NZ242270A

NZ242270A - Dna sequences coding for chitinase; vectors containing such sequences and its use in transforming hosts and biologically controlling chitin-containing plant pathogens

Info

Publication number: NZ242270A
Application number: NZ24227092A
Authority: NZ
Inventors: Jorn Dalgaard Mikkelsen; Kirsten Bojsen; Klaus K Nielsen; Lars Berglund
Original assignee: Sandoz Ltd
Priority date: 1991-04-08
Filing date: 1992-04-08
Publication date: 1994-07-26
Also published as: IE921104A1; EP0579709A1; AU1659992A; CA2048696A1; HU9302829D0; DK61691D0; AU659455B2; SK108193A3; CZ209293A3; WO1992017591A1; JPH06507070A; CA2048477A1; HUT67059A

Description

<div class="application article clearfix" id="description"> V 9 242 270 Priority Catc(o): .. 5?!. COO~lp'bt& i iJ' "*i ; 1 Cls^s: azMrf.k?, (o%>, t>x Qw?h?. CiJVVJ jl.<?,^v. 09!vJp&f. fo?ltu<f>2.l&ri s/oo 2 6 JUL 1994 Publication Date: P.O. Journal, No: .mz,. Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION PLANT CHITINASE GENE AND USE THEREOF ry^^rQF^fflCAtir gyECCQ-Jl/S, a Danish company of Langebrogade lf DIt-1411, Copenhagen K, DENMARK nereDy declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) ia 242270 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 chiti-nase 4" or an analogue of said DNA sequence encoding a polypeptide 5 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 10 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 yS-l,3-glucanase. In another aspect, the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet 15 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 >9-1,3-glucanase activity so as to result in an increased resistance to 20 chitin-containing plant pathogens. BACKGROUND OF THE INVENTION Most plants are susceptible to infection by pathogens such as microorganisms and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently 25 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 30 and /3-1,3-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 de- 829746BI.002/MKA/SPK/A36/1992 04 02 (followed by page 2) 242 27 2 fense mechanism of the plant is, however, normally delayed in relation 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 suffi-5 ciently 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 10 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 15 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 >3-1,3-glueanase have been shown 20 to be capable of enzymatic ally 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 , 3-glucanase activity has been observed in plant species such as tobacco, barley, potato, rice, maize, corn, bean, 25 tomato, cucumber, wheat germ, 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 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 3 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 5 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; 10 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 15 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 20 harbouring DNA sequences encoding plant pathogenesis-related proteins (PRP), e.g. chitinase and /3-1,3-glucanase. Pathogenesis-related proteins from sugar beet plants or transgenic sugar beet plants are not mentioned. EP 0 448 511 also relates to the transgenic plants comprising 25 recombinant DNA sequences encoding hydrolytic enzymes such as chitinases and glucanases. Additionally, the reference relates to compositions comprising hydrolytic enzymes such as a glucanase and chitinase for use for controlling plant pathogens. Chitinase or glucanase from sugar beet are not mentioned. 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 '24 2 2 7 0 4 Rousseau-Limouzin M. and Fritig B. (1991) describe Che production of basic and acidic PR-proceins in sugar beecs infected with Cercospora. betieols. and the serological relation of these PR-proteins to the PR-proteins la tobacco. The described PR-proteins are found to be 5 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. 10 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 Molecu-15 lar 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 20 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 che chitinase enzymes were not separately discussed. By the present invention a novel plant chitinase has been elucidated 25 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 30 In one aspect the present invention relates to a DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO.rl or an analogue thereof, the analogue being a DNA sequence encoding a 829746BI.002/MKA/SPK/A34/I992 04 06 42 270 i 5 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 5 ii) hybridizing with the DNA sequence shown in SEQ ID N0.:1 at 55*C utlder the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", or 10 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. 15 The chitinase 4 DNA sequence, SEQ ID NO.:l, 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 prepar-20 ed 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 25 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 30 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, 829746BI.002/MKA/SFK/A36/1992 04 06 242270 6 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 5 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 10 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 phyto-15 pathogenic 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 20 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 25 different chitinase and a /?-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. 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 sequence shown in SEQ ID NO.:l 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 5 one or more copies of a DNA sequence encoding a j3-l, 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 10 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 15 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 20 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 25 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. 829746BI.002/MKA/SPK/A36/1992 04 02 242 27 8 DETAILED DISCLOSURE OF THE INVENTION The chitinase 4 DNA sequence, SEQ ID NO.:l 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 5 "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 10 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 15 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 acti-20 vity 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 ^H-25 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 9 242 270 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 ^H-chitin and the resulting formation of mainly dimers of chitin in this assay. 5 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 10 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 15 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 20 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 25 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 10 A typical example o£ 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 5 with the chitinase 4 DNA sequence under the conditions specified in che "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 plane chici-10 nasas 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 belong-15 ing to the chicinase 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 20 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 che 25 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 SEQ ID N0.:2, i.e. the amino acid sequence of the mature chitinase 4 enzyme. It is 30 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 829746BI.002/MKA/SPK/A36/1992 04 06 242270 n 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 5 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 10 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 15 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 20 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 25 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, 30 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 N0.:1 and with the amino acid sequence shown 829746BI.002/MKA/SPK/A36/1992 04 02 242270 12 in SEQ ID NO.: 2 are further described and compared to other plant chitinases. The chitinase 4 DNA SEQ ID N0.:1 comprises a leader sequence (nucleotides 2-70) encoding 23 amino acid residues, a part 5 (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 10 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 15 NO:l) 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 N0.: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 20 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. 25 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-30 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 35 other basic plant chitinases from the hevein class. 829746BI.002/MKA/SPK/A36/1992 04 02 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 5 compared to the hevein structure. The functional domain contain 206 amino acid residues. In the basic chitinase from Nicotiana Cabaccum (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 10 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 15 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. 20 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 25 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-30 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 N0.:1 are the part encoding the hevein domain and especially the part encoding the 829746BI.002/MKA/SPK/A36/1992 04 02 242270 14 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 5 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 enzyme may be used in heterologous gene construct in transformation in plants, in particular sugar beet plants, when the encoded polypeptides are 10 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.:l and encoding the hevein domain and the functional domain of the 15 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 se quence , 20 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 25 sequence comprising nucleotides 175-793 of the chitinase 4 DNA sequence shown in SEQ ID NO.:l 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, 829746BI.002/MKA/SPK/A36/1992 04 02 242270 15 Bii) hybridizes with a DNA probe prepared from said DNA se quence , Biii) encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or 5 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. 10 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 se-15 quences) 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 se-20 quences 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 geno-25 mic 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 30 sequence of the chitinase 4 cDNA shown in SEQ ID N0.: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. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 16 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 5 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.:l. Possible 5' regulatory sequences of the chitinase 4 gene are shown in Examples 17 and 18 below. 10 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, 15 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 20 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 N0.:2. On the basis of the 3D-structure of the chitinase 4 enzyme which may 25 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 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 17 so as to obtain a modified mode of action of the enzyme, e.g. wich respect Co 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 5 by use of well-known principles of protein engineering, such as site-directed 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 10 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 en2yme 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 15 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 che chitinase 4 enzyme encoded by a DNA sequence of the invention, a DNA sequence encoding the modified chitinase 4 20 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 ^-1-3-glucanases be used in the construction 25 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. 30 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 che presence of the degree of complementarity between the 35 amino acid sequence of a given polypeptide and the amino acid 829746BI.002/MKA/SPK/A36/1992 04 06 42 270 18 sequence of another polypeptide baing 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 5 of homology between different genes, especially between the coding regions, may thus be used to assess che degree of familarity becveen 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 che basis 10 of mature proteins, i.e. wichout Caking any leader sequence into account. In accordance herewith, the present invention relaces 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 15 sequence SEQ ID N0.:1 and at the most 40% homologous with Che sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO.:11. The minimum degree of homology of at least 602 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 20 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 che chitinase 4 class. Of course, a higher degree of homology with the chitinase 4 enzyme 25 and therefor a lower degree of homology with the chitinase 1 enzyme reflects an even higher similarity herewith and accordingly, che 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 30 enzyme encoded by Che DNA sequence SEQ ID NQ.:1 and/or at the most 38% such as at the most 35% homologous with the sugar beet chitinase 1 enzyme encoded by che DNA sequence SEQ ID NO.:11. An example of a DNA sequence encoding a polypeptide being about 75% homologous to the sugar beet chicinase 4 enzyme and at che most 40% 35 homologous to the sugar beet chitinase 1 enzyme is the genomic DNA 829746BI.002/MKA/SPK/A36/1992 04 06 9A2 27 19 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 thac sugar beet chitinase 4 isolated 5 from sugar beet leaves is recognized by an antibody raised against this sugar beet chitinase, but not by an antibody raised against the sugar beat 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 10 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 again3t sugar beet chitinase 4, but not 15 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 20 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 25 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. 30 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 35 sequence is inserted, and when "deletion" is performed one or more 829746BI.002/MKA/SPK/A36/1992 04 06 242270 20 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. 5 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 10 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 15 conventional one-letter amino acid code) consisting of the amino acids No's. 179-200 of SEQ ID N0.: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 20 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 25 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 30 active site. A further interesting DNA sequence encoding a part of the chitinase 4 enzyme is a DNA sequence encoding the polypeptide having the follow- 829746BI.002/MKA/SPK/A36/1992 04 02 242270 21 ing 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 5 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 4-10 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 15 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 20 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 25 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 inven-30 tion is a subsequence of the chitinase 4 DNA sequence of SEQ ID NO.:l encoding a polypeptide comprising the hevein domain of the sugar 829746BI.002/MKA/SPK/A36/1992 04 02 242270 22 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 5 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 establish-10 ing 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 15 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.:l encoding the 20 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 25 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 30 epitopes are expected to be found among the hydrophilic peptides of the chitinase 4 amino acid sequence SEQ ID N0.:2, because these peptides seem to be substantially different from peptide parts of other chitinases than sugar beet chitinase 4. Antibodies (either monoclonal, monospecific or polyspecific) may be prepared by use of conven-35 tional methods, e.g. as described in the Materials and Methods sec- 829746BI.002/MKA/SPK/A36/1992 04 02 24 2 2 70 23 tion 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 5 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) 10 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. 15 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. 20 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 25 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, 30 barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea. 829746BI.002/MKA/SPK/A36/1992 04 02 §4 2 2 24 The terras "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 iso-5 lated, 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 10 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 15 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 20 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 subse-25 quences 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 subse-30 quences 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 polypep-35 tide of the invention has the advantage that it may be easily pro- 829746BI.002/MKA/SPK/A36/1992 04 02 242270 25 duced in large quantities by use of well known conventional recombinant productions techniques, e.g. as described in Sambrook et al., 1990, and that it may be obtained in a form which is free from impurities normally associated with the naturally occurring sugar beet 5 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 10 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 15 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. 20 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 25 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 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 2422 26 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 5 composition comprising a polypeptide having the antifungal activity of the sugar beet chitinase 4 in admixture with an acidic chitinase and a basic £-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 10 effect will be observed in general when the sugar beet chitinase 4 is used in combination with other chitinases and /3-1,3-glucanases, preferably of plant origin. Thus, in another important aspect, the present invention relates to a genetic construct comprising 15 one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID N0.: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 20 chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having 0-1,3-glucanase activity, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA se-25 quences into functional polypeptides. The polypeptides with chitinase or /9-1,3-glucanase activity is preferably of plant origin. The chitinase and /9-1,3-glucanase activity may be determined as explained in the section "Materials and Methods" below. 30 Of particular interest is a genetic construct comprising 829746BI.002/MKA/SPK/A36/J992 04 02 242271 27 one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID N0.:1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding an acidic chitinase 5 having a pi equal to or less than 4.0, and one or more copies of a DNA sequence encoding a basic /J-l, 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 se-10 quences 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 15 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 20 capable of hydrolyzing ^H-chitin into mainly hexamers. In the present context, the term "basic yS-1,3-glucanase" means a fi-1,3-glucanase having a pi of more than 9.0. Preferably, the basic /3-1,3-glucanase is one which is capable of hydrolyzing glucan into mainly dimers, e.g. as determined by the %-laminarin assay described 25 in the Materials and Methods section below. The basic /9-1,3-glucanase is preferably of plant origin. Examples of a suitable basic /3-l,3-glucanase are basic /9-1,3-glucanases derived from tobacco (Shinshi et al., 1990), barley (Fincher et al. , 1986) or sugar beet, e.g. the basic sugar beet /9-1,3-glucanase 4, the DNA sequence of which is 30 shown in SEQ ID N0.:9 or an analogue thereof encoding a basic >9-1, 3-glucanase having a pi of at least 9.0 and preferably being capable of hydrolyzing ^H-laminarin into mainly dimers of /3-1,3-glucan. The 829746BI.002/MKA/SPK/A36/1992 04 02 242270 28 basic sugar beet /3-1,3-glucanase 4 is different from other plant /3-1,3-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 /3-1,3-glucanase 4 may in part be 5 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 10 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 15 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 £-1,3-glucanase results in an increased antifungal activity as compared to the antifungal activity of each of the constituents. The 20 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 ^-1,3-glucanase and sugar beet chitinase 4, respectively. When the acidic chitinase is one which hydrolyses chitinase primarily into hexamers (as compared to chiti-25 nase 4 which primarily hydrolyses chitin into dimers) and the basic 0-1,3-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 30 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 1,3-glucanase, e.g. a basic £-1,3-glucanase, is believed to be due to the fact that such combination will attack both the chitin and glucan constituents 35 of the cell wall of phytopathogenic fungi and also parts of the cell 829746BI.002/MKA/SPK/A36/1992 04 02 242 2 70 29 wall in which the chitin and glucan constituents are intimately cross-linked to one another. The >3-1,3-glucanase further serves to remove the outer glucan layer covering the chitin structure of chitin containing plant pathogens, e.g. phytopathogenic fungi, resulting in 5 an exposure of the chitin structure to the enzymatic action of the chitinase. DNA sequences encoding the second chitinase referred to above and the 0-i, 3-glucanase may be obtained, e.g. from already known sources, or may be identified and isolated from natural sources, e.g. by use 10 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 15 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. 