IE921104A1 - A plant chitinase gene and use thereof - Google Patents

Abstract

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

Description

FIELD OF THE INVENTION The present invention relates to a DNA sequence encoding the sugar beet chitinase referred to in the following as the sugar beet chitinase 4 or an analogue of said DNA sequence encoding a polypeptide having the antifungal activity of sugar beet chitinase 4, as well as to a genetic construct useful for the construction of genetically transformed plants having an increased resistance to plant pathogens containing chitin, such as phytopathogenic fungi, as compared to untransformed plants. The genetic construct comprises and is capable of LO expressing the DNA sequence of the invention, preferably in combination with a DNA sequence encoding a second chitinase different from sugar beet chitinase 4 and a DNA sequence encoding a β-1,3-glucanase. In another aspect, the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet L5 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 /9-1,3glucanase activity so as to result in an increased resistance to chitin-containing plant pathogens.

BACKGROUND OF THE INVENTION Most plants are susceptible to infection by pathogens such as microorganisms and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently economical loss to farmers. The plants respond to infection with several defense mechanisms including phytoalexins, deposition of lignin-like material, accumulation of cell wall hydroxyproline - rich glycoproteins, pathogenesis related proteins (PR-proteins) and increase in the activity of several lytic enzymes such as chitinases and /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 de829746BI.002/MKA/SPK/A36/1992 04 02 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 suffi5 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 retarded epidemic establishment of the infecting fungi in plant crops and thus an overall reduction in the effect of the infection.

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

Chitinase and /3-1,3-glucanase activity has been observed in plant species such as tobacco, barley, potato, rice, maize, corn, bean, 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 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 and the use thereof in the construction of transgenic plants having an increased resistance to various microorganisms such as fungi.

EP 0 292 435 relates basically to the regeneration of fertile Zea mays plants and mentions, inter alia, that a tobacco chitinase gene may be introduced in the plant in order to make it resistant to pathogens. Chitinase genes of other sources and other plants than Zea mays are not mentioned.

EP 0 290 123, WO 88/00976 and US 4 940 840 disclose the use of chitinases of bacterial origin in the construction of transgenic plants; chitinase of plant origin is not mentioned or alternatively only mentioned in general terms.

WO 90/07001 discloses DNA constructs comprising a high level promoter operably linked to a DNA sequence encoding a plant chitinase, which constructs are used in the transformation of plants so as to achieve overexpression of chitinase in the plant and thereby conferring resistance to plant pathogenic fungi. The only plant chitinase exemplified is a bean chitinase.

EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304 and EP 0 418 695 disclose the construction of transgenic plants harbouring DNA sequences encoding plant pathogenesis - related proteins (PRP), e.g. chitinase and β-l,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 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.

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 Rouaseau-Linouzin K. and Frltlg B. (1991) describe the production of basic and acidic R-protains in sugar beets infected with Careoapora betieola and the serological relation cf these R-protalns to the Rpcotelns in tobacco. The described R-proteins are found co ba serological related to tobacco R-proteins whereas the sugar beet chit lease 4 of the preaent invention does not show a serological relationship to astj known chitinass, confer below. Bo inforaation about the anino acid sequence or nucleotide sequence of any of the R-proteine is given.

In conclusion, none of the above cited, publications disclose eny sugar beet chitlnaae enzyne or the use thereof in the construction of transgenic plants.

At the fhytocheaioal Society of Europe International Synpoeiun, Rorwlch, United Klngdc·, April 11-13, 1989: BLocheaistry and ttolecu15 lar Biology of Plant: Pathogen Interactions, the present inventors disclosed the isolation and purification of $ chitlnaae isoenzynes, Including chitlnaae 4, froa the leaves of sugar beet plants infected by Caraoapora betieola, e phytopathogenie chi tin-containing fungi.

Ihe chitlnaae isoenzynes were characterised by their noleculer weight end kinetics cf chitin hydrolysis. Chitlnaae preparations were indicated to he capable of hydrolysing newly synthesized chitin in the cell well of the growing fungi. Bo further characterization wee reported and the chitlnaae enzynea were not separately discussed.

By the present invention e novel plant chitlnaae has been elucidated which, either «Iona or in coobinatlon with other pathogenesis-related proteins, shows praaleing results in the inhibition of chitin-containing fungi.

BRIEF DISCLOSURE OF IHE UTOBNIIOB In one aspect the present invention relates to a DBA sequence conprlsing the sugar beet chitlnaae 4 DBA sequence shown in SEQ ID MO. :1 or an analogue thereof, the analogue being a MA sequence encoding a BSWBLaea/MXA/SnVAX/ISSB 0« DC polypeptide having th* antifungal activity of th* sugar beat chiti· nase 4 as defined herein and 1) being a characteristic part of the DKA sequence ahovn in SBQ ID 110.:1, or li) hybridising with the DNA sequence chows in SBQ ID HO.;1 al 55 *C under the conditions specified in the Materials and Methods” section under the heading Identification of DMA belonging to the chitinaaa 4 gene fanlly”, or iii) encoding a polypeptide having the aslno acid sequence of the sugar beet chitinass 4 shown in $BQ ZD 1(0.:2, or iv) encoding a polypeptide being recognized by an antibody raised against sugar beat chltlnase 4.

IS The chitinasa 4 DMA sequence, SBQ ID 110.:1, shown in the Sequence listing below was detemined on the basis of a cDKA clone Isolated fro· S sugar beet cDKA library prepared ee described in the Materiel and Methods section below on the basis of hybridization with a vary specific oligonucleotide probe. The oligonucleotide probe was prepar20 ed on the basis of s tryptic peptide produced frost a substantially pure sugar beat chitinasa 4 obtained as described in Material! and Methods and in Bxaaple 1 below. The procedure used for isolating the chitinaae 4 DNA sequence is outlined is Bxaaple 4 below.

Prior to the present invention, the aaino acid sequence of the sugar beet chltlnase 4 enzyse or the DMA sequence encoding sugar beet chitinasa 4 had not been reported, and so indication had been given that It could be intereating to lock for these sequences. In fact, the initial analysis of auger beet chitinaae 4, which revealed an enzyne with a snail functional donaio, suggested that the chitinaae enzyse had a low ehitls affinity and thus low enzysatlc activity.

Thus the enzyne did not seea to be of particular interest.

The elucidation of the aalno acid aequance cf the sugar beet chitinase 4 wes an laportsnt step in the analysis of the enzyse. Thus, tawatan/MKA/sre/AM/im w » from the amino acid sequence it was clear that the sugar beet chitinase 4 belongs to the plant chitinases of the hevein class in that it contains a leader sequence, a hevein domain and a functional (catalytic) domain. Hevein is a lectin which binds to chitin, and the hevein domain of the enzyme is the part of the enzyme which is expected to bind to chitin and chitin-containing structures, e.g. of phytopathogenic fungi .

By hydrophobic clustering analysis using the method according to Gaboriaud et al., 1987, the primary structure of chitinase 4 has been found to be more compact than the structures of other plant chitinases belonging to the sugar beet chitinase 2 class (as described in further detail below). It is anticipated that the compact structure of chitinase 4 is an advantage in order to allow the enzyme to get access to chitin structures, e.g. in the cell walls of phyto15 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 has been experimentally verified.

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

Accordingly, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence comprising the chitinase 4 DNA 829746BI.002/MKA/SPK/A36/1992 04 02 sequence shown in SEQ ID NO. :1 or an analogue thereof as defined above or a subsequence thereof (further defined below), one or more copies of a DNA sequence encoding a second chitinase different from the sugar beet chitinase 4, and one or more copies of a DNA sequence encoding a β-1,3-glucanase, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.

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

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

The chitinase 4 DNA sequence or an analogue thereof, and in particular a specific subsequence thereof (which will be further discussed below), may also be used in the isolation of DNA sequences belonging to the chitinase 4 gene family as defined above. Also, the chitinase 4 DNA sequence or an analogue thereof or a genetic construct of the invention may be used in a method of preparing a polypeptide, e.g. a recombinant sugar beet chitinase enzyme, or a polypeptide mixture having a potent antifungal activity. The polypeptide or polypeptide mixture may by prepared by use of recombinant DNA techniques and may be used in the antifungal treatment of various products, especially food products. 829746BI.002/MKA/SPK/A36/1992 04 02 DETAILED DISCLOSURE OF THE INVENTION The chitinase 4 DNA sequence, SEQ ID NO.:1 encodes the basic sugar beet chitinase 4 enzyme, the amino acid sequence of which also appears from SEQ ID NO.:2. In the present context, the terms chitinase 4 and sugar beet chitinase 4 are used interchangeably.

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

In accordance herewith, the term the antifungal activity of the sugar beet chitinase 4 denotes the characteristic bifunctional activity of the enzyme, i.e. the combination of chitinase activity and lysozyme activity found in the sugar beet chitinase 4.

The term chitinase activity denotes the enzyme's ability to decompose chitin and chitin-containing structures and the chitinase acti20 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 H25 chitin by chitinase 4 to result in mainly dimers of chitin is monitored.

The biological assay may be carried out using any of the 3 different methods described in Materials and Methods herein under the heading Antifungal activity. When a positive result is obtained in any of these methods, i.e. the observance of destruction of the hyphae walls and retardation of the growth of the fungal hyphae, it is taken as evidence of biological chitinase 4 activity. 829746BI.002/MKA/SPK/A36/1992 04 02 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.

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 obtained from the nucleotide sequence of the chitinase 4 DNA sequence or which has a nucleotide sequence corresponding to a part of the chitinase 4 DNA sequence and which encodes a polypeptide having retained the antifungal activity of sugar beet chitinase 4. Typically, the characteristic part comprises a subsequence of the chitinase 4 DNA sequence, the subsequence being either a consecutive stretch of nucleotides of the chitinase 4 DNA sequence or being composed of one or more separate nucleotide sequences of the chitinase 4 DNA sequence. In order to allow the polypeptide encoded by the characteristic part of the chitinase 4 DNA sequence to retain its characteristic antifungal activity, the part will normally be only a small number of nucleotides shorter than the chitinase 4 DNA sequence, e.g. 1-50, such as 1-25 nucleotides shorter. 829746BI.002/MKA/SPK/A36/1992 04 02 1« A typical example of a characteristic part of th* chitinase 4 DMA sequence include* tha nucleotides encoding the active aita of chitinase 4.

The analogue defined in ii) above I* a SKA sequence lAieh hybridizes 5 with tha chitinase 4 DMA sequence under ths conditions specified in die Material* and Methods section below under die heeding Identification of DBA belonging to the chitinase 4 gene family. The conditions defined for the hybridization co cake piece are based on hybridization experiment* carried out with a nuaber of known plant ehitl10 nazes and auger beet chitinase 4 and is further described in Example li below.

In the present context, any DHA sequence hybridizing with the chttinsse 4 DHA sequence under tha hybridization conditions specified in the above cited part of Material and Methods is defined as belong* ing to the chitinase 4 gene family and is contemplated to encode a polypeptide having the structure end antifungal activity of tha sugar beet chitinase 4. Furthermore, when the polypeptides produced from such DMA sequences reset with antibodies raised against auger beet chitinase 4, it is a strong indication that the polypeptide encoded by the DMA sequence in question belongs to the sugar beet chitinase 4 serological class. Such DMA 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 DMA sequences, a.g. as described below. In tho following, DMA sequences belonging to the chitinase 4 gene family arc also termed chitinase 4 related DMA sequences.

The analogue defined in ill) shove is a DHA sequence which encodes a polypeptide comprising the amino acid sequence shown in SBQ IS MO.:2, i.e. the amino ecid sequence of the mature chitinase 4 enzyme. It is well known that Cha same amino acid may ba encoded by various codons, the codon usage being related, inter alia, to the preference of the or ganiaa In question express ing the nucleotide sequence. Thus, one or mors nucleotides or codons of the chitinase 4 DMA sequence of ths invention aay ba exchanged by others wfaicb, when expressed, result in i»746BLan/MXA/SPI/A36/OT2 « M a polypeptide identical to or substantially identical to the polypeptide encoded by the chitinase 4 DNA sequence in question.

The analogue defined in iv) above is a DNA sequence encoding a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4. In the present context, the term is recognized by is used interchangeably with binds to. As it is described in Example 3 below, it has been found that the sugar beet chitinase 4 enzyme belongs to a new serological class of basic chitinases hitherto not reported in the literature. A recent serological analysis of a rape L0 seed chitinase has revealed a close serological resemblance between this chitinase and sugar beet chitinase 4, indicating that the analyzed rape seed chitinase belongs to the same new class of basic chitinases.

The antibody to be used in determining the serological relationship between the polypeptide encoded by the chitinase 4 DNA sequence of the invention and a polypeptide encoded by a DNA sequence of another origin may be a monospecific polyclonal antibody or a monoclonal antibody. A particularly suitable antibody is a monoclonal or polyclonal antibody prepared against one or more characteristic epitopes encoded by the chitinase 4 DNA sequence. Such epitopes are explained in further detail below.

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

In the following, the composition of the chitinase 4 DNA sequence and each of the domains of the chitinase 4 enzyme encoded by the DNA sequence shown in SEQ ID NO.:1 and with the amino acid sequence shown 829746BI.002/MKA/SPK/A36/1992 04 02 in SEQ ID NO.:2 are further described and compared to other plant chitinases.

The chitinase 4 DNA SEQ ID N0.:i comprises a leader sequence (nucleotides 2-70) encoding 23 amino acid residues, a part (nucleotides 71-174) encoding a hevein domain of 35 amino acid residues and a part (nucleotides 175-793) encoding a functional domain of 206 amino acid residues. The N-terminal part of the mature polypeptide chain is blocked and it has not been possible to determine the sequence by conventional amino acid sequencing methods. However, based on comparison with the DNA sequences of a wheat germ agglutinin (WGA-A) and a potato chitinase and based on an analysis by electrospray mass spectrometry (vide Example 4), the start codon of the chitinase 4 DNA sequence has been deduced. Comparison between the leader sequence from chitinase 4 DNA (SEQ ID N0: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 NO.:1) are missing. Thus, while the leader sequence of the genomic chitinase 4 consists of 24 amino acid residues (SEQ ID NO..’4), the leader sequence from chitinase 4 consists of 24 amino acid residues although the almost full· length chitinase form cDNA is missing the first amino acid Met (SEQ ID NO.:2) .

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

Sugar beet chitinase 4 is a basic chitinase belonging to the hevein class. However, it is distinctly different from the other basic chitinases of this class. Whereas chitinases from bean, tobacco, tomato, potato, pea, poplar, barley (T and K) and sugar beet (chitinase 2) have molecular weights of 32-38 kDa (vide Example 10) , chiti30 nase 4 is smaller with a molecular weight of about 26 kDa (as determined for the mature enzyme). In addition, since antibodies raised against chitinase 4 do not recognize the other basic chitinases described above (vide Example 10), it is evident that chitinase 4 also belong to a different serological class than all other basic plant chitinases from the hevein class. 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 compared to the hevein structure. The functional domain contain 206 amino acid residues. In the basic chitinase from Nicotians tabaccum (cv, Havanna) (Shinshi et al., 1989), the hevein domain consists of 43 amino acid residues and the functional domain contains 263 amino acid residues. Although the hevein domain (i.e the chitin binding domain) of chitinase 4 is shorter than that of the tobacco chitinase, chitinase 4 has a binding affinity which is of a similar magnitude as that of the other basic chitinases belonging to the hevein class. For comparison, very poor or no binding is observed when chitinases from the non-hevein class are examined. This class of chitinases does not contain the hevein domain, but only the functional domain. The homology between the functional domains of the hevein class and the nonhevein class is very high. In addition, polyclonal antibodies raised against the chitinases from the hevein-class recognize the chitinases from the non-hevein class.

In general, the specific activity of the non-hevein class, the acidic chitinase from tobacco and the basic chitinase C from barley (Kragh Κ. Μ., Thesis, 1990) are approximately 6-fold lower than that of the hevein class chitinases.

Since the functional domain in chitinase 4 contains only 206 amino acid residues as compared to the 263 amino acid residues of the functional domain of the basic tobacco chitinase, a decrease in the specific activity was expected. Chitinase 4, however, performs extremely well and was by the present inventors shown to be superior to chitinase T, K, and C from barley (results not shown) when ana30 lyzed by the radiochemical enzyme assay described in Material and Methods below.

From the above explanation, it will be clear that the most important parts of the chitinase 4 DNA sequence shown in SEQ ID NO.:1 are the part encoding the hevein domain and especially the part encoding the 829746BI.002/MKA/SPK/A36/1992 04 02 functional domain of the enzyme. While the presence of a leader sequence in most cases is a prerequisite for allowing the polypeptide expressed from the DNA sequence to be transported out of the cell in which it is produced, the nature and origin of the particular leader sequence to be used may vary and need not be the leader sequence naturally associated with the chitinase 4 enzyme. Additionally, the leader sequence naturally associated with the chitinase 4 enzyme may be used in heterologous gene construct in transformation in plants, in particular sugar beet plants, when the encoded polypeptides are targeted to the extracellular space.

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

The term analogue is referred to as a DNA sequence which either Ai) Aii) Aiii) Aiv) is a characteristic part of said DNA sequence, hybridizes with a DNA probe prepared from said DNA sequence , encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or 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.:1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence The term analogue refers to a DNA sequence which Bi) is a characteristic part of said DNA sequence. 829746BI.002/MKA/SPK/A36/1992 04 02 Bii) hybridizes with a DNA probe prepared from said DNA sequence , Biii) encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or Biv) encodes a polypeptide which is recognized by an antibody raised against a polypeptide encoded by said sequence.

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

In a further aspect, the present invention relates to a DNA sequence comprising a sugar beet chitinase 4 gene. In the present context, the term gene is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream se15 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 se20 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 geno25 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 sequence of the chitinase 4 cDNA shown in SEQ ID NO.:1, (the comparisons are shown in Fig. 24) it is contemplated that nucleotides 356358 of the chitinase 4 gene sequence constitute the start codon of the chitinase 4 gene. 829746BI.002/MKA/SPK/A36/1992 04 02 Based on a comparison with the chitinase 76 sequence (comprising one intron) and the DNA sequence of chitinase 1 shown in SEQ ID NO.:11 (comprising two introns), it is believed that the chitinase 4 gene comprises only one intron starting at nucleotide 398 downstream of the ATG start codon. The position of the intron is believed to correspond to a position between nucleotides 395 and 396 in the chitinase 4 cDNA sequence shown in SEQ ID NO.:1.

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

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

The contemplated position of the active site of the sugar beet chitinase 4 enzyme has been revealed by comparison to the active site of other known enzymes catalyzing the hydrolysis of other oligosaccharides such as explained in Example 16 below. Thus, it is believed that the active site of the sugar beet chitinase 4 is constituted by amino acid residues 183 (Asp) and 189 (Glu) in SEQ ID NO.:2.

On the basis of the 3D-structure of the chitinase 4 enzyme which may be elucidated by use of conventional x-ray crystallography analysis and the amino acid sequence of the enzyme, it will be possible to predict parts of the enzyme which are responsible for the enzyme's specific properties. Thus, in addition to the active site disclosed above, also the specific amino acids of the enzyme responsible for its substrate specificity and substrate binding may be envisaged or elucidated. Also, the amino acid residues forming the epitopes of the enzyme may be elucidated. On the basis of the knowledge of such specific amino acids it is possible to specifically modify the enzyme 829746BI.002/MKA/SPK/A36/1992 04 02 ac aa co obtain a modified mods of action of the enryme. a.g. vlth re «pact to an increased catalytic activity, an improved. i.e. broadened, eubstxete spaclficlty, aa improved substrate, e.g. chi tin, binding or a nidified epitope. Such modifications nay be accomplished by uae of wall-lmown principles of protein engineering, such aa sit·* directed mutagenesis, e.g. aa described in Example 16 below.

As an exaaple, Che replacement of one oz «ore of the Trp residues In position 169, 204 snd 206 with Tyr residues is expected to change the binding of the substrate (chitin) to tho catalytic site and perhaps Che substrate specificity. Likewise, changes of tbs aalno acid residues constituting the active «its or aad.no acid residues which fore the structure of tho folded enzyme are expected cc influence, e.g., the catalytic activity, substrate specificity and/or substrate binding nay be found to result in improved properties of the resulting modified enzyme. Of course, the nature of the modification to be carried out will depend on tha daalrad result, i.e. ths specific desired function of the resulting modified enzyne.

Corresponding to the chitinase 4 enzyne encoded by a DNA sequence cf the invention, a DNA sequence encoding ths modified chitinase 4 enzyne nay either alone cr in coobinatlon with DNA sequences encoding other proteins, e.g. pathogenesis related proteins, such es theuoatin, oseothin and/or xesoatin (Vlegers, 1991) or thlonin (Bohlaann et «1., 1968), cercropln (J. Jaynes, 1969) or other enzymes such as chitinases and £-l-3-glucanases ba used in ths construction of a genetically transformed plant, preferably a sugar beet plant, having s particularly high and advantageous antifungal activity.

Also, ths nodl/led chitinase 4 enzyme nay prove to be a particular interesting component of an antifungal composition as described below.

Within a gene family, a high degree of homology between coding regions of the gems ia expected, whereas lose homology is expected between ηση-ooding regions. Between different gene families, tha homology may vary considerably. Tha term homology* is used here to denote the presence of tha degree of coegrtenentarlty between tha amino acid sequence of a given polypeptide end tha amino acid «9^auaVMKAAK/A3f/inz m at sequsnoa of another polypeptide being analyzed ee determined by use of the computer prog»· by Myere end Miller, version 1.05, September 1990, using the comparison matrix: Genetic code, the Open Gap Cost 6 end the Unit Gap coat 1. 9ee eleo Myers and Miller, 1988. The degree of homology between different genes, especially between the coding regions, nay thus bs used to assess ths degree of fomllarlty between different genes. The anino acid sequences may be deduced fro· a DMA. sequence or nay bs obtained by conventional amino add saquaneing methods. The degree of homology is preferably determined on the basis of mature proteins, i.e. without taking any leader sequence into account.

In accordance herewith, the present Invention relates to a DNA sequence encoding a chitinase isoenzyme which is at least 60X homologous with the auger beat chitinase A enzyme encoded by the DNA sequence SBQ ID BO. :1 and at the most AOX homologous with the sugar beet chltlnese 1 encoded by the DMA sequence shown in SEQ ID 90.:11. The minimum degree of homology of et least 60X has been determined on the basis of sn analysis of a repo seed chitinase (based on the mature protein) which has been shown co belong to the sugar beet chitinase A serological class (see Example 11). The degree of homology of AOX with chitinase 1 (which does not belong to the chitinase 4 class) reflects the minimal degree which Is expected to be acceptable for a polypeptide belonging to the chitinase A class.

