WO1992017591A1

WO1992017591A1 - A plant chitinase gene and use thereof

Info

Publication number: WO1992017591A1
Application number: PCT/DK1992/000108
Authority: WO
Inventors: Jørn Dalgaard MIKKELSEN; Kirsten Bojsen; Klaus K. Nielsen; Lars Berglund
Original assignee: Sandoz Ltd.
Priority date: 1991-04-08
Filing date: 1992-04-07
Publication date: 1992-10-15
Also published as: AU1659992A; CA2048696A1; HU9302829D0; IE921104A1; NZ242270A; DK61691D0; CZ209293A3; CA2048477A1; SK108193A3; AU659455B2; EP0579709A1; HUT67059A; JPH06507070A

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 β-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

A PLANT CHITINASE GENE AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to a DNA sequence encoding the sugar beet chitinase referred to in the following as "the sugar beet chitinase 4" or an analogue of said DNA sequence encoding a polypeptide having the antifungal activity of sugar beet chitinase 4, as well as to a genetic construct useful for the construction of genetically transformed plants having an increased resistance to plant pathogens containing chitin, such as phytopathogenic fungi, as compared to untransformed plants. The genetic construct comprises and is capable of expressing the DNA sequence of the invention, preferably in combination with a DNA sequence encoding a second chitinase different from sugar beet chitinase 4 and a DNA sequence encoding a β-1,3-glucanase. In another aspect, the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet plant, from which a polypeptide having the antifungal activity of the sugar beet chitinase 4 is expressed in an increased amount as compared to the untransformed plant, preferably in combination with a polypeptide having chitinase activity and a polypeptide having β-1,3-glucanase activity so as to result in an increased resistance to chitin-containing plant pathogens.

BACKGROUND OF THE INVENTION

Most plants are susceptible to infection by pathogens such as micro-organisms and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently economical loss to farmers. The plants respond to infection with several defense mechanisms including phytoalexins, deposition of lignin-like material, accumulation of cell wall hydroxyproline-rich glycoproteins, pathogenesis related proteins (PR-proteins) and increase in the activity of several lytic enzymes such as chitinases and β-1,3-glucanases. Some of these responses can be induced not only directly by infection, but also by exposure of the plant to elicitors isolated from fungal cell walls, and in some cases by exposure to exogenous chemicals such as ethylene. The full capacity of the defense mechanism of the plant is, however, normally delaved in rela tion to the onset of infection, and thus, the plant may be severely injured before its defense mechanism reaches its maximum capacity. Also, the defense mechanism of the plant may not in itself be sufficiently strong to effectively combat the infectious organism. Therefore, a normal and necessary procedure is to treat infected plants or plants susceptible to infection with a chemical, e.g. a fungicide, either as a prophylactic treatment or shortly after infection.

However, the use of a chemical treatment is neither desirable from an ecological nor from an economic point of view and it would be desirable to be able to enhance the defense of the host plant itself by introducing new and/or improved genes by genetic engineering. A further advantageous effect of this strategy would be the immediate inhibition of the fungal attack which is obtained, leading to a retarded epidemic establishment of the infecting fungi in plant crops and thus an overall reduction in the effect of the infection.

The cell walls of many phytopathogenic fungi contain chitin and glucan, the chitin constituting the major component of the tips of the hyphae. The enzymes chitinase and β- 1,3-glucanase have been shown to be capable of enzymatically digesting the fungal cell walls so as to result mainly in soluble dimers or oligomers of N-acetyl-D-glucosamine and D-glucose.

Chitinase and β- 1 ,3-glucanase activity has been observed in plant species such as tobacco, barley, potato, rice, maize, corn, bean, tomato, cucumber, wheat germv rape seed and pea and it has been shown that the chitinase activity increases in response to infection with most phytopathogenic fungi.

Plant chitinases have been purified and characterized from crop plants such as tobacco, barley, corn, tomato, bean and pea, and cDNA and genomic clones have been obtained therefrom. Plant chitinases are reviewed by Bol and Linthorst, 1990 and Boiler, 1988.

Several publications have discussed bacterial and plant chitinases and the use thereof in the construction of transgenic plants having an increased resistance to various microorganisms such as fungi. EP 0 292 435 relates basically to the regeneration of fertile Zea mays plants and mentions, inter alia, that a tobacco chitinase gene may be introduced in the plant in order to make it resistant to pathogens. Chitinase genes of other sources and other plants than Zea mays are not mentioned.

EP 0 290 123, WO 88/00976 and US 4 940 840 disclose the use of chitinases of bacterial origin in the construction of transgenic plants; chitinase of plant origin is not mentioned or alternatively only mentioned in general terms. WO 90/07001 discloses DNA constructs comprising a high level promoter operably linked to a DNA sequence encoding a plant chitinase, which constructs are used in the transformation of plants so as to achieve overexpression of chitinase in the plant and thereby conferring resistance to plant pathogenic fungi. The only plant chitinase exemplified is a bean chitinase.

EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304 and

EP 0 418 695 disclose the construction of transgenic plants

harbouring DNA sequences encoding plant pathogenesis-related proteins (PRP), e.g. chitinase and β-1,3-glucanase. Pathogenesis-related proteins from sugar beet plants or transgenic sugar beet plants are not mentioned.

EP 0 448 511 also relates to transgenic plants comprising

recombinant DNA sequences encoding hydrolytic enzymes such as chitinases and glucanases. Additionally, the reference relates to compositions comprising hydrolytic enzymes such as glucanase and chitinase for use for controlling plant pathogens. Chitinase or glucanase from sugar beet are not mentioned.

WO 91/06312 discloses a composition for protecting a harvested crop comprising endoenzymes such as glucanase or chitinase. No particular source of chitinase or glucanase is mentioned. Rousseau-Limouzin M. and Fritig B. (1991) describe the production of basic and acidic PR-proteins in sugar beets infected with Cercospora beticola and the serological relation of these PR-proteins to the PR- proteins in tobacco. The described PR-proteins are found to be serological related to tobacco PR-proteins whereas the sugar beet chitinase 4 of the present invention does not show a serological relationship to any known chitinase, confer below. No information about the amino acid sequence or nucleotide sequence of any of the PR-proteins is given. In conclusion, none of the above cited publications disclose any sugar beet chitinase enzyme or the use thereof in the construction of transgenic plants.

At the Phytochemical Society of Europe International Symposium, Norwich, United Kingdom, April 11-13, 1989: Biochemistry and Molecular Biology of Plant: Pathogen Interactions, the present inventors disclosed the isolation and purification of 6 chitinase isoenzymes, including chitinase 4, from the leaves of sugar beet plants infected by Cercospora beticola , a phytopathogenic chitin-containing fungi. The chitinase isoenzymes were characterized by their molecular weight and kinetics of chitin hydrolysis. Chitinase preparations were indicated to be capable of hydrolyzing newly synthesized chitin in the cell wall of the growing fungi. No further characterization was reported and the chitinase enzymes were not separately discussed.

At the APS/CPS (The American Phytopathological Society/The Canadian Phytopathology Society) 1990 Joint Meeting Program held August 4-8, 1990 in Michigan, USA and at the 5th International Symposium on the Molecular Genetics of Plant-Microbe Interactions held in Interlaken, Switzerland September 9-14, 1990, the present inventors presented an abstract and a poster showing the amino acid composition of various sugar beet chitinase isoenzymes, including chitinase 4, It was mentioned - for the first time - that two serologically different groups of chitinase enzymes are present in sugar beet plants. This statement was not further qualified. In the abstract and poster presented by the present inventors at the 8th Congress of the Mediterranean Phytopathological Union, held in Agadir, Morocco in October 28-November 3, 1990, it is mentioned that antibodies has been raised against wheat germ chitinase, three sugar beet chitinases and two β-1,3- glucanases and that N-terminal and partial amino acid sequencing have been performed on 5 of the total of 9 purified chitinase isoenzymes. Only the N-terminal sequence of the sugar beet chitinase isoenzyme 2 was disclosed.

In the Annual Report 1989 from the Center for Plant Biotechnology (Denmark) which issued on August 20, 1990, the above results are disclosed and it is furthermore disclosed that one of the genes encoding a sugar beet chitinase has been isolated and characterized, inter alia by nucleotide sequencing. However, although it is not directly expressed in this publication, this gene encodes chitinase 1 and not the sugar beet chitinase 4. In retrospect and in view of the disclosure of the present application, it is evident that this chitinase is not chitinase 4 because of the high degree of homology (about 50%) with other known sequences of chitinase genes from tobacco and bean. In contrast, as it appears from Example 11, there is a very low degree of homology between chitinase 4 and the other known chitinases.

None of the above cited references mention or indicate the amino acid sequence of the sugar beet chitinase 4 enzyme or of the DNA sequence encoding sugar beet chitinase 4, neither do they mention or indicate that it would be interesting to look for this sequence. In fact, the initial analysis of sugar beet chitinase 4, which revealed an enzyme with a small functional domain, suggested that the chitinase enzyme had a low chitin affinity and thus low enzymatic activity. Thus, the enzyme did not seem to be of any particular interest. Futhermore, non of the references mention or indicate the

synergistic antifungal effect which may be obtained when the sugar beet chitinase 4 enzyme is combined with an acidic chitinase and a basic β-1,3-glucanase. This synergistic antifungal effect is reported for the first time in connection with this application. By the present invention a novel plant chitinase has been elucidated which, either alone or in combination with other pathogenesis-related proteins, shows promising results in the inhibition of chitin-containing fungi.

BRIEF DISCLOSURE OF THE INVENTION

In one aspect the present invention relates to a DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO . : 1 or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 as defined herein and i) being a characteristic part of the DNA sequence shown in SEQ ID NO. :1, or ii) hybridizing with the DNA sequence shown in SEQ ID NO.:1 at 55°C under the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", or iii) encoding a polypeptide having the amino acid sequence of the sugar beet chitinase 4 shown in SEQ ID NO.:2, or iv) encoding a polypeptide being recognized by an antibody raised against sugar beet chitinase 4.

The chitinase 4 DNA sequence, SEQ ID NO.:1, shown in the Sequence Listing below was determined on the basis of a cDNA clone isolated from a sugar beet cDNA library prepared as described in the Material and Methods section below on the basis of hybridization with a very specific oligonucleotide probe. The oligonucleotide probe was prepared on the basis of a tryptic peptide produced from a substantially pure sugar beet chitinase 4 obtained as described in Materials and

Methods and in Example 1 below. The procedure used for isolating the chitinase 4 DNA sequence is outlined in Example 4 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 had not been reported, and no indication had been given that it could be interesting to look for these sequences. In fact, the initial analysis of sugar beet chitinase 4, which revealed an enzyme with a small functional domain, suggested that the chitinase enzyme had a low chitin affinity and thus low enzymatic activity. Thus the enzyme did not seem to be of any particular interest.

The elucidation of the amino acid sequence of the sugar beet chitinase 4 was an important step in the analysis of the enzyme. Thus, from the amino acid sequence it was clear that the sugar beet chitinase 4 belongs to the plant chitinases of the hevein class in that it contains a leader sequence, a hevein domain and a functional (catalytic) domain. Hevein is a lectin which binds to chitin, and the hevein domain of the enzyme is the part of the enzyme which is expected to bind to chitin and chitin-containing structures, e.g. of phytopathogenic fungi.

By hydrophobic clustering analysis using the method according to Gaboriaud et al., 1987, the primary structure of chitinase 4 has been found to be more compact than the structures of other plant chitinases belonging to the sugar beet chitinase 2 class (as described in further detail below) . It is anticipated that the compact structure of chitinase 4 is an advantage in order to allow the enzyme to get access to chitin structures, e.g. in the cell walls of phytopathogenic fungi.

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

The sugar beet chitinase 4 has been found to have a surprisingly high antifungal activity and have shown a particularly good inhibiting effect on the growth of phytopathogenic fungi. In addition, the use of a combination of the sugar beet chitinase 4 enzyme a second different chitinase and a β-1,3-glucanase in the control of phytopathogenic "fungi has been found to result in an even more improved antifungal activity as compared to the use of the sugar beet chitinase 4 alone. This synergistic antifungal effect is reported for the first time in connection with this application.

Accordingly, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence .comprising the chitinase 4 DNA sequence shown 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 different from the sugar beet chitinase 4, and one or more copies of a DNA sequence encoding a β- 1,3-glucanase, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.

The constituents 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 a genetically transformed plant having an increased resistance to chitin-containing plant pathogens such as phytopathogenic fungi as compared to plants which do not contain the construct such as untransformed or natural plants. The genetically transformed plants are advantageously prepared by use of a plant transformation vector harbouring the genetic construct of the invention.

The chitinase 4 DNA sequence or an analogue thereof, and in particular a specific subsequence thereof (which will be further discussed below), may also be used in the isolation of DNA sequences belonging to the chitinase 4 gene family as defined above. Also, the chitinase 4 DNA sequence or an analogue thereof or a genetic construct of the invention may be used in a method of preparing a polypeptide, e.g. a recombinant sugar beet chitinase enzyme, or a polypeptide mixture having a potent antifungal activity. The polypeptide or polypeptide mixture may by prepared by use of recombinant DNA techniques and may be used in the antifungal treatment of various products, especially food products.

DETAILED DISCLOSURE OF THE INVENTION

The chitinase 4 DNA sequence, SEQ ID NO . : 1 encodes the basic sugar beet chitinase 4 enzyme, the amino acid sequence of which also appears from SEQ ID NO . : 2. In the present context, the terms

"chitinase 4" and "sugar beet chitinase 4" are used interchangeably.

One characteristic feature of the chitinase 4 DNA sequence of the invention and an analogue thereof is that they encode a polypeptide having the antifungal activity of the sugar beet chitinase 4. The antifungal activity of the sugar beet chitinase 4 is characteristic in that it is a bifunctional activity constituted by a chitinase activity and a lysozyme activity. As far as the present inventors are aware, this bifunctional activity has hitherto not been reported for any other basic plant chitinase of the hevein class. In accordance herewith, the term "the antifungal activity of the sugar beet chitinase 4" denotes the characteristic bifunctional activity of the enzyme, i.e. the combination of chitinase activity and lysozyme activity found in the sugar beet chitinase 4.

The term "chitinase activity" denotes the enzyme's ability to decompose chitin and chitin-containing structures and the chitinase activity may be determined by 1) a biological assay and 2) a chemical assay. In the biological assay, the effect of chitinase 4 on growing hyphae of pathogenic fungi, i.e. the ability of chitinase 4 to destroy the hyphae walls and thereby retard the growth of the hyphae. is directly observed. In the chemical assay, the decomposition of ³H-chitin by chitinase 4 to result in mainly dimers of chitin is monitored. The biological assay may be carried out using any of the 3 different methods described in "Materials and Methods" herein under the heading "Antifungal activity". When a positive result is obtained in any of these methods, i.e. the observance of destruction of the hyphae walls and retardation of the growth of the fungal hyphae, it is taken as evidence of biological chitinase 4 activity.

The chemical assay may be carried out as described in "Materials and Methods" under the heading "The radiochemical chitinase assay".

Chitinase 4 activity is shown by hydrolysis of ³H-chitin and the resulting formation of mainly dimers of chitin in this assay.

The term "lysozyme activity" denotes the enzyme's fungal cell wall lysing ability. The lysozyme activity is determined by carrying out the lysozyme assay described in "Materials and Methods" under the heading "Lysozyme assay". It will be understood that the antifungal activity of the sugar beet chitinase 4 is a qualitative as well as a quantitative measure reflecting the ability of the polypeptide to destroy components e.g. chitin, of the hyphae walls of a phytopathogenic fungus thereby inhibiting or retarding the growth of the fungus. The analogue of the chitinase 4 DNA sequence is a DNA sequence having at least one of the properties i)-iv) listed above. The terms used to define the analogues of the invention are explained in further details below.

The term "characteristic part" as used in connection with the analogue defined in i) above denotes a nucleotide sequence which is obtained from the nucleotide sequence of the chitinase 4 DNA sequence or which has a nucleotide sequence corresponding to a part of the chitinase 4 DNA sequence and which encodes a polypeptide having retained the antifungal activity of sugar beet chitinase 4. Typically, the characteristic part comprises a subsequence of the chitinase 4 DNA sequence, the subsequence being either a consecutive stretch of nucleotides of the chitinase 4 DNA sequence or being composed of one or more separate nucleotide sequences of the chitinase 4 DNA sequence. In order to allow the polypeptide encoded by the characteristic part of the chitinase 4 DNA sequence to retain its characteristic antifungal activity, the part will normally be only a small number of nucleotides shorter than the chitinase 4 DNA sequence, e.g. 1-50, such as 1-25 nucleotides shorter.

A typical example of a characteristic part of the chitinase 4 DNA sequence includes the nucleotides encoding the active site of chitinase 4. The analogue defined in ii) above is a DNA sequence which hybridizes with the chitinase 4 DNA sequence under the conditions specified in the "Materials and Methods" section below under the heading "Identification of DNA belonging to the chitinase 4 gene family". The conditions defined for the hybridization to take place are based on hybridization experiments carried out with a number of known plant chitinases and sugar beet chitinase 4 and is further described in Example 11 below.

In the present context, any DNA sequence hybridizing with the chitinase 4 DNA sequence under the hybridization conditions specified in the above cited part of "Material and Methods" is defined as belonging to the chitinase 4 gene family and is contemplated to encode a polypeptide having the structure and antifungal activity of the sugar beet chitinase 4. Furthermore, when the polypeptides produced from such DNA sequences react with .antibodies raised against sugar beet chitinase 4, it is a strong indication that the polypeptide encoded by the DNA sequence in question belongs to the sugar beet chitinase 4 serological class. Such DNA sequences constituting part of the present invention may either comprise sequences isolated from natural sources, e.g. plants, synthetically produced sequences or may be synthetically modified DNA sequences, e.g. as described below. In the following, DNA sequences belonging to the chitinase 4 gene family are also termed "chitinase 4 related DNA sequences".

The analogue defined in iii) above is a DNA sequence which encodes a polypeptide comprising the amino acid sequence shown in SEO ID NO. : 2, i.e. the amino acid sequence of the mature chitinase 4 enzyme. It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia , to the preference of the organism in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the chitinase 4 DNA sequence of the invention may be exchanged by others which, when expressed, result in a polypeptide identical to or substantially identical to the polypeptide encoded by the chitinase 4 DNA sequence in question.

The analogue defined in iv) above is a DNA sequence encoding a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4. In the present context, the term "is recognized by" is used interchangeably with "binds to". As it is described in Example 3 below, it has been found that the sugar beet chitinase 4 enzyme belongs to a new serological class of basic chitinases hitherto not reported in the literature. A recent serological analysis of a rape seed chitinase has revealed a close serological resemblance between this chitinase and sugar beet chitinase' 4, indicating that the analyzed rape seed chitinase belongs to the same new class of basic chitinases. The antibody to be used in determining the serological relationship between the polypeptide encoded by the 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 prepared against one or more characteristic epitopes encoded by the chitinase 4 DNA sequence. Such epitopes are explained in further detail below.

The DNA sequences of the invention explained herein may comprise natural as well as synthetic DNA sequences, the natural sequence typically being derived directly from cDNA or genomic DNA, normally of plant origin, e.g. as described below. A synthetic sequence may be prepared by conventional methods for synthetically preparing DNA molecules, e.g. using the principles in solid or liquid phase DNA synthesis such as a DNA synthesizer 381 A (Applied Biosystems). Of course, also the DNA sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic and mixed genomic and synthetic origin.

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

The chitinase 4 DNA SEQ ID NO . : 1 comprises a leader sequence

(nucleotides 2-70) encoding 23 amino acid residues, a part

(nucleotides 71-174) encoding a hevein domain of 35 amino acid residues and a part (nucleotides 175-793) encoding a functional domain of 206 amino acid residues. The N-terminal part of the mature polypeptide chain is blocked and it has not been possible to determine the sequence by conventional amino acid sequencing methods. However, based on comparison with the DNA sequences of a wheat germ agglutinin (WGA-A) and a potato chitinase and based on an analysis by electrospray mass spectrometry (vide Example 4), the start codon of the chitinase 4 DNA sequence has been deduced.

Comparison between the leader sequence from chitinase 4 DNA (SEQ ID NO:1) and the leader sequence from the genomic chitinase 4 DNA (SEQ

ID NO.:3) shows that the two first nucleotides in the leader sequence from chitinase 4 DNA (SEQ ID NO.:1) are missing. Thus, while the leader sequence of the genomic chitinase 4 consists of 24 amino acid residues (SEQ ID NO.:4), the leader sequence from chitinase 4 consists of 24 amino acid residues although the almost full length chitinase form cDNA is missing the first amino acid Met (SEQ ID NO . : 2).

Plant chitinases may be divided into 3 different groups, the hevein class, the non-hevein class and the cucumber class. Sugar beet chitinase 4 is a basic chitinase belonging to the hevein class. However, it is distinctly different from the other basic chitinases of this class. Whereas chitinases from bean, tobacco, tomato, potato, pea, poplar, barley (T and K) and sugar beet (chitinase 2) have molecular weights of 32-38 kDa (vide Example 10), chiti nase 4 is smaller with a molecular weight of about 26 kDa (as determined for the mature enzyme). In addition, since antibodies raised against chitinase 4 do not recognize the other basic

chitinases described above (vide Example 10), it is evident that chitinase 4 also belong to a different serological class than all other basic plant chitinases from the hevein class.

The primary structure of the mature chitinase 4 as determined on the basis of its amino acid sequence contains 2 different domains: the hevein domain and the functional domain. At the N-terminal part of the polypeptide chain, 12 out of 35 amino acid residues are conserved compared to the hevein structure. The functional domain contain 206 amino acid residues. In the basic chitinase from 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 the tobacco chitinase, chitinase 4 has a binding affinity which is of a similar magnitude as that of the other basic chitinases belonging to the hevein class. For comparison, very poor or no binding is observed when chitinases from the non-hevein class are examined. This class of chitinases does not contain the hevein domain, but only the 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 the chitinases from the hevein-class recognize the chitinases from the non-hevein class.

In general, the specific activity of the non-hevein class, the acidic chitinase from tobacco and the basic chitinase C from barley (Kragh K. M., Thesis, 1990) are approximately 6-fold lower than that of the hevein class chitinases. Since the functional domain in chitinase 4 contains only 206 amino acid residues as compared to the 263 amino acid residues of the functional domain of the basic tobacco chitinase, a decrease in the specific activity was expected. Chitinase' 4, however, performs extremely well and was by the present inventors shown to be superior to chitinase T. K. and C from barley (results not shown) when ana lyzed by the radiochemical enzyme assay described in "Material and Methods" below.

From the above explanation, it will be 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 especially the part encoding the functional domain of the enzyme. While the presence of a leader sequence in most cases is a prerequisite for allowing the polypeptide expressed from the DNA sequence to be transported out of the cell in which it is produced, the nature and origin of the particular leader sequence to be used may vary and need not be the leader sequence naturally associated with the chitinase 4 enzyme. Additionally, the leader sequence naturally associated with the chitinase 4 enzvme may be used in heterologous gene construct in transformation in plants, in particular sugar beet plants, when the encoded polypeptides are targeted to the extracellular space.

In accordance herewith, a particularly interesting DNA sequence according to the present invention is a DNA sequence comprising nucleotides 71-793 of the chitinase 4 DNA sequence shown in SEQ ID NO.:1 and encoding the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

The term "analogue" is referred to as a DNA sequence which either

Ai) is a characteristic part of said DNA sequence,

Aii) hybridizes with a DNA probe prepared from said DNA sequence, Aiii) encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or

Aiv) encodes a polypeptide which is recognized by an antibody raised against a polypeptide encoded by said DNA sequence.

A still more interesting DNA sequence of the invention is a DNA sequence comprising nucleotides 175-793 of the chitinase 4 DNA se quence shown in SEQ ID NO.:1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence. The term "analogue" refers to a DNA sequence which

Bi) is a characteristic part of said DNA sequence, Bii) hybridizes with a DNA probe prepared from said DNA sequence,

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

Biv) encodes a polypeptide which is recognized by an antibody raised against a polypeptide encoded by said sequence.

The analogues as defined by the properties Ai)-Aiv) and Bi)-Biv) above are defined in a similar manner to the analogues of the chitinase 4 DNA sequence defined by the properties i)-iv) above.

In a further aspect, the present invention relates to a DNA sequence comprising a sugar beet chitinase 4 gene. In the present context, the term "gene" is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5'-upstream or 3'-downstream region. The 5'-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter. The 3' -downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.

An example of a DNA sequence of the invention comprising a chitinase 4 gene is the genomic sugar beet DNA sequence harboured in the genomic chitinase 4 clone (chit 4), the isolation of which is described in Example 4. The partial nucleotide sequence of the gene has been elucidated and is shown in SEQ ID NO.: 3. Based on comparison of the partial DNA sequence with the DNA sequence of the chitinase 76 gene shown in SEQ ID NO.:5 and further discussed below, and the nucleotide sequence of the chitinase 4 cDNA shown in SEQ ID NO.:1, (the comparisons are shown in Fig. 24) it is contemplated that nucleotides 356-358 of the chitinase 4 gene sequence constitute the start codon of the chitinase 4 gene.

Based on a comparison with the chitinase 76 sequence (comprising one intron) and the DNA sequence of chitinase 1 shown in SEQ ID NO.: 11 (comprising two introns), it is believed that the chitinase 4 gene comprises only one intron starting at nucleotide 398 downstream of the ATG start codon. The position of the intron is believed to correspond to a position between nucleotides 395 and 396 in the chitinase 4 cDNA sequence shown in SEQ ID NO.:1.

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

As mentioned above, the knowledge of the amino acid sequence of the sugar beet chitinase 4 makes it possible to analyze the enzyme and elucidate the important parts of the enzyme, this being done, e.g., on the basis of a comparison with the amino acid sequence of other known chitinases. An especially interesting part of the enzyme is, for instance, a part comprising the active site of the enzyme, a part comprising epitopes of the enzyme and a part responsible for the enzyme's substrate specificity and/or binding properties.

The contemplated position of the active site of the sugar beet chitinase 4 enzyme has been revealed by comparison to the active site of other known enzymes catalyzing the hydrolysis of other oligosaccharides such as explained in Example 16 below. Thus, it is believed that the active site of the sugar beet chitinase 4 is constituted by amino acid residues 183 (Asp) and 189 (Glu) in SEQ ID NO.:2. On the basis of the 3D-structure of the chitinase 4 enzyme which may be elucidated by use of conventional x-ray crystallography analysis and the amino acid sequence of the enzyme, it will be possible to predict parts of the enzyme which are responsible for the enzyme's specific properties. Thus, in addition to the active site disclosed above, also the specific amino acids of the enzyme responsible for its substrate specificity and substrate binding may be envisaged or elucidated. Also, the amino acid residues forming the epitopes of the enzyme may be elucidated. On the basis of the knowledge of such specific amino acids it is possible to specifically modify the enzyme so as to obtain a modified mode of action of the enzyme, e.g. with respect to an increased catalytic activity, an improved, i.e.

broadened, substrate specificity, an improved substrate, e.g. chitin, binding or a modified epitope. Such modifications may be accomplished by use of well-known principles of protein engineering, such as sitedirected mutagenesis, e.g. as described in Example 16 below.

As an example, the replacement of one or more of the Trp residues in position 169, 204 and 206 with Tyr residues is expected to change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity. Likewise, changes of the amino acid residues constituting the active site or amino acid residues which form the structure of the folded enzyme are expected to influence, e.g., the catalytic activity, substrate specificity and/or substrate binding may be found to result in improved properties of the resulting modified enzyme. Of course, the nature of the modification to be carried out will depend on the desired result, i.e. the specific desired function of the resulting modified enzyme.

Corresponding to the chitinase 4 enzyme encoded by a DNA sequence of the invention, a DNA sequence, encoding the modified chitinase 4 enzyme may either alone or in combination with DNA sequences encoding other proteins, e.g. pathogenesis related proteins, such as thaumatin, osmothin and/or zeamatin (Viegers, 1991) or thionin

(Bohlmann et al., 1988), cercropin (J. Jaynes, 1989) or other enzymes such as chitinases and β-1-3-glucanases be used in the construction of a genetically transformed plant, preferably a sugar beet plant, having a particularly high and advantageous antifungal activity.

Also, the modified chitinase 4 enzyme may prove to be a particular interesting component of an antifungal composition as described below. Within a gene family, a high degree of homology between coding regions of the genes is expected, whereas less homology is expected between non-coding regions. Between different gene families, the homology may vary considerably. The term "homology" is used here to denote the presence of the degree of complementarity between the amino acid sequence of a given polypeptide and the amino acid sequence of another polypeptide being analyzed as determined by use of the computer program by Myers and Miller, version 1.05, September 1990, using the 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 the coding regions, may thus be used to assess the degree of familarity between different genes. The amino acid sequences may be deduced from a DNA sequence or may be obtained by conventional amino acid sequencing methods. The degree of homology is preferably determined on the basis of mature proteins, i.e. without taking any leader sequence into account .

In accordance herewith, the present invention relates to a DNA sequence encoding a chitinase isoenzyme which is at least 60% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO.:1 and at the most 40% homologous with the sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO.: 11. The minimum degree of homology of at least 60% has been determined on the basis of an analysis of a rape seed chitinase (based on the mature protein) which has been shown to belong to the sugar beet chitinase 4 serological class. (see Example 11). The degree of homology of 40% with chitinase 1 (which does not belong to the chitinase 4 class) reflects the minimal degree which is expected to be acceptable for a polypeptide belonging to the chitinase 4 class. Of course, a higher degree of homology with the chitinase 4 enzyme and therefor a lower degree of homology with the chitinase 1 enzyme reflects an even higher similarity herewith and accordingly, the DNA sequence described above preferably encodes a chitinase isoenzyme which is at least 65%, e.g. at least 70% homologous, such as at least 75% or preferably 80% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO . : 1 and/or at the most 38% such as at the most 35% homologous with the sugar beet chitinase 1 enzyme encoded by the DNA sequence SEQ ID NO.: 11.

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

From Example 10 it is evident 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 the sugar beet chitinase 2. This is a very strong indication of the fact that the sugar beet chitinase 4 belongs to a different class of chitinases than the sugar beet chitinase 2 and thus that 2 different classes of sugar beet chitinases exist. It is contemplated that other polypeptides belonging to the chitinase 4 family will show a similar reaction pattern and accordingly, the present invention further comprises a DNA sequence which encodes a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4, but not by an antibody raised against sugar beet chitinase 2.

In a further aspect, the present invention relates to a modified DNA sequence comprising a DNA sequence as defined above comprising the chitinase 4 DNA sequence or gene or an 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 having retained the antifungal activity of the sugar beet chitinase 4 or having an increased antifungal activity as compared to the sugar beet chitinase 4. The polypeptide encoding by the modified DNA sequence has normally an amino acid sequence which is different from the amino acid sequence of the sugar beet

chitinase 4. It will be understood that a modified DNA sequence of the invention will be of importance in the preparation of novel polypeptides having an increased antifungal activity as compared to chitinase 4. When "substitution" is performed, one or more nucleotides in the full nucleotide sequence are replaced with one or more different nucleotides, when "addition" is performed, one or more nucleotides are added at either end of the full nucleotide sequence, when "insertion" is performed one or more nucleotides within the full nucleotide sequence is inserted, and when "deletion" is performed one or more nucleotides are deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it.

A modified DNA sequence may be obtained by well-known methods, e.g., by use of site-directed mutagenesis.

In a further aspect, the present invention relates to a subsequence of the chitinase 4 DNA sequence of SEQ ID NO . : 1 encoding a

polypeptide which need not, but which can have the antifungal activity of the sugar beet chitinase 4. Especially interesting subsequences of the chitinase 4 DNA sequence or of the genomic DNA sequence are subsequences comprising the nucleotide sequence defining the active site of the sugar beet chitinase 4 enzyme. An example of such a subsequence is a DNA sequence comprising the active site of the sugar beet chitinase 4 enzyme, e.g. the DNA sequence encoding the following peptide named peptide 4-22 (shown by use of the

conventional one-letter amino acid code) consisting of the amino acids No's. 179-200 of 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 the tryptic peptide 4-22 obtained from the purified sugar beet chitinase 4 as described in

Example 16 below. A DNA sequence encoding this polypeptide may be of significant importance for carrying out modifications of the active site with the aim of improving the antifungal activity of the resulting polypeptide. Furthermore, the DNA sequence may be fused to a part of another DNA sequence encoding an enzyme different from the sugar beet chitinase 4 or substituted with a part of such enzyme encoding the active site thereof with the aim of obtaining a hybrid enzyme having the 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 so as to provide a useful conformation around the active site.

A further interesting DNA sequence encoding a part of the chitinase 4 enzyme is a DNA sequence encoding the polypeptide having the following amino acid sequence consisting of the amino acids No's. 183-204 of 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 deduced from the DNA sequence of the genomic chitinase 76 clone shown in SEQ ID NO.: 5 and corresponds almost to the DNA sequence of the peptide 4-22 given above, except for the most important fact that the bolded D is an N in peptide 4-22. It is believed that the chitinase 76 derived polypeptide may have the same or nearly the same interesting properties and uses as the peptide 4-22. Two further interesting DNA sequences are the sequence encoding the following peptide consisting of the amino acids No's. 163-169 of 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 the amino acids No's. 201-224 of SEQ ID NO.: 2

T-A-F-W-F-W-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 below. The peptides comprises one and two Trp-residues, respectively. The Trp-residues are contemplated to be involved in the active site and/or substrate specificity of the chitinase 4 enzyme, e.g. as further discussed in Example 16 below. Analogues of these above mentioned 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 the catalytic and/or binding activities as that of the three above-mentioned peptides encoded by the chitinase 4 DNA subsequences may be very interesting.

Another example of an interesting subsequence according to the invention is a subsequence of the chitinase 4 DNA sequence of 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 in which at least one additional nucleotide has been inserted and which subsequence is encoding a polypeptide capable of binding to chitin as determined by affinity column chromatography on regenerated chitin prepared as described in "Materials and Methods" under the heading "Preparation of a chitin column".

Due to the fact that the hevein domain of the chitinase 4 enzyme is compact and believed to be very efficient, i.e. capable of establishing an intimate binding to chitin, this domain may prove to be very useful in the modification of chitinases, such as other plant chitinases, containing either a weak or no hevein domain with the aim of conferring a stronger chitin-binding capability to such chitinases. Examples of chitinase which could advantageously be modified by insertion of the DNA sequence encoding the hevein domain of sugar beet chitinase 4 are chitinases of the non-hevein class or cucumber class (e.g. the sugar beet chitinase SE disclosed herein).

A further interesting subsequence of the present invention is a subsequence of the chitinase 4 DNA sequence SEQ ID NO.:1 encoding the leader peptide of chitinase 4 or an 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 passenger polypeptide to which it is fused out of the cell in which the fused leader and passenger polypeptide is produced to be deposited in the extracellular space.

As explained above, epitopes of the sugar beet chitinase 4 enzyme may be used to raise monospecific polyclonal and monoclonal antibodies which are useful in identifying chitinase 4 isoenzymes belonging to the chitinase 4 serological class and for epitope mapping. Suitable epitopes are expected to be found among the hydrophilic peptides of the chitinase 4 amino acid sequence SEQ ID NO.:2, because these peptides seem to be substantially different from peptide parts of other chitinases than sugar beet chitinase 4. Antibodies (either monoclonai, monospecific or polyspecific) may be prepared by use of conventional methods, e.g. as described in the Materials and Methods section below on the basis of synthetically produced peptide parts of the sugar beet chitinase 4 enzyme. Based on a conventional computer analysis of the chitinase 4 DNA and amino acid sequence, the following possible epitopes of the sequence SEQ ID NO.: 2 have been identified:

Peptide 1 AGKRFYTRA (consisting of amino acids No's 87-95)

Peptide 2 CNPSKQYY (consisting of amino acids No's 153-160)

Peptide 3 IECNGGNS (consisting of amino acids No's 230-237)

Peptide 4 TARVGYYTQYCQ (consisting of amino acids No's 241-252)

These epitopes are believed to be particularly suitable for the production of monospecific antibodies to sugar beet chitinase 4.

