence of sugar beet beta-1,3-glucanase SEQ ID NO. 35: amino acid subsequence of the 3-16 amino acid sequence of sugar beet beta-1,3-glucanase SEQ ID NO. 36: DNA-5-primer named Oligo TG-1 constructed from the amino acid sequence of sugar beet beta-1,3-glucanase SEQ ID NO. 37: DNA-5-primer named Oligo TG-2 constructed from the amino acid sequence of sugar beet beta-1,3-glucanase SEQ ID NO. 38: DNA-3'-primer named Oligo TG-3 constructed from the amino acid sequences of tobacco and barley glucanase SEQ ID NO. 39: amino acid subsequence of an amino acid barley glucanase sequences used to construct Oligo TG-3 primers SEQ ID NO. 40: amino acid subsequence of an amino acid the sequence of tobacco glucanase used to construct the Oligo TG-3 primers SEQ ID c. 41: N-terminal amino acid sequence of the pea chitinase B amino acid sequence SEQ ID NO. 42: N-terminal amino acid sequence of the pea A1 amino acid sequence

SEQ ID NO: 43: N-terminal amino acid sequence of the pea chitinase A2 amino acid sequence

SEQ ID NO: 44: N-terminal amino acid sequence of the barley chitinase K amino acid sequence

SEQ ID NO: 45: N-terminal amino acid sequence of the amino acid sequence of barley chitinase T

SEQ ID NO: 46: amino acid subsequences of the active site of the tobacco chitinase amino acid sequence

SEQ ID NO: 47: amino acid subsequence of the active site of a tobacco chitinase polypeptide

SEQ ID NO: 48: amino acid sequence of the active site of a tobacco chitinase polypeptide

SEQ DI No. 49: DNA-5'-primer named KB-3 constructed in part from nucleotides No. 1-15 in SEQ ID No. 1 and nucleotides No. 471 - 485 in SEQ ID No. 5

SEQ ID NO: 50: a complementary DNA-3'-primer named KB-4 constructed from nucleotides Nos. 261 - 241 in

SEQ ID No. 1

SEQ ID NO: 51: a complementary DNA primer named 340 constructed in part from nucleotides Nos. 341 - 323 in SEQ ID NO: 1

Detailed Explanation of SEQ ID NOs: 1-12:

SEQ ID No. 1a SEQ ID No. 2

DNA sequence (SEQ ID No. 1) and deduced amino acid sequence (SEQ ID No. 2) of cDNA clone B15 chitinase 4 isolated from the lambdaZAP sugar beet cDNA library.

The sequence is 966 bp long and encodes a protein that has 264 amino acid residues in the polypeptide chain. The leader sequence consists of 23 amino acid residues and is followed by a hevein domain of 35 amino acid residues and a functional domain of 206 amino acid residues. Behind the termination codon, the cDNA has a 3 'flanking region of 158 bp with a putative polyadenylation signal at position 847 and a polyA terminus.

For comparison, the chitinase 4 gene, the partial nucleotide sequence of which is set forth in SEQ ID NO: 3, encodes a protein having 265 amino acid residues set forth in SEQ ID NO: 4. The leader sequence encoded by this gene consists of 24 amino acid residues. Thus, SEQ ID NO: 1 lacks nucleotides A and T and the first amino acid methionine is not present in the polypeptide sequence encoded by c DNA chitinase 4.

SEQ ID No. 3a SEQ ID No. 4

The partial DNA sequence (SEQ ID No. 3) and deduced amino acid sequence (SEQ ID No. 4) of a genomic clone encoding the chitinase 4 gene isolated from the sugar beet EMBL3 genomic library.

The sequence is 691 bp long and encodes the first 112 of the 265 amino acids of the chitinase 4 polypeptide chain. The leader sequence consists of 24 amino acid residues and is followed by a 35 amino acid hevein domain. The partially sequenced clone has a 355 bp 5'-non-coding region with the TATA-box (TATAAA) sequence located at position 285, which is 70 bp upstream from the ATG start codon.

SEQ ID No. 5 and SEQ ID No. 6

The DNA sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO: 6) of a genomic clone encoding the chitinase 76 gene isolated from the EMBL3 sugar beet genomic library.

The sequence is 1838 bp long and encodes a protein that has 268 amino acid residues in the polypeptide chain. The leader sequence consists of 24 amino acid residues and is followed by a hevein domain of 35 amino acid residues and a functional domain of 209 amino acid residues. The gene contains one intron, which is located at positions 875 to 1262. The exact location of this intron is based on pairing with the B15 chit 4 cDNA (Fig. 24). Intron boundaries contain conventional GT / AG sequences. The TATA-box (TATAAA) sequence is located at position 378, which is 90 bp upstream from the ATG start codon. The putative polyadenylation signal (AATAAA) is located at position 1725.

SEQ ID No. 7 and SEQ ID No. 8

The DNA sequence (SEQ ID No. 7) and the deduced amino acid sequence (SEQ ID No. 8) of the cDNA clone of acid chitinase SE isolated from the lambdaZAP sugar beet cDNA library.

The sequence is 1106 bp long and encodes a protein that has 293 amino acid residues in the polypeptide chain. The leader sequence consists of 25 amino acid residues and the functional domain of 268 amino acid residues. The cDNA clone has a 5 'non-coding region of 17 bp and a 3'-flanking region of 202 bp.

SEQ ID No. 9 and SEQ ID No. 10

The DNA sequence (SEQ ID No. 9) and the deduced amino acid sequence (SEQ ID No. 10) of the beta-1,3-glucanase 4 cDNA clone isolated from the lambdaZAP sugar beet cDNA library.

The sequence is 1249 bp long and encodes a protein that has 336 amino acid residues in the polypeptide chain. The cDNA clone has a 5 'non-coding region of 33 bp and a 3' flanking region of 182 bp containing the putative polyadenylation signal at position 1157 and a polyA terminus.

SEQ ID No. 11a SEQ ID NO. 12 Sequence DNA (SEQ ID C. 11) and derived amino acid new sequence (SEQ ID C. 12) genomic clone encoding gene chitinase 1 isolated of genomic libraries EMBG 3 sugar

beet.

The sequence is approximately 6.3 kb in length and encodes a protein that has 439 amino acid residues in the polypeptide chain. The leader sequence is considered to consist of 26 amino acid residues, followed by a hevein domain of 20 amino acid residues, a proline-rich domain of 132 amino acid residues, and a functional domain of 238 amino acid residues. The protein has a C-terminal extension of 23 amino acid residues, which probably directs the protein into the vacuole.

The sequence contains two introns at positions 2170-4618 and 4776-5406. The intron boundaries contain conventional GT / AG sequences. The TATA-box (TATAAA) sequence is located at position 1355 - 1360, which is approximately 70 bp upstream from the ATG start codon. The predicted polyadenylation signal (AATAAA) is located at position 6032.

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Material and methods

BIOLOGICAL MATERIAL:

Plants:

Seeds of Beta vulgaris variety Monova were sown in peat mixed with clay (Cycas) and placed in a growth chamber with eleven and thirteen hour day / night cycles, with a temperature of 25/18 ° C (day / night) and with relative humidity.

%. The light intensity was approximately 25,000 lux (Osram

HQI-T, 400 W / DH). Three weeks after sowing, the seedlings were transplanted individually into 12 cm plastic containers containing the same growth medium. Twice a day, the plants were watered with water containing 0.1% fertilizer: universal fertilizer Stjerne, 4: 1: 4 (Ν: P: K). Six weeks after sowing, the plants were prepared for infection experiments with Cercospora beticola.

The plants Nicotiana tabacum and Nicotiana benthamiana were obtained as described above.

Mushrooms:

An isolate of the fungus Cercospora beticola was used for infection experiments. Isolate F573 was obtained from the United States Department of Agriculture, Department of Agricultural Research, Fort Collins, Colorado, USA.

The C. nicotianae fungus isolate (ATCC 18366) was obtained from the American Type Culture Collection (ATCC).

Growth of Cercospora species:

The fungus was grown on solid growth medium in petri dishes. Sterile potato dextrose agar (Difco, 39 g / l) was used as growth medium. A piece of mycelia was placed in the center of a petri dish and the culture was incubated at room temperature for 4 weeks. Mycelium for spore induction was harvested by cutting off the entire carpet of mycelium, including part of the agar.

Sporulation of Cercospora species:

The mycelium was mixed with distilled water (1: 2) in a 50 ml sterile glass tube and homogenized using an Ultra Turrax T25 mixer operating at 8000 rpm for two minutes.

ml of homogenate was transferred to a petri dish containing solid sporulation medium. V-8 was used as the medium. It contained 200 ml of V-8 juice (Cambells, Italy), 800 ml of water, 3 g of calcium carbonate and 20 g of agar.

The suspension was allowed to settle for one hour. After air-drying the culture (for approximately one hour), the petri dishes were sealed, sealed and placed in an incubation chamber at 13 ° C and light (cold white) for 24 hours.

After seven days of incubation, the spores were harvested by pouring 10 ml of distilled water onto a petri dish and wiping the culture surface thoroughly with a sterile brush.

The resulting spore suspension contained approximately 100,000 spores per ml.

Cercospora species infections:

For inoculation, 12,500 spores were suspended in 1 ml of water containing 20 μg of Tween-20 surfactant. The suspension was applied using a chromatographic atomizer to the upper surface of the leaves of six week old sugar beet or tobacco (Nicotiana) plants until run-off. Without 88 immediately after inoculation, the plants were placed in a humid chamber at 30 ° C, 100% relative humidity and illumination (cold white) for 24 hours. After five days of incubation, the plants were transferred to a growth chamber at 30 ° C, 80% relative humidity and illumination for 24 hours. Approximately 10 days after inoculation, adult leaves developed necrotic spots indicating Cercospora infection. After inoculation, the sugar beet plants were harvested at specified intervals for:

i) purification of small amounts of chitinase 4, acid chitinase SE and beta-1,3-glucanase, ii) a time study examining the expression level of total enzyme activity using radiochemical assays and immunoblotting, iii) determining the expression level of each enzyme in transgenic plants using techniques mentioned under ii) above, and iv) isolating the mRNA for use in constructing a cDNA library.

Infection of tobacco plants (Nicotiana) with the root pathogen Rhizoctonia solani:

The Rhizoctonia solani isolate was obtained from Dr. K. Tzavella-Klonavi (Thessaloniki, Greece).

The Rhizoctonia solani inoculum was prepared on barley grains, which were soaked twice in a 1% potato dextrose suspension and autoclaved. The grains were inoculated with agar disks of a growing fungal culture, incubated for two weeks and then air dried.

Alternatively, Rhizoctonia solani disks growing on potato dextrose agar can be used directly as inocula.

The inoculum was mixed with potted soil in various concentrations, and transgenic plants were transplanted into the infected soil, which had previously been rooted for 14 days. The percentage of surviving plants can be determined after 1, 2 and 3 weeks, respectively, and after three weeks, the root damage is determined in the surviving plants. Alternatively, seeds of transgenic plants are sown directly into infected soil.

Protein extraction from 1 g of sugar beet leaf mass:

Purification of the small amount was performed as follows. 1 g of leaf material was homogenized using an Ultra-Turrax homogenizer in citrate buffer (0.1 M, pH 5, 2 ml / g tissue) containing 1 mM benzamidine, dithiothreitol and phenylmethylsulfonyl fluoride. The solids were removed by centrifugation at 15,000 g for 15 minutes. Prior to repeating the centrifugation, the enzyme-containing supernatant was transferred to another cuvette.

Extraction of proteins in large quantities from the leaf mass of sugar beet:

Large amounts of pure enzymes are required to determine the antifungal potential and amino acid sequence of the enzymes. To obtain a sufficient amount, i.e. of the order of mg, a large amount of chitinase 4, acid chitinase SE and beta-1,3-glucanase was purified from 2 kg of leaf mass of naturally infected sugar beet plants of the Monova variety. Naturally infected leaves bearing fifty or more necrotic lesions were collected in the field of a breeding station in Italy (Maribo-Italy, Bologna) and stored at 4 ° C until extraction of chitinase 4.

PREPARATION OF CKITINE COLUMN:

g of chitosan (from Protan, Sea Cure P, No. 709, Norway) was dissolved in 600 ml of 10% acetic acid. After 30 minutes, 600 ml of methanol was added slowly with stirring. The cloudy viscous solution was filtered twice to remove solids: first with glass wool and second with a sintered glass filter funnel. The filtrate was transferred to a beaker on a magnetic stirrer and 40 mL of acetic anhydride was added slowly with vigorous stirring. After about two minutes, the solution turned into a gel. The reaction was allowed to proceed for 10 minutes before the gel was cut into pieces with a spatula. The gel pieces were transferred to a mixer (Warring), covered with methanol and homogenized for two minutes at full power. Methanol, acetic acid and unreacted acetic anhydride were removed by filtration on a Buchner funnel using Whatman No. 1 filter paper. The filtrate was transferred to a beaker, 1 L of 1M sodium carbonate was added and the pH was adjusted to 9 by the addition of 6N sodium hydroxide. Then 50 ml of acetic anhydride was added slowly and the pH was adjusted to 9. The reaction was allowed to proceed for one hour before the resulting product was collected by filtration on a Buchner funnel. After washing vigorously with water, the product was equilibrated in 10 mM Tris buffer at pH 8.0 and then stored at 4 ° C. The yield was 700 ml of recovered chitin. The chitin column was prepared from regenerated chitin using standard Pharmacia procedures.

Preparation of radioactive colloidal chitin:

g of chitosan were acetylated with ³ H-labeled acetic anhydride as described for the synthesis of unlabeled chitin (see above). After intensive washing of the 1 H-labeled chitin on a Buchner funnel, this chitin was transferred to a beaker. 50 ml of concentrated HCl cooled to ice-cold was added, and the chitin was dissolved by stirring for five minutes at 0 ° C. The syrupy liquid was filtered on a sintered glass filter funnel and slowly poured into a vigorously stirred 50% aqueous ethanol solution to precipitate chitin in a highly dispersed state. The product was settled several times by centrifugation and resuspended in water to remove excess acid and ethanol. Finally, the colloidal chitin was suspended in 200 ml of water and sonicated for 5 x 1 minute at full power. 1 H-labeled chitin was stored at 4 ° C before use.

PREPARATION OF LAMINARINE COLUMN:

Divinyl sulfone-activated agarose (Mini-leak high, KEM-EN-TEC, Denmark) was used to immobilize laminarin (beta-1,3-glucan) (from Laminaria digitata, Sigma). 50 g of Mini-leak High was dispersed in 200 ml of 1M potassium phosphate (K-P) at pH 11, and 750 mg of laminarin dissolved in 5 ml of water was added. The reaction was allowed to proceed on a stirring table for 16 hours at 25 ° C. Unreacted divinyl sulfone groups were blocked by incubation with a solution of 5% mercaptoethanol in 1M K-β-buffer at pH 9.5. The reaction time was 16 hours at 25 ° C. The remaining mercaptoethanol was removed by vigorous washing of the gel on a Buchner funnel. Laminarin-Agarose was suspended in 20 mM Tris buffer at pH 8.0 and stored at 4 ° C. The laminamin column was prepared from Laminarin-Agarose using standard Pharmacia procedures.

Synthesis of 4 H-labeled laminarin:

Laminarin was radiolabeled by reduction with 4-labeled sodium borohydride. 500 mg of laminarin (from Laminaria digitata, Sigma) was dissolved in 2 ml of water, and purified by precipitation by adding 800 μl of sodium chloride solution (0.2 g / ml) followed by 8 ml of absolute ethanol. The precipitate was collected by centrifugation at 15,000 g for 5 minutes. The supernatant was discarded and the laminarin-containing pellet was dissolved in 4 ml of 0.1N sodium hydroxide. This solution was transferred to a reaction vessel containing 5 mCi 4 H-labeled sodium borohydride. After stirring for minutes at 25 ° C, 600 μl of 1M chloro / 3 hydrogen acid was added to remove unreacted H-labeled sodium borohydride. The reaction mixture was divided into 500 [mu] l portions, and 200 [mu] l of sodium chloride solution and 2 ml of absolute ethanol were added to each tube. After standing for 10 minutes at 0 ° C, the precipitate was collected by centrifugation at 15,000 g for 5 minutes. The H-labeled laminarin was dissolved in 500 [mu] l of water and the precipitation was repeated until the background radioactivity in the supernatant was less than 100 pulses per minute per 20 [mu] l. The labeled laminarin solution was stored at -20 ° C. The solution was diluted twenty-fold with water before use in the beta-1,3-glucanase assay.

HIGH PRESSURE LIQUID CHROMATOGRAPHY IN THE REVERSE PHASE:

A Kontron AG (Zurich, Switzerland) apparatus was used, consisting of two Type 420 pumps and a solvent mixer. Gradient control and data acquisition were performed on a 450-MT Data system (Kontron) according to the manufacturer's instructions. Proteins eluted from the Mono S column (see below) were subjected to reverse phase high performance liquid chromatography on either a VYDFAC RP4 column (0.46 x 15 cm, 10 μm particle size, The Separations Group, Hesperia, CA) or a Pole F column. (Du Pont de Nemours). The mobile system used for reverse phase high performance liquid chromatography (RP-HPLC) was buffer A: 0.1% trifluoroacetic acid in water and buffer B: 0.1% trifluoroacetic acid in acetonitrile.

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS:

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of crude plant extracts or partially purified chitinases was performed on an Easy-4 instrument (Kem-En-Tec, Denmark) using the Tricine SDS-PAGE system described by Schagger and von Jagow (1987). ). A total of 25 μg of protein was applied to each lane. Phast-System (Pharmacia) was used for the analysis of pure chitinase isoenzymes according to the manufacturer's instructions.

ENZYME TESTS:

Radiochemical chitinase test:

Chitinase activity was determined radiochemically using ³ H-chitin as substrate.

The specific activity of H-chitin was equal to the equivalent of 460 pulses per minute / nmol of N-acetyl-glucosamine (GlcNac)

3 (or 2.3 x 10 pulses per minute / mg 1 H-chitin). It was detected by scintillation measurement and colorimetric determination of GlcNac after complete hydrolysis of 4 H-chitin by crude chitinase preparations from sugar beet leaves and exochitinase from Serratia marcescens or Streptomyces griseus.

The test mixture contained a total of 200 [mu] l of en3 / zyme solution, 50 [mu] l of H-chitin suspension (emitting 100,000 pulses per minute) and 10 [mu] mol of sodium citrate (pH 5.0). After mixing, enzymatic hydrolysis of H-chitin was allowed to proceed at 40 ° C for 15 minutes before 300 μl of a 10% solution (w / v) of trichloroacetic acid was added. To reduce the background value, 100 μl of bovine serum albumin (10 mg / ml) was added, and then insoluble H-chitin was removed by centrifugation at 15,000 g for 5 minutes. Radioactivity in 300 [mu] l of supernatant was determined by scintillation counting.

Radiochemical beta-1,3-glucanase test:

Beta-1,3-glucanase activity was determined radiochemically using ^J H-labeled laminarin as a substrate.

The test mixture consisted of 50 [mu] l of enzyme extract, 50 [mu] l of 0.1 M sodium citrate pH 5.0 and 10 [mu] l of H-labeled laminarin (192,000 pulses per minute). Incubation was performed for 15 minutes at 40 ° C. 1000 [mu] l of absolute ethanol and 50 [mu] l of saturated sodium chloride solution were added to complete the reaction. After 10 minutes at 0 ° C, unreacted laminarin was removed by centrifugation at 10,000 g for 5 minutes. A 400 [mu] l portion of the supernatant was transferred to a scintillation cuvette. After the addition of 5 ml of PICO-FLUOR-40, the level of radioactivity was determined by measuring the scintillation of the liquid.

Lysozyme test:

The lysozyme activity of chitinase 4 was determined as described by Selstes et al. (1980). Lysozyme activity was measured on microtiter plates. Each well contained Micrococcus lysodeikticus cell walls suspended in 20 mM sodium phosphate buffer, pH 7.4, containing 1 mg / ml bovine serum albumin (BSA, bovine serum albumin). The initial absorbance at 450 nm was adjusted to 0.6 and then egg white lysozyme or plant chitinase 4 was added. The reaction was followed by measuring the decrease in absorbance at five minute intervals for 50 minutes.

Beta-glucuronidase (GUS) test:

When the GUS gene was used as a reporter gene in connection with the construction of genetically transformed plants according to the invention, the success of the transformation can be determined using the following GUS test described by Jefferson, 1987. Leaf tips were cut into thin sections (<0.5 mm). and incubated in a 2 mM solution of 5-bromo-4-chloro-3-indolyl-beta-glucuronide (x-gluc.) dissolved in 0.1 M sodium phosphate buffer pH 7.0 containing 0.5 mM potassium ferricyanide and 10 mM ethylenediaminetetraacetic acid (EDTA). Leaf sections were processed for 2-4 hours at 37 ° C, washed with water, and the intensity of the staining was determined by visual inspection under a microscope.

PURIFICATION OF ISOENZYME CHITINASES 2, 3 AND 4, ACID CHITINASES AND BETA-1,3-GLUCANASES:

Acidic and basic chitinase isoenzymes were purified together with beta-1,3-glucanases from sugar beet leaves as shown in the following diagram.

kg of sugar beet leaves

0.1 M sodium citrate mM DTT mM BAM, pH 5.0>

Heating, 50 ° C, 20 minutes 90% (NH ₄ ) ₂ SO ₄ dialysis homogenization centrifugation mM Tris-buffer pH 8.0 off

FF-Seoharose S i,

Laminarin-Acarose

FPLC, Mono-S

RP-HPLC beta-1,3-glucanase

FF-Sepharose Q - chitin column

FPLC, Mono-S

AND

RP-HPLC t

chitinase 2, 3 and 4

FF Sepharose Q

Chromatofokusing

AND

FPLC, Mono P acid chitinase SE

Sugar beet foxes were obtained from Italy (large quantities, see Biological material). In the following, each of the purification steps outlined below will be explained. The equipment and procedure used in each step is described below.

EXTRACTION OF PROTEINS FROM SUGAR BEET LEAVES INFECTED BY CERCOSPORA BETICOLA:

All steps were performed at 4 ° C. Centrifugation during the purification process was run at 20,000 g for 20 minutes in a Centrikon model H-401B centrifuge.

Preparation of cell-free extracts:

kg of Cercospora-infected leaves were homogenized in 4 l of sodium citrate buffer, pH 5.0, containing mM dithiothreitol (DTT), 1 mM benzamidine (BAM) (starting buffer) and 200 g Dowex 1x2 (100 μm / mesh). The homogenate was pressed through a double layer of nylon gauze with a pore size of 31 .mu.m, followed by centrifugation.

Heat and ammonium sulphate precipitation:

The supernatant fraction obtained after centrifugation was heated at 50 ° C for 20 minutes, and after cooling to 4 ° C, the precipitate was isolated by centrifugation. Solid ammonium sulfate was added to the supernatant until 90% saturation was reached. After centrifugation, the precipitated proteins were dissolved in the starting buffer at a ratio of 1 ml of buffer per 10 g of starting material.

PURIFICATION OF CHITINASES 2, 3 AND 4, ACID CHITINASES AND BETA-1,3-GLUCANASES BY COLUMN CHROMATOGRAPHY:

Chitinase and beta-1,3-glucanase isoenzymes were purified from the ammonium sulfate precipitated protein fraction. After dissolution, the protein solution was dialyzed against 10 mM Tris-buffer pH 8.0 containing 1 mM DTT and 1 mM BAM. Denatured proteins were removed by centrifugation and the supernatant was applied to the two columns indicated above, for example i) a 50 ml Fast Flow Sepharose Q (Pharmacia) and ii) a 100 ml chitin column (prepared as described above), which columns were connected in series. The columns are equilibrated with Tris buffer before loading 281 ml of sample. Unbound proteins, including beta-1,3-glucanase, were removed by extensive washing with baseline buffer. After disconnecting the Fast Flow Sepharose Q column, the chitinase was eluted from the chitin column with 20 mM acetic acid pH 3.2 containing 1 mM DTT. Acid chitinase SE was eluted from the Fast Flow Sepharose Q column with Tris buffer containing 0.5 M sodium chloride.

PURIFICATION OF BETA-1,3-GLUCANASE:

Separation of beta-1,3-glucanase using cation exchange chromatography:

Proteins that were not adsorbed on either the Fast Flow Sepharose Q or the chitin column were collected and concentrated to 60 ml by pressure dialysis using an Amicon PM-10 filter (Danver, MA, USA) (R j). dialysis against 20 mM sodium acetate buffer pH 4.2 containing 1 mM DTT and 1 mM BAM overnight, the protein solution was applied to a 50 ml Fast Flow Sepharose S column (Pharmacia) equilibrated with dialysis buffer. Non-adsorbed proteins were removed by washing with equilibration buffer. Bound proteins were eluted with 600 ml of starting buffer with a linear gradient from 0 to 0.5 M sodium chloride.

Three major peaks A, B and C of beta - -1,3-glucanase activity were observed. Peak B was further fractionated by affinity column chromatography on Laminarin-Agarose.

Peaks A and C were not further purified.

Purification of beta-1,3-glucanase on Laminarin-Agarose:

A ml column of Laminarin-Agarose was equilibrated with 10 mM Tris-buffer pH 8.0 containing 1 mM DTT and BAM each. The protein fractions from peak 3 were pooled, concentrated by pressure dialysis to 15 ml and dialyzed against Tris-buffer. After loading the sample onto a Laminarin-Agarose column, flow through the column was stopped for 20 minutes to allow beta-1,3-glucanase to bind to the affinity ligand. Unabsorbed proteins were removed by washing with Tris-buffer, beta-1,3-glucanase was washed with 1M sodium chloride in Tris-buffer.

Purification of four beta-1,3-glucanase isoenzymes by FPLC:

Fractions from the Laminarin-Agarose column with beta-1,3-glucanase activity were pooled, concentrated and dialyzed overnight against 20 mM acetate buffer pH 4.5. Proteins were separated on a cation exchange column (Mono S) (Pharmacia) using a high performance liquid chromatography (FPLC) system using a linear gradient of sodium chloride. Four major protein peaks were observed (see Figure 21), and all four hydrolyzed ³ H-labeled laminarin as a substrate in a radiochemical assay for beta-1,3-glucanase (see above).