20 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 25 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 30 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 35 the genetic construct. Also, when the genetic construct of the inven- 829746BI.002/MKA/SPK/A36/I992 04 02 242270 30 tion is too large, it may be difficult Co obtain a stable introduo tion thereof into the genome of the plant which may lead to excision of a part of or che entire genetic conscruct from the genome of che plant. Thus, the genetic construct should be adapted so chat the 5 expression products therefrom are generally acceptable Co Che hose 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 10 construct of the invention. With the fast increasing knowledge within the field of plane genetic engineering, improved transformation and biological containment techniques may be developed leading to the possibility of introducing larger foreign genetic fragments into a plant wichouc causing retarded growth, retarded yield or recom-15 binational 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 Che invention. Accordingly, it is preferred chat each of Che DNA sequences of the genetic construct of the invention is present in only one copy. The construction of a 20 genetic construcc 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 che chitinase 4 DNA sequence of the invention or an analogue thereof. Accordingly, it is contemplated that a genetic 25 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 (3-1,3-glucanase are present may show very potent antifungal effects when present in a genetically transformed plant of the invention. It 30 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. 829746BI.002/MKA/SPK/A36/1992 04 06 31 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 5 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 10 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 15 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 resulting nucleotide sequence serves to direct the polypeptide encoded 20 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 25 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 32 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 5 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 10 sequence of which is shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; and the N-terminal sequence of the 0-1,3-glucanase 4, the nucleotide and amino acid sequence of which is shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. Another interesting sequence is DNA subsequence from the sugar beet chitinase 1 encoding the proline 15 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 above-mentioned leader sequences are to be considered as non-limiting examples. 20 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 25 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 30 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 35 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 33 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 5 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 10 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 15 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 fi-i, 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 20 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 25 which is shown in SEQ ID NO.: 12, encoding the following polypeptide consisting of the amino acids No's 413-439 NLDGYRQTPFDWGLKKLQGARESWSSS* 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 30 ID NO.:2 N L R C * 829746BI.002/MKA/SPK/A36/1992 04 02 ^ t t "X a 34 the C-terminal sequence of a bean chitinase (PHA) encoding the following polypeptide shown in SEQ ID NO.:13 NLDCYSQTPFGNSLLLSDLVTSQ* the C-terminal sequence of a basic tobacco chitinase encoding the 5 following polypeptide shown in SEQ ID NO.:14 NLDCGNQRSFGNGLLVDTM* the C-terminal sequence of an acidic tobacco chitinase encoding the following polypeptide shown in SEQ ID NO.:15 NLDCYNQRNCFAG* 10 the C-terminal sequence of the barley chitinase CH26 encoding the following polypeptide shown in SEQ ID NO.:16 NLDCYSQRPFA*, or the C-terminal sequence of a basic /?-l,3-Glucanase from tobacco encoding the following polypeptide shown in SEQ ID NO.:17 15 GVSGGVWDSSVETNATASLVSEM 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 20 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 25 provided with a C-terminal sequence capable to transport the polypeptides expressed from the DNA sequences to the vacuole. 829746BI.002/MKA/SPK/A36/1992 04 02 24227 35 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 5 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 10 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 consti-15 tutive 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 20 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. se-25 quences 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 '42 270 36 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 5 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. 10 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 15 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 20 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 25 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, 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 37 promoter is a CaMV 19S promoter and a CaMV 355 promoter (Odell et al.. 1985). Other promoters may be derived from the Ti-plasmid such as the octo-pine synthase promoter, the nopaline synthase promoter (Herrera-5 Estrella et al., 1983), the mannopine synthase promoter, and promoters from other open reading frames in the T-DNA such as 0RF7. Further examples of suitable promoters are MAS/35S (Jatissen and Gardner, 1989), MAS dual Tr 1,2 (Velten et al., 1984) and a T-2 DNA gene 5 promoter (Konz and Schell, 198S). 10 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. 15 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 20 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, 25 Denmark, personal communication). Furthermore, the sugar beet promoters from the acidic chitinase SE, chitinase 1, chitinase 76 and chitinase 4 or 0-1,3-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-30 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 conn«cted to a transcrip- 8297468I.002/MKA/SPK/A36/1992 04 06 24227 38 tion 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 5 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 10 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 15 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 20 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. 25 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 N0:1 or an analogue or subsequence thereof, or a genetic construct of the invention. The vector may 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 39 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 5 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. 10 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 N0:1 or an analogue thereof or a chitinase gene or pseudogene 15 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 20 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. 25 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 94 2 2 40 pucida 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 5 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 /3-l,3-glucanase expression and a correspondingly increased antifungal activity. The recombinant production may be performed by use of 10 conventional techniques, e.g. as described by Sambrook et al., 1990. 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 15 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 20 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 25 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 con-30 struct 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 829746BI.002/MKA/SPK/A36/1992 04 02 422 41 chitinase active in destroying the cell walls of phytopathogenic fungi. Accordingly, the plant to be transformed by the genetic construct of the invention nay be a monocotyledonous as well as a dicotyledonous 5 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 geneti-10 cally 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 re-15 sistance 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 20 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. 25 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. 30 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. 829746BI.002/MKA/SPK. A36/1992 04 02 42 242270 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 5 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. 10 Verticillium alboatmm 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 de-15 stroys 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. 20 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 25 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 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 43 penetrate the cuticula. The growing raycelia produce haustoria that penetrate the walls of the host cells and invaginate their protoplasts. Ustilago maydis is a fungus with mainly intercellular growth, but 5 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 10 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 15 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 20 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 25 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 30 plasmids or plasmid derivatives of the bacteria Agrobacterium. The two best known Agrobacteria are Agrobacterium tumefaciens and Agro-bacterium rhizogenes (plasmids thereof are in the following termed pTi and pRi, respectively). The use of such plant transformation 829746B1.002/MKA/SPK/A36/1992 04 02 242270 44 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 5 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. 10 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 15 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 Agro- 20 bacterium 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. 25 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. 30 hygromycin or other known markers, e.g. the markers disclosed by Lindsey, 1990 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. 829746BI.002/MKA/SPK/A36/1992 04 02 2422 45 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 5 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 10 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 Agrobacterium strain, e.g. Agrobacterium tumefaciens. 15 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 20 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, 25 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 30 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. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 46 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 5 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 10 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 15 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 20 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 25 will be further explained in the following. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 47 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 5 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 10 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 15 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 20 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 25 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 30 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 35 described by Sambrook et al., 1990. For instance, to characterize 829746BI.002/MKA/SPK/A36/1992 04 02 242270 48 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 5 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:l or an analogue thereof, 10 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). 15 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 20 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 25 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. 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 49 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 pre-5 viously, 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 10 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 15 of the invention, e.g. DNA sequences encoding a polypeptide having chitinase or ^-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 20 (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 25 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 N0:1 or an ana-30 logue 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- 829746BI.002/MKA/SPK/A36/1992 04 02 50 242270 peptide encoded by the DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO:l 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 5 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:l or an 10 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 15 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 20 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 25 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 30 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 poly-35 peptide" is meant a polypeptide encoded by the chitinase 4 DNA se- 829746BI.002/MKA/SPK/A36/1992 04 02 242270 51 quence of the invention or an analogue thereof or a genetic construct of the invention having antifungal activity, i.e. chitinase activity and optionally >3-1,3-glucanase activity as defined above. The antifungal composition may in addition to the polypeptide encoded 5 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 /3-1,3-glucanase activity as defined above, contain one or several chemicals, e.g. fungicides, conventionally used in the combatting of fungi either 10 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 15 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, 20 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. 25 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 30 or to increase the amount released by secretion. The medium comprising a substantial amount of the antifungal polypep-tide(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 829746BI.002/MKA/SPK/A36/1992 04 02 242 270 52 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 5 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 10 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. 15 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 20 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 25 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 30 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 829746BI.002/MKA/SPK/A36/1992 04 02 242 2 53 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 5 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 10 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. 15 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 20 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 25 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 30 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 pm. 829746BI.002/MKA/SPK/A36/1992 04 02 54 242270 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 fxg 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 5 weight markers. The proteins were stained with silver. Fig. 3 shows the analysis of the water-soluble products released from ^H-chitin by chitinase 4. %-chitin was incubated with 4 pg chitinase 4 at 37°C for 0.25, 0.5, 3 and 24 hours. As a control ^H-chitin was incubated without enzyme at 37°C for 24 hours. The chito-10 oligosaccharides 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 15 by scintillation counting after cutting the TLC plate into pieces. Fig. 4 shows the lysozyme activity of chitinase 4. 1 /ig 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 fig of SE ("Sure Ellen") was used as a control, and (50 20 ng and 5/ig) lysozyme (lys) was used as standards. Fig. 5 shows the inhibition of the growth of Cercospora by a combination of chitinase 4, SE and glucanase 4 using the microscope slide bioassay. After 48 hours of incubation the cultures were stained with Calcofluor White and investigated under fluorescent light. 25 Fig. 5A shows the growth of the fungus when 20 pg of each of the enzymes chit 4, SE and glucanase 4 were added to the culture at time 0. \ 829746BI.002/MKA/SPK/A36/1992 04 02 24227 55 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 (absor-5 bance 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 10 culture. Curve B shows the growth of the fungus when 20 n1 of a chitinase containing fraction from the chitin-column was added at time 0. In curve C 20 ng of purified chitinase 4 was added to the culture at time 0. Fig. 7. is an autoradiography showing the effect of chitinase 4 on 15 chitin in the apex of Cercospora hyphae. Incorporation of ^H-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-acetylglu-coseamine into the cell wall in the apex of the fungal hyphae is seen 20 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. 25 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 30 0.7 ml/minute. 829746BI.002/MKA/SPK/A36/1992 04 02 24227 56 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. 5 Fig. 10 describes the two different serological classes of sugar beet, the chitinase 2 and chitinase 4 class. 5 /jg of both chitinase 2 (32 kD) and 4 (27 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) . 10 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 /ul probes of a plasmid preparation containing the chitinase sequences. 1 a chitinase 1 clone from sugar beet 15 2a chitinase 4 clone form sugar beet 3 a chitinase 76 clone form sugar beet 4 a chitinase clone from pea 5 a "SE" clone from sugar beet 6 a chitinase clone 1 from tobacco 20 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 25 hybridization buffer: 2 x SSC, 0.1% SDS, 10 x Denhardt's, 50 ngjml Salmon sperm DNA and a chitinase 4 cDNA sequence as probe and under washing conditions of 55°C, 2xSSC, 0.1% SDS in two times 15 minutes followed by two times 15 minutes lxSSC, 0.1% SDS, 55°C. 829746BI.002/MKA/SPK/A36/1992 04 02 242 2 57 Fig. 12 describes the induction of chitinase and 0-1,3-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 5 prepared. Enzyme activities of chitinase (Fig. 12A) and 0-1,3-glu-canase (Fig. 12B) were measured using the radiotracer assays with •^H-chitin and ^H-laminarin as the substrate, respectively. Fig. 13 describes the immuno-detection of sugar beet chitinase 2 and 4 and ^-1,3-glucanase 3 in protein extracts of Cercospora infected 10 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 (3-1,3-glucanase 3 (right) were employed. Fig. 14. Site directed mutagenesis of amino acids contemplated to 15 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 SDl, SD2, SD3, SD4 and SD5 primers are indicated by 20 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 N0.:4. Numbers in brackets denote the number of the corresponding 25 amino acid encoded from the chitinase 4 cDNA (SEQ ID NO.:2) SDl Trpl70->Tyr (169) TGG->TAC SD2 Glul90-»Gln (189) GAA-+CAA SD3 Aspl84-»Asn (183) GAT-AAT SD4 Trp207-^Tyr (206) TGG-+TAC SD5 Trp205-+Tyr (204) TGG->TAC The PCR products are digested with the relevant restriction enzymes 829746BI.002/MKA/SPK/A36/1992 04 02 242 27 58 and exchanged with the corresponding sequence in the chitinase 4 gene. Fig. 15. Construction of a hybrid /3-1,3-glucanase gene construct with a C-terminal extension from tobacco 5 Fig. 15A. A sugar beet cDNA /3-1,3-glucanase clone with an under lined tobacco C-terminal extension. Fig. 15B. 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. The arrows indicate the PCR primers; 10 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. 15C. Four annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a stop codon, a Smal 15 site and an Bglll .site. The fused gene product can be made by digesting the glucanase gene with Xbal and EcoRI and ligating it with the PCR product digested with Xbal and Dral and the annealed synthetic oligonucleotides digested with Smal and Bglll. 20 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. PCR primers which can be used to introduce a Smal site 25 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. 829746BI.002/MKA/SPK/A36/1992 04 02 59 242270 Fig. 16C. Four annealed synthetic oligonucleotides containing the sequence for the C-terminal extension, a changed stop codon, a Smal site and an EcoRI site. The fused gene product can be made by digesting the chitinase 4 gene 5 with BamHI and EcoRI and ligating it with the PCR product digested with BamHI and Smal and the annealed synthetic oligonucleotides digested with Smal and EcoRI. Fig. 17. Construction of the plant transformation vector pBKL4K4 containing the chitinase 4 DNA sequence shown in SEQ ID NO:l. The 10 boxed sequences indicate the B15 chitinase 4 cDNA, the enhanced 35S promoter and the 35S terminator sequences used for the construct. pB15K4.1 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 15 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 20 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 25 terminator. A polylinker containing several unique cloning sites is situated between the GUS and the NPTII genes. Fig. 18. Construction of the plant transformation vector pBKL4K4KSEl containing the DNA sequences encoding chitinase 4 and SE, respectively shown in SEQ ID N0:1 and SEQ ID NO:8. The boxed sequences indicate 30 the "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 829746BI.002/MKA/SPK/A36/1992 04 02 242270 60 is likewise pBluescript carrying almost the entire "SE" cDNA. The hatched boxes indicate the coding regions contained in the final product. ^'aGCTGTAC^' is an adaptor used for the KpnI-Hindlll ligation. pPS48 is mentioned in connection with Fig. 17. The construction 5 of the plant transformation vector harboring the chitinase 4 sequence (pBKL4K4) is described in Fig. 17. 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 10 gene, the enhanced 35S promoter and the 35S terminator sequences. pK76.1 is pUC19 carrying the Hindlll-EcoRI fragment encoding chitinase 76 in the Hindlll/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 15 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. 20 Fig. 20. PCR amplification of a part of the "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 25 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. 