Peptide 1 and Peptide 4 are believed to be the most suitable peptide sequences to be used in the production of monospecific antibodies to chitinase 4.

A DNA sequence comprising a subsequence of the present invention in which one or more nucleotides have been modified, e.g. as explained above, and having substantially retained the function and/or characteristics of the subsequence should be understood as being within the scope of the present invention.

As mentioned above, bacterial as well as plant chitinases exist. In the present context in which an important use of the DNA sequence of the invention is explained which is the construction of genetically transformed plants, the most interesting types of chitinases are believed to be plant chitinases, and accordingly it is preferred that the DNA sequence of the invention or an analogue or a subsequence thereof is of plant origin. Especially interesting plant chitinase DNA sequences are derived from a member of the family Chenopodiaceae, Solanaceae, Apiaceae, Brassicaceae, Cucurbitaceae or Fabaceae. Examples of such plants are corn, alfalfa, oat, wheat, rye rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea. The terms "sequence", "subsequence" and "analogue" as used herein with respect to sequences, subsequences and analogues according to the invention should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in isolated, purified, in vitro or recombinant form. The chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof as defined above and especially a single stranded DNA or RNA sequence which is substantially complementary to either strand of such a DNA sequence may be used to isolate corresponding sequences from other plants, whereupon they, if desirable, may be modified as described herein.

From the above explanation it will be clear that the chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may be fused to one or more second nucleotide sequences encoding a second polypeptide or part thereof under conditions which ensure that at least part of the DNA sequence of the invention is expressed in conjunction with the other nucleotide sequence(s), e.g. in the form of a fusion protein. For instance, a DNA sequence of the invention encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 enzyme may advantageously be fused to a C-terminal sequence encoding a signal peptide which gives rise to transport of the fusion protein expressed therefrom to specific organelles of the organism expressing the polypeptide. Signal peptides involving transport will be discussed in further detail below. Interesting subsequences of the chitinase 4 DNA sequence, such as those described above, e.g. a subsequence encoding the hevein domain and/or an epitope, may likewise be fused to DNA sequences encoding other proteins, such as enzymes, e.g. chitinases, in order to confer to the proteins the desirable properties of the polypeptides encoded by the subsequences of the chitinase 4 DNA sequence. Also within the invention is a polypeptide encoded by the chitinase 4 DNA sequence or an analogue or subsequence thereof as defined above, preferably in a non-naturally occurring or recombinant form. As compared to the naturally occurring chitinase 4 enzyme, the polypeptide of the invention has the advantage that it may be easily produced in large quantities by use of well known conventional recombinant productions techniques, e.g. as described in Sambrook et al., 1989, and that it may be obtained in a form which is free from impurities normally associated with the naturally occurring sugar beet chitinase 4. The polypeptide of the invention may be used as a constituent in an antifungal composition, e.g. as described below.

As it is explained above and in the examples to follow, the sugar beet chitinase 4 enzyme has been shown to have a number of advantageous properties including a surprisingly high antifungal activity as compared to other known chitinases such as other known sugar beet chitinases, probably due to its dual chitinase/lysozyme activity and its compact structure. Also, the strong hevein domain of the sugar beet chitinase 4 enzymes adds to its advantageous properties. Thus, the use of a DNA sequence encoding the sugar beet chitinase 4 or an analogue thereof encoding a polypeptide having the antifungal activity as defined above is expected to be very interesting in the construction of genetically modified plants having an increased resistance to phytopathogenic fungi as compared to untransformed plants. Accordingly, in another important aspect, the present invention relates to a genetic construct comprising

1) a promoter functionally connected to

2) a DNA sequence comprising a chitinase 4 DNA sequence or an analogue or a subsequence thereof as defined above and 3) a transcription terminator functionally connected to the DNA sequence. The genetic construct may be used in the construction of a genetically modified plant in order to produce a plant showing an increased antifungal activity as determined by the procedure given in Example 2 and thus an increased resistance towards phytopathogenic fungi.

Furthermore, it is contemplated that the genetic construct may be used in increasing the chitin-degrading capability of a plant. An example of a genetic construct as defined above is given in Example 18 below.

Furthermore, experiments have revealed (vide Example 2) that when phytopathogenic fungi (C. beticola and T. viride) are treated with a composition comprising a polypeptide having the antifungal activity of the sugar beet chitinase 4 in admixture with an acidic chitinase and a basic β-1,3-glucanase the growth rate of the fungal hyphae is drastically reduced and the number of germinating spores are decreased. In this connection, it is contemplated that the synergistic effect will be observed in general when the sugar beet chitinase 4 is used in combination with other chitinases and β-1,3-glucanases, preferably of plant origin.

Thus, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID NO. :1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide having the activity of a second chitinase different from the sugar beet chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having β-1,3-glucanase activity, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides. The polypeptides with chitinase or β- 1,3-glucanase activity is preferably of plant origin. The chitinase and β- 1,3-glucanase activity may be determined as explained in the section "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 shown in SEQ ID NO.:1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding an acidic chitinase having a pI equal to or less than 4.0, and one or more copies of a DNA sequence encoding a basic β- 1,3-glucanase having a pI of at least 9.0, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.

In the present context, an "acidic chitinase" is defined as a chitinase having a pi of less than 4.0. Preferably, the acidic chitinase is a chitinase which hydrolyses chitin into chitooligosaccharides of the hexamer type. The acidic chitinase is preferably of plant origin. Examples of such chitinases are cucumber lysozyme/chitinase and

Arabidopsis as well as the acidic sugar beet chitinase SE having the DNA sequence shown in SEQ ID NO.: 7 and the amino acid sequence shown in SEQ ID NO.: 8 or an analogue of said DNA sequence encoding an acidic chitinase having a pi of at the most 4.0 and preferably capable of hydrolyzing ³H-chitin into mainly hexamers .

In the present context, the term "basic β- 1 ,3-glucanase" means a β -1 ,3-glucanase having a pi of more than 9.0. Preferably, the basic β -1 ,3-glucanase is one which is capable of hydrolyzing glucan into mainly dimers, e.g. as determined by the ³H-laminarin assay described in the Materials and Methods section below. The basic β- 1,3-glucanase is preferably of plant origin. Examples of a suitable basic β-1,3- glucanase are basic β-1,3-glucanases derived from tobacco (Shinshi et al., 1990), barley (Fincher et al., 1986) or sugar beet. e.g. the basic sugar beet β-1,3-glucanase 4, the DNA sequence of which is shown in SEQ ID NO.: 9 or an analogue thereof encoding a basic β-1,3-glucanase having a pi of at least 9.0 and preferably being capable of hydrolyzing ³H-laminarin into mainly dimers of β-1,3-glucan. The basic sugar beet β-1,3-glucanase 4 is different from other plant β-1,3-glucanases in that it does not contain a C-terminal extension as appears from the amino acid sequence SEQ ID NO.: 10. The advantageous effect of using the basic sugar beet β-1,3-glucanase 4 may in part be due to this lacking C-terminal extension.

Another interesting sugar beet chitinase is the sugar beet chitinase 1 which shows a very low homology with the sugar beet chitinase 4 of the present invention, confer above. The DNA sequence of the sugar beet chitinase 1 is shown in SEQ ID NO.: 11. The DNA sequence is about 6.3 kb long and encodes a polypeptide having 439 amino acid residues. The polypeptide shown in SEQ ID NO.: 12 contains a leader sequence of 26 amino acid residues, a hevein domain of 20 amino acid residues and a C-terminal extension of 23 amino acids. Additionally, the sequence contains a most interest proline rich domain of 238 amino acids which forms and interest aspect of the present invention.

The experiments reported in Example 2 below show that the combination of the sugar beet chitinase 4 enzyme, an acidic chitinase and a basic β-1,3-glucanase results in an increased antifungal activity as compared to the antifungal activity of each of the constituents. The increased antifungal activity observed when using this specific combination is partly believed to be due to the different mode of action of the acidic chitinase, basic β-1,3-glucanase and sugar beet chitinase 4, respectively. When the acidic chitinase is one which hydrolyses chitinase primarily into hexamers (as compared to chitinase 4 which primarily hydrolyses chitin into dimers) and the basic β-1,3-glucanase is one which hydrolyses glucan primarily into dimers, it is believed that these different cleaving modes may be involved in the resulting advantageous total effect. Furthermore, the synergistic effect obtained when using a combination of the sugar beet chitinase 4, a polypeptide having the activity of a second chitinase different from chitinase 4, e.g. an acidic chitinase, and a polypeptide having the activity of a β-1,3-glucanase, e.g. a basic β-1,3-glucanase, is believed to be due to the fact that such combination will attack both the chitin and glucan constituents of the cell wall of phytopathogenic fungi and also parts of the cell wall in which the chitin and glucan constituents are intimately cross- linked to one another. The β-1,3-glucanase further serves to remove the outer glucan layer covering the chitin structure of chitin containing plant pathogens, e.g. phytopathogenic fungi, resulting in an exposure of the chitin structure to the enzymatic action of the chitinase.

DNA sequences encoding the second chitinase referred to above and the β-1,3-glucanase may be obtained, e.g. from already known sources, or may be identified and isolated from natural sources, e.g. by use of the techniques disclosed herein.

It will be understood that a large number of different genetic constructs as defined above may be designed and prepared. Without being an exhaustive list, elements of the genetic constructs which may be varied are the number of copies 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 connected to each DNA sequence, and the type of any other associated sequences, e.g. a C-terminal or N-terminal sequence (described below). Thus, genetic constructs of the present invention may vary within wide limits.

Normally, the combination of each of the above mentioned variable elements of the genetic construct to be chosen will depend, e.g. on the desired strength of the antifungal effect to be obtained which may be determined as a function of gene dosage and specific nucleotide sequence of each of the DNA sequences, and the type and strength of the promoter and terminator used for each DNA sequence. Also, expression in specific parts of the plant with respect to organs and intracellular and extracellular location may be varied with different types of promoter and terminator. However, in designing a genetic construct of the invention which is to be expressed in a given organism such as a plant, one must be aware of the possible toxic effect of a too high expression of one or more of the proteins encoded by the genetic construct which, e.g., may lead to a lower yield of the transformed organism, e.g. plant, as compared to an untransformed organism or an organism not containing the genetic construct. Also, when the genetic construct of the invention is too large, it may be difficult to obtain a stable introduction thereof into the genome of the plant which may lead to excision of a part of or the entire genetic construct from the genome of the plant. Thus, the genetic construct should be adapted so that the expression products therefrom are generally acceptable to the host organism.

The number of copies of the DNA sequences of the genetic construct of the invention together with the activity of the genes will determine the optimal number of copies of the DNA sequences of the genetic construct of the invention. With the fast increasing knowledge within the field of plant genetic engineering, improved transformation and biological containment techniques may be developed leading to the possibility of introducing larger foreign genetic fragments into a plant without causing retarded growth, retarded yield or recombinational events than what is at present possible.

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

As mentioned above, a significant antifungal effect is obtained from a protein encoded by the chitinase 4 DNA sequence of the invention or an analogue thereof. Accordingly, it is contemplated that a genetic construct of the invention, in which two copies of the chitinase 4 DNA sequence of the invention or an analogue thereof, and one copy of each of the DNA sequences encoding an acidic chitinase and a basic β-1,3-glucanase are present may show very potent antifungal effects when present in a genetically transformed plant of the invention. It is believed that such a genetic construct will not pose a too heavy burden on the plant in which it is harboured. Of course, also the choice of e.g. promoter used for each DNA sequence will influence the amount of protein expressed therefrom. This will be further explained below.

The genetic construct of the invention as described above may be present on one or several DNA fragments. Depending on the size of the genetic construct to be introduced in an organism such as a plant, in the case of a plant typically by means of a plant transformation vector, and the combination of promoters and transcription

terminators, it may be advantageous to introduce the construct by use of two or more plant transformation vectors, and accordingly it may be advantageous that the genetic construct is present on two or more DNA fragments. When the use of only one plant transformation vector is desirable, it is advantageous that the genetic construct is present on one DNA fragment.

When a polypeptide encoded by the DNA sequence of the invention is to be expressed in an organism, e.g. in a plant, it is desirable that the DNA sequence further comprises a nucleotide sequence encoding a leader sequence. The leader sequence may be the natural leader sequence, or a leader sequence derived from DNA encoding another protein. In any event, the leader sequence is to be functionally connected to the DNA sequence so that the polypeptide expressed from the resuiting nucleotide sequence serves to direct the polypeptide encoded by the DNA sequence out of the cell in which it is produced.

Depending of the nature of the leader sequence employed, the

polypeptide may be directed to specific locations of the organism in which it is produced, e.g. to lysosomes or vacuoles, or the passenger polypeptide may be excreted into the intracellular room. The leader sequence may be either N-terminally or C-terminally positioned.

The nature of the N-terminal sequence to be used will e.g. depend on the particular organism and the part thereof, e.g. the specific cell or tissue, in which the polypeptide encoded by the DNA sequence of the invention is to be produced and to which part of the same cell or another location in the organism the polypeptide is to be transported. A typical leader peptide has a core of hydrophobic amino acids and thus, a suitable leader sequence to be used in connection with the DNA sequence of the invention is a nucleotide sequence comprising a stretch of codons encoding hydrophobic amino acids.

Examples of a leader sequence to be used in the present 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 sequence of which is shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the N-terminal leader sequence of the genomic chitinase 76 clone, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 5 and SEQ ID NO : 6 , respectively; the N-terminal leader sequence of the acidic sugar beet chitinase SE, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 7 and SEQ ID NO: 8,

respectively; and the N-terminal sequence of the β-1,3-glucanase 4, the nucleotide and amino acid sequence of which is shown in SEQ ID N0:9 and SEQ ID NO:10, respectively. Another interesting sequence is DNA subsequence from the sugar beet chitinase 1 encoding the proline rich domain of the chitinase 1 gene comprising 132 amino acids and shown in SEQ ID NO: 12 which may also be used in the direction of the polypeptide to specific locations of the organism. The abovementioned leader sequences are to be considered as non- limiting examples.

As the above-mentioned leader sequences of the invention are all specific for sugar beet plants, these leader sequences may in another aspect of the invention be functionally connected to a DNA sequence different from the DNA sequences being part of the invention, and which DNA sequence is to be used in a transformation of a sugar beet plant. Such a DNA sequence may in particular be a DNA sequence which is not naturally present in sugar beet plant. The use of a leader sequence normally present in the sugar beet may be an advantage in a transformation of a sugar beet plant as such a leader sequence is known to function in a sugar beet. A leader sequence of the

invention may thus serve to direct the polypeptide expressed from the nucleotide sequence to specific locations of the cell or organism in which it is produced.

Another interesting subsequence in this aspect of the invention is the proline rich domain of the chitinase 1 shown in SEQ ID NO.:12 consisting of 132 amino acids. It is contemplated that the proline rich domain may be involved in the anchoring of the chitinase 1 protein to the cell wall after modification of the prolines to glycosylated hydroxyprolines, as in extensines. Thus, the subsequence containing the proline rich domain may be used when directing and obtaining a polypeptide at a desired location in the cell and/or organism in which the polypeptide is produced.

Furthermore, it may be advantageous that 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 a part of an organism in which it is to be deposited, e.g. in the vacuole. Thus, the same DNA sequence may be present with and without a C-terminal sequence in the same genetic construct. The C-terminal sequence may be the C-terminal extension normally associated with the DNA sequence, if any, or may be derived from the host in which the genetic construct is to be expressed or may be of another origin. This is especially relevant in connection with the chitinase 4 DNA sequence and the DNA sequence of the basic sugar beet β-1,3-glucanase 4 and the acidic chitinase SE all of which lack a C-terminal extension. In DNA sequences which normally comprises C-terminal extension, the natural C-terminal sequence can be replaced with another sequence.

Non-limiting examples C-terminal sequences to be included in a genetic construct of the invention are C-terminal sequences selected from the following sequences; the C-terminal sequence of sugar beet chitinase 1, the amino acid of which is shown in SEQ ID NO.: 12, encoding the following polypeptide consisting of the amino acids No's 413-439

N L D C Y R Q T P F D W G L K K LQ G A R E S W S S S * The C-terminal end of the sugar beet chitinase 4 encoding the following polypeptide consisting of the amino acids No's 261-264 of SEQ ID NO. :2

N L R C * the C-terminal sequence of a bean chitinase (PHA) encoding the following polypeptide shown in SEQ ID NO.: 13

N L D C Y S Q T P F G N S L L L S D L V T S Q * the C-terminal sequence of a basic tobacco chitinase encoding the following polypeptide shown in SEQ ID NO.:14 N L D C G N Q R S F G N G L LV D T M * the C-terminal sequence of an acidic tobacco chitinase encoding the following polypeptide shown in SEQ ID NO.: 15

N L D C Y N Q R N C F A G * the C-terminal sequence of the barley chitinase CH26 encoding the following polypeptide shown in SEQ ID NO.: 16

N L D C Y S Q R P F A *, or the C-terminal sequence of a basic β-1,3-Glucanase from tobacco encoding the following polypeptide shown in SEQ ID NO.: 17

G V S G G V W D S S V E T N A T A S LV S E M The choice of whether a C-terminal sequence is to be added to one or more of the DNA sequences of the genetic construct will be determined, e.g. on the basis of to which plant compartment the polypeptide expressed from the sequence is to be directed. Thus, when it is desirable to control a phytopathogenic fungus mainly present in the intercellular space of the plant, it may be desirable to avoid the use of a C-terminal sequence. When a phytopathogenic fungus mainly present intracellularly is to be controlled it may be desirable that most of or all of the DNA sequences of the genetic construct are provided with a C-terminal sequence capable to transport the polypeptides expressed from the DNA sequences to the vacuole.

As it will be apparent from the above explanation it is important to obtain a sufficient expression of the polypeptides encoded by the genetic construct in plants containing said construct in order to allow the polypeptides to exert their intended function, i.e. to exert their antifungal activity. One essential element in obtaining a sufficient expression is to provide a satisfactory regulation of the transcription and expression of the DNA sequence or gene from which the polypeptide is expressed.

The expression of each of the DNA sequences of the genetic construct of the invention or of a gene comprising such DNA sequences are accomplished by means of a regulatory sequence functionally connected to the DNA sequence or gene so as to obtain expression of said sequence or gene under the control of the inserted regulatory sequence. Typically, the regulatory sequence is a promoter which may be constitutive or regulatable.

The term "promoter" is intended to mean 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 functionally connected, allowing transcription to take place. The promoter is usually situated upstream (5') of the coding sequence. In its broader scope, the term "promoter" includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are. e.g. sequences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions,

A "constitutive promoter" is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place is fulfilled. The constitutive promoter may be enhanced.

A "regulatable promoter" is a promoter the function of which is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect 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 control of termination of transcription (transcription terminators) and removal of introns, as well as sequences responsible for polyadenylation, and for initiation of translation. When the regulatory sequence is to function in a plant, it is preferably of plant origin.

Factors regulating promoter activity may vary depending, inter alia, on the kind of promoter employed as well as on the organism in which it is to function. Tissue specific regulation may be regulated by certain intrinsic factors which ensure that genes encoding proteins specific to a given tissue are expressed. Examples of tissue specific promoters are leaf specific promoters such as the chlorophyll a/b promoter and the AHAS promoter, and further root specific, stem specific, seed specific and petal specific promoters. Also factors such as pathogenic attack or certain biological factors have been shown to regulate promoters. Furthermore, heat-response promoters and promoters involved in the developmental regulation of plants may be found to be of interest.

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

A preferred group of plant virus promoters are promoters which may be derived from a cauliflower mosaic virus (CaMV) . Such promoters are normally strong constitutive promoters. Examples of a preferred CaMV promoter is a CaMV 19S promoter and a CaMV 35S promoter (Odell et al., 1985).

Other promoters may be derived from the Ti-plasmid such as the octopine synthase promoter, the nopaline synthase promoter (HerreraEstrella et al., 1983), the mannopine synthase promoter, and promoters from other open reading frames in the T-DNA such as ORF7.

Further examples of suitable promoters are MAS/35S (Janssen and Gardner, 1989), MAS dual Tr 1,2 (Velten et al., 1984) and a T-2 DNA gene 5 promoter (Konz and Schell, 1986). The regulatory sequence may be a chitinase promoter, i.e. a promoter which is naturally found in connection with chitinase genes and involved in the transcription thereof. A chitinase promoter may be obtained from an isolated chitinase gene, e.g. an already known chitinase gene or a gene which may be identified and isolated e.g. by the methods disclosed herein. Typically, the chitinase promoter should be obtained from a plant which has been shown to have a fast response to pathogen challenge. In this connection, fast responses have been observed in pea and barley and it is contemplated that chitinase promoters from these plants may be useful for the present purpose. An example of such promoters is the chitinase promoter of pea (K. Vad, 1991). An example of another promoter which is contemplated to be useful in the present context is the sugar beet chitinase 1 promoter (SEQ ID NO: 11) and the sugar beet acetohydroxyacid synthase promoter (AHAS) (P. Stougard and K. Bojsen, Danisco A/S, Denmark, personal communication). Furthermore, the sugar beet promoters from the acidic chitinase SE, chitinase 1, chitinase 76 and chitinase 4 or β- 1 ,3-glucanase 4 may also be useful.

Optionally, and if desired, the natural promoter may be modified for the purpose, e.g. by modifications of the promoter nucleotide se quence so as to obtain a promoter functioning in another manner than the natural promoter, preferably activating the transcription of the gene earlier after the challenge with a pathogen or being stronger.

As stated above, each of the coding DNA sequences of the genetic construct of the invention is functionally connected to a transcription terminator. The transcription terminator serves to terminate the transcription of the 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 transcription terminators are a NOS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium (Herrera-Estrella et al., 1983), a 35S transcription terminator sequence of the cauliflower mosaic virus (Paszkowski et al., 1984), a PADG4 transcription terminator to the DNA gene 4 (Wing et al., 1989), and a PADG7 transcription terminator to the T-DNA gene 7.

One or more of the DNA sequences of the genetic construct of the invention may advantageously be functionally connected to an enhancer sequence which results in an increased transcription and expression of the DNA sequence(s). Suitable enhancer sequences and means for obtaining an increased transcription and expression are known in the art.

The specific promoters and the specific terminators, respectively, to be connected with each of the DNA sequences of the genetic construct may be the same or different. It may be an advantage to use different promoters and terminators, respectively, because then the risk of recombinational events, which may lead to excision of parts of or the entire genetic construct, are avoided. In a further aspect, the present invention relates to a vector which is capable of replicating in a host organism and which carries a DNA sequence of the invention comprising a chitinase 4 DNA sequence substantially as shown in SEQ ID NO:1 or an analogue or subsequence thereof, or a genetic construct of the invention. The vector may either be one which is capable of autonomous replication, such as a plasmid, or one which is replicated with the host chromosome, such as a bacteriophage or integrated into a plant genome via the border sequences of Ti vectors. For production purposes, the vector is an expression vector capable of expressing the DNA sequences in the organism chosen for the production. Thus, the expression vector is a vector which carries the regulatory sequences necessary for expression such as the promoter, an initiation signal and a termination signal, etc. These regulatory sequences may be the ones carried by the genetic construct of the invention. The vector may also be one used for identification and optionally isolation of chitinase genes or messengers from other organisms, e.g. other plants, for which purpose expression is not required. This may be done, e.g., as described below.

In a still further aspect, the present invention relates to an organism which carries and which 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 shown in SEQ ID NO:1 or an analogue thereof or a chitinase gene or pseudogene comprising said DNA sequence.

The term "inserted" indicates that the DNA sequence (or subsequence or analogue, or gene or pseudo-gene) has been inserted into the organism or an ancestor thereof by means of genetic manipulation, in other words, the organism may be one which did not naturally or inherently contain such a DNA sequence in its genome, or it may be one which naturally or inherently contains such a DNA sequence, but in a lower number so that the organism with the inserted DNA sequence or the inserted genetic construct has a higher number of such sequences than its naturally occurring counterparts.

The DNA sequence carried by the organism may be part of the genome of the organism, or may be carried on a vector as defined above which is harboured in the organism. The DNA sequence may be present in the genome or expression vector as defined above in frame with one or more second DNA sequences encoding a second polypeptide or part thereof so as to encode a fusion protein, e.g. as defined above.

The organism may be a higher organism such as a plant, or a lower organism such as a microorganism. A lower organism such as a bacterium, e.g. a gram-negative bacterium such as a bacterium of the genus Escherichia, e.g. E. coli, or of the genus Pseudomonas, e.g. P. putida and P. fluorescens, or a gram-positive bacterium such as of the genus Bacillus, e.g. B. subtilis, or a yeast such as of the genus Saccharomyces or a fungus, e.g. of the genus Aspergillus, is useful for producing a recombinant polypeptide as defined above. As many organisms inherently produce chitinase, the insertion of a DNA sequence or a genetic construct according to the present invention may lead to a considerably increased chitinase and optionally β-1,3-glucanase expression and a correspondingly increased antifungal activity. The recombinant production may be performed by use of conventional techniques, e.g. as described by Sambrook et al., 1989.

As it will be discussed in further detail below, a microorganism producing chitinase may be used in combating soil plant pathogens, i.e. pathogens present in the soil and responsible for retarded growth or death of the plant. Examples of such plant pathogens are soil fungi present in e.g. the rhizosphere.

Also, the organism may be a cell line, e.g. a plant cell line. Most preferably, the organism is a plant, i.e. a genetically modified plant such as will be discussed in further detail below. As mentioned above, the genetic construct is preferably to be used in modifying a plant. Accordingly, the present invention also relates to a genetically transformed plant comprising in its genome a genetic construct as defined above. The genetically transformed plant has an increased antifungal activity compared to a plant which does not harbour a genetic construct of the invention, e.g. an untransformed or natural plant or a plant which has been genetically transformed, but not with a genetic construct of the invention. Normally a constitutive expression of the polypeptides encoded by the genetic construct is desirable, but in certain cases it may be interesting to have the expression of the polypeptides encoded by the genetic construct regulated by various factors, for example by factors such as temperature, pathogens, and biological factors.

Chitinase genes have been found in monocotyledonous as well as dicotyledonous plants and have there been found to be expressed into chitinase active in destroying the cell walls of phytopathogenic fungi.

Accordingly, the plant to be transformed by the genetic construct of the invention may be a monocotyledonous as well as a dicotyledonous plant, since the genetic construct is expected to be active in such classes of plants. Non-limiting examples of monocotyledonous plants which may be transformed are corn, oat, wheat, rye, rice, barley and sorghum.

Non- limiting examples of dicotyledonous plants which may be genetically transformed are alfalfa, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea.

It will be apparent from the above disclosure, that the genetically transformed plant according to the invention has an increased resistance to chitin-containing plant pathogens such as phytopthogenic fungi and nematodes as compared to plants which have not been genetically transformed according to the invention or as compared to plants which do not harbour the genetic construct as defined above.

The most important chitin-containing plant pathogens to be controlled according to the invention are represented by phytopathogenic fungi. Phytopathogenic fungi differ in the way which they interact with their host plant during infection. Some species invade the plant via natural openings or wounded tissue and grow in between the plant cells, in the intercellular space, during the entire infection cycle. The fungal hyphae excrete toxins or enzymes that weaken or destroy the plant cells and thereby provide the fungus with cell constituents leaking out of the plant cells. Other fungal pathogens immediately destroy the host cells by penetrating the cell wall of healthy host cells and disintegrate their protoplasts.

Below are given some examples of chitin and glucan containing phytopathogenic fungi with different host interacting strategies, all of which are contemplated to be sensitive to the transgenic plants of the invention.

Cercospora spp. is a fungus the growth of which is restricted to the intercellular space. Conidia (i.e. spores) from the fungus germinate on the leaf surface and penetrate through the stomata of the leaves. Inside the leaf the plant cells close to the hyphae growing in the intercellular space are severely affected by the toxins excreted from the fungus. The toxins cause the plasma membrane to degrade, whereby the cell content leaks out into the intercellular space. Later in the infection cycle the plant cells collapse and necrotic areas containing dead plant cells and fungal mycelia emerge.

Verticillium alboatrum is a root pathogen which propagates in the intercellular space, but which penetrates through the openings made by the emergence of lateral roots, through mechanically injured areas or by direct penetration of hyphae through the tender root tissue in the regions of cell elongation or meristemic activity. The fungus destroys the parenchymatous cells and the tracery elements are mechanically plugged.

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

Colletotricum lindemuthianum causes "Bean anthracnose" . Conidia from this fungus germinate in a film of water in the infection court and the produced germ tube penetrates the cuticula and grows into the epidermal cells of bean leaves and pods. During the following infection, the fungus acts as a parasitic pathogen, penetrating living cells and causing disintegration of the protoplasts. Fusarium spp . is a typical soilborne fungus infecting the plants through the roots, where the hyphae penetrate the epidermal cells of young roots and invades the xylem of roots and stems. The vessels become plugged with granular material and surrounding cells of the outer phloem and cortex are destroyed.

Puccinia graminis causes "Stem rust" of wheat. The sporidia germinate on a film of water on the surface of the plant and the germ tubes penetrate the cuticula. The growing mycelia produce haustoria that penetrate the walls of the host cells and invaginate their protoplasts.

Ustilago maydis is a fungus with mainly intercellular growth, but occasionally penetrates the cell wall of host cells.

In a further aspect, the present invention relates to seeds, seedlings or plant parts obtained by growing the genetically transformed plant as described above. It will be understood that any plant part or cell derivable from the genetically transformed plant of the invention is to be considered within the scope of the present invention.

In recent years, a great effort has been focused on developing useful methods for constructing novel plants or plant cells having specific and desirable properties by transferring new genetic information encoding the desirable properties to the plant, and a number of such methods based on recombinant DNA technology and suitable plant transformation systems are now available. Usually, the genetic information is introduced into the plant by use of a vector system or by direct introduction, e.g. by use of the methods given by Herrera-Estrella 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, Thus, in another aspect, the present 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 the genetic construct into the genome of a plant such as a plant of the family Chienopodiaceae in particular of the genus Beta, especially Beta vulgaris.

Normally, plant transformation systems are based on the use of plasmids or plasmid derivatives of the bacteria Agrobacterium. The two best known Agrobacteria are Agrobacterium tumefaciens and Agrobacterium rhizogenes (plasmids thereof are in the following termed pTi and pRi, respectively). The use of such plant transformation systems is based on the ability of the bacteria Agrobacterium to transfer a specific piece of DNA (T-DNA) to a plant cell in a wounded area. In nature, the T-DNA is located between specific border DNA sequences on the pTi or pRi which further carries virulence genes necessary for the transfer of the T-DNA to the plant. The Agrobacterium transformation system mediates the transfer of any DNA sequence located between the "borders" and thus, it is possible to exchange the wild type Agrobacterium T-DNA with any desirable piece of DNA to be introduced into a plant.

Preferably, the plant transformation system of the invention is based on disarmed Agrobacteria harbouring derivatives of the pTi or pRi from which the wild type T-DNA has been removed. Normally, the vector system with which the plant is transformed comprises one or two plasmids. In the one-plasmid system (also termed a co- integrate vector system), the T-DNA of pTi or pRi has been removed and replaced by the DNA to be transferred into the plant cell by use of homologous recombination. In the two-plasmid system (also termed a binary vector system) both the T-DNA and the borders have been removed from the pTi or pRi. Introduction in the disarmed Agrobacterium of a small plasmid containing the DNA to be transferred between pTi or pRi identical borders and a suitable origin of replication, results in a vector system where the virulence functions are located on the disarmed pRi or pTi and the T-DNA and borders are located on another plasmid.

An example of a suitable plant transformation vector is pBI121 and derivatives thereof, e.g. as described by Jefferson 1987. Suitably, the vector to be used is provided with suitable markers, eucaryotic as well as procaryotic, e.g. genes encoding antibiotic resistance or herbicide resistance or glucoronidase (GUS), e.g.

hygromycin or other known markers, e.g. the markers disclosed by Lindsey, 1989 and Reynaerts et al., 1988. The marker is to be present so as to be able to determine whether the DNA insert has been inserted in the desired position in the plasmid and to be able to select plant cells transformed with the vector.

The use of more than one vector in one transformation event will according to the presently known plant transformation techniques normally require that different selective genes are present on each vector in order to be able to follow the success of the plant transformation.

In the construction of a transgenic plant using a plasmid such as a pTi or pRi or derivative thereof it is preferred that the genetic construct to be inserted in the plant is first constructed in a microorganism in which the plasmid can replicate and which is easy to manipulate. An example of a useful microorganism is E . coli , but other microorganisms having the above properties may be used. When a plasmid of a vector system as defined above has been constructed in E . coli , it is transferred, if necessary, into a suitable Agrobac terium strain, e.g. Agrobacterium tumefaciens .

The plasmid harboring the genetic construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A . tumefaciens , so as to obtain an Agrobacterium cell harboring the genetic construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified. This transformation may be performed in a number of ways, e.g. as described in An et al. (1988). Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher et al. (1980). Typically, a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive or brushing the plant with a steel brush (e.g. as described in Example 15). The wound is then inoculated with the Agrobacterium, e.g. in a suspension.

Alternatively, the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant. The inoculated plant or plant part is then subjected to selection and regeneration and grown on a suitable culture medium and allowed to develop into mature plants. This is accomplished by use of methods known in the art.

Other very suitable methods for transforming the plant is by use of sonication, electroporation (Joersbo, 1990) or particle gun methods, e.g. as described by Klein et al., 1989.

When genetically transformed plant cells are produced these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.

In accordance with well-known plant breeding techniques it will be understood that the production of a genetically transformed plant may be performed as a double transformation event (introducing the genetic construct in two transformation cycles) or may be associated with use of conventional breeding techniques. Thus, two genetically modified plants according to the present invention may be cross breeded in order to obtain a plant which contains the genetic construct of each of its parent plants. As will be understood from the introductory part of the present specification, the chitinase 4 DNA sequence of the present invention or an analogue thereof may be used for diagnostic purposes, which will be further explained in the following. Various types of diagnosis may be performed by use of the chitinase 4 DNA sequence of the invention. In a given example, chitinase messenger RNA's transcribed from a gene belonging to the chitinase 4 gene family may be qualitatively as well as quantitatively determined by hybridization to the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof under conditions suitable for said hybridization. Furthermore, genes belonging to the chitinase 4 gene family and present in an organism such as a plant may be identified and isolated by use of the DNA sequence of the invention, e.g. by screening a gene library of such an organism.

When the DNA sequence comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof is to be employed for diagnostic purposes, it will often be useful to provide it with a label which may be used for detection. Useful labels are known in the art and is, e.g. a fluorophore, a radioactive isotope, an isotope or a complexing agent such as biotin.

Also, the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof may be used in a method of isolating a gene or messenger belonging to or derived from the chitinase 4 gene family from an organism, e.g. a plant, in particular a dicotyledon, the method comprising hybridizing a nucleic acid containing sample obtained from a gene library or cDNA library from the organism with the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof, optionally in a labelled form, in a denatured form or an RNA copy thereof under conditions favorable to hybridization between the DNA sequence or RNA copy and the nucleic acid of the sample, and recovering the hybridized clone so as to obtain a gene or cDNA belonging to the chitinase 4 gene family of the organism.