Purification of beta-1,3-glucanase by reverse phase HPLC:

Purification was performed by applying FPLC-purified beta-1,3-glucanase to the Pole F high pressure liquid chromatography (HPLC) column described above on a reverse phase. Unabsorbed material (buffers, salt, etc.) was removed by washing with 10% acetonitrile in 0.1¾ trifluoroacetic acid (TFA). Proteins were eluted using a linear gradient of acetonitrile from 10 to 70%.

After this desalting and purification step, peaks 3 and 4 were prepared for i) N-terminal amino acid sequencing, ii) amino acid composition analysis (see Example 8), iii) tryptic digestion to obtain peptides, and iv) incubation in rabbits to generate semi-clonal antibodies.

PURIFICATION OF CHITINASES 2, 3 AND 4:

By washing the chitin column with 20 mM acetic acid, pH 3.2, 40 fractions of 10 ml with chitinase activity were obtained. The fractions were combined, adjusted to pH 4.5, concentrated to 15 ml and dialyzed against 20 mM sodium acetate buffer pH 4.5.

2 ml aliquots were applied to the above cation exchange column (Mono-S) using an FPLC system (Pharmacia). Unadsorbed material was removed by washing with acetate buffer. Elution of chitinase isoemzymes was performed with a linear gradient from 0 to 1M sodium chloride in acetate buffer. The elution procedure is shown in Figure 1. A VYDAC RP4 reverse phase HPLC column was used for further purification. The conditions were similar to those described above in connection with the purification of beta-1,3-glucanase.

PURIFICATION OF ACID CHITINASE IS:

Purification of acid chitinase SE by anion exchange chromatography:

Acid chitinase SE was eluted from the Fast Flow Sepharose Q column described above with Tris-buffer containing 0.5M sodium chloride as indicated in the purification scheme. Proteins were dialyzed against 10 mM Tris-HCl, pH 8.0 and applied to a 40 ml Fast Flow Sepharose Q column equilibrated with the same buffer. Proteins were washed with 800 ml of a solution with a linear gradient of sodium chloride from 0 to 0.5 mM NaCl. Fractions showing chitinase activity as determined by the radiochemical chitinase assay described above were pooled.

Purification of acid chitinase SE by chromatofocusing:

Protein fractions were dialyzed against 25 ml of Bis-Tris buffer adjusted to pH 7.0 with iminodiacetic acid. A 15 ml column of polybuffer Exchanger (Pharmacia, PBE74) was equilibrated with the same buffer and 50 ml of sample was applied. Unabsorbed proteins were removed by washing with Bis-Tris buffer.

Application of Polybuffer 74 adjusted to pH 3.6 created a linear gradient from 7 to 3.6 and resulted in the desorption of several proteins. Acid chitinase SE was still captured on the column at this pH, but was desorbed by adding 0.3M sodium chloride to Polybuffer 74.

Purification of acid chitinase SE by FPLC:

Protein fractions with high chitinase activity, as determined by the radiochemical chitinase assay described above, were pooled and dialyzed against 25 mM Bis-Tris buffer at 7.0. Proteins were loaded onto a Mono-P FPLC column (Pharmacia) equilibrated with Bis-Tris buffer. After initial washing with baseline buffer, the three acid chitinase SE isoenzymes were separated using a linear salt gradient to 0 to 0.3 M NaCl (Fig. 3).

ANALYSIS OF THE CHYSINASE 4 SUGAR BEET ENZYMATIC FISSION SCHEME:

[Mu] l of 1H-labeled quinine (50,000 pulses per minute) was incubated with 7 [mu] g of chitinase 4 (purified as described above) in 0.1M citrate buffer at 6.5. Total

- 101 C7 line co

O - C- cn cn > X p O what what CZ> f r— minutes ,, _30 minutes,

the volume was 300 μl. After this time (15 minutes, hour and 24 hours), the reaction was stopped by adding 300 μl of 10% (w / v) trichloroacetic acid (TCA). Unreacted polymer of 4 H-labeled chitin was removed, and 300 μl of the supernatant was subjected to thin layer chromatography (TLC) (Silica gel 60 H, Merck). The mobile phase was n-propanol / water / ammonia (70/30/1, v / v / v).

A standard of N-acetylglucosamine-derived oligosaccharides was created by acid hydrolysis of chitin (Rupley, 1964). This standard was used to localize the enzymatic cleavage products on the TLC layer. Areas of interest on the TLC layer were removed by scraping with a razor blade, and silica gel containing 1 H-labeled oligosaccharides was transferred to a scintillation cuvette. 10 ml of Dimilume scintillation fluid (Packard Instruments) was added and the radioactivity was determined with a liquid scintillation spectrophotometer.

ANTIFUNGAL ACTIVITY:

The inhibitory effect of chitinase 4 either alone or in combination with acidic chitinase SE and basic beta-1,3-glucanase 3 was observed on the growth of Cercospora beticola and Trichoderma reesei. Germination of spores and / or growth of hyphae of phytopathogenic fungi, such as Cercospora, in the presence of antifungal proteins can be analyzed by three different methods.

Procedure I is performed on microscope slides coated with a thin layer of medium and incubated with either buffer (control) or antifungal proteins in amounts of the order of. Spore germination or hyphal growth is analyzed by fluorescence microscopy after staining with Calcofluor White dye.

Procedure II is performed on microtiter plates containing growth medium, 10 or 100 Cercospora spores, and

102 buffer (control) or antifungal proteins. The plates are incubated at 25 ° C and then the optical density (at 620 nm) is determined at specified time intervals.

In Procedure III, radiological techniques are used in combination with autoradiography to demonstrate that chitin and beta-1,3-glucan are important components of the Cercospora cell wall, and that chitinase 4 can remove radioactivity stored in the tips of Cercospora hyphae.

Procedure I: Bioassay on microscope slides:

The microscope slides were coated with a thin layer of potato dextrose agar (PDA) and stored for 6 hours on moistened filter paper in petri dishes. 10 [mu] l of spore suspension (10,000 spores / ml) was placed in the center of the slide. To the spore suspension was added 10 [mu] l of 10 mM Tris-buffer, pH 8.0 or 10 [mu] l of a preparation containing 20 [mu] g of antifungal protein to be tested. The antifungal protein was dissolved in Tris buffer and filtered through a 0.22 .mu.m filter before mixing with the spore suspension. The petri dishes were sealed with adhesive tape and incubated for 24-48 hours at 30 ° C and full illumination. At the time of evaluation, the culture was stained with fluorescent dye Calcofluor White (0.05% (w / v) in water) for 10 minutes. Calcofluor White binds primarily to cell walls that contain emerging chitin structures, and this fluorescent dye can thus serve as a marker for the differentiation and growth of hyphal cell walls.

Procedure II: Bioassay on microtitre plates:

100 [mu] l of potato dextrose broth (PDB) liquid growth medium was placed in each well of a microtiter plate. The Cercospora spore suspension (100,000 / ml) was filtered twice through four layers

103 sterile gauze to remove small amounts of mycelial fragments. The spore suspension was diluted 1: 100 and 1: 1000 with sterile water, and then 100 / μl aliquots were transferred to microtiter wells. Antifungal proteins were dissolved in the same buffer and treated as described above for Procedure I. Bioassays were performed in five replicates for each dilution of fungal spores. The microtiter plates were sealed with adhesive tape to prevent evaporation and contamination. After incubation at room temperature on a stirrer operating at 50 rpm, the strip was removed and the absorbance at 620 nm was measured twice a day. Germination and fungal growth were monitored by measuring absorbance for 4 days. An absorbance versus time was constructed for each combination of antifungal protein and spore dilution.

Procedure III: Autoradiography:

Cercospora cultures were grown on a microscope slide as described in Procedure I. Liquid growth medium (PDB) containing ³ H-labeled N-acetylglucosamine was distributed uniformly to a one day old culture. After incubation for 20 minutes (radiolabeling), the reaction (growth and incorporation) was stopped by immersing the microscope slide in a 6% TCA solution (w / v). The preparation was dehydrated with ethanol (gradient 70 - 100%) and dried.

After radiolabeling, 50-100 .mu.l of a fraction containing chitinase 4 in 10 .mu.M Tris buffer pH 8.0 were added to half of the fixed and dehydrated culture. The microscope slide was placed on moistened filter paper in a petri dish. After closing the petri dish, the preparation was incubated for 20 hours at 30 ° C. The enzyme was stopped by immersing the slide in 6% TCA and the preparation was dehydrated with ethanol as described above.

The microscope slide was coated with an autoradiographic emulsion (Ilford K 5). After strong drying of the emulsion by a fan

104, the slide was placed in the dark for 7 days at 7 ° C and low relative humidity to allow exposure. The emulsion was developed by placing the slide in a Kodak D-19 developer, followed by fixation for 2 minutes and washing with running water for 10 minutes. After drying, the preparation was prepared for microscopic analysis of fungal hyphae.

PRODUCTION OF ANTIBODIES FOR USE IN SEROLOGICAL ANALYSIS:

Production of polyclonal antibodies against chitinase 2, 3 and 4:

Purified freeze-purified purified chitinases 2, 3 and 4 (obtained as described above) were dissolved in Tris-buffer (10 mM, pH 8.0) and diluted 1: 1 with Freund's incomplete adjuvant. Polyclonal antibodies were generated in rabbits according to standard Dakopatts (Denmark) procedures.

Production of monospecific polyclonal antibodies against chitinase peptides 4:

The procedure was performed as described in detail for the production of monospecific antibodies against AHAS peptides (Marcussen and Poulsen, 1991). Based on computer analysis of the amino acid sequence of chitinase 4, four peptides were selected according to the criteria of hydrophilicity and variability between chitinase 4 and other basic chitinases. Peptides were custom synthesized at Cambridge Research Biochemicals (UK). The structures were verified by mass spectroscopy and amino acid analysis to estimate purity.

Prior to immunization, the peptides were conjugated to diphtheria toxoid. The diphtheria toxoid serving as a carrier was converted to the corresponding sulfosuccinyl ester derivative by reaction with carbodiimide (EDC) followed by N-hydroxysulfosuccinimide. After condensation, there were four different peptide-toxoid conjugates

105 diphtheria purified by gel filtration on a Sephacryl column

S-300. The high molecular weight fractions were pooled, freeze-dried and dissolved in water. Immunization in rabbits was performed as described above for the production of polyclonal antibodies against chitinase 2 and 4.

SDS-PAGE and immunoblotting:

After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins for immunoblotting were transferred by semi-dry blotting to a 0.45 μm nitrocellulose membrane (Schleicher and Schuell, Germany). Antigens were probed with primary rabbit polyclonal antibodies raised against chitinase 2 and 4 (see above) and subsequently visualized using alkaline phosphatase-conjugated secondary antibodies (Dakopatts, Denmark) as described by Kynse-Andersen (1984).

Enhanced chemiluminescence (ECL) from Amersham was used to determine the level of expression in transgenic tobacco. After leaf mass extraction, 1 μg of protein was applied to each SDS-PAGE gel lane. After blotting, the nitrocellulose membrane was treated according to the Amersham procedure. Briefly, the nitrocellulose membrane was initially treated with 10% BSA, and then primary antibodies against sugar beet chitinase 4 diluted 1: 1000 were added. The antigen was detected by secondary antibodies conjugated to horseradish peroxidase. Detection reagent was added and after 2 minutes the protein bands were visualized on Hyperfilm-ECL.

ANALYSIS OF AMINO ACID COMPOSITION OF PURIFIED CHITINASE ISOENZYMES 2, 3 AND 4 AND BETA-1,3-GLUCANASES 3 AND 4:

After freeze-drying, the purified chitinase isoenzymes 2, 3 and 4 and beta-1,3-glucanase 3 and 4 were subjected to

106 amino acid analysis as described by Barkholt and Jensen (1989). A portion (4.2 .mu.g) of each of the chitinase isoenzymes and beta-1,3-glucanase, respectively, was incubated with 3,3-dithiopropionic acid to derivatize the cysteine residues prior to acid hydrolysis. The assay was repeated twice.

PREPARATION AND ANALYSIS OF THE AMINO ACID SEQUENCE OF TRYPTIC PEPTIDES CHITINASES 3, 4 AND SUGAR BEETS AND BETA-1, 3-GLUCANAS 3 AND 4:

Tryptic digestion:

After RP-HPLC as described above and freeze-drying, 100 μg of proteins were redissolved in 200 μl of 0.2 M Tris-HCl (pH 8.4) containing 7 M guanidine hydrochloride. 20 mM dithiothreitol was added and the protein was reduced for 4 hours at 37 ° C under nitrogen. 30 mM iodoacetamide was added and the reaction was allowed to proceed in the dark for 40 minutes at 25 ° C under nitrogen. Unreacted iodoacetamide was inactivated by adding 5 μl of beta-mercaptoethanol followed by incubation for 15 minutes at 25 ° C in the dark. The protein solution was dialyzed against 0.2M ammonium carbonate (pH 8.0) for 24 hours at 4 ° C in the dark using Eppendorf tubes with an inserted dialysis tube (10 kDa molecular weight cut-off, Servapore, Serva, Germany) placed under a pierced lid. . Then, the precipitated protein was pelleted by centrifugation for 5 minutes at 10,000 g, and the supernatant was transferred to another tube. The protein pellet was partially dissolved by adding a few particles of guanidine hydrochloride and incubated at 4 μg / ml.

of trypsin treated with tosyl-L-phenylalanine chloromethyl ketone (TPCK) in 20 [mu] l of ammonium carbonate (pH 8.0) at 40 [deg.] C. for 30 minutes. Finally, the supernatant and 6 μg of TPCK-treated trypsin were added. The digestion was allowed to proceed at 40 ° C

107 for 4 hours and was stopped by adding 20 μl of TFA. The peptide solution was subjected to RP-HPLC on a VYDAC C18 column (0.46 x x 15 cm, particle size 10 μm, The Separations Group, California) using the mobile system described above for RP-HPLC proteins (see Figure 4). The collected peptides were diluted three times with buffer A and rechromatographed on a Develosil C 18 g column (0.4 x 10 cm, particle size 5 μm, Dr. O Schou, Novo-Nordisk, Denmark) using the mobile system described above. Selected peptides were subjected to amino acid sequence analysis.

AMINO ACID SEQUENCE:

Amino acid sequencing of the peptides was performed on a Pulsed Liquid Phase Protein / Peptide Sequencer Model 477 with an On-line PTH-Amino Acid Analyzer Model 120 A HPLC from Applied Biosystems (CA, USA) connected according to the manufacturer's instructions.

BACTERIAL STEMS AND ENZYMES:

Restriction enzymes, Klenow polymerase and T4 DNA ligase were supplied by Boehringer Mannheim and used according to the manufacturer's instructions.

pBluescript was supplied by Stratagene (USA).

pUC19 was supplied by Boehringer Mannheim.

For subcloning in Escherichia coli, DNA transfer was performed using E. coli DK5alpha cells (from BRL) according to the manufacturer's instructions.

RNA ISOLATION FROM SUGAR BEET LEAVES:

RNA isolation was performed as described by Chirgwin et al.

(1976).

108

POLY-A-RNA PURIFICATION:

Poly-A-terminated RNA was purified by affinity chromatography on an oligo-dT cellulose column. 0.5 g of oligo-dT cellulose was mixed with 5 ml of 0.5M sodium hydroxide for 5 minutes (1 g of oligo-dT cellulose binds 1.2 mg of poly-A-RNA). The resulting mixture was neutralized with 1 mM Tris-pH buffer

7.5 to pH 7.5. A 1 cm column with a diameter of 1 cm was prepared and equilibrated with 20 ml of column buffer (500 mM sodium chloride, 10 mM Tris-buffer at 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA)). The RNA was denatured at 65 ° C for 5 minutes, and five times the volume of the column buffer was added to the RNA before column chromatography. The eluate was collected and chromatographed again. The column was washed with column buffer until 0 ^D 2go (optical density at 260 nm) reached 0.01 or less. Poly-A-RNA was washed with TE-buffer (Tris-buffer with EDTA) in 1 ml fractions and the RNA concentration for each of the fractions was determined at Οϋ ^ θθ. Fractions containing poly-A-RNA were pooled and mixed with 100 mM sodium chloride, and RNA was precipitated overnight with 2.5 times the volume of 96% ethanol at -20 ° C. Poly-A-RNA was isolated by centrifugation, dissolved in water at a concentration of 1 μg / μl and stored at -20 ° C. The yield was approximately 1-2% of the total RNA loaded on the column.

ISOLATION OF GENOME DNA FROM SUGAR BEET LEAVES:

Genomic DNA was isolated from sugar beet leaves, variety 60.159.838-131-4 (Dellaporta et al., 1983).

g of Cercospora-infected sugar beet leaves obtained as described above were ground in liquid nitrogen and frozen, and the frozen material was transferred to a 40 ml polyethylene centrifuge tube. 15 ml of extraction buffer (100 mM Tris-buffer pH 8.0, 50 mM EDTA and 500 mM sodium chloride) were added together with 1 ml of 20% sodium dodecyl sulfate (SDS) and after mixing, the mixture was incubated at 65 ° C after

109 for 20 minutes. 5 ml of 3M potassium acetate was added, the solution was mixed (vortexed) and incubated for 20 minutes on ice. Subsequently, the mixture was centrifuged for 20 minutes at 4 ° C and 25,000 g. The supernatant was filtered through 1-2 layers of miracloth filter cloth into a new centrifuge tube, 15 ml of isopropanol was added, and the mixture was incubated for 30 minutes at -20 ° C. C. After further centrifugation at 20,000 g for 15 minutes at 4 ° C, the pellet was washed with 70% ethanol and then dried briefly, then resuspended in 0.7 ml of X-TE buffer (50 mM Tris buffer, pH 8). 0 and 1 OmM EDTA). The suspension was transferred to an Eppendorf tube and centrifuged for 5 minutes. The supernatant was extracted twice with a mixture of phenol and chloroform. The DNA was precipitated by adding 75 μl of 3M sodium acetate and 500 μl of isopropanol, the mixture was mixed, and centrifuged for 30 seconds. The pellet was then dissolved in 400 [mu] l of water, the suspension was mixed with 100 mM sodium chloride and precipitated with 1 ml of 96% ethanol. The suspension was centrifuged for 5 minutes and the supernatant was discarded. The pellet was dried briefly and the DNA was dissolved in 200 [mu] l of TE buffer. DNA concentration was determined using absorbance at ^OD 260 ^ Ο ° 260 ^{= 1 =} 50 μg / DNA / ml. The DNA was stored at -20 ° C until use.

CONSTRUCTION OF SUGAR BEET CDNA LIBRARY:

Based on sugar beet mRNA isolated as described above, the lambdaZAP cDNA library was constructed using Stratagene Cloning Systems.

CONSTRUCTION OF THE GENOME DNA-LIBRARY OF SUGAR BEET:

Based on the genomic DNA of sugar beet obtained as described above, which was partially digested with SAU 3A, a genomic library of sugar beet was constructed by cloning

10 genomic DNA into the BamHI site of the EM3L3 vector. The library was constructed using Clontech.

CREATION OF BOARDS FOR SCREENING OF IMPORTANT DNA SEQUENCES OF LIBRARIES:

The titer of the library (either cDNA or genomic library) was determined as described by Sambrook et al. (1990), and for each g

10 phages were used for screening. For each of the 24.5 x x 24.5 mm plates, 2.5 x 10 6 phages were mixed with 3 ml of E-coli strain XL 1-Blue (in the case of the lambda ZAP sugar beet cDNA library) or LE392 (in the case of the genomic library sugar beet (EMBL3)) and grown on LB-medium with 10 mM magnesium sulphate and 0.2% maltose until Οϋθθθ = 1 · The mixture was allowed to stand at 37 ° C for 20-30 minutes.

Subsequently, 30 ml of 0.7% agarose overlay agar in LB-medium with 10 mM magnesium sulfate and 0.2% maltose (48 ° C) was added and the resulting mixture was plated.

24.5 x 24.5 mm containing 200 ml of LB-agar and allowed to grow overnight at 37 ° C.

TRANSFER OF PLATES TO NITROCELLULOSE FILTER IN THE NET:

Screening of recombinant lambdaZAP clones by in situ hybridization with individual plaques was performed as follows:

After growing overnight at 37 ° C, the plates were cooled to 5 ° C in about 15 minutes. Phages and DNA were transferred to a hybond-N nylon membrane (Amersham) by placing a dry filter on the cell layer. The phages were allowed to adsorb on the filter for 1 to 5 minutes. During adsorption, it was appropriate to label the filter and plate for needle orientation. If the filters were created repeatedly, the marks on the plate were filled with ink and it was then allowed to mark other filters with a similar mark.

1 1

The filters were then placed with the sand side up for 30 seconds on sheets of Whatman 3MM filter paper soaked in 0.5M sodium hydroxide and 1.5M sodium chloride. They were then washed after 30 seconds with each of the following solutions: 1) 0.5M sodium hydroxide, 1.5M sodium chloride,

2) 0.5M Tris-buffer, pH 7.5, 1.5M sodium chloride, 3) 2 x citrate buffer with sodium chloride (SSC) (modified Be.nton, 1977). The filters were air dried and illuminated with UV light for 3 minutes with the phage side up.

PREPARATION OF RADIOACTIVE PROBES FOR USE WHEN SEARCHING FOR CHITINASE 4 SUGAR BEET IN SUGAR BEET CDNA LIBRARIES:

Relevant oligonucleotides were labeled by phosphorylation with bacteriophage T4 polynucleotide kinase according to the procedure described by Sambrook et al. (1990). More specifically, oligonucleotides were synthesized without a phosphate group on them. „32 32 *

-terminals and were labeled with gamma-β z / gamma-β / ATP using the bacteriophage T4 polynucleotide kinase enzyme.

PURIFICATION OF RADIOACTIVELY LABELED OLIGONUCLEOTIDES BY COATING WITH ETHANOL:

After heat inactivation of the bacteriophage T4 polynucleotide kinase, 40 .mu.l of water was added to the tube and the contents were mixed thoroughly. Then 240 [mu] l of 5M ammonium acetate solution and 1 [mu] g of herring sperm DNA were added. The resulting mixture was mixed well and 750 μl of ethanol at ice temperature was added. The resulting mixture was again mixed thoroughly and stored for 30 minutes at 0 ° C.

The radiolabeled oligonucleotide was obtained by centrifugation at 12,000 g for 20 minutes at 4 ° C in a microcentrifuge. Using an automatic pipetting device equipped with a disposable tip, all supernatant (which contained most of the nonincorporate) was carefully removed.

2 gamma-P / ATP) and all free phosphorylation reactions. The resulting residue was water and 10 μl of 3M sodium acetate followed by 96% ethanol. The mixture was subjected to a cent minute at 4 ° C, dried and dissolved in

2-, _ P formed dissolved in was added rifugation after 200 / Ul of water during 100 _{/ U} 1 250 _{/ U} 1 time 20

OLIGONUCLEOTIDE HYBRIDIZATION OF CHITINASE 4 DNA BY FILTER HYBRIDIZATION:

The oligonucleotide hybridization procedure used eliminates the preferential denaturation of the A-T base pair over C-G and allows the stringency of the hybridization to be regulated only as a function of probe length. Hybridization was performed essentially as described by Wood et al. (1985). Nitrocellulose filters obtained as described above were surface soaked in 2 x SSC and subsequently prehybridized in hybridization buffer (6 x SSC, 1% BSA,% Ficoll 4000, 1% PVP, 50 μg / ml heat denatured

Salmon sperm DNA, 50 mM sodium phosphate, pH 6.8). Hybridization was performed for 4 hours at 37 ° C in a plastic bag with shaking. The filter was hybridized overnight in the same solution containing an additional radioactive nucleotide probe (23-mer chit 4 probe) at 37 ° C with shaking. Hybridization buffer with 1 x 10 pulses per minute / ml solution was used. The filter was washed three times in 6 x SSC at 4 ° C and then washed twice at 4 ° C in 6 x SSC for 30 minutes. Next, the filter was washed three times in TMAC-buffer (3M tetramethylammonium chloride, 50mM Tris-buffer pH 8.0, 2mM EDTA, 0.1% SDS) for 5 minutes at 37 ° C. (Tetramethylammonium chloride (TMAC) was prepared as a 5M stock solution. Since TMAC is hygroscopic, the actual molar concentration (c) must be determined from the refractive index (n) by the formula c = (n - 1.331) / 0.018). The filter was then washed twice in TMAC-buffer at 55 ° C for 20 minutes.

The filters were air dried at room temperature. The ink marks on the filters used to compare the autoradiographs with the filters and agar plates were marked

1 3 autoradiographic marks (Ultermit, Du Pont de Nemours). The filters were coated with Saran Wrap and the filters were exposed to X-ray sensitive films (AGFA CURIX RP2) for 16-70 hours at -70 ° C using image intensification.

Films were developed and positive plaques were identified by comparing spots on the film with spots on agar plates.

PLACE COLLECTION:

Agar fragments containing positive plaques were collected from the agar plate by gentle suction and tubes containing 500 [mu] l et al., 1990) were placed and one drop of Eppendorf tube was allowed to stand for hours at room temperature to allow phage particles to diffuse from the agar. 10 plaques were obtained.

into Eppendorf buffer (Sambrook

SM of phage chloroform. period 1 - 2

- 10 phages from each phage were then diluted with SM phage buffer and mixed with 200 μl of XL1 blue cells (Οϋθθθ = 1). The mixture was allowed to stand for 20 minutes at 37 ° C and 2.5 ml of overlaying agarose (48 ° C) was added. The mixture was poured into 9 cm diameter petri dishes and filter impressions were made for repeated screening.

Individual isolated positive plaques suitable for generating a pool of phages useful for in vivo excision were collected from agar plates according to the procedure described by Sambrook et al. (1990) using multiple plate creation and screening.

The phage stock was generated as described by Sambrook et al. (1990).

IN VIVO EXCIZE:

In vivo plaque excision was performed as described

1 4 in the In vivo Excision Protocol in the Instruction Manual (CAT No. 236201, August 30, 1988) for uncleaved Lambda ZA? II Clcning Kit, Stratagene Cloning Systems.