30 Fig. 21 describes the separation of sugar beet /3-1,3-glucanases 1, 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elu- 829746BI.002/M KA/SPK/A36/1992 04 02 2A 2 2 7 0 61 tion was performed with a linear gradient of NaCl. The absorbance was measured at 280 nm. Fig. 22 describes the construction of the plant transformation vector pBKL4K4KSElGl containing the DNA sequences encoding chitinase 4, SE 5 and 0-1,3-glucanase, respectively, and shown in SEQ ID NO:l, SEQ ID NO:7 and SEQ ID NO:9. The boxed sequences indicate the 0-1,3-glucanase cDNA, the enhanced 35S promoter and the 35S terminator. pGluc 1 is pBluescript carrying the 1249 bp EcoRI fragment encoding the 0-1,3-glucanase. The hatched box indicates the coding region. Plasmid 10 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 Kpnl) 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 15 Fig. 17 and Fig. 18. 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. 20 "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. 25 Fig. 24 A comparison between the DNA sequence of the chitinase 4 cDNA sequence shown in SEQ ID NO. :1 and the genomic clone chitinase 76 shown in SEQ ID N0.: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 and Fig. 24C should be 30 considered as one figure. : indicates identical nucleotides. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 62 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 5 position 62-64. : 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 10 observed in the non-coding 5' sequence. It is contemplated that some of these boxes may be of regulatory importance. 829746BI.002/MKA/SPK/A36/1992 04 02 242 2 63 SEQUENCE LISTING SEQ ID N0.: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 5 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 10 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 15 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 0-1,3-glucanase SEQ ID NO.: 10 the deduced amino acid sequence of the basic sugar 20 beet 0-1,3-glucanase SEQ ID NO.:ll. 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. 25 SEQ ID NO.:12 The amino acid sequence deduced from the coding region of the chitinase 1 gene. 829746BI.002/MKA/SPK/A36/1992 04 02 ^ 242 27 64 SEQ ID NO.:13: C-terminal amino acid sequence of a bean chitinase (PHA). SEQ ID N0.:14: C-terminal amino acid sequence of a basic tobacco chitinase. 5 SEQ ID N0.:15: C-terminal amino acid sequence of an acidic tobacco chitinase. SEQ ID N0.:16: C-terminal amino acid sequence of the barley chitinase CH26. SEQ ID NO.:17: C-terminal amino acid sequence of a basic 0-1,3 10 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. 15 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 20 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 25 (Triticum aestivum). 829746BI.002/MKA/SPK/A36/1992 04 02 242 27 65 SEQ ID NO.:24: N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from hevein (Hevea brasiliensis). SEQ ID NO.:25: N-terminal amino acid sequence of the amino acid 5 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). 10 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). 15 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 20 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 25 of a 0-1,3-glucanase from sugar beet. SEQ ID NO.:34: The amino acid subsequence 3-17 of the amino acid sequence of a 0-1,3-glucanase from sugar beet. 829746BI.002/MKA/SPK/A36/1992 04 02 24227 66 SEQ ID NO.:35: The amino acid subsequence 3-16 of the amino acid sequence of a 0-1,3-glucanase from sugar beet. SEQ ID NO.:36: DNA 5'primer named Oligo TG-1 constructed from the amino acid sequence of a 0-1,3-glucanase from sugar 5 beet. SEQ ID NO.:37: DNA 5'primer named Oligo TG-2 constructed from the amino acid sequence of a 0-1,3-glucanase from sugar beet. SEQ ID NO.:38: DNA 3'primer named Oligo TG-3 constructed from the 10 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. 15 SEQ ID N0.: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. 20 SEQ ID N0.:42: N-terminal amino acid sequence of the amino acid sequence from pea chitinase Al. SEQ ID N0.:43: N-terminal amino acid sequence of the amino acid sequence from pea chitinase A2. SEQ ID N0.:44: N-terminal amino acid sequence of the amino acid 25 sequence from barley chitinase K. SEQ ID N0.:45: N-terminal amino acid sequence of the amino acid sequence from barley chitinase T. 829746BI.002/MKA/SPK/A36/1992 04 02 242 270 67 SEQ ID NO.:46: Amino acid subsequence of the active site of the amino acid sequence from a chitinase from tobacco. SEQ ID N0.:47: Amino acid subsequence of the active site of the polypeptide from a chitinase from tobacco. 5 SEQ ID NO.:48: Amino acid sequence of the active site of the polypeptide from a chitinase from tobacco. SEQ ID N0.:49: DNA 5'primer named KB-3 constructed partly from nucleotides No's. 1-15 of SEQ ID NO.:l and nucleotides No's. 471-485 from SEQ ID NO.:5. 10 SEQ ID NO.:50: Complementary DNA 3'primer named KB-4 constructed from nucleotides No's. 261-241 of SEQ ID NO.:l. SEQ ID NO.:51: Complementary DNA primer named 340 constructed partly from the nucleotide numbers 341-323 of SEQ ID NO.:l. 829746BI.002/MKA/SPK/A36/1992 04 02 24 2 2 68 DETAILED EXPLANATION OF THE SEQUENCES SEQ ID NO'S:1-12 SEQ ID NO.:1 and SEQ ID N0.:2 The DNA sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of the B15 chitinase 4 cDNA clone isolated from a sugar beet 5 AZAP 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 amino acid residues. 10 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 N0.:4. The leader sequence encoded by 15 the gene consists of 24 amino acid residues. Thus, the SEQ ID N0:1 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 20 The partial DNA sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) of a genomic clone encoding the chitinase 4 gene isolated from 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 25 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. 829746BI.002/MKA/SPK/A36/1992 04 02 242270 69 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 5 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 10 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. 15 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 "SE" cDNA clone isolated from a sugar beet AZAP cDNA library The sequence is 1106 bp long and encodes a protein having 293 amino 20 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 25 The DNA sequence (SEQ ID NO.:9) and the deduced amino acid sequence (SEQ ID N0.:10) of a 0-1,3-glucanase 4 cDNA clone isolated from a sugar beet AZAP 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-30 coding region of 33 bp and a 182 bp 3' flanking region containing a 829746BI.002/MKA/SPK/A36/1992 04 02 24227 70 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 NO.:11) and deduced amino acid sequence (SEQ ID NO.:12) of a genomic clone encoding the chitinase 1 gene isolated 5 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 10 238 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 15 about 70 bp upstream of the ATG start codon. A putative poly A signal (AATAAA) is located at position 6032. 829746B1.002/MKA/SPK/A36/1992 04 02 242270 71 REFERENCES An G. et al. , 1986, Plant Physiol., Vol. 81, pp. 301-305 An G. et al. , 1988, Plant Molecular Biology Manual A3, 1-19, Kluwer Academic Publishers, Dordrecht 5 Barkardottir et al., 1987, Developmental Genetics, Vol. 8, pp. 495-511 Barkholt et al., 1989, Anal. 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Helgeson, pp. 203-208 Chirgwin et al., 1978, Biochemistry, Vol. 18, pp. 5294-5299 Feistner et al., Charting of rat pituitary peptides by plasma desorp-tion and electrospray mass spectrometry, Proceedings of the Nato Advanced Research workshop on Methods and Mechanisms for Producing 25 Ions from Large Molecules, Minaki, June 1990 829746BI.002/MKA/SPK/A36/1992 04 02 24 2 2 ^ 72 Fincher G. B. et al., Primary structure of the (1-+3, l->4)-/3-D-glucan 4-glucohydrolase from barley aleurone, 1986, Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 2081-2085 Fraley R. T. et al., Expression of bacterial genes in plant cells, 5 August 1983, Proc. Natl. Acad. Sci. USA, Genetics, Vol. 80, pp. 4803-4807 Fritsch E. F., Molecular Cloning, A laboratory manual, 1989, 2nd edition, Cold Spring Harbor Laboratory Press Herrera-Estrella L. et al., Expression of chimaeric genes transferred 10 into plant cells using a Ti-plasmid-derived vector, 1983, Nature, Vol. 303 Herrera-Estrella L. et al., Use of reporter genes to study gene expression in plant cells, 1988, Plant Molecular Biology Manual Bl, 1-22 15 Hoekema A. et al., 1983, Nature, Vol. 303, p. 179-180 Hooykaas P.J.J., Agrobacterium molecular genetics, 1988, Plant Molecular Biology Manual A4, 1-13 Horsch R. B. et al., 1986, Science, Vol. 227, pp. 1229-1231 Horsch R. B. et al., 1988, Leaf disc transformation, Plant Molecular 20 Biology Manual A9, 1-16 Jacobsen S. et al., 1990, Physiol. Plantarum, Vol. 79, p. 554 Janssen B.-J. et al., Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium, 1989, Plant Molecular Biology, Vol. 14, pp. 61-72 25 Jaynes J., Peptides to the rescue, 16 December 1989, New Scientist, pp. 42-44 829746BI.002/MKA/SPK/A36/1992 04 02 242270 73 Jefferson R.A., 1987, Plant Molecular Biology Reporter, Vol. 5, No. 4, pp. 387-405 Jefferson et al., 1987, EMBOJ., Vol. 6, No. 13, 3901-3907 Joersbo M. et al., Direct gene transfer to plant protoplasts by 5 electroporation by alternating, rectangular and exponentially decaying pulses, 1990, Plant Cell Reports, Vol. 8, pp. 701-705 Joersbo M. et al., Direct gene transfer to plant protoplasts by mild sonication, 1990, Plant Cell Reports, Vol. 9. pp. 207-210 Kay R. et al., Duplication of CaMV 35S Promoter Sequences Creates a 10 Strong Enhancer for Plant Genes, 1987, Science, Vol. 236, pp. 1299-1302 Klein T. M. et al., Advances in Direct Gene Transfer into Cereals, 1989, Genetic Engineering Principles and Methods, Vol. 11, Plenum Press, New York 15 Koncz C. et al., The promoter of Tl-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector, 1986, Mol Gen Genet, Vol. 204, pp. 383-396 Kragh K. M. et al., 1990, Plant Science, Vol. 71, p. 55 20 Kragh K. M., 1991, Chitinases and /9-1,3-glucanases in barley (Hordeum vulgar L.), Plant Biology Section, Riso National Laboratory, Ros-kilde, Denmark Kyhse-Andersen, 1984, J. Biochem. Biophys. Methods 10, pp. 203-209 Leah et al., 1987, Carlsberg Res. Commun. Vol. 52, pp. 31-37 25 Leah R. et al., Biochemical and Molecular Characterization of Three Barley Seed Proteins with Antifungal Properties, 1991, The Journal of Biological Chemistry, Vol. 266, No. 3, pp. 1564-1573 829746BI.002/MKA/SPK/A36/1992 04 02 242 27 74 Lindsey, K. and M. G. K. Jones, Selection of transformed cells, in Plant Cell Line Selection, Procedures and Applications, edited by Philip J. Dix, VCH, 1990 van Loon L. C. et al., Identification, purification, and characteriz-5 ation of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves, 1987, Plant Molecular Biology, Vol. 9, pp. 593-609 Marcussen J. et al., A Nondestructive Method for Peptide Bond Conjugation of Antigenic Haptens to a Diphtheria Toxoid Carrier, Exemplified by Two Antisera Specific to Acetolactate Synthase' 1991, 10 Anal.Biochem., Vol. 198, pp. 318-323 McNeil M. et al., Structure and function of the primary cell walls of plants, 1984, Ann. Rev. Biochem., Vol. 53, pp. 625-663 Metraux J.-P. et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 896-900 15 Mornon J. P. et al., Hydrophobic cluster analysis: an efficient new way to compare and analyze amino acid sequences, 1987, Elsevier Science Publishers B.V. (Biomedical Devision), Vol. 224, No. 1 Murashige T. et al., A revised medium for rapid growth and bioassay with tobacco cultures., 1962, Physiol Plant, Vol. 15, pp. 473-497 20 Myers et al., 1988, CABIOS, Vol. 4, pp. 11-17 Neuhaus J. M. et al., 1990, 5th Int. Symp. of The Mol. Genetic of Plant-Microbe Interaction, Interlaken, Switzerland, p. 218 Odell J. T. et al., Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter, 1985, Nature, 25 Vol. 313 Ooms G. et al., 1982, Plasmid no. 7, pp. 15-29 829746BI.002/MKA/SPK/A36/1992 04 02 2*227r 75 Paszkowski J. et al., Direct gene transfer to plants, 1984, The EMBO Journal, Vol. 3, No. 12, pp. 2717-2722 Reynaerts A. et al., 1988, Selectable and screenable markers, Plant Molecular Biology Manual A9, 1-16 5 Rogers S.G. et al., Use of cointegrating Ti plasmid vectors, 1988, Plant Molecular Biology Manual A2, 1-12 Samac et al., 1990, Plant Physiol., Vol. 93, pp. 907-914 Sambrook J., Molecular Cloning, A laboratory manual, 1990, 2nd edition, Cold Spring Harbor Laboratory Press 10 Saul M.W. et al., Direct DNA transfer to protoplasts with and without electroporation, 1988, Plant Molecular Biology Manual Al, 1-16 Schagger et al., Anal. Biochem., 1987, Vol. 166, pp. 368-379 Selstes M. E. et al., 1980, Anal. Biochem, Vol. 109, pp. 67-70 Shinshi H. et al., Agric. Biol. Chem. 47, 1455-1460, 1983 15 Shinshi H. et al., Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-/3-1,3-glucanase, 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 5541-5545 Shinshi H. et al., Structure of a tobacco endochitinase gene: 20 evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain, 1990, Plant Molecular Biology, Vol. 14, pp. 357-368 Sierks M. R. et al., Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Aspl76, Glul79 and Glul80 in the enzyme 25 from Aspergillus awamori, 1990, Protein Engineering, Vol. 3, No. 3, pp. 193-198 829746BI.002/MKA/SPK/A36/1992 04 02 242 27 76 Stougaard J. et al., 1986, Nature, Vol. 321, pp.669-674 Tomes D.T. et al., Direct DNA transfer into intact plant cells and recovery of transgenic plants via microprojectile bombardment, 1990, Plant Molecular Biology Manual A13, 1-22 5 Vad K. et al., 1991, Planta, 184, In Press Velten J. et al., Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens, 1984, The EMBO Journal, Vol. 3, No. 12, pp. 2723-2730 Viegers, A. J. et al., 1991, Mol. Plant Microbe Interaction, vol 4, 10 pp. 315-323, Waldron C. et al. , Resistance to hygromycin B - A new marker for plant transformation studies, 1985, Plant Molecular Biology, Vol. 5, pp. 103-108 Wing D. et. al., Conserved function in Nicotiana tabacum of a single 15 Drosophila hsp 70 promoter heat shock element when fused to a minimal T-DNA promoter, 1989, Mol Gen Genet, Vol. 219, pp. 9-16 Wood W. I. et al., 1985, PNAS, Vol. 82, p. 1585 829746BI.002/MKA/SPK/A36/1992 04 02 242270 77 MATERIALS AND METHODS Biological material Plants Seeds of Beta vulgaris, cv. "Monova", were sown in clay mixed peat 5 ("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 10 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. 15 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. 20 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/1) was used as growth medium. 25 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. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 78 Sporulation of Cercospora species 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. 5 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 CaC03 and 20 g agar. The suspension was allowed to settle for 1 hour. After airdrying the 10 culture (approximately 1 hour) the Petri dish was closed, sealed and placed in an incubation chamber at 13°C 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 15 of the culture with a sterile brush. The resulting spore suspension contained approximately 100,000 spores/ml. Infection with Cercospora species For inoculation, 12.500 spores were suspended in 1 ml of water con-20 taining 20 /xg 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 25 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 829746EX.002/MKA/SPK/A36/1992 04 02 242270 79 i) small scale purification of chitinase 4, the acidic chitinase "SE" and 0-1,3-glucanase, and ii) a time course study to determine the expression level of total enzyme activity using radiochemical assays and immunoblotting. 5 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 with the root pathogen Rhizoctonia solani 10 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 incubat-15 ed 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 20 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. 25 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 fluo-30 ride. Particulate matters were removed by centrifugation at 15,000 x 829746EX.002/MKA/SPK/A36/1992 04 02 242 2 80 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 5 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 0-1,3-glucanase was carried out from 2 kg of leaf material from naturally infected sugar beet plants, cv. "Monova". Naturally infected leaves carrying 50 or 10 more necrotic lesions were picked in the field at a breeding station in Italy (Maribo-Italy, Bologna) and stored at 4°C 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 dis-15 solved 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 anhy-20 dride 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. 25 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 Na2C03 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 30 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 829746EX.002/MKA/SPK/A36/1992 04 02 242270 81 column was prepared from the regenerated chitin by use of the conventional procedure according to Pharmacia. Preparation of radioactive colloidal chitin 2 g of chitosan was acetylated with ^H-labelled acetic anhydride as 5 described for the synthesis of unlabelled chitin (see above). After extensive washing of the ^H-labelled chitin on a Buchner funnel, it was transferred to a beaker. 50 ml of concentrated ice-cold HC1 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 10 slowly poured into vigorously stirred 50% aqueous ethanol to precipitate the chitin in a highly dispersed state. The residue was sedi-mented 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 5x1 minute at full 15 power. The %-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 (0-1,3-glucan) (from Laminaria digitata, Sigma). 50 g Mini-leak High was dispensed in 200 20 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 25°C on a shaking table. Unreacted divinylsulfone groups were blocked by incubation with a solution of 5% mercapto-ethanol in 1 M K-P-buffer at pH 9.5. The reaction time was 16 hours 25 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. 30 Synthesis of ^H-labelled laminarin Laminarin was labelled with radioactivity by reduction with %-label-led NaB-Hfy. 500 mg laminarin (from Laminaria digitata, Sigma) was 829746EX.002/MKA/SPK/A36/1992 04 02 242270 82 dissolved, in 2 ml H2O, and purified by precipitation by addition of 800 pi 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 5 dissolved in 4 ml of 0.1 N NaOH. This solution was transferred to a reaction wessel containing 5 mCi of NaB^H^. After stirring for 90 min. at 25°C, 600 pi of 1 M HC1 was added to destroy unreacted NaB^H^. The reaction mixture was divided into 500 pi aliquots and 200 pi of NaCl and 2 ml of absolute ethanol was added to each test tube. 10 After storage for 10 min. at 0°C, the precipitate was collected by centrifugation for 5 min. at 15.000 x g. The %-labelled laminarin was dissolved in 500 pi of H2O and the precipitation was repeated until the background level in the supernatant was less than 100 cpm per 20 pi. The labelled solution of laminarin was stored at -20°C. 15 Before use in the 0-1,3-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 20 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 x 15 cm; 10 pm particle size; The Separations Group, He-speria, California) column or a Poly F (Du Pont de Nemours) column. 25 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 30 Tricine SDS-PAGE system described by Schagger and von Jagow (1987). A total of 25 pg of protein was applied to each lane. Pure chitinase isoenzymes were analyzed on the Phast-System (Pharmacia) in accordance with the manufacturers instructions. 829746EX.002/MKA/SPK/A36/1992 04 02 242 27 83 Enzyme assays The radiochemical chitinase assay Chitinase activity was determined radiochemically with %-chitin as a substrate. 5 The specific activity of the -^H-chitin was 460 cpm/nmol N-acetyl-glucosamine (GlcNAc) equivalent (or 2,3 x 10^ cpm/mg %-chitin). It was determined by scintillation counting and colorimetric determination of GlcNAc after total hydrolysis of %-chitin by crude chitinase preparations from sugar beet leaves and exochitinase from serratia 10 marcescens or Streptomyces griseus. The assay mixture contained in a total volume of 200 pi of enzyme solution, 50 pi of ^H-chitin suspension (containing 100.000 cpm) and 10 pmol of sodium citrate (pH 5,0). After mixing, the enzymatic hydrolysis of ^H-chitin was allowed to take place at 40°C for 15 min. 15 before addition of 300 pi of 10 % (w/v) TCA. In order to decrease the background reading, 100 pi of bovine serum albumin (10 mg/ml) were added before the insoluble chitin was removed by centrifugation at 15.000 x g for 5 min. The radioactivity in 300 pi supernatant was determined by scintillation counting. 20 The radiochemical ft-1,3-glucanase assay /9-1,3-glucanase activity was determined radiochemically with ^H-labelled laminarin as substrate. The assay mixture consisted of 50 pi of enzyme extract, 50 pi of 0,1 M Na-citrate pH 5,0 and 10 pi of ^H-labelled laminarin (192.000 cpm). 25 Incubation was carried out for 15 min. at 40°C. To terminate the reaction, 1000 pi of abs. Ethanol and 50 pi of a saturated NaCl-solution was added. After 10 min. at 0°C, unreacted laminarin was removed by centrifugation at 10.000 x g for 5 min. An aliquot of 400 pi of supernatant was transferred to a scintillation vial. 5 ml of 30 PICO-FLUOR-40 were added and the amount of radioactivity was determined by a liquid scintillation counting. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 84 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 5 walls from Micrococcus lysodeikticus suspended in a 20 mM sodium phosphate buffer, pH 7.4, containing 1 rag/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. 10 /3-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. 15 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-/0-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 inten-20 sity recorded by visual inspection by microscopy. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 85 Purification of chitinase 2, 3 and 4, acidic chitinase "SE" and 0-1,3-glucanase isoenzymes Acidic and basic chitinase isoenzymes were purified together with 0-1,3-glucanases from sugar beet leaves as shown in the following flow 5 diagram. 