The identification and isolation of a gene or cDNA clone in a sample belonging to the chitinase 4 gene family by use of the chitinase 4 DNA sequence of the invention or an analogue thereof, in particular a subsequence thereof, may be based on standard procedures, e.g. as described by Sambrook et al., 1989. For instance, to characterize chitinase 4 related genes in other plants, it is preferred to employ standard Southern techniques.

The chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may also be used in a method of quantifying the amount of a chitinase 4 related messenger present in different tissues in an organism, e.g. a plant, the method comprising hybridizing a nucleic acid containing sample obtained from the organism with the chitinase 4 DNA sequence of the invention comprising a nucleotide sequence substantially as shown SEQ ID NO : 1 or an analogue thereof, especially a subsequence thereof, optionally in labelled form, in denatured form or an RNA copy thereof under conditions favorable to hybridization between the denatured DNA sequence or RNA copy and the RNA of the sample and determining the amount of hybridized nucleic acid (Barkardottir et al., 1987). The hybridization should be carried out in accordance with conventional hybridization methods under suitable conditions with respect to e.g. stringency, incubation time, temperature, the ratio between the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof to be used for the identification and the sample to be analyzed, buffer and salt concentration or other conditions of importance for the hybridization. The choice of conditions will, inter alia, depend 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 concentrations, low ionic strength of the buffer and higher temperatures, whereas a low degree of complementarity requires less stringent conditions, e.g. higher salt concentration, higher ionic strength of the buffer or lower temperatures, for the hybridization to take place. The support to which DNA or RNA fragments of the sample to be analyzed are bound in denatured form is preferably a solid support and may be any of the supports conventionally used in DNA and RNA analysis.

The DNA sequence used for detecting the presence of the chitinase 4 related gene is preferably labelled, e.g. as explained above, and the presence of hybridized DNA is determined by autoradiography, scintillation counting, luminescence, or chemical reaction.

Another approach for detecting the presence of a specific chitinase 4 related gene, e.g introduced by the genetic methods described previously, or a part thereof in an organism, e.g. a plant, in particular a dicotyledon, is to employ the principles of the well-known polymerase chain reaction, e.g. as described in the "Materials and Methods" section below.

The sample to be analyzed for the presence of a chitinase 4 related gene or part thereof in accordance with the methods outlined above may be taken from the group of plant parts consisting of leaves, stems, tubers, flowers, roots, sprouts, shoots and seeds.

The same principles as described above may be used in the isolation of DNA sequences to be used in the preparation of a genetic construct of the invention, e.g. DNA sequences encoding a polypeptide having chitinase or β- 1 ,3-glucanase activity.

Restriction fragment length polymorphisms (RFLP) are increasingly used to follow specific alleles of genes in various organisms. The alleles are either themselves followed or they are used as markers (unlinked or linked) in crosses involving other characteristics, e.g. pathogen resistance and morphological characteristics such as tuber colour. So far, the method has primarily been employed in humans, but it has also been employed in plants. It is contemplated that the chitinase 4 DNA sequence of the invention or a analogue thereof may be useful in RFLP-analysis of chitinase 4 related genes, especially in sugar beet.

In a further aspect the present invention relates to an antifungal composition comprising a polypeptide encoded by a DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO:1 or an analogue or subsequence thereof as defined above, or by a genetic construct of the invention as defined above and a suitable vehicle. In another embodiment, the present invention relates to an antifungal composition comprising a microorganism capable of expressing a poly peptide encoded by the DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO : 1 or an analogue or subsequence thereof as defined above, or by a genetic construct of the invention defined above and a suitable vehicle. Microorganisms suitable as constituents in an antifungal composition are mentioned above.

The antifungal composition according to the present invention may be prepared by a method comprising culturing a microorganism harbouring and being capable of expressing a DNA sequence of the invention comprising the chitinase 4 DNA sequence shown in SEQ ID NO:1 or an analogue or subsequence thereof or a genetic construct of the invention in an appropriate medium and under conditions which result in the expression of one or more antifungal polypeptides encoded by the DNA sequences, optionally rupturing the microorganisms so as to release their content of expressed antifungal polypeptide(s) into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide(s) to freeze-drying or spray-drying thereby obtaining an antifungal composition comprising the antifungal polypeptide(s). Alternatively, the antifungal proteins may be excreted to 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 the prolines by conventional methods, the medium may be used directly or after freeze drying.

The antifungal composition according to the invention may be used in combating or inhibiting the germination and/or growth of a phytopathogenic fungus in or on a plant or in any other material in which the presence of fungi is undesirable. This will be further discussed below.

The antifungal composition of the invention shall, of course, be adapted to its intended purpose, both with respect to the vehicle to be used and with respect to the form, in which the antifungal agent is present. By the term "antifungal agent" is meant the active constituent of the antifungal composition responsible for or involved in providing the antifungal activity. By the term "antifungal polypeptide" is meant a polypeptide encoded by the chitinase 4 DNA se quence of the invention or an analogue thereof or a genetic construct of the invention having antifungal activity, i.e. chitinase activity and optionally β- 1 ,3-glucanase activity as defined above.

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

Normally, the antifungal agent is in itself a microorganism or will be prepared by a microorganism. In most cases, the most easy and inexpensive way of preparing the antifungal composition will be to use the microorganism as such or the medium in which it is grown as the antifungal agent. The antifungal polypeptide(s) expressed from the microorganisms may be secreted into the medium, e.g. as a consequence of the action of a suitable signal peptide capable of directing the polypeptide out into the medium, or may be released from the microorganism by well known mechanical or chemical means. Before use, it may be advantageous to remove the microorganisms or any cell debris from the medium.

The medium may, in principle, serve as the vehicle for the antifungal agent, but it is preferred to add a further vehicle suited for the particular intended use. A culture of the microorganisms expressing the antifungal polypeptide(s) may be obtained as described above using methods known in the art. As mentioned above, it may be necessary or advantageous to subject the microorganism culture to a further treatment so as to release the content of the antifungal polypeptide(s) into the medium or to increase the amount released by secretion,

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

The microorganisms expressing the antifungal polypeptide(s) can be applied in various formulations containing agronomically acceptable vehicles, i.e. adjuvants or carriers, in dosages and concentrations chosen to maximize the beneficial effect of the microorganism. However, the microorganisms may also be distributed as such under circumstances allowing the microorganisms to establish themselves in the material to be treated. When the microorganism is a microorganism conventionally found in the soil, e.g. a rhizobacterium, it will generally be desirable that the transformed microorganism establishes itself in the soil so that it continuously may secrete the antifungal polypeptide(s) out into the soil surrounding the plant. It may be advantageous to add the microorganisms or the medium comprising the antifungal polypeptide(s) to pre-mixes, e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question. For such purposes it is convenient that the microorganisms or the medium is in a solid form, e.g. in a powdery form or in the form of a granule. The powdery form may be obtained by conventional means, e.g. by applying the microorganism on a particulate carrier by spray-drying or an equivalent method.

When the microorganism expressing the antifungal polypeptide(s) is to be used in a humid state it may be in the form of a suspension or dispersion, e.g. as an aqueous suspension.

In order to induce the chitinase activity of 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 above, the present invention further relates to a method of inhibiting the germination and/or growth of a chitin containing plant pathogen, such as phytopathogenic fungus, in or on a plant, which method comprises 1) transforming the plant or a 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 a part thereof, a seedling or seed from which the plant is to be propagated, or the medium on which it is grown with an antifungal composition as defined above,

While genetic transformation of plants is for most purposes are the preferred method, it may be an advantage to combine transformation with treatment of the plant with an antifungal composition of the invention. Since the genetic transformation is a time-consuming and in certain aspects difficult process, it may be an advantage to use a biologically based composition instead of or in addition to the conventionally used and from an environmental point of view undesirable chemical fungicides. In most cases the material to be treated with the antifungal composition of the invention is a plant. However, a number of chitin containing fungi exist which infect other materials than plants, e.g. food products such as bread or bread products, milk products cheese, meat, vegetables, cereals, in which the presence and growth of fungi are undesirable. It is contemplated that an antifungal composition according to the present invention may be used to control or combat such fungi. In this respect, it is contemplated that also beverages and containers (any part thereof) used for food products or beverages may be treated with an antifungal composition of the invention either as a prophylactic treatment or a combating treatment.

The present invention is further illustrated in the following sequences, examples and accompanying drawings, but not limited hereto.

The drawing:

Fig. 1 describes the purification of sugar beet chitinase 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elution of the proteins was performed with a linear gradient of NaCl. The absorbance was recorded at 280 μm. Fig. 2 describes the polypeptide pattern of sugar beet chitinase 2, 3 and 4 after purification on a Mono-S FPLC column. Lanes contain 50 μg of the following proteins. Lanes a and b, chitinase 4; lanes d and e, chitinase 3; lanes f and g, chitinase 2; and lanes c and h, molecular weight markers. The proteins were stained with silver.

Fig. 3 shows the analysis of the water-soluble products released from ³H-chitin by chitinase 4. -"H-chitin was incubated with 4 μg chitinase 4 at 37°C for 0.25, 0.5, 3 and 24 hours. As a control ³H-chitin was incubated without enzyme at 37°C for 24 hours. The chitooligosaccharides released were separated by TLC and identified by comparing their migration with that of N-acetylglucosamine (monomer) (Fig. 3A), chitobiose (dimer) (Fig. 3B), chitotriose (trimer) (Fig. 3C) and chitotetraose (tetramer) (Fig. 3D) standards. The

radioactivity representing the chitooligosaccharides was determined by scintillation counting after cutting the TLC plate into pieces.

Fig. 4 shows the lysozyme activity of chitinase 4. 1 μg of the enzyme was incubated with cell walls from Micrococcus lysodeikticus and the decrease in absorbance at 450 nm was recorded at specified time intervals. 1 μg of SE ("Sure Ellen") was used as a control, and (50 ng and 5μg) lysozyme (lys) was used as standards.

Fig. 5 shows the inhibition of the growth of Cercospora by a combination of chitinase 4, acidic chitinase SE and β-1,3-glucanase 4 using the microscope slide bioassay. After 48 hours of incubation the cultures were stained with Calcofluor White and investigated under fluorescent light.

Fig. 5A shows the growth of the fungus when 20 μg of each of the enzymes chitinase 4, acidic chitinase SE and β-1,3-glucanase 4 were added to the culture at time 0 Fig. 5B shows the growth of a control culture where no antifungal proteins have been added.

Fig. 6 shows the inhibition of growth of Cercospora by chitinases using the microtiter plate bioassay. The time course curves (absorbance at 620 nm) describe the growth of the fungus during the first 92 hours of incubation. The absorbance (an indication of the growth) was measured at 8 to 16 hours time intervals and each measurement is an average of 5 replicates, Curve A is a control curve showing the growth of Cercospora when no growth inhibitors were added to the culture. Curve B shows the growth of the fungus when 20 μl of a chitinase containing fraction from the chitin-column was added at time 0. In curve C 20 μg of purified chitinase 4 was added to the culture at time 0.

Fig. 7 is an autoradiography showing the effect of chitinase 4 on chitin in the apex of Cercospora hyphae. Incorporation of ³H- labelled N-acetylglucosamine into the hyphae of Cercospora beticola was performed by growing the fungus for 20 minutes on growth medium containing radioactive monomer of chitin. Incorporation of N-acetylglucoseamine into the cell wall in the apex of the fungal hyphae is seen as black dots.

Fig. 7A shows the hyphae before treatment with purified chitinase 4.

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

Fig. 8 shows the separation of tryptic peptides of chitinase 4 by reverse phase HPLC on a Vydac RP-18 column. The peptides were eluted with a linear gradient from 10% to 45% acetonitrile from 25 to 75 minutes. Buffer A was water, whereas B was acetonitrile. Both solvents contained 0.1% trifluoroacetic acid. The flow rate was

0.7 ml/minute. Fig. 9 shows the separation of three acidic SE chitinase isozymes on an anion exchange column (Mono P) by the FPLC system. The proteins were eluted with a linear sodium chloride gradient in a 25 mM Bis-Tris buffer at pH 7.0.

Fig. 10 describes the two different serological classes of sugar beet, the chitinase 2 and chitinase 4 class. 5 μg of both chitinase 2 (32 kD) and 4 (26 kD) were blotted on to the nitrocellulose membrane before reaction with antibody to sugar beet chitinase 2 (Fig. 10A) or antibody to sugar beet chitinase 4 (Fig. 10B).

Fig. 11. Hybridization of different chitinase genes with a chitinase 4 cDNA probe under specific hybridization conditions. The different chitinase genes were spotted on Hybond N-nylon membranes as 1 μl probes of a plasmid preparation containing the chitinase sequences.

1 a chitinase 1 clone from sugar beet

2 a chitinase 4 clone form sugar beet

3 a chitinase 76 clone form sugar beet

4 a chitinase clone from pea

5 an acidic chitinase SE clone from sugar beet

6 a chitinase clone 1 from tobacco

7 a chitinase clone 2 from tobacco

8 a chitinase clone 3 from tobacco

9 a chitinase clone from bean

10 a chitinase 4 like clone from rape seed.

The hybridization was carried out over night at 55°C in the following hybridization buffer: 2 × SSC, 0.1% SDS. 10 × Denhardt's. 50 μg/ml Salmon sperm DNA and a chitinase 4 cDNA sequence as probe and under washing conditions of 55°C. 2×SSC, 0.1% SDS in two times 15 minutes followed bv two times 15 minutes 1×SSC. 0.1% SDS, 55°C. Fig. 12 describes the induction of chitinase and β - 1,3-glucanase in sugar beet leaves after infection with Cercospora beticola . Plants were inoculated with a suspension of fungal spores. Leaves were harvested after specified time intervals and crude extracts were prepared. Enzyme activities of chitinase (Fig, 12A) and β-1,3-glucanase (Fig. 12B) were measured using the radiotracer assays with 3H-chitin and ³H -laminarin as the substrate, respectively.

Fig. 13 describes the immuno-detection of sugar beet chitinase 2 and 4 and β - 1 ,3-glucanase 3 in protein extracts of Cercospora infected sugar beet leaves. Lanes I and c contain protein extracts from infected and control plants, respectively. Antibodies raised against chitinase 2 (left), chitinase 4 (centre) and β- 1,3-glucanase 3 (right) were employed.

Fig. 14. Site directed mutagenesis of amino acids contemplated to form part of the active site of the chitinase 4 enzyme by the use of the PCR technique described in "Materials and Methods". SDO is used as 5' primers for all the suggested PCR-reactions. The sequence is indicated by the arrow and is chosen 5' to the unique BamHI site, The sequences for the SD1, SD2 , SD3 , SD4 and SD5 primers are indicated by arrows. For these 3' primers the complementary sequence with the indicated substitutions are used. The primers can be used for the following substitutions with reference to the genomic chitinase DNA sequence (SEQ ID NO.: 3) encoding the amino acid sequence shown in SEQ ID NO.:4. Numbers in brackets denote the number of the corresponding amino acid encoded from the chitinase 4 cDNA (SEQ ID NO.:2)

SD1 Trp170→Tyr (169) TGG→TAC

SD2 Glu190→Gln (189) GAA→CAA

SD3 Asp184→Asn (183) GAT→AAT

SD4 Trp207→Tyr (206) TGG→TAC

SDS Trp205→Tyr (204) TGG→TAC

The PCR products are digested with the relevant restriction enzymes and exchanged with the corresponding sequence in the chitinase 4 gene. Fig. 14A and Fig. 14B should be considered as one figure.

Fig. 15. Construction of a hybrid β-1,3-glucanase gene construct with a C-terminal extension from tobacco Fig. 15A. A sugar beet cDNA β-1,3-glucanase clone with an underlined tobacco C-terminal extension.

Fig. 15B and Fig. 15C. PCR primers which can be used to change the stop codon and to introduce a part of the C-terminal extension, a DraI site is created at the 3' end. The arrows indicate the PCR primers; for the 5' primer the sequence underneath the arrow is used, for the 3' primer the

complementary sequence with the indicated substitutions is used. Fig. 15B and Fig. 15C should be considered as one figure.

Fig. 15D. Four annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a stop codon, a SmaI site and an BglII site.

The fused gene product can be made by digesting the glucanase gene with XbaI and EcoRI and ligating it with the PCR product digested with XbaI and Dral and the annealed synthetic oligonucleotides digested with Smal and BglII.

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

Fig. 16A. Chitinase 4 with an underlined tobacco C-terminal extension. Fig. 16B and Fig. 16C. PCR primers which can be used to introduce a SmaI site near the stop codon in the chitinase 4 gene. The arrows indicate the PCR primers; for the 5' primer the sequence underneath the arrow is used, for the 3' primer the complementary sequence with the indicated substitutions is used. Fig. 16B and Fig. 16C should be considered as one figure.

Fig. 16D, Four annealed synthetic oligonucleotides containing the sequence for the C - terminal extens ion , a changed stop codon, a Smal site and an EcoRI site.

The fused gene product can be made by digesting the chitinase 4 gene with BamHI and EcoRI and ligating it with the PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI.

Fig. 17. Construction of the plant transformation vector pBKL4K4 containing the chitinase 4 DNA sequence shown in SEQ ID NO: 1. The boxed sequences indicate the B15 chitinase 4 cDNA, the enhanced 35S promoter and the 35S terminator sequences used for the construct. ρB15K4.1 is pBluescript carrying the 966 bp EcoRI fragment encoding the chitinase 4. The hatched boxes indicate the coding regions contained in the final product. Kb3 (=KB3) and Kb4 (=KB4) 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 given in Example 18 and shown in SEQ ID NO: 49 and SEQ ID NO: 50. Plasmid pPS48 carries a conventional 35S enhanced promoter and a conventional 35S terminator separated by a polylinker containing unique cloning sites. The plant transformation vector pBKL4 (a modification of pBin 19 Bevon, 1984) carries a right and a left T-DNA border sequence from the Agrobacterium Ti plasmid pTiT37, a GUS gene with a 35S promoter and a conventional NOS terminator, a conventional NPTII gene with a 35S promoter and a conventional OCS terminator. A polylinker containing several unique cloning sites is situated between the GUS and the NPTII genes. Fig. 17A and Fig. 17B should be considered as one figure.

Fig. 18. Construction of the plant transformation vector pBKL4K4KSE1 containing the DNA sequences encoding chitinase 4 and acidic

chitinase SE, respectively shown in SEQ ID NO: 1 and SEQ ID NO: 8. The boxed sequences indicate the acidic chitinase SE cDNA. the enhanced 35S promoter and the 35S terminator sequences also used in connection with the construct shown in Fig. 17. pSurl is pBluescript carrying the 5' end of the SE gene, pSE22 is likewise pBluescript carrying almost the entire SE cDNA. The hatched boxes indicate the coding regions contained in the final product. ^5' AGCTGTAC^3' is an adaptor used for the KpnI-HindIII ligation. pPS48 is mentioned in connection with Fig. 17. The construction of the plant transformation vector harboring the chitinase 4 sequence (pBKL4K4) is described in Fig. 17. Fig. 18A and Fig. 18B should be considered as one figure.

Fig. 19. Construction of the plant transformation vector pBKL4K76 containing the genomic chitinase 76 gene, the sequence of which is shown in SEQ ID NO : 5. The boxed sequences indicate the chitinase 76 gene, the enhanced 35S promoter and the 35S terminator sequences. pK76.1 is pUC19 carrying the HindIII-EcoRI fragment encoding chitinase 76 in the HindIII/EcoRI site of the pUC19 polylinker. The hatched 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 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 plant transformation vector pBKL4 is described in Fig. 17. Fig. 19A and Fig. 19B should be considered as one figure.

Fig. 20. PCR amplification. of a part of the acidic chitinase SE cDNA using mRNA as a template. mRNA was reverse transcribed using a primer consisting of oligo-dT linked to two restriction sites (270) (see Example 7). Amplification was carried out using a gene specific mixed oligonucleotide linked to a restriction site (XbaI-KB7) as the 5' primer and 270 as the 3' primer. A second round of amplification was then carried out 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 of 270 is shown in Example 7 and SEQ ID NO: 30. Fig. 21 describes the separation of sugar beet β - 1,3-glucanases 1, 2. 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elution was performed with a linear gradient of NaCl. The absorbance was measured at 280 run.

Fig. 22 describes the construction of the plant transformation vector pBKL4K4KSE1G1 containing the DNA sequences encoding chitinase 4, acidic chitinase SE and β- 1 ,3-glucanase, respectively, and shown in SEQ ID NO:1, SEQ ID NO: 7 and SEQ ID NO: 9. The boxed sequences indicate the β- 1 ,3-glucanase cDNA, the enhanced 35S promoter and the 35S terminator. pGluc 1 is pBluescript carrying the 1249 bp EcoRI fragment encoding the β - 1 ,3-glucanase. The hatched box indicates the coding region. Plasmid pPS48M is the same as pPS48 described in connection with the construct shown in Fig. 17, except that the plasmid is supplemented with two additional restriction sites (EcoRI and KpnI) at each site of the E35S-35St box. The construction of the plant transformation vector harboring the chitinase 4 and SE

sequences is described in Fig. 17 and Fig. 18. Fig. 22A and Fig. 22B should be considered as one figure.

Fig. 23 describes the immuno-detection of sugar beet chitinase 4 and the acidic chitinase in protein extracts from transgenic N . ben thaminana using the antibody raised against sugar beet chitinase 4, C = Control plants containing the GUS and NPT gene construct. SE = The acidic chitinase.

K76 = The genomic chitinase (see Fig. 19).

K4 = Chitinase 4 (see Fig. 17).

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

Fig. 24. A comparison between the DNA sequence of the chitinase 4 cDNA sequence shown in SEQ ID N0.:1 and the genomic clone chitinase 76 shown in SEQ ID NO.:5. The position of the chitinase 76 intron is easily seen at position 875 to 1262. The homology of the sequences is about 73%. The figures Fig. 24A, Fig. 24B, Fig. 24C, Fig. 24D, Fig. 24E and Fig, 24F should be considered as one figure.

: indicates identical nucleotides.

Fig. 25. A comparison between the amino acid sequence of the chitinase 4 cDNA sequence shown in SEQ ID NO .:2 and chitinase 76 shown in SEQ ID NO. :6. A homology of about 80% is seen. The extra 3 amino acids in chitinase 76 are the amino acids (Ser, Thr, Pro) in position 62-64. Fig. 25A and Fig. 25B should be considered as one figure.

: indicates identical amino acids.

Fig. 26. A comparison between the non-coding 5' sequences of the chitinase 4 and chitinase 76 genomic sequences shown in SEQ ID NO . : 3 and SEQ ID NO.:5, respectively. 8 boxes of strong homology is observed in the non-coding 5' sequence. It is contemplated that some of these boxes may be of regulatory importance. Fig. 26A and Fig. 26B should be considered as one figure.

SEQUENCE LISTING

SEQ ID NO.:1 the chitinase cDNA sequence (harbored in the cDNA sugar beet chitinase 4 clone B15)

SEQ ID NO.:2 the chitinase 4 amino acid sequence (harbored in the cDNA sugar beet chitinase 4 clone B15)

SEQ ID NO.: 3 the partial DNA sequence of the genomic chitinase 4 clone

SEQ ID NO.:4 the partial amino acid sequence of the genomic

chitinase 4 clone SEQ ID NO.: 5 the DNA sequence of the genomic clone chitinase 76

SEQ ID NO .:6 the deduced amino acid sequence of the genomic clone chitinase 76

SEQ ID NO.:7 the cDNA sequence of the acidic sugar beet chitinase

SE SEQ ID NO.:8 the deduced amino acid sequence of the acidic sugar beet chitinase SE

SEQ ID NO.:9 the cDNA sequence of the basic sugar beet β-1,3- glucanase

SEQ ID NO.:10 the deduced amino acid sequence of the basic sugar beet β- 1 ,3-glucanaεe

SEQ ID NO.:11. The DNA sequence of the entire sugar beet chitinase 1 gene including introns, promoter and leader sequence, and the amino acid sequence deduced from the coding region of the chitinase 1 gene. SEQ ID NO.:12 The amino acid sequence deduced from the coding region of the chitinase 1 gene. SEQ ID NO.:13: C-terminal amino acid sequence of a bean chitinase (PHA).

SEQ ID NO. :14: C-terminal amino acid sequence of a basic tobacco chitinase. SEQ ID NO. :15: C-terminal amino acid sequence of an acidic tobacco chitinase.

SEQ ID NO.:16: C-terminal amino acid sequence of the barley

chitinase CH26.

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

SEQ ID NO. :18: Amino acid sequence of a tryptic peptide of a

chitinase 3 from sugar beet.

SEQ ID NO.: 19: Amino acid sequence of a tryptic peptide of a

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

chitinase 3 from sugar beet.

SEQ ID NO.: 20: Amino acid sequence of a tryptic peptide of a

chitinase 3 from sugar beet.

SEQ ID NO.: 21: Amino acid sequence of a tryptic peptide of a

chitinase 3 from sugar beet.

SEQ ID NO.: 22: Amino acid sequence of a tryptic peptide of a

chitinase 3 from sugar beet.

SEQ ID NO.:23: N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from WGA-A (Triticum aestivum). SEQ ID NO.:24: N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from hevein (Hevea brasi liensis ) .

SEQ ID NO.:25: N-terminal amino acid sequence of the amino acid

sequence of a chitinase from bean (Phaseolus

vulgaris ) .

SEQ ID NO.:26: N-terminal amino acid sequence of the amino acid

sequence of a chitinase from tobacco (Nicotiana tabacum) . SEQ ID NO.:27: N-terminal amino acid sequence of the amino acid

sequence from chitinase 2 from sugar beet.

SEQ ID NO.: 28: DNA primer named KB-7 constructed partly from the

polypeptide sequence of the acidic chitinase from sugar beet (SEQ ID NO.:9). SEQ ID NO.:29: Complementary DNA primer named KB-9 constructed partly from the polypeptide sequence of the acidic chitinase from sugar beet (SEQ ID NO.:9).

SEQ ID NO.:30: Complementary DNA primer named Oligo-dT constructed from the general knowledge of polyA mRNA's SEQ ID NO.: 31: Amino acid sequence of a lysozyme/chitinase from

cucumber (Cucumis sativus ) .

SEQ ID NO.:32: Amino acid sequence of a lysozyme/chitinase from

Arabidopsis thaliana .

SEQ ID NO.:33: Amino acid subsequence 3-15 of the amino acid sequence of a β-1,3-glucanase from sugar beet.

SEQ ID NO.:34: The amino acid subsequence 3-17 of the amino acid

sequence of a β - 1 ,3-glucanase from sugar beet. SEQ ID NO. :35: The amino acid subsequence 3-16 of the amino acid sequence of a β-1,3-glucanase from sugar beet

SEQ ID NO.:36: DNA 5'primer named Oligo TG-1 constructed from the amino acid sequence of a β-1,3-glucanase from sugar beet.

SEQ ID NO.:37: DNA 5'primer named Oligo TG-2 constructed from the amino acid sequence of a β-1,3-glucanase from sugar beet.

SEQ ID NO.:38: DNA 3'primer named Oligo TG-3 constructed from the amino acid sequences of a glucanase from tobacco and barley.

SEQ ID NO.:39: Amino acid subsequence from the amino acid sequence of a glucanase from barley used to construct the primer Oligo TG-3. SEQ ID NO.:40: Amino acid subsequence from the amino acid sequence of a glucanase from tobacco used to construct the primer Oligo TG-3.

SEQ ID N0.:41: N-terminal amino acid sequence of the amino acid

sequence from Pea chitinase B. SEQ ID NO. :42: N-terminal amino acid sequence of the amino acid

sequence from pea chitinase A1.

SEQ ID NO.: 43: N-terminal amino acid sequence of the amino acid

sequence from pea chitinase A2.

SEQ ID NO.: 44: N-terminal amino acid sequence of the amino acid

sequence from barley chitinase K.

SEO ID NO. :45: N-terminal amino acid sequence of the amino acid

sequence from barley chitinase T. SEQ ID NO. :46: Amino acid subsequence of the active site of the amino acid sequence from a chitinase from tobacco.

SEQ ID NO.:47: Amino acid subsequence of the active site of the

polypeptide from a chitinase from tobacco. SEQ ID NO.:48: Amino acid sequence of the active site of the

polypeptide from a chitinase from tobacco.

SEQ ID NO.:49: DNA 5'primer named KB-3 constructed partly from

nucleotides No's. 1-15 of SEQ ID NO.:1 and nucleotides No's. 471-485 from SEQ ID NO.:5. SEQ ID NO.:50: Complementary DNA 3'primer named KB-4 constructed from nucleotides No's. 261-241 of SEQ ID NO.:1.

SEQ ID NO.: 51: Complementary DNA primer named 340 constructed partly from the nucleotide numbers 341-323 of SEQ ID NO.:1.

DETAILED EXPLANATION OF THE SEQUENCES SEQ ID NO'S:1-12

SEQ ID NO.: 1 and SEQ ID NO. :2

The DNA sequence (SEQ ID NO:1) and deduced ammo acid sequence (SEQ ID NO:2) of the B15 chitinase 4 cDNA clone isolated from a sugar beet λZAP cDNA library

The sequence is 966 bp long and encodes a protein having 264 amino acid residues in the polypeptide chain The leader sequence consists of 23 amino acid residues followed by a hevein domain of 35 amino acid residues and a functional domain of 206 ammo acid residues. After the stop codon the cDNA has a 158 bp 3' flanking region with a putative polyadenylation signal at position 847 and a poly A tail.

For comparison, the chitinase 4 gene the partial nucleotide sequence of which is shown in SEQ ID NO 3, encodes a protein having 265 amino acid residues shown in SEQ ID NO 4 The leader sequence encoded by the gene consists of 24 amino acid residues Thus, the SEQ ID NO: 1 are missing the nucleotide A and T and the first amino acid Met is not present in the polypeptide sequence encoded by the chitinase 4 cDNA

SEQ ID NO: 3 and SEQ ID NO:4 The partial DNA sequence (SEQ ID NO 3) and deduced ammo acid sequence (SEQ ID NO 4) of a genomic clone encoding the chitinase 4 gene isolated from a 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 followed by a hevein domain of 35 amino acids The partially sequenced clone has a 5' non-coding region of 355 bp with a TATA-box sequence (TATAAA) located at position 285. which is 70 bp upstream of 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 a sugar beet EMBL3 genomic library The sequence is 1838 bp long and encodes a protein having 268 amino acid residues in the polypeptide chain. The leader sequence consists of 24 amino acid residues followed by a hevein domain of 35 and a functional domain of 209 amino acid residues. The gene contains one intron which is located in position 875 to 1262. The exact location of this intron is based on an alignment with the B15 chit 4 cDNA

(Fig. 24). The intron borders contain the consensus GT/AG sequences. A TATA-box sequence (TATAAA) is located at position 378, which is 90 bp upstream of the ATG start codon. A putative poly-A signal (AATAAA) is located at position 1725. SEQ ID NO: 7 and SEQ ID NO: 8

The DNA sequence (SEQ ID NO: 7) and deduced amino acid sequence (SEQ ID NO: 8) of the acidic chitinase SE cDNA clone isolated from a sugar beet λZAP cDNA library

The sequence is 1106 bp long and encodes a protein having 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 a β- 1 ,3-glucanase 4 cDNA clone isolated from a sugar beet λZAP cDNA library

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

SEQ ID NO: 11 and SEQ ID NO: 12

The DNA sequence (SEQ ID N0.:11) and deduced amino acid sequence (SEQ ID NO.: 12) of a genomic clone encoding the chitinase 1 gene isolated from a sugar beet EMBG 3 genomic library.

The sequence is about 6.3 kb lang and encodes a protein having 439 amino acid residues in the polypeptide chain. The leader sequence is deduced to consist of 26 amino acid residues followed by a hevein domain of 20, a proline rich domain of 132 and a functional domain of 238 amino acid residues. The protein has a C-terminal extension of 23 amino acid residues which probably direct the protein to the vacuole.

The sequence contains two introns at position 2170-4618 and 4776-5406. The intron borders contain the consensus GT/AG sequences. A TATA box sequence (TATAAA) is located at position 1355-1360 which is about 70 bp upstream of the ATG start codon. A putative poly A signal (AATAAA) is located at position 6032.

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MATERIALS AND METHODS

Biological material

Plants

Seeds of Beta vulgaris, cv. "Monova", were sown in clay mixed peat ("Cycas") and placed in growth chamber with 11/13 hours day/night cycles, 25/18°C (day/night) and 70% rh. Light intensity was approximately 25000 lux ("Osram HQI-T", 400 W/DH). Three weeks after sowing the seedlings were replanted singly in 12 cm plastic pots containing the same growth medium. Twice a day the plants were supplied with water containing 0.1% fertilizer: "Stjerne" universal fertilizer, 4:1:4 (N:P:K). Six weeks after sowing the plants were ready for infection experiments with Cercospora beticola .

Nicotiana tabacum and N . benthamiana plants were obtained as described above. Fungi

An isolate of the fungus Cercospora beticola was used for infection experiments. The isolate, "F573", was obtained from United States Department of Agriculture, Agricultural Research Division, Fort Collins, Colorado, USA. An isolate of the fungus C . nicotianae (ATCC 18366) was obtained from the American Type Culture Collection.

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 plug of mycelia was placed in the center of the Petri dish and the culture was incubated at room temperature for 4 weeks. Mycelia for spore induction was "harvested" by cutting off the whole mycelia "mat" including some agar. Sporulation of Cercospora speci es

Mycelia was mixed with distilled water (1:2) in a 50 ml sterile glass tube and homogenized using a "Ultra Turrax T25" mixer operated at 8000 rpm for 2 minutes. 1 ml of the homogenate was transferred to a Petri dish containing solid sporulation medium. "V-8" was used as medium. It contained 200 ml "V-8" juice (Cambells, Italy), 800 ml water, 3 g CaCO₃ and 20 g agar.

The suspension was allowed to settle for 1 hour. After airdrying the culture (approximately 1 hour) the Petri dish was closed, sealed and placed in an incubation chamber at 13ºC and 24 hours light (cool white).

After 7 days of incubation the spores were harvested by pouring 10 ml distilled water onto the Petri dish and firmly brushing the surface of the culture with a sterile brush.

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

Infection wi th Cercospora species

For inoculation, 12.500 spores were suspended in 1 ml of water containing 20 μg of Tween-20. Using a chromatographic atomizer the suspension was applied to the upper leaf surface of six-week old sugar beet or Nicotiana plants until "run off". Immediately after inoculation the plants were placed in a "mist chamber" kept at 30°C, 100% rh and 24 hours light (cool white). After 5 days of incubation the plants were moved to a growth chamber kept at 30°C, 80% rh and 24 hours light. Approximately 10 days after inoculation necrotic spots developed on mature leaves showing that an infection with Cercospora had been established. After inoculation, the sugar beet plants were harvested at specific time intervals for i) small scale purification of chitinase 4, the acidic chitinase SE and β- 1 ,3-glucanase, and

ii) a time course study to determine the expression level of total enzyme activity using radiochemical assays and immunoblotting.

iii) determination of the expression level of each of the enzymes in transgenic plants using the above (ii) techniques,

vi) isolation of mRNA for use in the construction of a cDNA library.

Infection of Nicotiana plants wi th the root pathogen Rhizoctonia solani An isolate of R . solani was obtained from Dr. K. Tzavella-Klonavi (Saloniki, Greece).