Of course, a higher degree of homology with the chitinase A enzyme and therefor a lower degree of homology with the chitinase 1 enzyme reflects an even higher similarity herewith sad accordingly, the DMA sequence described above preferably encodes a chitinase isoenzyme which is at least 65X, e.g. at lease 70X homologous, such as at lease 75X or preferably 80X homologous with tbs sugar beet chitinase A enzyme encoded hy the DMA sequence SBQ ID B0. :1 and/or et the most 38X ouch as at the most 35X homologous with the sugar beat chitinase 1 enzyme encoded by the DBA sequence SBQ ID 110.:11.

An example of a DBA sequence encoding a polypeptide being about 7SX homologous to the sugar beee chitinase A enzyme and at the most AOX homologous to the sugar beat chitinase 1 enzyme is the genomic DBA aziTAffluxa/MZA/spiVAM/Ma « w sequence (chitinase 76. th· sequence of which ia shewn la SB] ID WO. :5) contained la tha genomic clone chitinase 76 obtained as described in Sxampls 9.

From Example 10 It ia evident that sugar beet chitinase A isolated 9 fro· sugar beet leaves la recognized by an antibody raised against thia sugar beet chitinase, hut not hy en antibody raised against the sugar beet chitinase 2. nils is a very strong indication of the fact that the sugar beet chitinase 4 belongs to a different class of chitinases than the sugar beet chitinase 2 and thus that 2 different classes of sugar beet chitinases exist. It ia contemplated that other polypeptides belonging to the chitinase 4 family will Show a similar reaction pattern and accordingly, the present invention further comprises a DMA sequence which encodes a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4, hut not by en antibody raised against sugar heat chitinase 2.

In a further aspect, the present invention relates to a modified EHA sequence comprising e OKA sequence es defined above comprising the chitinase 4 DMA sequence or gene or an analogue thereof In which et least one nucleotide has been deleted, substituted or modified or In which at least one additional nucleotide bee 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 ENA sequence has normally an amino acid sequence which Is different from the amino acid sequence of the auger beet chitinase 4. It will be understood that e modified SNA sequence of the invention vi 11 be of importance in the preparation of novel polypeptides having an increased antifungal activity as compared to chitinase 6.

When substitution*1 Is performed, one or bota nucleotides in tho full nucleotide sequence are replaced with one ox more different nucleotide·, when ’addition* ia performed, one or more nucleotides ere added at either end of the full nucleotide sequence, when ’insertion* ie performed one or more nucleotides within tb« full nucleotide sequence is Inserted, end when deletion is performed one or more BSMeBUB/MXAySFK/AB/UB W 94 nucleotides are deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it.

A modified DNA sequence may be obtained by well-known methods, e.g., by use of site-directed mutagenesis, In a further aspect, the present invention relates to a subsequence of the chitinase 4 DNA sequence of SEQ ID NO.:1 encoding a polypeptide which need not, but which can have the antifungal activity of the sugar beet chitinase 4. Especially interesting subsequences of the chitinase 4 DNA sequence or of the genomic DNA sequence are subsequences comprising the nucleotide sequence defining the active site of the sugar beet chitinase 4 enzyme. An example of such a subsequence is a DNA sequence comprising the active site of the sugar beet chitinase 4 enzyme, e.g. the DNA sequence encoding the following peptide named peptide 4-22 (shown by use of the conventional one-letter amino acid code) consisting of the amino acids No's. 179-200 of SEQ ID NO.:2 S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R This sequence is the amino acid sequence of the tryptic peptide 4-22 obtained from the purified sugar beet chitinase 4 as described in Example 16 below. A DNA sequence encoding this polypeptide may be of significant importance for carrying out modifications of the active site with the aim of improving the antifungal activity of the resulting polypeptide. Furthermore, the DNA sequence may be fused to a part of another DNA sequence encoding an enzyme different from the sugar beet chitinase 4 or substituted with a part of such enzyme encoding the active site thereof with the aim of obtaining a hybrid enzyme having the antifungal activity of sugar beet chitinase 4. Of course, the polypeptide chain of the hybrid enzyme should be able to fold in the correct manner so as to provide a useful conformation around the active site.

A further interesting DNA sequence encoding a part of the chitinase 4 enzyme is a DNA sequence encoding the polypeptide having the follow829746BI.002/MKA/SPK/A36/1992 04 02 ing amino acid sequence consisting of the amino acids No's. 183-204 of SEQ ID NO.:6 S-1-G-F-D-G-L-N-A-P-E-T-V-A-N-D-A-V-1 -A-F-K This polypeptide is deduced from the DNA sequence of the genomic chitinase 76 clone shown in SEQ ID NO.:5 and corresponds almost to the DNA sequence of the peptide 4-22 given above, except for the most important fact that the bolded D is an N in peptide 4-22. It is believed that the chitinase 76 derived polypeptide may have the same or nearly the same interesting properties and uses as the peptide 422.

Two further interesting DNA sequences are the sequence encoding the following peptide consisting of the amino acids No's. 163-169 of SEQ ID NO.:2 G-P-L-Q-I-T-W which is the tryptic peptide 4.19.3 of chitinase 4 and the DNA sequence encoding the tryptic peptide 4-26 consisting of the amino acids No's. 201-224 of SEQ ID NO.:2 T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-S-I which sequences are described in Example 16 below. The peptides comprises one and two Trp-residues, respectively. The Trp-residues are contemplated to be involved in the active site and/or substrate specificity of the chitinase 4 enzyme, e.g. as further discussed in Example 16 below. Analogues of these above mentioned subsequences in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which still have the catalytic and/or binding activities as that of the three above-mentioned peptides encoded by the chitinase 4 DNA subsequences may be very interesting.

Another example of an interesting subsequence according to the inven30 tion is a subsequence of the chitinase 4 DNA sequence of SEQ ID NO. :1 encoding a polypeptide comprising the hevein domain of the sugar 829746BI.002/MKA/SPK/A36/1992 04 02 beet chitinase 4 enzyme, or an analogue of said subsequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which subsequence is encoding a polypeptide capable of binding to chitin as determined by affinity column chromatography on regenerated chitin prepared as described in Materials and Methods under the heading Preparation of a chitin column.

Due to the fact that the hevein domain of the chitinase 4 enzyme is compact and believed to be very efficient, i.e. capable of establish10 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 insertion of the DNA sequence encoding the hevein domain of sugar beet chitinase 4 are chitinases of the non-hevein class or cucumber class (e.g. the sugar beet chitinase SE disclosed herein).

A further interesting subsequence of the present invention is a subsequence of the chitinase 4 DNA sequence SEQ ID NO. :1 encoding the leader peptide of chitinase 4 or an analogue thereof in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which is capable of directing a passenger polypeptide to which it is fused out of the cell in which the fused leader and passenger polypeptide is produced to be deposited in the extracellular space.

As explained above, epitopes of the sugar beet chitinase 4 enzyme may be used to raise monospecific polyclonal and monoclonal antibodies which are useful in identifying chitinase 4 isoenzymes belonging to the chitinase 4 serological class and for epitope mapping. Suitable epitopes are expected to be found among the hydrophilic peptides of the chitinase 4 amino acid sequence SEQ ID NO.:2, because these peptides seem to be substantially different from peptide parts of other chitinases than sugar beet chitinase 4. Antibodies (either monoclonal, monospecific or polyspecific) may be prepared by use of conven35 tional methods, e.g. as described in the Materials and Methods sec829746BI.002/MKA/SPK/A36/1992 04 02 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 identified: Peptide 1: AGKRFYTRA (consisting of amino acids No's 87-95) Peptide 2: CNPSKQYY (consisting of amino acids No's 153-160) Peptide 3: IECNGGNS (consisting of amino acids No’s 230-237) Peptide 4: TARVGYYTQYCQ (consisting of amino acids No's 241-252) These epitopes are believed to be particularly suitable for the production of monospecific antibodies to sugar beet chitinase 4. Peptide 1 and Peptide 4 are believed to be the most suitable peptide sequences to be used in the production of monospecific antibodies to chitinase 4.

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

As mentioned above, bacterial as well as plant chitinases exist. In the present context in which an important use of the DNA sequence of the invention is explained which is the construction of genetically transformed plants, the most interesting types of chitinases are believed to be plant chitinases, and accordingly it is preferred that the DNA sequence of the invention or an analogue or a subsequence thereof is of plant origin. Especially interesting plant chitinase DNA sequences are derived from a member of the family Chenopodiaceae, Solanaceae, Apiaceae, Brassicaceae, Cucurbitaceae or Fabaceae. Examples of such plants are corn, alfalfa, oat, wheat, rye rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea. 829746BI.002/MKA/SPK/A36/1992 04 02 The terms sequence, subsequence and analogue as used herein with respect to sequences, subsequences and analogues according to the invention should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in iso5 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 sequences from other plants, whereupon they, if desirable, may be modified as described herein.

From the above explanation it will be clear that the chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may be fused to one or more second nucleotide sequences encoding a second polypeptide or part thereof under conditions which ensure that at least part of the DNA sequence of the invention is expressed in conjunction with the other nucleotide sequence(s), e.g. in the form of a fusion protein. For instance, a DNA sequence of the invention encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 enzyme may advantageously be fused to a C-terminal sequence encoding a signal peptide which gives rise to transport of the fusion protein expressed therefrom to specific organelles of the organism expressing the polypeptide. Signal peptides involving transport will be discussed in further detail below. Interesting subse25 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 subse30 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 polypep35 tide of the invention has the advantage that it may be easily pro829746BI.002/MKA/SPK/A36/1992 04 02 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 chitinase 4. The polypeptide of the invention may be used as a constituent in an antifungal composition, e.g. as described below.

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

Accordingly, in another important aspect, the present invention relates to a genetic construct comprising 1) a promoter functionally connected to 2) a DNA sequence comprising a chitinase 4 DNA sequence or an analogue or a subsequence thereof as defined above and 3) a transcription terminator functionally connected to the DNA sequence.

The genetic construct may be used in the construction of a genetical ly modified plant in order to produce a plant showing an increased antifungal activity as determined by the procedure given in Example : 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 example of a genetic construct as defined above is given in Example 18 below.

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

Thus, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID NO.:1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide having the activity of a second chitinase different from the sugar beet chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having βL,3-glucanase activity, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA se25 quences into functional polypeptides.

The polypeptides with chitinase or /3-1,3-glucanase activity is preferably of plant origin. The chitinase and β-1,3-glucanase activity may be determined as explained in the section Materials and Methods below.

Of particular interest is a genetic construct comprising 829746BI.002/MKA/SPK/A36/1992 04 02 one or more chitinase 4 subsequence one or more having a pi one or more having a pi copies of a DNA sequence as defined above comprising the DNA sequence shown in SEQ ID NO.:1 or an analogue or thereof, copies of a DNA sequence encoding an acidic chitinase equal to or less than 4.0, and copies of a DNA sequence encoding a basic β-1,3-glucanase 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 se10 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 Examples of such chitinases are cucumber lysozyme/chitinase and Arabidopsis as well as the acidic sugar beet chitinase SE having the DNA sequence shown in SEQ ID NO.:7 and the amino acid sequence shown in SEQ ID NO.:8 or an analogue of said DNA sequence encoding an acidic chitinase having a pi of at the most 4.0 and preferably capable of hydrolyzing ^H-chitin into mainly hexamers.

In the present context, the term basic β-1,3-glucanase means a β1,3-glucanase having a pi of more than 9.0. Preferably, the basic β1,3-glucanase is one which is capable of hydrolyzing glucan into mainly dimers, e.g. as determined by the ^H-laminarin assay described in the Materials and Methods section below. The basic β-ί,3-glucanase is preferably of plant origin. Examples of a suitable basic /3-1,3glucanase are basic β-1,3-glucanases derived from tobacco (Shinshi et al., 1990), barley (Fincher et al., 1986) or sugar beet, e.g. the basic sugar beet /3-1,3-glucanase 4, the DNA sequence of which is shown in SEQ ID NO.:9 or an analogue thereof encoding a basic /3-1,3glucanase having a pi of at least 9.0 and preferably being capable of hydrolyzing ^H-laminarin into mainly dimers of β-1,3-glucan. The 829746BI.002/MKA/SPK/A36/1992 04 02 basic sugar beet β-1,3-glucanase 4 is different from other plant β1,3-glucanases in that it does not contain a C-terminal extension as appears from the amino acid sequence SEQ ID NO.:10. The advantageous effect of using the basic sugar beet β-1,3-glucanase 4 may in part be due to this lacking C-terminal extension.

Another interesting sugar beet chitinase is the sugar beet chitinase 1 which shows a very low homology with the sugar beet chitinase 4 of the present invention, confer above. The DNA sequence of the sugar beet chitinase 1 is shown in SEQ ID NO.:11. The DNA sequence is about LO 6,3 kb long and encodes a polypeptide having 439 amino acid residues. The polypeptide shown in SEQ ID NO.:12 contains a leader sequence of 26 amino acid residues, a hevein domain of 20 amino acid residues and a C-terminal extension of 23 amino acids. Additionally, the sequence contains a most interest proline rich domain of 238 amino acids which forms and interest aspect of the present invention.

The experiments reported in Example 2 below show that the combination of the sugar beet chitinase 4 enzyme, an acidic chitinase and a basic β-1,3-glucanase results in an increased antifungal activity as compared to the antifungal activity of each of the constituents. The increased antifungal activity observed when using this specific combination is partly believed to be due to the different mode of action of the acidic chitinase, basic β-1,3-glucanase and sugar beet chitinase 4, respectively. When the acidic chitinase is one which hydrolyses chitinase primarily into hexamers (as compared to chiti25 nase 4 which primarily hydrolyses chitin into dimers) and the basic β-1,3-glucanase is one which hydrolyses glucan primarily into dimers, it is believed that these different cleaving modes may be involved in the resulting advantageous total effect.

Furthermore, the synergistic effect obtained when using a combination of the sugar beet chitinase 4, a polypeptide having the activity of a second chitinase different from chitinase 4, e.g. an acidic chitinase, and a polypeptide having the activity of a /3 -1,3 - glucanase , e.g. a basic β-1,3-glucanase, is believed to be due to the fact that such combination will attack both the chitin and glucan constituents of the cell wall of phytopathogenic fungi and also parts of the cell 829746BI.002/MKA/SPK/A36/I992 04 02 wall in which the chitin and glucan constituents are intimately cross-linked to one another. The β-1,3-glucanase further serves to remove the outer glucan layer covering the chitin structure of chitin containing plant pathogens, e.g. phytopathogenic fungi, resulting in an exposure of the chitin structure to the enzymatic action of the chitinase.

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

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

Normally, the combination of each of the above mentioned variable elements of the genetic construct to be chosen will depend, e.g. on the desired strength of the antifungal effect to be obtained which may be determined as a function of gene dosage and specific nucleotide sequence of each of the DNA sequences, and the type and strength of the promoter and terminator used for each DNA sequence. Also, expression in specific parts of the plant with respect to organs and intracellular and extracellular location may be varied with different types of promoter and terminator.

However, in designing a genetic construct of the invention which is to be expressed in a given organism such as a plant, one must be aware of the possible toxic effect of a too high expression of one or more of the proteins encoded by the genetic construct which, e.g., may lead to a lower yield of the transformed organism, e.g. plant, as compared to an untransformed organism or an organism not containing the genetic construct. Also, when the genetic construct of the inven829746BI.002/MKA/SPK/A36/1992 04 02 cion ia too large, it may ba difficult to obtain a stable Introduction thereof Into the genoae of the pleat which nay lead to excision of a part of or the entire genetic construct fro· the genoae of the plant. Thus, the genetic construct should be adapted so that the expression products therefrom are generally acceptable to the host organism.

The nunber of copies of the DNA sequences of the genetic construct of tbe invention together with the activity of the genes will determine the optimal number of copies of the SKA sequences of the genetic construct of the invention. With the fast Increasing knowledge within the field of plant genetic engineering, improved transformation snd biological containment techniques may ba developed leading to the possibility of introducing larger foreign genetie fragments into a plant without causing retarded growth, retarded yield or recoils blnatlonal events then what ia at present possible.

At present, a genetic construct is preferred which contains only a few copies of the DBA sequence of tbe invention. Accordingly, lc is preferred chat each of tbe DNA sequences of tbs genetic construct of the invention is present in only one copy. The construction of a genetic construct containing one copy of each of the CHA sequences is illustrated ln the exeaples below.

As mentioned above, a significant antifungal effect is obtained from a protein encoded by the chitinase 4 DMA sequence of the invention or an analogue thereof. Accordingly, it la contenplated that a genetic construct cf the invention, in which two copies of the chitinase A DHA sequence of tbs invention or an analogue thereof, and one copy of each of the DNA sequences encoding an acidic chitinase and a basic fi1,3-glucanase are present may show very, potent antifungal effects when present in a genetically transformed plant of the Invention. It la believed that such a genetic construct will not pose a too heavy burden on tbe plant in which it is harboured. Of course, also the choice of a.g. promoter used for each DMA sequence will influence the amount of protein expressed therefrom. This yill be further explained below. tsneBun>/MXA/sn7A3e/tfn 0»« The genetic construct of the invention as described above may be present on one or several DNA fragments. Depending on the size of the genetic construct to be introduced in an organism such as a plant, in the case of a plant typically by means of a plant transformation vector, and the combination of promoters and transcription terminators, it may be advantageous to introduce the construct by use of two or more plant transformation vectors, and accordingly it may be advantageous that the genetic construct is present on two or more DNA fragments. When the use of only one plant transformation vector is desirable, it is advantageous that the genetic construct is present on one DNA fragment.

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

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

The nature of the N-terminal sequence to be used will e.g. depend on the particular organism and the part thereof, e.g. the specific cell or tissue, in which the polypeptide encoded by the DNA sequence of the invention is to be produced and to which part of the same cell or another location in the organism the polypeptide is to be transported. A typical leader peptide has a core of hydrophobic amino acids and thus, a suitable leader sequence to be used in connection with the DNA sequence of the invention is a nucleotide sequence comprising a stretch of codons encoding hydrophobic amino acids. 829746BI.002/MKA/SPK/A36/1992 04 02 Examples of a leader sequence to be used in the present context are the following leader sequences which are also part of the invention. These leader sequences are the N-terminal leader sequence of the sugar beet chitinase 1 enzyme, the nucleotide and amino acid sequence of which is shown in SEQ ID NO:11 and SEQ ID NO:12, respectively; the N-terminal leader sequence of the genomic chitinase 76 clone, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 5 and SEQ ID NO:6, respectively; the N-terminal leader sequence of the acidic sugar beet chitinase SE, the nucleotide and amino acid sequence of which is shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; and the N-terminal sequence of the β-l,3-glucanase 4, the nucleotide and amino acid sequence of which is shown in SEQ ID NO:9 and SEQ ID N0:10, respectively. Another interesting sequence is DNA subsequence from the sugar beet chitinase 1 encoding the proline rich domain of the chitinase 1 gene comprising 132 amino acids and shown in SEQ ID NO:12 which may also be used in the direction of the polypeptide to specific locations of the organism. The abovementioned leader sequences are to be considered as non-limiting examples .

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

Another interesting subsequence in this aspect of the invention is the proline rich domain of the chitinase 1 shown in SEQ ID NO.:12 consisting of 132 amino acids. It is contemplated that the proline rich domain may be involved in the anchoring of the chitinase 1 829746BI.002/MKA/SPK/A36/1992 04 02 protein to the cell wall after modification of the prolines to glycosylated hydroxyprolines, as in extensines. Thus, the subsequence containing the proline rich domain may be used when directing and obtaining a polypeptide at a desired location in the cell and/or organism in which the polypeptide is produced.

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

Non-limiting examples C-terminal sequences to be included in a genetic construct of the invention are C-terminal sequences selected from the following sequences: the C-terminal sequence of sugar beet chitinase 1, the amino acid of which is shown in SEQ ID NO.:12, encoding the following polypeptide consisting of the amino acids No's 413-439 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 ID NO.:2 N L R C * 829746BI.002/MKA/SPK/A36/I992 04 02 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* 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 β-1,3-Glucanase from tobacco encoding the following polypeptide shown in SEQ ID NO.:17 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 desirable to control a phytopathogenic fungus mainly present in the intercellular space of the plant, It may be desirable to avoid the use of a C-terminal sequence. When a phytopathogenic fungus mainly present intracellularly is to be controlled it may be desirable that most of or all of the DNA sequences of the genetic construct are provided with a C-terminal sequence capable to transport the polypep tides expressed from the DNA sequences to the vacuole. 829746BI.002/MKA/SPK/A36/1992 04 02 As it will be apparent from the above explanation it is important to obtain a sufficient expression of the polypeptides encoded by the genetic construct in plants containing said construct in order to allow the polypeptides to exert their intended function, i.e. to exert their antifungal activity. One essential element in obtaining a sufficient expression is to provide a satisfactory regulation of the transcription and expression of the DNA sequence or gene from which the polypeptide is expressed.

The expression of each of the DNA sequences of the genetic construct 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 consti15 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 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. se25 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 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 CM 02 A regulatable promoter is a promoter the function of which is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of each of the DNA sequences of the genetic construct of the invention.

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

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

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

A preferred group of plant virus promoters are promoters which may be derived from a cauliflower mosaic virus (CaMV). Such promoters are normally strong constitutive promoters. Examples of a preferred CaMV 829746BI.002/MKA/SPK/A36/1992 04 02 promoter 1« a CaMV 19S promoter end a CaHV 35S promoter (Odell ac al., 1985).

Other promoters may be derived from the Tl-plasmld auch aa the octo plna synthase promoter, tha nopsltne synthase promoter (Harrera5 Estrelle et al., 1983), tha manaopine aynthaaa promoter, and promoters from other open reading frames in the T-DNA such aa 0817.

Pur Chef examples of suitable promoters are MAS/33S (Janssen and Gardner, 1989), HAS dual Tr 1,2 (Veltsn et el., 1984) and a T-2 DBA gene 5 promoter (Kona and Schell. 198$), Tha regulatory sequence may be a chitinase promoter, I.e. a promoter which la naturally found In connection with chitinase genes snd Involved la 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 uay be identified and isolated e.g. by the methods disclosed herein. Typically, the chitinase promoter should be obtained froa a plant which has bean shown to have a fast response to pathogen challenge. In this connection, fast responses have been observed in pea and barley and it la contemplated that chitinase promoters from these plants may be useful for the present purpose. An example of such promoters is tha chitinase promoter of pea (K. Vad, 1991). An example of another promoter which la contemplated to be useful in the present context is the sugar beet chitinase 1 promoter (SBQ ID BO:11) and the sugar.beet acetohydroxyacid synthase promoter (AHAS) (Γ. Stougird and K. Bojaen, Daniseo h/S, Denmark, personal communication). Furthermore, the sugar beet promoters from the addle chitinase SS, chitinase 1, chitinase 76 and chitinase 4 or fi-ί,3-glucanase 4 may also be useful.