10 15 20 Roff 2 kg of sugar beet leaves 0.1 M Na-citrate 1 mM DTT 1 mM BAM, pH 5.0 Heat treatment, 50CC, 20 min. 90% (NH4)2S04 Dialysis FF-Sepharose S Laminarin-Agarose FF-Sepharose Q Chitin column FPLC, Mono-S RP-HPLC chitinase 2,3, and 4 Homogenization Centrifugation 10 mM Tris, pH 8.0 25 FF Sepharose Q I Chromato-focusing I FPLC, Mono P 4- acidic chitinase "SE" 30 FPLC, Mono-S 4. RP-HPLC I 0-1,3-glucanase 3 and 4 The sugar beet leaves were obtained in Italy (large scale, see "Bio logical Material"). In the following each of the purification steps 35 outlined below will be explained. The equipment and procedure used for each step are carried out as described below. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 86 Extraction of protein from Cercospora beticola infected sugar beet leaves All steps were performed at 4°C. Centrifugation was carried out at 20000 x g for 20 minutes in a Centrikon model H-401B centrifuge, 5 throughout the purification procedure. Preparation of cellfree-extracts 2 kg of Cercorspora infected leaves were homogenized in 4 1 Na-ci-trate buffer pH 5.0 containing 1 mM DTT (Dithiothreitol), 1 mM BAM (Benzamidine) (starting buffer) and 200 g Dowex 1x2 (100 pm/mesh 10 size. The homogenate was squeezed through a double layer of 31 pm mesh nylon gauze, before centrifugation. Precipitation with 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 15 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 0-20 1,3-glucanase by column chromatography Chitinase and 0-1,3-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 centri-25 fugation 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 in-30 eluding 0-1,3-glucanase were removed by extensive washing with the starting buffer. After disconnecting the Fast Flow Sepharose Q co- 829746EX.002/MKA/SPK/A36/1992 04 02 242270 87 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. 5 Purification of 0-1,3-glucanase Separation of 0-1,3-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.). 10 After dialysis overnight against 20 mM Na-acetate buffer at pH 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 15 linear gradient from 0 to 0.5 M NaCl in the starting buffer. Three major peaks A, B and C of 0-1,3-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 0-1,3-glucanase on Laminarin-Agarose 20 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 25 was stopped for 20 minutes to allow the 0-1,3-glucanase to bind to the affinity ligand. Unabsorbed protein was removed by washing with Tris buffer. 0-1,3-glucanase was eluted with 1 M NaCl in Tris buffer. Purification of 4 0-1,3-glucanase isoenzymes by FPLC Fractions from the Laminarin-Agarose column with 0-1,3-glucanase 30 activity were combined, concentrated and dialyzed overnight against a 829746EX.002/MKA/SPK/A36/1992 04 02 242270 88 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 substra-5 te in the radiochemical assay for /S-1,3-glucanase (see above). Purification of the (3-1,3 -glucanase on Reverse Phase HPLC The purification was achieved by injecting the FPLC-purified 0-1,3-glucanase into the above described Poly F reverse phase HPLC column. Non-adsorbed materials (buffers, salt etc.) were removed by washing 10 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 15 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 20 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 25 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 VYDAC RP4 HPLC column was employed. The conditions were similar to those described above in connection with the purification of 0-1,3-glucanase. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 89 Purification of acidic chitinase "SE" Purification of the acidic chitinase "SE" on anion-exchange chromatography The acidic chitinase "SE" was eluted from the above described Fast 5 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. 10 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" 15 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. 20 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 25 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 30 gradient from 0 to 0.3 M NaCl (Fig. 3). 829746EX.002/MKA/SPK/A36/1992 04 02 242270 90 Analysis of the enzymatic cleavage pattern of sugar beet chitinase 4 40 pi of ^H-labelled chitin (50,000 cpm) was incubated with 7 pg of chitinase 4 (purified as described above) in a 0.1 M citrate buffer 5 at pH 6.5. The total volume was 300 pi. After a specified time (15 min., 30 min., 1 hour and 24 hours) the reaction was stopped by the addition of 300 pi of 10% (w/v) TCA. The unreacted polymer of ^H-labelled chitin was removed, and an aliquot (300 pi) of the supernatant was applied to a thin layer chromatography (TLC) plate (Silica 10 gel 60 H, Merck). The mobile phase was n-propanol/l^O/NHj (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 15 plate. Zones of interest on the TLC plate were removed by scraping with a razor blade, and the silica gel containing the ^H-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 spectro-20 photometer. 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 25 0-1,3-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 pg quantities 30 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. 829746EX.002/MKA/SPK/A36/1992 04 02 2 2 91 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 inter- 5 vals. In method III, radiotracer techniques in combination with autoradiography are used to demonstrate that chitin and 0-1,3-glucan are important cell wall components in Cercospora and that chitinase 4 can remove radioactivity deposited in the hyphae tip of Cercospora. 10 Method I: Microscopy Slide 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 pi of a spore suspension (10.000 spores/ml) was placed in the center of the slide. 10 pi of a 10 mM Tris-buffer, pH 15 8.0 or 10 pi of a preparation containing 20 pg 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 pm filter before mixing with the spore suspension. The petridish was sealed with tape and incubated for 24-48 hours at 30°C 20 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 25 wall. Method II: Microtiter Plate Bioassay 100 pi 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 30 remove small amounts of mycelia fragments. The spore suspension was diluted 1:100 and 1:1000 with sterile water, before aliquots of 100 pi was transferred to the microtiter wells. The antifungal proteins were dissolved in the same buffer and treated as described above for 829746EX.002/MKA/SPK/A36/1992 04 02 24 2 27 0 92 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 5 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 10 Cercospora cultures were grown on a microscope slide as described in method I. Liquid growth medium (PDB) containing %-labelled N-ace-tylglucosamine 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% 15 (w/v) of TCA. The preparation was dehydrated in an ethanol gradient (70-100%) and dried. After the pulse labelling, 50-100 pi 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 20 on moistened filter paper in a petridish. After sealing the petri- dish, 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 25 (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 30 drying the preparation was ready for a microscope analysis of the hyphae of the fungus. 829746EX.002/MKA/SPK/A36/1992 04 02 270 93 Production of antibodies for use in serological analysis Production of polyclonal antibodies to chitinase 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 5 with Freunds incomplete adjuvant. Polyclonal antibodies were raised in rabbits according to conventional methods by Dakopatts (Denmark) . Production of monospecific polyclonal antibodies to chitinase 4 peptides The procedure was carried out as described in detail for the produc-10 tion of monospecific antibodies to AHA.S 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 15 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-sul-fosuccinyl-ester derivative by reaction with carbodiimide (EDC) 20 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 25 production of polyclonal antibodies to chitinase 2 and 4. SDS-PAGE and immunoblotting For immunoblotting, proteins were transferred by semi-dry blotting onto a 0.45 fim nitrocellulose membrane (Schleicher and Schuell, FGR) after separation by SDS-PAGE. The antigens were probed with primary 30 polyclonal rabbit antibodies raised against chitinase 2 and 4 (see above) and subsequently visualized using alcaline phosphatase con- 829746EX.002/MKA/SPK/A36/1992 04 02 242 270 94 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 5 of the leaf materials, 1 pg 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 10 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 0-1,3-glucanase 3 and 4 15 After freeze-drying, the purified chitinase isoenzymes 2,3 and 4 and 0-1,3-glucanase 3 and 4 were subjected to amino acid analysis as described by Barkholt and Jensen (1989). An aliquot (4.2 pg) of each of the chitinase isoenzymes and the 0-1,3-glucanase respectively were incubated with 3,3-dithiopropionic acid to derivatize the cysteine 20 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 0-1,3-glucanase 3 and 4 Tryptic digestion 25 After RP-HPLC as described above and freeze-drying, 100 pg of proteins were redissolved in 200 pi 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 30 the dark for 40 minutes at 25°C under nitrogen. Unreacted iodoacetamide was inactivated by addition of 5 pi of 0-mercaptoethanol followed by incubation for 15 minutes at 25CC in the dark. The protein 829746EX.002/MKA/SPK/A36/1992 04 02 242 270 95 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 5 centrifugation for 5 minutes at 10,000 x 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 pg TPCK-treated trypsin in 20 pi of ammonium carbonate (pH 8.0) at 40°C for 30 minutes. Finally the supernatant 10 and 6 pg 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 pi of TFA. The peptide solution was subjected to RP-HPLC on a VYDAC C18 column (0.46 x 15 cm; 10 pm particle size; The Separations Group, California) using the mobile system described above for RP-HPLC of 15 proteins (see Fig. 4). Collected peptides were diluted 3 times with buffer A and rechromatographed on a Develosil C^g column (0.4 x 10 cm; 5 pm particle size; Dr. 0 Schou, Novo-Nordisk, Denmark) using the mobile system described above. Selected peptides were subjected to amino acid sequence analysis. 20 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. 25 Bacterial strains and enzymes Restriction enzymes, Klenow polymerase and T4 DNA ligase were supplied by Boehringer Mannheim and used in accordance with the manufacturers ins true t ions. pBluescript was supplied by Stratagene (USA). 30 pUC19 was supplied by Boehringer Mannheim. 829746EX.002/MKA/SPK/A36/1992 04 02 H9 0 £ 4 l. c 96 For subcloning in E. coli, transfer of DNA was carried out using DH5a 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. 5 (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 10 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 15 added to the RNA before chromatography through the column. The "run-through" was collected and subjected to chromatography again. The column was washed with column buffer until OD26O 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 20 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 H20 at a concentration of 1 pg/pl and stored at -20°C. The yield was about 1-2% of total RNA applied to the column. 25 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 30 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) 829746EX.002/MKA/SPK/A36/1992 04 02 4 2 2 7 0 97 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 5 4°C, 25,000 x 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 x g for 15 min. at 4CC, the pellet was washed with 70% ethanol and thereafter dried briefly before being 10 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 min. The supernatant was extracted twice with phenol/chloroform. The DNA was precipitated by adding 75 pi of 3 M Na-acetate and 500 pi of iso-propanol, mixing and spinning for 30 se-15 conds. Afterwards, the pellet was dissolved in 400 pi 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 pi of TE buffer. The DNA concentration was determinated 20 using the absorbance at OD2go> where OD2go=l=50 pg 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 AZAP cDNA library was constructed by Stratagene Cloning Systems. 25 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. 30 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. (1990), and about 10® 829746EX.002/MKA/SPK/A36/1992 04 02 242270 98 phages were used for each screening. For each 24.5 x 24.5 mm plates, 2.5 x 10^ phages were mixed with 3 ml of the E. coli strain XL 1-Blue (in case of a sugar beet AZAP cDNA library) or LE392 (in case of the sugar beet genomic library (EMBL3)) and grown in LB medium with 10 mM 5 MgS04 and 0.2% maltose to an 0Dg00=^-- ^he 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 MgS04 and 0.2% maltose) (48°C) were added and the resulting mixture plated onto 24.5 x 24.5 mm plates containing 200 ml of LB agar and 10 allowed to grow overnight at 37°C. Transfer of plaques to nitrocellulose filter in situ The screening of AZAP 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 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 20 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 follow-25 ing solutions: 1) 0.5 M NaOH, 1.5 M NaCl, 2) 0.5 M Tris, pH 7.5, 1.5 M NaCl, 3) 2 x SSC (modified Benton, 1977). The filters were air dried and illuminated with UV for 3 minutes with the phage side upwards. 829746EX.002/MKA/SPK/A36/1992 04 02 142 270 99 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 5 in Sambrook et al. (1990). More specifically, the oligonucleotides were synthesized without a phosphate group at their 5' termini ends and were labelled with 7-^P from [7-^^P]ATP using the enzyme bacteriophage T4 polynucleotide kinase. Purification of radiolabelled oligonucleotides by precipitation with 10 ethanol After inactivation of the bacteriophage T4 polynucleotide kinase by heat, 40 pi of H2O was added to the tube, the content of which was subjected to thorough mixing. Then 240 pi of a 5 M solution of ammonium acetate and 1 pg of herring sperm DNA were added. The result-15 ing mixture was mixed well, and 750 pi 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 x g for 20 minutes at 4°C in a microfuge. Using an automatic 20 pipette device equipped with a disposable tip, all of the supernatant fluid (which contained most of the unincorporated [7-^^P]ATP) and any free ^P generated during the phosphorylation reaction were carefully removed. The resulting residue was redissolved in 100 pi of H2O and 10 pi of 3M sodium acetate and thereafter 250 pi of 96% ethanol were 25 added. The mixture was subjected to centrifugation for 20 minutes at 4°C, dried and redissolved in 200 pi of H2O. Oligonucleotide hybridization of chitinase 4 DNA by filter hybridization The oligonucleotide hybridization procedure used eliminates the 30 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 829746EX.002/MKA/SPK/A36/1992 04 02 242270 100 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 (6xSSC, 1% BSA, 1% Ficoll 4000, 1% PVP, 50 pg/ml of heat denaturated salmon sperm DNA, 5 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 10 rinsed three times in 6xSSC at 4°C and thereafter washed twice for 30 min. at 4°C in 6xSSC. 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 TMA.C is hydroscopic, 15 the actual molar concentration (c) must be determined from the refractive index (n) by the formula c-(n-l,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 20 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 25 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 pi of SM phagebuffer 30 (Sambrook et al., 1990) 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 10®-10^ phages per plaque were obtained. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 101 The phages were then diluted in SM phage buffer and mixed with 200 pi of XL1 blue cells (ODggo ~ 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 5 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 (1990) using several steps of replating and rescreening. 10 A phage stock was prepared according to the method of Sambrook et al. (1990). In vivo excision In vivo excision of plaques was performed as described in "In vivo excision Protocol" in the Instruction Manual (CAT# 236201, August 15 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 20 Sambrook et al. (1990), and was performed as follows: Bacterial strains (DH5a and XLl-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 x g. The pellet was resuspended 25 in 200 pi of Solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA) in 1.5 ml tubes. 400 pi 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 pi of 5 M KOAc pH 4.8 was added and subjected to thorough mixing. The resulting mixture was placed on ice for 10 minutes 30 and subsequently subjected to centrifugation at 15,000 x g for 10 minutes at 4°C. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 102 The supernatant (900 pi) was transferred to new tubes and 0.6 volume (540 pi) 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 x g and 4°G for 10 minu-5 tes and the supernatant was removed. The pellet was dissolved in 100 pi of TE and 100 pi of 5 M LiCl was added. The mixture was allowed to stand on ice for 5 minutes and subjected to centrifugation at 15,000 x g and 4°C for 10 minutes. The supernatant was transferred to new tubes and 500 pi of 96% etha-10 nol was added. The tubes were centrifugated at 15,000 x g and 4°C for 30 minutes and the supernatant was removed. The pellet was washed with 70% ethanol (about 100 pi) and dried. The dried pellet was redissolved in 50 pi of TE. DNA sequencing 15 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 pg of the relevant plasmid, 1 pi of 2 M NaOH, 2 mM EDTA, 1 pi of primer (100 pg/ml) and H2O up to 10 pi was 20 incubated at 85°C for 5 minutes and subsequently put on ice. The mixture was neutralized by adding 1 pi of 5 M NH^Ac 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 pi of H2O. 1.5 pi of 5 x conc. sequenase buffer was added. The mixture was placed at 25 37°C for 5 minutes. 4 pi of sequetide (Biotechnology Systems NEN® Research Products, Du Pont de Nemours) and 2 pi of sequenase (United States Biochemical) were added, resulting in a total volume of the mixture of 13.5 pi. The mixture was placed at room temperature for 5 minutes. 30 3.1 pi of the labelling reaction was transferred to each termination tube (G, A, T and C) containing 2.5 pi of the dNTP terminating mix- 829746EX.002/MKA/SPK/A36/1992 04 02 242 103 ture. The mixtures in each of the tubes were allowed to react for 5 minutes at 37°C and 4 nl of stop solution was added. The mixtures were then heated to 85°C and 2 pi of the heated mixture was applied onto a 6% sequencing gel (Gel-mix 6 from BRL). The gel was vacuum 5 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 10 instructions. More specifically, the following procedure was used: A sample comprising 25 ng (1-23 /i 1) of the DNA template to be labeled, 0-22 n 1 of H2O and 10 y.1 of random oligonucleotide primers (constituting a total volume of 33 /xl) were added to the bottom of a clean microcentrifuge tube. The reaction tubes were heated to 95-15 100°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: 20 10 fil of 5X primer buffer containing dATP, dGTP and dTTP. 5 fil of labeled nucleotide a-^^PdCTP (3000 Ci/mM) (Amersham), and 2 n1 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//al. The reaction components were mixed with the 25 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 /il of Stop Mix. The probes with the ^^P-labeled DNA were further purified using the Elutip™-D column system (Schleicher & Schuell). 829746EX.002/MKA/SPK/A36/1992 04 02 24 2 104 Then, the probe DNA was made ready for hybridization by mixing the proper amount of radioactive probe with 200 fil 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 -5 20 °C for up to one week and heated to 95-100°C 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 A-ZAP cDNA library were subjected to 10 prehybridization for 2 hours at 67°C under conventional prehybridi- zation conditions using a prehybridizing solution comprising 2 x SSC, 10 x Denhardt's, 0.1% SDS and 50 fig/ml salmon sperm DNA. Hybridization was carried out overnight using a hybridization solution identical to the prehybridization solution except for the fact 15 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 x 15 min. in 2 x SSC and 0.1% SDS, and 2 x 15 min. in 1 x SSC and 20 0.1% SDS. The positive plaques were identified as described under "Oligonucleotide hybridization of chitinase 4 DNA in filter hybridization". 829746EX.002/MKA/SPK/A36/1992 04 02 242270 105 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 5 "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 N0.: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. 10 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 15 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-20 ® 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 fi 1 of agarase (free of DNase, from Calbiochem) was added. The mixture was 25 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", 0-1,3-glucanase and chit 76 on the basis of sugar beet mRNA 30 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- 829746EX.002/MKA/SPK/A36/1992 04 02 2422 106 tion with a few modifications. The reverse transcription protocol was followed using the concentrations in the scheme below. Component volume Final conc. MgCl2 solution 4 pi 5 mM 10 x PCR buffer II 2 pi 1 mM dGTP 2 pi 1 mM dATP 2 pi 1 mM dTTP 2 pi 1 mM dCTP 2 pi 1 mM RNase Inhibitor 1 Ml 1 U/M1 Reverse Transcriptase 1 Ml 2.5 U//il primer 270 0.4 pi 2.5 pM. mRNA 3.6 pi 15 Total volume per sample 20 pi In the step cycle the following procedure was used. Segment 1: 42°C for 2 hours Segment 2: 99°C for 5 minutes 20 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: 829746EX.002/MKA/SPK/A36/1992 04 02 107 PCR cycles: no. of cycles °C time (min.) 1 98 5 5 6 5 addition of Taq polymerase and oil 1 94 1 37 2 50 1 10 72 40 5 94 1 37 2 72 10 30 94 1 15 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 20 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 25 no. of cycles °C time (min.) 35 94 1 1/2 60 1 1/2 72 4 30 1 72 7 829746EX.002/MKA/SPK/A36/1992 04 02 242270 108 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 5 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 10 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. 