An inoculum of R . solani was prepared on barley grains soaked twice in 1% of potato dextrose broth and autoclaved. The grains were inoculated with agar disks of a growing culture of the fungus and incubated for two weeks, after which they were airdried.

Alternatively, disks of R . solani growing on potato dextrose agar can be used directly as inoculum.

The inoculum was mixed into potting soil in different concentrations, and the transgenic plantlets which had been rooted for 14 days, were transplanted into the infected soil. The percentage of surviving plants may be recorded after 1, 2 and 3 weeks, respectively, and after 3 weeks the surviving" plants are assessed for root damage.

Alternatively, seeds from transgenic plants were sown directly in the infected soil. Extraction of protein from 1 g of sugar beet leaf material

More specifically, the small scale purification was carried out as follows. 1 g of leaf material was homogenized by a Ultra-Turrax homo-genizer in citrate buffer (0.1 M, pH 5, 2 ml/g tissue), containing 1 mM of both benzamidine, dithiothreitol and phenylmethylsuphonyl fluoride. Particulate matters were removed by centrifugation at 15,000 × g for 15 minutes. The supernatant comprising the enzymes was transferred to another test tube before the centrifugation was repeated.

Large scale extraction of proteins from sugar beet leaf material

To determine the antifungal potential and the amino acid sequence of the enzymes, large quantities of pure enzymes are required. To obtain sufficient quantities, i.e. mg quantities, a large scale purification of chitinase 4, the acidic chitinase SE and β- 1 ,3-glucanase was carried out from 2 kg of leaf material from naturally infected sugar beet plants, cv. "Monova" . Naturally infected leaves carrying 50 or more necrotic lesions were picked in the field at a breeding station in Italy (Maribo-Italy, Bologna) and stored at 4ºC until the extraction of chitinase 4 was carried out.

Preparation of a chitin column

30 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 slowly added while mixing. The cloudy viscous solution was filtered twice to remove particulate materials; first with glass wool and then with a sintered glass funnel. The filtrate was transferred to a beaker on a magnetic stirrer, and 40 ml of acetic anhydride was slowly added with extensive stirring. After approximately 2 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 Warring blender, covered with methanol, and homogenized for 2 minutes at full power. Methanol, acetic acid and unreacted acetic anhydride were removed by filtration in a Buchner funnel using Whatman No. 1 filter paper. The filtrate was transferred to a beaker, 1 1 of 1 M Na₂CO₃ was added and the pH was adjusted to 9 with 6 N NaOH . 50 ml of acetic anhydride was slowly added and the pH adjusted to 9. The reaction was allowed to take place for 1 hour before the final product was collected by filtering on a Buchner funnel. After extensive washing with water, the product was equilibrated in a 10 mM Tris buffer at pH 8.0 before storing at 4°C. The yield was 700 ml of regenerated chitin. A chitin column was prepared from the regenerated chitin by use of the conventional procedure according to Pharmacia.

Preparation of radioactive col loidal chi tin

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

Preparation of a Laminarin column

Divinylsulfone activated agarose (Mini-leak high, KEM-EN-TEC, Denmark) was employed to immobilize laminarin (β- 1 ,3-glucan) (from Laminaria digi tata , Sigma). 50 g Mini-leak High was dispensed in 200 ml 1 M potassium phosphate (K-P) at pH 11, and 750 mg laminarin dissolved in 5 ml H₂O was added. The reaction was allowed to proceed for 16 hours at 25ºC on a shaking table. Unreacted divinylsulfone groups were blocked by incubation with a solution of 5% mercaptoethanol in 1 M K-P-buffer at pH 9.5. The reaction time was 16 hours at 25°C. Residual mercaptoethanol was removed by excessive washing of the gel on a Buchner funnel. The Laminarin-Agarose was suspended in 20 mM Tris-buffer at pH 8.0, and stored at 4°C. A laminarin column was prepared from the Laminarin-Agarose using the conventional procedure according to Pharmacia. Synthesis of ³H- labelled laminarin

Laminarin was labelled with radioactivity by reduction with ³H-labelled NaB³H₄. 500 mg laminarin (from Laminaria digi tata , Sigma) was dissolved in 2 ml H₂O, and purified by precipitation by addition of 800 μl NaCl (0.2 g/ml) followed by 8 ml absolute ethanol. The precipitate was collected by centrifugation for 5 min. at 15.000 g. The supernatant was discarded and the pellet containing the laminarin was dissolved in 4 ml of 0.1 N NaOH. This solution was transferred to a reaction wessel containing 5 mCi of NaB³H₄. After stirring for 90 min. at 25°C, 600 μl of 1 M HCl was added to destroy unreacted

NaB³H₄. The reaction mixture was divided into 500 μl aliquots and 200 μl of NaCl and 2 ml of absolute ethanol was added to each test tube. After storage for 10 min. at 0ºC, the precipitate was collected by centrifugation for 5 min. at 15.000 × g. The H-labelled laminarin was dissolved in 500 μl of H₂O and the precipitation was repeated until the background level in the supernatant was less than 100 cpm per 20 μl. The labelled solution of laminarin was stored at -20°C. Before use in the β-1,3-glucanase -assay, the solution was diluted 20-fold with water.

Reverse Phase-HPLC

A Kontron AG (Zurich, Switzerland) instrument consisting of 2 model 420 pumps and a solvent mixer was used. Gradient control and data acquisition was performed by a Kontron model 450-MT Data system according to the manufacturers instructions. Proteins eluted from the Mono S column (see below) were subjected to RP-HPLC on either VYDAC RP4 (0.46 × 15 cm; 10 μm particle size; The Separations Group, Hesperia, California) column or a Poly F (Du Pont de Nemours) column. The mobile system used for RP-HPLC was buffer A: 0.1% TFA in water and buffer B: 0.1% TFA in acetonitrile.

SDS-PAGE

SDS-PAGE of crude plants extracts or partly purified chitinases were performed on an Easy-4 apparatus (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. Pure chitinase isoenzymes were analyzed on the Phast-System (Pharmacia) in accordance with the manufacturers instructions. Enzyme assays

The radiochemical chi tinase assay

Chitinase activity was determined radiochemically with ^H-chitin as a substrate.

The specific activity of the ³H-chitin was 460 cpm/nmol N-acetylglucosamine (GlcNAc) equivalent (or 2,3 × 10⁶ cpm/mg ³H-chitin). It was determined by scintillation counting and colorimetric determination of GlcNAc after total hydrolysis of ³H-chitin by crude chitinase preparations from sugar beet leaves and exochitinase from serratia marcescens or Streptomyces griseus .

The assay mixture contained in a total volume of 200 μl of enzyme solution, 50 μl of ³H-chitin suspension (containing 100,000 cpm) and 10 μmol of sodium citrate (pH 5,0). After mixing, the enzymatic hydrolysis of ³H-chitin was allowed to take place at 40°C for 15 min. before addition of 300 μl of 10 % (w/v) TCA. In order to decrease the background reading, 100 μl of bovine serum albumin (10 mg/ml) were added before the insoluble ³H-chitin was removed by centrifugation at 15.000 × g for 5 min. The radioactivity in 300 μl supernatant was determined by scintillation counting. The radiochemical β- 1 , 3 -glucanase assay β- 1,3-glucanase activity was determined radiochemically with ³H-labelled laminarin as substrate.

The assay mixture consisted of 50 μl of enzyme extract, 50 μl of 0,1 M Na-citrate pH 5,0 and 10 μl of ³H- labelled laminarin (192.000 cpm). Incubation was carried out for 15 min. at 40°C. To terminate the reaction, 1000 μl of abs. Ethanol and 50 μl of a saturated NaCl-solution was added. After 10 min. at 0°C, unreacted laminarin was removed by centrifugation at 10.000 × g for 5 min. An aliquot of 400 μl of supernatant was transferred to a scintillation vial. 5 ml of PICO-FLUOR-40 were added and the amount of radioactivity was determined by a liquid scintillation counting. Lysozyme assay

The lysozyme activity of chitinase 4 was determined by the method described by Selstes et al . (1980). More specifically, lysozyme activity was measured in microtiter plates. Each well contains cell walls from Micrococcus lysodeikticus suspended in a 20 mM sodium phosphate buffer, pH 7.4, containing 1 mg/ml of BSA. The initial absorbency at 450 nm was adjusted to 0.6 before addition of egg-white lysozyme or plant chitinase 4. The reaction was followed by measuring the decrease in absorbance at 5 min. intervals for 50 min. β-glucuronidase (GUS) -Assay

When GUS is employed as a reporter gene in connection with the construction of the genetically transformed plants according to the present invention, the success of the transformation may be determined by use of the following GUS-assay described by Jefferson, 1987. Leaf tips were sliced into thin sections (<0.5 mm) and incubated in a 2 mM solution of x-gluc. (5-bromo-4-chloro-3-indolyl-β-glucuronide) dissolved in 0.1 M sodium phosphate buffer pH 7.0 containing 0.5 mM potassium ferri cyanide and 10 mM EDTA. The leaf sections were treated for 2-4 hours at 37°C, rinsed with water and the staining intensity recorded by visual inspection by microscopy.

Purification of chitinase 2, 3 and 4, acidic chitinase SE and β- 1 ,3-glucanase isoenzymes

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

The sugar beet leaves were obtained in Italy (large scale, see "Bio- logical Material"). In the following each of the purification steps outlined below will be explained. The equipment and procedure used for each step are carried out as described below. Extraction of protein from Cercospora beticola infected sugar beet leaves

All steps were performed at 4°C. Centrifugation was carried out at 20000 × g for 20 minutes in a Centrikon model H-401B centrifuge, throughout the purification procedure.

Preparation of cel lfree - extracts

2 kg of Cercorspora infected leaves were homogenized in 4 1 Na-citrate buffer pH 5.0 containing 1 mM DTT (Dithiothreitol), 1 mM BAM (Benzamidine) (starting buffer) and 200 g Dowex 1×2 (100 μm/mesh size. The homogenate was squeezed through a double layer of 31 μm mesh nylon gauze, before centrifugation.

Precipi tation wi th heat and Ammoniumsulfate

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

Purification of chitinase 2, 3 and 4, acidic chitinase SE and β-1,3-glucanase by column chromatography

Chitinase and β- 1 , 3-glucanase isoenzymes were purified from the ammonium sulfate precipitated protein fraction. After solubilization, the protein solution was dialyzed against 10 mM Tris pH 8.0 containing 1 mM DTT and 1 mM BAM. Denatured proteins were removed by centrifugation and the supernatant was loaded on the above outlined two columns e.g. i) a 50 ml Fast Flow Sepharose Q (Pharmacia) and ii) a 100 ml Chitin column (prepared as described above), the columns being connected in series. The columns were equilibrated with the Tris buffer, before 281 ml of the sample were loaded. Unbound proteins ineluding β - 1,3-glucanase were removed by extensive washing with the starting buffer. After disconnecting the Fast Flow Sepharose Q co- lumn, the chitinase was eluted from the chitin column with 20 mM acetic acid, pH 3.2 containing 1 mM DTT. The acidic chitinase SE was eluted from the Fast Flow Sepharose Q column with the Tris-buffer containing 0,5 M NaCl. Purification of β- 1 ,3-glucanase

Separation of β- 1 , 3 -glucanase on Cation Exchange Chromatography

Proteins which were not adsorbed on either the Fast Flow Sepharose Q nor the chitin column were collected, and concentrated to 60 ml by pressure dialysis with an Amicon PM-10 filter (Danver, MA, U.S.A.). After dialysis overnight against 20 mM Na-acetate buffer at pll 4.2 containing 1 mM DTT and 1 mM BAM, the protein solution was loaded on to a 50 ml Fast Flow Sepharose S column (Pharmacia) equilibrated in the dialysis buffer, Unadsorbed proteins were removed by washing with the equilibration buffer. Bound proteins were eluted with a 600 ml linear gradient from 0 to 0.5 M NaCl in the starting buffer.

Three major peaks A, B and C of β - 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 β- 1 , 3 -glucanase on Laminarin -Agarose A 28 ml column of Laminarin-Agarose was equilibrated with a 10 mM

Tris buffer pH 8.0 containing 1 mM of both DTT and BAM. The protein fractions from peak B was combined, concentrated by pressure dialysis to 15 ml and dialyzed against the Tris buffer. After loading of the sample on the Laminarin-Agarose column, the flow through the column was stopped for 20 minutes to allow the β- 1 ,3-glucanase to bind to the affinity ligand. Unabsorbed protein was removed by washing with Tris buffer, β- 1,3-glucanase was eluted with 1 M NaCl in Tris buffer.

Purification of 4 β- 1 , 3 -glucanase isoenzymes by FPLC

Fractions from the Laminarin-Agarose column with β- 1,3-glucanase activity were combined, concentrated and dialyzed overnight against a 20 mM acetate buffer pH 4.5. The proteins were separated on a cation exchange column (Mono S) (Pharmacia) on the FPLC system using a linear NaCl gradient. Four major protein peaks were observed (see Fig. 21). They all four hydrolyzed the H-labelled laminarin substrate in the radiochemical assay for β- 1 , 3 -glucanase (see above).

Purification of the β-1,3-glucanase on Reverse Phase HPLC

The purification was achieved by injecting the FPLC-purified β-1,3-glucanase into the above described Poly F reverse phase HPLC column. Non-adsorbed materials (buffers, salt etc.) were removed by washing with 10% acetonitrile in 0.1% TFA (trifluoro acetic acid). Proteins were eluted by employing a linear gradient of acetonitrile from 10 to 70%.

After this desalting/purification step, peak 3 and 4 were ready for i) N-terminal amino acid sequencing, ii) amino acid composition analysis (see Example 8), iii) tryptic digestion to achieve peptides and iv) injecting into rabbits to produce polyclonal antibodies.

Purification of chitinase 2, 3 and 4

Elution of the chitin column with 20 mM acetic acid, pH 3.2, yielded 40 fractions (10 ml/fraction) with chitinase activity. The fractions were combined, adjusted to pH 4.5, concentrated to 15 ml and dialyzed against a 20 mM Na-acetate buffer at pH 4.5.

2 ml aliquots were loaded onto the above mentioned cation exchange column (Mono-S) by the FPLC system (Pharmacia). Non-adsorbed materials were removed by washing with the acetate buffer. Elution of the chitinase isoenzymes was achieved with a linear gradient from 0 to

1 M NaCl in the acetate buffer. The elution profile is shown in Fig. 1. For further purification, the reverse phase VΥDAC RP4 HPLC column was employed. The conditions were similar to those described above in connection with the purification of β-1,3-glucanase. Purification of acidic chitinase SE

Purification of the acidic chi tinase SE on anion- exchange chromatography

The acidic chitinase SE was eluted from the above described Fast Flow Sepharose Q column with the Tris buffer containing 0.5 M NaCl as shown in the purification scheme. The proteins were dialyzed against 10 mM Tris-HCl, pH 8.0, and loaded onto a 40 ml "Fast Flow Sepharose Q" column equilibrated with the same buffer. The proteins were eluted with a 800 ml linear sodium chloride gradient from 0 to 0,5 mM NaCl. Fractions containing chitinase activity as determined by the radiochemical chitinase assay described above were pooled.

Purification of acidic chitinase SE on Chromatofocusing

The protein fractions were dialyzed against 25 mM Bis-Tris, adjusted to pH 7.0 with iminodiacetic acid. A 15 ml "polybuffer Exchanger" column (Pharmacia; PBE 74) was equilibrated with the same buffer and 50 ml of the sample was loaded. Unabsorbed proteins were removed by washing with the Bis-Tris buffer.

Application of "Polybuffer 74" adjusted to pH 3.6, created a linear pH gradient from 7 to 3.6 and gave desorption of several proteins. The acidic chitinase SE was still retained on the column at this pH, but it was desorbed by addition of 0.3 M NaCl to the "Polybuffer

74".

Purification of acidic chitinase SE by FPLC

Protein fraction with high chitinase activity as determined by the radiochemical chitinase assay described above were pooled and dialyzed against 25 mM Bis-Tris at pH 7.0, The proteins were resolved on a Mono-P FPLC column (Pharmacia) equilibrated with the Bis-Tris buffer. After an initial wash with the starting buffer, three isoenzymes of acidic chitinase SE was separated using a linear salt gradient from 0 to 0.3 M NaCl (Fig. 3). Analysis of the enzymatic cleavage pattern of sugar beet chitinase 4

40 μl of ³H-labelled chitin (50,000 cpm) was incubated with 7 μg of chitinase 4 (purified as described above) in a 0.1 M citrate buffer at pH 6.5. The total volume was 300 μl. After a specified time (15 min., 30 min., 1 hour and 24 hours) the reaction was stopped by the addition of 300 μl of 10% (w/v) TCA. The unreacted polymer of ³H-labelled chitin was removed, and an aliquot (300 μl) of the supernatant was applied to a thin layer chromatography (TLC) plate (Silica gel 60 H, Merck). The mobile phase was n-propanol/H₂O/NH₃ (70/30/1; v/v/v).

A standard of N-acetylglucosamine-derived oligosaccharides was produced by acid hydrolysis of chitin (Rupley, 1964). This standard was used to localize the products from the enzymatic cleavage on the TLC plate. Zones of interest on the TLC plate were removed by scraping with a razor blade, and the silica gel containing the ³H-labelled oligosaccharides was transferred to a scintillation vial. 10 ml of scintillation liquid Dimilume (Packard Instruments) were added and the radioactivity was determined by a liquid scintillation spectrophotometer.

Antifungal activity

An inhibitory effect of sugar beet chitinase 4 has been observed on the growth of both Cercospora beticola and Trichoderma reesei either alone or in combination with the acidic chitinase SE and the basic β-1,3-glucanase 3. Germination of spores and/or growth of hyphae from phytopathogenic fungi, e.g. Cercospora, in the presence of antifungal proteins may be analyzed with three different methods.

Method I is carried out on microscope slides covered with a thin film of medium and incubated with either buffer (control) or μg quantities of the antifungal proteins. Germination of spores or growth of the mycelium is followed by staining with Calcofluor White before analysis by a fluorescent microscope. Method II is carried out in microtiter plates containing growth media, 10 or 100 spores from Cercospora, buffer (control) or the antifungal proteins. The plates are incubated at 25°C before the optical density (at 620 nm) is determined at specified time intervals.

In method III , radiotracer techniques in combination with autoradiography are used to demonstrate that chitin and β- 1 ,3-glucan are important cell wall components in Cercospora and that chitinase 4 can remove radioactivity deposited in the hyphae tip of Cercospora . Method I : Microscopy Sl ide Bioassay

The microscopy slides were covered with a thin layer of potato dextrose agar (PDA) and stored for 6 hours on moistened filter paper in petri dishes. 10 μl of a spore suspension (10.000 spores/ml) was placed in the center of the slide. 10 μl of a 10 mM Tris-buffer, pH 8.0 or 10 μl of a preparation containing 20 μg of the antifungal protein to be tested was applied to the spore suspension. The antifungal protein was dissolved in the Tris-buffer and filtered through a 0,22 μm filter before mixing with the spore suspension. The petridish was sealed with tape and incubated for 24-48 hours at 30°C and full light. At the time for evaluation, the culture was stained with the fluorescent dye Calcofluor White (0.05% (w/v) in water) for 10 min. Calcofluor White binds primarily to cell walls containing nascent structures of chitin, and the fluorescent dye may therefore serve as a marker for differentiation and growth of the hyphae cell wall.

Method II : Microti ter Plate Bioassay

100 μl of potato dextrose broth (PDB) liquid growth medium was placed in each well of a microtiter plate. A spore suspension of Cercospora (100,000/ml) was filtered twice through 4 layers of sterile gaze to remove small amounts of mycelia fragments. The spore suspension was diluted 1:100 and 1:1000 with sterile water, before aliquots of 100 μl was transferred to the microtiter wells. The antifungal proteins were dissolved in the same buffer and treated as described above for method I. The bioassays were carried out with 5 repeats for each dilution of the fungal spores. The microtiter plate was sealed with tape to avoid evaporation and contamination. After incubation at room temperature on an agitator operated with 50 rpm the tape was removed and twice a day, the absorbance was measured at 620 nm. The germination and growth of the fungus was followed for 4 days by measuring the absorbance. For each combination of antifungal protein and spore dilution the absorbance vs. time was plotted.

Method III - Autoradiography Cercospora cultures were grown on a microscope slide as described in method I. Liquid growth medium (PDB) containing ³H-labelled N-acetylglucosamine was distributed uniformly over a one day old culture. After incubation for 20 min. (pulse labelling), the reaction (growth/incorporation) was stopped by dipping the microscope slide in 6% (w/v) of TCA. The preparation was dehydrated in an ethanol gradient (70-100%) and dried.

After the pulse labelling, 50-100 μl of a fraction containing chitinase 4 in 10 mM Tris-buffer at pH 8.0 was distributed over one half of the fixed and dehydrated culture. The microscope slide was placed on moistened filter paper in a petridish. After sealing the petridish, the preparation was incubated at 30°C for 20 hours. The enzyme treatment was stopped by dipping the slide in 6% TCA and the preparation was dehydrated in ethanol as described above.

The microscope slide was coated with an autoradiographic emulsion (Ilford K 5). After drying the emulsion extensively with a "fan dryer" the slide was placed in the dark for 7 days at 7°C and low relative humidity for exposure. The emulsion was developed by placing the slide in Kodak D-19 developer for 10 minutes followed by fixation for 2 minutes and washed in running water for 10 minutes. After drying the preparation was ready for a microscope analysis of the hyphae of the fungus. Production of antibodies for use in serological analysis

Production of polyclonal antibodies to chi tinase 2 , 3 , and 4

Freezedried purified chitinase 2, 3 and 4 (obtained as described above) were dissolved in Tris buffer (10 mM, pH 8,0) and diluted 1:1 with Freunds incomplete adjuvant. Polyclonal antibodies were raised in rabbits according to conventional methods by Dakopatts (Denmark).

Production of monospecific polyclonal antibodies to chi tinase 4 peptides

The procedure was carried out as described in detail for the production of monospecific antibodies to AHAS peptides (Marcussen and

Poulsen, 1991). Based on computer analysis of the amino acid sequence for chitinase 4, four peptides were selected on the criteria of hydrophilicity and variability between chitinase 4 and other basic chitinases. Peptides were custom synthesized by Cambridge Research Biochemicals (UK) . The structures were verified by mass spectroscopy and amino acid analysis to estimate purity.

Before immunization the peptides were conjugated to diphtheria toxoid. The carrier, diphtheria toxoid was converted to the toxoid-sulfosuccinyl-ester derivative by reaction with carbodiimide (EDC) followed by N-hydroxy sulfosuccinimide. After the coupling, the four different peptide-diphtheria toxoid conjugates were purified by gel filtration on a Sephacryl S-300 column. The high molecular weight fractions were collected, freeze-dried and dissolved in water. Immunization in rabbits were performed as described above for the production of polyclonal antibodies to chitinase 2 and 4.

SDS-PAGE and immunob lotting

For immunoblotting, proteins were transferred by semi-dry blotting onto a 0.45 μm nitrocellulose membrane (Schleicher and Schuell, FGR) after separation by SDS-PAGE. The antigens were probed with primary polyclonal rabbit antibodies raised against chitinase 2 and 4 (see above) and subsequently visualized using alcaline phosphatase con jugated secondary antibodies (Dakopatts. Denmark; according to Kyhse-Andersen (1984).

To determine the expression level in transgenic tobacco, the ECL (enhanced chemiluminescence) from Amersham was used. After extraction of the leaf materials, 1 μg protein was applied to each lane of the SDS-PAGE gel. After blotting, the nitrocellulose membrane was treated according to Amershams protocol. In brief, the nitrocellulose membrane was initially treated with 10% BSA, before the primary antibodies to sugar beet chitinase 4 diluted 1:1000 was added. The antigen was detected with horse-radish peroxidase conjugated secondary antibodies. Detection reagent was added and after 2 minutes the protein bands are visualized on a Hyperfilm-ECL.

Analysis of the amino acid composition of the purified chitinase isoenzymes 2,3 and 4 and β- 1 ,3-glucanase 3 and 4 After freeze-drying, the purified chitinase isoenzymes 2,3 and 4 and β- 1 ,3-glucanase 3 and 4 were subjected to amino acid analysis as described by Barkholt and Jensen (1989). An aliquot (4.2 μg) of each of the chitinase isoenzymes and the β- 1 ,3-glucanase respectively were incubated with 3 , 3-dithiopropionic acid to derivatize the cysteine residues before acid hydrolysis. The determination was repeated twice .

Preparation and amino acid sequence analysis of tryptic peptides of sugar beet chitinase 3 and '4, SE and β- 1 ,3-glucanase 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 at 37°C for 4 hours 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 addition of 5 μl of β-mercaptoethanol followed by incubation for 15 minutes at 25°C in the dark. The protein solution was dialyzed against 0.2 M ammonium carbonate (pH 8.0) for 24 hours at 4°C in the dark using Eppendorf test tubes with dialysis tubing (10 kDa cut off; Servapore, Serva, FRG) inserted underneath a punctured lid. Thereafter, precipitated protein was pelleted by centrifugation for 5 minutes at 10,000 × g and the supernatant was transferred to another test tube. The protein pellet was partially solubilized by addition of a few particles of guanidine hydrochloride and incubated with 4 μg TPCK- treated trypsin in 20 μl of ammonium carbonate (pH 8.0) at 40°C for 30 minutes. Finally the supernatant and 6 μg of TPCK- treated trypsin were added. The digestion was allowed to take place at 40°C for 4 hours and stopped by addition of 20 μl of TFA. The peptide solution was subjected to RP-HPLC on a VYDAC C18 column (0.46 × 15 cm; 10 μm particle size; The Separations Group, California) using the mobile system described above for RP-HPLC of proteins (see Fig. 4). Collected peptides were diluted 3 times with buffer A and rechromatographed on a Develosil C₁₈ column (0.4 × 10 cm; 5 μm particle size; Dr. O Schou, Novo-Nordisk, Denmark) using the mobile system described above. Selected peptides were subjected to amino acid sequence analysis. Amino acid sequencing

Amino acid sequencing of the peptides was done with a Pulsed Liquid Phase Protein/Peptide Sequencer model 477 and a HPLC On-line PTH-Amino Acid Analyzer model 120 A from Applied Biosystems (CA, USA), according to the manufacturers instructions. Bacterial strains and enzymes

Restriction enzymes, Klenow polymerase and T4 DNA ligase were supplied by Boehringer Mannheim and used in accordance with the manufacturers instructions. pBluescript was supplied by Stratagene (USA). pUC19 was supplied by Boehringer Mannheim. For subcloning in E . col i , transfer of DNA was carried out using DH5α E . coli cells (from BRL) according to the manufacturers instructions.

Isolation of RNA from sugar beet leaves

Isolation of RNA was carried out as described by Chirgwin et al .

(1976).

Purification of poly-A RNA

The RNA with a poly-A tail was purified by affinity chromatography through an oligo-dT cellulose column. 0.5 g of oligo-dT cellulose was mixed in 5 ml of 0.5 M NaOH for 5 minutes (1 g of oligo-dT cellulose binds 1.2 mg of poly-A RNA). The resulting mixture was neutralized with 10 mM Tris pH 7.5 until pH reached 7.5. An 1 cm column with a diameter of 1 cm was made and equilibrated with 20 ml of column buffer (500 mM NaCl, 10 mM Tris pH 7.5 , 1 mM EDTA). The RNA was denatured at 65°C for 5 minutes, and 5 volumes of column buffer were added to the RNA before chromatography through the column. The "runthrough" was collected and subjected to chromatography again. The column was washed with column buffer until OD₂₆₀ reached 0.01 or less. The poly-A RNA was eluted with TE buffer in 1 ml fractions, and the RNA concentration for each of the fractions was determined at OD₂₆₀. The poly-A RNA-containing fractions were pooled and adjusted to 100 mM NaCl and the RNA was precipitated overnight with 2.5 volumes of 96% ethanol at -20°C. The poly-A RNA was spun and dissolved in H2O at a concentration of 1 μg/μl and stored at -20°C. The yield was about 1-2% of total RNA applied to the column. Isolation of genomic DNA from sugar beet leaves

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

2 g of Cercospora infected sugar beet leaves obtained as described above were ground in liquid N₂ and frozen, and the frozen material was transferred to a 40 ml polyethylene centrifuge tube. 15 ml of extraction buffer (100 mM Tris pH 8.0. 50 mM EDTA and 500 mM NaCl) were added together with 1 ml of 20% SDS and after mixing, the mixture was incubated at 65°C for 20 min. 5 ml of 3 M potassium acetate were added, the solution was mixed (vortex) and incubated for 20 min. on ice. Subsequently, the mixture was centrifugated for 20 min. at 4°C, 25,000 × g. The supernatant was filtered through 1-2 layers of miracloth into a new centrifuge tube, 15 ml iso-propanol was added and the mixture was incubated for 30 min. at -20°C. After another centrifugation at 20,000 × g for 15 min. at 4°C, the pellet was washed with 70% ethanol and thereafter dried briefly before being resuspended in 0.7 ml of X-TE buffer (50 mM Tris, pH 8.0 and 10 mM EDTA). The suspension was transferred to an eppendorf tube and centrifugated for 5 rain. The supernatant was extracted twice with phenol/chloroform. The DNA was precipitated by adding 75 μl of 3 M Naacetate and 500 μl of iso-propanol, mixing and spinning for 30 seconds. Afterwards, the pellet was dissolved in 400 μl of H₂O, and the suspension was adjusted to 100 mM NaCl and precipitated with 1 ml of 96% ethanol. The suspension was centrifugated for 5 min. and the supernatant removed. The pellet was dried briefly, and the DNA dissolved in 200 μl of TE buffer. The DNA concentration was determinated using the absorbance at OD₂₆₀, where OD₂₆₀=1=50 μg DNA/ml. The DNA was stored at -20°C until use.

Construction of a sugar beet cDNA library

On the basis of sugar beet mRNA isolated as described above a λZAP cDNA library was constructed by Stratagene Cloning Systems. Construction of a sugar beet genomic DNA library

On the basis of genomic sugar beet DNA obtained as described above, which had been partially digested with SAU 3A, a genomic sugar beet library was constructed by cloning the genomic DNA in the BamHI site of the vector EMBL3. The library was constructed by Clontech. Plating libraries for screening for relevant DNA sequences

The titer of the library (either of the cDNA or the genomic library) was determined according to Sambrook et al . (1989), and about 10⁶ phages were used for each screening. For each 24.5 × 24.5 mm plates, 2.5 × 10⁵ phages were mixed with 3 ml of the E . coli strain XL 1-Blue (in case of a sugar beet λZAP cDNA library) or LE392 (in case of the sugar beet genomic library (EMBL3)) and grown in LB medium with 10 mM MgSO₄ and 0.2% maltose to an OD₆₀₀=1. The mixture was allowed to stand at 37°C for 20-30 minutes.

Subsequently, 30 ml of top agar (0.7% agarose in LB medium with 10 mM MgSO₄ and 0.2% maltose) (48°C) were added and the resulting mixture plated onto 24.5 × 24.5 mm plates containing 200 ml of LB agar and allowed to grow overnight at 37°C.

Transfer of plaques to nitrocellulose filter in situ

The screening of λZAP recombinant clones by hybridization to single plaques in situ was done as follows.

After growth overnight at 37°C, the plates were cooled for about 15 minutes at 5°C. Phages and DNA were transferred to a hybond-N nylon membrane (Amersham) by placing the dry filter on the lawn of cells.

Phages were allowed to adsorb to the filter for 1 to 5 minutes.

During adsorption it was convenient to mark the filter and plate with a needle for orientation. If replicate filters were made, the marks on the plate were filled with ink, and it was then possible to mark the replicate filters with similar marks.

The filters were then placed with the "plaque side" upwards on Whatman 3MM filter paper sheets soaked with 0.5 M NaOH, 1.5 M NaCl for 30 seconds. They were then washed for 30 seconds in each of the following solutions: 1) 0.5 M NaOH, 1.5 M NaCl, 2) 0.5 M Tris, pH 7.5,

1.5 M NaCl, 3) 2 × SSC (modified Benton, 1977). The filters were air dried and illuminated with UV for 3 minutes with the phage side upwards. Preparation of radioactive probes for use in screening for sugar beet chitinase 4 in sugar beet cDNA libraries

Relevant oligonucleotides were labelled by phosphorylation with bacteriophage T4 polynucleotide kinase according to the method described in Sambrook et al. (1989). More specifically, the oligonucleotides were synthesized without a phosphate group at their 5' termini ends and were labelled with γ - ³²P from [γ-³²P]ATP using the enzyme bacteriophage T4 polynucleotide kinase.

Purification of radiolabelled oligonucleotides by precipitation with ethanol

After inactivation of the bacteriophage T4 polynucleotide kinase by heat, 40 μl of H₂O was added to the tube, the content of which was subjected to thorough mixing. Then 240 μl of a 5 M solution of ammonium acetate and 1 μg of herring sperm DNA were added. The resulting mixture was mixed well, and 750 μl of ice-cold ethanol were added. Again, thorough mixing was performed, and the resulting mixture was stored for 30 minutes at 0°C.

The radiolabelled oligonucleotide was recovered by centrifugation at 12,000 × g for 20 minutes at 4°C in a microfuge. Using an automatic pipette device equipped with a disposable tip, all of the supernatant fluid (which contained most of the unincorporated [γ-³²P]ATP) and any free ³²P generated during the phosphorylation reaction were carefully removed. The resulting residue was redissolved in 100 μl of H₂O and 10 μl of 3M sodium acetate and thereafter 250 μl of 96% ethanol were added. The mixture was subjected to centrifugation for 20 minutes at 4°C, dried and redissolved in 200 μl of H₂O.

Oligonucleotide hybridization of chitinase 4 DNA by filter hybridization

The oligonucleotide hybridization procedure used eliminates the preferential melting of A-T versus G-C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. The hybridization was carried out essentially as described by Wood et al . (1985). The nitrocellulose filters obtained as described above were wetted on the surface with 2xSSC and subsequently prehybridized in hybridization buffer (6×SSC, 1% BSA . 1% Ficoll 4000, 1% PVP, 50 μg/ml of heat denaturated salmon sperm DNA, 50 mM sodium phosphate, pH 6.8). The hybridization was performed at 37°C for 4 hours in a plastic bag with shaking. The filter was hybridized overnight in the same solution plus the radioactive oligonucleotide probe (the 23-mer chit 4 probe) at 37°C with shaking. A 1x10° cpm/ml solution of the hybridization buffer was used. The filter was rinsed three times in 6xSSC at 4°C and thereafter washed twice for 30 min. at 4°C in 6×SSC. Further, the filter was washed three times in TMAC-buffer (3 M Tetramethylammonium chloride, 50 mM Tris pH 8, 0, 2 mM EDTA, 0,1% SDS) for 5 min. at 37°C. (The tetramethylammonium chloride is made in a 5 M stock solution. Since TMAC is hydroscopic, 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 for 20 min. in TMAC-buffer at 55°C.

The filters were dried in the air at room temperature. Inkmarks on the filters serving to align the autoradiographs with the filters and the agar plates were marked with an autoradiography marker (Ultermit, Du Pont de Nemours). The filters were covered with Saran Wrap and an X-ray film (AGFA CURIX RP2) were exposed to the filters for 16-70 hours at -70°C with an intensifying screen. The films were developed and positive plagues were identified by aligning the dots on the film with those on the agar plates.