Optionally, and if desired, the natural promoter may be modified for the purpose, a.g. by modifications of the promoter nucleotide se30 quence so aa 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.

Aa stated above, each of the coding DNA sequences ef the genetic construct of ths invention is functionally connected to a transcripSim«BL0IB/MKA/SPK/A3«/mz 0« ac 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 sequences.

Specific examples of suitable transcription terminators are a NOS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium (Herrera-Estrella et al. , 1983), a 35S transcription terminator sequence of the cauliflower mosaic virus (Paszkowski et al., 1984), a PADG4 transcription terminator to the DNA gene 4 (Wing et al., 1989), and a PADG7 transcription terminator to the T-DNA gene 7.

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

The specific promoters and the specific terminators, respectively, to be connected with each of the DNA sequences of the genetic construct may be the same or different. It may be an advantage to use different promoters and terminators, respectively, because then the risk of recombinational events, which may lead to excision of parts of or the entire genetic construct, are avoided.

In a further aspect, the present invention relates to a vector which is capable of replicating in a host organism and which carries a DNA sequence of the invention comprising a chitinase 4 DNA sequence substantially as shown in SEQ ID N0:l or an analogue or subsequence thereof, or a genetic construct of the invention. The vector may either be one which is capable of autonomous replication, such as a plasmid, or one which is replicated with the host chromosome, such as a bacteriophage or integrated into a plant genome via the border sequences of Ti vectors. For production purposes, the vector is an expression vector capable of expressing the DNA sequences in the 829746BI.002/MKA/SPK/A36/1992 04 02 organism chosen for the production. Thus, the expression vector is a vector which carries the regulatory sequences necessary for expression such as the promoter, an initiation signal and a termination signal, etc. These regulatory sequences may be the ones carried by the genetic construct of the invention. The vector may also be one used for identification and optionally isolation of chitinase genes or messengers from other organisms, e.g. other plants, for which purpose expression is not required. This may be done, e.g., as described below.

In a still further aspect, the present invention relates to an organism which carries and which is capable of replicating or expressing an inserted DNA sequence as defined above, i.e. a chitinase 4 DNA sequence comprising a nucleotide sequence substantially as shown in SEQ ID NO:1 or an analogue thereof or a chitinase gene or pseudogene comprising said DNA sequence.

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

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

The organism may be a higher organism such as a plant, or a lower organism such as a microorganism. A lower organism such as a bacterium, e.g. a gram-negative bacterium such as a bacterium of the genus Escherichia, e.g. E. coli, or of the genus Pseudomonas , e.g. P. 829746BI.002/MKA/SPK/A36/1992 04 02 putida and P. fLuorescens, or a gram-positive bacterium such as of the genus Bacillus, e.g. B. subtilis, or a yeast such as of the genus Saccharomyces or a fungus, e.g. of the genus Aspergillus, is useful for producing a recombinant polypeptide as defined above. As many organisms inherently produce chitinase, the insertion of a DNA sequence or a genetic construct according to the present invention may lead to a considerably increased chitinase and optionally β-1,3glucanase expression and a correspondingly increased antifungal activity. The recombinant production may be performed by use of conventional techniques, e.g. as described by Sambrook et al., 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 soil fungi present in e.g. the rhizosphere.

Also, the organism may be a cell line, e.g. a plant cell line. Most preferably, the organism is a plant, i.e. a genetically modified plant such as will be discussed in further detail below.

As mentioned above, the genetic construct is preferably to be used in modifying a plant. Accordingly, the present invention also relates to a genetically transformed plant comprising in its genome a genetic construct as defined above. The genetically transformed plant has an increased antifungal activity compared to a plant which does not harbour a genetic construct of the invention, e.g. an untransformed or natural plant or a plant which has been genetically transformed, but not with a genetic construct of the invention. Normally a constitutive expression of the polypeptides encoded by the genetic construct is desirable, but in certain cases it may be interesting to have the expression of the polypeptides encoded by the genetic con30 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 chitinase active in destroying the cell walls of phytopathogenic fungi .

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

Non-limiting examples of dicotyledonous plants which may be geneti10 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 re15 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 according to the invention are represented by phytopathogenic fungi. Phytopathogenic fungi differ in the way which they interact with their host plant during infection. Some species invade the plant via natural openings or wounded tissue and grow in between the plant cells, in the intercellular space, during the entire infection cycle.

The fungal hyphae excrete toxins or enzymes that weaken or destroy the plant cells and thereby provide the fungus with cell constituents leaking out of the plant cells. Other fungal pathogens immediately destroy the host cells by penetrating the cell wall of healthy host cells and disintegrate their protoplasts.

Below are given some examples of chitin and glucan containing phytopathogenic fungi with different host interacting strategies, all of which are contemplated to be sensitive to the transgenic plants of the invention. 829746BI.0O2/MKA/SPK/A36/1992 04 02 Cercospora spp. is a fungus the growth of which is restricted to the intercellular space. Conidia (i.e. spores) from the fungus germinate on the leaf surface and penetrate through the stomata of the leaves. Inside the leaf the plant cells close to the hyphae growing in the intercellular space are severely affected by the toxins excreted from the fungus. The toxins cause the plasma membrane to degrade, whereby the cell content leaks out into the intercellular space. Later in the infection cycle the plant cells collapse and necrotic areas containing dead plant cells and fungal mycelia emerge.

Verticillium alboatrum is a root pathogen which propagates in the intercellular space, but which penetrates through the openings made by the emergence of lateral roots, through mechanically injured areas or by direct penetration of hyphae through the tender root tissue in the regions of cell elongation or meristemic activity. The fungus de15 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.

Colletotricum lindemuthianum causes Bean anthracnose. Conidia from this fungus germinate in a film of water in the infection court and the produced germ tube penetrates the cuticula and grows into the epidermal cells of bean leaves and pods. During the following infection, the fungus acts as a parasitic pathogen, penetrating living cells and causing disintegration of the protoplasts.

Fusarium spp. is a typical soilborne fungus infecting the plants through the roots, where the hyphae penetrate the epidermal cells of young roots and invades the xylem of roots and stems. The vessels become plugged with granular material and surrounding cells of the outer phloem and cortex are destroyed.

Puccinia graminis causes Stem rust of wheat. The sporidia germinate on a film of water on the surface of the plant and the germ tubes 829746BI.002/MKA/SPK/A36/1992 04 02 penetrate the cuticula. The growing mycelia produce haustoria that penetrate the walls of the host cells and invaginate their protoplasts .

Ustilago maydis is a fungus with mainly intercellular growth, but 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 invention is to be considered within the scope of the present invention .

In recent years, a great effort has been focused on developing useful methods for constructing novel plants or plant cells having specific and desirable properties by transferring new genetic information encoding the desirable properties to the plant, and a number of such methods based on recombinant DNA technology and suitable plant transformation systems are now available. Usually, the genetic information is introduced into the plant by use of a vector system or by direct introduction, e.g. by use of the methods given by Herrera-Estrella et al·., 1988, Rogers et al., 1988, Saul et al, 1988, An et al., 1988, Hooykaas, 1988, Horsch et al., 1988, Reynaerts et al., 1988, and Tomes et al., 1990.

Thus, in another aspect, the present invention relates to a transformation system comprising at least one vector which carries a genetic construct as defined above and which is capable of introducing the genetic construct into the genome of a plant such as a plant of the family Chienopodiaceae, in particular of the genus Beta, especially Beta vulgaris .

Normally, plant transformation systems are based on the use of plasmids or plasmid derivatives of the bacteria Agrobacterium. The two best known Agrobacteria are AgrobacCerium tumefaciens and Agrobacterium rhizogenes (plasmids thereof are in the following termed pTi and pRi, respectively). The use of such plant transformation 829746BI.002/MKA/SPK/A36/1992 04 02 systems is based on the ability of the bacteria Agrobacterium to transfer a specific piece of DNA (T-DNA) to a plant cell in a wounded area. In nature, the T-DNA is located between specific border DNA sequences on the pTi or pRi which further carries virulence genes necessary for the transfer of the T-DNA to the plant. The Agrobacterium transformation system mediates the transfer of any DNA sequence located between the borders and thus, it is possible to exchange the wild type Agrobacterium T-DNA with any desirable piece of DNA to be introduced into a plant.

Preferably, the plant transformation system of the invention is based on disarmed Agrobacteria harbouring derivatives of the pTi or pRi from which the wild type T-DNA has been removed.

Normally, the vector system with which the plant is transformed comprises one or two plasmids. In the one-plasmid system (also termed a co-integrate vector system), the T-DNA of pTi or pRi has been removed and replaced by the DNA to be transferred into the plant cell by use of homologous recombination. In the two-plasmid system (also termed a binary vector system) both the T-DNA and the borders have been removed from the pTi or pRi. Introduction in the disarmed Agro20 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.

An example of a suitable plant transformation vector is pBI121 and derivatives thereof, e.g. as described by Jefferson 1987.

Suitably, the vector to be used is provided with suitable markers, eucaryotic as well as procaryotic, e.g. genes encoding antibiotic resistance or herbicide resistance or glucoronidase (GUS), e.g. hygromycin or other known markers, e.g. the markers disclosed by Lindsey, 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 The use of more than one vector in one transformation event will according to the presently known plant transformation techniques normally require that different selective genes are present on each vector in order to be able to follow the success of the plant transformation.

In the construction of a transgenic plant using a plasmid such as a pTi or pRi or derivative thereof it is preferred that the genetic construct to be inserted in the plant is first constructed in a microorganism in which the plasmid can replicate and which is easy to L0 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.

The plasmid harboring the genetic construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harboring the genetic construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified. This transformation may be performed in a number of ways, e.g. as described in An et al. (1988) .

Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher et al. (1980). Typically, a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive or brushing the plant with a steel brush (e.g. as described in Example 15). The wound is then inoculated with the Agrobacterium, e.g. in a suspension. Alternatively, the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant. The inoculated plant or plant part is then subjected to selection and regeneration and grown on a suitable culture medium and allowed to develop into mature plants. This is accomplished by use of methods known in the art. 829746BI.002/MKA/SPK/A36/1992 04 02 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 known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.

In accordance with well-known plant breeding techniques it will be understood that the production of a genetically transformed plant may be performed as a double transformation event (introducing the genetic construct in two transformation cycles) or may be associated with use of conventional breeding techniques. Thus, two genetically modified plants according to the present invention may be cross breeded in order to obtain a plant which contains the genetic construct of each of its parent plants.

As will be understood from the introductory part of the present specification, the chitinase 4 DNA sequence of the present invention or an analogue thereof may be used for diagnostic purposes, which will be further explained in the following. 829746BI.002/MKA/SPK/A36/1992 04 02 Various types of diagnosis may be performed by use of the chitinase 4 DNA sequence of the invention. In a given example, chitinase messenger RNA's transcribed from a gene belonging to the chitinase 4 gene family may be qualitatively as well as quantitatively determined by hybridization to the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof under conditions suitable for said hybridization. Furthermore, genes belonging to the chitinase 4 gene family and present in an organism such as a plant may be identified and isolated by use of the DNA sequence of the invention, e.g. by screening a gene library of such an organism.

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

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

The identification and isolation of a gene or cDNA clone in a sample belonging to the chitinase 4 gene family by use of the chitinase 4 DNA sequence of the invention or an analogue thereof, in particular a subsequence thereof, may be based on standard procedures, e.g. as described by Sambrook et al·., 1990. For instance, to characterize 829746BI.002/MKA/SPK/A36/1992 04 02 chitinase 4 related genes in other plants, it is preferred to employstandard Southern techniques.

The chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may also be used in a method of quantifying the amount of a chitinase 4 related messenger present in different tissues in an organism, e.g. a plant, the method comprising hybridizing a nucleic acid containing sample obtained from the organism with the chitinase 4 DNA sequence of the invention comprising a nucleotide sequence substantially as shown SEQ ID NO:1 or an analogue thereof, especially a subsequence thereof, optionally in labelled form, in denatured form or an RNA copy thereof under conditions favorable to hybridization between the denatured DNA sequence or RNA copy and the RNA of the sample and determining the amount of hybridized nucleic acid (Barkardottir et al., 1987).

The hybridization should be carried out in accordance with conventional hybridization methods under suitable conditions with respect to e.g. stringency, incubation time, temperature, the ratio between the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof to be used for the identification and the sample to be analyzed, buffer and salt concentration or other conditions of importance for the hybridization. The choice of conditions will, inter alia, depend on the degree of complementarity between the fragments to be hybridized, i.e. a high degree of complementarity requires more stringent conditions such as low salt concentrations, low ionic strength of the buffer and higher temperatures, whereas a low degree of complementarity requires less stringent conditions, e.g. higher salt concentration, higher ionic strength of the buffer or lower temperatures, for the hybridization to take place .

The support to which DNA or RNA fragments of the sample to be analyzed are bound in denatured form is preferably a solid support and may be any of the supports conventionally used in DNA and RNA analysis.

The DNA sequence used for detecting the presence of the chitinase 4 related gene is preferably labelled, e.g. as explained above, and the 829746BI.002/MKA/SPK/A36/1992 04 02 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 pre5 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 of the invention, e.g. DNA sequences encoding a polypeptide having chitinase or /3-1,3-glucanase activity.

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

In a further aspect the present invention relates to an antifungal composition comprising a polypeptide encoded by a DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO :1 or an ana30 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 poly829746BI.002/MKA/SPK/A36/1992 04 02 peptide encoded by the DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO :1 or an analogue or subsequence thereof as defined above, or by a genetic construct of the invention defined above and a suitable vehicle. Microorganisms suitable as constituents in an antifungal composition are mentioned above.

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

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

The antifungal composition of the invention shall, of course, be adapted to its intended purpose, both with respect to the vehicle to be used and with respect to the form, in which the antifungal agent is present. By the term antifungal agent is meant the active constituent of the antifungal composition responsible for or involved in providing the antifungal activity. By the term antifungal poly35 peptide is meant a polypeptide encoded by the chitinase 4 DNA se829746BI.002/MKA/SPK/A36/I992 04 02 quence of the invention or an analogue thereof or a genetic construct of the invention having antifungal activity, i.e. chitinase activity and optionally β-1,3-glucanase activity as defined above.

The antifungal composition may in addition to the polypeptide encoded 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 β-1,3-glucanase activity as defined above, contain one or several chemicals, e.g. fungicides, conventionally used in the combatting of fungi either therapeutically or prophylactically.

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

The medium may, in principle, serve as the vehicle for the antifungal agent, but it is preferred to add a further vehicle suited for the particular intended use.

A culture of the microorganisms expressing the antifungal polypeptide (s) may be obtained as described above using methods known in the art. As mentioned above, it may be necessary or advantageous to subject the microorganism culture to a further treatment so as to release the content of the antifungal polypeptide (s) into the medium or to increase the amount released by secretion.

The medium comprising a substantial amount of the antifungal polypeptide(s) may be directly applied to the soil in which the plants are present or in which the plants are to be grown, or to the plants or 829746BI.002/MKA/SPK/A36/1992 04 02 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 the material to be treated. When the microorganism is a microorganism conventionally found in the soil, e.g. a rhizobacterium, it will generally be desirable that the transformed microorganism establishes itself in the soil so that it continuously may secrete the antifungal polypeptide(s) out into the soil surrounding the plant.

It may be advantageous to add the microorganisms or the medium comprising the antifungal polypeptide(s) to pre-mixes, e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question. For such purposes it is convenient that the microorganisms or the medium is in a solid form, e.g. in a powdery form or in the form of a granule. The powdery form may be obtained by conventional means, e.g. by applying the microorganism on a particulate carrier by spray-drying or an equivalent method.

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

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

Xn accordance with the above, the present invention further relates to a method of inhibiting the germination and/or growth of a chitin containing plant pathogen, such as phytopathogenie fungus, in or on a plant, which method comprises 829746BI.002/MKA/SPK/A36/1992 04 02 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 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.

L5 In most cases the material to be treated with the antifungal composition of the invention is a plant. However, a number of chitin containing fungi exist which infect other materials than plants, e.g. food products such as bread or bread products, milk products cheese, meat, vegetables, cereals, in which the presence and growth of fungi are undesirable. It is contemplated that an antifungal composition according to the present invention may be used to control or combat such fungi. In this respect, it is contemplated that also beverages and containers (any part thereof) used for food products or beverages may be treated with an antifungal composition of the invention either as a prophylactic treatment or a combating treatment.

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

The drawing: Fig. L describes the purification of sugar beet chitinase 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elution of the proteins was performed with a linear gradient of NaCl. The absorbance was recorded at 280 μπι. 829746BI.002/MKA/SPK/A36/1992 04 02 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 /ig of the following proteins. Lanes a and b, chitinase 4; lanes d and e, chitinase 3; lanes f and g, chitinase 2; and lanes c and h, molecular weight markers. The proteins were stained with silver.

Fig. 3 shows the analysis of the water-soluble products released from ^H-chitin by chitinase 4. ^H-chitin was incubated with 4 μg chitinase 4 at 37 °C for 0.25, 0.5, 3 and 24 hours. As a control -^Hchitin was incubated without enzyme at 37°C for 24 hours. The chito10 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 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 lysodeikt icus and the decrease in absorbance at 450 nm was recorded at specified time intervals. 1 ^g of SE (Sure Ellen) was used as a control, and (50 ng and 5^g) 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.

Fig. 5A shows the growth of the fungus when 20 μς 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 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 (absor5 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 culture. Curve B shows the growth of the fungus when 20 pi of a chitinase containing fraction from the chitin-column was added at time 0. In curve C 20 pg of purified chitinase 4 was added to the culture at time 0.

Fig. 7. is an autoradiography showing the effect of chitinase 4 on chitin in the apex of Cercospora hyphae. Incorporation of -'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-acetylglucoseamine into the cell wall in the apex of the fungal hyphae is seen as black dots .

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

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

Fig. 8 shows the separation of tryptic peptides of chitinase 4 by reverse phase HPLC on a Vydac RP-18 column. The peptides were eluted with a linear gradient from 102 to 452 acetonitrile from 25 to 75 minutes. Buffer A was water, whereas B was acetonitrile. Both solvents contained 0,12 trifluoroacetic acid. The flow rate was 0.7 ml/minute. 829746BI.002/MKA/SPK/A36/I992 04 02 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 BisTris buffer at pH 7.0.

Fig. 10 describes the two different serological classes of sugar beet, the chitinase 2 and chitinase 4 class. 5 pg 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. LOB).

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 pi probes of a plasmid preparation containing the chitinase sequences. a chitinase 1 clone from sugar beet 2a chitinase 4 clone form sugar beet a chitinase 76 clone form sugar beet a chitinase clone from pea a SE clone from sugar beet a chitinase clone 1 from tobacco 7a chitinase clone 2 from tobacco a chitinase clone 3 from tobacco a chitinase clone from bean a chitinase 4 like clone from rape seed.

The hybridization was carried out over night at 55°C in the following hybridization buffer: 2 x SSC, 0.1% SDS, 10 x Denhardt's, 50 pg/ml 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 Fig. L2 describes the induction of chitinase and β-1,3-glucanase in sugar beet leaves after infection with Cercospora beticola. Plants were inoculated with a suspension of fungal spores. Leaves were harvested after specified time intervals and crude extracts were prepared. Enzyme activities of chitinase (Fig. 12A) and /3-1,3-glucanase (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 β-1,3-glucanase 3 in protein extracts of Cercospora infected sugar beet leaves. Lanes I and c contain protein extracts from infected and control plants, respectively. Antibodies raised against chitinase 2 (left), chitinase 4 (centre) and β-1,3-glucanase 3 (right) were employed.

Fig. 14. Site directed mutagenesis of amino acids contemplated to form part of the active site of the chitinase 4 enzyme by the use of the PCR technique described in Materials and Methods. SDO is used as 5' primers for all the suggested PCR-reactions. The sequence is indicated by the arrow and is chosen 5' to the unique BamHI site. The sequences for the SDl, SD2, SD3, SD4 and SD5 primers are indicated by arrows. For these 3' primers the complementary sequence with the indicated substitutions are used. The primers can be used for the following substitutions with reference to the genomic chitinase DNA sequence (SEQ ID NO.:3) encoding the amino acid sequence shown in SEQ ID NO.:4. Numbers in brackets denote the number of the corresponding amino acid encoded from the chitinase 4 cDNA (SEQ ID NO.:2) SDl SD2 SD3 SD4 SD5 Trpl70-*Tyr Glul90-*Gln Aspl84- Trp207->Tyr Trp205-”Tyr (169) (189) (183) (206) (204) TGG->TAC GAA-CAA GAT-*AAT TGG->TAC TGG-+TAC The PCR products are digested with the relevant restriction enzymes 829746BI.002/MKA/SPK/A36/1992 04 02 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 Fig. 15A. A sugar beet cDNA /3-1,3-glucanase clone with an underlined 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; 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 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.

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 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 Fig. 16C. Four annealed synthetic oligonucleotides containing the sequence for the C-terminal extension, a changed stop codon, a Smai 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 Smai and the annealed synthetic oligonucleotides digested with Smai and EcoRI.

Fig. 17. Construction of the plant transformation vector pBKL4K4 containing the chitinase 4 DNA sequence shown in SEQ ID N0:l. The 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 oligonucleotides acting as primers in the polymerase chain reaction (PCR) using pB15K4.1 DNA as template. The DNA sequences of KB3 and KB4, respectively, are given in Example 18 and shown in SEQ ID NO:49 and SEQ ID NO:50. Plasmid pPS48 carries a conventional 35S enhanced promoter and a conventional 35S terminator separated by a polylinker containing unique cloning sites. The plant transformation vector pBKL4 (a modification of pBin 19 Bevon, 1984) carries a right and a left T-DNA border sequence from the Agrobacterium Ti plasmid pTiT37, a GUS gene with a 35S promoter and a conventional NOS terminator, a conventional NPTII gene with a 35S promoter and a conventional OCS terminator. A polylinker containing several unique cloning sites is situated between the GUS and the NPTII genes.

Fig. 18. Construction of the plant transformation vector pBKL4K4KSEl containing the DNA sequences encoding chitinase 4 and SE, respectively shown in SEQ ID NO:1 and SEQ ID NO:8. The boxed sequences indicate 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 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 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 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 in the polymerase chain reaction (PCR) using pK76.1 as template. The DNA sequences of KB3 and 340, respectively, are shown in Example 18 and shown in SEQ ID NO:49 and SEQ ID NO:51. Plasmid pPS48 was used in connection with Fig. 17. The plant transformation vector pBKL4 is described in Fig. 17.