15 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 20 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 25 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 isozymes 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 30 protein peak for each of the isozymes. This is further evidence for a homogeneous protein preparation for each of the basic chitinase isozymes. 829746EX.002/MKA/SPK/A36/1992 04 02 109 242 2 70 The molecular weights determined by SDS-PAGE for chitinase 2, 3 and 4 are 32, 27 and 27 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 5 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 produc-10 ing chitooligosaccharides or an exochitinase liberating only N-ace-tylglucosamine from the non-reducing end of chitin or chitooligosaccharides, the pattern of reaction products liberated by chitinase 4 from ^H-chitin was analyzed by TLC (Fig. 3). Irrespective of duration of incubation, N-acetylglucosamine was only a very minor reac-15 tion 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 ^H-chitin, chitinase 4 was also capable of hydrolyzing the cell walls of Micrococus 20 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 0-1,3-GLUCANASE ISOEN-25 ZYMES FROM SUGAR BEET LEAVES Three different bioassays were conducted to ascertain the in vitro antifungal activity of chitinase and 0-1,3-glucanase isoenzymes on the germination and growth of Cercospora beticola. In the same manner the antifungal activity of chitinases and 0-1,3-glucanases from other 30 sources or other isozymes from sugar beets may be determinated using either purified enzymes or extracts containing the enzymes. Also, the 829746EX.002/MKA/SPK/A36/1992 04 02 110 assays may be used to determine whether a given transgenic plant is within the scope of the present invention. Method I - Microscope slide Bioassay Spore cultures of Cercospora germinate and grow well on a thin film 5 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 fluores-10 cent 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 15 hyphae is drastically reduced. In Fig. 5 is shown the results when a combination of chitinase 4, "SE" and 0-1,3-glucanase 3 is applied to the culture. 60 pi of protein solution containing 20 pg of each antifungal proteins were applied to each microscope slide. When chitinase 4 was used alone or in combination with either 0-1,3-glucanase 3 20 or "SE" alone, the inhibitory effect was less pronounced. Neither 0-1,3-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 0-1,3-gluca-25 nase. Method II - Microtiter 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 Cer-30 cospora 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 pg per well) is included, the inial lag period is increased to 75 hours and the growth rate is decreased as 829746EX.002/MKA/SPK/A36/1992 04 02 242 270 ill 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 label-5 led with ^H-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 0-1,3-glucanase was added after the pulse labeling, the radioactivity deposited in the hyphae tip was effectively removed. The amounts of 10 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 15 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 20 growth inhibitor. Fractions which contain both chitinase and 0-1,3-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 25 the same manner from control plants. 829746EX.002/MKA/SPK/A36/1992 04 02 24227 112 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 5 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 10 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 se-15 quence encoding the sugar beet chitinase 4 without signal peptide is also shown. The cDNA sequence was obtained as described in Example 5 below. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 113 TABLE I Amino acid composition of barley, wheat, bean and sugar beet chitinases 2, 3 and 4 5 Amino acid Barley Wheat Bean S.B.2 S.B.3 S.B.4 cDM 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 10 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 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 20 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 25 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. 829746EX.002/MKA/SPK/A36/1992 04 02 24 2 270 Tryptic digestion of sugar beet chitinase 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 5 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. 10 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 absor-15 bance 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 se-20 quence 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 25 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 oligo-30 nucleotide probe to be used in the isolation of DNA encoding chitinase 4 (see Example 4 below). 829746EX.002/MKA/SPK/A36/1992 04 02 242270 TABLE II Tryptic peptides of chitinase 3 and 4 Chitinase 3: 5 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 10 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 15 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 N0.:18 3-16.1: shown in SEQ ID NO.:19 20 3-16.2: shown in SEQ ID N0.: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 N0.:2 4-19.3: consisting of amino acids No's 163-169 25 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 30 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). 829746EX.002/MKA/SPK/A36/1992 04 02 242270 116 Peptide 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 y ^ F w F W M N N Phe Trp Phe Trp Met Asn Asn TTT TGG TTT TGG ATG AAT AAT C C C C Using this gene probe, the expression cDNA library AZAP was screened 10 using the procedure given in "Materials and Methods" above. 8 cDNA clones were obtained from AZAP, 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 AZAP 15 library and the DNA sequence thereof is shown in SEQ ID NO.:l. On the basis of the cDNA sequence, a deduced amino acid sequence o£ 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% 20 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 25 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. 30 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 Gin. Hereafter the rest of the amino acid sequence 829746EX.002/MKA/SPK/A36/1992 04 06 242270 117 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 5 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 Gin is converted to the pyroglutamyl deriva-10 tive (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 15 2 and the following terminal amino acid sequence was found in chitinase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 118 TABLE III Comparison of the N-terminal amino acid sequence between different chitin binding proteins: 10 WGA-A: Hevein: Chit. Bean Chit. Tob. Chit. SB 2 Chit. SB 4 QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCGKG- -CQNGACWTS EQ**R*AGGKL*********W- *W**STDE**S PDHN**SN- *KD EQ**R*AGGAL**GGN****F - *W**STT****P* - -**SQ-*GG EQ**S*AGGAR*ASG****KF - *W**NTN****P* -N**SQ- *PG EL**N*AGGAL***G****** - *W**NTNP***N *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 15 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 N0.:2 EXAMPLE 5 20 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 25 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 30 was initiated with the primer used for screening of the AZAP library 829746EX.002/MKA/SPK/A36/1992 04 02 242270 119 (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 5 NO.:i) but only 34X homology to che entire chitinase 1 protein (vide SEQ ID NO.:li). The gene contains one intron which is located in position 875 to 1262. The exact location o£ this intron is based on an alignment with the chitinase 4 cDNA SEQ ID NO. ;i (Fig. 24). The intron borders contain the consensus GT/AG sequences. The chit 76 10 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 15 (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 20 N0.: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 25 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. 82J746EX002/MKA/SPK/A36/1W2 04 06 24227 120 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 5 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. Analy-10 sis 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 15 "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 labelled chitin. The major product of the enzymatic hydrolysis was the hexamers of chitin or higher homologous of chitin oligo saccharides. 20 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 25 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 30 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. 829746EX.002/MKA/SPK/A3G/1992 04 02 242270 L21 TABLE IV N-terminal: SE 22.5: 5 SE 23.0 SE 25.1 SE 26.1 SE 30.4 SE 31.1 10 N-terminal: consisting of amino acids No's 26-46 of S£Q ID NO.:8 SE 22.5: consisting of amino acids No's 98-109 of SEQ IB 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 15 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 ISOIATION AND CHARACTERIZATION OP THE cDNA FOR THE ACIDIC CHITINASE 20 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) 25 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 co chitinase 4. The nucleotide sequence of the gene probes are shown in Table V. S-Q-I-V-I-Y-w-G-Q-N-G-D-E-G-S-L-A-D-T-C-N v-l-l-s-i-g-g-g-a-g-g-y A-D-Y-L-W-N-T-Y N-N-F-P-C-Q-Y-D-T-S-A-D-N-L-L-S-S Y-G-G-V-M-L-W S-L-S-S-T-D-D-X-N-T-F-X-D-Y-L-W-N-T-Y T-T-V-Q-A-N-Q-I-F-L-G-L-P-A-S-T-D-A-A-G-S-G-F-1 827746EX002/MKA/SPK/A36/1992 04 06 242270 122 TABLE V NPPCQYDT KB- 7. 5'-GACTCTAGAAA$CC£CC£TG$CA$TA$GA$AC- 3' 5 Q A N Q 1 F KB-9. 5'-GGAGGATCCCA^GCGAAJCA^ATATT- 3' A T C C T 10 270. 5'-CCAAGCTTGAATTCTTTTTTTTTTTTTTTTTTTT-3' K3-7: shown in SEQ ID NO.:28 K8-9: shown in SEQ ID NO.:29 270: shown in SEQ ID NO.:30 15 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 subse-20 quent 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 N0.:7. 25 This cDNA was used to screen the A-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 30 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 AZAP library with a 122 bp EcoRI-Kpnl from the 5' end of the longest cDNA clone (SE22), gave a sequence containing the entire 5' end. The clones were ligated using the Kpnl site. The structural 829746EX.002/MKA/SPK/A36/1992 04 02 242 2 123 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 5 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 10 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. 829746EX.002/MKA/SPK/A36/1992 04 02 * 124 242270 TABLE VI SE22SAML MAAKIVS--VLFLISLLIFASFESSHGS--QIVIYWGQNGDEGSLADTCN 46 CUCUMBER MAAHKIT--TTLSIFFLLSSIFRSSDAA--GIAIYWGQNGNEGSLASTCA 46 ARABIDQPS MTNMTLRKHVIYFLFFISCSLSKPSDASRGGIAlYWGQNGNEGNLSATCA 50 « • • SE22SAML SGNYGTVILAFVATFGNGQTPALNLAGHCDPATN-CNSLSSDIKTCQOAG 95 CUCUMBER TGNYEFVNIAFLSSFGSGQAPVLNLAGHCNPDNNGCAFLSDEINSCKSQN 96 ARABIDOPS TGRYAYVNVAFLVKFGNGQTPELNLAGHCNPAANTCTHFGSQVKDCQSRG 100 . t », SE22SAML IKVLLSIGGGAGRYSLSSTDDANTFADYLWNTYLGGQSSTRPLGDAVLDG 145 CUCUMBER VKVLLSIGGGAGSYSLSSADDAKQVANFIWNSYLGGQSDSRPLGAAVLDG 146 ARABIDOPS IKVMLSLGGGIGNYSIGSREDAKVIADYLWNNFLGGKSSSRPLGDAVLDG 150 SE22SAML IDFDIESGDDRFWDDLARALAGHNNGQKTVYLSAAPOCPLPDASLSTAIA 195 CUCUMBER VDFDIESGSGQFWDVLAQELKHFGQ VILSAAPQCPIPDAHLDAAIK 192 ARABIDOPS IDFNIELGSPQHWDDLARTLSKFSHRGRKIYLTGAPQCPFPDRLMGSALN 200 SE22SAML TGLFDYVWVQFYNHPPCQYDT-SADNLLSSWNQWTT-VQANOIFLGLPAS 243 CUCUMBER TGLFDSVWVQFYNNPPCMFAD-NADNLLSSWHOWTA-FPTSKLYMGLPAA 240 ARABIDOPS TKRFDYVWIQFYNNPPCSYSSGNTQNLFDSWNKWTTSIAAQKFFLGLPAA 250 • •» »».••*»•••» ... .. SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS TDAA-GSGFIPADALTSQVLPTIKGSAKYGGVMLWSKAYD--SGYSSAIK 290 REAAPSGGFIPADVLISQVLPTIKASSNYGGVMLWSKAFD--NGYSDSIK 288 PEAA-DSGYIPPDVLTSQILPTLKKSRKYGGVMLWSKFWDDKNGYSSSIL 299 .•» » , »»*##•»»* , t SSV- 293 GSIG 292 ASV- 302 Consensus length: 304 Identity : 137 < 45.1*/.) Similarityt 106 ( 34.9%) SE22SAML: shown in SEQ ID NO.:8 Cucumber: shown in SEQ ID NO.:31 5 Arabidopsis: shown in SEQ ID NO.:32 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 10 al., 1990). It appears from this that there is a homology of about 45% when all tree segment are compared. When "SE" is compared with 829746EX.002/MKA/SPK/A36/1992 04 02 242270 125 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 /3-i, 3-GLUCANASES 3 AND 4 The sugar beet /3-1., 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 £-1,3-glucan. The amino acid composition of the sugar beet £-1,3-glucanase 3 and 4 isoenzymes are similar to the one given for 0-L, 3-glucanases 10 from tobacco and barley as shown in Table VII. 829746EX.002/MKA/SPK/A36/1992 04 02 * 10 15 20 25 242270 126 TABLE VII Amino acid composition of tobacco and sugar beet /3-1,3-glucanases Amino acid Tobaccoa) Sugar beet 3 Sugar beet 4 Barley Gs^3) 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., b> From Kragh et al., 1991 1983 829746EX.002/MKA/SPK/A36/1992 04 02 242270 127 SDS-PAGE of 0-1,3-glucanase The apparent molecular weight of >9-1,3-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 5 9.5. When analyzed by thin layer chromatography, the major reaction products liberated from laminarin after 24 hours of incubation with the two /3-1,3-glucanase isoenzymes 3 and 4 were the dimer, laminari-biose. This strongly suggests that the 0-1,3-glucanase 3 and 4 isozymes are endoglucanases. 10 Amino acid sequencing of 0-1,3-glucanase 3 and 4 The purified 0-1,3-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 15 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. 829746EX.002/MKA/SPK/A36/1992 04 02 128 242270 TABLE VIII Amino acid sequences for /3-1,3-glucanase 3 and 4 isolated from sugar beet leaves 5 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 (Q)-(Q)-(A)-(P)-(R) -N- -D- A- -E- -A- •G- -S-I-V-P-A-M-Q-N-1 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 N0.:10 20 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 829746EX.002/MKA/SPK/A36/1992 04 02 242270 129 EXAMPLE 9 ISOLATION AND CHARACTERIZATION OF THE cDNA FOR 0-1,3-GLUCANASE 3 AND 4 In the same manner as described above in connection with "SE", oligo-5 nucleotide probes corresponding to peptides from the £-1,3-glucanase 3 and 4 polypeptides were synehesiz&d. As 5'primer was used the following two sequences in the first round of PCR for isolation of )3-1,3-glucanase 4: Pep. 3-15: W, V, Q, N, N, (V)... 10 G Oligoseq. TG-1: 5'TGGGT$CA$Aa£aa£gT 3' (shown in SEQ ID NO.:36) C In che second round of PCR the following sequence was used as 5'pri-15 mer peptide 4-27.1 consisting of amino acids No's 120-125 of SEQ ID NO.:10 Pep. 4-27.1: N, E, I, M, P, N.... C G Oligoseq. TG-2: 5'AA^GA^ATAATGCC^AA (shown in SEQ ID NO.:37) 20 T T By comparing the amino acid sequences from /3-1,3-glucanases in barley (Fincher, 1986) and tobacco (Shinshi et *2.,1988), a consensus sequence was selected and used for construction of a 3'primer with che following consensus sequence: 25 Pep. seq: F,A,M,F,D/N,E. G Oligoseq. TG-3: 5'Tc£t£$AACAtJgc£aa (shown in SEQ ID NO.:38) C This sequence was used in the second PCR round whereas the 270 primer 30 used for cloning of "SE" was used in the first round. To Isolate a (}• 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 829746EX.002/MKA/SPK/A36/1992 04 06 242270 130 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 A-ZAP library to isolate clones harboring cDNA 5 encoding 0-1,3-glucanases 3 and 4, respectively. The cDNA sequences and the deduced amino acid sequence of 0-1,3-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 10 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 27 kDa) was separated by SDS-PAGE before immunoblotting the following results were observed (see Fig. 10). Chitinase 4 antibodies detect only an approximately 27 kDa protein 15 (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 27 kD protein of chitinase 4. This strongly demonstrates the presence of two serological different 20 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 cross-reactivity at all was observed. The above results suggest that sugar 25 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 30 distinct gene families. 829746EX.002/MKA/SPK/A36/1992 04 02 242 270 131 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 5 When the N-terminal amino acid sequence of sugar beet chitinase 2 was aligned with the following chitinases from bean, tobacco, pea Al, 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 10 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 15 Linthorst, 1990), chitinases T, K and C from barley and chitinase Al, 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 20 to sugar beet chitinases 2, e.g. a sugar beet chitinase 2 class chitinase. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 132 TABLE IX N-terminal amino acid sequence of chitinase isozymes belonging to the sugar beet chitinase 2 class: 5 Chitinase 2 ELCGNQAGGALCPNGLCCSQYCWCGNTNPYCGN Bean EQCGRQAGGALCPGGNCCSQFGWCGSTTDYCGP Tobacco EQCGSQAGGARCASGLCCSKFGW Pea B EQCGRQAGGArCPNNLCCSQYGY Pea Al EQCGNQAGGXVPPNG Pea A2 EQCGTQAGGALCPGGL Barley K EQXGSQAGGATCPNXLCCSRFG Barley T XQQGSQAGGATCPNXLCCSXFGW 15 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 AX: shown in SEQ ID NO.:42 20 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, 25 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, proeein 30 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 82974fi£X.002/MKA/5PK/A36/l992 04 06 242270 133 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 homol-5 ogy 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 10 examined at 55CC 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. 15 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 20 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 25 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- 829746EX.002/MKA/SPK/A36/1992 04 02 242 2 7 134 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 0D 600 (ODggo) was 0.5-1.0. The culture was cooled on ice and centrifuged for 5 minutes at 10.000 x g at 4°C. The superna-5 tant 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 jig of plasmid DNA was first 10 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 x rpm). The cryo tube was centrifuged for 30 sec. at 10.000 x g, 4°C. 15 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/1 kanamycin and incubated for 2 to 4 days at 28°C until colonies appeared. The presence of a proper plasmids in the bacteria are verified by restriction analysis of the extracted plasmid prior 20 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 25 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 vitro or in vivo. In the latter case, the leaves were 30 sterilized prior to transformation. Sterilization was performed by placing the leaves for 20 min. in a solution of 5% Ca-hypochlorite 829746EX.002/MKA/SPK/A36/1992 04 02 242270 135 containing 0.1 ml Tween 80 per 1 followed by washing 5 times in sterile water. In vitro plants were grown in containers on 1/2 shoot inducing medium (1/2 MS) (Murashige & Skoog, 1962). i The leaves were placed one at a time in a 14 cm Petri dish. They were 5 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 transfor-10 med 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 x rpm). Transformstion Transformation of the plant was done essentially as described by 15 R.B. Horsch et al. (1986). 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 20 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 /xM). On the day of transformation a piece of sterile filter paper was placed on the 25 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. 829746EX.002/MKA/SPK/A36/1992 04 02 24227 136 Selection/regeneration The leaf pieces were transferred to Petri dishes containing shoot-inducing MS-medium with 300 mg/1 of kanamycin and 800 mg/1 of carbe-nicillin and sub-cultivated every 4 weeks to the same medium. 5 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 sub-cultivated when needed. After approximately 2 weeks, the expression of the /3-glucoronidase activity using the GUS-assay (see "Materials and Methods") was performed on the leaf tips of green shoots. 10 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 15 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 de-20 scribed below. Seeds were germinated for 4 days in darkness on a substrate containing 0,7 g/1 of agarose and 2 g/1 of sucrose. The seedlings were then transferred to a Nunc container, containing 1/2 x MS substrate and cultured for 3 days in the light. The cotyledons were removed from the seedling, and the cotyledon explants were then 25 brushed on the petiole with a small brush containing a suspension (OD 660=1,0) of Agrobacterium 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/1 of BAP, 400 30 mg/1 of kanamycin, 800 mg/1 of carbenicillin and 500 mg/ml of cefo- 829746EX.002/MKA/SPK/A36/1992 04 02 24 2 2 7 137 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/1 of BAP, 400 mg/1 of kanamycin, and 800 mg/1 of carbenicillin as the substrate. The isolated shoots were transferred 5 to fresh substrates with 4 weeks intervals for selection and multiplication. Selected shoots were rooted on 1/2 MS substrate containing 1 mg/1 IBA. Tissue from tobacco have been transformed with a genetic construct containing either chitinase 1, chitinase 4, chitinase 76 and acidic 10 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 15 ANALYSIS OF CHITINASE AND 0-1,3-GLUCANASE IN TRANSGENIC PLANTS The expression levels for chitinase and 0-1,3-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 dif-20 ferent 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". 25 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 30 non-transformed plant. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 138 In the radiochemical assays, ^H-chitin or -^H-laminarin are used as substrates for either chitinase or 0-1,3-glucanase, respectively. Using standard curves of product formation vs. enzyme amount, the activity for both chitinase and 0-1,3-glucanase in crude plant ex-5 tracts can be determined. This is illustrated further in a time course experiment where the level of either chitinase (Fig. 12a upper part) or 0-1,3-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 0-10 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 as 15 0-1,3-glucanase in transgenic plants can easily be recorded. These techniques, however, do not differentiate between the various chitinase and 0-1,3-glucanase isozymes. Only the total enzyme activities for all the chitinase or all the 0-1,3-glucanase isoenzymes are determined. However, the presence of the various chitinase and 0-20 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 0-1,3-glucanase 3 recognized only one single protein in the Cercospora infected leaf material (Fig. 13). In contrast, no 25 antigen was detected in the control leaves. This is in agreement with the low constitutive level of expression observed in control plants for 0-1,3-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 30 antibodies detect a 26 and a 32 kDa band, whereas two proteins having molecular weights of 29 and 27 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. 