Picking plaques

Agar fragments containing positive plagues were picked from the agar plate using mild suction and placed in 500 μl of SM phagebuffer (Sambrook et al . , 1989) and 1 drop of chloroform contained in an eppendorf tube. The eppendorf tubes were allowed to stand for 1-2 hours at room temperature so as to allow the phage particles to diffuse out of the agar. About 10⁶ - 10⁷ phages per plaque were obtained. The phages were then diluted in SM phage buffer and mixed with 200 μl of XL1 blue cells (OD₆₀₀ = 1). The mixture was allowed to stand for 20 minutes at 37°C and 2.5 ml of top agarose (48°C) was added. The mixture was poured onto 9 cm Petri dishes and filter prints were made for rescreening.

A single well-isolated positive plague useful for making a phage stock to be used in the in vivo excision was picked from the agar plates according to the method described by Sambrook et al . (1989) using several steps of replating and rescreening. A phage stock was prepared according to the method of Sambrook et al .

(1989).

In vivo excision

In vivo excision of plaques was performed as described in "In vivo excision Protocol" in the Instruction Manual (CAT# 236201, August 30, 1988) for undigested Lambda ZAP II Cloning Kit, Stratagene Cloning Systems.

Preparation of plasmid DNA

Preparation of plasmid DNA was modified according to the method of Sambrook et al . (1989), and was performed as follows:

Bacterial strains (DH5α and XL1-Blue) harboring the plasmids were grown overnight in 5 ml of LB medium supplied with the relevant antibiotics and 5 ml of the overnight culture was harvested by centrifugation for 10 minutes at 3000 × g. The pellet was resuspended in 200 μl of Solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM

EDTA) in 1.5 ml tubes. 400 μl of Solution II (0,2 N NaOH, 1% SDS) was added, the mixture subjected to gentle mixing and placed on ice for 5 minutes. 300 μl of 5 M KOAc pH 4.8 was added and subjected to thorough mixing. The resulting mixture was placed on ice for 10 minutes and subsequently subjected to centrifugation at 15,000 × g for 10 minutes at 4°C. The supernatant (900 μl) was transferred to new tubes and 0.6 volume (540 μl) of icecold isopropanol was added. The resulting mixture was allowed to stand for 15 minutes at room temperature. The mixture was again subjected to centrifugation at 15,000 × g and 4°C for 10 minutes and the supernatant was removed.

The pellet was dissolved in 100 μl of TE and 100 μl of 5 M LiCl was added. The mixture was allowed to stand on ice for 5 minutes and subjected to centrifugation 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 centrifugated at 15,000 × g and 4°C for 30 minutes and the supernatant was removed. The pellet was washed with 70% ethanol (about 100 μl) and dried. The dried pellet was redissolved in 50 μl of TE.

DNA sequencing The plasmid DNA (double-stranded template) to be sequenced was purified by the above described method. Sequencing was performed as follows :

A mixture comprising about 2 μg of the relevant plasmid. 1 μl of 2 M NaOH, 2 mM EDTA, 1 μl of primer (100 μg/ml) and H₂O up to 10 μl was incubated at 85 °C for 5 minutes and subsequently put on ice.

The mixture was neutralized by adding 1 μl of 5 M NH₄Ac and then precipitated by adding 20 ml of 96% ethanol. The resulting mixture was spun for 30 minutes at 4°C and resuspended in 6 μl of H₂O. 1.5 μl of 5 × cone, sequenase buffer was added. The mixture was placed at 37°C for 5 minutes.

4 μl of sequetide (Biotechnology Systems NEN® Research Products, Du Pont de Nemours) and 2 μl of sequenase (United States Biochemical) were added, resulting in a total volume of the mixture of 13.5 μl. The mixture was placed at room temperature for 5 minutes. 3.1 μl of the labelling reaction was transferred to each termination tube (G, A. T and C) containing 2.5 μl of the dNTP terminating mix ture. The mixtures in each of the tubes were allowed to react for 5 minutes at 37°C and 4 μl of stop solution was added. The mixtures were then heated to 85°C and 2 μl of the heated mixture was applied onto a 6% sequencing gel (Gel-mix 6 from BRL). The gel was vacuum dried and exposed to an X-ray film.

Labelling of sugar beet SE DNA probes

DNA probes to be used in the isolation of the sugar beet acidic chitinase SE was labelled by use of the Stratagene oligolabelling kit prime IT, (Random Primer Kit) according to the manufacturers instructions. More specifically, the following procedure was used:

A sample comprising 25 ng (1-23 μl) of the DNA template to be labeled, 0-22 μl of H₂O and 10 μl of random oligonucleotide primers (constituting a total volume of 33 μl) were added to the bottom of a clean microcentrifuge tube. The reaction tubes were heated to 95-100°C in a boiling water bath for 5 minutes and then centrifuged briefly at room temperature to collect the liquid, which may have condensed on the cap of the tubes. The reaction tube containing the DNA sample in LMT agarose was placed at 37°C and the following reagents were added to the reaction tubes: 10 μl of 5X primer buffer containing dATP, dGTP and dTTP.

5 μl of labeled nucleotide α-³²PdCTP (3000 Ci/mM) (Amersham), and

2 μl of diluted T7 DNA Polymerase. The T7 DNA Polymerase was diluted in ice cold Enzyme Dilution Buffer immediately before use to a final concentration of 1 U/μl. The reaction components were mixed with the tip of a pipette.

The tubes were incubated at 37-40°C for between 2 and 10 minutes and subsequently, the reaction was stopped by the addition of 2 μl of Stop Mix. The probes with the ³²P-labeled DNA were further purified using the Elutip™-D column system (Schleicher & Schuell). Then, the probe DNA was made ready for hybridization by mixing the proper amount of radioactive probe with 200 μl of 10 mg/ml salmon sperm DNA. 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 95-100ºC in a boiling water bath for 5 minutes and cooled on ice before use.

Hybridization of SE-DNA

Filter prints obtained as described above under "Oligonucleotide hybridization" of the sugar beet λ-ZAP cDNA library were subjected to prehybridization for 2 hours at 67°C under conventional prehybridization conditions using a prehybridizing solution comprising 2 × SSC, 10 x Denhardt's, 0.1% SDS and 50 μg/ml salmon sperm DNA.

Hybridization was carried out overnight using a hybridization solution identical to the prehybridization solution except for the fact that a radioactive DNA probe prepared as described above had been added.

After hybridization, a washing procedure was carried out in accordance with the following scheme:

2 × 15 min. in 2 × SSC and 0.1% SDS, and 2 × 15 min. in 1 × SSC and 0.1% SDS.

The positive plaques were identified as described under "Oligonucleotide hybridization of chitinase 4 DNA in filter hybridization".

Identification of DNA belonging to the chitinase 4 gene family

To identify DNA belonging to the chitinase 4 gene family, hybridization of the DNA in question with a chitinase 4 probe was carried out using the hybridization procedure disclosed in "hybridization of SE- DNA" except for the fact that the hybridization is carried out at a temperature of 55 °C. The chitinase 4 probe may be the chitinase 4 DNA sequence shown in SEQ ID NO.:1. It is contemplated that a probe prepared on the basis of a characteristic part or a specific subsequence of the chitinase 4 DNA sequence as disclosed herein, e.g. a probe prepared on the basis of the peptide 4-26 may also be useful. To identify a DNA sequence hybridizing to a specific subsequence of the chitinase 4 DNA sequence and encoding a specific part of the chitinase 4 enzyme, the nucleotide probe is advantageously prepared on the basis of the amino acid sequence of said specific part or a subsequence thereof.

Excision of DNA from agarose gels

DNA fragments to be used, e.g. in the construction of genetic constructs according to the invention were isolated as follows.

The DNA was run on LMT (Low Melting Temperature) agarose (Sea Plaque^® GTG, FMC) in TAE (0.04 M Tris-acetate, 0.002 M EDTA) buffer. The DNA band was excised with a Pasteur pipette. To the excised DNA, 1 vol 200 mM NaCl, 10 mM EDTA was added. The gel was melted at 68°C for 10 min. and re-equilibrated to 37°C. Subsequently, 2U/100 μl of agarase (free of DNase, from Calbiochem) was added. The mixture was allowed to stand overnight at 37°C and was subsequently extracted twice with phenol and twice with chloroform, subjected to EtOH precipitation and finally resolubilized in H₂O,

PCR used for the amplification of cDNA encoding SE, β- 1 ,3-glucanase and chit 76 on the basis of sugar beet mRNA The preparation of a partial cDNA molecule was done by use of the Gene Amp^® RNA Amplification Reagent Kit (Perkin Elmer Cetus, USA). The PCR was performed in accordance with the manufacturer's instruc tion with a few modifications. The reverse transcription protocol was followed using the concentrations in the scheme below.

Component volume Final cone.

MgCl₂ solution 4 μl 5 mM

10 × PCR buffer II 2 μl 1 mM

dGTP 2 μl 1 mM

dATP 2 μl 1 mM

dTTP 2 μl 1 mM

dCTP 2 μl 1 mM

RNase Inhibitor 1 μl 1 U/μl

Reverse Transcr: Lptase 1 μl 2.5 U/μl

primer 270 0 .4 μl 2.5 μM

mRNA 3 .6 μl

Total volume per sample 20 μl

In the step cycle the following procedure was used.

Segment 1 42°C for 2 hours

Segment 2 99°C for 5 minutes

Segment 3 5°C for 5 minutes

The PCR protocol was followed except that the Taq polymerase was added later (see PCR cycles) 'and the temperature cycling was changed to the following:

PCR cycles: no. of cycles °C time (min.)

1 98 5

6 5

addition of Taq polymerase and oil

1 94 1

37 2

50 1

72 40

5 94 1

37 2

72 10

30 94 1

42 2

72 5

1 72 20

PCR used in the construction of genetic constructs of the invention and in site-directed mutagenesis on the basis of cloned DNA templates The preparation of the relevant DNA molecule was done by use of the Gene Amp™ DNA Amplification Reagent Kit (Perkin Elmer Cetus, USA) and in accordance with the manufactures instructions except for the temperature cycling. Here the following procedure was used:

PCR cycles no. of cycles °C time (min.)

35 94 1 1/2

60 1 1/2

72 4

1 72 7 EXAMPLE 1

PURIFICATION AND CHARACTERIZATION OF CHITINASE 2,3 AND 4

The method used for the synthesis of regenerated chitin has been specifically developed in order to make it possible to obtain a high yield of active chitinase 4. A high yield of active and pure chitinase is required in order to have sufficient protein material for i) determining the antifungal potential,

ii) preparing and analyzing tryptic peptides which makes it

possible to prepare an oligonucleotide probe suitable for isolation of DNA encoding a chitinase,

iii) producing monoclonal and polyclonal antibodies thereto.

The isolation and characterization of the DNA encoding the chitinase is necessary when the DNA is to be used for the construction of genetically modified plants having an increased chitinase activity. Also, a high amount of pure chitinase is required to make it possible to elucidate and characterize the important parts of the enzyme such as the active site.

The regenerated chitin was obtained by acetylating the free amino groups at low as well as at high pH as described above (as compared to the conventional method in which this synthesis is performed only at low pH). The new combined method was easier, faster and gave a much higher yield and a more stable product than the conventional method in which acetylation is carried out only at low pH.

The degree of purity of the enzymes was examined throughout the purification steps by SDS-PAGE on the Phast-System as described in "Materials and Methods". After separation on the Mono S FPLC column (Fig. 1) only a single silver stained band for each chitinase iso¬zymes 2,3 and 4 could be observed on the SDS-gel (Fig. 2). Further analysis by reverse phase HPLC on a VYDAC RP4 column gave only one protein peak for each of the isozymes. This is further evidence for a homogeneous protein preparation for each of the basic chitinase isozymes. The molecular weights determined by SDS-PAGE for chitinase 2, 3 and 4 are 32, 27 and 26 kDa, respectively (Fig. 2). By isoelectric focusing, the isoelectric points for chitinase 2, 3 and 4 were determined to 8.3, 9.0 and 9.1, respectively. Using the radiochemical chitinase assay described above, all three isoenzymes was found to have a broad pH optimum with maximum activity around 4.5. The specific activity for chitinase 4 is 480 nkat/mg protein, whereas that for chitinase 3 and 2 are 208 and 164 nkat/mg protein, respectively.

In order to determine whether chitinase 4 is an endochitinase producing chitooligosaccharides or an exochitinase liberating only N-acetylglucosamine from the non-reducing end of chitin or chitooligosaccharides, the pattern of reaction products liberated by chitinase 4 from ³H-chitin was analyzed by TLC (Fig. 3). Irrespective of duration of incubation, N-acetylglucosamine was only a very minor reaction product, whereas chitobiose, chitotriose and chitotetraose were the major product. This strongly implies that chitinase 4 is an endochitinase.

In addition to the catalytic activity exerted on ³H-chitin, chitinase 4 was also capable of hydrolyzing the cell walls of Micrococus lysodeicticus using the lysozyme assay described in "Materials and Methods" (see Fig. 4). This demonstrate, that chitinase 4 is a bifunctional enzyme having both chitinase and lysozyme activity.

EXAMPLE 2

ANTIFUNGAL ACTIVITY OF PURIFIED CHITINASE AND β- 1,3-GLUCANASE ISOENZYMES FROM SUGAR BEET LEAVES

Three different bioassays were conducted to ascertain the in vi tro antifungal activity of chitinase and β - 1 ,3-glucanase isoenzymes on the germination and growth of Cercospora beticola . In the same manner the antifungal activity of chitinases and β- 1 ,3-glucanases from other sources or other isozymes from sugar beets may be determinated using either purified enzymes or extracts containing the enzymes. Also, the assays may be used to determine whether a given transgenic plant is within the scope of the present invention.

Method I - Microscope slide B ioassay

Spore cultures of Cercospora germinate and grow well on a thin film of PDA on a microscope slide. The growth can be followed by light microscopic investigations of the number of germinating spores and the total/average mycelial growth. Furthermore, at any specific time the growth activity can be visualised by staining the culture with Calcofluor white followed by microscopic investigation under fluorescent light. The number of hyphae with fluorescent tips and the extension of the staining at the individual tip reflect the growth activity in the culture.

When proteins with strong antifungal activity are added, the number of germinating spores are decreased, and the growth rate of the hyphae is drastically reduced. In Fig. 5 is shown the results when a combination of chitinase 4, acidic chitinase SE and β-1,3-glucanase 3 is applied to the culture. 60 μl of protein solution containing 20 μg of each antifungal proteins were applied to each microscope slide. When chitinase 4 was used alone or in combination with either β-1,3-glucanase 3 or SE alone, the inhibitory effect was less pronounced. Neither β - 1 ,3-glucanase 3 nor SE had any significant inhibitory effect alone or when combined. However, as seen from Fig. 5 when all 3 enzymes were used together, a very strong inhibitory effect was seen indicating a synergistic effect between chitinase 4 SE and β-1,3-glucanase.

Method II - Microti ter plate Bioassay

The germination of spores and growth of the mycelium can be followed in a microtiter plate by measuring the absorbance (620 nm) at specified time intervals. In the control experiments, the growth of Cercospora is initiated after an approx. 40 hours lag period and increases almost linearly for the next 40-50 hours (curve A in Fig. 6). When pure chitinase 4 (5 μg per well) is included, the inial lag period is increased to 75 hours and the growth rate is decreased as compared to the control (curve C in Fig. 6). The eluate from the chitin column is shown as a comparison.

Method III - Autoradiography

In the third bioassay, the chitin in the hyphae cell wall was labelled with ³H-labelled N-acetylglucosamine. After a short pulse, the radioactivity was deposited in the tip of the fungal hyphae (see Fig. 7). When chitinase 4 alone or in combination with SE and β-1,3-glucanase was added after the pulse labeling, the radioactivity deposited in the hyphae tip wasa effectively removed. The amounts of enzymes is similar to that described in Method I (see above). This strongly indicates that the mode of action of chitinase 4 on the cell wall of Cercospora is specifically to hydrolyze the chitin fibers in the hyphae tip and thereby inhibit cell wall synthesis.

The following conclusions can be made on the bases of the above experiments:

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

The inhibition is primarily seen as a lag time for germination, the length of which depends of the strength and concentration of the growth inhibitor.

Fractions which contain both chitinase and β- 1 ,3-glucanase have a stronger inhibiting effect than chitinase alone.

Chitinase/glucanase fractions from Cercospora infected sugar beet plants have a stronger inhibiting effect than fractions purified in the same manner from control plants. EXAMPLE 3

AMINO ACID COMPOSITION AND PARTIAL AMINO ACID SEQUENCES OF THE PURIFIED CHITINASE ISOENZYMES 2, 3 AND 4

After freeze-drying, the amino acid composition of pure sugar beet chitinases 2, 3 and 4 were determined (see "Materials and Methods").

The results are shown in Table I. For comparison, the amino acid composition of chitinase from barley, wheat and bean (Leah et al ., 1987) are included in the Table. The amino acid composition of chitinase 2 is similar to that of bean chitinase in a number of amino acid residues, e.g. aspartic acid, proline, glycine, leucine, tyrosine, phenylalanine, valine and lysine. In contrast, chitinase 3 and 4 have a significant different amino acid composition than any of the other chitinases.

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

TABLE I

Amino acid composition of barley, wheat, bean and

sugar beet chitinases 2, 3 and 4

Amino acid Barley Wheat Bean S.B.2 S.B.3 S.B.4 cDNA

Aspartic acid 23 28 29 34.4 24.7 24.4 22

Threonine 13.8 22 22 16.2 13.0 12.8 12

Serine 17.7 24 26 21.0 24.8 24.8 24

Glutamic acid 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 10 8.6 14.4 14.3 14

Methionine 1.6 3 2 1.8 1.1 1.1 1

Isoleucine 10.8 9 11 11.9 10.9 11.0 11

Leucine 11.3 13 17 16.2 9.0 9.0 8

Tyrosine 11.9 14 15 17.3 12.7 12.7 12

Phenylalanine 12.7 14 13 11.5 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

Tryptophane 3.2 7 4 nd nd nd 3

MW (KD) 27 29 32 30.6 27.6 27.7 25.9

S.B.2 = sugar beet chitinase 2

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 protein, chitinase 4

nd = not determined. Tryptic digestion of sugar beet chi tinase 3 and 4

Analysis of the pure chitinase 4 enzyme has revealed that the N-terminal part of the enzyme is blocked Thus, by analysis of the mature chitinase 4 it was not directly possible to determine its amino acid sequence, and in order to get sufficient information about the enzyme with the eventual aim of being able to isolate and characterize the DNA by which it is encoded, it was chosen to subject the mature enzyme to tryptic digestion in order to obtain protein fragments (peptides) susceptible to amino acid sequencing The tryptic digestion of the purified chitinase enzymes was carried out as described in "Materials and Methods" above. The tryptic peptides were separated by reverse phase-HPLC on the Vydac RP-18 column mentioned above under the conditions specified in "Materials and Methods" see (Fig. 8). Peptides representing large peaks at an absorbance of 214 nm and displaying a high retention time (indicating long polypeptide chains) were selected for further purification on a Develosil RP-18 column.

The purified peptides were subjected to amino acid sequence analysis as described above in "Materials and Methods" and the amino acid sequence of each of the peptides is shown below in Table II.

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

One of the tryptic peptides proved to be very advantageous to form the basis for the construction of an oligonucleotide probe Thus, by analysis of the amino acid sequence of the tryptic peptide 4-26 it was found that use of this sequence in the construction of an oligonucleotide probe would require only few codon choices Thus, this peptide was chosen to form the basis of the construction of an oligonucleotide probe to be used in the isolation of DNA encoding chitinase 4 (see Example 4 below) TABLE II

Tryptic peptides of chitinase 3 and 4

Chitinase 3;

3-10.3 S-T-Y-C-Q-S-Y-A-A-F-P-P-N-P-S-K

3-16.1 A-C-V-T-H-E-T-G-H-F-C-Y-I-E-E-I-A-K

3-16.2 V-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-P-E-T-V-A-N-N-A-V-T-A-F-R Chitinase 4:

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

4-19.3 G-P-L-Q-I-T-W

4-22 S-I-G-F-D-G-L-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-P-S-S-G-G-G-S-V-S-S-L-V-T-D-A-F-F

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

3-10.3: shown in SEQ ID NO. 18

3-16.1: shown in SEQ ID NO. 19

3-16.2: shown in SEQ ID NO. 20

3-22.3: shown in SEQ ID NO. 21

3-23.3: shown in SEQ ID NO. 22

4-4.2: consisting of amino acids No's 244-250 of SEQ ID NO.:2

4-19.3: consisting of amino acids No's 163-169

4-22: No's 179-200

4-23: No's 37-52

4-24: No's 58-75

4-26: No's 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 very specific oligonucleotide gene probe was synthesized using a DNA synthesizer 381 A (Applied Biosystems). Peptide 4-26

Using this gene probe, the expression cDNA library λZAP was screened using the procedure given in "Materials and Methods" above. 8 cDNA clones were obtained from λZAP. and one of the clones was fully sequenced while the others were only partly sequenced. The sequencing was performed as described in "Materials and Methods" above. An almost full length cDNA clone, chit 4-B15, was obtained from the λZAP library and the DNA sequence thereof is shown in SEQ ID NO . : 1.

On the basis of the cDNA sequence, a deduced amino acid sequence of chitinase 4 was obtained SEQ ID NO.:2. The deduced amino acid sequence was aligned with the partial sequence obtained from the chitinase 4 protein (as described in Example 3 above) and an almost 100% identity was observed. This demonstrates that the isolated cDNA clone codes for the chitinase 4 polypeptides purified by the

chromatographic procedure described above. The chit 4-B15 cDNA clone is 966 bp long and encodes a protein having 264 amino acid residues in the polypeptide chain out of the 265 amino acids predicted for the chitinase 4 genomic DNA. The leader sequence consist probably of 23 amino acid residues (out of 24 amino acid residues as determined for the genomic chitinase 4 DNA, see below), followed by a hevein domain of 35 and a functional domain of 206 amino acid residues. After the stop codon the cDNA has a 158 bp 3' noncoding region. As mentioned in Example 3 above, it has not been possible to sequence the N-terminal amino acid sequence of chitinase 4 directly, because the N-terminal is blocked. However comparison with wheat germ agglutinin (WGA-A) and potato chitinase lead to the guess of the first amino acid being Gln. Hereafter the rest of the amino acid sequence of the chitinase 4 N-terminal was deduced from the DNA sequence to be Gln-Asn-Cys-Gly-Cys

The N-terminal sequence of chitinase 4 was further examined by determining the molecular weight (MW) of the mature chitinase 4 by electrospray mass spectrometry as described by G.J. Feistner et al., 1990. A MW of 25893.6 +/- 10 was observed. On the basis of the amino acid sequence, a MW of 25923 can be calculated. Given that the mature chitinase 4 contains 7 S-S-bridges (loss of 14 protons) and that the first amino acid residue Gln is converted to the pyroglutamyl derivative (loss of - NH2 - 15 MW), the calculated MW of the mature chitinase 4 is 25894. This is in agreement with the data observed by the electrospray mass spectrometric analysis and confirms the deduced N-terminal amino acid sequence given above for the mature chitinase 4.

The N-terminal amino acid sequence could be determined for chitinase 2 and the following terminal amino acid sequence was found in chitinase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala.

TABLE I I I

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

WGA-A: QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCGKG--CQNGACWTS

Hevein: EQ**R*AGGKL*********W-*W**STDE**SPDHN**SN-*KD

Chit. Bean: EQ**R*AGGAL**GGN****F-*W**STT****P*- -**SQ-*GG

Chit. Tob : EQ**S*AGGAR*ASG****KF-*W**NTN****P*-N**SQ-*PG

Chit. SB 2: EL**N*AGGAL***G******-*W**NTNP***N

Chit. SB 4: *N**C-A**LC*SRFGF*GSTDA***E*CREGP *^RS

* = amino acid residues identical to WGA-A

WGA-A: shown in SEQ ID NO.:23

Hevein: shown in SEQ ID NO.:24

Chit. Bean shown in SEQ ID NO. 25

Chit. Tob. shown in SEQ ID NO. 26

Chit. SB 2 shown in SEQ ID NO. 27

Chit. SB 4 consisting of amino acids No's 24-54 of SEQ ID NO.:2

EXAMPLE 5 ISOLATION AND CHARACTERIZATION OF THE SUGAR BEET GENOMIC CLONES CHIT

76 AND CHIT 4

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

The three hybridizing clones were characterized by restriction fragment analysis and sequencing. These analysis showed, that one of the clones contained a chitinase gene, now called chitinase 76, the sequence of which is shown in SEQ ID NO . : 5. Sequencing of this gene was initiated with the primer used for screening of the λZAP library (see Example 4), and continued with other primers complementary to sequences inside the chit 76 gene.

The chit 76 gene codes for a 268 amino acid long chitinase which has 80% homology to the chitinase 4 amino acid sequence (vide SEQ ID NO.:1) but only 34% homology to the entire chitinase 1 protein ( vide SEQ ID NO.:11). The gene contains one intron which is located in position 875 to 1262. The exact location of this intron is based on an alignment with the chitinase 4 cDNA SEQ ID NO.:1 (Fig. 24). The intron borders contain the consensus GT/AG sequences. The chit 76 intron is located exactly at the same position as the second intron in the chitinase 1 gene, when the amino acid sequences of chitinase 1 and chit 76 are aligned.

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

In a similar way a genomic clone encoding chitinase 4 was isolated. The DNA has been partially sequenced and about 350 nucleotides of the 5' noncoding region has been elucidated. About 340 nucleotides of the coding region has been sequenced. The sequence appear from the SEQ ID NO.: 3.

Alignment of the 5' noncoding regions from the two genomic genes show boxes of homology (e.g. chitinase 4 nucleotides 14-49, 60-122, 123-135, 159-173, 174-207 and 277-328, (Fig. 26).

Based on knowledge of the chit 4 B15 cDNA sequence and the partially sequenced genomic chitinase 4 gene, the rest of the gene can easily be sequenced. It is contemplated that the chitinase 4 gene comprises at least 1 intron, probably only 1 corresponding to that given in the same position as that of the chitinase 76 sequence. EXAMPLE 6

CHARACTERIZATION OF THE ACIDIC CHITINASE ISOENZYME SE AND DETERMINATION OF PARTIAL AMINO ACID SEQUENCE

The acidic chitinase SE was purified as described in "Materials and Methods" above.

After the final purification on the Mono P FPLC column three isozymes of SE could be resolved (see Fig. 9). By analysis on SDS-PAGE only a single protein band for each of the isozymes could be demonstrated. The same molecular weight of 29 kD was determined by SDS-PAGE. Analysis by isoelectric focusing an isoelectric point of approximately 3.0 was determined for the three isozymes of SE. This corresponds well to the theoretical isoelectric point which has been estimated to 3.87.

In contrast to the basic chitinases 2, 3 and 4, the acidic chitinase SE was not retained on the chitin-affinity column either at the usual condition, at pH 8 (see "Materials and Methods") nor at higher or lower pH. SE did, however, readily degrade the ³H-labelled chitin. The major product of the enzymatic hydrolysis was the hexamers of chitin or higher homologous of chitin oligo saccharides. Since the major product for chitinase 4 was the dimer, a different mode of action for SE is inferred. No lysozyme activity could 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 6 peptides were selected. The peptides were subjected to further purification in the same manner as the tryptic peptides of chitinase 4 described in Example 3 above and the amino acid sequence of the 6 peptides were determined. The peptides were selected using the same criteria as the ones used in connection with chitinase 4. The amino acid sequence of these peptides are shown in Table IV. In addition, the N-terminal amino acid sequence was also determined as shown in the Table IV. TABLE IV

N-terminal: S-Q-I-V-I -Y-W-G-Q-N-G-D-E-G-S-L-A-D-T-C-N

SE 22.5: V-L-L-S-I-G-G-G-A-G-G-Y

SE 23.0 A-D-Y-L-W-N-T-Y

SE 25.1 N-N-P-P-C-Q-Y-D-T-S-A-D-N-L-L-S-S

SE 26.1 Y-G-G-V-M-L-W

SE 30.4 S-L-S-S-T-D-D-X-N-T-F-X-D-Y-L-W-N-T-Y

SE 31.1 T-T-V-Q-A-N-Q-I-F-L-G-L-P-A-S-T-D-A-A-G-S-G-F-I

N-terminal: consisting of amino acids No's 26-46 of SEQ ID NO.:8

SE 22.5: consisting of amino acids No's 98-109 of SEQ ID NO.:8

SE 23.0: consisting of amino acids No's 121-128 of SEQ ID NO.:8

SE 25.1: consisting of amino acids No's 208-224 of SEQ ID NO.:8 SE 26.1: consisting of amino acids No's 271-277 of SEQ ID NO.:8

SE 30.4: consisting of amino acids No's 110-128 of SEQ ID NO.:8

SE 31.1: consisting of amino acids No's 229-252 of SEQ ID NO.:8

EXAMPLE 7

ISOLATION AND CHARACTERIZATION OF THE cDNA FOR THE ACIDIC CHITINASE ISOENZYME SE

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

A partial cDNA molecule was prepared in two steps using the PCR- technique and mRNA, the first step using the above mentioned primers KB7 and 270. The PCR- technique was performed as described above in "Materials and Methods". The cDNA synthesized was isolated on LTM agarose gel and the agarose was removed with agarase. For the subsequent PCR reaction the primers KB9 and 270 were used. The method is illustrated in Fig. 20. The product from the second PCR reaction was cloned in pUC 19 (Boehringer Mannheim) and sequenced.

The DNA sequence obtained for the partial cDNA molecule constituted by nucleotides 711-962 of the DNA sequence shown in SEQ ID NO.:7. This cDNA was used to screen the λ-ZAP cDNA library described in

"Materials and Methods" and 23 cDNA clones were obtained. The longest cDNA clone was sequenced using the method described in "Materials and Methods" above and was found to be 1070 bp long. The sequence is constituted by nucleotides 37-1106 of the DNA sequence shown in SEQ ID NO.: 7. As normally observed in connection with the isolation of cDNA, the entire cDNA was found to be difficult to isolate. Rescreening of the λZAP library with a 122 bp EcoRI -KpnI from the 5' end of the longest cDNA clone (SE22), gave a sequence containing the entire 5' end. The clones were ligated using the KpnI site. The structural gene has a 5' noncoding region of 17 bp, a leader sequence of 25 amino acid residues, a functional domain of 268 amino acid residues and a 3' noncoding region of 202 bp after the stop codon. The cDNA sequence and the amino acid sequence are shown in SEQ ID NO . : 7 and SEQ ID NO.:8, respectively.

When the amino acid sequence obtained from the N-terminal and the 6 tryptic peptides (107 residues) were compared an almost 100% agreement to the cDNA derived sequence were observed. This demonstrates that the isolated cDNA clone codes for the SE polypeptides purified by the chromatographic procedure described above. The cDNA contains the N-terminal as well as the C-terminal end of the mature protein. The N-terminal of the mature SE is apparent from Table IV.

Table VI shows an alignment of the amino acid sequence corresponding to the structural gene for the acidic chitinase SE and the amino acid sequence of a cucumber lysozyme/chitinase (EP 0 392 225 and Metraux, et al , 1989) and an Arabidopsis lysozyme/chitinase (Samac et al . , 1990). It appears from this that there is a homology of about 45% when all tree segment ire compared. When SE is compared with the cucumber lysozyme/chitinase a homology of about 60% was observed. EXAMPLE 8

CHARACTERIZATION AND DETERMINATION OF THE PARTIAL AMINO ACID SEQUENCE FOR THE SUGAR BEET β- 1 ,3-GLUCANASES 3 AND 4

The sugar beet β- 1 ,3-glucanases 3 and 4 were isolated from Cercospora infected sugar beet leaves as described in the above "Materials and Methods". They are basic proteins having a strong affinity for β-1,3-glucan. The amino acid composition of the sugar beet β- 1 ,3-glucanase 3 and 4 isoenzymes are similar to the one given for β- 1,3-glucanases from tobacco and barley as shown in Table VII.

TABLE VI I

Amino acid composition of tobacco and sugar beet β - 1,3-glucanases Ammo acid Tobacco^{a )} Sugar beet 3 Sugar beet 4 Barley Gs^b )

Aspartic A. 35 46.4 53.4 39

Threonine 10 12.6 12.1 14

Serine 23 25.0 27.4 23

Glutamic A. 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

Tryptophane 4 ND ND ND

MW (KD) 32 32.8 37.6 32 pI 9.9 9.5 9.5 9.8 a) Data taken from Shinshi H. et al., 1983

b) From Kragh et al., 1991 SDS-PAGE of β - 1 , 3 -glucanase

The apparent molecular weight of β- 1 ,3-glucanase 3 and 4 determined on a 10-15% gradient SDS-gel (Phast-System, Pharmacia) were 33 and 38 kDa, respectively. The isoelectric point was greater than or equal to 9.5. When analyzed by thin layer chromatography, the major reaction products liberated from laminarin after 24 hours of incubation with the two β- 1 ,3-glucanase isoenzymes 3 and 4 were the dimer, laminaribiose. This strongly suggests that the β- 1,3-glucanase 3 and 4 isozymes are endoglucanases. Amino acid sequencing of β - 1 , 3 -glucanase 3 and 4

The purified β - 1,3-glucanases 3 and 4 were subjected to tryptic digestion using the method described in the above "Materials and Methods" and selected peptides were further purified and sequenced as described in "Materials and Methods" and in Example 3 above. The peptides were selected on the basis of the same criteria as the ones used in connection with the selection of the tryptic peptides of chitinase 4 (see Example 3). The amino acid sequence of the peptides are shown in Table VIII.

TABLE VIII

Amino acid sequences for β-1,3-glucanase 3 and 4 isolated

from sugar beet leaves

Peptide 3-15 W-V-Q-N-N-V-V-P-Y

Peptide 3-17 (A) -G-A-P-N-V-P-I -V-V-S-E-S -G-W- P-S -A-G-G

Peptide 3-16 L-Q-G-K-V-S

Peptide 4-25.1 L-G-N-N-L-P-S-E-E-D-V-V-S-L-Y Peptide 4-26.3 L-D-Y-A-L-F

Peptide 4-27.1 Y-I-A-V-G-N-E-I-M-P-N-D-A-E-A-G-S-I-V-P-A-M-Q-N-V

(Q)-(Q)-(A)-(P)-(R)

Peptide 4-28.2 W-V-Q-N-N-V-V-P-Y

Peptide 4-40.1 G-A-P-N-V-P-I-V-V-S-E-S-G-X-P-S-A-G-G-N-A-A-S-F

Pep. 3-15: shown in SEQ ID NO. 33

Pep. 3-17: shown in SEQ ID NO. 34

Pep. 3-16: shown in SEQ ID NO. 35

Pep. 4-25.1 consisting of amino acids No's 37-51 of SEQ ID NO.:10

Pep. 4-26.3 consisting of amino acids No's 211-216 of SEQ ID NO.:10

Pep. 4-27.1 consisting of amino acids No's 115-139 of SEQ ID NO.:10

Pep. 4-28.2 consisting of amino acids No's 101-109 of SEQ ID NO.:10

Pep. 4-40.1 consisting of amino acids No's 249-272 of SEQ ID NO.:10 EXAMPLE 9

ISOLATION AND CHARACTERIZATION OF THE cDNA FOR β- 1,3-GLUCANASE 3 AND 4

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

Pep. 3-15: W, V, Q, N, N, (V) ...

In the second round of PCR the following sequence was used as 5'primer peptide 4-27.1 consisting of amino acids No's 120-125 of SEQ ID NO. :10

Pep. 4-27.1: N, E, I, M, P, N

By comparing the amino acid sequences from β- 1,3-glucanases in barley (Fincher, 1986) and tobacco (Shinshi et al ., 1988), a consensus sequence was selected and used for construction of a 3'primer with the following consensus sequence: Pep. seq: F,A,M,F,D/N,E.