PLASMID DNA PREPARATION:

Plasmid DNA preparation was modified according to the method described by Sambrook et al. (1990) and was carried out as follows:

Bacterial strains (DH5alpha and XLI-Blue) containing plasmids were grown overnight in 5 ml of LB-medium supplemented with appropriate antibiotics and 5 ml of this culture was centrifuged for 10 minutes at 3000 g. The pellet was resuspended in 200 μl of solution I (50 mM glucose, 25 mM Tris-buffer pH 8.0, 10 mM EDTA) in 1.5 ml tubes.

400 [mu] l of solution II (0.2N sodium hydroxide, 1% SDS) was added, the mixture was stirred gently and placed on ice for 5 minutes. Then 300 [mu] l of 5M potassium acetate, at 4.8, was added and the mixture was stirred vigorously. The resulting mixture was placed on ice for 10 minutes and then centrifuged at 15,000 g for 10 minutes at 4 ° C.

The supernatant (900 [mu] l) was transferred to new tubes and 0.6 times the volume (540 [mu] l) of ice-cold isopropanol was added. The resulting mixture was allowed to stand for 15 minutes at room temperature. The mixture was again centrifuged at 15,000 g and 4 ° C for 10 minutes, and the supernatant was discarded.

The pellet was dissolved in 100 [mu] l of TE buffer and 100 [mu] l of 5M lithium chloride was added. The mixture was allowed to stand on ice for 5 minutes and centrifuged at 15,000 g and 4 ° C for 10 minutes.

The supernatant was transferred to new tubes and 500 μl of 96% ethanol was added. The tubes were centrifuged at 15,000 g and 4 ° C for 30 minutes and the supernatant was discarded. The pellet was washed with 70% ethanol (approximately 100 μl) and dried. The dried pellet was resuspended in 50 [mu] l of TE buffer.

DNA SEQUENCE:

Plasmid DNA (double-stranded template) subjected to sequencing was purified as described above. Sequencing was performed as follows:

A mixture containing approximately 2 [mu] g of the appropriate plasmid, [mu] l of 2M sodium hydroxide, 2 [mu] M EDTA, 1 [mu] l of primer (100 [mu] g / ml) and made up to 10 [mu] l with water was incubated at 85 DEG C. for 5 minutes .

The mixture was neutralized by adding 1 μl of 5M ammonium acetate and then precipitated by adding 20 ml of 96% ethanol. The resulting mixture was centrifuged for 30 minutes at 4 ° C and suspended in 6 μl of water. 1.5 [mu] l of 5 * concentrated sequencing buffer was added. The mixture was placed at 37 ° C for 5 minutes.

4 [mu] l of sequetide (Biotechnology Systems NEN Research Products, Du Pont de Nemours) and 2 [mu] l of sequenase (United States Biochemical) were added. The resulting total volume of the mixture was 13.5 / Ul. The mixture was placed at room temperature for 5 minutes.

A 3.1 [mu] l labeling reaction containing 2.5 [mu] l of dNTP termination mixture was transferred to each termination tube (G, A, T and C). The mixtures in each of the tubes were allowed to react for 5 minutes at 37 ° C and 4 μl of termination solution was added. The mixtures were then heated to 85 ° C and / U of the heated mixtures were applied to a 6% sequencing gel (Gel-mix 6 from BRL). The gel was vacuum dried and exposed to an X-ray film.

MARKING OF DNA-PROBES WITH SUGAR BEET:

DNA probes for use in the isolation of acid chitinase SE

1 6 sugar beets were labeled using the Stratagene oligolabelling kit prime IT (Random Primer Kit) according to the manufacturer's instructions. Specifically, the following procedure was used:

A sample containing 25 ng (1-23 .mu.l) of DNA template to be labeled with 0-22 .mu.l of water and 10 .mu.l of random oligonucleotide primers with a total volume of 33 .mu.l was placed at the bottom of a clean microcentrifuge tube. The reaction tubes were heated to 95-100 ° C on a boiling water bath for 5 minutes and then centrifuged briefly at room temperature to collect liquid that could condense on the cap of the tubes. A reaction tube containing a DNA sample in low melting temperature agarose (LMT agarose) was placed at 37 ° C, and the following reagents were added to the reaction tubes:

/ Ul 5X primer-buffer containing dATP, dGTP and dTTP,

2 .mu.l of labeled alpha-PdCTP nucleotide (3000 Ci / mM) (Amersham), and .mu.l of diluted T7 DNA polymerase. T7 DNA polymerase was diluted to Enzyme Dilution Buffer to a final concentration of 1 U / μl immediately before use. The reactants were mixed with a pipette tip.

The tubes were incubated at 37-40 ° C for between

2 and 10 minutes and then the reaction was stopped by adding 32 μl of Stop Mix. P-labeled DNA probes were further purified using the Elutip-D column system (Schleicher and Schuell).

The DNA probe was then prepared for hybridization by mixing an appropriate amount of radioactive probe with 200 [mu] l of salmon sperm DNA (10 mg / ml). The mixture was heated to 95-100 ° C in a boiling water bath for 5 minutes and cooled on ice. The resulting probe was stored at 20 ° C for up to one week and heated to 95-100 ° C for 5 minutes in a boiling water bath for 5 minutes and cooled on ice.

1 7

SE-DNA HYBRIDIZATION:

Filter fingerprints obtained as described above as oligonucleotide hybridization of the lambdaZAP sugar beet cDNA library were prehybridized for 2 hours at 67 ° C under standard prehybridization conditions using a prehybridization solution containing 2 x SSC, 10 x Denhardts, 0.1% SDS and 50 μg / ml salmon sperm DNA.

Hybridization was performed overnight using a hybridization solution identical to the prehybridization solution, except that a radioactive probe prepared as described above was added.

After hybridization, the following washes were performed: twice after 15 minutes in 2 x SSC and 0.1% SDS and twice after 15 minutes in 1 x SSC and 0.1% SDS.

Positive plaques were identified as described above under the title Oligonucleotide Hybridization of DNA Chitinase 4 by Filter Hybridization.

DNA IDENTIFICATION OF THE GENE FAMILY CHITINASE 4:

To identify DNA belonging to the chitinase 4 gene family, hybridization of the test DNA to the chitinase 4 probe was performed using the hybridization procedure described in the SE-DNA Hybridization section, except that the hybridization was performed at 55 ° C. The chitinase 4 probe may be the chitinase 4 DNA sequence set forth in SEQ ID NO: 1. It is contemplated that the probe is prepared based on a characteristic portion or specific subsequence of the chitinase 4 DNA sequence as described herein, e.g., a probe prepared based on peptide 4-26. may also be appropriate. To identify a DNA sequence hybridizing to a specific subsequence of the chitinase 4 DNA sequence and encoding a specific portion of the chitinase 4 enzyme, the nucleotide probe is preferably prepared based on the amino acid sequence of that specific portion or subsequence thereof.

EXCISION OF DNA FROM AGAROS GELS:

DNA fragments for use, for example, in the construction of genetic constructs of the invention have been isolated as follows:

DNA was run on low melting point (LMT) agarose (Sea Plaque GTG, FMC) in TAE-buffer (0.04M Tris-acetate, 0.002M EDTA). The DNA band was excised with a Pasteur pipette. An equal volume of 200 mM sodium chloride, 10 mM EDTA was added to the excised DNA. The gel was melted at 68 ° C for 10 minutes and re-equilibrated at 37 ° C. Subsequently, 2U / 100 [mu] l of agarase (DNase - free, from

Calbiochem). The mixture was allowed to stand overnight at 37 ° C and was then extracted twice with phenol and twice with chloroform, subjected to ethanol precipitation and finally dissolved in water.

PCR USED TO AMPLIFY CDNA ENCODING BETA-1,3-GLUCANAS AND CHIT 76 BASED ON SUGAR BEET mRNA:

Preparation of the partial cDNA molecule was performed using the Gene Amp RNA Amplification Reagent Kit (Perkin Elmer Cetus, USA). The polymerase chain reaction (PCR) was performed according to the manufacturer's instructions with several modifications. The concentrations shown in the scheme below were used for reverse transcription:

1 9

Component

Volume

The resulting concentration

MgCl 2 solution 4 ui 5 mM 10 x PCR buffer 2 _{/ U1} 1 mM dGTP 2 _{/ U1} 1 mM dATP 2 1 mM dTTP 2 1 mM dCTP 2 1 mM RNase inhibitor 1 _U1 1 u, reverse transcriptase 1 _/Hive 2 .5 ' primer 270 0 , 4 / Ul 2 , 5 mRNA 3 , 6, ul

The total sample volume is 20 .mu.l

The following procedure was used in the cycle:

Part 1: 42 ° C for 2 hours

Part 2: 99 ° C for 5 minutes

Part 3: 5 ° C for 5 minutes.

The PCR procedure was followed, with Taq polymerase added later (see PCR cycles) and the temperature of the cycles changed as follows:

PCR cycles:

number of cycles ° C time (in minutes)

98 5

5 addition of Taq polymerase and oil

94 1

2

1

40

120

number of cycles ° C time (in minutes) 5 94 1 37 2 72 10 30 94 1 42 2 72 5 1 72 20

PCR USED IN THE CONSTRUCTION OF GENETIC STRUCTURES ACCORDING TO OF THE INVENTION AND MUTAGENESIS AT LOCAL MANAGEMENT CLONED DNA: BASED ON MATRIX The preparation of the respective DNA molecule was carried out using living kit Gene Amp DNA Amplification Reagent Kit (Per- Elmer Cetus, USA) in accordance with the guidelines manufacturer by that the cycle temperature has been changed. Was used the following tup: PCR cycles: number of cycles ° C time (in minutes) 35 94 1 1/2 60 1 1/2 72 4 1 72 7

121

Examples of embodiments of the invention

Example 1

Purification and characterization of chitinases 2, 3 and 4

The process used for the synthesis of regenerated chitinase has been specifically developed to allow a high yield of active chitinase 4 to be achieved. A high yield of active and pure chitinase is necessary to ensure sufficient protein material for

i) determination of the antifungal potential, ii) preparation and analysis of tryptic peptides, which makes it possible to prepare an oligonucleotide probe suitable for the isolation of DNA encoding chitinase, iii) production of monoclonal and polyclonal antibodies.

Isolation and characterization of DNA encoding chitinase is necessary if the DNA is to be used to construct genetically modified plants exhibiting increased chitinase activity. High amounts of pure chitinase are also necessary to find and characterize important parts of the enzyme, such as the active site.

Regenerated chitin was obtained by acetylation of free amino groups at low as well as high pH as described above (compared to conventional methods in which this synthesis is performed only at low pH). The new combined process was simpler, faster and gave much higher yields and more stable products than the conventional process, in which the acetylation is performed only at low pH.

The degree of enzyme purity was examined during the purification steps by SDS-PAGE on a Phast-System as described in Materials and Methods. After separation on a Mono S column for FPLC (Fig. 1), only a 122 d-silver band was observed on the SDS-gel (Fig. 2) for each of the chitinase isoenzymes 2, 3 and 4. Further analyzes by reverse phase HPLC on the column VYDAC RP4 gave only one protein peak for each of the isoenzymes, further evidence that homogeneous proteins of each of the basic chitinase isoenzymes were prepared.

Molecular weights were determined by SDS-PAGE for chitinase 2, 3 and 4 at 32, 27 and 26 kDa, respectively (Fig. 2). By isoelectric focusing, the isoelectric points for chitinase 2, 3 and 4 were determined to be 8.3, 9.0 and 9.1, respectively. Using the radiochemical chitinase assay described above, all three isoenzymes were found to have a broad pH optimum with a maximum activity of about 4.5. The specific activity of chitinase 4 is 480 nkat / mg protein, while chitinase 3 and 2 are 208 and 164 nkat / mg protein, respectively.

To determine whether chitinase 4 is a chitioligosaccharide-producing endochitinase or an exochitinase releasing only N-acetylglucosamine from the non-reducing end of chitin or chitooligosaccharides, a sample of reaction products released by chitinase 4 from 1 H-chitin was analyzed by thin layer chromatography (Fig. 3). Regardless of the incubation time, N-acetylglucosamine was only a very minor minor reaction product, while chitobiose, chitotriose and chitotetraose were the main products, leading to the conclusion that chitinase 4 is an endochitinase.

In addition to the catalytic activity on 1 H-chitin, chitinase 4 was also able to hydrolyze the cell walls of Micrococus lysodeicticus using the lysozyme assay described in Materials and Methods (see Figure 4). This indicates that chitinase 4 is a bifunctional enzyme that has both chitinase and lysozyme activity.

Example 2

Antifungal activity of purified chitinase and beta123

-1,3-chitinase isoenzymes from sugar beet leaves

Three different bioassays were performed to determine the in vitro antifungal activity of chitinase and beta-1,3-glucanase isoenzymes on the germination and growth of Cercospora beticola. In the same manner, the antifungal activity of chitinases and beta-1,3-glucanases from other sources or other sugar beet isoenzymes can be determined using either purified enzymes or enzyme-containing extracts. Assays can also be used to determine whether a given transgenic plant is within the scope of the invention.

Procedure I - Bioassay on microscope slides:

Cercospora spore cultures germinate well and grow on a thin layer of potato dextrose (PDA) agar on a microscope slide. Growth can be monitored by examining the number of germinating spores and total or average mycelial growth using a light microscope. Growth activity can be further visualized at any given time by staining the culture with Calcofluor white dye followed by microscopic examination. The number of hyphae with fluorescence below the fluorescent peaks and the extent of staining calibrate the growth activity of the culture.

If proteins with strong antifungal activity are added, the number of germinating spores is reduced and the growth rate of hyphae is significantly reduced. Figure 5 shows the results of applying a combination of chitinase 4, acid chitinase SE and beta-1,3-glucanase 3 to a culture. 60 [mu] l of a protein solution containing 20 [mu] g of each of the antifungal proteins was applied to each microscope slide. When chitinase 4 was used alone or in combination with either beta-1,3-glucanase alone or SE alone, the inhibitory effect was less pronounced. Neither beta-1,3-glucanase alone, SE alone, nor a combination thereof showed a significant inhibitory effect.

124

However, as can be seen from Figure 5, a very strong inhibitory effect was observed using all three enzymes at once, indicating a synergistic effect between chitinase 4, SE and beta-1,3-glucanase.

Procedure II - Bioassay on microtiter plates:

Spore germination and mycelial growth can be monitored in microtiter plates by measuring absorbance (at 620 nm) at specified time intervals. In control experiments, Cercospora begins to grow after approximately 40 hours in between and increases almost linearly for another 40-50 hours (curve

And in Fig. 6). If pure chitinase 4 (5 .mu.g per well) is present, the initial interval is extended to 75 hours and the growth rate is reduced compared with the control (curve C in FIG. 6). The eluate from the chitin column is given for comparison.

Procedure III - Autoradiography:

In the third bioassay, chitin in cell walls - 3 hyphae was labeled with H-labeled N-acetylglucosamine. After a short time, the radioactivity was deposited in the tips of the fungal hyphae (see Fig. 7). When chitinase 4 was added after radiolabeling, either alone or in combination with SE and beta-1,3-glucanase, the radioactivity deposited at the hyphal tips was effectively removed. The amount of enzymes is similar to that described in Procedure I (see above). This clearly shows that the mode of action of chitinase 4 on the cell wall of Cersospora consists in the hydrolysis of chitin fibers in the tips of hyphae and the consequent inhibition of cell wall synthesis.

Based on the above experiments, the following conclusions can be drawn:

It is possible to inhibit the growth of Cercospora in spore cultures by adding chitinase fractions from sugar beet leaves.

125

Inhibition of germination by its inhibitor.

is primarily observed as the length interval depends on the strength and concentration of the growth fraction, which contain both chitinase and beta-1,3-glucanase have a stronger inhibitory effect than chitinase alone.

Chitinase / glucanase fractions from sugar beet plants infected with Cercospora have a stronger inhibitory effect than fractions purified in the same way from control plants.

Example 3

Amino acid composition and partial amino acid sequences of purified chitinase isoenzymes 2, 3 and 4

After freeze-drying, the amino acid composition of pure sugar beet chitinase 2, 3 and 4 was determined (see section

Material and methods).

The results are shown in Table I. For comparison, the amino acid composition of barley, wheat and bean chitinases is also shown in the table (Leah et al., 1987). The amino acid composition of chitinase 2 is similar to that of bean chitinase at a number of amino acid residues, such as aspartic acid, proline, glycine, leucine, tyrosine, phenylalanine, valine and lysine. In contrast, chitinases 3 and 4 have a substantially different amino acid composition compared to all other chitinases.

The amino acid composition derived from the cDNA sequence encoding sugar beet chitinase 4 without signal peptides is also given below. The cDNA sequence was obtained as described in Example 5 below.

126

TA3JLKA I

Amino acid composition of barley, wheat, bean and chitinase 2, 3 and 4 sugar beet chitinases

Amino acid Barley Wheat Beans S.B.2 S.B.3 S.B. 4 cDNA Acid asparagus 23 28 29 34.4 24.7 24.4 22 Threonine 13.8 22 22 16.2 13.0 12.8 1 2 Serine 17.7 24 26 21, 0 24.8 24.8 24 Acid glutamic 18 20 22 24.9 22.1 21.0 18 Proline 17 15 20 17.1 10.3 10.2 9 Glycine 30.7 52 37 39.7 30.6 30.4 27 Alanine 37.3 27 26 28.0 28.2 28.5 26 Cysteine 7.2 12 16 16.9 16.8 16.9 15 Valine 12.5 14 1 0 8.6 14.4 14.3 14 Methionine 1.6 3 2 1.8 1.1 1.1 1 Isoleucine 10.8 9 1 1 11.9 10.9 11.0 1 1 Leucine 1 1.3 13 17 16.2 9.0 9.0 8 Tyrosine 11.9 1 4 15 17.3 12.7 12.7 1 2 Phenylalanine 12.7 1 4 13 1 1.5 18.3 18.1 17 Histidine 4.9 4 3 4.4 4.7 5.4 4 Lysine 6.9 8 8 8.7 4.3 3.1 3 Arginine 15.2 14 16 11.3 14.2 16.1 15 Tryptophan 3.2 7 4 ns ns ns 3 Molecular weight (KD) 27 29 32 30.6 27.6 27.7 25.9

Legend to Table I:

S.B.2 = sugar beet chitinase 2

127

S.B.3 = sugar beet chitinase 3

S.B.4 = sugar beet chitinase 4 cDNA = amino acid composition derived from the cDNA sequence encoding the mature chitinase protein 4 ns = not determined.

Tryptic cleavage of chitinases 3 and 4 of sugar beet:

Analysis of the pure enzyme chitinase 4 showed that the N-terminal part of the enzyme is blocked. Thus, analysis of mature chitinase 4 did not directly determine its amino acid sequence, and to obtain sufficient information about the enzyme for the ultimate goal of isolating and characterizing the DNA encoded, the mature enzyme was tryptically digested to obtain protein fragments (peptides) that can be subject to amino acid sequencing.

Tryptic digestion of purified chitinase enzymes was performed as described in Materials and Methods above. Tryptic peptides were separated by the above-mentioned reverse phase HPLC on a Vydac RP-18 column under the conditions specified in the Materials and Methods section (see Figure 8). Peptides representing large peaks at absorbance at 214 nm and showing a long residence time (indicating that they have long polypeptide chains) were selected for further purification on a Develosil RP-18 column.

Purified peptides were subjected to amino acid sequence analysis as described in Materials and Methods, and the amino acid sequence of each of the peptides is shown in Table II below.

When comparing the amino acid sequences of each of the peptides with the amino acid sequences of known chitinases (not from sugar beet), a low degree of homology was found.

One of the tryptic peptides proved to be very

128 suitable to form the basis for the construction of an oligonucleotide probe. Thus, analysis of the amino acid sequence of tryptic peptide 4-26 revealed that the use of this sequence to construct an oligonucleotide probe would require only a few codons to be selected. Thus, this peptide was chosen to form the basis for the construction of an oligonucleotide probe for use in isolating DNA encoding chitinase 4 (see Example 4 below).

TABLE II

Tryptic peptides of chitinase 3 and 4

Chicinasa 3:

3-LQ.3 S-T-Y-C-Q-S-Y-A-A-F-P-P-N-P-S-K

3-L6.1 A-C-V-T-H-E-T-G-H-F-C-Y-I-E-E-I-A-K

3-16.2 'J-G-Y-Y-T-Q-Y-C-Q-Q

3-22.3 G-P-L-Q-I-T-W

3- 23.3 S-I-G-F-D-G-L-N-A-F-E-T-V-A-M-N-A-V-T-A-F-R.

Chicinasa 4:

4- 4.2 V-G-Y-Y-T-Q-Y

4-1.9.3 G-F-L-Q-I-T-U ’

4-22 s-i-g-f-d-g-1-n-a-p-e-t-v-a-n-n-a-v-t-a-f-r

4-23 F-G-F-C-G-S-T-D-A-Y-C-G-E-G-C-R

4-24 S-F-S-S-G-C-G-S-V-S-S-L-V-T-D-A-F-F

4-26 T-A-F-W-F-V-M-N-N-V-H-S-V-I-V-N-C-Q-G-F-G-A-S-I

Legend k ¹ Tables II: 3-10.3 Yippee listed in SEQ ID C. 18 3-16.1 Yippee listed in SEQ ID C. 19 3-16.2 Yippee listed in SEQ ID C. 20

29

3-22.3 is shown in SEQ ID NO: 21

3- 23.3 is shown in SEQ ID No. 22

4- 4.2 consists of amino acids No. 244-250 in SEQ ID No. 2

4-19.3 consists of amino acids No. 163 - 169

4-22 of amino acids C. 179 - 200 4-23 of amino acids C. 37 - 52 4-24 of amino acids C. 58 - 75 4-26 of amino acids C. 201 - 224

Example 4

Isolation and characterization of the cDNA gene for chitinase 4

From the amino acid sequence obtained for peptide 4-26 (see Table II in Example 3), the following highly specific oligonucleotide gene probe was synthesized using a DNA synthesizer 381 A (Applied Biosystems).

Using a gene probe, the lambdaZAP expression cDNA library was screened using the procedure described above in Materials and Methods. Eight cDNA clones were obtained from lambdaZAP and one of these clones was fully sequenced while the others were only partially sequenced.

130

Sequencing was performed as described in the Materials and Methods section above. An almost full-length cDNA clone, chit 4-315, was obtained from the lambdaZAP library, and its DNA sequence is shown in SEQ ID NO: 1.

Based on the cDNA sequence, the deduced amino acid sequence of chitinase 4, shown in SEQ ID No. 2, was generated. The deduced amino acid sequence was compared to a partial sequence obtained from chitinase 4 protein (as described in Example 3 above) and almost 100% identity was observed. which demonstrates that the isolated cDNA clone encodes chitinase 4 polypeptides purified by the chromatographic procedures described above. The cDNA clone chit 4-B15 is 966 bp long and encodes a protein that has 264 amino acid residues in the polypeptide chain out of the 265 amino acid residues predicted for chitinase 4 of genomic DNA. The leader sequence probably consists of 23 amino acid residues (of the 24 amino acid residues determined for chitinase 4 genomic DNA, see below), followed by a hevein domain of 35 amino acid residues and a functional domain of 206 amino acid residues. After the termination codon, the cDNA has a 3-non-coding region 158 bp long.

As mentioned above in Example 3, it was not possible to sequence the N-terminal amino acid sequence of chitinase 4 directly because the N-terminus is blocked. However, compared to wheat germ agglutinin (WGA-A) and potato chitinase, glutamine was estimated to be the first amino acid. It was later deduced from the DNA sequence that the residue of the amino acid sequence of the N-terminus of chitinase 4 is Gln-Asn-Cys-Gly-Cys .....

The N-terminal sequence of chitinase 4 was further examined by determining the molecular weight (MW) of mature chitinase 4 by electrospray mass spectrometry as described by GJ Feistner et al., 1990. A molecular weight of 25893.6 +/- 10 was found. Based on the amino131 acid sequence, a molecular weight of 25923 can be calculated. Since mature chitinase 4 contains seven disulfide bridges (loss of 14 protons) and the first amino acid residue of Gln is converted to The pyroglutamyl derivative (loss of -NH ₂ , i.e. molecular weight reduction of 15) is the calculated molecular weight of mature chitinase 4 25894. This is consistent with the data obtained by analysis by electrospray mass spectrometry and confirms the correctness of the derived N-terminal amino acid sequence given above. for mature chitinase 4.

The N-terminal amino acid sequence of chitinase 2 can be determined, and thus the following terminal amino acid sequence has been found in chitinase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala.

TABLE III

Comparison of the N-terminal amino acid sequence of different chitin binding proteins

WGA-A: QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCGKG - CQNGACW7S Hevein: EQ ** r * AGGKL ********* W- * * W ** STD E ** S PDHN ** SN- * KD Chic. Beán: EQ ** R * AGGAL ** GGN **** F- * W ** STT **** P * - - ** SQ- * GG Chic. Tob. : EQ ** S * AGGAR * ASG **** KF- * W ** NTN **** P * -N ** SQ- * PG Chic. SB 2: EL ** N * AGGAL *** G ****** - * y ** jjTNP *** N Chic. SB 4: * n ** c-a ** lc * srfgf * gstda *** e * cregp-- - * RS ..... 1

Legend to Table III:

* = amino acid residues

WGA-A: shown in SEQ ID NO.

Hevein: shown in SEQ ID NO identical to WGA-A 23

132

Chit. Beán: Chit. Tob .: Chit. SB 2

Chit. SB 4:

(from bean chitinase), shown in SEQ ID No. 25 (from tobacco chitinase), shown in SEQ ID No. 26 (from sugar beet chitinase 2), shown in SEQ ID No. 27 (from sugar beet chitinase 4), consists of of amino acids No. 24-54 in SEQ ID No. 2

Example 5

Isolation and characterization of genomic clones of sugar beet chit 76 and chit 4

Screening of 500,000 clones from an amplified EMBL3 library containing sugar beet genomic inserts from partial digestion of Sau3A resulted in the isolation of three clones with cDNA chitinase 4 as a probe.