35 Similarly, the antibody to chitinase 4 detected the authentic chitin- 829746EX.002/MKA/SPK/A36/1992 04 02 242 27 139 ase antigen (27 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 N0.:8). The "3-D" structure of chitinase 4 and "SE" on the nitrocel-5 lulose 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 10 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 15 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 20 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, 25 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. 30 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, 27 and 25 kD, respectively. The reasons for the 35 triple bands are not known at present. It is, however, contemplated that chitinase 4 is i) not processed given rise to a protein 829746EX.002/MKA/SPK/A36/1992 04 02 242270 140 maintaining the signal peptide = the 29 kD band, ii) cleaved at the normal processing site at the amino acid sequence Leu Val Val Ala -Gin Asn Cys in chitinase 4 (amino acid position 23-24 in SEQ ID NO.:2) given rise to the 27 kD protein band, and iii) a second 5 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. 10 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 15 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. 20 When seeds of N. tabacurn 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 MUTAGENE-25 SIS 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 30 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 829746EX.002/MKA/SPK/A36/1992 04 02 242270 141 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 5 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 10 this connection. The active site of sugar beet chitinase 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 15 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 gluco-amylase from Aspergillus niger (Sierks et al., 1990) have elucidated the mode of action by which carbodiimide and NBS inactivates this 20 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 25 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 30 contemplated to be constituted by amino acid residue 183 (Asp) and 189 (Glu) in SEQ ID NO.:l (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 35 encoded by the genomic chitinase 4). In contrast, chitinase C from 829746EX.002/MKA/SPK/A36/1992 04 02 242270 142 barley arid 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). 5 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. 10 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. 15 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 20 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 25 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 30 (190) to Gin 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. 829746EX.002/MKA/SPK/A36/1992 04 02 242270 143 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 5 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 10 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 15 (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 Trpl69(170) of the chitinase 4 amino acid sequence is to be substituted by the amino acid Tyr, the following procedure may be 20 carried out: For the PCR reaction the 3' primer SDl 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, 1990). 25 When Glul89(190) is to be substituted with the amino acid Gin, the 3' primer SD2 is used (Fig. 14). When Aspl83(184) is to be substituted with the amino acid Asn, the 3' primer SD3 is used (Fig 14). 829746EX.002/MKA/SPK/A36/1992 04 02 242270 144 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 Trpl69(170). When Trp206(207) is to be substituted with the amino acid Tyr, the 3' 5 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-Ball fragment in the chitinase 4 gene as described above. 10 In a similar manner, other desirable modifications may be carried out. EXAMPLE 17 CONSTRUCTIONS OF GENETIC CONSTRUCTS WITH SUITABLE C-TERMINAL EXTENSION 15 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 20 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 0-1,3-glucanase cDNA with a 25 tobacco C-terminal extension which is underlined in the figure. 82?746EX.002/MKA/SPK/A36/1992 04 02 242270 145 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 5 the last part of the C-terminal extension, a stop codon, a Smal site and an EcoRI site. The C-terminal extension can be introduced by exchanging the Xbal-EcoRI fragment in the 0-1,3-glucanase gene with the PCR product digested with Xbal and Dral and the annealed synthetic oligonucleo-10 tides digested with Smal and EcoRI using conventional methods (Sambrook et al, 1990). 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 Smal 15 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 Smal site and a EcoRI site. The C-terminal extension can be introduced by exchanging the BamHI-20 EcoRI fragment with the PCR product digested with BamHI and Smal and the annealed synthetic oligonucleotides digested with Smal 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 25 0-1,3-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 N0.:12, the N-terminal sequence of the acidic chitinase SE shown in SEQ ID N0.:8, the N-terminal sequence of chitinase 4 shown 30 in SEQ ID N0.:2, the N-terminal sequence of chitinase 76 shown in SEQ ID NO.:6, the N-terminal sequence of 0-1,3-glucanase shown in SEQ ID 829746EX.002/MKA/SPK/A36/1992 04 02 242270 146 NO.:10 and the protein sequence of chitinase 1 shown in SEQ ID NO.:12. 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. 5 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 10 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) /3-glucoronidase (GUS) from E. coli equipped with a CaMV 35S 15 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 20 restriction sites (Hindlll and Bglll) 5' to the cDNA sequence. The oligonucleotide KB3 (shown in SEQ ID NO.:49): (5'CCGAAGCTTAGATCTAAACAACAACATGTCTTCT(J)T(J)GGACC3') — I 15 25 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 829746EX.002/MKA/SPK/A36/1992 04 02 242 2 70 147 The oligonucleotide KB4 (shown in SEQ ID N0.:50): (5' GCACACGTAGCGCTAGCTTGG3') | ** | 261 Nhel 241 5 was used as the 3' PCR primer (nucleotide 255 and 256 was interchanged in order to destroy the second Nhel 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 Hindlll and Nhel. The Hindlll-Nhel fragment from pB15 10 chit 4 was exchanged with the Hindlll-Nhel 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) : 340:(CATCGGAGGATCCACTACC) 15 | | 341 323 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 Nhel 20 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 Bglll after removal of the Klenow enzyme. The DNA fragment Bglll-EcoRI containing the entire chitinase 25 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-Smal (Fig. 17). The chitinase 4 gene with the enhanced 35S promoter and 35S terminator was transferred to the plant transformation vector 30 pBKL4 (Fig. 17) as a Hindlll fragment (Fig. 17). The resulting vector, pBKL4K4, harboured in an E. coli DH5a has been deposited with the Deutsche Samralung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300 Braunschweig (DSM) on 30 July, 1991 under 829746EX.002/MKA/SPK/A36/1992 04 02 ro ff a f IS L " 148 che 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 5 of the gene from the pSurl clone (EcoRI*Kpnl) with the rest of the gene from pSE22 (Kpnl-Hindlll) in the cloning vector pUCl9 (Fig. 18). The "SE" gene was subcloned in the Smal sice of pPS48 as a EcoRI-Hindlll fragment filled in with Klenow polymerase in the presence of all four nucleotides. The orientation of che gene with 10 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 Kpnl site of pBKL4K4 as a Hindlll fragment in che 15 presence of a Hindlll-Kpnl adapter (Fig. 18), The Hindlll fragment was furthermore cloned in the Hindlll 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 20 construct pBKIA, pBKL4K4, pBKL4KSE, or pBKLKK4KSE (Fig. 22). The full length cDNA clone (SEQ ID NO. :9) was digested with EcoRI and Bglll, 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 Smal site of pPS48Mod. The orientation of the gene with respect 25 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 35$ terminator is cloned in the EcoRI site of pBKIA, pBKL4K4, pBKL4KSE, 30 pBKL4K4KSE. 829746EX.002/MKA/SPK/A36/1992 W 0$ 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 fit Vingtoft (B) STREET: Sarikt Annae Plads 11 (C) CITY: Copenhagen (E) COUNTRY: Denmark (F) ZIP: DK-1021 COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFIWARE: Patentln Release #1.0, Version #1.25 CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: ATTORNEY/AGENT INFORMATION: (A) NAME: Plougmann, Ole (C) REFERENCE/DOCKET NUMBER: 329751MKA/SPK (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 45 33 11 05 66 (B) TELEFAX: 45 33 11 18 87 (C) TELEX: 18333 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENSIH: 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 (v) (vi) (viii) (vii) IMMEDIATE SOURCE: 2 '> ? 2 7 0 x i?>o (A) LIBRARY: sugar beet lairpda 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:l: G TCT TCT TTC GGA CCA ATC TTT GCC ATA CTC ATG GCA CIT GCT TCT 46 Ser Ser Phe Gly Pro lie Phe Ala lie Leu Met Ala Leu Ala cys 15 10 15 ATG TCA AGC ACC CIA GTT GTG GCT CAA AAC TGT GGA TGT GCC TCT AAT 94 Met Ser Ser Thr Leu Val Val Ala Gin Asn Cys Gly Cys Ala Ser Asn 20 25 30 TTA TGT TCT AGC CGA TIT GGT TTC TGT GGC TCC ACA GAC GCC TAC TGC 142 Leu Cys Cys Ser Arg Phe Gly Hie Cys Gly Ser Thr Asp Ala Tyr Cys 35 40 45 GGC GAG GGG TGC AGA GAA GGT CCT TGT AGA TCA COG TCT AGT 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 GOG TTC TTT AAT AGG ATC ATT 238 Gly Ser Val Ser Ser Leu Val Thr Asp Ala Hie Phe Asn Arg lie lie 65 70 75 AAC CAA GCT AGC GCT AGC TGT GCT GGT AAG AGA TTC TAC ACC AGG GCT 286 Asn Gin Ala Ser Ala Ser cys Ala Gly Lys Arg Phe Tyr Thr Arg Ala 80 85 90 95 GCC TTT TTG AGT GCT CTC AGA TTT TAT CCC CAG TTT GGT AGT GGA TCC 334 Ala Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gin Phe Gly Ser Gly Ser 100 105 110 TCC GAT GTC GIT AGG GGT GAA GTT GCT GCA TTC TTT GCC CAT CTC AOC 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 lie Glu Glu He Ala Lys Ser Thr 130 135 140 TAT TCT CAG TCA AGT GCA GCA TTT CCA TGC AAC CCA ACT AAG CAA TAC 478 Tyr Cys Gin Ser Ser Ala Ala Phe Pro Cys Asn Pro Ser Lys Gin Tyr 145 150 155 TAC GGA AGG GGG OCT CTT CAG ATC ACA TGG AAT TAT AAC TAC ATA OCA 526 Tyr Gly Arg Gly Pro Leu Gin lie Thr Trp Asn Tyr Asn Tyr lie Pro 160 165 170 175 GCT GGT CGA AGC ATT GGA TTT GAT GGT CTG AAT GCA CCA GAA ACA GTT 574 Ala Gly Arg Ser lie Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val 180 185 190 GCC AAC AAT GCC CTG ACT GCA TTC OGG ACA GCC TTC TGG TTT TGG ATG 622 242 27 X IS"i 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 GGG GCC AGC 670 Asn Asn Val His Ser Val lie Val Asn Gly Gin Gly Phe Gly Ala Ser 210 215 220 AIT CGA GCT ATC AAT GGA ATC GAA TGT AAT GGT GGT AAC TCT GCT GCT 718 lie Arg Ala lie Asn Gly lie Glu cys Asn Gly Gly Asn Ser Ala Ala 225 230 235 GIT ACT GCT OGT GIT GGG TAC TAT ACT CAG TAT TGT CAA CAG CIT GGC 766 Val Ihr Ala Arg Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin Leu Gly 240 245 250 255 GIT TOG OCA GGG AAT AAC CTC OGT TGC TAGflCAAATG GCTGGITTTC 813 Val Ser Pro Gly Asn Asn Leu Arg Cys 260 CDGGTCAGAA TTCACAAGGC TTAGTCAAAA GAAAAIAAAG AGAATTA3GT AAACTGTTCA 873 TTTCTCATGT AACITGCEAC TITGGACAAG CATEAAGITG G7ITAOGAGGC TTTATCCATA 933 AAGGAATGAA AAATAITATT TAAAAAAAAA AAA 966 (2) DEFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 264 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPITON: SEQ ID NO:2: Ser Ser Phe Gly Pro lie Phe Ala lie Leu Met Ala Leu Ala cys Met 15 10 15 Ser Ser Ihr Leu Val Val Ala Gin Asn Cys Gly cys Ala Ser Asn Leu 20 25 30 cys cys Ser Arg Phe Gly Phe cys Gly Ser Ihr 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 Ihr Asp Ala Phe Phe Asn Arg lie lie Asn 65 70 75 80 Gin Ala Ser Ala Ser Cys Ala Gly Lys Arg Phe Tyr Ihr Arg Ala Ala 85 90 95 Phe Leu Ser Ala Leu Arg Phe Tyr Pro Gin Phe Gly Ser Gly Ser Ser 100 105 110 Asp Val Val Arg Arg Glu Val Ala Ala Phe Phe Ala His Val Thr His * isa 115 120 125 Glu Ihr Gly His Phe Cys Tyr lie Glu Glu lie Ala Lys Ser Thr Tyr 130 135 140 Cys Gin Ser Ser Ala Ala Phe Pro Cys Asn Pro Ser Lys Gin Tyr Tyr 145 150 155 160 Gly Arg Gly Pro Leu Gin lie Thr Trp Asn Tyr Asn Tyr lie Pro Ala 165 170 175 Gly Arg Ser lie 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 lie Val Asn Gly Gin Gly Hie Gly Ala Ser lie 210 215 220 Arg Ala lie Asn Gly lie Glu Cys Asn Gly Gly Asn Ser Ala Ala Val 225 230 235 240 Thr Ala Arg Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin Leu Gly Val 245 250 255 Ser Pro Gly Asn Asn Leu Arg Cys 260 (2) INFORMATICS P0R SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 TTIACAIAAC AAGITCACTG AAOGGAGAAG ATAACIATGC 60 ATAIATATAA CAAAGGITTC TTCOTTCAT TTTCCITGAA CAAGTCAAAC TATNTACACC 120 24 2 2 S 153 AAATCATTGT CCAAAATAAA ATTAAATGTG TTGGCTAAGT CAAATTTGAA CACTTCTGAA 180 TGTATCTAAA ATATCTCCAT TCCCATCTTA TTAATTAGAA TACAAGTAAG CAAGTAGCCA 240 AACTAGTAAA CATTTCCTCA AAGIACCACC CITATAATTT TCIATATAAA CCCATATACA 300 AGTGTCTAGT TTOCTCATCC CATACATIAT ATTGTTCGCT TTAACATACT CCAAAATGTC 360 TTCTITOGGA CCAATCHTG CCATACTCAT GGCACTTGCT TGTATGTCAA GCACCCIAGT 420 TGTGGCTCAA AACIGTGGAT GTGCCTCTAA TTTATGTTGT AGCOGATTTG GTTTCTGTGG 480 CTCCACAGAC GCCTACTGOG GOGAGGGGTG CAGAGAAGGT CCITGTAGAT CACOGTCIAG 540 TGGTGGTGGT TCCGTCT0GA GITTGGTGAC CGATGCGITC TTTAATAGGA TCATEAACCA 600 AGCIAGCGCT AGCTGTGCIG GTAAGAGAIT CEACACCAGG GCIGCTTTTT TGAGTGCTCT 660 CAGATnTAT CCCCAGTITG GTAGTGGATC C 691 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENCJIH: 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 lie Phe Ala lie Leu Met Ala Leu Ala Cys 15 10 15 Met Ser Ser Thr Leu Val Val Ala Gin 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 lie lie 65 70 75 80 Asn Gin 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 Gin Phe Gly Ser Gly Ser 100 105 110 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: 24 2 2 70 15 ^ (A) LENGTH: 1838 base pairs (B) TYPE: nucleic acid (C) STTRANDHDNESS: 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 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 ATTAA'l'l'iT'A AATTGITGGT TGAAAATGTG TTAATGCCTC 60 TIGAGTCTTG ATACAACITA AAAACGGAGC CGGITGGGAA ACATITITAC ATGATAAGTT 120 CAGITAAOSA AGAACCGTAA TGGACCCATA TACAACAAAA TATCCGTCGT TTCATl'iTCC 180 TTGAACAAGTT CAAACTAATA ACAOGAATCA TTQ3ATAAAA TGACTGGCCA AAGTCAAATG 240 TCAACTCGAN AIAAGAAAAG ACATOGAGIC AAATGTCAAC ATl'i'llAAACG TATCAAACAA 300 TAATTCCAIT CACATCCCAC TATACAACTA GCTAACTCGT AAGCTTCTTC CCTAAATCAC 360 CTATCITTCA TITTCrATAT AAACTCATCT TCAAGTGTCT AG'ITICCACA ACCCACTCAT 420 TGCITCAAAA GrrrccrcTAc TAGTCTACTA TCATTCTACr CCTCCAAAAT GTCTTCTCIT 480 GGACcrnrr TGGCTATACT TATAGCAGIT GCCDGTATGT CTAGCACCCT GGITCTGGCr 540 CAAAACIGTG GCiUiGOCIC TGGTTTATGC TGTAGCAGAT ATGGITACIG OGGCACCACA 600 GCTGCCTACT GCGGCACTGG GIGCCAGCAA GGTCCITGIT CCTCAACGCC ATCCACCCOG 660 AGTGGTGGTG TTTCGGTCCC AAGTl'lGGTG ACCGATGCAT TCl'lTAATGG AATCATTAAC 720 CAAGCAAGCI CIAGCTGTGC TGGTAAGAGC TTCTACACTA GGTCTGCTTT CTTGAGTGCT 780 CTCAGITCIT ATCCTCAGIT TGGTAGIGGA TCCTCOGATG AGGTTAAACG TGAAGTTGCT 840 GCCiTl'lTiG CTCATGCGAC GCATGAAACT GGAOGTAAGT GTTAACATTA TTAATGCCTC 900 CITTGATAGA AITGAAATOG AATAAAATCT TCTTCCCCGC TCATTTGOGC GCACITAGCT 960 AITCAGCIAA TCTTATTGIT TTATGTCAAT CATTCTGTCT TAATTATl'lT TTCTAATTGA 1020 GAATTGTGTC TAAATCTATT ATGTGGATTG CAAACCAATA ATATTGAGTG AOGTATAATG 1080 xi i fe '•< ✓ ' \ t 4 o < /,"V " V !■ . 2 3 F F B !994 ^ 242270 lS6 GTAAAAGAAA TGAGAGCAAA AGATTTGAAA TTAATTGAAA CTAGiTi'lTA GITTGCTAGT 1140 TAAAACIGAT TTAATTCATA TTATTATGIT AAGITGAATT AAGCGATACC TAAATCAAAG 1200 GGAATGCATT GAGTTACAGA AAAATATATA CTCAGCIGAT CAATTGAACT TGTGTCTTGT 1260 AGATiTTiGC TACATAGAGG AGA3TGCCAA ATCAACCTAT TGTCAGTCGA GCACAACATG 1320 GCCATGCACC ACAAATAAGC AATACTACGG ACGTGGGCCT CTCCAAATCA CATGGAACTA 1380 CAACTAOGGA CCAGCAGGTC GAAGCATTGG ATTTGAIGGT TTCAATGCAC CTGAAACAGT 1440 TGCCAATGAT GCTGTTATCG CCITTAAGAC AGCCITCDGG TiT'iGGATGA ACAATGTCCA 1500 CTCTCGAAIT GTCTCOGGCA AAGGGITTGG CTOCACCATT CXSAGCTATCA ATGGAGGTGA 1560 AIGTGGTGGC GGGAACACAC OGGCGGTCAA OGCTOGTGTT AGGtTACTATA CTCAGTATTG 1620 CAATCAGCIT GGTCTTTCAC CIGGGAAIAA CCrCTCITGC TAGTCACATA ATOGAAGTGT 1680 TTCCATGGTC ACAAITTACA AGICITAGAC TCITAGTAIA AGGAAAATAA AAATACAATC 1740 AAGGGAACTC ACTiUITlTC TTAGCCAGTA AGGGAAATAT GCATCACTTT GTAATTTAIA 1800 TATATTTCAT AGTCTTAOGG CCTATTAATA GGGATAOG 1838 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (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 He Leu lie Ala Val Ala Cys 15 10 15 Met ser Ser Thr Leu Val Val Ala Gin Asn Cys Gly Cys Ala Ser Gly 20 25 30 Leu cys Cys Ser Arg Tyr Gly Tyr Cys Gly Thr Thr Ala Ala Tyr Cys 35 40 45 f *2 3 FEB 3994:. * 2270 Ser Gly Gly Val Ser Val Pro Ser Leu Val Ihr Asp Ala Phe Phe Asn 65 70 75 80 Gly lie lie Asn Gin 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 Gin 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 lie Glu Glu lie Ala 130 135 140 Lys Ser Thr Tyr Cys Gin Ser Ser Thr Thr Trp Pro Cys Thr Thr Asn 145 150 155 160 Lys Gin Tyr Tyr Gly Arg Gly Pro Leu Gin lie Thr Trp Asn Tyr Asn 165 170 175 Tyr Gly Pro Ala Gly Arg Ser lie Gly Phe Asp Gly Leu Asn Ala Pro 180 185 190 Glu Thr Val Ala Asn Asp Ala Val lie Ala Phe Lys Thr Ala Phe Trp 195 200 205 Phe Trp Met Asn Asn Val His Ser Arg lie Val Ser Gly Lys Gly Phe 210 215 220 Gly Ser Thr lie Arg Ala lie 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 Gin Tyr Cys Asn 245 250 255 Gin 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: roonova (F) TISSUE TYPE: leaf (vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet lampda-ZAP cDNA library (B) CLONE: "SE" cDNA clone g 151 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 18..896 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AOGTACCCAA AACAAGC ATG GCA GCC AAA ATA GTG TCA GIT CIA TTC CTG Met Ala Ala Lys lie Val Ser Val Leu Phe Leu 15 10 ATT TCT CTC TTA ATC TTT GCT TCA TTC GAG TCC TCT CAT GGC TCC CAA lie Ser Leu Leu lie Hie Ala Ser Hie Glu Ser Ser His Gly Ser Gin 15 20 25 ATT GTC ATA TAC TGG GGC CAA AAT GGT GAT GAA GGA AGT CIT GCT GAC lie Val lie Tyr Trp Gly Gin Asn Gly Asp Glu Gly Ser Leu Ala Asp 30 35 40 ACT TGT AAC TCC GGA AAC TAC GGT ACC GTG ATC CIA GCT TTC GEA GCT Thr Cys Asn Ser Gly Asn Tyr Gly Thr Val lie Leu Ala Phe Val Ala 45 50 55 ACC TIT GGT AAC GGG CAA AOC COG GOG CTG AAC TTA GCT GGG CAC TGT Thr Phe Gly Asn Gly Gin Thr Pro Ala Leu Asn Leu Ala Gly His Cys 60 65 70 75 GAC CCT GCT ACA AAT TGT AAC AGT CTG AGC AGT GAC ATC AAA ACA TGC Asp Pro Ala Thr Asn Cys Asn Ser Leu Ser Ser Asp lie Lys Thr Cys 80 85 90 CAA CAG GCA GGC ATT AAG GTA CTC CTC TCT ATA GGA GCT GCT GCC GGA Gin Gin Ala Gly lie Lys Val Leu Leu Ser He Gly Gly Gly Ala Gly 95 100 105 GGC TAT TCT CIT TCC TCA ACC GAT GAT GCA AAC ACA TTT GCT GAT TAC Gly Tyr Ser Leu Ser Ser Thr Asp Asp Ala Asn Thr Phe Ala Asp Tyr 110 115 120 CTC TGG AAC ACT TAT CIT GGG GGT CAG TCC AGC ACC OGA CCC CIT GGA Leu Trp Asn Thr Tyr Leu Gly Gly Gin Ser Ser Thr Arg Pro Leu Gly 125 130 135 GAT GCA GTT TIG GAT GGT ATT GAT TTC GAT ATC GAG AGT GCT GAT GGC Asp Ala Val Leu Asp Gly lie Asp Phe Asp lie Glu Ser Gly Asp Gly 140 145 150 155 AGA TIT TGG GAT GAC CIA GCT AGA GCA TTG GCA GGT CAT AAC AAT GCT Arg Phe Trp Asp Asp Leu Ala Arg Ala Leu Ala Gly His Asn Asn Gly 160 165 170 CAG AAA ACA GIG TAC TTA TCA GCA GCT OCT CAA TGT CCC TTG CCA GAT Gin Lys Thr Val Tyr Leu Ser Ala Ala Pro Gin Cys Pro Leu Pro Asp 175 180 185 GCC AGC TTA AGC ACT GCC ATA GCC ACA GGC CIA TTC GAC TAT GTA TGG Ala Ser Leu Ser Thr Ala lie Ala Thr Gly Leu Phe Asp Tyr Val Trp 190 195 200 I 2 270 20 ISS GTT CAG TIC TAC AAT AAC CCC CCT TGT CAA TAT GAT ACC AGC GCT GAT Val Gin Phe Tyr Asn Asn Pro Pro Cys Gin Tyr Asp Ihr Ser Ala Asp 205 210 215 AAT CTC TTG AGC TOG TGG AAC CAG TGG ACC ACA GTA CAA GCT AAC CAG Asn Leu Leu Ser Ser Trp Asn Gin Trp Thr Thr Val Gin Ala Asn Gin 220 225 230 235 ATC TTC CTC GGA CIA CCA GCA TCA ACT GAT GCT GCC GGC AGT GGT TIT lie Phe Leu Gly Leu Pro Ala Ser Thr Asp Ala Ala Gly Ser Gly Phe 240 245 250 ATT CCA GCA GAT GCT CIT ACA TCT CAA GTC CIT CCC ACT ATC AAG GGT lie Pro Ala Asp Ala Leu Thr Ser Gin Val Leu Pro Thr lie Lys Gly 255 260 265 TCT GCT AAA TAT GGA GGA GTC ATG CIA TGG TCA AAG GCA TAT GAC AGT Ser Ala Lys Tyr Gly Gly Val Met Leu Trp Ser Lys Ala Tyr Asp Ser 270 275 280 GGG TAC AGC AGT GCT ATT AAA AGC AGT GTT TAATTTAAAT TACEAGTGTA Gly Tyr Ser Ser Ala lie Lys Ser Ser Val 285 290 TCCAAAGATA TAGAIACAAA ATAAG7TTATA GAGATACATC AAAAAACCAT CITAGTTTTA AATHTTTAT GCACCACAAA AGCITGTAAT ACIAATATAC TATTATCAIA AATGGCTTAT TGCCTOGCTA TMTTTGGTG ATTATEATAT ACACAGTIAC AACTTOGCAA TTATGCGACT CnTCIAAAA 674 722 770 818 866 916 976 1036 1096 1106 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 lie Val Ser Val Leu Phe Leu lie Ser Leu Leu lie 15 10 15 Phe Ala Ser Phe Glu Ser Ser His Gly Ser Gin lie Val lie Tyr Trp 20 25 30 Gly Gin Asn Gly Asp Glu Gly Ser Leu Ala Asp Thr Cys Asn Ser Gly 35 40 45 Asn Tyr Gly Thr Val lie Leu Ala Phe Val Ala Thr Phe Gly Asn Gly 50 55 60 Gin Thr Pro Ala Leu Asn Leu Ala Gly His Cys Asp Pro Ala Thr Asn 65 70 75 80 42270 M 15^ cys Asn Ser Leu Ser Ser Asp lie Lys Ihr Cys Gin Gin Ala Gly lie 85 90 95 Lys Val Leu Leu Ser lie Gly Gly Gly Ala Gly Gly Tyr Ser Leu Ser 100 105 110 Ser Thr Asp Asp Ala Asn Ihr Phe Ala Asp Tyr Leu Trp Asn Ihr Tyr 115 120 125 Leu Gly Gly Gin Ser Ser Ihr Arg Pro Leu Gly Asp Ala Val Leu Asp 130 135 140 Gly lie Asp Phe Asp lie 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 Gin Lys Thr Val Tyr 165 170 175 Leu Ser Ala Ala Pro Gin Cys Pro Leu Pro Asp Ala Ser Leu Ser Ihr 180 185 190 Ala lie Ala Ihr Gly Leu Phe Asp Tyr Val Trp Val Gin Phe Tyr Asn 195 200 205 Asn Pro Pro Cys Gin Tyr Asp Ihr Ser Ala Asp Asn Leu Leu Ser Ser 210 215 220 Trp Asn Gin Trp Ihr Ihr Val Gin Ala Asn Gin lie Phe Leu Gly Leu 225 230 235 240 Pro Ala Ser Ihr Asp Ala Ala Gly Ser Gly Phe lie Pro Ala Asp Ala 245 250 255 Leu Ihr Ser Gin Val Leu Pro Ihr lie 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 lie Lys Ser Ser Val 290 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 1249 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE IYPE: 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 2 54 102 150 198 246 294 342 390 438 486 534 582 630 24 18" I toO (B) CLONE: beta-1,3-glucanase cDNA clone (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 34.. 