Fig. 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 and 270 as the 3' primer. A second round of amplification was then carried out using another gene specific mixed oligonucleotide linked to a restriction site (BamHI-KB9) as the 5' primer and 270 as the 3' primer. The DNA sequence of 270 is shown in Example 7 and SEQ ID NO:30.

Fig. 21 describes the separation of sugar beet /3-1,3-glucanases 1, 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elu829746BI.002/MKA/SPK/A36/1992 04 02 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 and β-1,3-glucanase, respectively, and shown in SEQ ID N0:l, SEQ ID NO:7 and SEQ ID NO:9. The boxed sequences indicate the /3-1,3-glucanase cDNA, the enhanced 35S promoter and the 35S terminator. pGluc 1 is pBluescript carrying the 1249 bp EcoRI fragment encoding the β1,3-glucanase. The hatched box indicates the coding region. Plasmid pPS48M is the same as pPS48 described in connection with the construct shown in Fig. 17, except that the plasmid is supplemented with two additional restriction sites (EcoRI and 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 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. benthaminana using the antibody raised against sugar beet chitinase 4.

C = Control plants containing the GUS and NPT gene construct.

SE = The acidic chitinase.

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

K4 = Chitinase 4 (see Fig. 17).

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

Fig. 24 A comparison between the DNA sequence of the chitinase 4 cDNA sequence shown in SEQ ID NO.:1 and the genomic clone chitinase 76 shown in SEQ ID NO. :5. The position of the chitinase 76 intron is easily seen at position 875 to 1262, The homology of the sequences is about 73%. The figures Fig. 24A, Fig. 24B and Fig. 24C should be considered as one figure. indicates identical nucleotides. 829746BI.002/MKA/SPK/A36/1992 04 02 Fig. 25 A comparison between the amino acid sequence of the chitinase 4 cDNA sequence shown in SEQ ID NO.:2 and chitinase 76 shown in SEQ ID NO.:6. A homology of about 80% is seen. The extra 3 amino acids in chitinase 76 are the amino acids (Ser, Thr, Pro) in position 62-64. : indicates identical amino acids.

Fig. 26 A comparison between the non-coding 5' sequences of the chitinase 4 and chitinase 76 genomic sequences shown in SEQ ID NO.:3 and SEQ ID NO.:5, respectively. 8 boxes of strong homology is observed in the non-coding 5' sequence. It is contemplated that some of these boxes may be of regulatory importance. 829746BI.002/MKA/SPK/A36/1992 04 02 SEQUENCE LISTING SEQ ID NO.:1 the chitinase cDNA sequence (harbored in the cDNA sugar beet chitinase 4 clone B15) SEQ ID NO.:2 the chitinase 4 amino acid sequence (harbored in the 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 LO SEQ ID NO.:5 the DNA sequence of the genomic clone chitinase 76 SEQ ID NO.:6 the deduced amino acid sequence of the genomic clone chitinase 76 SEQ ID NO.:7 the cDNA sequence of the acidic sugar beet chitinase SE SEQ ID NO.:8 the deduced amino acid sequence of the acidic sugar beet chitinase SE SEQ ID NO.:9 the cDNA sequence of the basic sugar beet /3-1,3glucanase SEQ ID NO.:10 the deduced amino acid sequence of the basic sugar 20 beet /3-1,3-glucanase SEQ ID NO.:11. The DNA sequence of the entire sugar beet chitinase 1 gene including introns, promoter and leader sequence, and the amino acid sequence deduced from the coding region of the chitinase 1 gene.

SEQ ID NO.:12 The amino acid sequence deduced from the coding region of the chitinase 1 gene. 829746BI.002/MKA/SPK/A36/1992 64 SEQ ID NO. :13: C-terminal amino acid sequence of a bean chitinase (PHA). SEQ ID NO. : 14: C-terminal amino acid sequence of a basic tobacco chitinase. 5 SEQ ID NO. :15: C-terminal amino acid sequence of an acidic tobacco chitinase. SEQ ID NO. : 16: C-terminal amino acid sequence of the barley chitinase CH26. LO SEQ ID NO. :17: C-terminal amino acid sequence of a basic /3-1,3 glucanase from tobacco . SEQ ID NO. :18: Amino acid sequence of a tryptic peptide of a chitinase 3 from sugar beet. SEQ ID NO. :19: Amino acid sequence of a tryptic peptide of a chitinase 3 from sugar beet. 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. 20 SEQ ID NO. :21: Amino acid sequence of a tryptic peptide of a chitinase 3 from sugar beet. SEQ ID NO. :22: Amino acid sequence of a tryptic peptide of a chitinase 3 from sugar beet. 25 SEQ ID NO. :23: N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from WGA-A (Triticum aestivum). 829746BI.002/MKA/SPK/A36/1992 CD 02 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 (N icotiana tabacum).

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

SEQ ID NO.:28: DNA primer named KB-7 constructed partly from the polypeptide sequence of the acidic chitinase from sugar beet (SEQ ID NO.:9).

L5 SEQ ID NO.:29: Complementary DNA primer named KB-9 constructed partly from the polypeptide sequence of the acidic chitinase from sugar beet (SEQ ID NO.:9).

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

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

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

SEQ ID NO.:34: The amino acid subsequence 3-17 of the amino acid sequence of a β-l,3-glucanase from sugar beet. 829746BI.002/MKA/SPK/A36/1992 04 02 SEQ ID NO.:35: The amino acid subsequence 3-16 of the amino acid sequence of a /3-1,3-glucanase from sugar beet.

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

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

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

SEQ ID NO.:41: N-terminal amino acid sequence of the amino acid sequence from Pea chitinase B.

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

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

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

SEQ ID NO.:45: N-terminal amino acid sequence of the amino acid sequence from barley chitinase T. 829746BI.002/MKA/SPK/A36/1992 04 02 SEQ ID NO.:46: Amino acid subsequence of the active site of the amino acid sequence from a chitinase from tobacco.

SEQ ID NO.:47: Amino acid subsequence of the active site of the polypeptide from a chitinase from tobacco.

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

SEQ ID NO.:49: DNA 5'primer named KB-3 constructed partly from nucleotides No's. 1-15 of SEQ ID NO.:1 and nucleotides No's. 471-485 from SEQ ID NO.:5.

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

SEQ ID NO.:51: Complementary DNA primer named 340 constructed partly from the nucleotide numbers 341-323 of SEQ ID NO. :1. 829746BI.002/MKA/SPK/A36/1992 04 02 DETAILED EXPLANATION OF THE SEQUENCES SEQ ID NO'S :1-12 SEQ ID NO.:1 and SEQ ID NO.:2 The DNA sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO: 2) of the B15 chitinase 4 cDNA clone isolated from a sugar beet 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.

After the stop codon, the cDNA has a 158 bp 3' flanking region with a putative polyadenylation signal at position 847 and a poly A tail.

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

SEQ ID NO :3 and SEQ ID NO:4 The partial DNA sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID N0:4) of a genomic clone encoding the chitinase 4 gene isolated from a sugar beet EMBL3 genomic library The sequence is 691 bp long and encodes the first 112 of the 265 amino acids of the chitinase 4 polypeptide chain. The leader sequence consists of 24 amino acid residues followed by a hevein domain of 35 amino acids. The partially sequenced clone has a 5' non-coding region of 355 bp with a TATA-box sequence (TATAAA) located at position 285, which is 70 bp upstream of the ATG start codon. 829746BI.002/MKA/SPK/A36/1992 04 02 SEQ ID NO :5 and SEQ ID NO:6 The DNA sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6) of a genomic clone encoding the chitinase 76 gene isolated from a sugar beet EMBL3 genomic library The sequence is L838 bp long and encodes a protein having 268 amino acid residues in the polypeptide chain. The leader sequence consists of 24 amino acid residues followed by a hevein domain of 35 and a functional domain of 209 amino acid residues. The gene contains one intron which is located in position 875 to 1262. The exact location of this intron is based on an alignment with the B15 chit 4 cDNA (Fig. 24). The intron borders contain the consensus GT/AG sequences.

A TATA-box sequence (TATAAA) is located at position 378, which is 90 bp upstream of the ATG start codon. A putative poly-A signal (AATAAA) is located at position 1725.

SEQ ID NO:7 and SEQ ID NO:8 The DNA sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:8) of the SE cDNA clone isolated from a sugar beet AZAP cDNA library The sequence is 1106 bp long and encodes a protein having 293 amino acid residues in the polypeptide chain. The leader sequence consists of 25 amino acid residues and the functional domain of 268 amino acid residues. The cDNA clone has a 5' non-coding region of 17 bp and a 3' flanking region of 202 bp.

SEQ ID NO:9 and SEQ ID NO:10 The DNA sequence (SEQ ID NO.:9) and the deduced amino acid sequence (SEQ ID NO. :10) of a /3-1,3-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' non30 coding region of 33 bp and a 182 bp 3' flanking region containing a 829746BI.002/MKA/SPK/A36/1992 04 02 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 from a sugar beet EMBG 3 genomic library.

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

The sequence contains two introns at position 2170-4618 and 47765406. The intron borders contain the consensus GT/AG sequences. A TATA box sequence (TATAAA) is located at position 1355-L360 which is about 70 bp upstream of the ATG start codon. A putative poly A signal (AATAAA) is located at position 6032. 829746BI.002/MKA/SPK/A36/1992 04 02 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 Barkardottir et al., 1987, Developmental Genetics, Vol. 8, pp. 495511 Barkholt et al., 1989, Anal. Biochem., Vol. 117, pp. 318-322 Benton et al., 1977, Science, Vol. 196, p. L80 Bevan, 1984, Nuc. Acid. Res. 12, p. 8711 Bohlmann H. et al., Leaf-specific thionins of barley - a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants, The EMBO Journal, Vol. 7, No. 6, pp. 1559-1565 Bol J. F. et al., Plant pathogenesis-related proteins induced by virus infection, L990, Annu. Rev. Phytopathol., Vol. 28, pp. 113-38 Boiler Τ., Ethylene and the regulation of antifungal hydrolases in plants, 1988, Oxford Survey of Plant Molecular & Cell Biology, Vol. 5, pp. 145-174 Butcher D. N. et al., Tissue Culture Methods for Plant Pathologists, 1980, eds.; D. S. Ingrams and J. P. 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 Ions from Large Molecules, Minaki, June 1990 829746BI.002/MKA/SPK/A36/1992 04 02 Fincher G. B. et al. , Primary structure of the (1-3,1—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, August 1983, Proc. Natl. Acad. Sci. USA, Genetics, Vol. 80, pp. 4803-4807 Fritsch E. F., Molecular Cloning, A laboratory manual, L989, 2nd edition, Cold Spring Harbor Laboratory Press Herrera-Estrella L. et al., Expression of chimaeric genes transferred L0 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 Bi, 1-22 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 Jaynes J., Peptides to the rescue, 16 December 1989, New Scientist, pp. 42-44 829746BI.002/MKA/SPK/A36/1992 04 02 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 decay- ing 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 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 Koncz C. et al., The promoter of T^-DNA gene 5 controls the tissuespecific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector, 1986, Moi Gen Genet, Vol. 204, pp. 383-396 Kragh Κ. M. et al., 1990, Plant Science, Vol. 71, p. 55 Kragh Κ. Μ. , 1991, Chitinases and β-1,3-glucanases in barley (Hordeum vulgar L.), Plant Biology Section, Rise National Laboratory, Roskilde, Denmark Kyhse-Andersen, 1984, J. Biochem. Biophys. Methods 10, pp. 203-209 Leah et al., 1987, Carlsberg Res. Commun. Vol. 52, pp. 31-37 Leah R. et al., Biochemical and Molecular Characterization of Three Barley Seed Proteins with Antifungal Properties, L991, The Journal of Biological Chemistry, Vol. 266, No. 3, pp. 1564-1573 829746BI.002/MKA/SPK/A36/1992 04 02 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 characteriz5 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, Anal.Biochem. , Vol. 198, pp. 318-323 McNeil M. et al., Structure and function of the primary ceil 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 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 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, Vol. 313 Ooms G. et al. , 1982, Plasmid no. 7, pp. 15-29 829746BI.002/MKA/SPK/A36/1992 04 02 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 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 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-/31,3-glucanase, 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 5541-5545 Shinshi H. et al., Structure of a tobacco endochitinase gene: 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 from Aspergillus awamori, 1990, Protein Engineering, Vol. 3, No. 3, pp. 193-198 829746BI.002/MKA/SI’K/A36/1992 W 02 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 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, 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 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 Ll/13 hours day/night cycles, 25/18°C (day/night) and 70% rh. Light intensity was approximately 25000 lux (Osram HQI-T, 400 W/DH). Three weeks after sowing the seedlings were replanted singly in 12 cm plastic pots containing the same growth medium. Twice a day the plants were supplied with water containing 0.1% fertilizer: Stjerne universal fertilizer, 4:1:4 (N:P:K). Six weeks after sowing the plants were ready for infection experiments with Cercospora beticola.

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

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

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

Growth of Cercospora species The fungus was grown on solid growth medium in Petri dishes. Sterile Potato Dextrose Agar (Difco, 39 g/1) was used as growth medium.

A plug of mycelia was placed in the center of the Petri dish and the culture was incubated at room temperature for 4 weeks. Mycelia for spore induction was harvested by cutting off the whole mycelia mat including some agar. 829746EX.002/MKA/SPK/A36/1992 04 02 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. 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 CaCO-j 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 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 con20 taining 20 pg of Tween-20, Using a chromatographic atomizer the suspension was applied to the upper leaf surface of six-week old sugar beet or Nicotiana plants until run off. Immediately after inoculation the plants were placed in a mist chamber kept at 30°C, 100% rh and 24 hours light (cool white). After 5 days of incubation the plants were moved to a growth chamber kept at 30°C, 80% rh and 24 hours light. Approximately 10 days after inoculation necrotic spots developed on mature leaves showing that an infection with Cercospora had been established. After inoculation, the sugar beet plants were harvested at specific time intervals for 829746EX.002/MKA/SPK/A36/1992 04 02 i) small scale purification of chitinase 4, the acidic chitinase SE and β-1,3-glucanase, and ii) a time course study to determine the expression level of total enzyme activity using radiochemical assays and immunoblotting. iii) determination of the expression level of each of the enzymes in transgenic plants using the above (ii) techniques. vi) isolation of mRNA for use in the construction of a cDNA library.

Infection of Nicotians plants with the root pathogen Rhizoctonia solan! 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 12 of potato dextrose broth and autoclaved. The grains were inoculated with agar disks of a growing culture of the fungus and incubat15 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 transplanted into the infected soil. The percentage of surviving plants may be recorded after 1, 2 and 3 weeks, respectively, and after 3 weeks the surviving plants are assessed for root damage. Alternatively, seeds from transgenic plants were sown directly in the infected soil.

Extraction of protein from 1 g of sugar beet leaf material More specifically, the small scale purification was carried out as follows. 1 g of leaf material was homogenized by a Ultra-Turrax homogenizer in citrate buffer (0.1 M, pH 5, 2 ml/g tissue), containing 1 mM of both benzamidine, dithiothreitol and phenylmethylsuphonyl fluo30 ride. Particulate matters were removed by centrifugation at 15,000 x 829746EX.002/MKA/SPK/A36/1992 04 02 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 β-L,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 LO 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 g of chitosan (from Protan; Sea Cure P, No. 709, Norway) was dis15 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 anhy20 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.

Methanol, acetic acid and unreacted acetic anhydride were removed by filtration in a Buchner funnel using Whatman No. i filter paper. The filtrate was transferred to a beaker, 1 1 of 1 Μ Ν32<30β was added and the pH was adjusted to 9 with 6 N NaOH. 50 ml of acetic anhydride was slowly added and the pH adjusted to 9. The reaction was allowed to take place for 1 hour before the final product was collected by filtering on a Buchner funnel. After extensive washing with water, the product was equilibrated in a 10 mM Tris buffer at pH 8.0 before storing at 4°C. The yield was 700 ml of regenerated chitin. A chitin 829746EX.002/.MKA/SPK/A36/1992 04 02 column was prepared from the regenerated chitin by use of the conven tional procedure according to Pharmacia.

Preparation of radioactive colloidal chitin 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 JH-labelled chitin on a Buchner funnel, it was transferred to a beaker. 50 ml of concentrated ice-cold HCl was added, and the chitin was dissolved by stirring for 5 minutes at 0°C The syrupy liquid was filtered through a sintered glass funnel and slowly poured into vigorously stirred 50% aqueous ethanol to precipi tate the chitin in a highly dispersed state. The residue was sedimented by centrifugation and resuspended in water several times to remove excess acid and ethanol. Finally the colloidal chitin was suspended in 200 ml of water and sonicated for 5x1 minute at full power. The ^H-labelled chitin was stored at 4°C before use.

Preparation of a Laminarin column Divinylsulfone activated agarose (Mini-leak high, KEM-EN-TEC, Denmark) was employed to immobilize laminarin (β-1,3-glucan) (from Laminaria digitata, Sigma). 50 g Mini-leak High was dispensed in 200 ml 1 M potassium phosphate (K-P) at pH 11, and 750 mg laminarin dissolved in 5 ml H2O was added. The reaction was allowed to proceed for 16 hours at 25°C on a shaking table. Unreacted divinylsulfone groups were blocked by incubation with a solution of 5% mercaptoethanol in 1 M K-P-buffer at pH 9.5. The reaction time was 16 hours at 25°C. Residual mercaptoethanol was removed by excessive washing 0 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 proce dure according to Pharmacia.

Synthesis of JH-Labelled laminarin Laminarin was labelled with radioactivity by reduction with ^H-label led NaBJH4. 500 mg laminarin (from Laminaria digitata, Sigma) was 829746EX.0O2/MKA/SPK/A36/1992 04 02 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 dissolved in 4 ml of 0.1 N NaOH. This solution was transferred to a reaction wessel containing 5 mCi of NaB^Hp. After stirring for 90 min. at 25°C, 600 pi of 1 M HCl 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.

After storage for 10 min. at 0°C, the precipitate was collected by centrifugation for 5 min. at 15.000 x g. The -^Η-labelled laminarin was dissolved in 500 pi of Η£θ and the precipitation was repeated until the background level· in the supernatant was less than 100 cpm per 20 μΐ. The labelled solution of laminarin was stored at -20°C.

Before use in the /3-1,3-glucanase-assay, the solution was diluted 20fold with water.

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

The mobile system used for RP-HPLC was buffer A: 0.1% TFA in water and buffer Β: 0.Π TFA in acetonitrile.

SDS-PAGE SDS-PAGE of crude plants extracts or partly purified chitinases were performed on an Easy-4 apparatus (Kem-En-Tec, Denmark) using the Tricine SDS-PAGE system described by Schagger and von Jagow (1987). A total of 25 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 Enzyme assays The radiochemical chitinase assay o Chitinase activity was determined radiochemically with JH-chitin as a substrate .

The specific activity of the ^H-chitin was 460 cpm/nmol N-acetylZ· O glucosamine (GlcNAc) equivalent (or 2,3 x 10° cpm/mg JH-chitin). It was determined by scintillation counting and colorimetric determination of GlcNAc after total hydrolysis of -^Η-chitin by crude chitinase preparations from sugar beet leaves and exochitinase from serratia marcescens or Streptomyces griseus.

The assay mixture contained in a total volume of 200 μΐ of enzyme solution, 50 μΐ of JH-chitin suspension (containing 100.000 cpm) and 10 μπιοί 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. before addition of 300 μΐ of 10 % (w/v) TCA. In order to decrease the background reading, 100 μΐ of bovine serum albumin (10 mg/ml) were added before the insoluble ^H-chitin was removed by centrifugation at 15.000 x g for 5 min. The radioactivity in 300 μΐ supernatant was determined by scintillation counting.

The radiochemical β-1,3-glucanase assay β-1,3-glucanase activity was determined radiochemically with JHlabelled laminarin as substrate.

The assay mixture consisted of 50 μΐ of enzyme extract, 50 μΐ of 0,L M Na-citrate pH 5,0 and 10 μΐ of ^H-labelled laminarin (192.000 cpm).

Incubation was carried out for 15 min. at 40°C. To terminate the reaction, 1000 μΐ of abs. Ethanol and 50 μΐ of a saturated NaClsolution 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 μΐ of supernatant was transferred to a scintillation vial. 5 ml of PICO-FLUOR-40 were added and the amount of radioactivity was determined by a liquid scintillation counting. 829746EX.002/MKA/SPK/A36/1992 04 02 Lysozyme assay The lysozyme activity of chitinase 4 was determined by the method described by Selstes et al. (1980). More specifically, lysozyme activity was measured in microtiter plates. Each well· contains cell walls from Micrococcus lysodeikticus suspended in a 20 mM sodium phosphate buffer, pH 7.4, containing 1 mg/ml of BSA. The initial absorbency at 450 nm was adjusted to 0.6 before addition of egg-white lysozyme or plant chitinase 4. The reaction was followed by measuring the decrease in absorbance at 5 min. intervals for 50 min. β-glucuronidase (GUS)-Assay When GUS is employed as a reporter gene in connection with the construction of the genetically transformed plants according to the present invention, the success of the transformation may be determined by use of the following GUS-assay described by Jefferson, 1987.

Leaf tips were sliced into thin sections (<0.5 mm) and incubated in a 2 mM solution of x-gluc. (5-bromo-4-chloro-3-indolyl-/3-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 inten20 sity recorded by visual inspection by microscopy. 829746EX.002/MKA/SPK/A36/1992 04 02 Purification of chitinase 2, 3 and 4, acidic chitinase SE and β1,3-glucanase isoenzymes Acidic and basic chitinase isoenzymes were purified together with β1,3-glucanases from sugar beet leaves as shown in the following flow diagram. kg of sugar beet leaves 0.1 M Na-citrate mM DTT 1 mM BAM, Heat Dialysis Roff FF-Sepharose S Laminarin-Agarose FPLC, Mono-S 1 RP-HPLC I β-ϊ., 3-glucanase 3 and 4 pH 5.0 v treatment, 50°C, 20 min. 90% (NH4)2SO4 v FF-Sepharose Q Chitin column FPLC, Mono-S i RP-HPLC chitinase 2,3, and 4 Homogenization Centrifugation mM Tris , pH 8.0 FF Sepharose Q 1 Chromatofocusing FPLC, Mono P 1 acidic chitinase SE The sugar beet leaves were obtained in Italy (large scale, see Biological Material). In the following each of the purification steps outlined below will be explained. The equipment and procedure used for each step are carried out as described below. 829746EX.002/MKA/SPK/A36/1992 04 02 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, throughout the purification procedure.