Three hybridizing clones were characterized by restriction fragment analysis and sequencing. These analyzes showed that one of the clones contained the chitinase gene, now designated chitinase 76, the sequence of which is set forth in SEQ ID NO: 5. Sequencing of this gene was started with the primer used to screen the lambdaZAP library (see Example 4), and continued with other primers. complementary to sequences within the chit 76 gene.

The chit 76 gene encodes a 268 amino acid chitinase that exhibits 80% homology to the amino acid sequence of chitinase 4 (see SEQ ID NO: 1), but only 34% homology to the entire chitinase 1 protein (see SEQ ID NO: 11). The gene contains a single intron located at positions 875 to 1262. The exact location of this intron is based on comparison with the chitinase 4 cDNA shown in SEQ ID NO: 1 (see Figure 24). Intron boundaries contain conventional GT / AG sequences. Intron chit 76 is located at exactly the same site as the second intron of the chitinase 1 gene when comparing amino acid

133 chitinase 1 and chit 76 sequences.

The TATA-box (TATAAA) sequence is located at position 378, which is 90 bp upstream from the ATG translation start codon. The polyadenylation signal (AATAAA) is located at position 1725 in SEQ ID NO: 5.

The genomic clone encoding chitinase 4 was isolated in a similar manner. The DNA was partially sequenced and approximately 350 nucleotides of the 5-non-coding region were determined. Approximately 340 nucleotides of the coding region were sequenced. The sequence is apparent from SEQ ID NO: 3.

Comparison of the 5-non-coding regions of the two genomic genes showed the presence of homologous portions (e.g. nucleotides 14-49, 60-122, 123-135, 159-173, 1774-207 and 277-328 chitinase 4) (see FIG. 26).

Based on knowledge of the chit 4 B15 cDNA sequence and the partially sequenced chitinase 4 genomic gene, the remainder of the gene can be easily sequenced. The chitinase 4 gene is thought to contain at least one intron, and probably only one of these introns corresponds to an intron listed at the same position as in the chitinase 76 sequence.

Example 6

Characterization of acid chitinase SE isoenzymes and determination of partial amino acid sequence

Acid chitinase SE was purified as described in Materials and Methods above.

After final purification on a Mono P column for FPLC, three SE isoenzymes can be observed (see Figure 9). When analyzed by SDS-PAGE, only one protein band was shown for each of the isoenzymes. The same molecular weight of 29 kD was determined by SDS-PAGE. By isoelectric focusing analysis, the isoelectric focus was determined for three SE isoenzymes.

34 point approximately 3.0, which corresponds well to the theoretically determined isoelectric point estimated at 3.87.

In contrast to basic chitinases 2, 3 and 4, acid chitinase SE was not retained on the chitin affinity column either under normal conditions, at pH 8 (see Materials and Methods) or at higher or lower pH. However, SE easily degraded 1H-labeled chitin. The main product of enzymatic hydrolysis was hexamers derived from chitin or higher homologues of chitin oligosaccharides. Since the main product of chitinase 4 was dimers, a different mode of action is expected in SE. No lysozyme activity can be determined for SE at pH 4-9.

The purified enzyme was subjected to tryptic digestion as described in Materials and Methods and in Example 3 above for chitinase 4, and six peptides were selected. The peptides were subjected to further purification in the same manner as the tryptic peptides of chitinase 4, as described in Example 3 above, and the amino acid sequence of these six peptides was determined. Peptides were selected using the same criteria as in conjunction with chitinase 4. The amino acid sequences of these peptides are listed in Table IV. In addition, the N-terminal amino acid sequence was also determined as shown in Table IV.

TABLE IV

N-terminus: S-Q-1-7-1 -Y - '«- G-Q-N-C-D- E-G-S - L-A-Q SE 22.5: 7-L-L-S-I-G-C-G-A-C-G-Y SE 23.0 A-D-Y-L-w-N-1-ϊ SE 25.L K - N -? -? - C - 0 - Y - D - T - S - A - D - K - L - L - S - S SE 26.L Y-G-G-Y-M-L-W SE 30.i S-L-S-S-I-D-D-X-N-T-F-X-D-Y-L-w-N-I SE 3L.L I-T-V-Q-A-N-Q-I-F-L-G-L-P-A-S-I-D-A

135

Legend to the Table IV: N-! end: consists of of amino acids C. 26 - 46 v SEQ ID No. 8 SE 22.5: consists of of amino acids C. 98 - 109 ' v; SEQ ID No. 8 SE 23.0: consists of of amino acids C. 121 - 128 in SEQ ID no. 8 SE 25.1: consists of of amino acids C. 208 - 224 in SEQ ID no. 8 SE 26.1: consists of of amino acids C. 271 - 277 in SEQ ID No. 8 SE 30.4: consists of of amino acids C. 1 10 - 128 in SEQ ID no. 8 SE 31.1: consists of of amino acids C. 229 - 252 in SEQ ID no. 8

Example 7

Isolation and characterization of cDNA for the acidic chitinase isoenzyme SE

Based on the amino acid sequences of the tryptic peptides listed in Table IV, two subsequences were selected from the peptides SE 25.1 and SE 31.1 (Table IV) for oligonucleotide synthesis because they had the best codons. PCR primers KB 7 (SE 25.1), which is given in SEQ ID No. 28, KB-9 (SE 31.1), which is shown in SEQ ID No. 29, and oligo-dT primer (270), which is shown in SEQ ID NO: 30, were prepared in the same manner as described in Example 4 in relation to chitinase 4. The nucleotide sequences of the gene probes are shown in Table V.

TABLE V

U _? ? C 0 Y D I č »3 / -GACTC j ..- i.GAAAÁGcýCcý úG £ CA ^ i.A (* GAÁAC- 3 o> / - f

K3 - 9. 5 '- GGAGCATCCCAÝGCGAA? CAÝΑΤΑΠ - 3 ’A C

T T c

210 5 '- CCAAGC i .GAAT -CTTTTTTTTTTTTTTTTTTTTTT · 3 ‘

36

Legend to Table V:

KB-7: stated in SEQ ID C. 28 KB-9: stated in SEQ ID C. 29 270 stated in SEQ ID C. 30.

The partial cDNA molecule was prepared in two steps using mRNA and PCR techniques, using the above-mentioned primers KB7 and 270 in the first step. PCR was performed as described in Materials and Methods above. The synthesized cDNA was isolated on a low melting point agarose gel and the agarose was removed with agarase. Primers K39 and 270 were used for the subsequent PCR reaction. The procedure is shown in Figure 20. The product from the second PCR reaction was cloned into pUC 19 (Boehringer Mannheim) and sequenced.

The DNA sequence obtained for the cDNA partial molecule consisted of nucleotides 711-962 of the DNA sequence shown in SEQ ID NO: 7. This cDNA was used to screen the lambdaZAP cDNA library as described in Materials and Methods and 23 cDNA clones were obtained. The longest cDNA clone was sequenced using the procedure described in Material and Methods above and was found to be 1070 bp long. The sequence consists of nucleotides 37-1106 of the DNA sequence set forth in SEQ ID NO: 7. As is normally observed in connection with cDNA isolation, it has been difficult to isolate the entire cDNA. A new screening of the lambdaZAP library with a 122 bp EcoRI-KpnI fragment from the 5-terminus of the longest cDNA clone (SE22) provided a sequence containing the entire 5-terminus. Clones were ligated using the KpnI site. The structural gene has a 5-non-coding region of 17 bp, a leader sequence of 25 amino acid residues, a functional domain of 268 amino acid residues, and a 3-non-coding region of 202 bp after the termination codon. The cDNA sequence and amino acid sequence are set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

137

Comparison of the amino acid sequence obtained from the N-terminus and the six tryptic peptides (107 residues) showed almost 100% identity with the sequence derived from the cDNA, proving that the isolated cDNA clone encodes SE polypeptides purified by the chromatographic procedure described above. The cDNA contains both the N-terminus and the C-terminus of the mature protein. The N-terminus of the mature SE is shown in Table IV.

TABLE VI

ΊΑί-.L * J C w X Ξ Ξ A. =. A 3 Z 2 2 r a A KIV S - »’ L F ZAAnKIT - - 77'_Ξ. “. 7NK7LP.XHV XY

AS “ΞΞΞΗΰΞ ~ ΣIYWGCNwZ ΞΖ · Ξ '» ΑΖ7ΙΗ Ξ Σ7 = ΞΞ3Aa --ΣΣA ΐYXGGHGSE39La973a Ξ’-ΞλΡΞΣ aSRGG Σ A Σ YWGGHGN EGíiLSA 7GA

ΓΑΓ.Σ. IJCGhSEP. aRaBIGG?

GNYG7V Σ LAr V a 7F3N3G7? A LHLaGHGZ. = Α7.Ν-ΣΝΞΕΞΞΖ Σ X73GG aG 3S Y EF V N Σ AFLEEFGsGG AP VLHLAGříCHPOHHGCAFLS3Ξ Σ ΗΞΣΧΞ - Η GRYAYVNVAFLVKFGNGGTPELHLAGHCíiPAANYCTKFGECVKOCrt

ΞΞ22Ξα. * 0

CUCUXSER arabiogps: XVLL9IGGGaGRYE! _SS700aN7Fa0YLWNTY'_GGG9 = 7R? LG0aVL3G VKVLLSIGGGAGSYSLSSAĎOaKGV AHFI '»HSYLGGGS32RPLGAAVGXXXXXGGGGXXXXXXX

GEGGsAhL

GUCUXSER aRaSIGGPS

IDFOIESCDGRFWDSLARALAGHNNGCXTVYLSAAPCCPLPDASLSTAlA

VDFD Σ ESGSGGFWOVLaGELXNFGZ --- v τ L.SA aPGC? AND? 2AHLOA aIK

Σ37 NIELGSPGHWOĎLARTLsXFSHRGRKIYLGGAPCCPFPORL .-. GsAl-N = E22SAř! L TGLFDYVVVaFYNNPPCCYOT-SADNLLSSWNGWTT-VCANCIFLGLPAS CUCUMBEP. 7GLFDSVWVGFYNNPPC «FA0-NADNLLSSWNCWTA-F? TSXLYhCL.? AA ARABÍDCPS 7KRF0YVWICFYNNPPCSYSSGNTCNLFDSWNKWTTSIAACXFFL.CLPAA

SE223A7.L

CUCUMBER arabidg?

TDAA-GSGFIPADAL7SCVLP7IXGSAXYGGV «LWSKAY2--5GYSsAIK HEAAPSGGFIPADVLISGVLPTXXaSSNYGGVMLWSKArD - NGYSOS3K PEAA-DSGYIPPDVLTSGILPTLXKSRKYCGVMLWSXFWDDKNGYSSSIL Ό about ^1.1 mm noo (j ti) in oi '<tn υ» d><I »R« ρ in (gi least oj

- '-i -i-ill (I (1 r <f ((j _r j

Ξ Ξ 2 2 Ξ A íí!

ARA =: 33. =

SSV- 293 3ΞΣ3 292 A3V- 3C3

138

Legend to Table VI:

Conventional length: 304

Identity: 137 (45.1%)

Similarity: 106 (34.9%)

SE22SAML: shown in SEQ ID NO: 8

Cucumber: set forth in SEQ ID NO: 31

Arabidopsis: set forth in SEQ ID NO: 32

Table VI shows a comparison of the amino acid sequence corresponding to the structural gene for acidic chitinase SE and the amino acid sequence of cucumber lysozyme / chitinase (European Patent 0 392 225 and Metraux et al., 1989) and Arabidopsis thaliana lysozyme / chitinase (Samac et al., 1990). It is clear from the table that when comparing all three segments, the homology is approximately 45%. When comparing SE with cucumber lysozyme / chitinase, approximately 60% homology was observed.

Example 8

Characterization and determination of the partial amino acid sequence of beta-1,3-glucanases 3 and 4 of sugar beet

Sugar beet beta-1,3-glucanases 3 and 4 were isolated from sugar beet leaves infected with Cercospora as described above in the Materials and Methods section. These are basic proteins that have a strong affinity for beta-1,3-glucan. The amino acid composition of the sugar beet isoenzymes beta-1,3-glucanases 3 and 4 is similar to that of tobacco and barley beta-1,3-glucanases, as seen in Table VII.

39

TABLE VII

Amino acid composition of beta-1,3-glucanases from tobacco, barley and sugar beet

Amino acid Tobacco ^a) Sugar beet 3 Sugar beet 4 τ - z, b) Barley Gs Acid asparagus 35 46.4 53.4 39 Threonine 10 12.6 12.1 14 Serine 23 25.0 27.4 23 Acid glutamic 20 23.4 26.7 20 Proline 19 18.4 21.7 15 Glycine 26 27.8 32.2 31 Alanine 20 31.5 35.1 43 Cysteine 1 0 0 0.7 Valine 18 21.3 25.6 18 Methionine 7 5.1 6.6 4.8 Isoleucine 17 15.9 19.2 14.9 Leucine 23 22.7 27.0 22.1 Tyrosine 16 13.5 15.4 15.4 Phenylalanine 13 12.8 14.6 12.9 Histidine 5 3.1 1, 9 1.2 Lysine 13 12.9 16.2 9.7 Arginine 12 12.7 15.0 12.9 Tryptophan 4 NS NS NS Molecular weight (KD) 32 32.8 37.6 32 pi 9.9 9.5 9.5 9.8

140

Legend to Table VII:

^(a ) data from Shinshi H. et al., 1983

b) data from Kragn et al., 1991

NS means not determined

SDS-PAGE of beta-1,3-glucanase:

The molecular weights of beta-1,3-glucanase 3 and 4 determined on an SDS-gel with a gradient of 10-15% (Phast-System Pharmacia) were 33 and 38 kDa, respectively. The isoelectric point was equal to or greater than 9.5. In thin layer chromatography analysis, the major reaction product released from laminarin after 24 hours of incubation with two beta-1,3-glucanase isoenzymes 3 and 4 was laminarinbiose dimer, indicating that beta-1,3-glucanase isoenzymes 3 and 4 are endoglucanases.

Amino acid sequencing of beta-1,3-glucanases 3 and 4:

Purified beta-1,3-glucanases 3 and 4 were subjected to tryptic digestion using the procedure described above in the Materials and Methods section and selected peptides were further purified and sequenced as described in the Materials and Methods section and in Example 3 above. Peptides were selected based on the same criteria used in connection with the selection of chitinase 4 tryptic peptides (see Example

3). The amino acid sequences of the peptides are listed in Table VIII.

141

TABLE VIII

Amino acid sequences of tryptic peptides from beta-1,3-glucanase 3 and 4 isolated from sugar beet leaves

Pepcid 3-L5 V-V-Q-N’-N-7-V -? - Y Papcid 3-L7 (A) -G-A-P-N-V-P-I-V-V-S-E-S-G-V-P-S-A-G-G Pepcid 3-L6 L-Q-G-K-V-S Pepcid 4-25.L L-G-II-N-L -? - S-E-E-D-V-V-S-L-Y Pepcid 4-26.3 L-D-Y-A-L-P Pepcid 4-27.L Y-I-A-V-G-N-E-I-M-P-N-D-A-E-A-G-S-I-V-P-A-M-O-N (Q) - (Q) - (a) - (?) - (R) Pepciď 4-28.2 v-v-q-n-n-v-v-p-y Pepcid 4-40.i G-A-P-i-V-P-I-V-V-S-E-S-G-X-P-S-A-G-G-N-A-A-S-F

Legend to Table VIII:

Peptide 3-15 is listed in SEQ ID No. 33 Peptide 3-17 is listed in SEQ ID No. 34 Peptide 3-16 is listed in SEQ ID NO: 35 Peptide 4-25.1 consists of of amino acids C. 37 - 51 of SEQ ( ID No. 10 Peptide 4-26.3 consists of of amino acids C. 21 1 - 216 of SEQ ID No. 10 Peptide 4-27.1 consists of of amino acids C. 1 1 5 - 139 of SEQ ID

No. 10

142

Peptide 4-28.2 consists of amino acids No. 101-109 of SEQ ID No. 10

Peptide 4-40.1 consists of amino acids No. 249-272 of SEQ ID No. 10.

Example 9

Isolation and characterization of beta-1,3-glucanase cDNA 3 and 4

Oligonucleotide probes corresponding to peptides from beta-1,3-glucanase 3 and 4 polypeptides were synthesized in the same manner as described above in connection with SE. In the first round of PCR for the isolation of beta-1,3-glucanase 4, the following two sequences were used as the 5-primer:

Peptide 3-15: W, V, Q, Ν, Ν, (V) ...

G

Oligosequence TG-1: 5'tGGGT ^ CA ^ AaJaa ^ GT 3 'i C L.

c (shown in SEQ ID No. 36)

In the second round of PCR, the following sequence corresponding to the part of peptide 4-27.1 consisting of. amino acids No. 120-125 of SEQ ID No. 10

Peptide 4-27.1: ..... Ν, Ε, I, Μ, P, N ....

C

Oligosequence TG-2: 5'AA ^ GA ^ ATAATGCC ^ AA J- A C

T (shown in SEQ ID No. 37)

By comparing the amino acid sequences of barley beta-1,3-glucanases (Fincher, 1986) and tobacco (Shinshi et al.,

143

1988), a conventional sequence was selected and used to construct a 3'-primer with the following conventional sequence:

Peptide sequence: F, A, M, F, D / N, E.

G, g ca τ a

Oligosequence TG-3: 5 TC ^ T ^ AACATIGC ^ AA A IL · A (j c

(shown in SEQ ID No. 38)

This sequence was used in the second round of PCR, while primer 270 used to clone SE was used in the first round. Primer TG-1 was used to isolate beta-1,3-glucanase 3 clone, as peptide 4-28.2 is the same as peptide 3-15 (see Table VII in Example 8). This primer was used as a 5-primer for both PCR reactions. Oligonucleotides TG-3 and 270 were used as 3'-primers for the test and second rounds of PCR, respectively.

The resulting PCR products were used to screen the lambdaZAP sugar beet cDNA library described above to isolate clones containing cDNAs encoding beta-1,3-glucanase 3 and 4, respectively. The cDNA sequence and deduced amino acid sequence of beta-1,3-glucanase 4 is shown in SEQ ID No. 9 and SEQ ID No. 10, respectively.

Example 10

Serological characterization of chitinas 2 and 4 sugar beet

The serological relationship between chitinase 2 and 4 was analyzed by immunoblotting. When a protein sample containing both chitinase 2 (molecular weight 32 kDa) and 4 (molecular weight 26 kDa) was separated before immunoblotting by SDS-PAGE, the following

144 results (see Fig. 10). Anti-chitinase 4 antibodies only detect a protein with a molecular weight of approximately 26 kDa (chitinase 4), but not a protein with a molecular weight of 32 kDa (chitinase 2 isoenzyme 2), although it is also present on the same nitrocellulose membrane. In contrast, anti-chitinase 2 antibodies recognize only a protein with a molecular weight of 32 kDa (chitinase 2), but not a protein with a molecular weight of 26 kDa (chitinase 4). This proves the presence of two serologically different groups of chitinas. The observation was further documented by analysis of pure chitinase 2 and 4 antigens by immunoblotting. Anti-chitinase 4 antibodies detect only chitinase 4, while anti-chitinase 2 antibodies only recognize chitinase 2, and no cross-reactivity was observed. The above results suggest that sugar beet contains two different classes of basic chitinas. This observation is also supported by the information obtained from the amino acid sequencing and amino acid composition (see Table I in Example 3 above) of basic chitinas 2 and 4. The difference shows that the genes encoding chitinas 2 and 4 form two different gene families.

As far as the inventors are aware, the fact that there are two different classes of basic sugar beet chitinas has not yet been reported in the literature.

Definition of sugar beet chitinase class 2:

When comparing the N-terminal amino acid sequence of sugar beet chitinase 2 with the following chitinases from beans, tobacco, pea A1, pea A2, pea B (Vad et al., 1991), barley T (Jacobsen et al., 1990) and barley K ( Kragh et al., 1990), strong homology was observed between these basic chitinases (see Table IX), suggesting that these chitinases belong to the same class of chitinases. This was further demonstrated by serological cross-reactivity using antibodies raised against sugar beet chitinase 2.

145

This antibody recognized not only sugar beet chitinase 2, but also chitinase P (27.5 kDa), Q (28.5 kDa), Ch. 32 and Ch. 34 from tobacco (Bol and Linhorst, 1,990), chitinases Τ, K and C from barley and chitinase A1, A2 and B from peas. When antibodies raised against barley chitinase K or wheat germ chitinase were used, similar serological cross-reactivity was observed. The chitinases described above have therefore been defined as belonging to the class of chitinases serologically related to sugar beet chitinase 2, for example the class of sugar beet chitinase 2 chitinases.

TABLE IX

N-terminal amino acid sequence of chitinase isoenzymes belonging to sugar beet chitinase class 2

Chitinasa 2 ELCGNQAGGALCPNGLCCSQYGWCGNTNPYCGN Beans EQCGRQAGGALCPGGNCCSQFGWCGSTTDYCGP Tobacco EQCGSQAGGARCASGLCCSKFGW Peas B EQCGRQAGGATCPNNLCCSQYGY Peas A1 EQCGNQAGGXVPPNG Peas A2 EQCGTQAGGALCPGGL Barley K EQXGSQAGGATCPNXLCCSRFG Barley T XQQGSQAGGATCPNXLCCSXFGW

Legend to Table IX:

The N-terminal sequence of chitinase 2 is shown in SEQ ID No. 27 Bean: The N-terminal sequence of bean chitinase, consisting of amino acids No. 1-33 in SEQ ID No. 25 Tobacco: The N-terminal sequence of tobacco chitinase, consisting of amino acids No. 1-23 in SEQ ID NO: 26 Pea B: The N-terminal sequence of pea chitinase B is shown in SEQ ID NO: 41

46

AI games: N-terminal sequence chitinase AI peas, is stated in SEQ ID No. 42 Peas A2: N-terminal sequence chitinase A2 peas, is stated in SEQ ID No. 43 Barley K: N-terminal sequence chitinase TO barley, is stated in SEQ ID No. 44 Barley T: N-terminal sequence chitinase T barley, is stated in SEQ ID No. 45. 9 Definition of chitinase class 4 sugar beet:

Using antibodies raised against sugar beet chitinase 4, none of the chitinases of the chitinase 2 class described above were recognized. Chitinase 4 of sugar beet therefore belongs to a new class of chitinas not yet identified in plant species other than sugar beet. However, current research has suggested that chitinases belonging to the same new class occur in rapeseed. Thus, protein extracts from rapeseed obtained in a manner similar to that described above for sugar beet chitinases 4 were reacted with the above polyclonal antibodies against sugar beet chitinase 4 (see Rasmussen et al., 1992).

Example 11

Examination of homology between cDNA of chitinase 4 and DNA of other chitinases using hybridization techniques

In addition to examining homology between mature enzymes, homology between cDNA encoding the chitinase 4 enzyme and DNA encoding other chitinase enzymes was examined using the hybridization technique described above in Material and Methods entitled Identification of DNA Belonging to the Chitinase 4 Gene Family.

It can be seen from Figure 11 that when examined at 55 ° C

147 is a very low degree of homology between cDNA encoding the sugar beet chitinase 4 enzyme and DNA encoding chitinases from other plants such as peas, tobacco and beans, as well as DNA from sugar beet encoding the chitinase 1 and SE enzymes. Thus, these results further suggest that the enzyme chitinase 4 belongs to a new class of chitinases.

The high degree of homology between the cDNA encoding the chitinase 4 enzyme and the DNA encoding the rapeseed chitinase demonstrated by the high degree of DNA hybridization further indicates that the genes encoding the sugar beet chitinase 4 and the genes encoding the rapeseed chitinase are substantially homologous and therefore belong to the same gene class, which is supported by the results reported in Example 10, which show a high degree of serological homology between mature enzymes from the two plants.

Example 12

Transformation of bacterial cells

Agrobacterium tumefaciens (strain LBA 4404, Ooms et al., 1982) was transformed with the plant transformation vector pBKL4K4, the preparation of which is described in Example 18, using the freeze / thaw method as essentially described by An a. coll. (1988). The bacteria to be transformed were cultured in LB-medium, pH 7.4, overnight at 28 ° C and 280 rpm before the freeze / thaw procedure. The next day, the bacteria were subcultured in 50 ml of LB-medium, pH 7.4, and grown for approximately 4 hours until Οθ ₆₀₀ reached 0.5 1.0. The culture was cooled on ice and centrifuged for 5 minutes at 10,000 g and 4 ° C. The supernatant was discarded and the bacteria were carefully suspended in 1 ml of 20 mM calcium chloride at ice temperature. 0.1 ml of the bacterial suspension was pipetted into ice-cryo-tubes

148 and bacteria were frozen in liquid nitrogen and stored at -80 ° C.

For bacterial transformation, 1 .mu.g of plasmid DNA was first added to cryo-tubes with frozen bacteria. The bacteria were incubated for 5 minutes in a 37 ° C water bath, 1 ml of LB-medium, pH 7.4 was added to the cryo-tubes and the mixture was incubated for 4 hours at room temperature with gentle agitation (on a shaker, 100 rotations per minute). The cryo-tube was centrifuged for 30 seconds at 10,000 g and 4 ° C. The supernatant was removed and the bacteria were resuspended in 0.1 ml of LB-medium, pH 7.4. The bacteria were plated (YMB) with 50 mg / L kanamycin and incubated for 2-4 days at 28 ° C until colonies appeared. The presence of suitable plasmids in the bacteria was verified before using the bacteria for plant transformation by restriction analysis of the extracted plasmids.

In a similar manner, bacterial transformation can be performed with other genetic constructs of the invention, for example, as shown in Figures 17, 18, 19 and 22 and explained in Example 18.

Example 13

Preparation of genetically transformed tobacco plants (Nicotiana benthamiana and Nicotiana tabacum)

Plant material:

Leaves from plants to be genetically transformed were obtained from plants grown in vitro or in vivo. In the latter case, the leaves were sterilized before transformation. Sterilization was performed by placing the leaves for 20 minutes in a 5% calcium hypochlorite solution containing 0.1 ml of Tween surfactant.

149 to 1 followed by five washes in sterilized water. In vitro plants were grown in pots on 1/2 shoot-inducing medium (1/2 MS) (Murashige and Skoog, 1962).