This sequence was used in the second PCR round whereas the 270 primer used for cloning of SE was used in the first round. To isolate a β -1,3-glucanase 3 clone, the TG-1 primer was used since peptide 4-28.2 = peptide 3-15 (see Table VII in Example 8). This primer was used as the 5' primer for both the PCR reactions. As the 3' primer, the TG-3 and 270 oligonucleotides were used for the first and second round of PCR, respectively.

The resulting PCR products were employed to screen the above described sugar beet cDNA λ-ZAP library to isolate clones harboring cDNA encoding β- 1 ,3-glucanases 3 and 4, respectively. The cDNA sequences and the deduced amino acid sequence of β - 1 ,3-glucanase 4 are shown in SEQ ID NO.: 9 and SEQ ID NO.: 10, respectively.

EXAMPLE 10

SEROLOGICAL CHARACTERIZATION OF SUGAR BEET CHITINASES 2 AND 4 The serological relationship between chitinase 2 and 4 was analyzed by immunoblotting. When a protein sample containing both chitinase 2 (MW 32 kDa) and 4 (MW 26 kDa) was separated by SDS-PAGE before immunoblotting the following results were observed (see Fig. 10).

Chitinase 4 antibodies detect only an approximately 26 kDa protein (chitinase 4), but not the 32 kDa protein (chitinase 2 isozyme) although it is also present on the same nitrocellulose membrane. In contrast chitinase 2 antibody recognizes only a 32 kDa protein

(chitinase 2), but not the 26 kD protein of chitinase 4. This strongly demonstrates the presence of two serological different groups of chitinases. This observation is further substantiated with the immunoblotting analysis of the pure chitinase 2 and 4 antigens. Antibodies to chitinase 4 detect only chitinase 4, whereas antibodies directed against chitinase 2 only recognize chitinase 2 and no crossreactivity at all was observed. The above results suggest that sugar beet contain two different classes of basic chitinases. This observation is also supported by the information obtained from the amino acid sequencing and the amino acid composition (see Table I in Example 3 above) of the basic chitinases 2 and 4. The difference indicates that the genes coding for chitinase 2 and 4 constitute two distinct gene families. As far as the present inventors are aware, the fact that two different classes of basic sugar beet chitinases exist has hitherto not been reported in the literature.

Definition of sugar beet chitinase 2. class When the N-terminal amino acid sequence of sugar beet chitinase 2 was aligned with the following chitinases from bean, tobacco, pea A1, pea A2, pea B (Vad et al ., 1991), barley T (Jacobsen et al . , 1990), and barley K (Kragh et al., 1990), a strong homology between these basic chitinases were observed (see Table IX). This suggests that these chitinases belong to the same chitinase class. This was further substantiated by serological cross reactivity carried out with antibodies raised against sugar beet chitinase 2. This antibody recognized not only sugar beet chitinase 2, but in addition also chitinase P (27.5 kD), Q (28.5 kD), Ch. 32 and Ch, 34 from tobacco (Bol and Linthorst, 1990), chitinases T, K and C from barley and chitinase A1, A2 and B from Pea. When antibodies raised against barley chitinase K or wheat germ chitinase were employed, similar serological cross reactivities were observed. Therefore the chitinases described above were defined as belonging to a chitinase class serologically related to sugar beet chitinases 2, e.g. a sugar beet chitinase 2 class chitinase.

TABLE IX

N-terminal amino acid sequence of chitinase isozymes belonging to the sugar beet chitinase 2 class:

Chitinase 2 ELCGNQAGGALCPNGLCCSQYGWCGNTNPYCGN

Bean EQCGRQAGGALCPGGNCCSQFGWCGSTTDYCGP

Tobacco EQCGSQAGGARCASGLCCSKFGW

Pea B EQCGRQAGGATCPNNLCCSQYGY

Pea A1 EQCGNQAGGXVPPNG

Pea A2 EQCGTQAGGALCPGGL

Barley K EQXGSQAGGATCPNXLCCSRFG

Barley T XQQGSQAGGATCPNXLCCSXFGW Chitinase 2: shown in SEQ ID NO.:27

Bean: consisting of the amino acids No's 1-33 of SEQ ID NO.:25

Tobacco: consisting of the amino acids No's 1-23 of SEQ ID NO.: 26

Pea B: shown in SEQ ID NO.: 41

Pea A1: shown in SEQ ID NO. :42

Pea A2 : shown in SEQ ID NO.: 43

Barley K: : shown in SEQ ID NO. :44

Barley T: shown in SEQ ID NO.: 45

Definition of a sugar beet chitinase 4 class

When antibodies raised against sugar beet chitinase 4 was employed, none of the chitinases from the chitinase 2 class described above could be recognized. Chitinase 4 from sugar beets thus belongs to a new chitinase class so far not detected in other plant species than sugar beets. However, recent studies have indicated that chitinases belonging to the same new class exist in rape seed. Thus, protein extracts of rape seed obtained by a method similar to the one outlined above for sugar beet chitinases were shown to react with the above mentioned polyclonal antibodies directed against chitinase 4 from sugar beets (see Rasmussen et al., 1992 EXAMPLE 11

EXAMINATION OF THE HOMOLOGY BETWEEN THE CHITINASE 4 cDNA AND OTHER CHITINASES USING THE HYBRIDIZATION TECHNIQUE

Besides examining the homology between the mature enzymes, the homology between the cDNA encoding the chitinase 4 enzyme and DNA encoding other chitinase enzymes was examined using the hybridization technique described in the above "Materials and Methods" under the heading "Identification of DNA belonging to the chitinase 4 gene family".

It appears from Fig. 11 that there is a very low degree of homology examined at 55°C between the cDNA encoding the sugar beet chitinase 4 enzymes and DNA encoding chitinases from other plants such as pea, tobacco and beans as well as DNA form sugar beet encoding the chitinase 1 and SE enzymes. These results therefore further indicate that the chitinase 4 enzyme 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 chitinase from rape seed chitinase shown by the high degree of DNA hybridization further indicates that the genes encoding chitinase 4 in sugar beets and the genes encoding the chitinases in rape seed are significantly homologous and thus belong to the same gene class. This is supported by the results disclosed in Example 10 showing a high degree of serological homology between the mature enzymes from the two plants.

EXAMPLE 12

TRANSFORMATION OF BACTERIA CELLS Agrobacterium tumefaciens (the 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 a freeze/thaw method essentially as described by An et al , (1988). For the freeze/thaw method the bacteria to be transformed were cultivated in LB- medium, pH 7.4, overnight at 28°C, 280 rpm. The next day the bacteria were subcultivated in 50 ml of LB-medium, pH 7.4, and grown for about 4 hours until OD 600 (OD₆₀₀) was 0-5-1.0. The culture was cooled on ice and centrifuged for 5 minutes at 10.000 × g at 4°C. The supernatant was removed and the bacteria were carefully suspended in 1 ml of icecold 20 mM CaCl₂. 0.1 ml of the bacteria suspension was pipetted off in icecold cryo tubes and the bacteria were frozen in liquid nitrogen and maintained at -80°C.

For transformation of the bacteria 1 μg of plasmid DNA was first added to a cryo tube with the frozen bacteria. The bacteria were incubated in a 37°C water bath for 5 minutes, 1 ml of LB-medium, pH 7.4, was added to the cryo tube, and the mixture was incubated for 4 hours at room temperature using mild agitation (agitation table, 100 × rpm). The cryo tube was centrifuged for 30 sec. at 10.000 × g, 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 on to a YMB-dish with 50 mg/l kanamycin and incubated for 2 to 4 days at 28ºC until colonies appeared. The presence of a proper plasmids in the bacteria are verified by restriction analysis of the extracted plasmid prior to the use of the bacteria in the transformation of the plants.

In a similar manner, bacterial transformation with other genetic constructs of the invention may be performed, e.g. as shown in Figs. 17, 18, 19, and 22 and explained in Example 18.

EXAMPLE 13 PREPARATION OF GENETICALLY TRANSFORMED TOBACCO (Nicotiana benthamiana and N . tabacum) PLANTS

Plant material

Leaves from plants to be genetically transformed were obtained from plants grown in vi tro or in vivo . In the latter case, the leaves were sterilized prior to transformation. Sterilization was performed by placing the leaves for 20 min. in a solution of 5% Ca-hypochlorite containing 0.1 ml Tween 80 per 1 followed by washing 5 times in sterile water. In vi tro plants were grown in containers on 1/2 shoot inducing medium (1/2 MS) (Murashige & Skoog, 1962).

The leaves were placed one at a time in a 14 cm Petri dish. They were then cut into squares of about 1 cm , all 4 sides consisting of tissue which had been cut. Any cut tissue which had been bleached by hypochlorite sterilization was removed.

Cultivation of bacteria

24 hours before transformation a suspension of Agrobacteria transformed as described above was started by inoculating 2-3 ml media with appropriate antibiotics with the transformed Agrobacteria. The bacteria are grown at 28°C with agitation (300 × rpm).

Transformation

Transformation of the plant was done essentially as described by R.B. Horsch et al . (1985). The bacteria culture was diluted 50x with 1/10 MS immediately before transformation. Approximately 10 ml of the diluted bacteria suspension was poured into a 9 cm Petri dish, and the leaf pieces were dipped in this suspension for about 15 min. The leaf pieces were then removed and excess bacteria suspension was removed with sterile filter paper.

Co-cultivation

The day before transformation co-cultivation Petri dishes containing 1/10 MS medium were coated with acetosyringone (200 μM). On the day of transformation a piece of sterile filter paper was placed on the co-cultivation dishes, and the leaf pieces which had been dipped in the bacteria suspension were placed upside down on the filter paper. The leaf pieces were incubated in a growth chamber in weak light, e.g. 12 hours of light and 12 hours of darkness for 2-3 days. Selection/regeneration

The leaf pieces were transferred to Petri dishes containing shootinducing MS-medium with 300 mg/1 of kanamycin and 800 mg/l of carbenicillin and sub-cultivated every 4 weeks to the same medium. Shoots which appear on shoot- inducing MS-medium 300 k/c dishes were transferred to containers with 1/2 MSO 300 k/c. The shoots were subcultivated when needed. After approximately 2 weeks, the expression of the β-glucoronidase activity using the GUS-assay (see "Materials and Methods") was performed on the leaf tips of green shoots. Planting out

Genetically transformed shoots formed roots and the resulting plants which were GUS-positive were planted out in a growth chamber in water soaked compost. They were then covered with plastic bags and grown for about 1 week, after which the two corners of the plastic bags were cut off. After another week the plastic bags were removed.

EXAMPLE 14

PREPARATION OF GENETICALLY TRANSFORMED SUGAR BEETS PLANTS BY MEANS OF TRANSFORMATION WITH BACTERIA

Transformation was carried out using cotyledonary explants as described below. Seeds were germinated for 4 days in darkness on a substrate containing 0,7 g/l of agarose and 2 g/l of sucrose. The seedlings were then transferred to a Nunc container, containing 1/2 × MS substrate and cultured for 3 days in the light. The cotyledons were removed from the seedling, and the cotyledon explants were then brushed on the petiole with a small brush containing a suspension (OD 660=1,0) of Agrobac terium transformed as described above in Example 12. The cotyledons were then co-cultivated for 4 days on a substrate containing 1/10 MS substrate. The transformed explant were transferred to a MS substrate supplemented with 0,25 mg/l of BAP, 400 mg/l of kanamvcin. 800 mg/l of carbenicillin and 500 mg/ml of cefo taxime and the explants were incubated for 14 days on this substrate. The regenerated shoots were then transferred to containers with MS containing 0.25 mg/l of BAP, 400 mg/l of kanamycin, and 800 mg/l of carbenicillin as the substrate. The isolated shoots were transferred to fresh substrates with 4 weeks intervals for selection and multiplication. Selected shoots were rooted on 1/2 MS substrate containing 1 mg/l IBA.

Tissue from tobacco have been transformed with a genetic construct containing either chitinase 1, chitinase 4, chitinase 76 and acidic chitinase SE and the selective markers, NPT-II and GUS. Selection of the callus and shoots on kanamycin has proved that the obtained tissue expresses the GUS marker and thus that the transformation has occurred.

EXAMPLE 15 ANALYSIS OF CHITINASE AND β- 1,3-GLUCANASE IN TRANSGENIC PLANTS

The expression levels for chitinase and β- 1,3-glucanase isoenzymes can be evaluated either by measuring the total enzyme activity by the two radiochemical assays, by measuring the antifungal activity using the biological methods I-III or by measuring the level of the different isoenzymes by immunoblotting using specific antibodies, all of the methods being described above in "Materials and Methods". The final test of the resulting transgenic plants is the analysis of their degree of resistance to phytopathogenic fungi using the infection system described in "Materials and Methods". Using the biological methods I-III, the antifungal activity of the enzymes in the genetically transformed plants can be determined. A retarded growth of the fungi hyphae shows that the transformation has resulted in a plant having an improved tolerance i.e. an increased antifungal activity to the phytopatogenic fungi compared to a non-transformed plant. In the radiochemical assays, H-chitin or ³H-laminarin are used as substrates for either chitinase or β-1,3-glucanase, respectively. Using standard curves of product formation vs. enzyme amount, the activity for both chitinase and β-1,3-glucanase in crude plant extracts can be determined. This is illustrated further in a time course experiment where the level of either chitinase (Fig. 12a upper part) or β-1,3-glucanase (Fig. 12b lower part) is quantified in sugar beet leaves at specified time intervals after infection with C.

beticola. Although the enzyme level of both the chitinase and the β-1,3-glucanase is very low in the control plant it is readily determined by the very sensitive radiochemical techniques. In the infected plants, an enhanced production of both enzymes was first observed 8-9 days after the infection with the fungal pathogen.

With these techniques, the constitutive level of chitinase as well asβ-1,3-glucanase in transgenic plants can easily be recorded.

These techniques, however, do not differentiate between the various chitinase and β-1,3-glucanase isozymes. Only the total enzyme activities for all the chitinase or all the β-1,3-glucanase isoenzymes are determined. However, the presence of the various chitinase and β-1,3-glucanase isoenzymes can easily be detected separately by analyzing the crude protein extracts by immunoblotting after separation by SDS-PAGE.

The antibody to β-1,3-glucanase 3 recognized only one single protein in the Cercospora infected leaf material (Fig. 13). In contrast, no antigen was detected in the control leaves. This is in agreement with the low constitutive level of expression observed in control plants for β-1,3-glucanase using the radiochemical assay. When antibodies raised against either chitinase 2 or 4 were employed, two major protein bands were induced in the infected leaf tissues. Chitinase 2 antibodies detect a 26 and a 32 kDa band, whereas two proteins having molecular weights of 29 and 26 kDa were observed with the chitinase 4 antibody. When purified chitinases were analyzed by SDS-PAGE and immunoblotting, the protein bands recognized by chitinase 2 antibodies were chitinase 1 (26 kDa) and chitinase 2 (32 kDa), respectively. Similarly, the antibody to chitinase α detected the authentic chitin ase antigen (26 kDa), but in addition also the SE antigen (29 kDa). This was unexpected since no amino acid sequence homology between chitinase 4 and SE has been observed (see SEQ ID NO.: 2 and SEQ ID NO.:8). The "3-D" structure of chitinase 4 and SE on the nitrocellulose membrane may create sufficient epitope recognition to allow the antigen-antibody interaction between the SE antigen and the chitinase 4 antibody. The reaction between the SE antigen and the chitinase 4 antibody was only pronounced when the antibody solution is diluted 1:100 or 1:200. A much weaker reaction was observed when the antibody is diluted 1:5000 or 1:10,000.

Transgenic tobacco plants (Nicotiana tabacum and/or N . benthamiana ) were transformed with either chitinase 4, chitinase 76, the acidic chitinase or chitinase 4 + the acidic chitinase. After selection on kanamycin and regeneration, the transformed plants were examined with respect of i) GUS activity, II) expression of chitinase genes, and iii) degree of resistance against C. nicotiana or R . solani . The transgenic plants expressed GUS-activity in variable amounts. Only plants with high GUS-activity were subjected to further analysis. The expression of the chitinase gene products were analysed by

immunoblotting using the ECL-system described in "Materials and

Methods" and the antibody raised against chitinase 4. In leaf extract from N . benthamiana , transformed with only NPT and GUS, no protein band could be detected by this antibody (see Fig. 23, lane "C").

Transgenic plants containing the constructs, the acidic chitinase, the genomic chitinase 76, chitinase 4, and the double gene construct chitinase 4 + the acidic chitinase, showed a strong positive reaction (see lanes SE, K76, K4, K4 + SE, respectively). To evaluate the level of expression 10 pg of chitinase 4 isolated from sugar beet was included, lane Std in Fig. 23. A broad protein band was observed in extracts from transgenic plants with the chitinase 4 or chitinase 76 gene constructs. When smaller amounts of proteins were applied to the various lanes of the SDS-PAGE, this band could be resolved into three distinct protein bands, having MW of 29, 26 and 25 kD, respectively. The reasons for the triple bands are not known at present. It is, however, contemplated that chitinase 4 is i) not processed given rise to a protein maintaining the signal peptide = the 29 kD band, ii) cleaved at the normal processing site at the amino acid sequence Leu Val Val Ala Gln Asn Cys in chitinase 4 (amino acid position 23-24 in SEQ ID NO.:2) given rise to the 26 kD protein band, and iii) a second putative tobacco processing site is localized at the amino acid sequence Ala Ser Ala Ser - Cys Ala (position 85-86 in SEQ ID NO.: 2). This cleavage site may give rise to the 25 kD polypeptide band. In addition to malfunctinal processing of sugar beet chitinase 4 in transgenic tobacco, the translocation of chitinase 4 was inhibited. In sugar beet, this basic chitinase 4 is deposited in the

extracellular space. In transgenic tobacco, cytochemical analysis, demonstrate clearly that sugar beet chitinase 4 is localized intracellularly.

Preliminary experiments to examine the degree of resistance of transgenic tobacco plants against R. solani and C. nicotiana have been performed. The transgenic plants with the chitinase 4 (10 plants) and chitinase 4 + the acidic chitinase (4 plants) showed less disease symptoms, whereas the control plants (10 plants) containing the GUS and NPT genes were severely infected with C. nicotiana. When seeds of N. tabacum containing the chitinase 4 gene construct were germinated in R. solani infected soil, the survival and growth were improved as compared to the seed from non-transgenic plants.

EXAMPLE 16

MODIFICATION OF THE SUGAR BEET CHITINASE 4 BY SITE DIRECTED MUTAGENESIS

Site directed mutagenesis on a DNA sequence encoding the sugar beet chitinase 4, e.g. the chitinase 4 gene, may be carried out by use of PCR reactions (described in "Materials and Methods" under the heading "PCR used in the construction of genetic constructs of the invention and in site-directed mutagenesis on the basis of cloned DNA templates") using specific 3' and 5' primers for each site directed mutagenesis. The choice of the specific 3' and 5' primers to be used depend on the position in the DNA sequence in which the modification is to be carried out.

Typically, suitable amino acids to be modified, either by substitution, deletion or insertion are selected on the basis of an analysis of the amino acid sequence of the mature chitinase 4 enzyme, optionally in combination with an analysis of the enzyme's 3-D structure. Especially amino acids forming part of the active site of the enzyme or of epitopes thereof as well as amino acids of importance for substrate specificity and substrate binding are of interest in this connection.

The active si te of sugar beet chi tinase 4

The position of the essential amino acid residues included in the active site of chitinase 4 have been tentatively identified by the following observations. Firstly, recent investigations with barley chitinase C demonstrated that chemical modification with carbodiimide and N-bromosuccinimide (NBS) completely inhibits the enzymatic activity (results not shown). Similar experiments carried out with glucoamylase from Aspergillus niger (Sierks et al . , 1990) have elucidated the mode of action by which carbodiimide and NBS inactivates this enzyme. Carbodiimide is covalently linked to the three essential acidic groups (glutamic and aspartic acid residues) constituting the catalytic site of glucoamylase. NBS oxidizes Trp residues important in either stabilizing the transition state intermediate of the catalysis or Trp residues involved in substrate binding at a distance from the catalytic site. The experiments with chitinase C indicate that three acidic and two Trp-residues are very important constituents of the active site. Secondly, by comparison to the active sites of other enzymes which hydrolyze oligosaccharide chains including the glucoamylase described above, the active site of chitinase 4 is contemplated to be constituted by amino acid residue 183 (Asp) and 189 (Glu) in SEQ ID NO.:1 (corresponding to amino acid residue 184 and 190 in the amino acid sequence encoded by the genomic chitinase 4 amino acid sequence. The number given below in brackets denotes the number of the amino acid from the corresponding amino acid sequence encoded by the genomic chitinase 4). In contrast, chitinase C from barley and all other plant chitinases of the same serological class (the sugar beet chitinase 2 class) 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 position of the two important Trp residues involved in the active site of chitinase C have not been elucidated. Since chitinase 4 only contain three Trp residues in contrast to the 6 present in chitinase C, the important Trp residues may be more easily identified in chitinase 4. The two acidic residues 183 Asp and 189 Glu of SEQ ID NO: 2 (184 and 190, respectively) forming the active site of chitinase 4 is

contained in the peptide 4-22: SIGFDGLNAPETVANNAVTAFR. Important Trp-residues of the active sites may be contained in peptide 4-19.3:

GPLQITW and peptide 4-26: TAFWFWMNNVHSVIVNGQGFGASI. The active site of the chitinase 4 differs from the active sites of other plant chitinases, e.g. tobacco, which has the following corresponding amino acid sequences AIGVDLLNNPDLVATDPV shown in SEQ ID NO.: 46, GPIQISH shown in SEQ ID NO.: 47 and SALWFWMTPQSP shown in SEQ ID NO. :48 (Shinshi et al., 1987), and it would be interesting to look at the specific amino acids residues of chitinase 4 which differ from the corresponding amino acids residues of tobacco in order to obtain further information about the active site and possibly identify suitable modifications resulting in improved properties of the modified enzyme. The acidic amino acid residues and the Trp residues are contemplated to be particularly interesting in this respect.

Accordingly, an interesting modification is one in which the glutamic acid in position 189 (190) is substituted with aspargine and/or the aspartic acid in position 183 (184) are substituted with glutamine. Changing the carboxyl groups Asp 183 (184) to Asn and for Glu 189 (190) to Gln in chitinase 4 are in itself expected to have a

negative influence on the enzymatic activity, but is contemplated to result in further knowledge of the mode of action of the chitinase 4 enzyme. The substitution of Trp in positions 169. 204 and 206 (170, 205 and 207, respectively) to Tyr may change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate

specificity. The scheduled substitution given above is only shown as examples, and numerous changes is inferred to achieve a more potent antifungal chitinase. This may be accomplished by site-directed mutagenesis e.g. using the method outlined below.

Site directed mutagenesis

For all the PCR reactions suggested here primers are chosen either themselves containing restriction sites or being located near restriction sites in a manner creating the possibility of exchanging the PCR product with a corresponding sequence in the gene by restriction enzyme digestion followed by ligation of the relevant fragments.

The 5' primer to be used in the following examples is termed SD 0 (see Fig. 14). The number in brackets denotes the number of the corresponding amino acid residue encoded by the genomic chitinase 4 DNA sequence.

When Trp169(170) of the chitinase 4 amino acid sequence is to be substituted by the amino acid Tyr, the following procedure may be carried out:

For the PCR reaction the 3' primer SD1 is used (see Fig. 14).

The resulting PCR product (from bp 301 to 538) is digested with BamHI and PvuII and interchanged with the corresponding fragment of the chitinase 4 gene by conventional methods (Sambrook et al . , 1989). When Glu189(190) is to be substituted with the amino acid Gln, the 3'primer SD2 is used (Fig. 14).

When Asp183(184) is to be substituted with the amino acid Asn, the 3' primer SD3 is used (Fig 14). The PCR products are digested with BamHI and BspMII and interchanged with the BamHI -BspMII fragment of the chitinase 4 gene in a similar manner as described above for exchange of Trp169(170).

When Trp206(207) is to be substituted with the amino acid Tyr, the 3' primer SD4 is used (Fig. 14).

When Trp204(205) is to be substituted with the amino acid Tyr, the 3' primer SD5 is used (Fig. 14).

PCR products are digested with BamHI and Ball and interchanged with the BamHI -BalI fragment in the chitinase 4 gene as described above. In a similar manner, other desirable modifications may be carried out.

EXAMPLE 17

CONSTRUCTIONS OF GENETIC CONSTRUCTS WITH SUITABLE C-TERMINAL EXTENSION C-terminal amino acid sequences found in connection with various plant chitinases and glucanases are exemplified in the specification and are believed to prove useful in modification of one or more of the antifungal enzymes encoded by the genetic constructs according to the present invention which do not comprise a C-terminal extension so as to allow these enzymes to be translocated to the vacuole.

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

Fig. 15a illustrates the sugar beet β-1,3-glucanase cDNA with a tobacco C-terminal extension which is underlined in the figure. Fig. 15b illustrates PCR primers which can be used to change the stop codon and to introduce a part of the C-terminal extension, a Dral site is created at the 3' end.

Fig. 15c illustrates 4 annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a stop codon. a SmaI site and an EcoRI site.

The C-terminal extension can be introduced by exchanging the XbaI- EcoRI fragment in the β- 1 ,3-glucanase gene with the PCR product digested with XbaI and DraI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI using conventional methods (Sambrook et al ., 1989).

Fig 16a illustrates the chitinase 4 gene with a tobacco C-terminal extension (the underlined sequence in the figure).

Fig 16b illustrates PCR primers which can be used to introduce a SmaI site near the stop codon in the chitinase 4 gene.

Fig 16c illustrates four annealed synthetic oligonucleotides containing the sequence for the C-terminal extension, a changed stop codon, a SmaI site and a EcoRI site.

The C-terminal extension can be introduced by exchanging the BamHI-EcoRI fragment with the PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI likewise using conventional methods.

Likewise other C-terminal sequences like the ones exemplified in the description can be added to the chitinase 76, chitinase 4, SE and β-1,3-glucanase sequences. The N-terminal sequence may in a similar manner be exchanged with other N-terminal sequences. Of particular interest may be the N-terminal sequence of chitinase 1 shown in the SEQ ID NO.: 12, the N-terminal sequence of the acidic chitinase SE shown in SEQ ID NO.:8, the N-terminal sequence of chitinase 4 shown in SEQ ID NO.:2, the N-terminal sequence of chitinase 76 shown in SEQ ID N0.:6. and the N-terminal sequence of β - 1,3-glucanase shown in SEQ ID NO.:10. Other interesting N-terminal sequences of the mature protein may be the ones shown in Table III, or in Table IX, or the proline rich region from the sugar beet chitinase 1 shown in SEQ ID NO . : 1.

EXAMPLE 18

Genetic constructs

The excised recombinant pBluescript containing the chitinase 4 cDNA gene (B15 chitinase 4) was subcloned in order to supply the gene with an enhanced 35S promoter and a 35S terminator. This construct was transferred to the plant transformation vector pBKL4 containing a NPTII and a GUS gene. pBKL4 is a derivative of the A. tumefaciens Ti-plasmid pBI121 (Bevan et al., 1984), in which the genes between the left and right borders have been replaced with the following genes: 1) β-glucoronidase (GUS) from E. coli equipped with a CaMV 35S promoter and a Nopaline Synthase terminator (NOS) and 2) Neomycin phosphotransferase (NPT) from E. coli equipped with a CaMV 35S promoter and an Octopine Synthase (OCS) terminator.

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

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

containing the two restriction sites, a ribosome binding site, the

ATG (underlined) and the first 15 nucleotides of the B15 chit 4 clone (nucleotide 8 and 10 were mixed (

) nucleotides due to the fact that the KB3 primer was used for the chit 76 clone as well) was used as the 5' PCR primer and the oligonucleotide KB4 The oligonucleotide KB4 (shown in SEQ ID NO.:50):

was used as the 3' PCR primer (nucleotide 255 and 256 was interchanged in order to destroy the second NheI site).

The PCR product was extracted twice with phenol and twice with chloroform and EtOH precipitated. After resuspension in H₂O the DNA was digested with HindIII and Nhel. The HindIII-NheI fragment from pB15 chit 4 was exchanged with the HindIII-NheI PCR fragment (Fig. 17).

The construct was sequenced with the T7 sequencing primer (corresponding to the pBluescript T7 promoter) and primer 340 (shown in SEQ ID NO. :51) :

and it was confirmed that the entire exchanged sequence was correct. Furthermore, in the 5' sequence the original nucleotide 8 was a T and nucleotide 10 was a C as in the pB15 chit 4 clone and both the NheI sites at position 245 and 251 were still present.

The construct was digested with EcoRI and a fill in reaction was performed with Klenow enzyme in the presence of dATP and TTP, the construct was further digested with BglII after removal of the Klenow enzyme. The DNA fragment BglII-EcoRI containing the entire chitinase 4 sequence was cloned into the vector pPS48 containing an enhanced

35S promoter and a 35S terminator. The chitinase 4 gene was inserted in the correct orientation by digesting the pPS48 vector with BamHI -SmaI (Fig. 17). The chitinase 4 gene with the 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, harboured in an E. coli DH5α has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb. D-3300 Braunschweig (DSM) on 30 July. 1991 under the provisions of the Budapest Treaty under the accession number DSM 6635.

The SE gene was then introduced into the construct pBKL4K4 (Fig. 17). A full length SE gene was constructed by combining the 5' end of the gene from the pSur1 clone (EcoRI-KpnI) with the rest of the gene from pSE22 (KpnI-HindIII) in the cloning vector pUC19 (Fig.

18). The SE gene was subcloned in the Smal site of pPS48 as a EcoRI-HindIII fragment filled in with Klenow polymerase in the presence of all four nucleotides. The orientation of the gene with respect to the enhanced 35S promoter and 35S terminator was examined by restriction enzyme analysis and further confirmed by sequence analysis.

The SE gene with the enhanced 35S promoter and 35S terminator was cloned in the KpnI site of pBKL4K4 as a HindIII fragment in the presence of a HindIII-KpnI adapter (Fig. 18). The HindIII fragment was furthermore cloned in the HindIII site of pBKL4.

Similarly to the chitinase 4, the chitinase 76 gene was cloned in pBKL4 (Fig. 19).

In a similar manner, the glucanase gene can be introduced into the construct pBKL4 , pBKL4K4 , pBKL4KSE, or pBKLKK4KSE (Fig. 22). The full length cDNA clone (SEQ ID NO.:9) was digested with EcoRI and BglII, the sticky ends were filled in with Klenow polymerase in the presence of all four dNTP's. The glucanase gene is then subcloned in the SmaI site of pPS48Mod. The orientation of the gene with respect to the enhanced 35S promoter and the 35S terminator, respectively, may be examined by restriction enzyme analysis and further confirmed by sequence analysis.

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

pBKL4K4KSE. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Dalgaard Mikkelsen, Joern

Bojsen, Kirsten

Nielsen, Klaus K.