Preparation of cellfree-extracts kg of Cercorspora infected leaves were homogenized in 4 1 Na-citrate buffer pH 5.0 containing 1 mM DTT (Dithiothreitol), 1 mM BAM (Benzamidine) (starting buffer) and 200 g Dowex 1x2 (100 μιη/mesh size. The homogenate was squeezed through a double layer of 31 μπι mesh nylon gauze, before centrifugation.

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

Purification of chitinase 2, 3 and 4, acidic chitinase SE and β20 1,3-glucanase by column chromatography Chitinase and β-1,3-glucanase isoenzymes were purified from the ammonium sulfate precipitated protein fraction. After solubilization, the protein solution was dialyzed against 10 mM Tris pH 8.0 containing 1 mM DTT and 1 mM BAM. Denatured proteins were removed by centri25 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 in30 eluding β-1,3-glucanase were removed by extensive washing with the starting buffer. After disconnecting the Fast Flow Sepharose Q co829746EX.002/MKA/SPK/A36/1992 04 02 lumn, the chitinase was eluted from the chitin column with 20 mM acetic acid, pH 3.2 containing 1 mM DTT. The acidic chitinase SE was eluted from the Fast Flow Sepharose Q column with the Tris-buffer containing 0,5 M NaCl.

Purification of /3-1,3-glucanase Separation of β-1,3-glucanase on Cation Exchange Chromatography Proteins which were not adsorbed on either the Fast Flow Sepharose Q nor the chitin column were collected, and concentrated to 60 ml by pressure dialysis with an Amicon PM-10 filter (Danver, MA, U.S.A.).

After dialysis overnight against 20 mM Na-acetate buffer at 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 linear gradient from 0 to 0.5 M NaCl in the starting buffer.

Three major peaks A, B and C of β-1,3-glucanase activity were observed. Peak B was further fractionated by affinity column chromatography on Laminarin-Agarose. Peaks A and C were not further purified.

Purification of β-1,3-glucanase on Laminarin-Agarose A 28 ml column of Laminarin-Agarose was equilibrated with a 10 mM Tris buffer pH 8.0 containing 1 mM of both DTT and BAM. The protein fractions from peak B was combined, concentrated by pressure dialysis to 15 ml and dialyzed against the Tris buffer. After loading of the sample on the Laminarin-Agarose column, the flow through the column was stopped for 20 minutes to allow the β-1,3-glucanase to bind to the affinity ligand. Unabsorbed protein was removed by washing with Tris buffer, β-1,3-glucanase was eluted with 1 M NaCl in Tris buffer.

Purification of 4 β-1,3-glucanase isoenzymes by FPLC Fractions from the Laminarin-Agarose column with /3-1,3-glucanase activity were combined, concentrated and dialyzed overnight against a 829746EX.002/MKA/SPK/A36/1992 04 02 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 substra5 te in the radiochemical assay for β-l,3-glucanase (see above).

Purification of the β-1,3-glucanase on Reverse Phase HPLC The purification was achieved by injecting the FPLC-purified /3-1,3glucanase into the above described Poly F reverse phase HPLC column. Non-adsorbed materials (buffers, salt etc.) were removed by washing with 102 acetonitrile in 0.12 TFA (trifluoro acetic acid). Proteins were eluted by employing a linear gradient of acetonitrile from 10 to 702.

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

Purification of chitinase 2, 3 and 4 Elution of the chitin column with 20 mM acetic acid, pH 3.2, yielded 40 fractions (10 ml/fraction) with chitinase activity. The fractions were combined, adjusted to pH 4.5, concentrated to 15 ml and dialyzed against a 20 mM Na-acetate buffer at pH 4.5. ml aliquots were loaded onto the above mentioned cation exchange column (Mono-S) by the FPLC system (Pharmacia). Non-adsorbed materials were removed by washing with the acetate buffer. Elution of the chitinase isoenzymes was achieved with a linear gradient from 0 to 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 /3-1,3 - glucanase . 829746EX.002/MKA/SPK/A36/1992 04 02 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, Fractions containing chitinase activity as determined by the radiochemical chitinase assay described above were pooled.

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

Application of Polybuffer 74 adjusted to pH 3.6, created a linear pH gradient from 7 to 3.6 and gave desorption of several proteins.

The acidic chitinase SE was still retained on the column at this pH, but it was desorbed by addition of 0.3 M NaCl to the Polybuffer 74 .

Purification of acidic chitinase SE by FPLC Protein fraction with high chitinase activity as determined by the radiochemical chitinase assay described above were pooled and dialyzed against 25 mM Bis-Tris at pH 7.0. The proteins were resolved on a Mono-P FPLC column (Pharmacia) equilibrated with the Bis-Tris buffer. After an initial wash with the starting buffer, three isoenzymes of acidic chitinase SE was separated using a linear salt gradient from 0 to 0.3 M NaCl (Fig. 3). 829746EX.002/MKA/SPK/A36/1992 04 02 Analysis of the enzymatic cleavage pattern of sugar beet chitinase 4 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 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 ^Hlabelled chitin was removed, and an aliquot (300 pi) of the supernatant was applied to a thin layer chromatography (TLC) plate (Silica gel 60 H, Merck). The mobile phase was n-propanoi/H20/NH3 (70/30/1; v/v/v).

A standard of N-acetylglucosamine-derived oligosaccharides was produced by acid hydrolysis of chitin (Rupley, 1964). This standard was used to localize the products from the enzymatic cleavage on the TLC plate. Zones of interest on the TLC plate were removed by scraping with a razor blade, and the silica gel containing the JH-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 spectro20 photometer.

Antifungal activity An inhibitory effect of sugar beet chitinase 4 has been observed on the growth of both Cercospora betieola and Trichoderma reesei either alone or in combination with the acidic chitinase SE and the basic β-L,3-glucanase 3. Germination of spores and/or growth of hyphae from phytopathogenie 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 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 ()4 02 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 inter5 vals.

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

Method I: Microscopy 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 μΐ of a spore suspension (10.000 spores/ml) was placed in the center of the slide. 10 μΐ of a 10 mM Tris-buffer, pH 8.0 or 10 μΐ 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 and full light. At the time for evaluation, the culture was stained with the fluorescent dye Calcofluor White (0.05% (w/v) in water) for 10 min. Calcofluor White binds primarily to cell walls containing nascent structures of chitin, and the fluorescent dye may therefore serve as a marker for differentiation and growth of the hyphae cell wall.

Method II: 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 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 method I. The bioassays were carried out with 5 repeats for each dilution of the fungal spores. The microtiter plate was sealed with tape to avoid evaporation and contamination. After incubation at room temperature on an agitator operated with 50 rpm, the tape was removed and twice a day, the absorbance was measured at 620 nm. The germination and growth of the fungus was followed for 4 days by measuring the absorbance. For each combination of antifungal protein and spore dilution the absorbance vs. time was plotted.

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

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

The microscope slide was coated with an autoradiographic emulsion (Ilford K 5). After drying the emulsion extensively with a fan dryer the slide was placed in the dark for 7 days at 7°C and low relative humidity for exposure. The emulsion was developed by placing the slide in Kodak D-19 developer for 10 minutes followed by fixation for 2 minutes and washed in running water for 10 minutes. After drying the preparation was ready for a microscope analysis of the hyphae of the fungus. 829746EX.002/MKA/SPK/A36/1992 04 02 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 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 produc10 tion of monospecific antibodies to AHAS peptides (Marcussen and Poulsen, 1991). Based on computer analysis of the amino acid sequence for chitinase 4, four peptides were selected on the criteria of hydrophilicity and variability between chitinase 4 and other basic chitinases. Peptides were custom synthesized by Cambridge Research Biochemicals (UK). The structures were verified by mass spectroscopy and amino acid analysis to estimate purity.

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

SDS-PAGE and immunoblotting For immunoblotting, proteins were transferred by semi-dry blotting onto a 0.45 μπι nitrocellulose membrane (Schleicher and Schuell, FGR) after separation by SDS-PAGE. The antigens were probed with primary polyclonal rabbit antibodies raised against chitinase 2 and 4 (see above) and subsequently visualized using alcaline phosphatase con829746EX.002/MKA/SPK/A36/1992 04 02 jugated secondary antibodies (Dakopatts, Denmark) according to KvhseAndersen (1984).

To determine the expression level in transgenic tobacco, the ECL (enhanced chemiluminescence) from Amersham was used. After extraction 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 LOX BSA, before the primary antibodies to sugar beet chitinase 4 diluted 1:1000 was added. The antigen was detected with horse-radish peroxidase conjugated secondary antibodies. Detection reagent was added and after 2 minutes the protein bands are visualized on a Hyperfilm-ECL.

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

Preparation and amino acid sequence analysis of tryptic peptides of sugar beet chitinase 3 and 4, SE and β-1,3-glucanase 3 and 4 Tryptic digestion After RP-HPLC as described above and freeze-drying, 100 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 the dark for 40 minutes at 25°C under nitrogen. Unreacted iodoacetamide was inactivated by addition of 5 pi of /3-mercaptoethanol followed by incubation for 15 minutes at 25°C in the dark. The protein 829746EX.002/MKA/SPK/A36/1992 04 02 solution was dialyzed against 0.2 M ammonium carbonate (pH 8.0) for 24 hours at 4°C in the dark using Eppendorf test tubes with dialysis tubing (10 kDa cut off; Servapore, Serva, FRG) inserted underneath a punctured lid. Thereafter, precipitated protein was pelleted by centrifugation for 5 minutes at 10,000 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 ^g TPCK-treated trypsin in 20 μΐ of ammonium carbonate (pH 8.0) at 40°C for 30 minutes. Finally the supernatant and 6 μg of TPCK-treated trypsin were added. The digestion was allowed to take place at 40°C for 4 hours and stopped by addition of 20 μΐ of TFA. The peptide solution was subjected to RP-HPLC on a VYDAC C18 column (0.46 x 15 cm; 10 μm particle size; The Separations Group, California) using the mobile system described above for RP-HPLC of proteins (see Fig. 4). Collected peptides were diluted 3 times with buffer A and rechromatographed on a Develosil C^g column (0.4 x 10 cm; 5 μπι particle size; Dr. 0 Schou, Novo-Nordisk, Denmark) using the mobile system described above. Selected peptides were subjected to amino acid sequence analysis.

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

Bacterial strains and enzymes Restriction enzymes, Klenow polymerase and T4 DNA ligase were supplied by Boehringer Mannheim and used in accordance with the manufacturers instructions. pBluescript was supplied by Stratagene (USA). pUCl9 was supplied by Boehringer Mannheim. 829746EX.002/MKA/SPK/A36/1992 04 02 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. (1976).

Purification of poiy-A RNA The RNA with a poly-A tail was purified by affinity chromatography through an oligo-dT cellulose column. 0.5 g of oligo-dT cellulose was mixed in 5 ml of 0.5 M NaOH for 5 minutes (1 g of oligo-dT cellulose binds 1.2 mg of poly-A RNA). The resulting mixture was neutralized with LO mM Tris pH 7.5 until pH reached 7.5. An 1 cm column with a diameter of 1 cm was made and equilibrated with 20 ml of column buffer (500 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA). The RNA was denatured at 65°C for 5 minutes, and 5 volumes of column buffer were added to the RNA before chromatography through the column. The runthrough was collected and subjected to chromatography again. The column was washed with column buffer until· OD260 reached 0.01 or less. The poly-A RNA was eluted with TE buffer in 1 ml fractions, and the RNA concentration for each of the fractions was determined at θθ260· The poly-A RNA-containing fractions were pooled and adjusted to 100 mM NaCl and the RNA was precipitated overnight with 2.5 volumes of 96% ethanol at -20°C. The poly-A RNA was spun and dissolved in H2O at a concentration of 1 μg/μl and stored at -20°C. The yield was about 1-2% of total RNA applied to the column.

Isolation of genomic DNA from sugar beet leaves Genomic DNA was isolated from sugar beet leaves (of the variety 60.159.838-131-4) (Dellaporta et al., 1983). g of Cercospora infected sugar beet leaves obtained as described above were ground in liquid N2 and frozen, and the frozen material was transferred to a 40 ml polyethylene centrifuge tube. 15 ml of extraction buffer (100 mM Tris pH 8.0, 50 mM EDTA and 500 mM NaCl) 829746EX.002/MKA/SPK/A36/1992 04 02 were added together with 1 mi of 202 SDS and after mixing, the mixture was incubated at 65°C for 20 min. 5 ml of 3 M potassium acetate were added, the solution was mixed (vortex) and incubated for 20 min. on ice. Subsequently, the mixture was centrifugated for 20 min. at 4°C, 25,000 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 4°C, the pellet was washed with 702 ethanol and thereafter dried briefly before being resuspended in 0.7 ml of X-TE buffer (50 mM Tris, pH 8.0 and L0 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 μΐ of 3 M Naacetate and 500 μΐ of iso-propanol, mixing and spinning for 30 se15 conds. Afterwards, the pellet was dissolved in 400 μΐ of H2O, and the suspension was adjusted to 100 mM NaCl and precipitated with L ml of 962 ethanol. The suspension was centrifugated for 5 min. and the supernatant removed. The pellet was dried briefly, and the DNA dissolved in 200 μΐ of TE buffer. The DNA concentration was determinated using the absorbance at OD26O· where OD26q=1=5O 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.

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

Plating libraries for screening for relevant DNA sequences The titer of the library (either of the cDNA or the genomic Library) was determined according to Sambrook et al. (1990), and about 10θ 829746EX.002/MKA/SPK/A36/1992 04 02 phages were used for each screening. For each 24.5 x 24.5 mm plates, 2.5 x 103 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 MgSO^ and 0.2% maltose to an ODgQQ=l. The mixture was allowed to stand at 37°C for 20-30 minutes.

Subsequently, 30 ml of top agar (0.7% agarose in LB medium with 10 mM MgSO^ 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 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 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 follow25 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 Preparation of radioactive probes for use in screening for sugar beet chitinase 4 in sugar beet cDNA Libraries Relevant oligonucleotides were labelled by phosphorylation with bacteriophage T4 polynucleotide kinase according to the method described in Sambrook et al. (1990). More specifically, the oligonucleotides were synthesized without a phosphate group at their 5' termini ends and were labelled with γ-^^ρ from [y-^pjATP 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 pipette device equipped with a disposable tip, all of the supernatant Ο Π fluid (which contained most of the unincorporated [γ- ZP]ATP) and any free ^2p 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 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 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 02 100 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, 50 mM sodium phosphate, pH 6.8). The hybridization was performed at 37°C for 4 hours in a plastic bag with shaking. The filter was hybridized overnight in the same solution plus the radioactive oligonucleotide probe (the 23-mer chit 4 probe) at 37°C with shaking. A 1x10^ cpm/ml solution of the hybridization buffer was used. The filter was rinsed three times in 6xSSC at 4°C and thereafter washed twice for 30 min. at 4°C in 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 TMAC is hydroscopic, the actual molar concentration (c) must be determined from the refractive index (n) by the formula c=(n-1,331)/0,018). The filter was then washed twice for 20 min. in TMAC-buffer at 55°C.

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

Picking plaques Agar fragments containing positive plagues were picked from the agar plate using mild suction and placed in 500 pi of SM phagebuffer (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.0O2/MKA/SPK/z\36/1992 04 02 101 The phages were then diluted in SM phage buffer and mixed with 200 μΐ of XL1 blue cells (OD^qq - 1). The mixture was allowed to stand for 20 minutes at 37°C and 2.5 ml of top agarose (48°C) was added. The mixture was poured onto 9 cm Petri dishes and filter prints were made for rescreening.

A single well-isolated positive plague useful for making a phage stock to be used in the in vivo excision was picked from the agar plates according to the method described by Sambrook et al (1990) using several steps of replating and rescreening.

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 , 1988) for undigested Lambda ZAP II Cloning Kit, Stratagene Cloning Systems.

Preparation of plasmid DNA Preparation of plasmid DNA was modified according to the method of Sambrook et al. (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 in 200 μΐ of Solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA) in 1.5 ml tubes. 400 μΐ 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 μΐ of 5 M KOAc pH 4.8 was added and subjected to thorough mixing. The resulting mixture was placed on ice for 10 minutes and subsequently subjected to centrifugation at 15,000 x g for L0 minutes at 4°C. 829746EX.002/MKA/SPK/A36/1992 04 02 L02 The supernatant (900 μΐ) was transferred to new tubes and 0.6 volume (540 μΐ) 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°C for 10 minu5 tes and the supernatant was removed.

The pellet was dissolved in 100 μΐ of TE and 100 μΐ 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 μΐ of 96% etha10 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 μΐ) and dried. The dried pellet was redissolved in 50 μΐ of TE.

DNA sequencing The plasmid DNA (double - stranded template) to be sequenced was purified by the above described method. Sequencing was performed as follows : A mixture comprising about 2 μg of the relevant plasmid, 1 μΐ of 2 M NaOH, 2 mM EDTA, 1 μΐ of primer (100 μg/ml) and H2O up to 10 μΐ was incubated at 85°C for 5 minutes and subsequently put on ice.

The mixture was neutralized by adding 1 μΐ 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 μΐ of H2O. 1.5 μΐ of 5 x cone, sequenase buffer was added. The mixture was placed at 37°C for 5 minutes. μΐ of sequetide (Biotechnology Systems NEN® Research Products, Du Pont de Nemours) and 2 μΐ of sequenase (United States Biochemical) were added, resulting in a total volume of the mixture of 13.5 μΐ.

The mixture was placed at room temperature for 5 minutes. 3.1 μΐ of the labelling reaction was transferred to each termination tube (G, A, T and C) containing 2.5 μΐ of the dNTP terminating mix829746EX.002/MKA/SPK/A36/1992 04 02 L03 ture. The mixtures in each of the tubes were allowed to react for 5 minutes at 37°C and 4 pi 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 dried and exposed to an X-ray film.

Labelling of sugar beet SE DNA probes DNA probes to be used in the isolation of the sugar beet acidic chitinase SE was labelled by use of the Stratagene oligolabelling kit prime IT, (Random Primer Kit) according to the manufacturers instructions. More specifically, the following procedure was used: A sample comprising 25 ng (1-23 pi) of the DNA template to be labeled, 0-22 pi of H20 and 10 pi of random oligonucleotide primers (con stituting a total volume of 33 pi) were added to the bottom of a clean microcentrifuge tube. The reaction tubes were heated to 9515 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: pi of 5X primer buffer containing dATP, dGTP and dTTP. pi of labeled nucleotide Q-^^pdcTP (3000 Ci/mM) (Amersham), and pi 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/pl. The reaction components were mixed with the tip of a pipette.

The tubes were incubated at 37-40°C for between 2 and 10 minutes and subsequently, the reaction was stopped by the addition of 2 pi 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 02 104 Then, the probe DNA was made ready for hybridization by mixing the proper amount of radioactive probe with 200 μΐ 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 λ-ΖΑΡ cDNA library were subjected to prehybridization for 2 hours at 67°C under conventional prehybridization conditions using a prehybridizing solution comprising 2 x SSC, 10 x Denhardt's, 0.12 SDS and 50 /ig/ml salmon sperm DNA.

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

After hybridization, a washing procedure was carried out in accordance with the following scheme: x 15 min. in 2 x SSC and 0.12 SDS, and 2 x 15 min. in 1 x SSC and 0.12 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 02 105 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 SE-DNA except for the fact that the hybridization is carried out at a temperature of 55°C. The chitinase 4 probe may be the chitinase 4 DNA sequence shown in SEQ ID NO.:1. It is contemplated that a probe prepared on the basis of a characteristic part or a specific subsequence of the chitinase 4 DNA sequence as disclosed herein, e.g. a probe prepared on the basis of the peptide 4-26 may also be useful. To identify a DNA sequence hybridizing to a specific subsequence of the chitinase 4 DNA sequence and encoding a specific part of the chitinase 4 enzyme, the nucleotide probe is advantageously prepared on the basis of the amino acid sequence of said specific part or a subsequence thereof.

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

The DNA was run on LMT (Low Melting Temperature) agarose (Sea Plaque20 ® 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 μΐ of agarase (free of DNase, from Calbiochem) was added. The mixture was allowed to stand overnight at 37°C and was subsequently extracted twice with phenol and twice with chloroform, subjected to EtOH precipitation and finally resolubilized in H2O.

PCR used for the amplification of cDNA encoding SE, β-1,3-glucanase and chit 76 on the basis of sugar beet mRNA The preparation of a partial cDNA molecule was done by use of the Gene Amp® RNA Amplification Reagent Kit (Perkin Elmer Cetus, USA).

The PCR was performed in accordance with the manufacturer's instruc829746EX.002/MKA/SPK/A36/1992 04 02 106 tion with a few modifications. The reverse transcription protocol was followed using the concentrations in the scheme below.

Component volume Final cone.

MgCl2 solution 4 pi 5 mM 10 x PCR buffer II 2 pi 1 mM dGTP 2 pi 1 mM dATP 2 pi i mM dTTP 2 pi 1 mM dCTP 2 pi 1 mM RNase Inhibitor 1 pi 1 U/pl Reverse Transcriptase 1 Ml 2.5 U/pl primer 270 0.4 pi 2.5 pM mRNA 3.6 pi 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 time (min.) PGR cycles : no. of cycles °C 98 6 addition of Taq polymerase and oil 94 72 94 94 42 72 PCR used in the construction of genetic constructs of the invention and in site-directed mutagenesis on the basis of cloned DNA templates The preparation of the relevant DNA molecule was done by use of the Gene Amp™ DNA Amplification Reagent Kit (Perkin Elmer Cetus, USA) and in accordance with the manufactures instructions except for the temperature cycling. Here the following procedure was used: PCR cycles no. of cycles °C time (min.) 94 1 1/2 60 1 1/2 72 4 30 1 72 7 829746EX.002/MKA/SPK/A36/1992 04 02 108 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 yield of active chitinase 4. A high yield of active and pure chitinase is required in order to have sufficient protein material for i) determining the antifungal potential, ii) preparing and analyzing tryptic peptides which makes it possible to prepare an oligonucleotide probe suitable for isolation of DNA encoding a chitinase, iii) producing monoclonal and polyclonal antibodies thereto.

The isolation and characterization of the DNA encoding the chitinase is necessary when the DNA is to be used for the construction of genetically modified plants having an increased chitinase activity.

Also, a high amount of pure chitinase is required to make it possible to elucidate and characterize the important parts of the enzyme such as the active site.