The leaves were placed in 14 cm diameter petri dishes, and then cut into squares approximately 1 cm in area, all sides of which were formed by dissected tissue. All cut tissue that was decolorized by calcium hypochlorite sterilization was removed.

Bacterial culture:

hours before transformation, a suspension of Agrobacterium transformed as described above was established by inoculating 2-3 ml of medium with appropriate antibiotics transformed Agrobacterium. The bacteria were grown at 28 ° C with stirring (300 rpm).

Transformation:

Plant transformation was performed essentially as described by R.B. Horsch et al. (1986). The bacterial culture was diluted 50-fold with 1/10 MS immediately before transformation. Approximately 10 ml of the diluted bacterial suspension was poured into a 9 cm diameter petri dish, and pieces of leaves were immersed in this suspension for approximately 15 minutes. The leaf pieces were then removed and excess bacterial suspension was removed with sterile filter paper.

Co-cultivation:

The day before transformation, co-culture Petri dishes containing 1/10 MS medium were coated with acetosyringone (200 μM). On the day of transformation, a sheet of sterile filter paper and pieces of leaves were placed on the co-culture dishes.

50, which had previously been immersed in the bacterial suspension, were placed upside down on the filter paper. Pieces of leaves were incubated in a growth chamber under low light, for example 12 hours of light and 12 hours of darkness, for 2-3 days.

Selection and regeneration:

Pieces of leaves were transferred to petri dishes containing MS-medium inducing shoot formation with 300 mg / l kanamycin and 800 mg / l carbenicillin and subcultured in the same medium every four weeks.

Shoots that appeared on plates with MS-medium inducing shoot formation of 300 k / c were transferred to containers with 1/2 MSO 300 k / c. Shoots were subcultured when needed. After approximately two weeks, the expression of beta-glucuronidase activity was determined on the leaf tips of the green shoots using the GUS test (see Materials and Methods).

Planting:

Genetically transformed shoots formed roots and the resulting plants, which were GUS-positive, were planted in a growth chamber in water-saturated compost. They were then covered with plastic bags and grown for about one week, and then the two corners of the plastic bags were cut. After another week, the plastic bags were removed.

Example 14

Preparation of genetically transformed sugar beet plants by transformation using bacteria

Transformation was performed using uterine

151 explants as described below. The seeds were germinated for 4 days in the dark on a substrate containing 0.7 g / l agarose and 2 g / l sucrose. The seedlings were then transferred to a vessel (Nunc) containing 1/2 x MS-substrate and cultured under light for 3 days. The uteri were removed from the seedlings and the uterine explants were rubbed on the petiole with a small brush containing a suspension (Οϋθ ^ θ - 1/0) of Agrobacterium transformed as described above in Example 12. The uteri were then co-cultured for 4 days on a substrate containing 1 / 10 MS-substrate. Transformed explants were transferred to MS-substrate supplemented with 0.25 mg / l BAP, 400 mg / l kanamycin, 800 mg / l carbenicillin and 500 mg / ml cefotaxime and incubated on this substrate for 14 days. The regenerated shoots were then transferred to vessels containing MS-substrate containing 0.25 mg / l BAP, 400 mg / l kanamycin and 800 mg / l carbenicillin as substrate. The isolated shoots were transferred to fresh substrate at four week intervals for selection and propagation. Selected shoots were rooted on 1/2 MS-substrate containing 1 mg / l IBA.

Tobacco tissue was transformed with a genetic construct containing chitinase 1, chitinase 4, chitinase 76 and acidic chitinase SE and selective markers NPT-II and GUS. Selection of callus and shoots on kanamycin showed that the obtained tissue expresses the GUS marker and that therefore transformation took place.

Example 15

Analysis of chitinase and beta-1,3-glucanase in transgenic plants

The level of expression of chitinase and beta-1,3-glucanase isoenzymes can be determined either by measuring total enzyme activity by two radiochemical assays, by measuring antifungal

152 activity using biological procedures I-III or by measuring the level of various isoenzymes by immunoblotting using specific antibodies, all of which were described above in the Materials and Methods section. The final test of the resulting transgenic plants is the analysis of the degree of their resistance to phytopathogenic fungi using the infectious system described in the Materials and Methods section.

Using biological methods I - III, the antifungal activity of enzymes in genetically transformed plants can be determined. The retardation of the growth of fungal hyphae indicates that the transformation has created a plant that has improved tolerance, i.e. increased antifungal activity against phytopathogenic fungi compared to an untransformed plant.

In radiochemical tests, 1H-chitin and 1H-laminarin are used as substrates for chitinase and beta-1,3-glucanase, respectively. Using standard product formation curves versus enzyme amount, both chitinase and beta-1,3-glucanase activity can be determined in crude plant extracts. This is further evidenced by a time test in which the level of chitinase (Fig. 12A) or beta-1,3-glucanase (Fig. 12B) in sugar beet leaves is determined at specified time intervals after Cercospora beticola infection. Although the level of both chitinase and beta-1,3-glucanase enzyme in the control plant is very low, it is easily determined by very sensitive radiochemical techniques. In infected plants, increased production of both enzymes was observed for the first time 8-9 days after infection with the fungal pathogen.

Using these techniques, the constitutive level of both chitinase and beta-1,3-glucanase in transgenic plants can be easily determined.

However, these techniques do not differentiate between different chitinase and beta-1,3-glucanase isoenzymes. May be

153 determined only the total enzyme activity of all chitinase or all beta-1,3-glucanase isoenzymes. However, the presence of different chitinase and beta-1,3-glucanase isoenzymes can be easily determined separately by analysis of crude protein extracts by immunoblotting after separation by SDS-PAGE.

The anti-beta-1,3-glucanase 3 antibody recognized only a single protein in the leaf material infected with Cercospora (Fig. 13). In contrast, no antigen was detected in the control sheets. This is consistent with the low constitutive level of beta-1,3-glucanase expression observed in control plants using a radiochemical assay. When antibodies raised against either chitinase 2 or 4 were used, two major protein bands were generated in infected leaf tissues. Anti-chitinase 2 antibodies detected the 26 and 32 kDa bands, while proteins having molecular weights of 29 and 26 kDa were detected by the anti-chitinase 4 antibody. Analysis of purified chitinases by SDS-PAGE and immunoblotting showed that the protein bands recognized by anti-chitinase 2 antibodies were chitinases. 1 (26 kDa) and chitinase 2 (32 kDa), respectively. Similarly, anti-chitinase 4 antibodies detected authentic chitinase antigen (26 kDa), but also SE antigen (29 kDa), which was unexpected as no homology of the amino acid sequences of chitinase 4 and SE was observed (see SEQ ID NO: 2 and SEQ ID NO: 2). No. 8). The spatial structure of chitinase 4 and SE on the nitrocellulose membrane may be sufficient for epitope recognition to allow antigen-antibody interaction between SE antigen and anti-chitinase 4 antibody. The reaction between SE antigen and anti-chitinase 4 antibody was evident only at 1: 100 dilution or 1: 200. A much weaker reaction was observed when the antibodies were diluted 1: 5000 or 1: 10000.

Transgenic tobacco plants (Nicotiana tabacum and / or Nicotiana benthamiana) were transformed with either chitinase

154

4, chitinase 76, acid chitinase or chitinase 4 together with acid chitinase. After kanamycin selection and regeneration, the transformed plants were examined for i) GUS activity, ii) chitinase gene expression, and iii) degree of resistance to C. nicotiana and R. solani. Transgenic plants expressed GUS-activity in various amounts. Only plants with high GUS activity were subjected to further analyzes. The expression of chitinase gene products was analyzed by immunoblotting using the ECL system described in Materials and Methods and antibodies raised against cnitinase 4. In Nicotiana benthamiana leaf extracts transformed with NPT and GUS only, no protein band was detected with this antibody (see Figure 23, lane C). Transgenic plants containing the acid chitinase, genomic chitinase 76, chitinase 4 and double chitinase 4 + acid chitinase gene constructs showed a strong positive reaction (see lanes SE, K76, K4, K4 + SE). To evaluate the expression level, 10 pg of chitinase 4 isolated from sugar beet was also included in the experiment (Std pathway in Fig. 23).

A broad protein band was observed in extracts from transgenic plants with chitinase 4 or chitinase 76 gene constructs. If smaller amounts of protein were applied to the different SDS-PAGE lanes, this band could be divided into three different protein bands having molecular weights of 29, 26 and 25 kD, respectively. The reasons for creating triple bands are not yet known. However, it is assumed that chitinase 4 i) is unmodified, resulting in a protein that retains the signal peptide and forms a band with a molecular weight of 29 kD, ii) is cleaved at the normal site of modification in the amino acid sequence Leu Val Val Ala - Gin Asn Cys chitinase 4 (amino acids at positions 23-24 in SEQ ID NO: 2), which forms a protein band with a molecular weight of 26 kD, and iii) has a second apparent tobacco modification site in the amino acid sequence Ala Ser Ala Ser - Cys Ala (position 85-86 in SEQ

55

ID No. 2), and this cleavage site results in a polypeptide band with a molecular weight of 25 kD. In addition to the malformation of sugar beet chitinase 4 in transgenic tobacco, translocation of chitinase 4 was inhibited. In sugar beet, this basic chitinase 4 is stored in the extracellular space. In transgenic tobacco, cytochemical analyzes have clearly shown that sugar beet chitinase 4 is localized intracellularly.

Preliminary experiments were performed to investigate the degree of resistance of transgenic tobacco plants to R. solani and C. nicotiana. Transgenic plants with chitinase 4 (10 plants) and chitinase 4 + acid chitinase (4 plants) showed fewer symptoms of the disease, while control plants (10 plants) containing the GUS and NPT genes were seriously infected with C. nicotiana.

When germinating Nicotiana tabacum seeds containing the chitinase 4 gene construct in soil infected with Rhizoctonia solani, improved survival and growth were observed compared to seeds of non-transgenic plants.

Example 16

Modification of sugar beet chitinase 4 by site-directed mutagenesis

Site-directed mutagenesis on a DNA sequence encoding sugar beet chitinase 4, for example the chitinase 4 gene, can be performed using a PCR reaction (as described in PCR) used in the construction of genetic constructs of the invention and site-directed mutagenesis based on clones of cloned DNA) using specific 3- and 5-primers for each site-directed mutagenesis. The choice of specific 3'- and S'-primers to be used depends on the position in the DNA sequence where the modification is to be performed.

56

Typically, suitable amino acids to be modified, either by substitution, deletion or insertion, are selected based on amino acid sequence analysis of the mature chitinase 4 enzyme, optionally in combination with analysis of the three-dimensional structure of the enzyme. Of particular interest in this context are amino acids forming part of the active site of the enzyme or its epitopes, as well as amino acids important for substrate specificity and substrate binding.

Active site of chitinase 4 sugar beet:

The position of the essential amino acid residues that are part of the active site of chitinase 4 was preliminarily identified by the following observations. First, current studies of barley chitinase C have shown that chemical modification with carbodiimide and N-bromosuccinimide (NBS) completely inhibits enzymatic activity (results not shown). Similar experiments with glucoamylase from Aspergillus niger (Sierks et al., 1990) elucidated the way in which carbodiimide and N-bromosuccinimide inactivate this enzyme. Carbodiimide is covalently attached to three essential acidic groups (glutamic and aspartic acid residues) forming the glucoamylase catalytic site. N-bromosuccinimide oxidizes tryptophan residues important for either stabilization of the catalysis transition stage intermediate or tryptophan residues involved in substrate binding outside the catalytic site. Experiments with chitinase C suggest that three acidic residues and two tryptophan residues are very important components of the active site. Second, compared to the active sites of other enzymes that hydrolyze oligosaccharide chains, including the glucoamylase described above, the active site of chitinase 4 is believed to consist of amino acid residues 183 (Asp) and 189 (Glu) in SEQ ID NO. 1 (corresponding to amino acid residues 184 and 190 in the amino acid sequence encoded by genomic chitinase 4. The numbers in parentheses indicate the amino acid number from the corresponding amino acid sequence encoded by genomic chitinase 4). In contrast, barley chitinase C and all other plant chitinases of the same serological class (sugar beet chitinase class 2) have three aspartic acid residues (corresponding to amino acid residues 183, 189 and 194 of chitinase 4 (SEQ ID

No. 2)) in the active site (184, 190 and 195, respectively). The location of two important tryptophan residues that are part of the active site of chitinase C has not yet been elucidated. Because chitinase 4 contains only three tryptophan residues as opposed to the six residues present in chitinase C, important tryptophan residues can be more easily identified in the case of chitinase 4.

Two acidic residues 183 (Asp) and 189 (Glu) in SEQ ID NO: 2 (184 and 190, respectively) forming the chitinase active site are contained in peptides 4-22: SIGFDGLNAPETVANNAVTAFR. Important tryptophan residues of the active site may be included in peptides 4-19.3: GPLQITW and peptides 4-26: TAFWFWMNNVHSVIVNGQGFGASI.

The active site of chitinase 4 differs from the active sites of other plant chitinases, for example from tobacco, which has the following corresponding amino acid sequences AIGVDLLNNPDLVATDPV, which is set forth in SEQ ID NO: 46, GPIQISH, which is set forth in SEQ ID NO: 47 and SALWFWMTPQSP, which is set forth in SEQ ID NO: 48 (Shinshi et al., 1987), and it would be interesting to focus on specific amino acid residues of chitinase 4 that differ from the corresponding amino acid residues of tobacco chitinase in order to obtain additional information about the active site and possibly identify suitable modifications that would result in improved properties of the modified enzyme. In this respect, acidic amino acid residues and tryptophan residues are believed to be of particular interest.

158

Accordingly, an interesting modification is a modification in which glutamic acid at position 189 (190) is substituted with asparagine and / or aspartic acid at position 183 (184) is substituted with glutamine. It is believed that changing the carboxyl groups of aspartic acid at position 183 (184) to asparagine and glutamic acid at position 189 (190) to glutamine in chitinase 4 will in itself have a negative effect on enzymatic activity, but it is expected that thus gaining further knowledge about the mode of action of the enzyme chitinase 4.

Substitution of tryptophan at positions 169, 204 and 206 (170,

205 and 207, respectively) to tyrosine may alter the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity. The indicated substitutions mentioned above serve only as examples and many changes can be deduced to achieve a more effective antifungal chitinase. They can be performed by site-directed mutagenesis, for example, using the procedure described below.

Site-directed mutagenesis:

For all PCR reactions indicated herein, the primers are selected to either contain restriction sites themselves or to be located near the restriction sites in a manner that allows the PCR product to be exchanged with the corresponding sequence in the gene by restriction enzyme digestion followed by ligation of appropriate fragments.

The 5-primer used in the following examples is designated SD 0 (see Fig. 14). The number in parentheses indicates the order of the corresponding amino acid residue encoded by the genomic chitinase 4 DNA sequence.

If tryptophan at position 169 (170) of the amino acid sequence of chitinase 4 is to be substituted with the amino acid tyrosine, this can be done as follows:

The 3-primer SD1 was used for the PCR reaction (see Fig.

159

14).

The resulting PCR product (from bp 301 to 538) was digested with BamHI and PvuII and exchanged with the corresponding fragment of the chitinase 4 gene using conventional procedures (Sambrook et al., 1990).

If glutamic acid at position 189 (190) is to be substituted with the amino acid glutamine, the 3-primer SD2 is used (see Figure 14).

If aspartic acid at position 1 83 (184) is to be substituted with the amino acid asparagine, the 3'-primer SD3 is used (see Figure 14).

The PCR products were digested with BamHI and BspMII and exchanged with the BamHI-BspMII fragment of the chitinase 4 gene in a similar manner.

like 169 (170). described above for tryptophan exchange in position If he has be tryptophan in position 206 (207) substitute amino acid tyrosine, 3-primer SD4 is used (see Fig. 14). If he has be tryptophan in position 204 (205) substitute

amino acid tyrosine, the 3-primer SD5 is used (see Figure 14).

The PCR products were digested with BamHI and BalI and exchanged with the BamHI-BalI fragment in the chitinase 4 gene as described above.

Other desired modifications may be made in a similar manner.

Example 17

Construction of genetic constructs with suitable C-terminal extension

The C-terminal amino acid sequences found in association with various plant chitinases and glucanases are exemplified in the specification and are believed to be useful in modifying one or more antifungal enzymes encoded by the genetic constructs of the invention that do not contain a C-terminal extension. in order to allow the translocation of these enzymes into the vacuole.

The C-terminal extension may be introduced into DNA sequences encoding one or more antifungal proteins of the invention by any suitable technique, such as PCR.

Giant. 15A shows a sugar beet beta-1,3-glucanase cDNA with a C-terminal tobacco extension, which is underlined in the figure.

Giant. 15B, C show PCR primers that can be used to change the termination codon and introduce a portion of the C-terminal extension. The Dral site is created at the 3-end.

Giant. 15D shows four thermally hybridized synthetic oligonucleotides containing the last part of the C-terminal extension, a termination codon, a SmaI site, and an EcoRI site.

The C-terminal extension can be introduced by exchanging the XbaI-EcoRI fragment in the beta-1,3-glucanase gene for a PCR product digested with XbaI and Dral and thermally hybridized synthetic oligonucleotides digested with SmaI and EcoRI, using conventional procedures (Sambrook et al., 1990). .

Giant. 16A shows the chitinase 4 gene with the C-terminal extension of tobacco, which is underlined in the figure.

Giant. 16B, C show PCR primers that can be used to introduce a SmaI site near the termination codon of the chitinase 4 gene.

Giant. 16D shows four thermally hybridized synthetic oligonucleotides containing a C-terminal extension sequence, an altered termination codon, a SmaI site, and a SmaI site.

EcoRI.

161

The C-terminal extension can be introduced by exchanging the BamHI-EcoRI fragment for a PCR product digested with BamHI and SmaI and thermally hybridized synthetic nucleotides digested with SmaI and EcoRI using conventional procedures.

Additional C-terminal sequences, similar to those described above, may be joined to the sequences of chitinase 76, chitinase 4, SE and beta-1,3-glucanase. The N-terminal sequence can be exchanged for other N-terminal sequences in a similar manner. Of particular interest may be the N-terminal sequence of chitinase 1 set forth in SEQ ID NO: 12, the N-terminal sequence of acid chitinase SE set forth in SEQ ID NO: 8, the N-terminal sequence of chitinase 4 set forth in SEQ ID NO: 2, the N-terminal the chitinase 76 sequence set forth in SEQ ID NO: 6 and the beta-1,3-glucanase N-terminal sequence set forth in SEQ ID NO: 10. Other interesting N-terminal sequences of the mature protein may be the sequences set forth in Table III or Table IX, or the proline-rich region of sugar beet chitinase 1 set forth in SEQ ID NO: 1.

Example 18

Genetic constructs

Excised recombinant pBluescript containing the cDNA gene of chitinase 4 (chitinase 4 B15) was subcloned to provide the gene with a 35S enhanced promoter and a 35S terminator. This construct was transferred into the pBKL4 plant transformation vector containing the NPTII and GUS genes. pBKL4 is a derivative of the Ti-plasmid pBI121 of Agrobacterium tumefaciens (Bevan et al., 1984), in which the genes between the left and right borders were exchanged for the following genes: 1) beta-glucuronidase (GUS) from Escherichia coli equipped with the CaMV 35S promoter and nopalin terminator -synthase (NOS) 2) neomycin phosphotransferase (NPT) from Escherichia coli equipped with the CaMV 35S promoter and the octopin162 terminator

-synthase (OCS).

More specifically, a PCR amplification reaction was performed to introduce an ATG site, a ribosome binding site, and two restriction sites (HindIII and BglII) 5 to the DNA sequence.

KB3 oligonucleotide (shown in SEQ ID NO: 49):

( ⁵ CCGAAGCTTAGATCTAAACAACAACATGTCTTCT (£) T (T) GGACC ³ )

Oligonucleotide KB3, containing two restriction sites, a ribosome binding site, ATG (underlined) and the first fifteen nucleotides of clone B15 chit 4 (nucleotides 8 and 10 were mixed nucleotides ( _c ), because primer KB3 was also used for clone chit 76), was used as 5'-PCR primer and oligonucleotide KB4

Oligonucleotide KB4 (shown in SEQ ID NO: 50):

( ⁵ GCACACGTAGCGCTAGCTTGG ³ ) ** |

261 NheI 241 was used as a 3'-PCR primer (nucleotides 255 and 256 were altered to destroy the second NheI site).

The PCR product was extracted twice with phenol and twice with chloroform and precipitated with ethanol. After resuspension in water, the DNA was digested with HindIII and NheI. The HindIII-NheI fragment from pB155 chit 4 was exchanged for the HindIII-NheI PCR fragment (Fig. 17).

The construct was sequenced using scintillation

163 of primer T7 (corresponding to the pBluescript promoter T7) and primer 340 (shown in SEQ ID NO: 51):

340: (CATCGGAGGATCCACTACC)

341 323

The sequence confirmed that the entire sequence exchanged was correct. Furthermore, in the 5'-sequence, the original nucleotide 8 was T and nucleotide 10 was C as in clone pB15 chit4, and both NheI sites at positions 245 and 251 were still present.

The construct was digested with EcoRI and the loading reaction was performed with Klenow enzyme in the presence of dATP and TTP, and after removal of Klenow enzyme, the construct was further digested with BglII. The BglII-EcoRI DNA fragment containing the complete chitinase 4 sequence was cloned into the pPS48 vector containing the amplified 35S promoter and 35S terminator. The chitinase 4 gene was inserted in the correct orientation by digestion of the vector pPS48 with BamHI and SmaI (Fig. 17). The chitinase 4 gene with an enhanced 35S promoter and 35S terminator was transferred to the plant transformation vector pBKL4 (Fig. 17) as a HindIII fragment (Fig. 17). The resulting vector, pBKL4K4 contained in E. coli DH5alpha, was deposited with the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmBH, Mascheroder Weg 1b. D-3300 Braunschweig (DSM)) on 30 July 1991 under the provisions of the Budapest Treaty under number DSM 6635.

The SE gene was then introduced into the pBKL4K4 construct (Fig.

17). The full-length SE gene was constructed by combining the 5-terminus of the gene from clone pSurl (EcoRI-KpnI) with the remainder of the gene from pSE22 (KpnI-HindIII) in the cloning vector pUC19 (Fig. 1).

18). The SE gene was subcloned into the SmaI site of pPS48 as an EcoRI-HindIII fragment filled in with Klenow polymerase at all four nucleotides. The orientation of the gene relative to the enhanced 35S promoter and 35S terminator was examined by restriction enzyme analysis and further confirmed by sequencing analysis.

The SE gene with the enhanced 35S promoter and 35S terminator was cloned into the KpnI site of pBKL4K4 as a HindIII fragment, in the presence of the HindIII-KpnI adapter (Fig. 18). The HindIII fragment was further cloned into the HindIII site of p3KL4.

Similar to chitinase 4, the chitinase 76 gene was cloned into pBKL4 (Fig. 19).

In a similar manner, a glucanase gene can be introduced into the pBKL4, pBKL4K4, pBKL4KSE or pBKL.4K4KSE construct (Fig. 22).

The full-length cDNA clone (SEQ ID NO: 9) was digested with EcoRI and BglII, and the sticky ends were supplemented with Klenow polymerase in the presence of all four dNTPs. The glucanase gene was then subcloned into the SmaI site of pPS48Mod. The orientation of the gene relative to the enhanced 35S promoter and 35S terminator, respectively, can be determined by restriction enzyme analysis and further confirmed by sequencing analysis.

The glucanase gene with an enhanced 35S promoter and a 35S terminator is cloned into the EcoRI site of pBKL4, pBKL4K4, pBKL4KSE, PBKL4K4KSE.