Berglund, Lars

(ii) TITLE OF INVENTION: A plant chitinase gene and use thereof

(iii) NUMBER OF SEQUENCES: 21

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Plougmann & Vingtoft

(B) STREET: Sarikt Annae Plads 11

(C) CITY: Copenhagen

(E) COUNTRY: Denmark

(F) ZIP: DK-1021

(v) COMPUTER READABLE POEM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Plougmann, Ole

(C) REFERENCT/DOCKET NUMBER: 329751MKA/SEK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 45 33 11 05 66

(B) TELEFAX: 4533 1118 87

(C) TELEX: 18333

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 966 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(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: sugar beet lampda ZAP cDNA library

(B) CLONE: B15 chitinase 4 cDNA clone

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 2..793

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

G TCT TCT TTC GGA CCA ATC TTT GCC ATA CTC ATG GCA CTT GCT TGT 46 Ser Ser Phe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala Cys

1 5 10 15

ATG TCA AGC ACC CTA GTT GTG GCT CAA AAC TGT GGA TGT GCC TCT AAT 94 Met Ser Ser Thr Leu Val Val Ala Gln Asn Cys Gly Cys Ala Ser Asn

20 25 30

TTA TGT TGT AGC CGA TTT GGT TTC TGT GGC TCC ACA GAC GCC TAC TGC 142 Leu Cys Cys Ser Arg Phe Gly Phe Cys Gly Ser Thr Asp Ala Tyr Cys

35 40 45

GGC GAG GGG TGC AGA GAA GGT CCT TCT AGA TCA COG TCT ACT GGT GGT 190 Gly Glu Gly Cys Arg Glu Gly Pro Cys Arg Ser Pro Ser Ser Gly Gly

50 55 60

GGT TCC GTG TOG AGT TTG GTG ACC GAT GCG TTC TTT AAT AGG ATC ATT 238 Gly Ser Val Ser Ser Leu Val Thr Asp Ala Phe Phe Asn Arg Ile Ile

65 70 75

AAC CAA GCT AGC GCT AGC TGT GCT GCT AAG AGA TTC TAC ACC AGG GCT 286 Asn Gln Ala Ser Ala Ser Cys Ala Gly Lys Arg Phe Tyr Thr Arg Ala

80 85 90 95

GCC TTT TTG ACT GCT CTC AGA TTT TAT CCC CAG TTT GGT AGT GGA TCC 334 Ala Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gln Phe Gly Ser Gly Ser

100 105 110

TCC GAT GTC GTT AGG GGT GAA GTT GCT GCA TTC TTT GCC CAT GTC ACC 382 Ser Asp Val Val Arg Arg Glu Val Ala Ala Phe Phe Ala His Val Thr

115 120 125

CAT GAA ACT GGA CAT TTT TGC TAC ATA GAG GAG ATT GCA AAG TCA ACC 430 His Glu Thr Gly His Phe Cys Tyr Ile Glu Glu Ile Ala Lys Ser Thr

130 135 140

TAT TGT CAG TCA AGT GCA GCA TTT CCA TGC AAC CCA ACT AAG CAA TAC 478 Tyr Cys Gln Ser Ser Ala Ala Phe Pro Cys Asn Pro Ser Lys Gln Tyr

145 150 155

TAC GGA AGG GGG CCT CTT CAG ATC ACA TGG AAT TAT AAC TAC ATA CCA 526 Tyr Gly Arg Gly Pro Leu Gln Ile Thr Trp Asn Tyr Asn Tyr Ile Pro

160 165 170 175

GCT GGT CGA AGC ATT GGA TTT GAT GCT CTG AAT GCA CCA GAA ACA GTT 574 Ala Gly Arg Ser Ile Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val

180 185 190

GCC AAC AAT GCC GTG ACT GCA TTC OGG ACA GCC TTC TGG TTT TGG ATG 622 Ala Asn Asn Ala Val Thr Ala Phe Arg Thr Ala Phe Trp Phe Trp Met

195 200 205

AAC AAT GTC CAC TCT GTT ATC GTC AAT GGC CAA GGG TTC AGC 670 Asn Asn Val His Ser Val Ile Val Asn Gly Gln Gly Phe Gly Ala Ser

210 215 220

ATT CGA GCT ATC AAT GGA ATC GAA TGT AAT GCT GGT AAC TCT GCT GCT 718 Ile Arg Ala Ile Asn Gly Ile Glu Cys Asn Gly Gly Asn Ser Ala Ala

225 230 235

GTT ACT GCT CGT GTT GGG TAC TAT ACT CAG TAT TCT CAA CAG CTT GGC 766 Val Thr Ala Arg Val Gly Tyr Tyr Thr Gln Tyr Cys Gln Gln Leu Gly

240 245 250 255

GTT TCG CCA GGG AAT AAC CTC CGT TGC TAGTCAAATG GCTGGTTTTC 813

Val Ser Pro Gly Asn Asn Leu Arg Cys

260

CTGGTCAGAA TTCACAAGGC TTAGTCAAAA GAAAATAAAG AGAATTAICT AAACTGTTCA 873

TTTCTCATGT AACTTGCTAC TTTGGACAAG CAITAAGTTG GTTAOGAGGC TITATCCAIA 933

AAGGAATGAA AAATATTATT TAAAAAAAAA AAA 966

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENCTH: 264 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ser Ser Phe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala Cys Met

1 5 10 15

Ser Ser Thr Leu Val Val 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 Pro Cys Arg Ser Pro Ser Ser Gly Gly Gly

50 55 60

Ser Val Ser Ser Leu Val Thr Asp Ala Phe Phe Asn Arg Ile Ile Asn

65 70 75 80

Gln Ala Ser Ala Ser Cys Ala Gly Lys Arg Phe Tyr Thr Arg Ala Ala

85 90 95

Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gln Phe Gly Ser Gly Ser Ser

100 105 110

sp Val Val Arg Arg Glu Val Ala Ala Phe Phe Ala His Val Thr His 115 120 125

Glu Thr Gly His Phe Cys Tyr Ile Glu Glu Ile Ala Lys Ser Thr Tyr

130 135 140

Cys Gln Ser Ser Ala Ala Phe Pro Cys Asn Pro Ser Lys Gln Tyr Tyr

145 150 155 160

Gly Arg Gly Pro Leu Gln Ile Thr Trp Asn Tyr Asn Tyr Ile Pro Ala

165 170 175

Gly Arg Ser Ile Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val Ala

180 185 190

Asn Asn Ala Val Thr Ala Phe Arg Thr Ala Phe Trp Phe Trp Met Asn

195 200 205

Asn Val His Ser Val Ile Val Asn Gly Gln Gly Phe Gly Ala Ser Ile

210 215 220

Arg Ala Ile Asn Gly Ile Glu Cys Asn Gly Gly Asn Ser Ala Ala Val

225 230 235 240

Thr Ala Arg Val Gly Tyr Tyr Thr Gln Tyr Cys Gln Gln Leu Gly Val

245 250 255

Ser Pro Gly Asn Asn Leu Arg Cys

260

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 691 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(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: sugar beet EMBL3 genomic library

(B) CLONE: genomic chitinase 4 clone

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 356..691

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAGCTTATTG TCCAAATAAT TTTACATAAC AAGTTCAGTG AAOGGAGAAG ATAACTATCC 60

ATATATATAA CAAAGGTTTC TTCCTTTCAT TTTCCTTGAA CAAGTCAAAC TATNTACACC 120 AAATCATTGT CCAAAATAAA ATTAAATGTG TTGGCTAAGT CAAATTTGAA CACTTCTGAA 180

TGTATCTAAA ATΑTCTCCAT TCCCATCTIA TTAAITAGAA TACAAGTAAG CAAGTΑGCCA 240

AACEAGTAAA CATTTCCTCA AAGTACCACC CTTATAATTT TCTATATAAA CCCATATACA 300

AGTGTCTACT TTCCTATCC CATACATTAT ATTCGTTGGCTTTAACATACT CCAAAATGTC 360

TTCTTTCGGA CCAATCTTTG CCATACTCAT GGCACTTGCT TGTATGTCAA GCACCCTAGT 420

TGTGGCTCAA AACTGTGGAT GTCCCTCTAA TTTATCTTGT AGCCVATTTG GTTTCTGTGG 480

CTCCACAGAC GCCTACTGCG GCGAGGGGTG CAGAGAAGGT C CTTGTAGAT CACCGTCTAG 540

TGGTGGTGGT TCCGTGTOGA GTTTGGTGAC CGATGCGTTC TTTAATAGGA TCATTAACCA 600

AGCTAGCGCT AGCTCTGCTG GTAAGAGATT CTACACCAGG GCTGCTTTTT TGAGTGCTCT 660 CAGATTTTAT CCCCAGTTTG GTAGTGGATC C 691 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 112 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ser Ser Phe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala Cys 1 5 10 15

Met Ser Ser Thr Leu Val Val Ala Gln Asn Cys Gly Cys Ala Ser Asn

20 25 30

Leu Cys Cys Ser Arg Phe Gly Phe Cys Gly Ser Thr Asp Ala Tyr Cys

35 40 45

Gly Glu Gly Cys Arg Glu Gly Pro Cys Arg Ser Pro Ser Ser Gly Gly

50 55 60

Gly Ser Val Ser Ser Leu Val Thr Asp Ala Phe Phe Asn Arg Ile Ile 65 70 75 80

Asn Gln Ala Ser Ala Ser Cys Ala Gly Lys Arg Phe Tyr Thr Arg Ala

85 90 95

Ala Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gln Phe Gly Ser Gly Ser

100 105 110

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1838 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(B) STRAIN: monσva

(F) TISSUE TYPE: leaf

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: sugar beet EMBL3 genomic library

(B) CLONE: genomic clone chitinase 76

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(469..874, 1263..1660)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ACTATATATA AATTGATCAT ATTAATTTTA AATTGTTGGT TGAAAATGTG TTAATGCCTC 60

TTGAGTCTTG ATACAACTTA AAAACGGAGC CGGTTGGGAA ACATTTTTΑC ATGATAAGTT 120

CAGTTAACGA AGAACCGTAA TGGACCCATA TACAACAAAA TATCCGTCGT TTCATTTTCC 180

TTGAACAAGT CAAACTAATA ACACGAATCA TTCGATAAAA TGACTGGCCA AAGTCAAATG 240

TCAACTCGAN ATAAGAAAAG ACATCGAGTC AAATGTCAAC ATTTTAAACG TATCAAACAA 300

TAATTCCATT CACATCCCAC TATACAACTA GCTAACTCGT AAGCTTCTTC CCTAAATCAC 360

CTATCTTTCA TTTTCTATAT AAACTCATCT TCAAGTGTCT AGTTTCCACA ACCCACTCAT 420

TGCTTCAAAA GTTCCTCTAC TAGTCTACTA TCATTGTACT CCTCCAAAAT GTCTTCTCTT 480

GGACCTTTTT TGGCTATACT TATAGCAGTT GCCTGEATGT CTAGCACCCT GGTTCTGGCT 540

CAAAACTGTG GCTGTGCCTC TGGTTTATGC TGTAGCAGAT ATGGTTACTG CGGCACCACA 600

GCTGCCTACT GCGGCACTGG GTGCCAGCAA GGTCCTTGTT CCTCAACGCC ATCCACCCCG 660

AGTGGTGGTG TTTCGGTCCC AAGTTIGGTG ACCGATGCAT TCTTTAATGG AATCAITAAC 720

CAAGCAAGCT CTAGCTGTGC TGGTAAGAGC TTCTACACTA GGTCTGCTTT CTTGAGTGCT 780

CTCAGTTCTT ATCCTCAGTT TGGTAGTGGA TCCTCCGATG AGGTTAAACG TGAAGTTGCT 840

GCCTTTTTTG CTCATGCGAC GCATGAAACT GGACGTAACT GTTAACATTA TTAATGCCTC 900

CTTTGATAGA ATTGAAATCG AATAAAATCT TCTTCCCCGC TCATTTGCGC GCACTTAGCT 960

ATTCAGCTAA TCTTATTGTT TTATGTCAAT CATTCTGTCT TAATTATTTT TTGTAATTGA 1020

GAATTGTGTC TAAATCTATT ATGTGGATTG CAAACCAATA ATATTGAGTG ACGTATAATG 1080 CTAAAAGAAA TGAGAGCAAA AGATTTGAAA TTAATTGAAA CTAGTTTTTA GTTTGCTACT 1140

TAAAACTGAT TTAATTCATA TTATTATCTT AAGTTGAATT AAGCGATACC TAAATCAAAG 1200

GGAATGCATT GAGTTACAGA AAAATATATA CTCAGCTGAT CAATTGAACT TGTGTGTTGT 1260

AGATTTTTGC TACATAGAGG AGATTGCCAA ATCAACCTAT TGTCAGTCGA GCACAACATG 1320

GCCATGCACC ACAAATAAGC AATACTACGG ACCTGGGCCT CTCCAAATCA CATGGAACTA 1380

CAACTACGGA CCAGCAGGTC GAAGCATTGG ATTTGATGGT TTGAATGCAC CTGAAACAGT 1440

TGCCAATGAT GCTCTTATCG CCTTTAAGAC AGCCTTCTGG TTTTGGATGA ACAATGTCCA 1500

CTCTCGAATT GTCTCCGGCA AAGGGTTTGG CTCCACCATT CGAGCTATCA ATGGAGGTGA 1560

ATGTGGTGGC GGGAACACAC CGGCGGTCAA CGCTCGTGTT AGGTACTATA CTCAGTATTG 1620

CAATCAGCTT GGTGTTTCAC CTGGGAATAA CCTCTCTTGC TAGTCACATA ATCGAAGTGT 1680

TTCCATGGTC ACAATTTACA AGTCTTAGAC TCTEAGTATA AGGAAAATAA AAATACAATC 1740

AAGGGAACTG ACTTCTTTTC TTAGCCACTA AGGGAAATAT GCATCACTTT GTAATTTATA 1800

TATATTTCAT AGTCTTACGG CCTATTAATA GGGATACG 1838 (2) INFORMATION FOR SEQ 3D NO:6:

(i) SEQUENCE CHARACTERICTICS:

(A) LENGTH: 268 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(B) STRAIN: monova

(F) TISSUE TYPE: leaf

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: sugar beet EMBL3 genomic library

(B) CLONE: chitinase 76 genomic clone

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Ser Ser Leu Gly Pro Phe Leu Ala Ile Leu Ile Ala Val Ala Cys 1 5 10 15

Met Ser Ser Thr Leu Val Val Ala Gln 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 Gln Gln Gly Pro Cys Ser Ser Thr Pro Ser Thr Pro

50 55 60 Ser Gly Gly Val Ser Val Pro Ser Leu Val Thr Asp Ala Phe Phe Asn 65 70 75 80

Gly Ile Ile Asn Gln Ala Ser Ser Ser Cys Ala Gly Lys Ser Phe Tyr

85 90 95

Thr Arg Ser Ala Phe Leu Ser Ala Leu Ser Ser Tyr Pro Gln Phe Gly

100 105 110

Ser Gly Ser Ser Asp Glu Val Lys Arg Glu Val 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

Lys Ser Thr Tyr Cys Gln Ser Ser Thr Thr Trp Pro Cys Thr Thr Asn 145 150 155 160

Lys Gln Tyr Tyr Gly Arg Gly Pro Leu Gln Ile Thr Trp Asn Tyr Asn

165 170 175

Tyr Gly Pro Ala Gly Arg Ser Ile Gly Phe Asp Gly Leu Asn Ala Pro

180 185 190

Glu Thr Val Ala Asn Asp Ala Val Ile Ala Phe Lys Thr Ala Phe Trp

195 200 205 Phe Trp Met Asn Asn Val His Ser Arg Ile Val Ser Gly Lys Gly Phe 210 215 220

Gly Ser Thr Ile Arg Ala Ile Asn Gly Gly Glu Cys Gly Gly Gly Asn 225 230 235 240

Thr Pro Ala Val Asn Ala Arg Val Arg Tyr Tyr Thr Gln Tyr Cys Asn

245 250 255 Gln Leu Gly Val Ser Pro Gly Asn Asn Leu Ser Cys

260 265

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(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: sugar beet lairpda-ZAP cDNA library

(B) CLONE: "SE" cDNA clone (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 18..896

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ACGTACCCAA AACAAGC ATG GCA GCC AAA ATA GTG TCA GIT CTA TTC CTG 50

Met Ala Ala Lys Ile Val Ser Val Leu Phe Leu

1 5 10

ATT TCT CTC TTA ATC TTT GCT TCA TTC GAG TCC TCT CAT GGC TCC CAA 98 Ile Ser Leu Leu Ile Phe Ala Ser Phe Glu Ser Ser His Gly Ser Gln

15 20 25

ATT GTC ATA TAC TGG GGC CAA AAT GCT GAT GAA GGA ACT CTT GCT GAC 146 Ile Val Ile Tyr Trp Gly Gln Asn Gly Asp Glu Gly Ser Leu Ala Asp

30 35 40

ACT TCT AAC TCC GGA AAC TAC GGT ACC CTG ATC CTA GCT TTC CTA GCT 194 Thr Cys Asn Ser Gly Asn Tyr Gly Thr Val Ile Leu Ala Phe Val Ala

45 50 55

ACC TTT GCT AAC GGG CAA ACC CCG GCG CTG AAC TTA GCT GGG CAC TCT 242 Thr Phe Gly Asn Gly Gln Thr Pro Ala Leu Asn Leu Ala Gly His Cys

60 65 70 75

GAC CCT GCT ACA AAT TGT AAC ACT CTG AGC ACT GAC ATC AAA ACA TGC 290 Asp Pro Ala Thr Asn Cys Asn Ser Leu Ser Ser Asp Ile Lys Thr Cys

80 85 90

CAA CAG GCA GGC ATT AAG GTA CTC CTC TCT ATA GGA GCT GCT GCC GGA 338 Gln Gln Ala Gly Ile Lys Val Leu Leu Ser Ile Gly Gly Gly Ala Gly

95 100 105

GGC TAT TCT CTT TCC TCA ACC GAT GAT GCA AAC ACA TTT GCT GAT TAC 386 Gly Tyr Ser Leu Ser Ser Thr Asp Asp Ala Asn Thr Phe Ala Asp Tyr

110 115 120

CTC TGG AAC ACT TAT CTT GGG GCT CAG TCC AGC ACC CGA CCC CTT GGA 434 Leu Trp Asn Thr Tyr Leu Gly Gly Gln Ser Ser Thr Arg Pro Leu Gly

125 130 135

GAT GCA GTT TTG GAT GGT ATT GAT TTC GAT ATC GAG ACT GCT GAT GGC 482 Asp Ala Val Leu Asp Gly Ile Asp Phe Asp Ile Glu Ser Gly Asp Gly

140 145 150 155

AGA TTT TGG GAT GAC CTA GCT AGA GCA TTG GCA GCT CAT AAC AAT GCT 530 Arg Phe Trp Asp Asp Leu Ala Arg Ala Leu Ala Gly His Asn Asn Gly

160 165 170

CAG AAA ACA GTG TAC TTA TCA GCA GCT CCT CAA TGT CCC TTG CCA GAT 578 Gln Lys Thr Val Tyr Leu Ser Ala Ala Pro Gln Cys Pro Leu Pro Asp

175 180 185

GCC AGC TTA AGC ACT GCC ATA GCC ACA GGC CTA TTC GAC TAT GTA TGG 626 Ala Ser Leu Ser Thr Ala Ile Ala Thr Gly Leu Phe Asp Tyr Val Trp

190 195 200 GTT CAG TTC TAC AAT AAC CCC CCT TGT CAA TAT GAT ACC AGC GCT GAT 674 Val Gln Phe Tyr Asn Asn Pro Pro Cys Gln Tyr Asp Thr Ser Ala Asp

205 210 215

AAT CTC TTG AGC TCG TGG AAC CAG TGG ACC ACA GTA CAA GCT AAC CAG 722 Asn Leu Leu Ser Ser Trp Asn Gln Trp Thr Thr Val Gln Ala Asn Gln

220 225 230 235

ATC TTC CTC GGA CTA CCA GCA TCA ACT GAT GCT GCC GGC AGT GGT TTT 770 Ile Phe Leu Gly Leu Pro Ala Ser Thr Asp Ala Ala Gly Ser Gly Phe

240 245 250

ATT OCA GCA GAT GCT CTT ACA TCT CAA GTC CTT CCC ACT ATC AAG GCT 818 Ile Pro Ala Asp Ala Leu Thr Ser Gln Val Leu Pro Thr Ile Lys Gly

255 260 265

TCT GCT AAA TAT GGA GGA GTC ATG CTA TGG TCA AAG GCA TAT GAC AGT 866 Ser Ala Lys Tyr Gly Gly Val Met Leu Trp Ser Lys Ala Tyr Asp Ser

270 275 280

GGG TAC AGC ACT GCT ATT AAA AGC ACT CTT TAATTTAAAT TACTAGTGTA 916 Gly Tyr Ser Ser Ala Ile Lys Ser Ser Val

285 290

TCCAAAGATA TAGATACAAA ATAAGTTAIA GAGATACATC AAAAAACCAT CTTAGTTTTA 976

AATTTTTAT GCACCACAAA AGCTTGTAAT ACTAATATAC TATTATCATA AATGGCTTAT 1036

TGCCTCGCTA TATTTTGGTG ATTATTATAT ACACAGTTAC MCTTCGCAA TTATGCGAGT 1096

CTTTCTAAAA 1106

(2) INFORMATION FOR SEQ 3D NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 293 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Ala Ala Lys Ile Val Ser Val Leu Phe Leu Ile Ser Leu Leu Ile

1 5 10 15

Phe Ala Ser Phe Glu Ser Ser His Gly Ser Gln Ile Val Ile Tyr Trp

20 25 30

Gly Gln Asn Gly Asp Glu Gly Ser Leu Ala Asp Thr Cys Asn Ser Gly

35 40 45

Asn Tyr Gly Thr Val Ile Leu Ala Phe Val Ala Thr Phe Gly Asn Gly

50 55 60

Gln Thr Pro Ala Leu Asn Leu Ala Gly His Cys Asp Pro Ala Thr Asn

65 70 75 80 Cys Asn Ser Leu Ser Ser Asp Ile Lys Thr Cys Gln Gln Ala Gly Ile 85 90 95

Lys Val Leu Leu Ser Ile Gly Gly Gly Ala Gly Gly Tyr Ser Leu Ser

100 105 110

Ser Thr Asp Asp Ala Asn Thr Phe Ala Asp Tyr Leu Trp Asn Thr Tyr

115 120 125

Leu Gly Gly Gln Ser Ser Thr Arg Pro Leu Gly Asp Ala Val Leu Asp 130 135 140

Gly Ile Asp Phe Asp Ile Glu Ser Gly Asp Gly Arg Phe Trp Asp Asp 145 150 155 160

Leu Ala Arg Ala Leu Ala Gly His Asn Asn Gly Gln Lys Thr Val Tyr

165 170 175

Leu Ser Ala Ala Pro Gln Cys Pro Leu Pro Asp Ala Ser Leu Ser Thr

180 185 190

Ala Ile Ala Thr Gly Leu Phe Asp Tyr Val Trp Val Gln Phe Tyr Asn

195 200 205

Asn Pro Pro Cys Gln Tyr Asp Thr Ser Ala Asp Asn Leu Leu Ser Ser 210 215 220

Trp Asn Gln Trp Thr Thr Val Gln Ala Asn Gln Ile Phe Leu Gly Leu 225 230 235 240

Pro Ala Ser Thr Asp Ala Ala Gly Ser Gly Phe Ile Pro Ala Asp Ala

245 250 255

Leu Thr Ser Gln Val Leu Pro Thr Ile Lys Gly Ser Ala Lys Tyr Gly

260 265 270

Gly Val Met Leu Trp Ser Lys Ala Tyr Asp Ser Gly Tyr Ser Ser Ala

275 280 285

Ile Lys Ser Ser Val

290

(2) INFORMATION FOR SEQ 3D NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1249 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(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: sugar beet lampda-ZAP cDNA library (B) CLONE: beta-1,3-glucanase cDNA clone

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 34..1041

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

AATTTTGTTT ATTCTTAGAG TTATTTCTTC ACA ATG AGG CTA ATT AGC ACA ACT 54

Met Arg Leu Ile Ser Thr Thr

1 5

TCT GCA GTT GCT ACT TTG CTG TTT CTT GTA GTA ATT CTA CCT ACT ATT 102 Ser Ala Val Ala Thr Leu Leu Phe Leu Val Val Ile Leu Pro Ser Ile

10 15 20

CAA CTG ACA GAG GCA CAA ATT GGC CTA TCT AAC GGG AGA CTA GGC AAC 150 Gln Leu Thr Glu Ala Gln Ile Gly Val Cys Asn Gly Arg Leu Gly Asm

25 30 35

AAC TTA CCT TCC GAG GAA GAT GTT CTA AGC TTG TAC AAG TCG AGG GGA 198 Asn Leu Pro Ser Glu Glu Asp Val Val Ser Leu Tyr Lys Ser Arg Gly

40 45 50 55

ATA ACG AGG ATG AGA ATC TAT GAC CCT AAC CAA CGG ACC CTC CAA GCG 246 Ile Thr Arg Met Arg Ile Tyr Asp Pro Asn Gln Arg Thr Leu Gln Ala

60 65 70

GIT AGA GGA TOG AAT ATA GGG CTA ATC GTC GAT GTC CCT AAG CGT GAC 294 Val Arg Gly Ser Asn Ile Gly Leu Ile Val Asp Val Pro Lys Arg Asp

75 80 85

CTA AGG TCA CTC GGC TCC GATGCTGGGGCTGCGTCTOCTTGG GTC CAA 342 Leu Arg Ser Leu Gly Ser Asp Ala Gly Ala Ala Ser Arg Trp Val Gln

90 95 100

AAC AAT GTA CTC CCT TAC GCG TCT AAT ATT CGA TAC ATA GCA CTTGGT 390 Asn Asn Val Val Pro Tyr Ala Ser Asn Ile Arg Tyr Ile Ala Val Gly

105 110 115

AAT GAA ATA ATG CCT AAT GAT GCC GAG GCA GGG TCA ATT GTC COG GCC 438 Asn Glu Ile Met Pro Asn Asp Ala Glu Ala Gly Ser Ile Val Pro Ala

120 125 130 135

ATG CAA AAT GTC CAA AAT GCC CTT CGA TCA GCT AAT TTA GCT GGT AGA 486 Met Gln Asn Val Gln Asn Ala Leu Arg Ser Ala Asn Leu Ala Gly Arg

140 145 150

ATT AAA GTC TCT ACC GCG ATA AAA ACT GAC CTC CTT GCT AAC TTC CCT 534 Ile Lys Val Ser Thr Ala Ile Lys Ser Asp Leu Val Ala Asn Phe Pro

155 160 165

CCC TCT AAA GCT GTT TTT ACT TCT TCA TCA TAC ATG AAT CCA AIT GIT 582 Pro Ser Lys Gly Val Phe Thr Ser Ser Ser Tyr Met Asn Pro Ile Val

170 175 180

AAC TTC CTT AAA AAT AAC AAT TCA CCT TIG TTA GCC AAC ATT TAC CCT 630 Asn Phe Leu Lys Asn Asn Asn Ser Pro Leu Leu Ala Asn Ile Tyr Pro 185 190 195

TAC TTT TCT TTC AIT GGC ACC CCA AGT ATG OCT CTA GAT TAT GCA CTC 678 Tyr Phe Ser Phe Ile Gly Thr Pro Ser Met Arg Leu Asp Tyr Ala Leu

200 205 210 215

ITT ACT TCA CCT AAT GCC CAA GTT AAT GAT AAT GCT TTA CAA TAC CAA 726 Phe Thr Ser Pro Asn Ala Gln Val Asn Asp Asn Gly Leu Gln Tyr Gln

220 225 230

AAT GTC TTT GAT GCT TTA CTA GAC ACT GTG TAT GCG GCC TTA GCG AAG 774 Asn Val Phe Asp Ala Leu Val Asp Thr Val Tyr Ala Ala Leu Ala Lys

235 240 245

GCC GCT GCC CCC AAT GTG CCG AIT GTT GTG TCC GAG ACT GGG TGG OCT 822 Ala Gly Ala Pro Asn Val Pro Ile Val Val Ser Glu Ser Gly Trp Pro

250 255 260

TCG GCT GCT GCT AAT GCT GCT AGT TTT TCT AAC GCG GGG ACT TAT TAC 870 Ser Ala Gly Gly Asn Ala Ala Ser Phe Ser Asn Ala Gly Thr Tyr Tyr

265 270 275

AAG GGC TTA ATT GCT CAT GTA AAG CAA GGA ACT CCC CTG AAG AAA GGA 918 Lys Gly Leu Ile Gly His Val Lys Gln Gly Thr Pro Leu Lys Lys Gly

280 285 290 295

CAA GCA ATT GAG GCA TAT TTG TTT GCT ATG TTT GAT GAG AAC CAA AAG 966 Gln Ala Ile Glu Ala Tyr Leu Ehe Ala Met Phe Asp Glu Asn Gln Lys

300 305 310

GCT GGA GCT ATT GAG AAC AAT TTT GGA CTG TTT ACT CCC AAT AAA CAG 1014 Gly Gly Gly Ile Glu Asn Asn Phe Gly Leu Phe Thr Pro Asn Lys Gln

315 320 325

CCA AAA TAC CAA CTC AAT TTC AAT AAT TGAAACTACT TTAATTGCCT 1061

Pro Lys Tyr Gln Leu Asn Phe Asn Asn

330 335

AGEATATATA TATATATGCT AATATGTTCT ATGTAGTEAT GTCATCTACA TATATAATAA 1121

GTGAAATCAA ACACCOGATC ATAGACTAAA ATTGTAATAA AAGATCCTCC TGTTGTAATA 1181

TTATCCTAGC TGCAATAATA TTTACTCTTA TATAGAGATC TTGTGAAAAA AAAAAAAAAA 1241

AAAAAAAA 1249

(2) INFOPMATION FOR 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 DEBCRIPTION: SEQ 3D NO:10:

Met Arg Leu Ile Ser Thr Thr Ser Ala Val Ala Thr Leu Leu Phe Leu 1 5 10 15

Val Val Ile Leu Pro Ser Ile Gln Leu Thr Glu Ala Gln Ile Gly Val

20 25 30

Cys Asn Gly Arg Leu Gly Asn Asn Leu Pro Ser Glu Glu Asp Val Val

35 40 45

Ser Leu Tyr Lys Ser Arg Gly Ile Thr Arg Met Arg Ile Tyr Asp Pro 50 55 60

Asn Gln Arg Thr Leu Gln Ala Val Arg Gly Ser Asn Ile Gly Leu Ile 65 70 75 80

Val Asp Val Pro Lys Arg Asp Leu Arg Ser Leu Gly Ser Asp Ala Gly

85 90 95

Ala Ala Ser Arg Trp Val Gln Asn Asn Val Val Pro Tyr Ala Ser Asn

100 105 110

Ile Arg Tyr Ile Ala Val Gly Asn Glu Ile Mst Pro Asn Asp Ala Glu

115 120 125

Ala Gly Ser Ile Val Pro Ala Met Gln Asn Val Gln Asn Ala Leu Arg 130 135 140

Ser Ala Asn Leu Ala Gly Arg Ile Lys Val Ser Thr Ala Ile Lys Ser 145 150 155 160

Asp Leu Val Ala Asn Phe Pro Pro Ser Lys Gly Val Phe Thr Ser Ser

165 170 175

Ser Tyr Met Asn Pro Ile Val Asn Phe Leu Lys Asn Asn Asn Ser Pro

180 185 190

Leu Leu Ala Asn Ile Tyr Pro Tyr Phe Ser Phe Ile Gly Thr Pro Ser

195 200 205

Met Arg Leu Asp Tyr Ala Leu Phe Thr Ser Pro Asn Ala Gln Val Asn 210 215 220

Asp Asn Gly Leu Gln Tyr Gln Asn Val Phe Asp Ala Leu Val Asp Thr 225 230 235 240

Val Tyr Ala Ala Leu Ala Lys Ala Gly Ala Pro Asn Val Pro Ile Val

245 250 255

Val Ser Glu Ser Gly Trp Pro Ser Ala Gly Gly Asn Ala Ala Ser Phe

260 265 270

Ser Asn Ala Gly Thr Tyr Tyr Lys Gly Leu Ile Gly His Val Lys Gln

275 280 285

Gly Thr Pro Leu Lys Lys Gly Gln Ala Ile Glu Ala Tyr leu Phe Ala 290 295 300

Met Phe Asp Glu Asn Gln Lys Gly Gly Gly Ile Glu Asn Asn Phe Gly 305 310 315 320 Leu Phe Thr Pro Asn Lys Gln Pro Lys Tyr Gln Leu Asn Phe Asn Asn

325 330 335

(2) INFORMATTON FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6313 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(B) STRAIN: Monσva

(F) TISSUE TYPE: leaf

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: sugar beet EMBL3 genomic library

(B) CLONE: genomic chitinase 1 clone

(ix) FEATURE:

(A) NAME/KEY: unsure

(B) LOCATION: 3214..4227

(D) OTHER INFORMATION: /note= "Approximately 1000 base pairs"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(1428..2171, 4621..4776, 5408..5824)

(xi) SEQUENCE DESCRIPTION: SEQ 3D NO:11:

TCTAGAGAGA GAAAAAACAA CGCCATGTGA CGGTTGGGCT GGGACAACTT CGACCCGCTA 60

ATATEAATGG GCGGATATGG ACAACTCTTG AACCCCTCCC TGGCACCTAG AGGTGGCTAT 120

GGGCCGGCCC GGCATGATTT GGGACCGCTC GAGGCCCGAC CCGTTGTCCG CTACGTGGCC 180

GCGGGTCTGT CATGGTCAAG AAAATTTATT GAAACTAAAT ATTAAATTTA TTTAACCGGT 240

AATTAGTTAA CCTGATCATT TTTTCCAAAA TACCTCAAAA TTATTGAAAC TAAATATTAA 300

ATTTAAATTG AGAATGTTTT TGTCAAGAGA ATCAIAGITA AAAGGAAAAT TTGGCAAAAA 360

ATTTTTTTTT AOGAGITCAT TTTTGTGAAA AAAMCCTTA TAAAGCATTT TTTGCGAAAA 420

CAAACAAAAA TCAAATTTTT TTTGGGAAAA CAAACCAAAA TCAAAATTAT ITTTGCAAGA 480

TGAAACCTAA CCACCACTAC CATGCATCAA TCCCCAACCC CTCCCTCCAC CCCCTCGGGG 540

CACCCACACC CCCCTTCGCC CCTACCCTAT CTCCACTCTA TTCTTCCCTC CTECACGCCT 600CCTCTCTCTC CTCTCCCTTC GTCCAACCCC CCCCCCCCCCC AACCGTTGTG CCACTCAGAA 660 OGCACCCCAC CGCTGCGCCA CTCAAAACCC CCAACCCTTG CGCCACCCAG AAACAAIGGT 720

TTT GTTCTT CGATITTTGA TAGTTTCCGT TTCATTTGTG GTGGTAGTGG ATAGTGGGCC 780

GTGTTTCGTG GTGGTGGTGG GGGTTTCATG GTGTTATGGT CATGTGATGG TGGGGGTATC 840

ATGTTGTTAT GGTCATGTGA TCGTGGGTTG CGGAGGGGAA GCTGCGGAGG GGGTGGGGTG 900

GACTGTGGTT GGGGGGGTGG ACAAAAGGAG AGGAGAGGGG GGAGGGAGCT AGGGAAGAAG 960

GGAGAGGAGA GAGACTAGGG CTGAAGGGGG CTGGGGGACT ACCGCCTGAG AAGGTGTAGG 1020

CAGGGGTTGG GGTTGCTGGT GGGGGTGGCT GGGGCTTGGG CGCAAGTGTT GGTAGTGGTG 1080

GCTAGAGGTG GTCAATGCCG GAAATTCTAT GGTCAACGCC AGAAACGACA CTAGATTTGT 1140

TTTCGAAAAA AAATTTGATC TTGATTTGTT TTTACAAAAA ATAATTTATA AGATTTTTTT 1200

CGCAAAAGTG AACTCGTAAA AGTTTTTTTA AAACAAATTT TCCTAGTTAA AATCATTCTT 1260

TTCACTTTTT ACTATCTTAC ATACATAAAT TTTGATAGAT ATCTCTCTTA AATAAGCTTG 1320

TCATTTCTCC CTTATCCCAA AGACCCAAAG CCTCTATAAA TTGGCTACAA ACTCTCCTCA 1380

CAGTCACACA TGACACAACG CAAGTTTGAA AGGAAAAACA AGAGGTGATG AAAATAAAAA 1440

CCTCTCCEAG TTTTCTACEA GGATEAATAT GTTTAGCTCT AGTCCTCCTA CTAGGAGAAG 1500

GTGTACAATG TGGGCGGCAA TGCAACACAA CCGATACTAA TTGTCTTTCC GGTTGCTCAG 1560

TCGGCCGCCC ATCACGTCCG ACACCTCCTC GTCCTCCCAC CCCGAGACCG CCACTCCTC 1620

GTCCTCCCAC CCCGAGACCG CCACCTCCTC GTCCTCCTAC CCCGAGACCA CCACCACCEA 1680

CACCAAGACC ACCACCTCCT CGTCCTCCEA CCCCGAGACC ACCACCACCT CCTACACCAA 1740

GACCACCACC TCCTCGTCCT OCTACCCCAA GACCGCCACC ACCTCCTACA CCAAGACCAC 1800

CACCTCCTCC TACACCAAGA CCACCACCTC CTAGTCCTCC TACCCCTAGA CCACCACCAC 1860

CACCACCTCC TAGTCCTCCT ACCCCAAGCC CACCATCTCC TCCTAGCCCT GAACCACCAA 1920

CTCCGCCCGA ACCTACGCCA CCAACTCCEA CACCACCAAC TCATCTEACT GACATAATCT 1980

CTGAAGAAAT GTTTAATGAA TTCCTCTTGA ACCGCATTCA GCCACGTTGT CCTGGTAGAT 2040

GGTTCTACAC TTACCAGGCT TTCATTACTG CAGCTGAAAC CTTCCCTGAG TTTGGTAATA 2100

CTGGGAATGA TGAAATTAGA AAGAGAGAAA TTGCTGCTTT CTTTGGACAG ACCTCTCATG 2160

AAACCTCTGG TTCATTCTTC TACTTCTCAT TCTTCTTTAC TTCCGATCTG CTTCΑCTTTA 2220

CTAACATGCA TGTTTTCATA CTATATOGTA TATTTATAGA TGAACAACTA GTACCTTATT 2280

ATTTGCTAGT GCCTACTTCC TAGTCTTTAT TCTTTGTCAA TCTAATGATC TTTATAGTTT 2340

ATGTTGGACA CAATAAATTA TATAATCTEA ACGTEAAGTT TAGACGATAT GAATTATTTC 2400 TTTTTCACGT ATCTACCCTA GTTATTATAG GTTGTEATTA CCACTTTTTT TΆCTTCEACT 2460

ATTTTGACTT TTGACCATAT CGATTCTTEA TGCATAAGAC ATATATAATA TATAAGATCT 2520 TGTTGTATTA TTTACTTCGA TATATATATT TTGTTGGATT ATGCTCCAAA ATTAGTCAAG 2580