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

The degree of purity of the enzymes was examined throughout the purification steps by SDS-PAGE on the Phast-System as described in Materials and Methods. After separation on the Mono S FPLC column (Fig. 1) only a single silver stained band for each chitinase 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 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 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 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 produc10 ing chitooligosaccharides or an exochitinase liberating only N-acetylglucosamine from the non-reducing end of chitin or chitooligosaccharides, the pattern of reaction products liberated by chitinase 4 from H-chitin was analyzed by TLC (Fig. 3). Irrespective of duration of incubation, N-acetylglucosamine was only a very minor reac15 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 lysodeicticus using the lysozyme assay described in Materials and Methods (see Fig. 4). This demonstrate, that chitinase 4 is a bifunctional enzyme having both chitinase and lysozyme activity.

EXAMPLE 2 ANTIFUNGAL ACTIVITY OF PURIFIED CHITINASE AND β-1,3-GLUCANASE ISOEN25 ZYMES FROM SUGAR BEET LEAVES Three different bioassays were conducted to ascertain the in vitro antifungal activity of chitinase and β-1,3 -glucanase isoenzymes on the germination and growth of Cercospora beticola. In the same manner the antifungal activity of chitinases and β-L,3-glucanases from other sources or other isozymes from sugar beets may be determinated using either purified enzymes or extracts containing the enzymes. Also, the 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 cent light. The number of hyphae with fluorescent tips and the exten sion of the staining at the individual tip reflect the growth activity in the culture.

When proteins with strong antifungal activity are added, the number of germinating spores are decreased, and the growth rate of the hyphae is drastically reduced. In Fig. 5 is shown the results when a combination of chitinase 4, SE and β-l,3-glucanase 3 is applied to the culture. 60 μΐ of protein solution containing 20 of each antifungal proteins were applied to each microscope slide. When chitinase 4 was used alone or in combination with either β-l,3-glucanase or SE alone, the inhibitory effect was less pronounced. Neither β1,3-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 indicat ing a synergistic effect between chitinase 4 SE and β-i,3-gluca25 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 Cer30 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 02 111 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 label5 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 /3-1,3glucanase was added after the pulse labeling, the radioactivity deposited in the hyphae tip was effectively removed. The amounts of enzymes is similar to that described in Method I (see above). This strongly indicates that the mode of action of chitinase 4 on the cell wall of Cercospora is specifically to hydrolyze the chitin fibers in the hyphae tip and thereby inhibit cell wall synthesis.

The following conclusions can be made on the bases of the above experiments: It is possible to inhibit the growth of Cercospora in spore cultures by addition of chitinase fractions from sugar beet leaves.

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

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

Chitinase/glucanase fractions from Cercospora infected sugar beet plants have a stronger inhibiting effect than fractions purified in the same manner from control plants. 829746EX.002/MKA/SPK/A36/1992 04 02 112 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 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 self 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 02 113 TABLE I Amino acid composition of barley, wheat, bean and sugar beet chitinases 2, 3 and 4 Amino acid Barley Wheat Bean S.B.2 S.B.3 S.B.4 cDNA Aspartic acid 23 28 29 34.4 24.7 24.4 22 Threonine 13.8 22 22 16.2 13.0 12.8 12 Serine 17.7 24 26 21.0 24.8 24.8 24 Glutamic acid 18 20 22 24.9 22.1 21.0 18 Proline 17 15 20 17.1 10.3 10.2 9 Glycine 30.7 52 37 39.7 30.6 30.4 27 Alanine 37.3 27 26 28.0 28.2 28.5 26 Cysteine 7.2 12 16 16.9 16.8 16.9 15 Valine 12.5 14 10 8.6 14.4 14.3 14 Methionine 1.6 3 2 1.8 1.1 1.1 1 Isoleucine 10.8 9 11 11.9 10.9 11.0 11 Leucine 11.3 13 17 16.2 9.0 9.0 8 Tyrosine 11.9 14 15 17.3 12 . 7 12.7 12 Phenylalanine 12.7 14 13 11.5 18.3 18.1 17 Histidine 4.9 4 3 4.4 4.7 5.4 4 Lysine 6.9 8 8 8.7 4.3 3.1 3 Arginine 15.2 14 16 11.3 14.2 16.1 15 Tryptophane 3.2 7 4 nd nd nd 3 MW (KD) 27 29 32 30.6 27.6 27.7 25.9 S.B.2 = sugar beet chitinase 2 S.B.3 = sugar beet chitinase 3 S.B.4 = sugar beet chitinase 4 cDNA = amino acid composition derived from the cDNA sequence encoding the mature protein, chitinase 4 nd = not determined. 829746EX.002/MKA/SPK/A36/1992 04 02 114 Tryptic digestion of sugar beet chitinase 3 and d Analysis of the pure chitinase 4 enzyme has revealed that the Nterminal part of the enzyme is blocked. Thus, by analysis of the mature chitinase 4 it was not directly possible to determine its amino acid sequence, and in order to get sufficient information about the enzyme with the eventual aim of being able to isolate and characterize the DNA by which it is encoded, it was chosen to subject the mature enzyme to tryptic digestion in order to obtain protein fragments (peptides) susceptible to amino acid sequencing.

The tryptic digestion of the purified chitinase enzymes was carried out as described in Materials and Methods above. The tryptic peptides were separated by reverse phase-HPLC on the Vydac RP-18 column mentioned above under the conditions specified in Materials and Methods see (Fig. 8). Peptides representing large peaks at an absor15 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 se20 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 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 oligo30 nucleotide probe to be used in the isolation of DNA encoding chitinase 4 (see Example 4 below). 829746EX.002/MKA/SPK/A36/1992 04 02 115 TABLE II Tryptic peptides of chitinase 3 and 4 Chitinase 3: 3-10.3 S-T-Y-C-Q-S-Y-A-A-F-P-P-N-P-S-K 3-16.1 A-C-V-T-H-E-T-G-H-F-C-Y-I-E-E-I-A-K 3-16.2 V-G-Y-Y-T-Q-Y-C-Q-Q 3-22.3 G-P-L-Q-I-T-W 3-23.3 S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R Chitinase 4: 4-4.2 V-G-Y-Y-T-Q-Y 4-19.3 G-P-L-Q-I-T-W 4-22 S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R 4-23 F-G-F-C-G-S-T-D-A-Y-C-G-E-G-C-R 4-24 S-P-S-S-G-G-G-S-V-S-S-L-V-T-D-A-F-F 4-26 T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-S-I 3-10.3: shown in SEQ ID NO. :18 3-16.1: shown in SEQ ID NO. :19 3-16.2: shown in SEQ ID NO. :20 3-22.3: shown in SEQ ID NO. :21 3-23.3: shown in SEQ ID NO. :22 4-4.2: consisting of amino acids No's 244-250 of SEQ ID NO.:2 4-19.3: consisting of amino acids No's 163-169 4-22: No' s 179-200 4-23: No' s 37-52 4-24: No' s 58-75 4-26: No's 201-224 EXAMPLE 4 ISOLATION AND CHARACTERIZATION OF THE cDNA GENE FOR CHITINASE 4 From the amino acid sequence obtained for peptide 4-26 (see Table II in Example 3), the following very specific oligonucleotide gene probe was synthesized using a DNA synthesizer 381 A (Applied Biosystems). 829746EX.002/MKA/SPK/A36/1992 04 02 11$ Peptide 4-26 T-A-T-W-P-V-M-W-R-V-H-S-V-I-V-B-G-Q-G-P-G-A-S-I Using thia gene probe, tha expression cDNA library AZAP ves screened 10 using the procedure given in Materials and Methods above. 8 cDMA clones were obtained froa A2AF, snd one of the donee vas fully aaquatkeed while the others were only partly sequenced. The sequencing vas parforaed as described In Materials and Methods* above. An alaost full length cDNA clone, chit 4-B15, vas obtained froa the AZAP library and Che DXA ee quence thereof le shown in SBQ ID 80.:1.

On the basis of the cDNA sequence, a deduced aalno acid sequence of chitinase 4 was obtained SBQ ID NO. :2. The deduced aalno acid sequence vas aligned with the partial sequence obtained froa the chitinase 4 protein (ee described in Bxeaple 3 above) and an alaost 100Z Identity was observed. Thia deaonstretes that the isolated αΟΝλ clone codes for the ehitinase 4 polypeptides purified by the chroaatographic procedure described above. The chit 4-B15 cDNA clone Is 966 bp long and encodes a protein having 264 aalno acid residues la Che polypeptide chain out of tha 265 aalno acids predicted for the chitinase 4 genoalc DNA. Tha leader sequence consist probably of 23 aalno acid residues (out of 24 aalno acid raslduea aa deterained for tho genoaia chitinase 4 MA, sea below), followed by e hevein donain of 33 and a functional doaain of 206 aalno acid residues. After the stop codon the cDMA has a 138 bp 3’ noneodiug region.

Aa aentloned In Bxeaple 3 above, it has not been possible to sequence tE« N-tarnInal aalno acid sequence of chitinase 4 directly, because the M-cexalnal ia blocked. However coaperlsoa with wheat gaza agglutinin (WGA-A) and potato ehltinaaa lead to the guess of the first aalno acid being Gin. Hereafter the rest of the aalno add sequence WWdEJUm/MKATSri/Ali/»» ¢( « 117 of the chitinase 4 N-terminal was deduced from the DNA sequence to be Gin-Asn-Cys-Gly-Cys.......

The N-terminal sequence of chitinase 4 was further examined by determining the molecular weight (MW) of the mature chitinase 4 by electrospray mass spectrometry as described by G.J. Feistner et al., 1990. A MW of 25893.6 +/- 10 was observed. On the basis of the amino acid sequence, a MW of 25923 can be calculated. Given that the mature chitinase 4 contains 7 S-S-bridges (loss of 14 protons) and that the first amino acid residue Gin is converted to the pyroglutamyl deriva10 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 Nterminal amino acid sequence given above for the mature chitinase 4.

The N-terminal amino acid sequence could be determined for chitinase 2 and the following terminal amino acid sequence was found in chitinase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala. 829746EX.002/MKA/SPK/A36/1992 04 02 118 TABLE III Comparison of the N-terminal amino acid sequence between different chitin binding proteins: WGA-A: Hevein: Chit. Bean: Chit. Tob. : Chit. SB 2: Chit. SB 4: QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCGKG--CQNGACWTS EQ**R*AGGKL*********W-*W**STDE**SPDHN**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 Heveir : shown in SEQ ID NO. 24 Chit. Bean: shown in SEQ ID NO. Chit. Tob. : shown in SEQ ID NO. Chit. SB 2: shown in SEQ ID NO. Chit. SB 4: consisting of amino :25 :26 :27 acids No's 24-54 of SEQ ID NO.:2 EXAMPLE 5 ISOLATION AND CHARACTERIZATION OF THE SUGAR BEET GENOMIC CLONES CHIT 76 AND CHIT 4 Screening of 500,000 clones from the amplified EMBL3 library containing genomic sugar beet inserts from a partial Sau3A digestion, resulted in the isolation of three clones with the chitinase 4 cDNA as probe.

The three hybridizing clones were characterized by restriction fragment analysis and sequencing. These analysis showed, that one of the clones contained a chitinase gene, now called chitinase 76, the sequence of which is shown in SEQ ID NO.:5. Sequencing of this gene was initiated with the primer used for screening of the AZAP library 829746EX.002/MKA/SPK/A36/1992 04 02 119 (sea Bxsaple 4), sad contiouad with other primers complementary to sequences inside the chic 76 gaaa.

Tit· chic 76 gene codas for a 268 amino acid long chitinase which has 80X homology co Che chitinase 4 amino acid sequence (vide SBQ ZD HO. :1) hut only 34X homology t» the entire chitinase 1 protein (vide SBQ ID 90.:11). The gene contains one intron which ia located in position 875 to 1262. The exact location of Chi* intron le baaed on en alignment with the chitinase 4 cDNA SBQ ID 90.:1 (Pig. 24). The intron borders contain the consensus CT/AG sequences. The chit 76 Intron is located exactly et Che same position as the second intron in the chitinase 1 gene, when Che amine acid sequences of chitinase 1 snd chit 76 are aligned.

A TAIA-box sequence (TATAAA) is located at position 378, which Is 90 bp upstream for tha ATG start codon for translation. A poly-A signal (AATAAA) is located at position 1725 in S1Q ID 90.:5.

In a similar way a genomic clone encoding chitinase 4 was isolated. The DHA has been partially sequenced and about 350 nucleotides of the 5' conceding region has bean elucidated. About 340 nucleotides of the coding region has been ssqusneed. The sequence appear from the SBQ ID 90.: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, 123135, 159-173, 174-207 and 277-328, (Pig. 26).

Baaed on knowledge of tha chit 4 B15 cDRA sequence and the partially sequenced genomic chitinase 4 gene, the rest of the gene can easily he sequenced. It ie contemplated that ths chitinase 4 gene comprises at least 1 intron, probably only 1 corresponding to that given in the same position as that of tha chitinase 76 sequence. eraoxooVMKA/srK/Ax/im w w 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. Analy10 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 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 JH-labelled chitin. The major product of the enzymatic hydrolysis was the hexamers of chitin or higher homologous of chitin oligo saccharides.

Since the major product for chitinase 4 was the dimer, a different mode of action for SE is inferred. No lysozyme activity could be determined for SE at pH 4-9.

The purified enzyme was subjected to tryptic digestion as described in Materials and Methods and in Example 3 above for chitinase 4 and 6 peptides were selected. The peptides were subjected to further purification in the same manner as the tryptic peptides of chitinase 4 described in Example 3 above and the amino acid sequence of the 6 peptides were determined. The peptides were selected using the same criteria as the ones used in connection with chitinase 4, The amino acid sequence of these peptides are shown in Table IV. In addition, the N-terminal amino acid sequence was also determined as shown in the Table IV. 829746EX.0O2/MKA/SPK/A36/1992 04 02 121 TABLE IV B-camlnal SS 22.5: SS 23.0 SS 25.1 SB 26.1 SS 30.4 SI 31.1 S-Q-I-V-I-Y-B-C-Q-B-O-D-E-C-S-VA-D-T-C-H v-L-L-s-x-c-c-c-A-e-e-T AD-Y-L-W-B-T-Y B-R-P-P-C-Q-Y-D-T-S-A-D-B-L-L-S-S Y-C-C-V-M-L-B S-L-S-8-T-D-D-X-B-T-F-X-D-Y-L-V-B-T-Y T-T-V-Q-A-B-Q-I-F-L-C-L-P-A-S-T-D-A-A’C-S-C-P-I R-terainal: consisting of aaino acids Bo’a 26-46 of SBQ ID BO. :8 SE 22.3 SE 23.0 SE 25.1 SX 26.1 SE 30.4 SE 31.1 consisting of aalno acids Bo's 98-109 of 8BQ ID BO.:8 consisting of aalno adds Bo's 121-128 of SBQ XD B0. :8 consisting of aalno adds Bo'a 208-224 of SEQ ZD NO. :8 cons is ting of aalno adds Bo's 271-277 of SBQ ID DO. :8 consisting of ulna adds Bo's 110-128 of SBQ ID B0. :8 consisting of aalno adds Bo’s 229-252 of SBQ ID B0. :8 EXAMPLE 7 ISOLATION ABD CHARACTERIZATION OF THE cDNA FOB THE ACIDIC CHITINASE ISOEHZYMB SE* On the basis of the aalno add sequence of the tryptic peptides listed in Table IV two subsequence· froa the peptides SX 25.1 and SB 31.1 (Table IV) were selected for the synthesis of aired oligonucleotides as they had the best codons. Ihe PCR priaer· KB 7 (SB 25.1) shown In SEQ ID 80.:28, KB-9 (SB 31.1) shown in SBQ ID 80.:29, and the oligo-dl priaer ¢270} shown la SBQ ID 80.: 30, ware prepared in the seae aanner as described in Bxaaple 4 in relation to chitinase 4. Ihe nucleotide sequence of the gene probes ere shown in Table V. wa«x«ia/iiKA/SFKZA36/iJsa μ m 122 TABLE V NPPCQYDT kb - 7. 5'-gactctagaaaJcc^cc^tgJca^taJgaJac- 3' Q A N Q I F KB- 9. 5'-GGAGGATCCCA^GCGAAjCA^ATATT- 3' 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 A partial cDNA molecule was prepared in two steps using the PCRtechnique 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 subse20 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 pUG 19 (Boehringer Mannheim) and sequenced.

The DNA sequence obtained for the partial cDNA molecule constituted by nucleotides 711-962 of the DNA sequence shown in SEQ ID NO.:7.

This cDNA was used to screen the λ-ΖΑΡ cDNA library described in Materials and Methods and 23 cDNA clones were obtained. The longest cDNA clone was sequenced using the method described in Materials and Methods above and was found to be 1070 bp long. The sequence is constituted by nucleotides 37-1106 of the DNA sequence shown in SEQ ID NO.:7. As normally observed in connection with the isolation of cDNA, the entire cDNA was found to be difficult to isolate. Rescreening of the 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 02 123 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 SEQ ID NO .:8 , respectively.

When the amino acid sequence obtained from the N-terminal and the 6 tryptic peptides (107 residues) were compared an almost 100% agreement to the cDNA derived sequence were observed. This demonstrates that the isolated cDNA clone codes for the SE polypeptides purified by the chromatographic procedure described above. The cDNA contains the N-terminal as well as the C-terminal end of the mature protein. The N-terminal of the mature SE is apparent from Table IV. 829746EX.002/MKA/SPK/A36/1992 04 02 124 TABLE VI SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS SE22SAML CUCUMBER ARABIDOPS MAAKIVS--VLFLISLLIFASFESSHGS--ΟΙVIYWGONGDEGSLADTCN MAAHKIT--TTLSIFFLLSSIFRSSDAA--GIAIYWGONGNEGSLASTCA MTNMTLRKHVIYFLFFISCSLSKPSDASRGG1AIYWGONGNEGNLSATCA . . . .. · ·.·»··#·».·». *..*·.

SGNYGTVILAFVATFGNGQTPALNLAGHCDPATN-CNSLSSDIKTCQQAG TGNYEFVNIAFLSSFGSGQAPVLNLAGHCNPDNNGCAFLSDEINSCKSQN TGRYAYVNVAFLVKFGNGOTPELNLAGHCNPAANTCTHFGSOVKDCOSRG 100 IKVLLSIGGGAGRYSLSSTDDANTFADYLWNTYLGGOSSTRPLGDAVLDG 145 VKVLLSIGGGAGSYSLSSADDAKQVANFIWNSYLGGOSDSRPLGAAVLDG 146 IKVMLSLGGGIGNYSIGSREDAKVIADYLWNNFLGGKSSSRPLGDAVLDG 150 IDFDIESGDGRFWDDLARALAGHNNGQKTVYLSAAPOCPLPDASLSTAIA 195 VDFDIESGSGQFWDVLAQELKNFGQ----VILSAAPQCPIPDAHLDAAIK 192 IDFNIELGSPQHWDDLARTLSKFSHRGRKIYLTGAPOCPFPDRLMGSALN 200 .»»,·· ·. . · · * ·. . » .. .

TGLFDYVWVQFYNNPPCQYDT-SADNLLSSWNQWTT-VQANQIFLGLPAS 243 TGLFDSVWVQFYNNPPCHFAD-NADNLLSSWNOWTA-FPTSKLYMGLPAA 240 TKRFDYVWIOFYNNPPCSYSSGNTQNLFDSWNKWTTSIAAQKFFLGLPAA 250 * »» **.·**»»**» ... . .. ♦*..·*·.··. ......»··».

TDAA-GSGFIPADALTSQVLPTIKGSAKYGGVMLWSKAYD--SGYSSAIK 290 REAAPSGGFIPADVLISQVLPTIKASSNYGGVMLWSKAFD--NGYSDSIK 288 PEAA-DSGYIPPDVLTSQILPTLKKSRKYGGVMLWSKFWDDKNGYSSSIL 299 . » * SE22SAML ssv- 293 Consensus length: 304 CUCUMBER GSIG 292 Identity : 137 ( 45.1%) ARABIDOPS ASV- . ». 302 Similarity: 106 ( 34.9%) SE22SAML: shown in SEQ ID NO.:8 Cucumber: shown in SEQ ID NO.:31 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, eC al, 1989) and an Arabidopsis lysozyme/chitinase (Samac et al. , 1990). It appears from this that there is a homology of about 45% when all tree segment are compared. When SE is compared with 829746EX.002/.MKA/SPK/A36/1992 04 02 125 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 β-1,3-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 /3-1,3glucan. The amino acid composition of the sugar beet /3-1,3-glucanase 3 and 4 isoenzymes are similar to the one given for /3-i,3-glucanases from tobacco and barley as shown in Table VII. 829746EX.002/MKA/SPK/A36/1992 04 02 126 TABLE VII Amino acid composition of tobacco and sugar beet /3-1,3- glucanases Amino acid Tobacco2) Sugar beet 3 Sugar beet 4 Barley Gs^) Aspartic A. 35 46.4 53.4 39 Threonine 10 12.6 12.1 14 Serine 23 25.0 27.4 23 Glutamic A. 20 23.4 26.7 20 Proline 19 18.4 21.7 15 Glycine 26 27.8 32.2 31 Alanine 20 31.5 35.1 43 Cysteine 1 0 0 0.7 Valine 18 21.3 25.6 18 Methionine 7 5.1 6.6 4.8 Isoleucine 17 15.9 19.2 14.9 Leucine 23 22.7 27.0 22.1 Tyrosine 16 13.5 15.4 15.4 Phenylalanine 13 12.8 14.6 12.9 Histidine 5 3.1 1.9 1.2 Lysine 13 12.9 16.2 9.7 Arginine 12 12.7 15.0 12.9 Tryptophane 4 ND ND ND MW (KD) 32 32.8 37.6 32 pi 9.9 9.5 9.5 9.8 a) Data taken from Shinshi H. et al., 1983 b) From Kragh et al., 1991 829746BX.002/MKA/SPK/A36/1992 04 02 127 SDS-PAGE of β-1,3-glucanase The apparent molecular weight of β-1,3-glucanase 3 and 4 determined on a 10-15% gradient SDS-gel (Phast-System, Pharmacia) were 33 and 38 kDa, respectively. The isoelectric point was greater than or equal to 9.5. When analyzed by thin layer chromatography, the major reaction products liberated from laminarin after 24 hours of incubation with the two β-1,3-glucanase isoenzymes 3 and 4 were the dimer, laminaribiose. This strongly suggests that the β-l,3-glucanase 3 and 4 isozymes are endoglucanases.