165

Sequence overview

Information about SEQ ID No. 1:

(i) sequence characteristics:

(A) length: 966 base pairs (B) type: nucleic acid (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova (F) tissue type: leaf (vii) immediate source:

(A) library: lambdaZAP beet cDNA library (B) clone: cDNA-clone B15 chitinase 4 (ix) properties:

(A) name / key: CDS (B) location: 2..793 (xi) sequence representation: SEQ ID NO: 1:

G TCT TCT TTC GGA CCA ATC ΊΊΤ GCC ΑΤΆ CIC ATG GCA CIT GCT TCT Ser Ser Fhe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala Cys 15 10 15

ATG TCA AGC ACC CIA GIT GTC GCT CAA AAC TCT GGA TCT GCC TCT AAT

Met Ser Ser Ihr Leu Val Val Ala Gin Asn Cys Gly Cys Ala Ser Asn

25 30

TTA TCT TCT AGC CGA ITT GGT TTC TCT GGC TCC ACA GAC GCC TAC TCC

Leu Cys Cys Ser Arg Fhe Gly Phe Cys Gly Ser Ihr Asp Ala Tyr Cys

40 45 sugar

142

6 6

GGC GAG GGG TGC AGA GAA GCT CCT TCT AGA TCA CCG TCT ACT GCT GCT 190 Gly Glu Gly 50 Cys Arg Glu Gly Pro Cys Arg Ser Pro Ser Ser Gly Gly 55 60 GCT TCC GTC TCG ACT TTG GTG ACC GAT GCG TTC TTT AAT AGG ATC ATT 233 Gly Ser Wall Ser Ser Leu Wall Thr Asp Ala Phe Phe Asn Arg Ile Ile 65 70 75 AAC CAA GCT AGC GCT AGC TCT GCT GCT AAG AGA TTC TRAY ACC AGG GCT 236 Asn Gin Ala Ser Ala Ser Cys Ala Gly Lys Arg Fhe Tyr Thr Arg Ala 80 85 90 95 GCC TCT TTG ACT GCT CTC AGA TTT MELT CCC CAG TTT GCT ACT GGA TCC 334 Ala Phe Leu Ser Ala Leu Arg Phe Tyr For Gin Phe Gly Ser Gly Ser 100 105 110 TCC GAT CTC GIT AGG CCT GAA CTT GCT GCA TTC TTT GCC CAT CTC ACC 382 Ser Asp Wall Wall Arg Arg Glu Wall Ala Ala Phe Phe Ala His Wall Thr 115 120 125 CAT GAA ACT GGA CAT TTT TGC TRAY AIA GAG GAG ATT GCA AAG TCA ACC 430 His Glu Thr Gly His Phe cys Tyr Ile Glu Glu Ile Ala Light Ser Thr 130 135 140 MELT TCT CAG TCA ACT GCA GCA ΊΊΤ CCA TGC AAC CCA ACT AAG CAA TAC 478 Tyr Cys Gin Ser Ser Ala Ala Fhe For Cys Asn For Ser Light Gin Tyr 145 150 155 TRAY GGA AGG GGG CCT CTT CAG ATC ACA TGG AAT MELT AAC TRAY ATA CCA 526 Tyr Gly Arg Gly For Leu Gin Ile Thr irp Asn Tyr Asn Tyr Ile For 160 165 170 175 GCT GCT CGA AGC ATT GGA ITT GAT CCT CTG AAT GCA CCA GAA ACA CTT 574 Ala Gly Arg Ser Ile Gly Phe Asp Gly Leu Asn Ala For Glu Thr Wall 180 185 190 GCC AAC AAT GCC GTG ACT GCA TTC CGG ACA GCC TTC TGG TTT TGG ATC 622 Ala Asn Asn Ala Wall Thr Ala Phe Arg Thr Ala Phe Tro Phe Trp Sword 155 200 205 AAC AAT CTC CAC TCT CTT CAA G \ jG -ττ »γ ~ GGG GCC ACC 670 Asn Asn Wall His Ser Wall Ile Wall Asn Gly Gin Gly C-lv Ala Ser 210 215 220 ATT CGA GCT ATC AAT GGA ATC GAA TCT i Τ ' GCT AAC TCT GCT 713 Ile Arg Ala Ile Asn Gly Ile Glu Cys Asn C-iy Gly . ”J3l 4 Ser Ala Ala 225 220 235 GCT ACT C-CT CCT CTT G ^ J <X TRAY MELT A.CT CAC- MELT -TV »·· .'Her. CAA CAG C?*' G ^ jG 7čo Wall Thr Ala Wall Gly Tyr ./4. Her Gin Tvr · <= Gin Gin Leu Gi'Z 240 245 250 255

167

GIT TCG CCA GGG AAT AAC CTC CGT TC-C TA.GTCAAATG GCTCGTTTTC Val Ser Pro Gly Asn Asn Leu Arg C / s

250

CTGGTCAGAA TTCAOAGGC TTA.GTCAAAA GAAAATAAAG AGAATTATGT AAACTG7TCA. TITCTCATGr AACTTGCTAC TTTGGACAAG CATTAAGTTG GTTACGAGGC TTTÁTCCA.ZA AAGGAATCAA AAATATTATT TAAAAAAAAA. AAA.

Information about SEQ ID No. 2:

(i) sequence characteristics:

(A) length: 264 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein

(xi) sequence representation: SEQ ID No. 2: Ser Phe Gly For Ile Phe Ala Ile Leu Met Ala Leu Ala Czs Met 1 5 10 15 What*· M ^ iii - Ser Thr Leu Wall Wall Ala GLn Asn Cys Gly Cys Ala Ser Asn Leu 20 25 30 Cys Cys Ser Arg Phe Gly Phe Cys Gly Ser Thr Asp Ala Tyr Cys Gly 35 40 45 Glu Gly Cys Arg Glu Gly For Cys Arg Ser Fro Ser Gly Gly Gly 50 55 50 Ser Val Ser Ser Leu Wall Thr Asp Ala Phe Asn Arg Ile Ile Asn 55 70 75 30 C-ln Ala Ser Ala Ser Cys Ala Gly Light Arg > _s Tvt Arg Ala Ala 35 90 95 Phe Leu Ser A_La Leu Arg Phe Tyt For Gin Phe Gly Ser Gly Ser Ser 100 105 110 Asp Val Val Am Arg Glu Wall Ala ALst Val Thr His

168

115 120 125 Glu Thr Gly His Fhe Cys 130 Tyr Ile Glu Glu Ile Ala 135 140 Lys Ser Thr Tyr Cys 145 Gin Ser Ser Ada Ada 150 Phe Fro Cys Asn Pro Ser 155 Lys Gin Tyr Tyr 160 Gly Arg Gly Pro Leu Gin 165 Ile Thr 'Trp Asn Tyr Asn 170 Tyr Ile Fro Ada 175 Gly Arg Ser Ile Gly Fhe 130 Asp Gly Leu Asn Ala Pro 135 Glu Thr Val Ala 190 Asn Asn Ada Val Thr Ada 195 Fhe Arg Thr Ada Fhe Tro 200 Fhe Trp Met Asn 205 Asn Val His Ser Val Ile 210 Val Asn Gly Gin Gly Fhe 215 220 Gly Ala Ser Ile Arg 225 Ala Ile Asn Gly Ile 230 Glu Cys Asn Gly Gly Asn 235 Ser Ada Ada Val 240 Thr Ala Arg Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin Leu Gly Val

245 250 255

Ser Pro Gly Asn Asn Leu Arg Cys 260

Information about SEQ ID No. 3:

(i) sequence characteristics:

(A) length: 691 base pairs (B) type: nucleic acid (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova (F) tissue type: leaf

169 (vii) immediate source:

(A) library: EMBL3 sugar beet genomic library (B) clone: chitinase 4 (ix) genomic clone properties:

(A) name / key: CDS (B) location: 356..691 (xi) sequence representation: SEQ ID NO: 3:

AA.GCITATTG TCCAAA.IAAT THACATAAC

ΑΣλΓΑΊΑΤΑΑ CAAAGGíITC TTCCnTCAT

AAATCATTGT CCAAAATAAA ATTAAATGIG

TGTATCTAAA ATATCICCAT TCCCATCITA

AACTAGTAAA CATTTCCTCA AAjGIACCA.CC

AGTGTCTAGT TTCCICATCC CATACATTAT

TTCITTC3GA CCAATCITTC CCATACTCAT

TGTGGCICAA AACTGTGGAT GIGCCICTAA.

CICCACAGAC C-CCIACTGCG GCGAGGGGTG

TGGTGGIGGT TCQGPGICGA GTTTGGIGAC

AGCTAGCGCT AGCTGIGCIG GTAAGAGAIT

CAGATITTAT CCCCAGTTIG GTAGIGGATC

AAdGITCAGTG AACGGAGAAG ATAACTATCC 60

TTTCCITGAA CAACTCAAA.C TAINTACACC 120

TTGCCIAAGr CAAATTTGAA CACITCTGAA. 130

TAΑΑΤΓΑΧΞΑΑ TAOAGTAA.G CA4G7AGCCA. 240

CITATAATTT TCTATAIAAA CCCAIAIACA 300

ATTGITCGCT TTAACATACT CCAAAA.TGTC 360

GGCACITGCT TGTATGTCAA GCACCCZAGI 420

TTIATGITGI AGCOGATTTG GITTCIGIGG 430

CAGAGAAGGT CCTIGIAGAO? CACCGICZAG 540

CGATGCGTTC TTOATAGCA TCATTAACCA 6C0

CTACACCAGG GCTCCÍITIT ICAGICCICI '660

C 691

Information about SEQ ID No. 4:

(i) sequence characteristics:

(A) length: 112 amino acids (B) type: amino acid

170 (D) topology: linear (ii) molecule type: peptide (xi) sequence representation: SEQ ID NO: 4:

Met Ser Ser Phe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala 1 5 10 -D Met Ser Ser Thr Leu 20 Wall Wall Ala Gin Asn 25 cys Gly Cys Ala 30 Ser Leu Cys Cys 35 Ser Arg Phe Gly Phe 40 Cys Gly Ser Thr ASD 45 * Ala Tyr Cys Gly Glu 50 Gly Cys Arg Glu C-lv 55 For Cys Arg Ser Fro 60 Ser Ser C-ly C-Iy Gly 65 Ser wall Ser Ser Leu 70 Wall Thr Asp Ala Phe 75 Phe Asn Arg Ile ZLs 30 Asn Gin Ala Ser Ala 85 Ser Cys Ala Gly Lys SO Arg Phe Tyt Thr «Tg 95 A_i.a Ala Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gin Phe Gly Ser O/ '--OF Ser

100 105 110

Information about SEQ ID No. 5:

(i) sequence characteristics:

(A) length: 1838 base pairs (B) type: nucleic acid (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova

171 (F) tissue type: leaf (vii) immediate source:

(A) library: EMBL3 sugar beet genomic library (B) clone: chitinase genomic clone 76 (ix) properties:

(A) name / key: CDS (B) location: linked (469..874, 1263..1660) (xi) sequence representation: SEQ ID NO: 5:

ACLATAIATA MTIGAICAT ΑΓΓΑΑΓΠΤΑ AATTCTTGGT TGAAAMCTG TLAkTC-CCIC 60

TIGAGTCTIG AIACAACITA AAAAOGGSGC OGGITGGGAA AOTITITAC AIGAdAAGIT 120

CAGnAAOGA AGAACCXJIAA TGGACCCAIA TACAACAAAA TATCaSTOGI TZCATTITCC 130

TIGAACAAGI CAAACLAATA ACAOGAATCA Γ2ΑΓΑΑΑΑ TGACIGGCCA AAGTC4AAZC- 240

TCAACTOGAN ATAAGAAAAG ACATCGAGTC AAATCTCAAC ΑΓΓΠΑΑΑΟΟ TATCAAACAA 300

TAAITCCAIT CACATCCCAC lAIACAACIA GCTAACTOGT AAGCTTCITC CCIAAAICAC 360 dATCTITCA ΊΤΓΓΟΑΙΑΤ AAACTCATCr TCAACTCTCT AGITTCCACA ACCCACICAT 420

TGCTTCAAAA CTTCCTCTAC TAGTCIACIA TCATTCTACT CCICCAAAAT CTCITCTCTT 430

GGACCITTTT TCTCTAIACT TAIAGCACTT GCCICTATCT CIAGCACCXT GCTTCTGGCT 540

CAAAACICTG GCICTGCCTC TGCTTTAIGC TCTAGCAGAT AICCTTACTG CGGCACCACA 600

C-CIGCCTACT GOSGCACIGG GIGOOGCAA GCTCCTICTi 'CCTCAACGCC ATCCACCCCG 660

ACTGCTCCTC- TTTCCCTCCC AACTTTGCTG ACCGAIGCAT TCTTIAAIGG AATCAITAAC 720

CAAGC-A.GCT CTAGCIGTGC TGGTAAGAGC TTCTACACTA GCTCTCCTTT CTTGACTCCT 730

CTCACTTCTI ATCCTCACTT TCCTACTGGA TCCTCCGATG AGCTTAAACG TGAAGITCCT 340

GCC'1'1'ÍTTTG CTCATGCGAC GCAIGAAACT GGACCTAACT CTTAACAITA TTAMGCCTC 300

CTTTGAZACA ATTGAAATCS AATAAAATCT TCTTCCCCGC TCAITTC-CGC GCACTTAC-CT 360 <Γί »γ *» · * Λ ι ^ γ * · »·» ρτ nry ^ rr · v · γJ! »' ^TT 7 ¹ X ¹

G3AITCTCTC TAAATCTATT ATCTGCATTG GAAA.CCAATA ATAITGACTG ACCTAT.AATG 1030

172

GTAA-AGAAA IGAGAGCAAA AGAITTGAnA ΤΤΑΑΤΙΌΑΑΑ CTAGITITTA GTTTGCTAGT 1140

TAAAACTGAT TTAATTCATA TTATTAIUIT AAGTTCAATT AAGCGATACC TAAATCAAAG 1200

C-GAATCCATT GAGITACAGA AAAATATATA CTCAGCTCAT CAATTCAACT TCTCTCTTGT 1260

AGA-ITTITCC TA.CA.TAGAGG AGA.ITCCCAA ATCAACCTAT TGTCAGTCGA GCACAACATC 1320

GCCAZGCA.CC ACAAATAAGC AATACTACGG ACGTGGGCCT CTCCAAATCA CATCGAACTA. 1330

CAACTA.CGGA CCA.GCAGGTC GAAGCATTCG ATITGATGCT TIGAATCCAC CTGAAA.CA.G? 1440

TCTCAATGAT CCTGITATCG CCTTTAAGAC AGCCTTCTCG TTTTGGATCA ACAATGTCCA 1 = 00

CTCTCGAATT GTCTCOSGCA AAGGGTTTGG CTCCACCATT OGAGCTAICA ATCGAGGTCA 1560

ATGTCGTCGC GGGAACA.CAC OGGCGGTCAA OGCTCGIGIT AGGTACTAIA CTCAGTAITC 1520

OATCAGCTT GGiGiTlCAC CTGGGAAIAA CCTCTCTIGC TAGICACAIA ATCGAAGIGT 1530

TTCCATCGTC A.CAATTTACA AGTCTTAGAC TCTTAGTATA AGGAAAATAA AAATA.CAATC ’1740

AAGGGAACTG ACTIGimC TTAGCCAGTA. AGGGAAATAT GCAICACTTT GTAATTTATA 1300

TATATTTCAT AGTCTTACGG CCTA.TTAATA. GGGATACG 1333

Information about SEQ ID No. 6:

(i) sequence characteristics:

(A) length: 268 amino acids (B) type: amino acid (C) topology: linear (ii) molecule type: peptide (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova (C) tissue type: leaf (vii) immediate source:

173 (A) library: EMBL3 sugar beet genomic library (B) clone: chitinase genomic clone 76

(xí) sequence representation: SEQ ID No. 6: Met Ser Ser Leu Gly Pro Phe Leu Ala Ile Leu lie Ala Val Ala Cys 1 5 * 10 15 Met Ser Ser Thr Leu Val Val Ala Gin Asn Cys Gly Cys Ala Ser Gly 20 25 30 Leu Cys Cys Ser Arg Tyr Gly Tyr Cys Gly Thr Thr Ala Ala Tyr Cys 35 40 45 Gly Thr Gly Cys Gin Gin Gly Pro Cys Ser Ser Thr Pro Ser Thr Pro 30 55 60 Ser Giv C-lv Val Ser Val Pro Ser Leu Val Thr Asp Ala Phe Phe Asn 65 70 75 30 Gly lie lie Asn Gin Ala Ser Ser Ser Cys Ala Gly Lys Ser Phe Tyr 35 90 95 Thr Arg Ser Ala Phe Leu Ser Ala Leu Ser Ser Tyr Pro Gin Phe Gly 100 105 110 Ser Gly Ser Ser Asp C-lu Val Lys Arg Glu Wall Ala Ala Phe Phe Ala 115 '120 125 His Ala Thr His Glu Thr Glu His Phe Cys Tyr Ile Glu Glu Ile Ala 130 135 140 Lvs Ser Thr Tyr Cys Gin Ser Ser Thr Thr Trp Pro Cys Thr Thr Asn 145 150 155 160 Light Gin Tyr Tyr Gly Arg Gly Pro Leu Gin Ile Thr Trp Asn Tyr Asn 165 * 170 175 Tyr Giv Pro Ala Gly Arg Ser Ile Gly Phe Aso Gly Leu Asn Ala Pro 130 135 190 Glu Thr Val Ala Asn Asp Ala Val Ile Ala Phe Lys Thr Ala Fhe Tro 195 ’2C0 205 Phe Tru Met Asn Asn Val His Ser Arg Ile Wall Ser Gly Lys Giv Fhe 210 215 220 Gly Ser Thr lie Arg Ala lie Asn Gly Giv Glu Cys Gly Gly Gly Asn 225 230 235 240

174

Thr Pro Ala Val Asn Ala Arg Val Arg Tyr 'Tyr Thr Gin Tyr Cys Asn 245 250 255

Gin Leu Gly Val Ser Pro Gly Asn Asn Leu Ser Cys 250 265

Information about SEQ

ID No. 7:

(i) sequence characteristics:

(A) length: 1106 base pairs (B) type: nucleic acid (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova (F) tissue type: leaf (vii) immediate source:

(A) library: lambdaZAP sugar beet cDNA library (B) clone: cDNA-clone SE (ix) properties:

(A) name / key: CDS (B) location: 1 8. . 896 (xi) sequence representation: SEQ ID NO: 7:

• Λ / W »’ *.-V-V- ·· * * Λ * <

-5-Λ-Α.

ATG GCA GCC A-A ATA GIG TCA GIT CIA TTC GIG Mez Ala Ala Lvs Ile Val Ser Val Leu Phe Leu

1 * 5 10

175

Ai'2 Z2.S C ¹ O '' ^ TTA Leu 15 ATC lle -rrrrn L · - Phe Ala Ser TTC GAG TCC TCT CAT GGC TCC CAA 98 Ser Leu Fhe 20 Glu Ser Ser His Gly 25 Ser Gin GCC - TCC- CCC n · * · * <-n U-ΓΛ ΓΛ * CCT GAT GAA CCA ACT FEELING GCT GAC 146 xle Wall Tle 30 T * and. Tr? Gly Gin Asn 35 Gly Asp Glu Gly Ser 40 Leu Ala Asp ACT TCT AAC TCC CCA AA.C TAC CCT ACC GTG ATC CTA. CCT TTC CTA CCT 194 Thr Cys 45 Asn Ser C-ly Asn Tyr Gly 50 Thr Wall lle Leu 55 Ala Phe Val Ala ACC '£ ^l 1 CCT AAC GCC GAA ACC CCC- CCC CTG AAC TTA CCT CCC CAC TGT 242 Thr 60 Gly Asn Gly Gin 65 Thr Pro Ala Leu Asn 70 Leu Ala Gly His Cys 75 GAC CCT GCT ACA AAT TCT AAC ACT CTG ACC ACT GAC ATC AAA ACA TCC 290 Asp For Ala Thr Asn 80 Cys Asn Ser Leu Ser 35 Ser Asp Tle Light Thr cys 50 CAA GAG GCA CCC ATT AAG GTA CTC CTC TCT ATA GGA CCT GCT GCC CCA 33S Gin Gin Ala Gly 95 Tle Light Val Leu Leu 100 Ser Tle Giy Giy Gly 105 Ala Gly CCC MELT TCT CTT TCC TCA ACC GAT GAT GCA AAC ACA TTT GCT GAT ZAC 336 Gly Tyr Ser 110 Leu Ser Ser Thr Asp 115 Asp Ala Asn Thr Phe 120 Ala Asp Tyr crc TGG AAC ACT TAT CIT CCC CCT GAG TCC ACC ACC CGA CCC CIT GGA 434 Leu Trp 125 Asn Thr Tyr Leu Gly Gly 130 Gin Ser Ser Thr 135 Arg For Leu Gly GAT GCA GTT TTG GAT CCT ATT GAT TTC GAT ATC GAG ACT GAT CCC 432 Asp 140 Ala Wall Leu Asp Gly 145 Tle Asp Phe Asp lle 150 Glu Ser Gly Asp Gly 155 AGA 'ITT TGG GAT GAC CTA GCT AGA CCA TTG GCA CCT CAT AAC AAT GCT 530 Arg Fhe Tip Asp Asp ISO Leu .Ala Arg Ala Leu 155 Ala Gly His Asn Asn Glv 170 CAC AAA ACA GTG TRAY TTA TCA CCA CCT CCT GAA. TCT CCC TTG CGA GAT 573 Gin Light Thr Wall 175 Tyr Leu Ser Ala Ala ISO For Gin Cys For Leu 185 For Asp GGC ACC ACC ACT ATA CCC AGA CCC CT- TTC GAC MELT CTA TCC 625 Ala Ser Leu * -0 Ser Thr Ala Tle Ala 155 Thr Giy Leu Phe Aso 200 Tyr Val Trp

176

Wall CAG TIC -T »^ A * -p T-.C itrti AAC CCC CCT TCT CAA TAT GAT ACC AGC GCT GAT 674 Gin 205 Phe Tyr Asn Asn For 210 For Cys C-ln Tyr Asp Thr 215 Ser Ala Asp .-A.7 CTC TTC AC-C TCG TCG AAC CAG TCG ACC ACA GTA CAA GCT AAC CAG 722 Asr * Leu Leu Ser Ser Trp Asn Gin Trp Thr Your Wall Gin Ala Asn Gin 220 225 220 225 »FcT CTC C-CA CTA CCA C-CA TCA ACT GAT GCT GCC GGC ACT GCT TTT 770 Ile Phe Leu Gly Leu Fro Ala Ser Thr Asp Ala Ala Gly Ser Gly Fhe 240 245 250 AT? CCA C-CA GAT GCT FEELING ACA TCT CAA CTC FEELING CCC ACT ATC AAG GCrZ 313 ΞΣβ For Ala Asp Ala Leu Thr Ser Gin Wall Leu For Your Ile Light Gly 255 260 265 TCT CCT AAA MELT GGA GGA GTC ATC CTA TCG TCA AAG GCA MELT GA.C ACT 366 Ser Ala Light Tyr Gly Gly Wall Met Leu Ttp Ser Light Ala Tyr Asp Ser 270 275 280 GGG TRAY AC-C AGT GCT ATT AAA AGC ACT GIT ΤΑΑΤΠΆΑΑΤ TACTAGTCTA or 5 Gly Tyr Ser Ser Ala Ile Light Ser Ser Wall

235 290

TCCAAA.GATA TA.GATA.CAAA ATAAGTTATA GAGATACATC AAAAAACCAT CTLAGTITTA

ΑΑΤΓΤΓΓΤΑΤ C-CACCACAAA AjGCTTCDAT ACTAATATAC TAITATCAIA AATCGCTTAT

TGCCTCGCTA TSTITTCGIG ΑΤΓΑΊΤΑΤΑΤ ACACACTTAC aacttcccaa ttatc-cgagt

CITTCZJtAAA

1025

1095 nos

Information about SEQ ID No. 8:

(i) sequence characteristics:

(A) length: 293 amino acids (B) type: amino acid (C) topology: linear (ii) molecule type: protein} ΊΊ (xi) sequence representation: SEQ ID NO: 8:

Met Ala Ala Light Ile Wall Ser Val Leu Phe Leu Ile Ser Leu Leu Ile AND 5 10 15 ? he Ala Ser Fhe Glu Ser Ser His Gly Ser Gin Ile Wall Ile Tyr TTo 20 25 30 Giy Gin Asn Gly Asp Glu Gly Ser Leu Ala Asp Thr Cys Asn Ser Giy 35 40 45 Asn Gly Thr Val Ile Leu Ala Phe Wall Ala Thr Phe Gly Asn Giy 50 55 60 Gin Thr Fro Ala Leu Asn Leu Ala Gly His Cys Asp For Ala Thr Asn 65 70 75 30 Cys Asn Ser Leu Ser Ser Asp Ile Light Thr Cys Gin Gin Ala Glv Ile 35 90 95 Lys Val Leu Leu Ser Ile Gly Giy Giy Ala Gly Giy Tyr Ser Leu Ser 100 105 110 Ser Thr Asp Asp Ala Asn Thr Phe Ala Asp Tyr Leu Tip Asn Thr Tyr 115 120 125 Leu Giy Gly Gin Ser Ser Thr Aru For Leu Glv Asn Ala Val Leu Asp 130 135 140 Gly Ile Asp Phe Asp Ile Glu Ser Gly Asp Giy Aru Fhe Trp Asp Asp 145 150 155 160 Leu Ala Arg Ala Leu Ala Gly His Asn Asn Gly Gin Light Thr Val Tyr 165 170 175 Leu Ser Ala Ala For Gin Cys Pro Leu For Asp Ala Ser Leu Ser Thr 130 135 190 Ala Ile Ala Thr Gly Leu Phe Asp Tyr Wall Trp Wall Gin Fhe Tyr Asn 155 200 205 Asn Fro For Cys Gin Tyr Asp Thr Ser Ala Asp Asn Leu Leu Ser Ser 210 215 220 Trp Asn Gin Trp Thr Thr Val Gin Ala Asn C-ln Ile Phe Leu Gly Leu 225 230 235 240 Pro Ala Thr Asp Ala Ala Gly Ser Gly Fhe Ile For Ala Aso Ala 245 250 255

178

Leu Ser Gin Wall Leu For Thr Ile Light Gly Ser Ala Light Tyf Gly 250 265 270 Gly Wall Met Leu Trp Ser Light Ala Tyr Asp Ser Gly Tyr Ser Ser Ala 275 230 2Š5 Ile Light Ser Ser Wall 2S0

Information about

SEQ ID NO

9:

(i) sequence characteristics:

(A) length: 1249 base pairs (B) type: nucleic acid (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (vi) original source:

(A) organism: Beta vulgaris (B) strain: monova (F) tissue type: leaf (vii) immediate source (A) library: cDNA-library of lambdaZAP sugar beet (B) clone: cDNA-clone beta-1,3- glucanase (ix) properties:

(A) name / key: CDS (B) location: 34 .. 1 041 (xi) sequence representation: SEQ ID NO: 9:

ιιτητσπτ attcttagag ttatitcttc aca atg agg cta at? agc ac-. act Met Arg Leu Ile Ser Thr Thr