1041 (Xi) SEQUENCE DESCRIPTION: SE1Q ID NO: 9: AATnTGTTT ATTCITAGAG TTATTTCITC ACA ATG AGG CIA ATT AGC ACA ACT Met Arg Leu lie Ser Thr Thr 1 5 TCT GCA GTT GCT ACT TTG CTG TIT CIT GTA GTA ATT CIA OCT AGT AIT Ser Ala Val Ala Thr Leu Leu Phe Leu Val Val lie Leu Pro Ser lie 10 15 20 CAA CIG ACA GAG GCA CAA ATT GGC GTA TGT AAC GGG AGA CEA GGC AAC Gin Leu Thr Glu Ala Gin lie Gly Val Cys Asn Gly Arg Leu Gly Asn 25 30 35 AAC TTA CCT TCC GAG GAA GAT GIT GIA AGC TTG TAC AAG TOG AGG GGA 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 OCT AAC CAA CGG AOC CTC CAA GOG lie Ihr Arg Met Arg lie Tyr Asp Pro Asn Gin Arg Thr Leu Gin Ala 60 65 70 GTT AGA GGA TOG AAT ATA GGG CTA ATC GTC GAT GTC CCT AAG OGT GAC Val Arg Gly Ser Asn lie Gly Leu lie Val Asp Val Pro Lys Arg Asp 75 80 85 CIA AGG TCA CTC GGC TCC GAT GCT GGG GCT GOG TCT OGT TGG GTC CAA Leu Arg Ser Leu Gly Ser Asp Ala Gly Ala Ala Ser Arg Trp Val Gin 90 95 100 AAC AAT GTA GTC CCT TAC GOG TCT AAT ATT OGA TAC ATA GCA GTT GGT Asn Asn Val Val Pro Tyr Ala Ser Asn lie Arg Tyr lie Ala Val Gly 105 110 115 AAT GAA ATA ATG CCT AAT GAT GCC GAG GCA GGG TCA AIT GTC COG GCC Asn Glu lie Met Pro Asn Asp Ala Glu Ala Gly Ser lie Val Pro Ala 120 125 130 135 ATG CAA AAT GTC CAA AAT GCC CIT CGA TCA GCT AAT TTA GCT GGT AGA Met Gin Asn Val Gin Asn Ala Leu Arg Ser Ala Asn Leu Ala Gly Arg 140 145 150 ATT AAA GTC TCT ACC GOG ATA AAA AGT GAC CTC GIT GCT AAC TTC CCT lie Lys Val Ser Thr Ala lie Lys Ser Asp Leu Val Ala Asn Phe Pro 155 160 165 CCC TCT AAA GGT GIT TIT ACT TCT TCA TCA TAC ATG AAT CCA AIT GTT Pro Ser Lys Gly Val Phe Thr Ser Ser Ser Tyr Met Asn Pro lie Val 170 175 180 AAC TTC CIT AAA AAT AAC AAT TCA OCT TIG TTA GCC AAC AIT TAC OCT Asn Phe Leu Lys Asn Asn Asn Ser Pro Leu Leu Ala Asn lie Tyr Pro ?4 2 2 M lb V 185 190 195 TAC TTT TCT TTC AIT GGC ACC CCA AGT ATG OGT CIA GAT TAT GCA CTC 678 Tyr Phe Ser Phe lie Gly Thr Pro Ser Met Arg Leu Asp Tyr Ala Leu 200 205 210 215 TTT ACT TCA CCT AAT GCC CAA GIT AAT GAT AAT GCT TTA CAA TAC CAA 726 Phe Thr Ser Pro Asn Ala Gin Val Asn Asp Asn Gly Leu Gin Tyr Gin 220 225 230 AAT GTC TTT GAT GCT TEA GTA GAC ACT GTG TAT GOG GCC TEA GOG 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 COG ATT GTT GTG TCC GAG ACT GGG TGG CCT 822 Ala Gly Ala Pro Asn Val Pro lie Val Val Ser Glu Ser Gly Trp Pro 250 255 260 TOG GCT GGT GGT 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 AIT GGT CAT GTA AAG CAA GGA ACT CCC CTG AAG AAA GGA 918 Lys Gly Leu lie Gly His Val Lys Gin Gly Thr Pro Leu Lys Lys Gly 280 285 290 295 CAA GCA ATT GAG GCA TAT TTG TTT GCT ATG TIT GAT GAG AAC CAA AAG 966 Gin Ala lie Glu Ala Tyr Leu Phe Ala Met Hie Asp Glu Asn Gin Lys 300 305 310 GGT GGA GGT ATT GAG AAC AAT TTT GGA CTG TTT ACT CCC AAT AAA CAG 1014 Gly Gly Gly lie Glu Asn Asn Phe Gly Leu Phe Thr Pro Asn Lys Gin 315 320 325 CCA AAA TAC CAA CTC AAT TTC AAT AAT TGAAAGTACT TEAATTGCCT 1061 Pro Lys Tyr Gin Leu Asn Hie Asn Asn 330 335 AGTATATAIA TATATATGCI AATATGTTGT ATGTAGITAT GTCATCTACA TATATAAIAA 1121 GTGAAATCAA ACACCOGATC ATAGACIAAA ATTCTAATAA AAGATCCTCC TGTTGTAATA 1181 TTATCCIAGC TGCAATAATA TTTACTCITA TAIAGAGATC TTGIGAAAAA AAAAAAAAAA 1241 AAAAAAAA 1249 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 336 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Arg Leu lie Ser Thr Thr Ser Ala Val Ala Thr Leu Leu Phe Leu ?4 10 15 Val Val lie Leu Pro Ser lie Gin Leu Ihr Glu Ala Gin lie 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 lie Ihr Arg Met Arg lie Tyr Asp Pro 50 55 60 Asn Gin Arg Thr Leu Gin Ala Val Arg Gly Ser Asn lie Gly Leu lie 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 Gin Asn Asn Val Val Pro Tyr Ala Ser Asn 100 105 110 lie Arg Tyr lie Ala Val Gly Asn Glu lie Met Pro Asn Asp Ala Glu 115 120 125 Ala Gly Ser lie Val Pro Ala Met Gin Asn Val Gin Asn Ala Leu Arg 130 135 140 Ser Ala Asn Leu Ala Gly Arg lie Lys Val Ser Ihr Ala lie Lys Ser 145 150 155 160 Asp Leu Val Ala Asn Hie Pro Pro Ser Lys Gly Val Phe Thr Ser Ser 165 170 175 Ser Tyr Met Asn Pro lie Val Asn Phe Leu Lys Asn Asn Asn Ser Pro 180 185 190 Leu Leu Ala Asn lie Tyr Pro Tyr Phe Ser Phe lie Gly Thr Pro Ser 195 200 205 Ifet Arg Leu Asp Tyr Ala Leu Phe Thr Ser Pro Asn Ala Gin Val Asn 210 215 220 Asp Asn Gly Leu Gin Tyr Gin 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 lie 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 lie Gly His Val Lys Gin 275 280 285 Gly Thr Pro Leu Lys Lys Gly Gin Ala lie Glu Ala Tyr Leu Phe Ala 290 295 300 Met Phe Asp Glu Asn Gin Lys Gly Gly Gly lie Glu Asn Asn Phe Gly 305 310 315 320 24 2 27 US ibS Leu Phe Ihr Pro Asn Lys Gin Pro Lys Tyr Gin Leu Asn Phe Asn Asn 325 330 335 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: Monova (F) TISSUE TYPE: leaf (Vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet EMBL3 genomic library (B) CLONE: genomic chitinase 1 clone (ix) FEAIURE: (A) NAME/KEY: unsure (B) LOCATION: 3214..4227 (D) OTHER INFORMATION: /note= 1 'Approximately 1000 base pairs" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join(1428..2169 , 4619..4775, 5407..5824) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TCTAGAGAGA GAAAAAACAA CGCCATGDGA CGGITGGGGT GGGACAAGTT OGACCCGCIA 60 AIAITAATGG GOGGATATGG ACAACTCITG AACCCCTCCC TGGCACCEAG AGGIGGGTAT 120 GGGCCGGCCC GGCATGATTT GGGACOGCTC GAGGCCOGAC COGTIGTCOG CEACGIGGCC 180 GCGGGTCTGT CATGGTCAAG AAAATTTATT GAAACTAAAT ATEAAA3TTA ITTAACCGGT 240 AATIAGTEAA CCTGATCATT 'iTi'iOCAAAA TACCICAAAA TTATIGAAAC TAAAIAITAA 300 ATTTAAATTG AGAATGnTT TGTCAAGAGA ATCATAGITA AAAGGAAAAT TIGGCAAAAA 360 Ai'l'lTlTl'lT AOGAGTTCAT 'lTiTGlGAAA AAAAACCTTA TAAAGCATTT T37IGOGAAAA 420 CAAACAAAAA TCAAA'H'HT TTTGGGAAAA CAAACCAAAA TCAAAATIAT TTTTGCAAGA 480 TGAAACCTAA CCACCACTAC CATGCATCAA TCCCCAACCC CTCOCTOCAC CCCCTOGGGG 540 CACCCACACC COCCTTCGCC CCTACCCIAT CTCCACTCTA TRJETCCCTC CTTCACGCCT 600 CCICTCTCTC CTCTCCCTTC GTCCAACCCC CCCCCCCCCC AACCGTTGIG CCACTCAGAA 660 ig Vb Mr OGCACCCCAC OGCIGCGCCA CTCAAAACCC CCAACOGTTC OGCCACCCAG AAACAATCGT 720 TITOGTrCIT OGATmTGA TAGTITCOC?r TTCATTTGTG GIGGIAGTGG ATAGIGGGCC 780 GTCTTTOCTG CTGGTGGTGG GGGTITCATG GTGTEATGGT CATGTGATGG TGGGGGTATC 840 ATGTTGTIAT GGTCATCTGA TCGTGGGITG CGGAGGGGAA GCTGOGGAGG GGGIGGGGTG 900 GACTGTGGTT GGGGGGGTGG ACAAAAGGAG AGGAGAGGGG GGAGGGAGCT AGGGAAGAAG 960 GGAGAGGAGA GAGAGTAGGG GTGAAGGGGG GIGGGGGACT ACOGCCTGAG AAGG1CTAGG 1020 CAGGGGTTGG GGTIGCTGGT GGGGGTGGGT GGGGGTTGGG GGCAAGTGTT GGTAGIGGDG 1080 GCTAGAGGIG GICAATGCOG GAAATTCEAT GGTCAAOGCC AGAAAOGACA CTAGATTIGT 1140 TITOGAAAAA AAA3TTGATC TTGATTTGTT TITACAAAAA ATAATTEATA AGAITl'l'lTi' 1200 OGCAAAAGTG AACTOGTAAA AGTl'lTiTlA AAACAAA3TT TCCIAGTTAA AATGATTCTT 1260 TTCACmrr ACTATGTTAC ATACATAAAT TTTGATAGAT ATCICrCXTA AATAAGCTTG 1320 TCATTICrOC CITATCCCAA AGACOCAAAG CCTCEAIAAA TTGGCEACAA ACTCTCCTCA 1380 CAGTCACACA TGACACAAOG CAAGITIGAA AGGAAAAACA AGAGGTGATG AAAATAAAAA 1440 CCICTOCEAG TnTCTACTA GGATEAATAT (JTTEAGCICT AGTCCTCCTA CTAGGAGAAG 1500 GTGTACAATG TGGGOGGCAA TGCAACACAA COGATACTAA TTCTCTITCC GGTTGCTCAG 1560 TCGG033CCC ATCAOGTCCG ACACCICCTC GTCCTCCCAC CCGGAGACCG CXACXJTCCTC 1620 GTCCTCCCAC CCCGAGACOG CCACCTCCTC GTCCTCCTAC OOOGAGACCA CCA.CCACCTA 1680 CACCAAGACC ACCACCTCCT aJTCCICCIA CCCOGAGACC ACCACCACCT CCTACACCAA 1740 GACCACCACC TCCTOGTCCT CCTACCCCAA GACCGCCACC ACCTCCTACA CCAAGACCAC 1800 CACCTCCTCC TACAOCAAGA CCACCACCTC CTAGTCCTCC TACCCCIAGA CCACCACCAC 1860 CACCACCTCC TAGTCCTCCT ACCCCAAGCC CACCATCTCC TCCTAGCCCT GAACGACCAA 1920 CTCCGCCCGA ACCEACGCCA CCAACTCCIA CACCACCAAC TCAIdTACT GACATAATCT 1980 CIGAAGAAAT GTITAATGAA TTOCTCITGA ACOGCATTCA GCCAOGTTGT CCTGGfTAGAT 2040 GGTTCTACAC TIACCAGGCT TTCATTACTG CAGCTGAAAC CITCCCTGAG TTTGGTAATA 2100 CTGGGAATGA TGAAATTAGA AAGAGAGAAA TTGCIGCnT CITTGGACAG AOCTCICAIG 2160 AAACCTCTGG TICATTCITC TACTTCTCAT TCTTCTITAC TTOGGATCTG CTTCACITTA 2220 CTAACATGCA TGTITJLCATA CTATATaTTA TATTTAIAGA TGAACAAGTA GTAOGITATT 2280 ATTTGCTAGT GCCTACTTCC TAGIGITTAT TCTTTGTCAA TCTAATGATC TTTATAGTIT 2340 ATCITGGACA CAATAAATTA TATAATCTTA AOGTEAAGIT TAGACGATAT GAATTATTTC 2400 ■"*2270 TITTTCACGT ATCTACCCTA GITATTATAG GTTGTTATTA CCAGTTITET TACITCTACr 2460 ATnTGACTT TTGACCATAT GGATTCTITA TGCATAAGAC ATATATAATA TATAAGATCT 2520 TGTTGTATEA TTTACITOGA TATATATATT TICTTGGATT ATGCTCCAAA ATTAGrTCAAG 2580 TITTCCTAAT AGITATGATT AGTTGCIA3T AATTAGITAG TGGGTTAACA AGTAGTGGTC 2640 TAGATAATGG TCTAGCTAGT GGGATAGCAA GTAAGTAGTA GGCTACATAG TGGACATGTT 2700 GTEAGTAGTG CnTUTATGC CTATITAAAG ATGGITITGT TTATTCATAA TGTGTACATG 2760 AAAAATATAT AAAAAATGTA GTCnTCTAA TCTCTTCAAG TTCTCTTOCr CCTCTAAAAA 2820 TTCTACATGG TATCAGAGCT CCAGGTTAGA TCOGGGAAAG GGAAAGAGAT GAAGCAAAAA 2880 AAAGAAAAGA AAAAAAAGAG AGAGATAGAG AAAGAAAGAG AGTA1TAAAA ACAAAATTGA 2940 GTAAAAAGAA ACAAGCAATG CAGCTTGATT TCATTGTTTG TGAGCAATTT TTTGTITGAG 3000 TGAAA1T1T1' TGTGTTEAAC CATGAAACTA ATGGTTGGGT GTGATGrlGIT GTTGGGCICC 3060 CnTTAGCCC ATGAAAATGG ATITEAATCC TCTGGAAAAA AGGGCATAAA AAATTGflTIG 3120 AGTTGAAGAT CGAGTAATCT TTAOGAGTGT GTGGAAGTOG TACICAAOGC AGAGAOGAIT 3180 TCCAAAGTIG 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 NNNNNNNNNN 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 242 27 3#lbfe> nnnnnnnnnn nnnnnnnnnn nnnnnnntct agattgacit tgtaagttaa acitaccaaa 4260 tcitgaiata tactaattta caaagtatat tataagctaa gtcgtgatgt ctccctccat 4320 crrrrrrrm ctacttgtta oottttcit aaatgaatgt ctcattitac acactacatt 4380 GCTTEA3TIG CATTGGTAAC TATGACCCAT CAAAAAATAA CAATATTCCC dttai'l'i'it 4440 CTCATCITCr TAACITEAAC TCTCAAATAT tigttatttt CAACAAAGTT AACTCTCAAA 4500 TATTTGTCAT AATAATGIGA AATCTTOGGT TGATAAAGTG TCCAAAACAA AATGGTGTCA 4560 TTTAAAAAGA AAAATGAGAG GACATATATA CTAATGGATT TITGAATTGA CAAIATAGGA 4620 GAACOSAOOG CACAACATGG AOCATITAGA TGGGGGEATT GTTTCATAGA agaaatigga 4680 gcoggccctc TCAGCCAAIA TTGTGCAOOC TCTGTAGAAT GGCCITGCAT TOCTGGGAGG 4740 THTACIATG GTOGTCGAOC AGTCCAACTT ACCIGGTAAG TACTCCCTCC GTITCCAAAAT 4800 ATAG7ITCICA TTnCCITlT TTCACAGEAA TTEA1GCAAG TAGAA1ATAA GAGGGTAGGT 4860 AAAGAITnT THTIATTTA AATAAATGTT GTATGGGAAA AGATGAnTT aggagagaga 4920 GTGGAGAATA ATEAGTGAAA GAGCATEAAT TCTAACAITE TGGTEGAATA AATAAAGGAA 4980 AAAACAAATT CAAGAAGCEA AAGTAATGAG GGCACAGGIT TECEAGACAA ATEAOGGAAA 5040 AATGTGGAAC TAAATATGAA AATGGGAACT ATAITITGAG ACACNCAAAA TAAAAATGGG 5100 AACTATATTT TGGGAOGGAG GGACTATEAT TATATTAGCT TACICCTATT ACITGCATOG 5160 CATGTCCAAA TTITEATTGT TCATAGAAAA GTCATTITCA AGAAITTTGC TATTOGAOGT 5220 CTIAAAATIT TEEACTACGC TEECEAATEA CATATTTTTA TAGTGTACIT AITITATACC 5280 TITCCATITC TTCrCi'ITlT (XTKXTITC CITCACITAA gtl'lTAACTT GATACATATA 5340 GCIAGCAAAA TTATCITAGG TATTTTAGCT AATITAAAAT TETEGGEAAT GATAAATAAT 5400 TTGCAGGAAT TTCAACIATG GAAAGCAGGT GAAGCACITA GGTTTGGAGC TCCEATTCAA 5460 CCCAGACATA CTAGCACATG ACCCAGEEAT TECEEEOGAA ACTGCAATTT GGTTTTGGAT 5520 GACTCCTGAA GGAAACAAGC dTCITCCCA TGAAGTCATA ACTGGGCAAT GGACACCAAC 5580 TOCTGCAGAC ATAGCTOGCA ACAGATTGOC TGGATATGGT TEAATCACAA ATATTITEAA 5640 TGGTGCTEEA GAATGOGGCA CTCATGGACC AGATAAIAGA GGGGAAAATC GAATTCAGIT 5700 TTACCAGAGA TACIGEGATC TECEAGA1GT TAGCEATGGA GATAACCITG ATTGCEAOOG 5760 TCAAACTCCC TTEGATTGGG GTCTEAAAAA ACTTCAGGGA GCEAGAGAAT CATGGECX3TC 5820 GAGCEAAAAT TATAOGCATG CATGTAGTCT CTAAGTCCAT ACATEATIGT CITCATGOGT 5880 GEATGAIAJT GAGTAAGTTG GTATGTTCAA AAATATG7TGG TGTCEGAAAA TATGCAAACA 5940 # 2 270 GMCCAGCAA TAAGTAATAA GCAAGGITTA CTTGCACCAA ATCTGGATCT AAirGTICTA TGTTOCTATT GTATGCTAAT GAATAAAGTT TGICTIGTAT TGCACCITAT TGATATTAAT TTITCATATT CAGGOGTITA CAATCATAAG GGACCATTGA. ACATGCAGIT GAGTEAGfTCT TIAATATGGT GTTCAAGAAG ATAGAAATGA GGAATGAAOG TACTCTATAT TATAAGAGAC TACIAGIGTT TGCEATDCTT ACACCIAAAA AAGCTCTATG AGATTACATT TACATTATGG TTAATGTCIA COG (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 CTTCIAGTGA TTGCATIATC GATTACICTA AGTATGGAAA GTITAGTCAG TCAAAAGGTC 6000 6060 6120 6180 6240 6300 6313 m (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Lys lie Lys Thr Ser Pro Ser Phe Leu Leu Gly Leu lie Cys Leu 15 10 15 Ala Leu Val Leu Leu Leu Gly Glu Gly Val Gin Cys Gly Arg Gin 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 "Ihr 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 \bZ Pro Pro Pro Pro Ser Pro Pro Thr Pro Ser Pro Pro Ser Pro Pro Ser 145 150 155 160 Pro Glu Pro Pro Ihr Pro Pro Glu Pro Ihr Pro Pro Ihr Pro Ihr Pro 165 170 175 Pro Ihr His Leu Ihr Asp lie lie Ser Glu Glu Met Phe Asn Glu Phe 180 185 190 Leu Leu Asn Arg lie Gin Pro Arg Cys Pro Gly Arg Itp Phe Tyr Ihr 195 200 205 Tyr Gin Ala Phe lie Ihr Ala Ala Glu Ihr Phe Pro Glu Phe Gly Asn 210 215 220 Ihr Gly Asn Asp Glu lie Arg Lys Arg Glu lie Ala Ala Phe Phe Gly 225 230 235 240 Gin Ihr Ser His Glu Ihr Ser Gly Glu Pro Ihr Ala Gin His Gly Pro 245 250 255 Phe Ihr Trp Gly Tyr cys Phe lie Glu Glu lie Gly Ala Gly Pro Leu 260 265 270 Ser Gin Tyr cys Ala Pro Ser Val Glu Trp Pro Cys lie Arg Gly Arg 275 280 285 Phe Tyr Tyr Gly Arg Gly Pro Val Gin Leu Ihr Trp Asn Phe Asn Tyr 290 295 300 Gly Lys Gin Val Lys His Leu Gly Leu Asp Leu Leu Phe Asn Pro Asp 305 310 315 320 lie Val Ala His Asp Pro Val lie Ser Phe Glu Ihr Ala lie Trp Phe 325 330 335 Trp Met Ihr Pro Glu Gly Asn Lys Pro Ser Ser His Glu Val lie Ihr 340 345 350 Gly Gin Trp Ihr Pro Ihr Pro Ala Asp lie Ala Arg Asn Arg Leu Pro 355 360 365 Gly Tyr Gly Leu lie Ihr Asn lie Phe Asn Gly Ala Leu Glu Cys Gly 370 375 380 Ihr His Gly Pro Asp Asn Arg Gly Glu Asn Arg lie Gin Phe Tyr Gin 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 Gin Ihr Pro Phe Asp Trp Gly Leu Lys Lys Leu Gin Gly Ala 420 425 430 Arg Glu Ser Trp Ser Ser Ser 435 (2) INFORMATION FOR SEQ ID NO: 13: 24227 lb^ (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 23 amino acids (B) TYPE: amino acid (D) TOPODDGY: 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 Gin Thr Pro Phe Gly Asn Ser Leu Leu Leu 15 10 15 Ser Asp Leu Val Thr Ser Gin 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 ID NO: 14: Asn Leu Asp Cys Gly Asn Gin Arg Ser Phe Gly Asn Gly Leu Leu Val 15 10 15 Asp Thr Met (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 32 l"70 (iii) HYPOTHETICAL: 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 Gin Arg Asn Cys Phe Ala Gly 15 10 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin Arg Pro Phe Ala 15 10 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: 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 15 10 15 Ala Ser Leu Val Ser Glu Met 242 270 sa n i 20 (2) INFORMATICS FOR SBQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 16 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: 18: Ser Thr Tyr Cys Gin Ser Tyr Ala Ala Phe Pro Pro Asn Pro Ser Lys 15 10 15 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPODDGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Ala cys Val Thr His Glu Thr Gly His Hie Cys Tyr lie Glu Glu lie 15 10 15 Ala Lys (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris 242270 na (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin 15 10 (2) INFORMATION FOR SBQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin lie Thr Trp 1 5 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 lie Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val Ala Asn Asn 15 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 242270 28 1"13 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (Vi) ORIGINAL SOURCE: (A) ORGANISM: Triticum aestivum (Xi) SEQUENCE DESCRIPTION: SBQ ID NO: 23: Gin Arg Cys Gly Glu Gin Gly Ser Asn Met Glu Cys Pro Asn Asn Leu 15 10 15 Cys Cys Ser Gin Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr cys Gly 20 25 30 Lys Gly Cys Gin Asn Gly Ala Cys Trp Thr Ser 35 40 (2) INFORMATION FOR SBQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: Hevea brasiliensis (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Glu Gin cys Gly Arg Gin Ala Gly Gly Lys Leu cys Pro Asn Asn Leu 15 10 15 Cys Cys Ser Gin Trp Gly Trp Cys Gly Ser Thr Asp Glu Tyr Cys Ser 20 25 30 Pro Asp His Asn Cys Gin Ser Asn Cys Lys Asp 35 40 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 41 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ae im- (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Leu Cys Pro Gly Gly Asn 15 10 15 cys Cys Ser Gin Phe Gly Tcp Cys Gly Ser Thr Ihr Asp Tyr Cys Gly 20 25 30 Pro Gly Cys Gin Ser Gin Cys Gly Gly 35 40 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin Cys Gly Ser Gin Ala Gly Gly Ala Arg Cys Ala Ser Gly Leu 15 10 15 Cys Cys Ser Lys Hie Gly Trp Cys Gly Asn Thr Asn Glu Tyr Cys Gly 20 25 30 Pro Asp Asn Cys Gin Ser Gin Cys Pro Gly 35 40 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO 24 2 2 70 19 115 (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (xi) SEQUENCE DESCRIPTION: SBQ ID NO: 27: Glu Leu Cys Gly Asn Gin Ala Gly Gly Ala Leu Cys Pro Asn Gly Leu 15 10 15 Cys Cys Ser Gin Tyr Gly Trp Cys Gly Asn Ihr Asn Pro Tyr Cys Gly 20 25 30 Asn (2) INFORMATION FOR SBQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) T0P0IT3GY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: priner (KB-7), constructed from beta vulgaris (Xi) SEQUENCE DESCRIPTION: SBQ ID NO: 28: GACICIAGAA AYOCRCCRYG YCAKIAYGAY AC 32 (2) INFORMATION FOR SBQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: 2422 SB \~H& GGAGGATCCC ARRCNAAYCA RATHTT 26 (2) INFORMATION FOR SBQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: CCAAGCITGA ATTC.T1THT TTTTTTTTTT TTTT 34 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 lie Thr Thr Thr Leu Ser lie Phe Phe Leu Leu 15 10 15 Ser Ser lie Phe Arg Ser Ser Asp Ala Ala Gly lie Ala lie Tyr Trp 20 25 30 Gly Gin Asn Gly Asn Glu Gly Ser Leu Ala Ser Thr Cys Ala Thr Gly 35 40 45 Asn Tyr Glu Phe Val Asn lie Ala Phe Leu Ser Ser Phe Gly Ser Gly 50 55 60 Gin 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 lie Asn Ser cys Lys Ser Gin Asn 85 90 95 242270 5% til Val Lys Val Leu Leu Ser lie Gly Gly Gly Ala Gly Ser Tyr Ser Leu 100 105 110 Ser Ser Ala Asp Asp Ala Lys Gin Val Ala Asn Phe lie Trp Asn Ser 115 120 125 Tyr Leu Gly Gly Gin Ser Asp Ser Arg Pro Leu Gly Ala Ala Val Leu 130 135 140 Asp Gly Val Asp Phe Asp lie Glu Ser Gly Ser Gly Gin Phe Trp Asp 145 150 155 160 Val Leu Ala Gin Glu Leu Lys Asn Phe Gly Gin Val lie Leu Ser Ala 165 170 175 Ala Pro Gin Cys Pro lie Pro Asp Ala His Leu Asp Ala Ala lie Lys 180 185 190 Ihr Gly Leu Phe Asp Ser Val Trp Val Gin Phe Tyr Asn Asn Pro Pro 195 200 205 cys ifet Phe Ala Asp Asn Ala Asp Asn Leu Leu Ser Ser Trp Asn Gin 210 215 220 Trp Thr Ala Phe Pro Ihr Ser Lys Leu Tyr Met Gly Leu Pro Ala Ala 225 230 235 240 Arg Glu Ala Ala Pro Ser Gly Gly Phe lie Pro Ala Asp Val Leu lie 245 250 255 Ser Gin Val Leu Pro Ihr lie 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 lie Lys 275 280 285 Gly Ser lie Gly 290 (2) INFORMATION FOR SBQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 302 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: Arabidopsis thaliana (xi) Met SEQUENCE DESCRIPTION: SBQ ID NO: 32: Thr Asn Met Thr Leu Arg Lys His Val lie Tyr Phe Leu Phe Phe 242270 yet ns i 5 10 15 lie Ser Cys Ser Leu Ser Lys Pro Ser Asp Ala Ser Arg Gly Gly lie 20 25 30 Ala lie Tyr Trp Gly Gin 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 Gin 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 Gin Val Lys Asp Cys 85 90 95 Gin Ser Arg Gly lie Lys Val Met Leu Ser Leu Gly Gly Gly lie Gly 100 105 110 Asn Tyr Ser lie Gly Ser Arg Glu Asp Ala Lys Val lie 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 lie Asp Hie Asn lie Glu Leu Gly Ser Pro 145 150 155 160 Gin His Trp Asp Asp Leu Ala Arg Thr Leu Ser Lys Phe Ser His Arg 165 170 175 Gly Arg Lys lie Tyr Leu Thr G]y Ala Pro Gin 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 lie Gin Phe Tyr Asn Asn Pro Pro Cys Ser Tyr Ser Ser Gly Asn Thr 210 215 220 Gin Asn Leu Phe Asp Ser Trp Asn Lys Trp Thr Thr Ser lie Ala Ala 225 230 235 240 Gin Lys Phe Hie Leu Gly Leu Pro Ala Ala Pro Glu Ala Ala Asp Ser 245 250 255 Gly Tyr lie Pro Pro Asp Val Leu Thr Ser Gin lie 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 lie Leu Ala Ser Val 290 295 300 (2) INFORMATION FOR SBQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: 2 2 70 S3 I'M (A) LENGIH: 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 Gin Asn Asn Val Val Pro Tyr 1 5 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 lie Val Val Ser Glu Ser Gly Trp Pro 15 10 15 Ser Ala Gly Gly 20 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin Gly Lys Val Ser 1 5 2422 3* I 'SO (2) INFORMATION FOR SBQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 frcm beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TGGGINCARA AYAAYGT 17 (2) INFORMATION FOR SBQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 frcm beta vulgaris (Xi) SEQUENCE DESCRIPTION: SBQ ID NO: 37: AAYGARATHA TGOCNAA 17 (2) INFORMATION FOR SEQ ID NO: 38: (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-3), constructed frcm N. tabacum and H. vulgare (xi) SEQUENCE DESCRIPTION: SBQ ID NO: 38: 3£ Itfl TCRTYRAACA TNGCRAA (2) INFORMATICS FOR SBQ 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: SBQ ID NO: 39: Phe Ala Met Phe Asp Glu 1 5 (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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: m 242270 I S3 (A) ORGANISM: Pisum sativum (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Thr Cys Pro Asn Asn Leu 15 10 15 Cys cys Ser Gin Tyr Gly Tyr 20 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin Cys Gly Asn Gin Ala Gly Gly Xaa Val Pro Pro Asn Gly 15 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 Gin cys Gly Thr Gin Ala Gly Gly Ala Leu cys Pro Gly Gly Leu 15 10 15 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: 242 27 3S" I S3 (A) LENGIH: 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 Gin Xaa Gly Ser Gin Ala Gly Gly Ala Ihr Cys Pro Asn Xaa Leu 15 10 15 Cys Cys Ser Arg Phe Gly 20 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 23 amino acids (B) TYPE: amino acid (D) T0P0L3GY: 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 Gin Gin Gly Ser Gin Ala Gly Gly Ala Ihr Cys Pro Asn Xaa Leu 15 10 15 Cys Cys Ser Xaa Phe Gly Trp 20 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: -142270 X «SM- (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Ala lie Gly Val Asp Leu Leu Asn Asn Pro Asp Leu Val Ala Ihr Asp 15 10 15 Pro Val (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 lie Gin lie Ser His 1 5 (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 Gin Ser Pro 15 10 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 2422 3? >2$ (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (Vi) ORIGINAL SOURCE: (A) ORGANISM: primer (KB-3), constructed from beta vulgaris (xi) SEQUENCE DESCRIPTION: SBQ ID NO: 49: CCGAAGCITA GATCEAAACA ACAACATGTC TTCIYTYGGA CC 42 (2) INFORMATION FOR SBQ 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 21 (2) INFORMATION FOR SBQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 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 frcm beta vulgaris, chitinase 4 (xi) SEQUENCE DESCRIPTION: SBQ ID NO:51: CATOGGAGGA TCCACTACC 19 </div>