Amino acid sequencing of β-l,3-glucanase 3 and 4 The purified β-1,3-glucanases 3 and 4 were subjected to tryptic digestion using the method described in the above Materials and Methods and selected peptides were further purified and sequenced as described in Materials and Methods and in Example 3 above. The peptides were selected on the basis of the same criteria as the ones used in connection with the selection of the tryptic peptides of chitinase 4 (see Example 3). The amino acid sequence of the peptides are shown in Table VIII. 829746EX.002/MKA/SPK/A36/I992 04 02 128 TABLE VIII Amino acid sequences for /9-1,3-glucanase 3 and 4 isolated from sugar beet leaves Peptide 3-15 W-V-Q-N-N-V-V-P-Y Peptide 3-17 (A)-G-A-P-N-V-P-I-V-V-S-E-S-G-W-P-S-A-G-G Peptide 3-16 L-Q-G-K-V-S Peptide 4-25.1 L-G-N-N-L-P-S-E-E-D-V-V-S-L-Y 10 Peptide 4-26.3 L-D-Y-A-L-F Peptide 4-27.1 Y-I-A-V-G-N-E-I-M-P-N-D-A-E-A-G-S-I-V-P-A-M-Q-N- (Q)-(Q)-(A)-(P)-(R) Peptide 4-28.2 W-V-Q-N-N-V-V-P-Y 15 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 Pep . 3 - L7 : shown Pep. 3-16: shown in SEQ ID NO.:33 in SEQ ID NO,:34 in SEQ ID NO.:35 Pep . 4-25.1: consisting of amino acids No's 37-51 of SEQ ID NO. : 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 02 129 EXAMPLE 9 ISOLATION AMD CHARACTERIZATION OF THE cDNA FOR β-l ,3-CLUCANASg 3 AMD A In the tai Banner aa described above In connection with “SB*, oligo5 nucleotide probee correepoodlng to peptide· fro· the d-l,3-glueanaaa end 4 polypeptide· were synthesized. As 5'prlaer was used the following two sequences in the first round of PCR for isolation of β1.3- glucanase 4: Pep. 3-15: Β. V, Q. Β, N, 0?)..10 0 Ollgoseq. TO-l: 5'TGOGT^CA^AaJaaJcT 3* (shown In SBQ ID HO. :36) C Tn the second round of PCR thn following sequence was used «* 5*pti15 war peptide 4-27.1 consisting of aaino acids Mo's 120-125 of SBQ ID MO.:10 Pep. 4-27.1: .....Β, Β, I, Μ, Ρ, B....

Ollgoseq. TC-2: 5'AA$GA£ATAATGCC§AA (shown in SBQ ID HO.:37) T T By comparing the aaino acid sequences froa /)-1,3-glucanases in barley (Fincher, 1986) and tobacco (Shinehi at el., 1988), a consensus se* quence wae selected and used for construction of e 3'priaer with the following consensus sequence: Pep. seq: P,A,K.F,D/H,E. β Ollgoseq. TC-3: 5'Tc£t$4ACAi{0C$AA (shown ln SBQ ID HO. :38) e This sequence wee used in the second PCR round whereas the 270 priaer 30 used for cloning of aSB* was used in the fizet round. To laolate a β1.3- gluesnase 3 clone, the TC-1 priaer was used since peptide 4*28.2 - peptide 3-15 (see Table VII ia Bxaaple 8). This priaer was used as the 5' priaer for both the PCR reaction·. As the 3' priaer, the TG-3 npMocum/MKA/SK/Aa/ttn m os 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 λ-ΖΑΡ library to isolate clones harboring cDNA encoding /3-1,3-glucanases 3 and 4, respectively. The cDNA sequences and the deduced amino acid sequence of β-1,3-glucanase 4 are shown in SEQ ID NO.:9 and SEQ ID NO.:10, respectively.

EXAMPLE 10 SEROLOGICAL CHARACTERIZATION OF SUGAR BEET CHITINASES 2 AND 4 The serological relationship between chitinase 2 and 4 was analyzed by immunoblotting. When a protein sample containing both chitinase 2 (MW 32 kDa) and 4 (MW 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 (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 groups of chitinases. This observation is further substantiated with the immunoblotting analysis of the pure chitinase 2 and 4 antigens. Antibodies to chitinase 4 detect only chitinase 4, whereas antibodies directed against chitinase 2 only recognize chitinase 2 and no crossreactivity at all was observed. The above results suggest that sugar beet contain two different classes of basic chitinases. This observation is also supported by the information obtained from the amino acid sequencing and the amino acid composition (see Table I in Example 3 above) of the basic chitinases 2 and 4. The difference indicates that the genes coding for chitinase 2 and 4 constitute two distinct gene families. 829746EX.002/MKA/SPK/A36/1992 04 02 131 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 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 these chitinases belong to the same chitinase class. This was further substantiated by serological cross reactivity carried out with antibodies raised against sugar beet chitinase 2. This antibody recognized not only sugar beet chitinase 2, but in addition also chitinase P (27.5 kD) , Q (28.5 kD), Ch. 32 and Ch. 34 from tobacco (Bol and Linthorst, 1990), chitinases Τ, 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 to sugar beet chitinases 2, e.g. a sugar beet chitinase 2 class chitinase. 829746EX.002/MKA/SPK/A36/1992 04 02 132 ZABLE IX N-teralnal «also «eld asqusnco of chitinase leocywes belonging to tho sugar boot chitin**· 2 clo*·; flMMnss· 2 KXjCCNQACCALOHClJCCSQTCWCCimPTOGN Been BQCGttQAOGALCPGCHCCSQPGWCCSTTDYCGP Tobacco BQC03QACG6KCASGLCCSKP6W Pea B B^CGRQAGCATCFIQfLCCSQYGT Pea Al BQCGHQAQfflWPINC Pea A2 BQCCTQACCAICPGGL Barley K EQECsqAGGATCPHXLCCSare Barley T XQQCSQAGGATCFHXUJGSXreW IS Chitin**· 2: shown la SEQ ID SO. :27 Bean: consisting of the aaino acids Mo's 1*33 ot SBQ ID NO,;25 Tobacco: consisting of tho aaino odds Mo's 1-23 of SEQ ID NO. :26 Pea £: shown In SBQ ID NO. :41 Foa Al: shown in SBQ ID NO.:42 Paa A2: shown in SBQ ID NO.:43 Barley K:: shown In SEQ ID SO. :44 Barley T: shown In SEQ ID NO. :45 Definition of a sugar boot chitinase 4 class When antibodies raised against auger hoot chitinase 4 wee eaployed, none of tbs chitinases fra· the ehitinaao 2 class described above could be recognised. Chitin*·· 4 froa sugar boots thus belong· to e new ehitinaao class so far not detected in other plant species than sugar beets. However, recent studios have indicated that chitinases belonging to th· mm now dose exist in rape seed. Thus, protein extracts ot rape seed obtained by e notbod elailar co the one outlined above for auger boat chitinase· wero shown to react with the above eendonod polyclonal antibodies directed against chitinase 4 fro· sugar beets (see Kasnuasen et el., 1992 8B746EXJlB/MK>VSrR/A3a/I«B U H 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 homol5 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 examined at 55°C between the cDNA encoding the sugar beet chitinase 4 enzymes and DNA encoding chitinases from other plants such as pea, tobacco and beans as well as DNA form sugar beet encoding the chitinase 1 and SE enzymes. These results therefore further indicate • that the chitinase 4 enzyme belongs to a new class of chitinases.

The high degree of homology between the cDNA encoding the chitinase 4 enzyme and the DNA encoding the chitinase from rape seed chitinase shown by the high degree of DNA hybridization further indicates that the genes encoding chitinase 4 in sugar beets and the genes encoding the chitinases in rape seed are significantly homologous and thus belong to the same gene class. This is supported by the results disclosed in Example 10 showing a high degree of serological homology between the mature enzymes from the two plants.

EXAMPLE 12 TRANSFORMATION OF BACTERIA CELLS Agrobacterium tumefaciens (the strain LBA 4404, Ooms et al., 1982) was transformed with the plant transformation vector, pBKL4K4, the preparation of which is described in Example 18, using a freeze/thaw method essentially as described by An et al, (1988). For the freeze/thaw method the bacteria to be transformed were cultivated in LB829746EX.002/MKA/SPK/A36/1992 04 02 134 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 OD 600 (OD^qq) 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 superna5 tant was removed and the bacteria were carefully suspended in 1 ml of icecold 20 mM CaC^· 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 pg of plasmid DNA was first added to a cryo tube with the frozen bacteria. The bacteria were incubated in a 37°C water bath for 5 minutes, 1 ml of LB-medium, pH 7.4, was added to the cryo tube, and the mixture was incubated for 4 hours at room temperature using mild agitation (agitation table, 100 x rpm). The cryo tube was centrifuged for 30 sec. at 10.000 x g, 4°C.

The supernatant was removed and the bacteria were resuspended in 0.1 ml of LB-medium, pH 7.4. The bacteria were plated on to a YMB-dish with 50 mg/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 to the use of the bacteria in the transformation of the plants.

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

EXAMPLE 13 PREPARATION OF GENETICALLY TRANSFORMED TOBACCO (Nicotiana benthamiana and N. tabacum) PLANTS Plant material Leaves from plants to be genetically transformed were obtained from plants grown in vitro or in vivo. In the latter case, the leaves were sterilized prior to transformation. Sterilization was performed by placing the leaves for 20 min. in a solution of 5% Ca-hypochlorite 829746EX.002/MKA/SPK/A36/1992 04 02 135 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).

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 hours before transformation a suspension of Agrobacteria transfor10 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).

Trans format ion Transformation of the plant was done essentially as described by 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 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 μΜ). On the day of transformation a piece of sterile filter paper was placed on the co-cultivation dishes, and the leaf pieces which had been dipped in the bacteria suspension were placed upside down on the filter paper.

The leaf pieces were incubated in a growth chamber in weak light, e.g. 12 hours of light and 12 hours of darkness for 2-3 days. 829746EX.002/MKA/SPK/A36/1992 04 02 136 Select ion/regeneration The leaf pieces were transferred to Petri dishes containing shootinducing MS-medium with 300 mg/1 of kanamycin and 800 mg/1 of carbenicillin and sub-cultivated every 4 weeks to the same medium.

Shoots which appear on shoot-inducing MS-medium 300 k/c dishes were transferred to containers with 1/2 MSO 300 k/c. The shoots were subcultivated when needed. After approximately 2 weeks, the expression of the β-glucoronidase activity using the GUS-assay (see Materials and Methods) was performed on the leaf tips of green shoots.

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

EXAMPLE L4 PREPARATION OF GENETICALLY TRANSFORMED SUGAR BEETS PLANTS BY MEANS OF TRANSFORMATION WITH BACTERIA Transformation was carried out using cotyledonary explants as de20 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 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 mg/1 of kanamycin, 800 mg/1 of carbenicillin and 500 mg/ml of cefo829746EX.002/MKA/SPK/A36/I992 04 02 137 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 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 chitinase SE and the selective markers, NPT-II and GUS. Selection of the callus and shoots on kanamycin has proved that the obtained tissue expresses the GUS marker and thus that the transformation has occurred.

EXAMPLE 15 ANALYSIS OF CHITINASE AND β-1,3-GLUCANASE IN TRANSGENIC PLANTS The expression levels for chitinase and /3-1,3-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 dif20 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.

Using the biological methods I-III, the antifungal activity of the enzymes in the genetically transformed plants can be determined. A retarded growth of the fungi hyphae shows that the transformation has resulted in a plant having an improved tolerance i.e. an increased antifungal activity to the phytopatogenic fungi compared to a non-transformed plant. 829746EX.0O2/MKA/SPK/A36/1992 04 02 138 In the radiochemical assays, -^Η-chitin or ^H-laminarin are used as substrates for either chitinase or /3-1,3-glucanase, respectively. Using standard curves of product formation vs. enzyme amount, the activity for both chitinase and β-1,3-glucanase in crude plant ex5 tracts can be determined. This is illustrated further in a time course experiment where the level of either chitinase (Fig. 12a upper part) or /3-1,3-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 β10 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 /3-1,3-glucanase in transgenic plants can easily be recorded.

These techniques, however, do not differentiate between the various chitinase and /3-1,3-glucanase isozymes. Only the total enzyme activities for all the chitinase or all the β-ί,3-glucanase isoenzymes are determined. However, the presence of the various chitinase and β20 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 /3-1,3-glucanase 3 recognized only one single protein in the Cercospora infected leaf material (Fig. 13). In contrast, no antigen was detected in the control leaves. This is in agreement with the low constitutive level of expression observed in control plants for /3-1,3-glucanase using the radiochemical assay. When antibodies raised against either chitinase 2 or 4 were employed, two major protein bands were induced in the infected leaf tissues. Chitinase 2 antibodies detect a 26 and a 32 kDa band, whereas two proteins having molecular weights of 29 and,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.

Similarly, the antibody to chitinase 4 detected the authentic chitin829746EX.002/MKA/SPK/A36/1992 04 02 139 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 NO.:8). The 3-D structure of chitinase 4 and SE on the nitrocel5 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 the antibody is diluted 1:5000 or 1:10,000.

Transgenic tobacco plants (Nicotiana tabacum and/or N. benthamiana) were transformed with either chitinase 4, chitinase 76, the acidic chitinase or chitinase 4 + the acidic chitinase. After selection on kanamycin and regeneration, the transformed plants were examined with respect of i) GUS activity, II) expression of chitinase genes, and iii) degree of resistance against C. nicotiana or R. solani. The transgenic plants expressed GUS-activity in variable amounts. Only plants with high GUS-activity were subjected to further analysis. The expression of the chitinase gene products were analysed by immunoblotting using the ECL-system described in Materials and Methods and the antibody raised against chitinase 4. In leaf extract from N. benthamiana, transformed with only NPT and GUS, no protein band could be detected by this antibody (see Fig. 23, lane ”C). Transgenic plants containing the constructs, the acidic chitinase, the genomic chitinase 76, chitinase 4, and the double gene construct chitinase 4 + the acidic chitinase, showed a strong positive reaction (see lanes SE, K76, K4, K4 + SE, respectively). To evaluate the level of expression 10 pg of chitinase 4 isolated from sugar beet was included, lane Std in Fig. 23.

A broad protein band was observed in extracts from transgenic plants with the chitinase 4 or chitinase 76 gene constructs. When smaller amounts of proteins were applied to the various lanes of the SDSPAGE, this band could be resolved into three distinct protein bands, having MU of 29, 27 and 25 kD, respectively. The reasons for the triple bands are not known at present. It is, however, contemplated that chitinase 4 is i) not processed given rise to a protein 829746EX.002/MKA/SPK/A36/1992 04 02 140 maintaining the signal peptide = the 29 kD band, ii) cleaved at the normal processing site at the amino acid sequence Leu Vai 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 putative tobacco processing site is localized at the amino acid sequence Ala Ser Ala Ser - Cys Ala (position 85-86 in SEQ ID NO.:2). This cleavage site may give rise to the 25 kD polypeptide band. In addition to malfunctinal processing of sugar beet chitinase 4 in transgenic tobacco, the translocation of chitinase 4 was inhibited.

In sugar beet, this basic chitinase 4 is deposited in the extracellular space. In transgenic tobacco, cytochemical analysis, demonstrate clearly that sugar beet chitinase 4 is localized intracellularly.

Preliminary experiments to examine the degree of resistance of transgenic tobacco plants against R. solani and C. nicotiana have been performed. The transgenic plants with the chitinase 4 (10 plants) and chitinase 4 + the acidic chitinase (4 plants) showed less disease symptoms, whereas the control plants (10 plants) containing the GUS and NPT genes were severely infected with C. nicotiana.

When seeds of N. tabacum containing the chitinase 4 gene construct were germinated in R. solani infected soil, the survival and growth were improved as compared to the seed from non-transgenic plants.

EXAMPLE 16 MODIFICATION OF THE SUGAR BEET CHITINASE 4 BY SITE DIRECTED MUTAGENE25 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 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 02 141 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 of the amino acid sequence of the mature chitinase 4 enzyme, optionally in combination with an analysis of the enzyme's 3-D structure. Especially amino acids forming part of the active site of the enzyme or of epitopes thereof as well as amino acids of importance for substrate specificity and substrate binding are of interest in this connection.

The active 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 chitinase C demonstrated that chemical modification with carbodiimide and N-bromosuccinimide (NBS) completely inhibits the enzymatic activity (results not shown). Similar experiments carried out with glucoamylase from Aspergillus niger (Sierks et al.,1990) have elucidated the mode of action by which carbodiimide and NBS inactivates this enzyme. Carbodiimide is covalently linked to the three essential acidic groups (glutamic and aspartic acid residues) constituting the catalytic site of glucoamylase. NBS oxidizes Trp residues important in either stabilizing the transition state intermediate of the catalysis or Trp residues involved in substrate binding at a distance from the catalytic site. The experiments with chitinase C indicate that three acidic and two Trp-residues are very important constituents of the active site. Secondly, by comparison to the active sites of other enzymes which hydrolyze oligosaccharide chains including the glucoamylase described above, the active site of chitinase 4 is contemplated to be constituted by amino acid residue 183 (Asp) and 189 (Glu) in SEQ ID NO.:1 (corresponding to amino acid residue 184 and 190 in the amino acid sequence encoded by the genomic chitinase 4 amino acid sequence. The number given below in brackets denotes the number of the amino acid from the corresponding amino acid sequence encoded by the genomic chitinase 4). In contrast, chitinase C from 829746EX.002/MKA/SPK/A36/1992 04 02 142 barley and all other plant chitinases of the same serological class (the sugar beet chitinase 2 class) have three aspartic acid residues (corresponding to amino acid residues 183, 189 and 194 of chitinase 4 (SEQ ID NO:2)) in the active site (184, 190 and 195, respectively).

The position of the two important Trp residues involved in the active site of chitinase C have not been elucidated. Since chitinase 4 only contain three Trp residues in contrast to the 6 present in chitinase C, the important Trp residues may be more easily identified in chitinase 4 .

The two acidic residues 183 Asp and 189 Glu of SEQ ID NO:2 (184 and 190, respectively) forming the active site of chitinase 4 is contained in the peptide 4-22: SIGFDGLNAPETVANNAVTAFR. Important Trpresidues of the active sites may be contained in peptide 4-19.3: GPLQITW and peptide 4-26: TAFWFWMNNVHSVIVNGQGFGASI.

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

Accordingly, an interesting modification is one in which the glutamic acid in position 189 (190) is substituted with aspargine and/or the aspartic acid in position 183 (184) are substituted with glutamine. Changing the carboxyl groups Asp 183 (184) to Asn and for Glu 189 (190) to 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 02 143 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 carried out: For the PCR reaction the 3' primer SD1 is used (see Fig. 14).

The resulting PCR product (from bp 301 to 538) is digested with BamHI and PvuII and interchanged with the corresponding fragment of the chitinase 4 gene by conventional methods (Sambrook et al, 1990).

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 02 144 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.

In a similar manner, other desirable modifications may be carried out.

EXAMPLE 17 CONSTRUCTIONS OF GENETIC CONSTRUCTS WITH SUITABLE C-TERMINAL EXTENSION C-terminal amino acid sequences found in connection with various plant chitinases and glucanases are exemplified in the specification and are believed to prove useful in modification of one or more of the antifungal enzymes encoded by the genetic constructs according to the present invention which do not comprise a C-terminal extension so as to allow these enzymes to be translocated to the vacuole.

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

Fig. 15a illustrates the sugar beet β-1,3-glucanase cDNA with a tobacco C-terminal extension which is underlined in the figure. 829746EX.002/MKA/SPK/A36/1992 04 02 145 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 XbalEcoRI fragment in the β-1,3-glucanase gene with the PCR product digested with Xbal and Dral and the annealed synthetic oligonucleo10 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 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 BamHI20 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 β-1,3-glucanase sequences. The N-terminal sequence may in a similar manner be exchanged with other N-terminal sequences. Of particular interest may be the N-terminal sequence of chitinase 1 shown in the SEQ ID NO.:12, the N-terminal sequence of the acidic chitinase SE shown in SEQ ID NO.:8, the N-terminal sequence of chitinase 4 shown in SEQ ID NO.:2, the N-terminal sequence of chitinase 76 shown in SEQ ID NO. :6, the N-terminal sequence of β-1,3-glucanase shown in SEQ ID 829746EX.002/MKA/SPK/A36/1992 04 02 146 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.

EXAMPLE 18 Genetic constructs The excised recombinant pBluescript containing the chitinase 4 cDNA gene (B15 chitinase 4) was subcloned in order to supply the gene with an enhanced 35S promoter and a 35S terminator. This construct was transferred to the plant transformation vector pBKL4 containing a NPTII and a GUS gene. pBKL4 is a derivative of the A. tumefaciens Ti-plasmid pBI121 (Bevan et al., 1984), in which the genes between the left and right borders have been replaced with the following genes: 1) β-glucoronidase (GUS) from E. coli equipped with a CaMV 35S promoter and a Nopaline Synthase terminator (NOS) and 2) Neomycin phosphotransferase (NPT) from £. coli equipped with a CaMV 35S promoter and an Octopine Synthase (OCS) terminator.

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

The oligonucleotide KB3 (shown in SEQ ID NO.:49): (5'CCGAAGCTTAGATCTAAACAACAACATGTCTTCT(J)T(J)GGACC 3') 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 (J) 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 02 147 The oligonucleotide KB4 (shown in SEQ ID NO.:50): (5'GCACACGTAGCGCTAGCTTGG3') I ** I 261 Nhel 241 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 H20 the DNA was digested with Hindlll and Nhel. The Hindlll-Nhel fragment from pB15 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) | | 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 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 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 BamHISmal (Fig. 17). The chitinase 4 gene with the enhanced 35S promoter and 35S terminator was transferred to the plant transformation vector pBKL4 (Fig. 17) as a Hindlll fragment (Fig. 17). The resulting vector, pBKL4K4, harboured in an E, coli DH5q has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-33OO Braunschweig (DSM) on 30 July, L991 under 829746EX.002/MKA/SPK/A36/1992 04 02 148 the provisions of the Budapest Treaty under the socaasion number OSH 6633.

The SE” gene wee then Introduced into the construct pBKL4K4 (Fig. 17) . A full length SB gene waa constructed by coeblning the S' end 5 of the gene froa the pSuri clone (EcoRI-Kpnl) with the rest of the gene froa pSE22 (Kpnl-Hindlll) In the cloning vector pUC19 (Fig. 18) . The SI* gene waa subclonod in the Seal site of pPS48 se a BcoRI-HindTII fragment filled in with Klenov polymerase in the presence of all four nucleotidee. The orientation of the gene with respect to the enhanced 358 promoter and 358 terainator was examined by restriction enzyme analysis and further confirmed by sequence analysis.