5

179

TCT GCA GTT C-CT AGT TTG CTG ΤΤΓ CTT GTA GTA ATT CTA. CCT AGT ATT 102 Ser Ma Wall Ala Thr Leu Leu Phe Leu Val Wall Ile Leu Pro Ser Ile 10 15 20 CAA CTG ACA GAG GCA CAA ATT GGC GTA TGT AAC GGG AGA CTA of"·/*/-" AAC 150 Gin Leu Thr Glu Ala Gin Ile Gly Val Cys Asn Gly Arg Leu Gly Asn 25 30 35 AAC TTA CCT TCC GAG GAA GAT GIT GTA AGC TTG TRAY AAG TCG AGG GCA 198 Asn Leu For Ser Glu Glu Asp Wall Val Ser Leu Tvr Light Ser Arg Gly 40 45 50 55 ATA ACG AGG ATG AGA ATC MELT GAC CCT AAC CAA. CGG ACC CTC CAA GCG 246 Ile ΊΪ1Τ Arg Met Arg Ile THE Asp Pro Asn Gin Arg Thr Leu Gin Ala 60 65 70 GTT AGA GGA TCG AAT ATA GGG CLA ATC GTC GAT GTC CCT AAG CGT CAC 294 Wall Arg Gly Ser Asn Ile Gly Leu Ile Val Asp Wall For Lys Arg Asp 75 80 85 CTA AGG TCA crc GGC TCC GAT GCT GGG GCT GCG TCT GGT TC-G GTC CAA. 342 Leu Arg Ser Leu Gly Ser Asp Ala Gly Ala and Ta Ser Arg Tra Wall C-ln 90 95 100 AAC AAT GTA GTC CCT TRAY GOG TCT AAT ATT CCA TAC ATA GCA GIT GGT 390 Asn Asn Val Val For Tyr Ala Ser Asn Ile Arg Tyr Ile Ala Wall Gly 105 110 115 AAT GAA ATA ATG CCT AAT GAT GCC GAG GCA GGG TCA ATT GTC CCG CCC 433 Asn Glu Ile Met For Asn Asp Ala Glu Ala Gly Ser Ile Val For Ala 120 125 130 135 ATG CAA AAT GTC CAA AAT GCC CTT CGA. TCA GCT AAT TTA GCT GGT AGA 435 Met Gin Asn Wall Gin Asn Ala Leu Arg Ser Ala Asn Leu Ala Gly Arg 140 145 150 ATT AAA GTC TCT ACC GCG ATA AAA AGT CAC CTC GTT GCT AAC TTC CCT 534 Ile Light Wall Ser Thr Ala Ile Light Ser Asp Leu Wall Ala Asn Phe For 155 160 165 / -ν '* - · tct AAA GGT GTT ITT ACT TCT TCA TCA TRAY ATG AAT CCA ATT 532 For Ser Lvs Gly Wall Phs Thr Ser Ser Ser Tyr Met Asn Pro Ile Wall 170 175 180 AAC TTC FEELING AAA AAT AAC TCA CCT TTG TTA GCC AAC ATT TRAY 530 Asn Phe Leu Light Asn Asn Asn Ser Pro Leu Leu Ala Asn Ile Tyr ? T3

180

195

/ - · TTT TCT TTC ATT ACC A <ri. ' * -FV ..iU CGT CTA GAT MELT GCA CTC -x.7 _ “T.e r — U v • C - -> Ser Xst: Arg Leu Asp Tyr Ala Leu 23 0 205 210 215 TTT AC 1 ' • ^ V “· X / - / ·> 5 * · »· /-"-in· CAA rr— * · * ·· » ΛΛ * CAT AA.T GGT TTA CAA TRAY C ^ A Fhe Thr Ser Fro .-οΞΠ Aia '• wí ^ JTk Wall Λ5 * Asp Asn Gly Leu Gin Tyr Gin 220 225 230 AA.T GTC TTT CAT GCT TTA GCA CAC GIG Τ — Φ GCG GCC TTA GCG AAG Asn Wall Fhe A = o Ala Leu Wall Asp Thr Wall Tyr There Ala Leu Ala Light 235 240 245 GCC GCC CCC AAT GTC- CCG GTT GLG TCC GAG AGT GGG TGG CCT IH Gly Ada Fro Asn Wall Fro Ile Wall Wall Ser Glu Ser Gly Trp For 250 255 260 TGG GCT GGT GGT —'Τ ’ GGT AGT 'TTT dY-d AAC GCG GGG ACT MELT TRAY Ser There Gly c-iy Asn There There Ser Fhe Ser Asn There Gly Thr Tyr Tyr 265 270 275 AAG C-GC TTA ATT GGT CAC GCA AAG CAA GCA ACT CCC CTG AAG AAA GCA Lvs Gly Leu Tis Gly His Wall Light Gin Gly Thr For Leu Light Lvs Glv 230 235 290 295 CAA GCA ACT CAG GCA CAC CCG GCT ATG anus CAT GAG AAC CAA AAG Gin Ala Tle Glu There Tyr Leu Fhe There Met Fhe Asp Glu Asn Gin Light 300 305 310 GGT GGA GGT ACT GAG AA.C AAC TTT GCA CTG ACT CCC AAC AAA CAG Gly Gly Gly Ile Glu Asn Asn Fhe Gly Leu Phe Thr For Asn Lvs Gin 315 320 325 CCA AAA CAC CAA CTC AAC TTC AAC AAC TCAAAGTAGT TCAACIGCCT Fro Light Tyr C-ln Leu Asn Fhe Asn Asn

67í

726

774

822

370

913

330 335

AGTACACACA TMACATGCT AATATGTTGT ACGTAGTT.AT GTCATCTACA ΈΑΤΑΤΑΑΤΑΑ.

GTCAAACCAA ACACCCCACC ACACACTAAA ACTGTAACAA AACATCCTCC TGTTGTAATA

TTACCCTAGC TGCAAIAATA TTT.ACTCTTA TATACAGATC TTGTGAAAAA AAAAAAAAAA

ΔΔΔΔΔΔΔΔ

1014

1061

1121

1131

1241

1249

181

Information about SEQ ID No. 10:

(i) sequence characteristics:

(A) length: 336 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence representation: SEQ ID NO: 10:

Met ; Arg 'Leu . Ile Ser • Tl ·.,. • Thr Ser • Ais . Wall . Ala Your 'Leu . Leu Fhe Leu 1 5 10 15 wall Wall Ile Leu Pro Ser Ile Gin Leu Thr Glu Ala Gin Ile Gly Wall 20 25 30 Cys Asn C-iy Arg Leu Gly Asn Asn Leu Ser Glu Glu Asp Wall Wall 35 40 45 Ser Leu Tyr Light Ser Λ «—r Gly Ile Your Arg Met Arg Ile Tyr Aso For 50 55 60 Asn Gin Arg Your Leu C-ln Ala Wall Arg Gly Ser Asn Ile Gly Leu Ile 63 70 75 30 Wall Aso Wall For Lvs Arg Asp Leu Arg Ser Leu Gly Ser Asp Ala C-ly 35 90 95 Ala Ala Ser Arg Trp Wall Gin Asn Asn Wall Wall For Tyr Ala Ser Asn 100 105 110 Ile Arg Tyr Ile Ala wall Gly Asn Glu Ile Met For Asn Asp Ala Glu 115 120 125 Ala Gly Ser Ile Val For Ala Met Gin Asn Wall Gin Asn Ala Leu Arg 130 135 140 Ser Ala Asn Leu Ala Gly Aro Badly Light Wall Ser Your Ala Ile Light Ser 145 150 155 160 Asp Leu Wall Ala Asn Phe Fro For Ser Light Gly Wall Fhe Your Ser Ser 165 170 175 Ser lyr Asn Pro lie Wall Asn Phe Leu Light Asn Asn Asn Ser For 130 135 190

182

Leu Leu Ala Asn Ile lir Pro Tr Phe Ser ? hs Ile Gly Your For Ser 195 200 205 Met Arg Leu Asp Tyr Ala Leu Phe Your Ser For Asn Ala Gin Wall Asn 210 215 220 Asp Asn Gly Leu Gin Tyr Gin Asn Val Phe Asp Ala Leu Wall Asp Thr 225 220 235 240 Wall Tyr Ala Ala Leu Ala List A_a Gly Ala For Asn Val For Ile Wall 245 250 255 Wall Ser Glu Ser Gly Tro Pro Ser Ala Gly Gly Asn Ala Ala Ser Phe 250 255 270 Ser Asn Ala GLv Your Tyr Tyr Lys Gly Leu Ile Gly His Wall Light Gin 275 2SG 285 C-iy Your Pro Leu Lys Lys Glv Gin Ala Ile Glu Ala Tyr Leu Phe Ala 290 295 300 Met Phe Asn Glu Asn Gin Lys Gry Gly Gly Ile Glu Asn Asn Phe Gly 305 310 315 220 Leu Phe Ihr Pro Asn Lys Gin Pro L ’/ s Τ'-ΊΓ Gl. Lsti - ~ n Phe Asn Asn 325 330 335

Information about SEQ ID No. 11:

(i) sequence characteristics:

(A) length: 6313 base pairs (B) type: nucleic acids (C) number of strands: double-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (B) strain: Monova

183 (F) tissue type: leaf (vii) immediate source:

(A) library: EMBL3 sugar beet genomic library (B) clone: chitinase 1 (ix) genomic clone properties:

(A) name / key: uncertain (B) location: 3214..4227 (D) other information: note = approximately 1000 base pairs (ix) properties:

(A) name / key: CDS (B) location: connected (1428..2171, 4621..4776,

5408..5824) (xi) representation of the sequence: SEQ ID No. 11:

TCTAGAGAGA

AZA2TAATCG

GGG0C2GCQC

GCGGGTCIGT

AAZTAdTAA

ATTLAAATTG

ΑΤΓΠΊΤΠΤ

CAAA.C.-AAAA.

TCAAA.CC7AA f ^ *** ’*» No. C * »!. C*'*

CCTCTCTCTC

GAAAAAACAA

GCSGAIATGG

G ^ aiGATIT

CAIGGTCAAG

CCOGATCATT

AGAATGITET

ACGAGITCAT team ’

CSCCATGIGA

ACAACTCTTG

GGGACCGCIC

ΑΑΑΑΤΠΑ2Τ

TmCCAAAA

TGTCAA.GAGA

TITIGTGAAA.

ACCACTAC CATGCATC-A

GGGITGGGGT

AACCCCTCCC

GAGGCCCGAC

GAAACTAAAT

TACCTCAAAA

ATCA3AGTZA

AAAAA.CC ^T 'ZA

GAAACCAAAA.

GGGACAAGIT CEACCCSCIA

TGGCACCIAG AjGCTGGGZAT

CCSTTCTCCS CIACGIGGCC

ΑΤΏΑΑΤΙΤΑ TTTAA.CCGG7

TOCTGAAAC TAAATASTAA.

AAAC-GAAAAT TTGGCAAAAA.

¹ 1 ¹ ·! '! ¹ ί yrr r Λ Λ Λ

V * Ρ'Ζ * Q. . ₍ ν—

AACCG7TGIG CCACICA

120

130

240

300

360

420

430

540

600

660

84

CGCACCCCAC CGCTGOGCCA CTCAAAACCC CGAACGGTTG

TTTCSTTCIT CGATmTGA TAGrnTCCGT TTCATnGTG

GTGTTTCGTG OTGGTGGTCG GGGTITCATG GTGITAIGGT

ATGITGITAT GGTCATGIGA TOGTGGGiTG CGGAGGGGAA

GACTGTGGTT GGGGGGCTGG ACAAAAGGAG AGGAGAGGGG

GGAGAGGAGA GAGAGTAGGG CTGAAGGGGG CTGCGCGACT

CAGGGGITGG GGTTGCTGGT GGGGGIGGGT GGGGGTIGGG

GCTSGAGGIG GICAATGCOG GAAAITCTAT GGTCAACGCC

TTTOGAAAAA AAATTIGATC TTGATTTGIT TTIACSAAAA

OGCAAAAGTG AACTCGTAAA ΑύΤΠ'ΓΠΤΑ AAACAAAITT

TTCACITITT ACTATGITAC AIACAIAAAT TTIGATAGAT

TCAITTCTCC CTTATCOCAA AGACCCAAAG CCTCTATAAA

GAGICACACA TGACAGAAOG CAACTTTGAA ACGAAAAAGA

CCTCTCCTAG TITLE GGATTAAIAT GITIAGCTCI

GTGIACAATG TGGGOGGCAA TGCAACACAA CCGATACTAA

TOGGCOGCCC ATCACGTCGS ACACCTCCTC GTCCTCCCAC

GTCCTCCCAC CCCGAGACCG CCACCTCCTC GIGCTCCIAC

CACCAAGACC ACCA.CCTCCT CGTCCTCCIA CCCCGAGACC

GACCACCACC TCCTCGTCCT CCIACCCCAA GACCGCGACC

CACCTCCTCC TACACCAAGA CCACCACCTC CT.AGTCCTCC

CACCACCTCC TAGTCCTCCT ACCCCAAGCC CACCATCTCC

CTCOGCCCGA ACCIACGCCA CCAACICCTA CACCACCAAC

CTGAAGAAAT GITTAAIGAA TTCCTCTTGA ACCGCACTCA

GGTTCIACAC TTACC4CGCT TTCATIACTG CAGCTGAAAC

CTGGGAAIGA TGAAAITAGA AAGAGAGAAA 77 /> - / - V— · * / CTGG7ACTGG A7AG73GGCC “τύ · ^ ^^ 'λΖλΖλΖΌ' ™” -

790

SCO

XACUT-Gi

AGCGAAG-A.G 960 .AAGG7G7AGG 1020 GGIAGIGG7G 1030 CTSGAT77G7 1140 agactttttt 1200 AATG4I7CTT 1250 AAIAAGCT7C- 1320 ACTCTCCTGA 1330 ΑΑΑΑΏΑΑΑΑ 1440 CIAGGAGAAG 1500 GGTTGCTC4G 1550 CCACCTCCTC 1520 CCACCACCTA 1630 CCIACACCAA 1740 CCAAGACCAC 1300 CCACCS.CCAC 1360 GAACCACCAA 1920 GACATSATCT 1930 CCTGGIAGAT 2040 TTTGGTAAZS. 2100 -> 0 * '“' Z. 2150

185

AAACCTCTGG TTCAITCITC TACTTCTCAT TCTTCITTAC TTCCGATCTG CTTCAC77TA 2220 CTAACATGCA TGTTITCATA CTATATCGTA TATTTATAGA TGAOACLA GTACGTTA? 2230 ATTTGCTAGT GCCTACTTCC TACTCTTTAT TCTTTGTCAA TCTAATGAZC TTTATAGT2T 2240 ATGTTGGACA CAATAAATTA TATAATCTTA ACCTTAAGTT TAGACGATAT GAATTATTTC 2400 TTTTTCACGT ATCTACCCTA GTTAx.ATAC- CTTCTTATTA ccagtttttt TACTTCTACT » 2460 ATTTTGACTT TTGACCATAT CGATTCTTIA TGCATAAGAC ATATATAAIA TATAAGATCT 2520 I TGTTGTATTA TTTACTTCGA TATATATATT 'TTGTTGGATT ATC-CTCCAAA ATTAGTCAAG 2530 TTTTCCTAAT AGTTAT2ATT AGTTGCTATT AATTAGTTAG TGGGTTAACA AGTAGTGGTC 2640 TAGATAATGG TCTAGCTAGT GGGATAGCAA GTAAGTAGTA GGCTACATAG TGGACATGTT 2700 GTL-JGIAGIG CITIGISTGC CLATTLVAG ATGGTTTIGT TTATTCATAA TGIGTACATG 2760 AAAAATATAT AAAAAATGIA GTCITTCIAA TCTCITCAAG TTCTCTTCCT CCTCZAAAAA 2320 TTCIACCGG TATCAGAGCT CCSGGTTAGA TCCGGGAAAG GGAAAGAGAT GAAGCAAAAA 2330 AAAGAAAAGA AAAAAAAGAG AGAGAZAGAG AAAGAAAGAG AGTATTAAAA ACAAAAITGA 2940 CTAAAAAGSA ACAAGCAAiTG CAGCTTGATT TCAITCITTG TGAGCAATIT TTIGTTTGAG 3000 TGAAATTTTT TGIGITIAAC CATGAAAGTA ATGGITGGGT CTGAICíGTT GITGGGCICC 3060 CTTTTAGCCC MGAAAATCG ATTTTTAATCC TGTGGAAAAA AGGGGOAAA AAAUGITIG 3120 AGTTGAAGAT OSGIAATCT TTAjOGAGICT GIGGAAGICG TACICAACGC AGAGACGATT 3130 TCCAAAGITG TACTACTGGA TGTGGTCAAA ΊΠΝΝΝΝΝΝΝ NNNNNNNNNN NNNNNNNNH 3240 NNNNNNNřiNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360 _k NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNIINřiNříNNN NNNNNNNNNN NNNNNNNNNN 3420 NNNNNNUNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3430 NNřiNNNNřiNN NNNNNNNNNN NNNNNNNNÍ1N NNNNNNNNNN NNNNNNNNNN NNNNNNNNNJ 3540 NNNNNNNNNN nnnnnnnnnn NNNNNNNNNN nnnnnnnnnn NNNNNNNNNNN NNNNNNNNNN 3600 vtNNNNřtttNNN NNNřÍNNř3ttJN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNřlNNN 3660

186

ΝΝΝΪ (ΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ϊβϊίβββηί Μββββββββί 3720 ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ βίβίΝΝΝΪίϊΐΝ ΝΝΝΝΝϊββίΝΝ 3730 Μ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝίίΜ ^ ίίΜί ΝΝΙβίϊβιΝΝΙΟί 3340 ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ NNNNNNNNřiN ΝΝϊίΝΝΝϊίΝϊβί ΝΝΝΝΝΝ’ϊίΝ? Who 39CO * ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ íaaacaiNNTiN NNNNNIOiMuí NNNNNNNNNN ' 3560 Í ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ JÍNNNNNNNNN ΝΝΝΝΪδΙΝΝΝΝ NNNNNřiNNNU 4020 ¹ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΪΪΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ 4030 ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ϊΝΝΝΝΝΝδβϊΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝί <Ν 4140 'ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ 4200 N ΝΝΝΝΝΝΝΝΝΝ NNNNNNNTCT AGA7TGACTT ΊΟΤΑΑΟΣΊΑΑ ACITACCAAA 4260 TROUBLESHOOTING TROUBLESHOOTING GTCGTGA.TGT C7CCCTCC .-. T 4320 CrillTITTA CTACITGTTA CATTTTTCTT AAATGAATGT CTCATTTTAC ACACIACA-- 4330 GCrnAITIG CATTGGIAAC TATGACCCAT CAAAAAATAA C4ATAZTCCC ...Α ..... 4440 'CTCATCITCT TAACTTTAAC TCTCAAATAT TTGTTATTTT CAACAAAGIT AACTCTCAAA 4500 TATTTGTCAT AATAATGTGA AATCTTCGCT TGATAAACTG TCOAAACAA AATGCTCTCA * -Τ- » TTTAAAAAGA AAAATGAGAG GACATATATA CTAATGGATT TTIGAATTGA CAATATAGGA 4620 GAACCGACCG CACAACAIGG ACCATITACA TGGGGCTATT GITTCATAGA AGAAAZTGGA 4630 GCOGGCOCTC TCAGCCAAIA TTGIGCACCC TCTGTAGAAT GGCCTIGCAT TGGTGGGAGG 4740 TTITACTATG GTCGTGGACC AGTOCAACIT ACCTGGIAAG TACICOCTCC GTTCCAAAAT 4300 AIAGTTCTCA TITTCCTITT TICACACTAA TTIATGCAAG TAGAAIAIAA GAGGCTAGGT 4360 ⁴ AAAGAITTTT ΤΤΊΤΤΑΤΊΤΑ AATAAATCTT CTATGGGAAA AGATGAITTT AGGAGAGAGA 4520 CTGGAGAAIA. ATIAGIGAAA GAGCATTAAT TCTAACATTT TGGITGAATA AAIAAAGGAA 4530 AAAACAAATT CAAGAAGCTA AACTAATGAG GGCACAGCTT TICTAGACAA AITACGGAAA 5040 AATCTGGAAC TAAAIATGAA AATGGGAACT ATATTTTGAG ACAOíCAAAA TAAAAAZGGG 5100 AACIATATIT TGGGAOGGAG GGAGLATTAT TALATTAGCT TACTCCTATT ACTTGCATCG 5160 CATCTCCAAA TTTTTATTGT TCATAGAAAA CTCAITTTCA AGAATTTTGC TATTCGACGT 5220

187 σΓΓΑΑΑΑΙΊΤ TITACIAOGC TTICTAATTA CATAITIITA TAGPSLACTT ATCTLAIACG 5230

TTTCCATITC TTCTCimT CCITCCnTC CITGACTTAA σΠΤΏΑσίτ GAXACATATA 5340

GCTAGCAAAA TTATCITAGG TAITITAGCT ΑΑΤΠΑΑΑΑΤ THTGGTAAT GATAAAIAAT 5400

TTGCAGGAAT TTCAACIATG GAAAGCAGGT GAAC-CACITA GGTTIGGACC TCCLAITCAA 5450

CCCAGACATA GTAGCACATG ACCCAGTIAT TTCTETCGAA ACIGCAATIT GGTTTTGGAT 5520

GACTCCIGAA GGAAACAAGC CITCITCCCA TGAAGTCATA ACIGGGCAAT GGACACGAAC 5530

TCCIGCAGAC ATAGCTCGCA ACAGATTGCC TGGAIATGGT TIAATCACAA. ATA.TTTTTAA. 5640

TGGTGCITTA GAATGCGGCA CTCATGGACC AGATAATAGA GGGGAAAAIC GAATICAGIT 5700

TTACCAGAGA TACIGTGATC TTCTAGATGT TAGCTATGGA GATAACCITG ATIGCTACCG 5760

TCAAACTCCC TTTGATIGGG GTCITAAAAA ACITCAGGGA GCIAGAGAAT CATGGTCGTC 5320

GAGCTAAAAT TATA.OGCATG CATCTAGTCT CZAAGICCAX ACATTAHGT CTTCATGCGT 5330

CLATGATATT GAGTAAGTTG GTATGTTCAA AAAECCTGG TGTCTGAAAA TATGCAAACA 5340

GAACCAGCAA TAAGTAATAA GCAAGGITTA CITGCACCAA ATCTGGATCT GITCTAGTGA 6000

AA.TTGnGTA.TGTTCGTATT GTATGGTAAT GAAIAAAGTT TCTGITGTAT TIGCATIATC 6060

TGCACCTTAT TGATATTAAT TITTCATATT CAGGOGTTTA CAATCATAAG GATTACTGTA 5120

GGACCATTGA ACATGCAGTT GAGITAGICI TTAATATGGT GITCAAGAAG AGTATGGAAA 6130

AIAGAAATGA GGAATGAAOG TACTCTATAT TATAAGAGAC TACTAGTGTT GITTAGICAG 6240

TGCIATTGTT ACACCIAAAA AAGCTCIATG AGATTACATT TACATTATGG TCAAAAGGTC 6300

TTAATGTCIA CCG 6313

Information about SEQ ID No. 12:

(i) sequence characteristics:

(A) length: 439 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein

188 (vi) original source:

(A) organism: Beta vulgaris (B) strain: Monova (F) tissue type: leaf

(xi) sequence representation: SEQ ID NO. 12: Met 1 Lys Ile Lys Thr Ser Pro Ser Phe Leu Leu Gly 5 10 Leu Ile C / s Leu 15 Ala Leu Val Leu Leu Leu Gly Glu Gly Val Gin Cys 20 25 Gly Arg Gin Cys 30 Asn Your Thr 35 Asp Ihr Asn Cys Leu Ser Gly Cys Ser 40 Val Gly Arg Pro 45 Ser Arg Fro 50 Ihr Pro Pro Arg Pro Pro Ihr Pro Arg 55 60 Pro Pro Pro Pro Arg 65 Pro Pro Your Pro Arg Pro Pro Pro Arg Pro 70 75 Pro Ihr Pro Arg 80 Fro Fro Fro Pro Ihr Fro Arg Pro Pro Fro Fro Arg 85 90 Pro Pro Your Pro 95 Arg Pro Pro Pro Pro Pro Ihr Pro Arg Pro Fro Pro 100 105 Pro Arg Fro Fro 110 Your Pro Arg 115 Pro Pro Pro Pro Pro Ihr Pro Arg Fro 120 Pro Pro Pro Pro 125 Your For Arg 130 Pro Pro Pro Fro Ser Fro Pro Thr Pro 135 140 Arg Pro Fro Pro For 145 Pro Pro Pro Ser Pro Pro Thr Pro Ser Pro Pro 150 135 Ser Pro Pro Ser 150 Fro Glu Pro Pro Thr Pro Pro Glu Pro Ihr Pro Fro 153 170 Your Pro Your Pro 175 Fro Thr Kis Leu Thr Asp Ile Ile Ser Glu Glu Met 130 '135 Phe Asn Glu Phe 190 Leu Leu Asn 155 Arg Ile Gin Pro Arg C / s Pro Gly Arg Trp Phe Tyr Ihr 2GQ * * 205 Tvr Gin Ala 210 Phe Ile Ihr Ala Ala Glu Ihr Phe Pro 215 220 Glu Phe Gly Asn

189

Thr Gly 225 Asn Asp Glu Ile 230 Arg Light Arg Glu Ile Ala Ala Phe Phe Glv 235 240 Gin Ihr Ser His Glu Thr Ser Gly Glu Pro Thr Ala Gin His Gly For 245 250 255 Fhe Thr Trp Glv Tyr Cys Fhe Ile Glu Glu Ile Gly Ala Gly Pro Leu 260 265 270 Ser Gin Tyr Cys Ala Pro Ser Wall Glu Trp Pro Cys Ile Arg C-iv 'v -.- r 275 230 285 Fhe Tyr Tyr Gly Arg Gly For Wall C-ln Leu Ihr Trp Asn Fhe Asn Tr 290 295 300 Gly Lys Gin Wall Lys His Leu Gly Leu Aso Leu Leu Phe Asn Pro Aso 305 310 315 320 Ile Val Ala His Asp Pro Wall Ile Ser Phe Glu Ihr Ala Ile Tro Phe 325 330 335 Trp Met Your For Glu Gly Asn Light Pro Ser Ser His Glu Val Ile Your 340 345 350 Gly Gin Tr? Your Pro Ihr For Ala Asp Ile Ala Arg Asn Arg Leu For 355 360 365 Gly Tyr Gly Leu Ile Ihr Asn Ile Phe Asn Glv Ala Leu Glu C / s Glv 370 375 380 Thr His Gly For Asp Asn Arg Gly Glu Asn Arg Ile Gin Phe T / r Gin 335 390 355 400 Arg Tyr Cys Asp Leu Leu Asp Wall Ser T / r Gly Asp Asn Leu Asp Gly 405 410 “415 Tyr Arg Gin Thr Pro Fhe Asp Trp Gly Leu Lys Lys Leu Gin Glv Ala 420 425 430 Arg Glu Ser Trp Ser Ser Ser

435

Information about SEQ ID No. 13:

(i) sequence characteristics:

(A) length: 23 amino acids (B) type: amino acid

190 (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical

(in) fragment type: C-terminus (vi) original source: (A) organism: Phaseolus vulgaris (xi) sequence representation: SEQ ID NO: 13:

Asn Leu Aso Cys Tyr Ser Gin Thr Pro Fhe Gly Asn Ser Leu Leu Leu 1 * 5 10 15

Ser Asp Leu Val Thr Ser Gin 20

Information about SEQ ID No. 14:

(and) sequence characteristics: (A) length: 19 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide

(iii) is not hypothetical

(iv) not anti-sense (in) fragment type: C-terminus (vi) original source:

(A) organism: Nicotiana tabacum

191 (xi) sequence representation: SEQ ID NO: 14:

Asn Leu Asp Cys Gly Asn Gin Arg Ser Fhe Gly Asn Gly Leu Leu Va ^{1 15} 10 15

Asp Thr Met l

Information about SEQ ID No. 15:

(i) sequence characteristics:

(A) length: 13 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: C-terminus (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 15:

Asn Leu Aso Cys Tyr Asn Gin Arg Asn Cys Phe Ala Gly 1 * 5 10

Information about SEQ ID No. 16:

(i) sequence characteristics:

192 (A) length: 11 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: C-terminus (vi) original source:

(A) organism: Hordeum vulgare (xi) sequence representation: SEQ ID NO: 16:

Asn Leu Asp Cys Tyr Ser Gin Arg Pro Phe Ala 1 5 10

Information about SEQ ID No. 17:

(i) sequence characteristics:

(A) length: 23 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: C-terminus (vi) original source (A) organism: Nicotiana tabacum (xi) sequence illustration: SEQ ID NO: 17:

Gly Val Ser Gly Gly Val Trp Asp Ser Ser Val Glu Thr Asn 1 5 10

Ala Thr Ala Ser Leu Val Ser Glu Met

20

193

Information about

SEQ ID NO.