TTTTCCTAAT AGTTATGATT AGTTGCTATT AATTAGTTAG TGGGTTAACA AGTAGTGGTC 2640

TAGATAATGG TCTAGCTAGT GGGATAGCAA GTAAGEAGTA GGCTACATAG TGGACATGTT 2700

GTTACTACTG CTTTCTATGC CTATTTAAAG ATGGTTTTCT TTATTCATAA TGTGTACATG 2760

AAAAATATAT AAAAAATGTA GTCTTTCTAA TCTCTTCAAG TTCTCTTCCT CCTCTAAAAA 2820

TTCTACATGG TATCAGAGCT CCAGGTTAGA TCCGGGAAAG GGAAAGAGAT GAAGCAAAAA 2880

AAAGAAAAGA AAAAAAAGAG AGAGATAGAG AAAGAAAGAG AGTATEAAAA ACAAAATEGA 2940

GTAAAAAGAA ACAAGCAATG CAGCTTGATE TCATTGTTTG TGAGCAATTT TTTGTTTGAG 3000

TGAAATTTTT TCTCTTTAAC CATGAAAGTA ATGGTEGGCT GTGATGTGTT GTTGGGCTCC 3060

CTTTTAGCCC ATGAAAATGG ATTTTAATCC TGTGGAAAAA AGGGCATAAA AAATTGTTTG 3120

AGTTGAAGAT CGACTAATCT TTACGAGECT GTGGAAGTCG TACTCAACGC AGAGACGATT 3180

TCCAAAGTTG TAGTAGTGGA TGTGGTCAAA TTTNNNNNNN NNNNNNNNNN NNNNNNNNNN 3240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4200 NNNNNNNNN NNNNNNNNNN NNNNNNNTCT AGATTGACTT TGTAAGTTAA ACTTACCAAA 4260

TCTTGATATA TAGTAATTTA CAAAGTATAT TATAAGCTAA GTCGTGATGT CTCCCTCCAT 4320

CTITTTTTTA CTACTTGTEA CAITTTTCTE AAATGAATCT CTCATTTTAC ACACTACATE 4380

GCTTTATTTG CΑTTGGTAAC TATGACCCAT CAAAAAAIAA CAATATECCC CTTTATTTTT 4440

CTCATCTECT TAACTTEAAC TCTCAAATAT TTCTTATTTE CAACAAAGTT AACTCTCAAA 4500

TATTTGTCAT AATAATCTGA AATCTTCGGT TGATAAAGTG TCCAAAACAA AATGGTGTCA 4560

TTEAAAAAGA AAAATGAGAG GACATATATA CEAATGGATE TTTGAATEGA CAATATAGGA 4620

GAACCGACCG CACAACATGG ACCATTTACA TGGGGCTATT GITECATAGA AGAAATEGGA 4680

GCCCGGCCCTC TCAGCCAATA TTGTGCACCC TCTGTAGAAT GGCCTTGCAT TCGTGGGAGG 4740

TTTTACTATG GTCGTGGACC AGTCCAACTT ACCTGGTAAG TACTCCCTCC GTTCCAAAAT 4800

ATAGTTCTCA TTTTCCTTTE TECACACTAA TTEATGCAAG TAGAATATAA GAGGGTAGGT 4860

AAAGATTTTT TTTTEATTTA AATAAATCTT GTATGGGAAA AGATGATTTT AGGAGAGAGA 4920

CTGGAGAATA ATTAGTGAAA GAGCATTAAT TCTAACATTE TGGTTGAAIA AAIAAAGGAA 4980

AAAACAAATT CAAGAAGCEA AAGEAATGAG GGCACAGGTT TTCTAGACAA ATEAOGGAAA 5040

AATGTGGAAC TAAATATGAA AATGGGAACT ATATTTTGAG ACACNCAAAA TAAAAATGGG 5100

AACEATATTT TGGGACGGAG GGAGTATEAT TATATEAGCT TACTCCTATE ACTTGCATCG 5160

CATCTCCAAA TTTTTATECT TCATAGAAAA GTCATTTTCA AGAATTTEGC TATECGACGT 5220

CTEAAAATTT TTTACEACGC TTTCTAATEA CATATTTTTA TAGTGTACTT ATTTTATACC 5280

TTTCCATTTC TTCTCTTTTT CCTTCCTTTC CTTCACTTAA GTTTTAACTT GATACATATA 5340

GCTAGCAAAA TTATCTTAGG TATTTTAGCT AATTEAAAAT TTTTGGTAAT GATAAATAAT 5400

TEGCAGGAAT TTCAACTATG GAAAGCAGCT GAAGCACTTA GGTTTGGACC TCCTATTCAA 5460

CCCAGACATA GEAGCACATG ACCCAGETAT TTCTETCGAA ACTGCAATEE GGTTTEGGAT 5520

GACTCCTGAA GGAAACAAGC CTTCTTCCCA TGAAGECATA ACTGGGCAAT GGACACCAAC 5580

TCCTGCAGAC ATAGCTCGCA ACAGATTGCC TGGATATGGT TTAATCACAA ATATTTTTAA 5640

TGGTGCTTTA GAATGCGGCA CTCATGGACC AGATAATAGA GGGGAAAATC GAATECAGTT 5700

TTACCAGAGA TACTCTGATC TECTAGATGT TAGCTATGGA GATAACCTTG ATEGCTACCG 5760

TCAAACTCCC TTTGATTGGG GTCTTAAAAA ACTECAGGGA GCTAGAGAAT CATGGTCGTC 5820

GAGCTAAAAT TATACGCATG CATGTAGTCT TAAGTCCAT ACATEATEGT CTTCATGCGT 5880

CTATGATATT GAGTAAGTEG GTATGTTCAA AAATATCTGG TGTCTGAAAA TATGCAAACA 5940 GAACCAGCAA TAAGTAATAA GCAAGGTTTA CTTGCACCAA ATCTGGATCT GTECTAGTGA 6000 AATTGTEGTA TGTTCGTATT CTATGGTAAT GAATAAAGTT TGTGTTGTAT TTGCATTATC 6060 TGCACCTEAT TGATATEAAT TTTECATATE CAGGGGTTTA CAATCATAAG GATEACTGTA 6120 GGACCATEGA ACATGCAGEE GAGTTAGTCT TTAATATGCT GTTCAAGAAG AGTATGGAAA 6180 ATAGAAATGA GGAATGAAOG TACTCTATAT TATAAGAGAC TACTAGTCTT GTTTAGTCAG 6240 TGCTATTCTT ACACCTAAAA AAGCTCTATG AGATTACATT TACATTATGG TCAAAAGGTC 6300 TTAATGTCTA CCG 6313 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISEICS:

(A) LENGTH: 439 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(B) STRAIN: Monova

(F) TISSUE TYPE: leaf

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Lys Ile Lys Thr Ser Pro Ser Phe Leu Leu Gly Leu Ile Cys Leu 1 5 10 15

Ala Leu Val Leu Leu Leu Gly Glu Gly Val Gln Cys Gly Arg Gln Cys

20 25 30

Asn Thr Thr Asp Thr Asn Cys Leu Ser Gly Cys Ser Val Gly Arg Pro

35 40 45

Ser Arg Pro Thr Pro Pro Arg Pro Pro Thr Pro Arg Pro Pro Pro Pro

50 55 60

Arg Pro Pro Thr Pro Arg Pro Pro Pro Pro Arg Pro Pro Thr Pro Arg 65 70 75 80

Pro Pro Pro Pro Thr Pro Arg Pro Pro Pro Pro Arg Pro Pro Thr Pro

85 90 95

Arg Pro Pro Pro Pro Pro Thr Pro Arg Pro Pro Pro Pro Arg Pro Pro

100 105 110

Thr Pro Arg Pro Pro Pro Pro Pro Thr Pro Arg Pro Pro Pro Pro Pro

115 120 125

Thr Pro Arg Pro Pro Pro Pro Ser Pro Pro Thr Pro Arg Pro Pro Pro

130 135 140 Pro Pro Pro Pro Ser Pro Pro Thr Pro Ser Pro Pro Ser Pro Pro Ser 145 150 155 160

Pro Glu Pro Pro Thr Pro Pro Glu Pro Thr Pro Pro Thr Pro Thr Pro

165 170 175

Pro Thr His Leu Thr Asp Ile Ile Ser Glu Glu Met Phe Asn Glu Phe

180 185 190

Leu Leu Asn Arg Ile Gln Pro Arg Cys Pro Gly Arg Trp Phe Tyr Thr

195 200 205

Tyr Gln Ala Phe Ile Thr Ala Ala Glu Thr Phe Pro Glu Phe Gly Asn 210 215 220

Thr Gly Asn Asp Glu Ile Arg Lys Arg Glu Ile Ala Ala Phe Phe Gly 225 230 235 240 Gln Thr Ser His Glu Thr Ser Gly Glu Pro Thr Ala Gln His Gly Pro

245 250 255

Phe Thr Trp Gly Tyr Cys Phe Ile Glu Glu Ile Gly Ala Gly Pro Leu

260 265 270

Ser Gln Tyr Cys Ala Pro Ser Val Glu Trp Pro Cys Ile Arg Gly Arg

275 280 285 Phe Tyr Tyr Gly Arg Gly Pro Val Gln Leu Thr Trp Asn Phe Asn Tyr 290 295 300

Gly Lys Gln Val Lys His Leu Gly Leu Asp Leu Leu Phe Asn Pro Asp 305 310 315 320 Ile Val Ala His Asp Pro Val Ile Ser Phe Glu Thr Ala Ile Trp Phe

325 330 335

Trp Met Thr Pro Glu Gly Asn Lys Pro Ser Ser His Glu Val Ile Thr

340 345 350

Gly Gln Trp Thr Pro Thr Pro Ala Asp Ile Ala Arg Asn Arg Leu Pro

355 360 365

Gly Tyr Gly Leu Ile Thr Asn Ile Phe Asn Gly Ala Leu Glu Cys Gly 370 375 380

Thr His Gly Pro Asp Asn Arg Gly Glu Asn Arg Ile Gln Phe Tyr Gln 385 390 395 400

Arg Tyr Cys Asp Leu Leu Asp Val Ser Tyr Gly Asp Asn Leu Asp Gly

405 410 415

Tyr Arg Gln Thr Pro Phe Asp Trp Gly Leu Lys Lys Leu Gln Gly Ala

420 425 430

Arg Glu Ser Trp Ser Ser Ser

435

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: C-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Phaseolus vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Asn Leu Asp Cys Tyr Ser Gln Thr Pro Phe Gly Asn Ser Leu Leu Leu 1 5 10 15

Ser Asp leu Val Thr Ser Gln

20

(2) INFORMATION FOR SEQ 3D NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: C-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ 3D NO:14:

Asn Leu Asp Cys Gly Asn Gln Arg Ser Phe Gly Asn Gly Leu Leu Val 1 5 10 15

Asp Thr Met

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYRDTHETICAL: NO

(v) FRAGMENT TYPE: C-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Asn Leu Asp Cys Tyr Asn Gln Arg Asn Cys Phe Ala Gly

1 5 10

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: C-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hordeum vulgare

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Asn Leu Asp Cys Tyr Ser Gln Arg Pro Phe Ala

1 5 10

(2) INFORMATION FOR SEQ 3D NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHE3TCAL: NO

(v) FRAGMENT TYPE: C-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Gly Val Ser Gly Gly Val Trp Asp Ser Ser Val Glu Thr Asn Ala Thr 1 5 10 15

Ala Ser Leu Val Ser Glu Met (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPCTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Ser Thr Tyr Cys Gln Ser Tyr Ala Ala Phe Pro Pro Asn Pro Ser Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTEDRISTTCS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHEITCAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Ala Cys Val Thr His Glu Thr Gly His Phe Cys Tyr Ile Glu Glu Ile 1 5 10 15

Ala Lys

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHEEICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPnON: SEQ ID NO:20:

Val Gly Tyr Tyr Thr Gln Tyr Cys Gln Gln

1 5 10

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Gly Pro Leu Gln Ile Thr Trp

1 5

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Ser Ile Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val Ala Asn Asn 1 5 10 15

Ala Val Thr Ala Phe Arg

20

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Triticum aestivum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn Asn Leu 1 5 10 15

Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr Cys Gly

20 25 30

Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser

35 40

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHEITCAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-termirial

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hevea brasiliensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Glu Gln Cys Gly Arg Gln Ala Gly Gly Lys Leu Cys Pro Asn Asn Leu 1 5 10 15

Cys Cys Ser Gln Trp Gly Trp Cys Gly Ser Thr Asp Glu Tyr Cys Ser

20 25 30

Pro Asp His Asn Cys Gln Ser Asn Cys Lys Asp

35 40

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Phaseolus vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Glu Gln Cys Gly Arg Gln Ala Gly Gly Ala Leu Cys Pro Gly Gly Asn 1 5 10 15

Cys Cys Ser Gln Phe Gly Trp Cys Gly Ser Thr Thr Asp Tyr Cys Gly

20 25 30

Pro Gly Cys Gln Ser Gln Cys Gly Gly

35 40

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Glu Gln Cys Gly Ser Gln Ala Gly Gly Ala Arg Cys Ala Ser Gly Leu 1 5 10 15

Cys Cys Ser Lys Phe Gly Trp Cys Gly Asn Thr Asn Glu Tyr Cys Gly

20 25 30

Pro Asp Asn Cys Gln Ser Gln Cys Pro Gly

35 40

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Glu Leu Cys Gly Asn Gln Ala Gly Gly Ala Leu Cys Pro Asn Gly Leu 1 5 10 15

Cys Cys Ser Gln Tyr Gly Trp Cys Gly Asn Thr Asn Pro Tyr Cys Gly

20 25 30

Asn

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (KB-7), constructed from beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

GACTCTAGAA AYCCRCCRYG YCARTAYGAY AC 32

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (KB-9), constructed from Beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGAGGATCCC ARRCNAAYCA RATHTT 26

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (270)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

CCAAGCTTGA ATTCTTTETE TTTTTTTTTT TTTT 34

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTTCS:

(A) LENGTH: 292 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Cucumis sativus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Met Ala Ala His Lys Ile Thr Thr Thr Leu Ser Ile Phe Phe Leu Leu 1 5 10 15

Ser Ser Ile Phe Arg Ser Ser Asp Ala Ala Gly Ile Ala Ile Tyr Trp

20 25 30

Gly Gln Asn Gly Asn Glu Gly Ser Leu Ala Ser Thr Cys Ala Thr Gly

35 40 45

Asn Tyr Glu Phe Val Asn Ile Ala Phe Leu Ser Ser Phe Gly Ser Gly

50 55 60

Gln Ala Pro Val Leu Asn Leu Ala Gly His Cys Asn Pro Asp Asn Asn 65 70 75 80

Gly Cys Ala Phe leu Ser Asp Glu Ile Asn Ser Cys Lys Ser Gln Asn

85 90 95 Val Lys Val Leu Leu Ser Ile Gly Gly Gly Ala Gly Ser Tyr Ser Leu 100 105 110

Ser Ser Ala Asp Asp Ala Lys Gln Val Ala Asn Phe Ile Trp Asn Ser

115 120 125

Tyr Leu Gly Gly Gln Ser Asp Ser Arg Pro Leu Gly Ala Ala Val Leu 130 135 140

Asp Gly Val Asp Phe Asp Ile Glu Ser Gly Ser Gly Gln Phe Trp Asp 145 150 155 160

Val Leu Ala Gln Glu Leu Lys Asn Phe Gly Gln Val Ile Leu Ser Ala

165 170 175

Ala Pro Gln Cys Pro Ile Pro Asp Ala His Leu Asp Ala Ala Ile Lys

180 185 190

Thr Gly Leu Phe Asp Ser Val Trp Val Gln Phe Tyr Asn Asn Pro Pro

195 200 205

Cys Met Phe Ala Asp Asn Ala Asp Asn Leu Leu Ser Ser Trp Asn Gln 210 215 220

Trp Thr Ala Phe Pro Thr Ser Lys Leu Tyr Met Gly Leu Pro Ala Ala 225 230 235 240

Arg Glu Ala Ala Pro Ser Gly Gly Phe Ile Pro Ala Asp Val Leu Ile

245 250 255

Ser Gln Val ILeu 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 Tyr Ser Asp Ser Ile Lys

275 280 285

Gly Ser Ile Gly

290

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 302 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHEEICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Thr Asn Met Thr Leu Arg Lys His Val Ile Tyr Phe Leu Phe Phe 1 5 10 15 Ile Ser Cys Ser Leu Ser Lys Pro Ser Asp Ala Ser Arg Gly Gly Ile

20 25 30

Ala Ile Tyr Trp Gly Gln Asn Gly Asn Glu Gly Asn Leu Ser Ala Thr

35 40 45

Cys Ala Thr Gly Arg Tyr Ala Tyr Val Asn Val Ala Phe Leu Val Lys 50 55 60

Phe Gly Asn Gly Gln Thr Pro Glu Leu Asn Leu Ala Gly His Cys Asn 65 70 75 80

Pro Ala Ala Asn Thr Cys Thr His Phe Gly Ser Gln Val Lys Asp Cys

85 90 95 Gln Ser Arg Gly Ile Lys Val Met Leu Ser Leu Gly Gly Gly Ile Gly

100 105 110

Asn Tyr Ser Ile Gly Ser Arg Glu Asp Ala Lys Val Ile Ala Asp Tyr

115 120 125

Leu Trp Asn Asn Phe Leu Gly Gly Lys Ser Ser Ser Arg Pro Leu Gly 130 135 140

Asp Ala Val Leu Asp Gly Ile Asp Phe Asn Ile Glu Leu Gly Ser Pro 145 150 155 160 Gln His Trp Asp Asp Leu Ala Arg Thr Leu Ser Lys Phe Ser His Arg

165 170 175

Gly Arg Lys Ile Tyr Leu Thr Gly Ala Pro Gln Cys Pro Phe Pro Asp

180 185 190

Arg Leu Met Gly Ser Ala Leu Asn Thr Lys Arg Phe Asp Tyr Val Trp

195 200 205 Ile Gln Phe Tyr Asn Asn Pro Pro Cys Ser Tyr Ser Ser Gly Asn Thr 210 215 220

Gln Asn Leu Phe Asp Ser Trp Asn Lys Trp Thr Thr Ser Ile Ala Ala 225 230 235 240 Gln Lys Phe Phe Leu Gly Leu Pro Ala Ala Pro Glu Ala Ala Asp Ser

245 250 255

Gly Tyr Ile Pro Pro Asp Val Leu Thr Ser Gln Ile Leu Pro Thr Leu

260 265 270

Lys Lys Ser Arg Lys Tyr Gly Gly Val Met Leu Trp Ser Lys Phe Trp

275 280 285

Asp Asp Lys Asn Gly Tyr Ser Ser Ser Ile Leu Ala Ser Val

290 295 300

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARCTERISTICS: (A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Trp Val Gln Asn Asn Val Val Pro Tyr

1 5

(2) INFORMATION FOR SEQ 3D NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Ala Gly Ala Pro Asn Val Pro Ile Val Val Ser Glu Ser Gly Trp Pro 1 5 10 15

Ser Ala Gly Gly

20

(2) INFORMATION FOR SEQ 3D NO:35:

(i) SEQUENCE CHARACTERISTTCS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

leu Gln Gly Lys Val Ser

1 5 (2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (TG-1), constructed from beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

TGGGTNCARA AYAAYGT 17

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (TG-2), constructed from beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

AAYGARATHA TGCCNAA 17

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENCTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (TG-3) , constructed from N. tabacum

and H. vulgare

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TCRIYRAACA TNGCRAA 17

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hordeum vulgare

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Phe Ala Met Phe Asp Glu

1 5

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Phe Ala Met Phe Asn Glu

1 5

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Pisum sativum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Glu Gln Cys Gly Arg Gln Ala Gly Gly Ala Thr Cys Pro Asn Asn Leu 1 5 10 15

Cys Cys Ser Gln Tyr Gly Tyr

20

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pisum sativum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Glu Gln Cys Gly Asn Gln Ala Gly Gly Xaa Val Pro Pro Asn Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pisum sativum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Glu Gln Cys Gly Thr Gln Ala Gly Gly Ala Leu Cys Pro Gly Gly Leu 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CΗARACTERISTICS: (A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hordeum vulgare

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Glu Gln Xaa Gly Ser Gln Ala Gly Gly Ala Thr Cys Pro Asn Xaa Leu 1 5 10 15

Cys Cys Ser Arg Phe Gly

20

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hordeum vulgare

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Xaa Gln Gln Gly Ser Gln Ala Gly Gly Ala Thr Cys Pro Asn Xaa Leu 1 5 10 15

Cys cys Ser Xaa Phe Gly Trp

20

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Ala Ile Gly Val Asp Leu Leu Asn Asn Pro Asp Leu Val Ala Thr Asp 1 5 10 15

Pro Val

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Gly Pro Ile Gln Ile Ser His

1 5

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Ser Ala Leu Trp Phe Trp Met Thr Pro Gln Ser Pro

1 5 10

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTTCS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (KB-3), constructed from beta vulgaris

(xi) SEQUENCE DESCRIPTION: SEQ NO:49:

CCGAAGCTTA GATCTAAACA ACAACATGEC TTCTYTYGGA CC

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer (KB-4), constructed from beta vulgaris, chitinase 4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

GCACACGTAG CGCTAGCTTG G

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: primer, constructed from beta vulgaris,

chitinase 4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

CATCGGAGGA TCCACTACC

Claims

1. A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO:.1 or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 as defined herein and i) being a characteristic part of the DNA sequence shown in SEQ ID NO: .1, or ii) hybridizing with the DNA sequence shown in SEQ ID NO:.1 at 55°C as defined in and under the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", or iii) encoding a polypeptide having the amino acid sequence of the sugar beet chitinase 4 shown in SEQ ID NO:.2, or iv) encoding a polypeptide being reactive with an antibody raised against sugar beet chitinase 4.

2. A DNA sequence according to claim 1, comprising nucleotides 71-793 of the chitinase 4 DNA sequence shown in SEQ ID NO:.1 and encoding the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

3. A DNA sequence according to claim 1, comprising nucleotides 175-793 of the chitinase 4 DNA sequence shown in SEQ ID NO:.1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

4. A DNA sequence comprising a sugar beet chitinase 4 gene.

5. A DNA sequence encoding a chitinase isoenzyme which is at least 60% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO:.1 and at the most 40% homologous with the sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO: .11.

6. A DNA sequence according to claim 5 which encodes a chitinase isoenzyme which is at least 65% homologous, e.g. at least 70% homologous, such as at least 75% or preferably at least 80%

homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO : .1 and/or at the most 38% such as at the most 35% homologous with the sugar beet chitinase 1 enzyme encoded by the DNA sequence SEQ ID NO:.11.

7. A DNA sequence according to claim 5 or 6 comprising the genomic chitinase 76 sequence shown in SEQ ID NO : .5.

8. A DNA sequence according to any of claims 1-7 encoding a

polypeptide which reacts with an antibody raised against sugar beet chitinase 4, but not with an antibody raised against sugar beet chitinase 2.

9. A modified DNA sequence comprising a DNA sequence as defined in any of claims 1-8 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 having retained the antifungal activity of the sugar beet chitinase 4 or having an increased antifungal activity as compared to the sugar beet chitinase 4.

10. A subsequence of the chitinase 4 DNA sequence of SEQ ID NO:.1 comprising a DNA sequence encoding a polypeptide comprising the active site of the sugar beet chitinase 4 enzyme, e.g. a DNA sequence encoding the following peptide S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R or a polypeptide comprising a part of the sugar beet chitinase 4 enzyme which is involved in the active site of the sugar beet chitinase 4, e.g. a DNA sequence encoding the peptide

G-P-L-Q-I-T-W or the peptide

T-A- F-W- F-W-M-N-N-V-H- S -V- I -V-N-G-Q-G-F-G-A- S - I which Is involved in the active site or an 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 have the same catalytic and/or binding activities as that of said peptides.

11. A subsequence of the chitinase 4 DNA sequence of 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 in which at least one additional nucleotide has been inserted and which subsequence is encoding a polypeptide capable of binding to chitin as determined by affinity column chromatography on regenerated chitin prepared as described in "Materials and Methods" under the heading "Preparation of a chitin column".

12. A subsequence of the chitinase 4 DNA sequence SEQ ID NO:.1 encoding the leader peptide of chitinase 4 or an 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 passenger polypeptide to which it is fused out of the cell in which the fused leader and passenger polypeptide is produced, to be deposited in the

extracellular space.

13. A subsequence of the chitinase 4 DNA sequence SEQ ID NO:.1 encoding one or more of the following epitopes of the sugar beet chitinase 4 enzyme

Peptide 1: AGKRFYTRA

Peptide 2: CNPSKQYY

Peptide 3: IECNGGNS

Peptide 4: TARVGYYTQYCQ

14. A DNA sequence according to any of the preceding claims which is of plant origin.

15. A DNA sequence according to claim 14 which is derived from a member of the family Chenopodiaceae, Solanaceae, Apiaceae,

Brassicaceae, Cucurbitaceae or Fabaceae.

16. A DNA sequence according to claim 15 which is derived from a corn, alfalfa, oat, wheat, rye, rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, bean, chenille, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce and pea.

17. A polypeptide encoded by a DNA sequence according to any of the preceding claims.

18. A genetic construct comprising

1) a promoter functionally connected to 2) a DNA sequence as defined in any of claims 1-16 comprising a chitinase 4 DNA sequence or an analogue or a subsequence thereof and

3) a transcription terminator functionally connected to the DNA sequence.

19. A genetic construct comprising one or more copies of a DNA sequence as defined in any of claims 1-16 comprising the chitinase 4 DNA sequence shown in SEQ ID NO:.1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide having the activity of a second chitinase different from the sugar beet chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having β- 1,3-glucanase activity. each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.

20. A genetic construct according to claim 19 in which the DNA sequence encoding the second chitinase encodes an acidic chitinase having a pl equal to or less than 4.0 and preferably being capable of cleaving ³H-chitin into mainly chito hexamers, and/or the DNA sequence encoding the β- 1 ,3-glucanase encodes a basic β- 1 , 3 - glucanase having a pl of at least 9.0 and preferably being capable of cleaving ³H-laminarin into mainly dimers of β- 1 ,3-glucans.

21. A genetic construct according to any of claims 19 or 20, in which the second chitinase and the β- 1,3-glucanase are of plant origin.

22. A genetic construct according to claim 21 in which the DNA sequence encoding the acidic chitinase is the DNA sequence of SEQ ID NO:.7 encoding an acidic sugar beet chitinase SE having the amino acid sequence shown in SEQ ID NO: .8 or an analogue of said DNA sequence encoding an acidic chitinase having a pI of at the most 4.0 and being capable of hydrolysing ³H-chitin into mainly hexamers, and/or the DNA sequence shown in SEQ ID NO : . 9 encoding a basic β- 1 , 3 -glucanase is the DNA sequence encoding the basic sugar beet β-1,3-glucanase 4 having the amino acid sequence shown in SEQ ID NO:.10 or an analogue thereof encoding a basic β-1 ,3-glucanase having a pI of at least 9.0 and being capable of hydrolysing ³H-laminarin into mainly dimers of β- 1 ,3-glucans.

23. A genetic construct according to any of claims 18-22 in which the promoter is a constitutive or regulatable promoter.

24. A genetic construct according to claim 23 wherein the

constitutive promoter is selected from the group consisting of plant promoters, bacterial promoters or plant virus promoters.

25. A genetic construct; according to claim 24, in which the promoter is selected from the group consisting of the sugar beet

Acetohydroxyacid synthase promoter (AHAS), the sugar beet chitinase 1 promoter, the sequence of which appears from SEQ ID NO:.11 26. A genetic construct according to any of claims 18-25, in which the N-terminal leader sequence is selected from the group consisting of the coding regions of the sugar beet chitinase 1, the sequence of which appears from SEQ ID NO:.11, the sugar beet chitinase 4, the sequence of which appears from SEQ ID NO:.1, the sugar beet β-1,3-glucanase, the sequence of which appears from SEQ ID NO : .9 , the sugar beet chitinase 76, the sequence of which is shown in SEQ ID N0.:5, and the acidic chitinase SE from sugar beet, the sequence of which appears from SEQ ID NO : .7.

27. A genetic construct according to any of claims 18-26 which contains the DNA subsequence from the sugar beet chitinase 1 encoding the proline rich region.

28. A genetic construct according to claim 24, in which the promoter is selected from the group consisting of a NOS promoter and an OCS promoter of the opine synthase genes of Agrobacterium.

29. A genetic construct according to claim 24, in which the promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) promoter such as a CaMV 19S promoter or a CaMV 35S promoter, a MAS/35S, MAS dual Tr 1,2 and a T-2 DNA gene 5 promoter.

30. A genetic construct according to claim 23 wherein the

regulatable promoter is regulatable by at least one factor selected from the group consisting of a growth factor, a chemical factor, a biological factor, and a physical factor.

31. A genetic construct according to claim 23 in which the promoter is a tissue specific promoter.

32. A genetic construct according to any of claims 18-31 wherein the transcription terminator is selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences, fungal transcription terminators and plant virus terminator sequences.

33. A genetic construct according to claim 32, in which the

transcription terminator is selected from the group consisting of a NOS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium , a CaMV 35S transcription terminator sequence, a PADG4 transcription terminator to the DNA gene 4, a PADG7

transcription terminator to the T-DNA gene 7.

34. A genetic construct according to any of claims 18-33 in which at least one of the DNA sequences of the construct is functionally connected to an enhancer sequence which results in an increased transcription and expression of the DNA sequence (s).

35. A genetic construct comprising a DNA sequence encoding a polypeptide, which DNA sequence is linked to a DNA subsequence encoding a N-terminal leader sequence selected from the group consisting of the sugar beet chitinase 4 N-terminal sequence shown in SEQ ID NO:.1, the sugar beet β-1,3-glucanase N-terminal sequence shown in SEQ ID NO:.9, the sugar beet acidic chitinase SE N-terminal sequence shown in SEQ ID NO:.7 and the sugar beet chitinase 1 N-terminal sequence shown in SEQ ID NO:.11.

36. A genetic construct according to claim 35, for use in the transformation of a plant, in particular a sugar beet plant.

37. A genetic construct according to any of claims 18-36 which contains the DNA subsequence from the sugar beet chitinase 1 encoding the proline rich domain.

38. A vector which is capable of replicating in a host organism and which carries a DNA sequence as defined in any of claims 1-16, or a genetic construct as defined in any of claims 18-37.

39. A host organism harboring a vector as defined in claim 38.

40. A host organism according to claim 39, which is capable of replicating or expressing the DNA sequence as defined in any of claims 1-16 or the genetic construct as defined in any of claims 18-37.

41. A host organism which in its genome carries a DNA sequence according to any of claims 1-16 or a genetic construct according to any of claims 18-37 and which is capable of replicating or expressing the DNA sequence or the genetic construct.

42. A host organism according to any of claims 39-41 which is a microorganism such as bacteria or yeast.

43. A host organism according to any of claims 39-41 which is a plant cell or a protoplast.

44. A genetically transformed plant comprising in its genome a genetic construct according to any of claims 18-37.

45. A genetically transformed plant according to claim 44 which is selected from the group of monocotyledonous plants consisting of corn, oat, wheat, rice, barley, rye and sorghum.

46. A genetically transformed plant according to claim 45 which is selected from the group of dicotyledonous plants consisting of alfalfa, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, chenille, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce and pea.

47. A genetically transformed plant according to any of claims 44-46 having an increased resistance to a chitin containing plant pathogen as compared to a plant which does not harbour the genetic construct as defined in any of claims 18-37.

48. A genetically transformed plant according to claim 47, having increased resistance to a phytopathogenic fungus or a nematode.

49. A genetically transformed plant according to claim 47 having increased resistance to phytopathogenic fungi of the genus

Cercospora, Rhizoctonia , Fusarium, Cladosporium , Phytophthora , Phoma , Sclerotonia , Ascochγta , Pyrenophora , Helmithosporium , Ustilago , Puccinia , Ramularia , Botrytis or Verticillium.

50. A genetically transformed plant according to claim 49 having increased resistance to phytopathogenic fungi selected from the group consisting of Rhizoctonia solani , Cercospora beticola , Cercospora nicotianae , Cladosporium herbarium , Phytophthora megasperma ,

Sclerotonia sclerotiorum, Ramularia beticola , Botrytis cinerea and Phoma lingam.

51. Seeds, seedlings or plant parts obtained by growing the

genetically transformed plant according to any of claims 44-49.

52. A transformation system comprising at least one vector which carries a genetic construct according to any of claims 18-37 and which is capable of introducing the genetic construct into the genome of a plant.

53. The transformation system according to claim 52 which comprises a binary or a co-integrate vector system.

54. The transformation system according to claim 52 or 53, which contains a virulence function capable of effecting the transformation of the plant and at least one border part of a T-DNA fragment, the border part being located on the same plasmid as the genetic

construct.

55. The transformation system according to any of claims 52-54, which comprises an Agrobacterium tumefaciens Ti or an Agrobacterium rhizogenes Ri plasmid or a derivative thereof.

56. A microorganism capable of infecting a plant and harboring a transformation system according to any of claims 52-55.

57. The microorganism according to claim 56 which is an Agrobacterium spp.

58. A method of producing a genetically transformed plant having increased resistance to chitin containing plant pathogens such as phytopathogenic fungi as compared to a natural plant, comprising transferring a genetic construct according to any of claims 18-37 into the genome of the plant so as to obtain a genetic material comprising the construct, and subsequently regenerating the genetic material into a genetically transformed plant.

59. The method according to claim 58 in which the genetic construct is transferred into the plant by means of a microorganism according to claim 56 or 57.

60. The method according to claim 58, in which the genetic construct is transferred into the plant or into a part thereof by direct introduction of naked DNA by injection, sonication or

electroporation.

61. An antifungal composition comprising a polypeptide encoded by the DNA sequence as defined in any of claims 1-16, or by a genetic construct as defined in any of claims 18-37 and a suitable vehicle.

62. An antifungal composition according to claim 61 comprising a chemical, e.g a fungicide, conventionally used in the therapeutic and/or prophylactic treatment of fungi.

63. A method of preparing an antifungal composition comprising culturing a microorganism according to claim 39-42 in an appropriate medium and under conditions which result in the expression of one or more polypeptides encoded by the DNA sequence according to any of claims 1-16 or the genetic construct according to any of claims 18-37, optionally rupturing the microorganisms so as to release their content of expressed polypeptide(s) into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide(s) to freeze-drying or spray-drying thereby obtaining an antifungal composition comprising the polypeptide (s) encoded by said DNA sequence or said genetic

construct.

64. A method according to the claim 63, in which the antifungal proteins are excreted into the medium and optionally purified from the medium.

65. A method of inhibiting the germination and/or growth of a chitin containing plant pathogen such as a phytopathogenic fungus in a plant which method comprises

1) transforming the plant or a part thereof with a genetic construct as defined in any of claims 18-37 and regenerating the resulting transformed plant or plant part into a genetically transformed plant and/or

2) treating the plant or a part thereof, a seedling or seed from which the plant is to be propagated, or the medium on which the plant is grown with a composition as defined in claim 61.

66. A method according to claim 65, wherein the composition according to claim 61 or prepared by the method according to claim 62 has been added to water or a nutrient composition supplied to the plant.

67. A method of biologically controlling the germination and/or growth of a chitin containing plant pathogen such as a

phytopathogenic fungus present on a material comprising treating the material with a culture of microorganisms as defined in claim 39-42 under conditions allowing the culture of microorganism to establish itself on the material to be treated.

68. A method according to claim 67 wherein the microorganism is a Pseudomonas spp., or a Streptomyces spp. or another microorganism conventionally used for biological pest control.

69. A method according to claim 66 or 67, wherein the material is a plant.

70. A method of inhibiting the germination and/or growth of a fungi on a material, comprising treating the material with an antifungal composition according to claim 61 or 62 or prepared by the method according to claim 63 or 64.

71. A method according to claim 70, wherein the material to be treated is a food product such as bread, a beverage, a food product constituent such as cereal, or any part of a container for a food product, a beverage or a food product constituent.

72. The plant transformation vector pBKL4K4 harbored in the E . coli strain DH5α deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) on 30 July, 1991 under the provisions of the Budapest Treaty under accession number DSM 6635.