Claims

1. 24 2 2 70 I Sb WHAT WE CLAIM IS:- 1 • An isolated DNA sequence comprising the sugar beet chitinase 4 DNA. sequence shown in SEQ ID NO: .1 or an analoque thereof, the analogue being a DNA sequence encoding a polypeptide'having che antifungal activity of 5 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 10 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 che amino acid sequence of the 15 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 N0:.l and encoding 20 the hevein domain and the functional domain of che 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:.l encoding the functional domain of the sugar beet chitinase 4 enzyme, or an 25 analogue of said DNA sequence.

4. An isolated DNA sequence comprising a sugar beet chitinase 4 gene.

5. A DNA sequence encoding a chitinase isoenzyme which is at lease 601 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 30 sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO:.11. V.V£ * ;'"V V 329746CL001/MKA/SPK/A36/1992 04 02 ^,Prft . '0! V * / fc nr - 24227 feso l *§"7

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 5 sequence SEQ ID N0:.l 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. 10 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 15 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 20 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 25 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 829746CL.001/MKA/SPK/A36/1992 04 02 242270 fe&J. 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 5 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 N0:.l 10 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 15 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 N0:.l encoding the leader peptide of chitinase 4 or an analogue thereof in 20 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 25 extracellular space.

13. A subsequence of the chitinase 4 DNA sequence SEQ ID N0:.l encoding one or more of the following epitopes of the sugar beet chitinase 4 enzyme Peptide 1: 30 Peptide 2: Peptide 3: Peptide 4: AGKRFYTRA CNPSKQYY IEGNGGNS TARVGYYTQYCQ 829746CL.001 /MKA/SPK/A36/1992 04 02 189

14. - An isolated DNA sequence according to any of the preceding claims which is of plant origin.

15. An isolated DNA sequence according to claim 14 which is derived frcm a member of the family Chenopodiaceae, Solanaceae, Apiaceae, 5 Brassicaceae, Cucurbitaceae or Fabaceae.

16. An isolated 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

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 15 2) an isolated 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 20 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 N0:.l 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 25 chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having /9- a 1,3-glucanase activity, , \ t. . / 10 pea. 829746CL001/MKA/SPK/A36/1992 04 02 •'24MAR!9W-£.i 242270 •bay 1^0 each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.

20. A genetic construct according to claim 19 in which the DNA 5 sequence encoding the second chitinase encodes an acidic chitinase having a pi equal to or less than 4.0 and preferably being capable of cleaving ^H-chitin into mainly chito hexamers, and/or the DNA sequence encoding the ,5-1 glucanase having a pi of at least 10 cleaving ^H-laminarin into mainly , 3-glucanase encodes a basic >5-1,3-9.0 and preferably being capable of dimers of /3-1,3-glucans.

21. A genetic construct according to any of claims 19 or 20, in which the second chitinase and the £-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 15 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 •'H-chitin into mainly hexamers, and/or the DNA sequence shown in SEQ ID NO:.9 encoding a basic /S-1,3- 20 glucanase is the DNA sequence encoding the basic sugar beet /9-l,3- glucanase 4 having the amino acid sequence shown in SEQ ID NO:.10 or an analogue thereof encoding a basic /?-l,3-glucanase having a pi of at least 9.0 and being capable of hydrolysing -^H-laminarin into mainly dimers of p-1,3-glucans. 25

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. 829746CL.001/MKA/SPK/A36/1992 04 02 24 2 2 70 # 191

25. A genetic construct according to claim 24, in which the promoter is selected from che group consisting of the sugar beet Acetohydroxyacid synthase promoter (AHAS), the sugar beet chitinase L promoter, the sequence of which appears from SEQ ID NO:. 1.1

26. 5 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 che sugar beet chitinase 1, the sequence of which appears from SEQ ID NO:.11, che sugar beet chitinase 4, che sequence of which appears from SEQ ID NO:,l, the sugar beet /3-l,3-10 glucanase, the sequence of which appears from SEQ ID NO:.9, the sugar beet chitinase 76, che sequence of which is shown in SEQ ID N0.:5, and the acidic chitinase SE from sugar beet, che sequence of which appears from SEQ ID NO:.7.

27. A genetic construct according co any of claims 18-26 which 15 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 Agrobaccerium.

29. 20 29. A genetic conscrucc according co claim 24, in which che 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 25 regulatable promoter is regulatable by at least one factor selected from che 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. 30

32. A genetic construct according to any of claims 18-31 wherein the transcription terminator is selected from che group consisting of 829746CL.001/MKA/SPK/A36/1992 06 1*2 3 FEB 19%f; 24 2 2 70 192 plane transcription terminator sequences, bacterial transcription terminator sequences, fungal transcription terminators and. plane virus terminator sequences.

33. A genetic construct according to claim 32, in which the transcription terminator is selected from the group consisting o£ 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 Co the T-DNA gene 7.

34. A genetic construct according to any of claims 3.8-33 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 N0:.l, the sugar beet 0-1,3-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 ehe 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. / H (, t; 'V ^2 3 FEB 2994^ 829746CL.00 l/MKA/SPK/A36/1992 04 06 \ •bStf 1^3 242270

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. 5 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 10 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. 15 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 20 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 25 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. 829746CL.001 /MKA/SPK/A36/1992 04 02 *37 242270

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, Ascochyta, Pyrenophora, Helmithosporium, Ustilago, 5 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, 10 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 15 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. 20 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. 25 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. 829746CL.001/MKA/SPK/A36/1992 04 02 242 2 l^S

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 5 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. 10

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 15 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. 20 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 25 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 30 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 829746CL.001 /MKA/SPK/A36/1992 04 02 £S£ < ^ (0 2422 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 5 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 10 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 15 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 20 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. 25 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. 829746CL.001/MKA/SPK/A36/1992 04 02 242 27

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. 4 10

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 DH5a 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. 829746CL.001/MKA/SPK/A36/1992 04 02 242270 -198-

73. A DNA sequence as claimed in claim 1 and substantially as herein described with reference to the Drawings and Examples.

74. A polypeptide as claimed in claim 17 and substantially as herein described with reference to the Drawings and Examples.

75. A genetic construct as claimed in claim 18 and substantially as herein described with reference to the Drawings and Examples.

76. A vector as claimed in claim 38 and substantially as herein described with reference to the Drawings and Examples.

77. A host organism as claimed in claim 39 and substantially as herein described with reference to the Drawings and Examples.

78. A genetically transformed plant as claimed in claim 44 and substantially as herein described with reference to the Drawings and Examples.

79. Seeds, seedlings or plant parts as claimed in claim 51 and substantially as herein described with reference to the Drawings and Examples.

80. A method as claimed in claim 58 and substantially as herein described.

81. A method as claimed in claim 63 and substantially as herein described.

82. A method as claimed in claim 65 and substantially as herein described.

83. The plant transformation vector as claimed in claim 72 and substantially as herein described. DANISCO A/S By Their Attorneys BALDWIN SON & CAREY ABSTRACT f 4 2 2 A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO.:l 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 )9-l,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 transformed sugar beet plants, having an increased resistance to chitin-containing fungi as compared to untransformed plants. »- - N.Z. PATENT OFFICE -8 APR 1992