18:

(i) sequence characteristics:

(A) length: 16 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 18:

Ser Thr Tyr Cys Gin Ser Tyr Ala Ala Phe Pro Pro Asn Pro 1 5 10

Ser Lys 15

Information about SEQ ID No. 19:

(i) sequence characteristics:

(A) length: 18 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris

194 (xi) representation of the sequence: SEQ ID NO: 19:

Ala Cys Val Thr His Glu Thr Gly His Phe Cys Tyr Ile Glu 1 5 10

Glu Ile Ala Lys 1 5

Information about SEQ ID No. 20:

(i) sequence characteristics:

(A) length: 10 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 20:

Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin 1 5 10

Information about SEQ ID No. 21:

(i) sequence characteristics:

(A) length: 7 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical

195 (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 21:

Gly Pro Leu Gin lle Thr Trp

5

Information about SEQ ID No. 22:

(i) sequence characteristics:

(A) length: 22 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 22:

Ser lle Gly Phe Asp 1 5 Asn Asn Ala Wall Thr 15

Gly Leu Asn Ala

Ala Phe Arg 20

Pro Glu Thr Val Ala 10

Information about SEQ ID No. 23:

(i) sequence characteristics:

(A) length: 43 amino acids (B) type amino acid (D) topology: linear

196

(ii) molecule type: peptide (iii) is not hypothetical (iv) not anti-sense (IN) fragment type: N-terminus (vi) original source:

(A) organism: Triticum aestivum (xi) sequence representation: SEQ ID NO: 23:

C-ln Arg cys Gly Glu GLn Gly Ser Asn Met Glu Cys For Asn Asn Leu 1 5 10 15 Cys Cys Ser GLn iyr Gly Tyr Cys Gly Met C-ly Gly Asp Tyr Cys Gly 20 25 30 Light Gly Czs Gin Asn Gly Ala cys 'TV * - * Thr Ser

40

Information about SEQ ID No. 24:

(i) sequence characteristics:

(A) length: 43 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (iv) not anti-sense (v) fragment type: N-terminus (vi) original source:

(A) organism: Hevea brasiliensis (xi) sequence representation: SEQ ID NO: 24:

197

Glu Gin Cys Gly Arg Gin Ala Gly Gly Lys Leu Cys Pro Asn Asn Leu

5 * 10 15

Cys Cys Ser Gin Tm Gly Trp C / s Gly Ser Thr Asn Glu Tvr Cvs Ser '25 * 30

Pro Asn His Asn Cys Gin Ser Asn Cys Lvs Asn * 35 * 40

Information about SEQ ID No. 25:

(i) sequence characteristics:

(A) length: 41 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: N-terminus (vi) original source:

(A) organism: Phaseolus vulgaris

(xi ) 2 : name arable land sec vence: SEQ ID C. 25 Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Leu Cys For Gly Gly Asn 1 5 10 15 Cys Cys Ser Gin Phe Gly Trp Cys Gly Ser Thr Thr Asn Tvr Cys Gly 20 25 30 For Gly Cys Gin Ser Gin Cys Gly Gly 35 40

198

Information about SEQ ID No. 26:

(i) sequence characteristics:

(A) length: 42 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: N-terminus (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 26:

Glu Gin Cys Gly Ser Gin Ala Gly Gly Ala Arg Cys Ala Ser Gly Leu 1 5 10 15 Cys cys Ser Light Fhe Gly Trp Cys Gly Asn Your Asn Glu Tyr Cys Gly 20 25 30 For Asp Asn Cys Gin Ser C-ln Cys For Gly

40

Information about SEQ ID No. 27:

(i) sequence characteristics:

(A) length: 33 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: N-terminus

199 (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 27:

Glu 1 Leu Cys Gly Asn Gin Ala Gly 5 Gly Ala Leu Cys Pro 10 Asn Gly Leu 15 * Cys Cys Ser Gin Tyr Gly Trp Cys Glv Asn Thr Asn Pro Tyr Cys Gly 20 25 * 30 Asn

Information about SEQ ID No. 28:

(i) sequence characteristics:

(A) length: 32 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (iv) is not anti-sense (vi) original source:

(A) organism: primer (KB-7) constructed from Beta vulgaris (xi) Sequence representation: SEQ ID NO: 28:

GACTCTAGAA AYCCRCCRYG YCARTAYGAY AC

200

Information about

SEQ ID NO.

29

(i) sequence characteristics:

(A) length: 26 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (iv) is not anti-sense (vi) original source:

(A) organism: primer (KB-9), constructed from Beta vulgaris (xi) Sequence representation: SEQ ID NO: 29:

GGAGGATCCC ARRCNAAYCA

RATHTT

Information about SEQ ID No. 30:

(i) sequence characteristics:

(A) length: 34 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (ii) molecule type: cDNA (iii) is hypothetical (vi) original source:

(A) organism: primer (270) (xi) sequence representation: SEQ ID NO: 30:

CCAAGCTTGA ATTCTTTTTT TTTTTTTTTT TTTT

201

Information about SEQ ID NO: 31: (and) sequence characteristics: (A) length: 292 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) is not hypothetical (iv) not anti-sense (vi) original source:

(A) organism: Cucumis sativus

(xi) z : illustration sequence: SEQ ID C. 31 Met Ala Ala His Light lle Thr Thr Thr Leu Ser lle Fhe Fhe Leu Leu 1 5 10 15 Ser Ser lle Phe Arg Ser Ser Asp Ala Ala Giy lle Ala lle Tyr lip 20 25 30 Gly Gin Asn Gly Asn Glu Giy Ser Leu Ala Ser Your Cvs Ala Your Giy 35 40 45 Asn Tyr Glu Fhe Wall Asn lle Ala Fhe Leu Ser Ser Fhe Gly Ser Gly 50 55 60 Gin Ala For Wall Leu Asn Leu Ala Gly His Cys Asn For Asp Asn Asn 65 70 75 80 Gly cys Ala Phe Leu Ser Asp Glu lle Asn Ser Cys Light Ser Gin Asn 35 90 95 Wall Light Wall Leu Leu Ser lle Gly Gly Gly Ala Gly Ser Tyr Ser Leu 100 105 110 Qpr Ser Ala Asp Asp Ala Light Gin Wall Ala Asn Phe lle Tip Asn Ser 115 120 125 Tyr Leu Gly Gly Gin Ser Asp Ser Arg For Leu Gly Ala Ala Val Leu 130 135 140 Asp Gly Val Asp Phe Aso lle Glu Ser Gly Ser Gly Gin Fhe Trp Asp 145 150 155 160

202

Wall Leu Ala Gin Glu 165 Leu Light Asn Phe Gly 170 Gin Wall Ile Leu Ser 175 Ala Ala For Gin Cys 180 For Ile For Asp Ala 185 His Leu Asp Ala Ala 190 Ile Light Thr Gly Leu 195 Phe Asp Ser Wall Trp 200 Wall Gin Phe Tyr Asn 205 Asn For Cys Met 210 Phe Ala Asp Asn Ala 215 Asp Asn Leu Leu Ser 220 Ser 'Trp Asn GLn

fxj ', í'

Trp Thr Ala Phe Pro Thr Ser Lys Leu Tyr Met Gly Leu Fro Ala 225 230 * 235

Arg Glu Ala Ala Pro Ser Gly Gly Phe Tle Pro Ala Aso Val Leu Tle

245 '250' 255

Ser Gin Val Leu Pro Thr Ile Lys Ala Ser Ser Asn Tyr Gly Gly Val

260 265 270

Met Leu Trp Ser Lys Ala Phe Asp Asn Gly Tvr Ser Aso Ser Ile Lys 275 280 * 285 'Gly Ser Ile Gly 290

Information about SEQ ID No. 32:

(i) sequence characteristics:

(A) length: 302 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (iv) not anti-sense (vi) original source:

(A) organism: Arabidopsis thaliana (xi) sequence representation: SEQ ID NO: 32:

203

Met Thr Asn Met Thr Leu Arg Light His Wall Ile Tyr Phe Leu 1 5 10 15 Ile Ser Cys Ser Leu Ser Light For Ser Asp Ala Ser Arg Gly Gly Ile 20 25 30 Ala Ile TVr Trp Gly Gin Asn Gly Asn Glu Gly Asn Leu Ser Ala Thr 35 40 45 Cys Ala Thr Gly Arg Tyr Ala Τ '» Wall Asn Wall Ala Fhe Leu Wall Light 50 55 60 Fhe Gly Asn Gly Gin Thr For Glu Leu Asn Leu Ala Gly His Cys Asn 65 70 75 80 For Ala Ala Asn Thr cys Thr His Phe Gly Ser Gin Wall Light Asp Cys 85 90 95 ' Gin Ser Arg Gly Ile Light Wall Met Leu Ser Leu Gly Gly Gly Ile Gly 100 105 110 Asn Tyr Ser Ile Gly Ser Arg Glu Asp Ala Light Wall Ile Ala Asp Tyr 115 120 125 Leu Trp Asn Asn Fhe Leu Gly Gly Light Ser Ser Ser Arg For Leu Gly 130 135 140 Asp Ala Wall Leu Asp Gly Ile Asp Phe Asn Ile Glu Leu Gly Ser For 145 150 155 160 Gin His Trp Asp Asp Leu Ala Arg Thr Leu Ser Light Fhe Ser His Arg 165 170 175 Gly Arg Light Ile Tyr Leu Thr Gly Ala For Gin Cys For Phe Fro Asp ISO 185 190 Arg Leu Met Gly Ser Ala Leu Asn Thr Light Arg Phe Asp Tyr Wall tr 195 200 205 Ile Gin Phe Tyr Asn Asn For For cys Ser Tyr Ser Ser Gly Asn Thr 210 215 220 Gin Asn Leu Phe Asp Ser Trp Asn Light Ttp Thr Thr Ser Ile Ala Ala 225 230 235 240 Gin Light Phe Phe Leu Gly Leu For Ala Ala For Glu Ala Ala Asp Ser 245 250 255

Gly Tyr Ile Pro Pro Asp Val Leu Thr Ser Gin Ile Leu Pro Thr Leu 260 265 270

204

Lvs Lys Ser Arg Lys Tyr Gly Gly Val Met Leu Trp Ser Lys Phe 275 280 235

Asp Asp Lys Asn Gly Tyr Ser Ser Ser Ser Ile Leu Ala Ser Val 250 * 295 300

Information about SEQ ID No. 33:

(i) sequence characteristics:

(A) length: 9 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 33:

Trp Val Gin Asn Asn Val Val Pro Tyr 1 5

Information about SEQ ID No. 34:

(i) sequence characteristics:

(A) length: 20 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 34:

205

Ala Gly Ala For Asn Val Pro lle Val Val Ser Glu 1 5 10 Trp For Ser Ala Gly Gly 15 20

Information about SEQ ID No. 35:

(i) sequence characteristics:

(A) length: 6 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Beta vulgaris (xi) sequence representation: SEQ ID NO: 35:

Leu Gin Gly Lys Val Ser 1 5

Information about SEQ ID No. 36:

(i) sequence characteristics:

(A) length: 17 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (ii) molecule type: DNA (genomic)

206 (iii) is a hypothetical (vi) original source:

(A) organism: primer (TG-1) constructed from Beta vulgaris (xi) Sequence representation: SEQ ID NO: 36:

TGGGTNCARA AYAAYGT 1 7

Information about SEQ ID No. 37:

(i) sequence characteristics:

(A) length: 17 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear

(ii) molecule type: DNA (genomic) (iii) is hypothetical (vi) original source: (A) organism: primer (TG-2), engineered Beta vulgaris (xi) sequence representation: SEQ ID NO: 37:

AAYGARATHA TGCCNAA

Information about SEQ ID No. 38:

(i) sequence characteristics:

(A) length: 17 base pairs (B) type: nucleic acid

207 (C) number of strings: single-string

(D) topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (vi) original source: (A) organism: primer (TG-3), constructed from Nicotiana tabacum and Hordeum vulgar

(xi) sequence shown: SEQ ID NO: 38:

TCRTYRAACA TNGCRAA

Information about SEQ ID No. 39: (and) sequence characteristics: (A) length: 6 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) is not hypothetical (in) fragment type: N-terminus (vi) original source:

(A) organism: Hordeum vulgare (xi) sequence shown: SEQ ID NO: 39:

Phe Ala Met Phe Asp Glu 1 5

208

Information about SEQ ID No. 40:

(i) sequence characteristics:

(A) length: 6 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 40:

Phe Ala Met Phe Asn Glu 1

Information on (i) (ii) (iii) (v) (vi) (xi)

SEQ ID NO: 41:

sequence characteristics:

(A) length: 23 amino acids (B) type: amino acid (D) topology: linear molecule type: peptide is not hypothetical fragment type: N-terminus original source:

(A) organism: Pisum sativum sequence representation: SEO ID No. 41:

Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Thr Cys Pro Asn 1 5 10

Asn Leu Cys Cys Ser Gin Tyr Gly Tyr

209

15 20 Information about SEQ ID NO: 42: (and) sequence characteristics: (A) length: 15 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) is not hypothetical (in) fragment type: N-terminus (vi) original source: (A) organism: Pisum sativum (xi) sequence representation: SEQ ID NO.

Glu Gin Cys Gly Asn Gin Ala Glv Gly Xaa Val Pro Pro Asn 1 5 10

Gly

Information about SEQ ID No. 43:

(i) sequence characteristics:

(A) length: 16 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: N-terminus

210 (vi) original source:

(A) organism: Pisum sativum (xi) sequence representation: SEQ ID NO: 43:

Glu Gin Cys Gly Thr Gin Ala Gly Gly Ala Leu Cya Pro Gly 1 5 10

Gly Leu

Information about SEQ ID No. 44: (and) sequence characteristics: (A) length: 22 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) is not hypothetical (in) fragment type: N-terminus (vi) original source:

(A) organism: Hordeum vulgare (xi) sequence representation: SEQ ID NO: 44:

Glu Gin Xaa Gly Ser Gin Ala Gly Gly Ala Thr Cys Pro Asn 1 5 10 Xaa Leu Cys Cys Ser Arg Phe Gly 15 20

Information about SEQ ID No. 45:

(i) sequence characteristics:

I - 211 - (A) length: 23 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (v) fragment type: N-terminus (vi) original source:

(A) organism: Hordeum vulgare (xi) sequence representation: SEQ ID NO: 45:

Xaa Gin Gin Gly Ser Gin Ala Gly Gly Ala THr Cys Pro Asn 1 5 10 Xaa Leu Cys Cys Ser Xaa Phe Gly Trp 15 20

Information about SEQ ID No. 46:

(i) sequence characteristics:

(A) length: 18 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 46:

Ala Ile Gly Val Asp Leu Leu Asn Asn Pro Asp Leu Val Ala 1 5 10

Thr Asp Pro Val

Information about

SEQ ID NO.

212 (i) sequence characteristics:

(A) length: 7 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 47:

Gly Pro Ile Gin Ile Ser His 1 5

Information about SEQ ID No. 48:

(i) sequence characteristics:

(A) length: 12 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) not hypothetical (vi) original source:

(A) organism: Nicotiana tabacum (xi) sequence representation: SEQ ID NO: 48:

Ser Ala Leu Trp Phe Trp Met Thr Pro Gin Ser Pro 1 5 10

213

Information about SEQ ID No. 49:

(i) sequence characteristics:

(A) length: 42 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (vi) original source:

(A) organism: primer (KB-3), constructed from Beta vulgaris (xi) Sequence representation: SEQ ID NO: 49:

CCGAAGCTTA GATCTAAACA ACAACATGTC TTCTYTYGGA CC 42

Information about SEQ ID No. 50:

(i) sequence characteristics:

(A) (B) (C) (D) length: 21 base pairs type: nucleic acid number of strands: single-stranded topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (iv) is anti-sense (vi) original source:

(A) organism: primer (KB-4), constructed from

Beta vulgaris, chitinase 4

214

(xi) sequence representation: SEQ ID NO. 50 GCACACGTAG CGCTAGCTTG G Information about SEQ ID NO: 51: (and) sequence characteristics: (A) length: 19 base pairs (B) type: nucleic acid (C) number of strings: single-string (D) topology: linear (ii) molecule type: DNA (genomic) (iii) is hypothetical (iv) is anti-sense (vi) original source: (A) organism: primer, engineered vulgaris, chitinases 4 (xi) sequence representation: SEQ ID NO. 51

from Beta

Claims (3)

PATENT CLAIMS 1. A DNA sequence comprising the sugar beet chitinase 4 DNA sequence set forth in SEQ ID NO: 1 or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the antifungal activity of the aforementioned chitinase, and i) is a characteristic portion of the DNA sequence set forth in SEQ ID NO: 1; or ii) hybridizes to the DNA sequence set forth in SEQ ID NO: 1; or iii) encodes a polypeptide having the amino acid sequence of sugar beet chitinase 4 set forth in SEQ ID NO. 2, or iv) encodes a polypeptide that reacts with an antibody raised against chitinase 4 sugar beet. 2. DNA sequence according to claim 1, characterized in that it comprises nucleotides 71-793 of the sequence The chitinase 4 DNA of SEQ ID NO: 1 encodes the hevein and functional domains of the sugar beet chitinase 4 enzyme, or an analog of this DNA sequence, or comprises nucleotides 175-793 of the chitinase 4 DNA sequence of SEQ ID NO: 1. 1 encoding a functional domain of a sugar beet chitinase 4 enzyme, or an analog of this DNA sequence. 3. A DNA sequence that encodes a chitinase isoenzyme that is at least 60% homologous to the chitinase 4 sugar beet encoded by the DNA sequence of SEQ ID NO: 1a at most 40% homologous to the chitinase 1 sugar beet encoded by the DNA sequence shown in SEQ ID No. 11. 4. A modified DNA sequence characterized by: II comprising a DNA sequence as defined in any one of claims 1-3 in which at least one nucleotide has been deleted, substituted or modified, or into which at least one additional nucleotide has been inserted to encode a polypeptide that retains the antifungal activity of chitinase 4 sugar beet or exhibits increased antifungal activity compared to chitinase 4 sugar beet. 5. A chitinase 4 DNA sequencing sequence of SEQ ID NO: 1 comprising a DNA sequence encoding the following peptide S-I-G-F-D-G-L-N-A-P-E-T-A-N-N-A-V-T-A-F-R or peptide G-P-L-Q-I-T-W or peptide TAFWFWMNNVHSVIVNGQGFG-ASI which peptides refer to the chitinase active site, or analogs thereof, in which at least one nucleotide has been deleted, substituted or modified, or into which at least one additional nucleotide has been inserted, and which exhibit the same catalytic and / or binding activities as above peptides. 6. The chitinase 4 DNA sequence of SEQ ID NO: 1, which codes for a chitinase 4 leader peptide, or analogue thereof, wherein at least one nucleotide has been deleted, substituted or modified, or into which at least one additional inIII nucleotide has been joked. and which is capable of directing the passenger polypeptide to which it is fused, out of the cell in which the fused leader and passenger polypeptide are formed, for storage in the extracellular space. 7. The chitinase 4 DNA sequence of SEQ ID NO: 1, which codes for one or more of the following epitopes of the chitinase 4 enzyme of sugar beet Peptide 1: AGKRFYTRA Peptide 2: CNPSKQYY Peptide 3: IECNGGNS Peptide 4: TARVGYYTQYCQ 8. DNA sequence according to any of claims 1 - 7, v y z water. learning s e that is plant orig- 9. Polypeptide, v y z n and seeing s e t í πι, that is encoded by the sequence DNA according to any of the claims 1 - 8. team 10. The genetic construct that it contains marked c and s e 1) a promoter operably linked to a 2) a DNA sequence as defined in any one of claims 1-8, comprising a DNA sequence of chitinase 4 or an analogue or subsequence thereof, and 3) a transcription terminator operably linked to this DNA sequence. 11. A genetic construct comprising one or more copies of a DNA sequence as defined in any one of claims 1-8, comprising a DNA sequence. IV chitinase 4 shown in SEQ ID No. 1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide that exhibits activity of a second chitinase distinct from a sugar beet chitinase 4, and / or one or more copies of a DNA sequence encoding a polypeptide it exhibits beta-1,3-glucanase activity, each of which DNA sequences are operably linked to a promoter and transcriptional terminator capable of expressing DNA sequences into functional polypeptides. 12. The genetic construct of claim 11, comprising a DNA sequence of SEQ ID NO: 7 encoding a sugar beet SE chitinase having the amino acid sequence shown in SEQ ID NO: 8, or an analog of the above DNA sequence encoding an acidic acid sequence. a chitinase having a pI of at most 4.0 and capable of hydrolyzing 1 H-chitin predominantly to hexamers, and / or a DNA sequence shown in SEQ ID No. 9, encoding a basic beta-1,3-glucanase 4 of a sugar beet having an amino acid The sequence shown in SEQ ID No. 10, or an analogue thereof, encoding a basic beta-1,3-glucanase having a pI of at least 9.0 and capable of hydrolyzing 1 H-laminarin predominantly to beta-1,3-glucan dimers. A genetic construct according to any one of the claims 11-12, characterized in that the N-terminal leader sequence is selected from the group consisting of sugar beet chitinase 1 coding regions, the sequence of which is evident from SEQ ID NO. 11, sugar beet chitinase 4, the sequence of which is evident from SEQ ID NO. 1, sugar beet beta-1,3-glucanases, the sequence of which is apparent from SEQ ID No. 9, sugar beet chitinase 76, the sequence of which is shown in SEQ ID No. 5, and acidic chitinases SE from sugar beet, whose sequence is is evident from SEQ ID No. 7. A vector that is capable of replication in a host organism and which carries a DNA sequence as defined in any one of claims 1-8, or a genetic construct as defined in any one of claims 11-13. 15. A host organism comprising a vector as defined in claim 14. A genetically transformed plant comprising in its genome a genetic construct according to any one of claims 11-13. 17. The genetically transformed plant of claim 16, selected from the group of monocotyledons comprising corn, oats, wheat, rice, barley, rye and sorghum, or from the group of dicotyledons including alfalfa, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, chenille, tomato, potato, soy, oilseed rape, cabbage, peppercorn, lettuce and peas. A genetically transformed plant according to any one of claims 16 or 17, characterized in that it exhibits increased resistance to a plant pathogen containing chitin, as compared to a plant that does not contain a genetic construct as defined in any one of claims 11-13. 19. Seeds, seedlings or plant parts, characterized in that they have been obtained by growing a genetically transformed plant according to any one of claims 16-18. 20. A transformation system characterized by e VI, comprising at least one vector that carries the genetic construct of any one of claims 11-13 and which is capable of introducing the genetic construct into the genome of a plant. 21. A transformation system according to claim 20, comprising a binary or co-integrated vector system. 22. A method of generating a genetically transformed plant which exhibits increased resistance to chitin-containing plant pathogens, such as phytopathogenic fungi, as compared to a native plant, comprising introducing the genetic construct of any one of claims 11-13 into the genome of the plant. by obtaining genetic material containing the construct and subsequently regenerating the genetic material into a genetically transformed plant. An antifungal composition comprising a polypeptide encoded by a DNA sequence as defined in any one of claims 1-8, or a genetic construct as defined in any one of claims 11-13, and a suitable vehicle. 24. A method of making an antifungal composition comprising culturing the microorganism of claim 15 in a suitable medium and under conditions causing expression of one or more polypeptides encoded by the DNA sequence of any one of claims 1-8, or a genetic construct according to any one of claims 11 13, optionally disrupting the microorganisms to release their expressed polypeptide or polypeptide content into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide or polypeptides to drying VII by freeze-drying or spray-drying, to obtain an antifungal composition comprising a polypeptide or polypeptides encoded by the aforementioned DNA sequence or the aforementioned genetic construct. 25. A method for inhibiting germination and / or growth of a plant pathogen containing chitin, such as a phytopathogenic fungus, in a plant, comprising: 1) transforming the plant or part thereof with a genetic construct as defined in any one of claims 11 13, and regenerating the resulting transformed plant or part of plant into a genetically transformed plant; and / or 2) treating the plant or part thereof, the seedling or seed from which the plant is to grow or the medium on which the plant is grown with a composition as defined in claim 23. 26. The transformation vector for pBKL4K4 contained in the strain Escherichia coli DH5alpha deposited in the German Collection of Microorganisms and Cell Cultures - Deutsche Sammlung von Microorganisms and Zellkulturen GmBH (DSM) on 30 July 1991 under the terms of the Budapest Agreement under number DSM 6635.