The SI* gene with the enhanced 35S promoter and 35S terainator was cloned in the Kpnl site of pBKLAK4 as a Hindlll fragment in the presence of a Hindlll-Kpnl adapter (Fig. 18). The Hindlll fragment was furthermore cloned in the Hindlll alto of pBKIA.

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

In a similar aanner, the glucanase gens can be Introduced into the construct pBKIA, p8KLAK4. pBKLAKSE, or pBKLXK4KSE (Fig. 22).

The full length cDKA done (SBQ ID )10.:9) was digested with EcoBI and Bglll, the sticky ends were filled In with Klenov polymerase in the presence of ell four dHTP's. The glucanase gens ia then subcloned in the Seal site of pPS48Mod. The orientation of the gene with respect to the enhanced 33S proeioter and the 358 terainator, respectively, ay be by restriction enzyme analysis and further confirmed by sequence analysis.

The glucanase gene with the enhanced 3SS promoter and fhe 35S terminator ia cloned in the EcoRI alee of pBKIA, pBKL4K4, pBKL&KSE, pBKL4K4KSI. cmeExaaa/MKA/VK/AM/iM » « SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Dalgaard Mikkelsen, Joem Bojsen, Kirsten Nielsen, Klaus K.

Berglund, Lars (ii) TITLE OF INVENTION: A plant chitinase gene and use thereof (iii) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Plougmann & Vingtoft (B) STREET: Sankt Annae Plads 11 (C) CITY: Copenhagen (E) COUNTRY: Denmark (F) ZIP: DK-1021 (v) OOMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Plougmann, Ole (C) REFERENCE/DOCKET NUMBER: 329751MKA/SPK (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 45 33 11 05 66 (B) TET .EFAX: 45 33 11 18 87 (C) TELEX: 18333 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 966 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (B) STRAIN: monova (F) TISSUE TYPE: leaf (vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet lampda ZAP cDNA library (B) CLONE: B15 chitinase 4 cDNA clone (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..793 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: G TCT TCT TTC GGA CCA ATC TTT GCC ATA CIG ATG GCA CIT GCT TGT 46 Ser Ser Phe Gly Pro lie Phe Ala lie Leu Met Ala Leu Ala Cys 15 ATG TCA AGC ACC CTA GIT GIG GCT CAA AAC TGT GGA TGT GCC TCT AAT 94 Met Ser Ser Thr Leu 20 Val Val Ala Gin Asn 25 Cys Gly Cys Ala Ser Asn 30 ΊΤΑ TGT TGT AGC CGA ITT GGT TTC TGT GGC TCC ACA GAC GCC TAC TGC 142 Leu Cys Cys Ser Arg 35 Phe Gly Phe Cys Gly 40 Ser Thr Asp Ala Tyr Cys 45 GGC GAG GGG TGC AGA GAA GGT CCT TGT AGA TCA COG TCT AGT GGT GGT 190 Gly Glu Gly Cys Arg 50 Glu Gly Pro Cys Arg 55 Ser Pro Ser 60 Ser Gly Gly GGT TCC GIG TOG AGT TIG GIG AOC GAT GCG TTC TTT AAT AGG ATC ATT 238 Gly Ser 65 Val Ser Ser Leu Val 70 Thr Asp Ala Hie Phe 75 Asn Arg lie He AAC CAA GCT AGC GCT AGC TGT GCT GGT AAG AGA TTC TAC ACC AGG GCT 286 Asn 80 Gin Ala Ser Ala Ser Cys 85 Ala Gly Lys Arg 90 Hie Tyr Thr Arg Ala 95 GCC TTT TIG AGT GCT CTC AGA ΤΊΤ TAT CCC CAG TTT GGT AGT GGA TCC 334 Ala Phe Leu Ser Ala 100 Leu Arg Phe Tyr Pro 105 Gin Phe Gly Ser Gly Ser 110 TCC GAT GTC GTT AGG CGT GAA GTT GCT GCA TTC TTT GCC CAT GTC ACC 382 Ser Asp Val Val Arg Arg Glu 115 Val Ala Ala 120 Phe Phe Ala His Val Thr 125 CAT GAA ACT GGA CAT TTT TGC TAC ATA GAG GAG ATT GCA AAG TCA ACC 430 His Glu Thr 130 Gly His Phe Cys Tyr lie Glu 135 Glu He Ala 140 Lys Ser Thr TAT TGT CAG TCA AGT GCA GCA ΠΤ CCA TGC AAC CCA ACT AAG CAA TAC 478 Tyr Cys 145 Gin Ser Ser Ala Ala 150 Phe Pro Cys Asn Pro 155 Ser Lys Gin Tyr TAC GGA AGG GGG CCT CIT CAG ATC ACA TGG AAT TAT AAC TAC ATA CCA 526 Tyr 160 Gly Arg Gly Pro Leu Gin 165 lie Thr Trp Asn 170 Tyr Asn Tyr He Pro 175 GCT GGT CGA AGC ATT GGA ITT GAT GGT CIG AAT GCA CCA GAA ACA GTT 574 Ala Gly Arg Ser lie 180 Gly Phe Asp Gly Leu 185 Asn Ala Pro Glu Thr Val 190 GCC AAC AAT GCC GIG ACT GCA TTC OGG ACA GCC TTC TGG TIT TGG ATG 622 1 .bl Ala Asn Asn Ala Val Ihr Ala Phe Arg Thr Ala Phe Trp Phe Trp Met 195 200 205 AAC AAT GTC CAC TCT GIT ATC GTC AAT GGC CAA GGG TTC GGG GCC AGC Asn Asn Val His Ser Val Ile Val Asn Gly Gin Gly Phe Gly Ala Ser 210 215 220 ATT OGA GCT ATC AAT GGA ATC GAA TCT AAT GCT GCT AAC TCT GCT GCT lie Arg Ala lie Asn Gly Ile Glu Cys Asn Gly Gly Asn Ser Ala Ala 225 230 235 GIT ACT GCT CGT GIT GGG TAC TAT ACT CAG TAT TCT CAA CAG CIT GGC Val Ihr Ala Arg Val Gly Tyr Tyr Ihr Gin Tyr Cys Gin Gin Leu Gly 240 245 250 255 GIT TOG CCA GGG AAT AAC CTC CGT TCC TAGTCAAATC GCIGGITITC Val Ser Pro Gly Asn Asn Leu Arg Cys 260 CTGGTCAGAA TTCACAAGGC TTAGTCAAAA GAAAATAAAG AGAATTATGT AAACTGTTCA TTTCTCATCT AACTTCCTAC TTIGGACAAG CATTAACTTC GTTAOGAGGC TITATCCATA AAGGAATGAA ΑΑΑΤΑΊΤΑΊΤ TAAAAAAAAA AAA 670 718 766 813 873 933 966 (2) INFORMATICS FOR SBQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 264 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ser Ser Phe Gly Pro Ile Phe Ala Ile Leu Met Ala Leu Ala Cys Met 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 Ile Ile Asn 70 75 80 Gin Ala Ser Ala Ser Cys Ala Gly Lys Arg Phe Tyr Thr Arg Ala Ala 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 115 120 125 Glu Thr Gly His Phe Cys Tyr He Glu Glu He Ala Lys Ser Thr Tyr 130 135 140 Cys Gin 145 Ser Ser Ala Ala 150 Phe Pro Cys Asn Pro Ser Lys 155 Gin Tyr Tyr 160 Gly Arg Gly Fro Leu 165 Gin He Thr Trp Asn 170 Tyr Asn Tyr lie Pro 175 Ala Gly Arg Ser lie Gly 180 Phe Asp Gly Leu Asn 185 Ala Pro Glu Thr 190 Val Ala Asn Asn Ala 195 Val Thr Ala Phe Arg Thr Ala 200 Phe Trp Phe 205 Trp Met Asn Asn Val 210 His Ser Val He Val Asn Gly Gin 215 Gly Phe Gly 220 Ala Ser He Arg Ala 225 He Asn Gly He 230 Glu Cys Asn Gly Gly Asn Ser 235 Ala Ala Val 240 Thr Ala Arg Val Gly Tyr Tyr Thr Gin Tyr Cys Gin Gin Leu Gly Val 245 250 255 Ser Pro Gly Asn Asn Leu Arg Cys 260 (2) INFORMATION FOR SBQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 691 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (B) STRAIN: monova (F) TISSUE TYPE: leaf (vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet EMBL3 genomic library (B) CLONE: genomic chitinase 4 clone (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 356..691 ATATATATAA CAAAGGITTC TTCCITTCAT TTTCCITGAA CAAGICAAAC TATNTACACC 120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAGCTTATTG TCCAAATAAT TTTACATAAC AAGTTCAGTG AACGGAGAAG ATAACTATCC AAATCATIGT CCAAAATAAA ATTAAATGIG TTGGCTAAGT CAAAITTGAA CACTTCIGAA TCTATCTAAA ATATCTCCAT TCCCATCTTA TTAATTAGAA TACAAGTAAG CAAGTAGCCA AACTAGTAAA CATITCCTCA AAGTACCACC CTTATAATTT TCTATATAAA CCCATATACA AGTGTCTAGT TTCCTCATCC CATACATTAT ATTGTTGGCT TTAACATACT CCAAAATGTC TTCITTOGGA CCAATCITIG CCATACTCAT GGCACTTGCT TCTAIGTCAA GCACCCTAGT TGrlGGCTCAA AACIGTGGAT GTGCCTCTAA TTEATGITGT AGCOGAITIG GTTTCIGTGG CTCCACAGAC GCCTACIGCG GGGAGGGGTG CAGAGAAGGT CCTTCTAGAT CACCGTCTAG TGGrTGGTGGT TCCGTGTOGA GTTIGG7IGAC OGATGCGTTC TTTAATAGGA TCATTAACCA AGCTAGOGCT AGCIGTGCIG GTAAGAGATT CTACACCAGG GCIGCTITIT TGAGTGCTCT CAGAITITAT OOCCAGTHG GTAGTCGAIC C (2) INFORMATION FOR SBQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 112 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 180 240 300 360 420 480 540 600 660 691 Met 1 Ser Ser Phe Gly 5 Pro lie Hie Ala He 10 Leu Met Ala Leu Ala 15 Cys Met Ser Ser Thr 20 Leu Val Val Ala Gin Asn 25 Cys Gly Cys Ala 30 Ser Asn Leu Cys Cys 35 Ser Arg Phe Gly Phe 40 Cys Gly Ser Thr Asp 45 Ala Tyr Cys Gly Glu 50 Gly Cys Arg Glu Gly 55 Pro Cys Arg Ser Pro 60 Ser Ser Gly Gly Gly 65 Ser Val Ser Ser Leu 70 Val Thr Asp Ala Hie 75 Phe Asn Arg lie He 80 Asn Gin Ala Ser Ala 85 Ser Cys Ala Gly Lys Arg 90 Phe Tyr Thr Arg 95 Ala Ala Phe Leu Ser Ala Leu Arg Hie Tyr Pro Gin Phe Gly Ser Gly Ser 100 105 110 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1838 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (B) STRAIN: 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 AATIGATCAT ΑΊΤΑΑΤΠΤΑ ITGAGTCnG ATACAACITA AAAACGGAGC CAGITAACGA AGAACCGTAA TGGACCCATA ITGAACAAGT CAAACTAATA ACAOGAATCA TCAACTOGAN ATAAGAAAAG ACATCGAGTC TAATTCCATT CACATCCCAC TATACAACTA CTATCTITCA ΤΠΤΟΓΑΤΑΤ AAACTCATCT TGCITCAAAA GITCCTCTAC TAGTCTACTA GGACCmTT TGGCTATACT TATAGCAGIT CAAAACIGTG GCIGTGCCTC TGGTTTATGC GCIGCCTACT GCGGCACIGG GIGCCAGCAA ACTGGTGGTG nTCGGTCCC AAGTITGGIG CAAGCAAGCT CTAGCIGTGC TGGTAAGAGC CTCAGTTCTT ATCCTCAGTT TGGTAGTGGA GCCTTTITTG CTCATGCGAC GCATGAAACT CTITGATAGA ATIGAAATCG AATAAAATCT ATTCAGCTAA ICTTAITGIT TTATGTCAAT GAATIGTGTC TAAATCTATT ATCTGGATTG AAHGHGGT TGAAAATGTG TTAATGCCTC OGGTIGGGAA ACAHTITAC ATGATAAGTT TACAACAAAA TATCCGTOGT TTCAHTTCC TTCGATAAAA TGACTGGCCA AAGTCAAATG AAATGTCAAC ATHTAAAOG TATCAAACAA GCTAACTCGT AAGCTTCTTC CCTAAATCAC TCAAGICTCT AGTITCCACA ACCCACTCAT TCATTGTACT CCTCCAAAAT GTCTTCTCTT GOCIGTATGT CTAGCACCCT GGTTGTGGCT TGTAGCAGAT ATGCTTACTG CGGCACCACA GGTQCTIGIT OCTCAAOGCC ATCCACCCOG ACCGATGCAT TCTTTAATGG AATCA1TAAC TTCTACACTA GGTCTGCTTT CTTGAGIGCT TCCTCCGATG AGGTTAAAOG TGAAGTTGCT GGAGGTAAGT CTTAACATTA TTAATGCCTC TCITCCCCGC TCATITGCGC GCACITAGCT CATTCIGTCT ΤΑΑΊΤΑΤΠΤ TIGTAATTGA CAAAOCAATA ATATIGAGIG ACGTATAATG 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 GfTAAAAGAAA TGAGAGCAAA AGATITGAAA TTAAITGAAA CTAGl'ITITA GTTTGCTAGT TAAAACTGAT TTAATTCATA TTATTATGTT AAGfTTGAATT AAGCGATACC TAAATCAAAG GGAATGCATT GAGTTACAGA AAAATATATA CTCAGCTGAT CAAITGAACT TGTGTGTTCT AGATTTTTGC TACATAGAGG AGATTGCCAA ATCAACCTAT TGTCAGTOGA GCACAACATG GCCATCCACC ACAAATAAGC AATACTAOGG AOGTGGGCCT CTCCAAATCA CATGGAACTA CAACTAOGGA CCAGCAGGTC GAAGCATTGG ATTIGATGGT TTGAATGCAC CIGAAACAGT IGCCAATGAT GCIGTTATOG CCTITAAGAC AGCCTTCIGG TITIGGATGA ACAATGTCCA CTCTCGAATT GTCTCOGGCA AAGGGTTTGG CTCCACCATT OGAGCTATCA ATGGAGGTGA ATGTGGTGGC GGGAACACAC CGGCGGTCAA CGCTCG7IGTT AGGTACTATA CTCAGTATIG CAATCAGCIT GGTGfHTCAC OTGGGAATAA CCTCTCTTGC TAGTCACATA ATCGAAOTGT TTCCATGGTC ACAATITACA AGTCITAGAC TCTTAGTATA AGGAAAATAA AAATACAATC AAGGGAACTG ACTIGT1T1C TTAGCCAGTA AGGGAAATAT GCATCACTTT GTAATITATA TATATITCAT AGTCTTACGG CCTAITAATA GGGATAOG (2) INFORMATION FOR SBQ 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 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1838 (xi) SEQUENCE DESCRIPTICN: SEQ ID NO: 6: Met Ser Ser Leu Gly Pro Phe Leu Ala He Leu lie Ala Val Ala Cys 1 5 10 15 Met Ser Ser Thr Leu Val Val Ala Gin Asn Cys Gly Cys Ala Ser Gly 20 25 30 Leu Cys Cys Ser Arg Tyr Gly Tyr Cys Gly Thr Thr Ala Ala Tyr Cys 35 40 45 Gly Thr Gly Cys Gin Gin Gly Pro Cys Ser Ser Thr Pro Ser Thr Pro 50 55 60 Ser 65 Gly Gly Val Ser Val Pro Ser Leu Val Ihr Asp Ala Phe Phe Asn 70 75 80 Gly He 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 Fhe Phe Ala 115 120 125 His Ala Ihr His Glu Ihr Glu His Phe Cys Tyr He Glu Glu He Ala 130 135 140 Lys Ser Ihr Tyr Cys Gin Ser Ser Ihr Ihr Trp Pro Cys Ihr Ihr Asn 145 150 155 160 Lys Gin Tyr Tyr Gly Arg Gly Pro Leu Gin He Ihr Trp Asn Tyr Asn 165 170 175 Tyr Gly Pro Ala Gly Arg Ser He Gly Phe Asp Gly Leu Asn Ala Pro 180 185 190 Glu Thr Val Ala Asn Asp Ala Val He Ala Phe Lys Ihr Ala Phe Trp 195 200 205 Phe Trp Met Asn Asn Val His Ser Arg He Val Ser Gly Lys Gly Phe 210 215 220 Gly Ser Thr He Arg Ala He Asn Gly Gly Glu Cys Gly Gly Gly Asn 225 230 235 240 Thr Pro Ala Val Asn Ala Arg Val Arg Tyr Tyr Ihr Gin Tyr Cys Asn 245 250 255 Gin Leu Gly Val Ser Pro Gly Asn Asn Leu Ser Cys 260 265 (2) INFORMATICS FOR SBQ 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 (gencenic) (Vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (B) STRAIN: monova (F) TISSUE TYPE: leaf (vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet lampda-ZAP cDNA library (B) CLONE: SE cDNA clone (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 18..896 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ACCTACCCAA AACAAGC ATG GCA GCC AAA ATA GIG TCA GIT CTA TTC CIG Met Ala Ala Lys lie Val Ser Val Leu Phe Leu 1 5 10 ATT TCT CTC ΊΤΑ ATC TIT GCT TCA TTC GAG TCC TCT CAT GGC TCC CAA He Ser Leu Leu He Phe Ala Ser Phe Glu Ser Ser His Gly Ser Gin 15 20 25 ATT CTC ATA TAC TGG GGC CAA AAT GGT GAT GAA GGA ACT CIT GCT GAC He Val He Tyr Trp Gly Gin Asn Gly Asp Glu Gly Ser Leu Ala Asp 30 35 40 ACT TCT AAC TCC GGA AAC TAC GCT ACC GTG ATC CTA GCT TIC CTA GCT Thr Cys Asn Ser Gly Asn Tyr Gly Thr Val He Leu Ala ihe Val Ala 45 50 55 ACC ΊΤΓ GCT AAC GGG CAA ACC COG GCG CIG AAC TTA GCT GGG CAC TCT 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 ACT CIG AGC AGT GAC ATC AAA ACA TCC Asp Pro Ala Thr Asn cys Asn Ser Leu Ser Ser Asp He Lys Thr Cys 80 85 90 CAA CAG GCA GGC ATT AAG CTA CTC CTC TOT ATA GGA GCT GCT GCC GGA Gin Gin Ala Gly He 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 ΊΤΤ 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 GCT CAG TCC AGC ACC GGA CCC CIT GGA Leu Trp Asn Thr Tyr Leu Gly Gly Gin Ser Ser Thr Arg Pro Leu Gly 125 130 135 GAT GCA GIT TTG GAT GGT ATT GAT TIC GAT ATC GAG ACT GGT GAT GGC Asp Ala Val Leu Asp Gly He Asp Phe Asp He Glu Ser Gly Asp Gly 140 145 150 155 AGA TIT TGG GAT GAC CTA GOT AGA GCA TTG GCA GCT 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 GOT COT CAA TCT 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 CTA TTC GAC TAT GTA TGG Ala Ser Leu Ser Thr Ala He Ala Thr Gly Leu Phe Asp Tyr Val Trp 190 195 200 146 194 242 290 338 386 434 482 530 578 626 GTT CAG TTC TAC AAT AAC Asn CCC CCT Pro Pro 210 TGT CAA TAT GAT ACC AGC GCT GAT 674 Val Gin Phe 205 Tyr Asn Cys Gin Tyr Asp 215 Thr Ser Ala Asp AAT CTC TTG AGC TOG TGG AAC CAG TGG ACC ACA GTA CAA GCT AAC CAG 722 Asn Leu Leu Ser Ser Trp Asn Gin Trp Thr Thr Val Gin Ala Asn Gin 220 225 230 235 ATC TTC CTC GGA CTA CCA GCA TCA ACT GAT GCT GCC GGC AGT GGT TTT 770 He 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 818 He Pro Ala Asp Ala Leu Thr Ser Gin Val Leu Pro Thr He Lys Gly 255 260 265 TCT GCT AAA TAT GGA GGA GTC ATG CTA TGG TCA AAG GCA TAT GAC AGT 866 Ser Ala Lys Tyr Gly Gly Val Met Leu Trp Ser Lys Ala Tyr Asp Ser 270 275 280 GGG TAC AGC AGT GCT ATT AAA AGC AGT GIT ΤΑΑΠΤΑΑΑΤ TACTAGTGTA 916 Gly Tyr Ser Ser Ala He Lys Ser Ser Val 285 290 TCCAAAGATA TAGATACAAA ATAAGITATA GAGATACATC AAAAAACCAT CITAG'iTJTA 976 ΑΑΤΊΤΓΤΤΑΤ GCACCACAAA AGCITCTAAT ACTAATATAC TATTATCATA AATGGCITAT 1036 TGCCTGGCTA TATTTIGGTG ΑΊΤΑΊΤΑΤΑΤ ACACAGTTAC AACTTOGCAA TTATGOGAGT 1096 CrTTCTAAAA 1106 (2) INFORMATION FOR SBQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 293 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Ala Ala Lys lie Val Ser Val Leu Phe Leu He Ser Leu Leu He 15 10 15 Hie Ala Ser Phe Glu Ser Ser His Gly Ser Gin He Val He 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 He Leu Ala Hie 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 Cys Asn Ser Leu Ser Ser Asp Ile Lys Ihr Cys Gin Gin Ala Gly Ile 85 90 95 Lys Val Leu Leu Ser Ile Gly Gly Gly Ala Gly Gly Tyr Ser Leu Ser 100 105 110 Ser Ihr Asp Asp Ala Asn Ihr Phe Ala Asp Tyr Leu Trp Asn Thr Tyr 115 120 125 Leu Gly Gly Gin Ser Ser Ihr Arg Pro Leu Gly Asp Ala Val Leu Asp 130 135 140 Gly 145 Ile Asp Phe Asp Ile Glu Ser Gly Asp Gly Arg Phe Trp Asp Asp 150 155 160 Leu Ala Arg Ala Leu Ala Gly His Asn Asn Gly Gin Lys Ihr 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 Ile 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 Ile 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 Ile Lys Gly Ser Ala Lys Tyr Gly 260 265 270 Gly Val Met Leu Trp Ser Lys Ala Tyr Asp Ser Gly Tyr Ser Ser Ala 275 280 285 Ile Lys Ser Ser Val 290 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1249 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (B) STRAIN: monova (F) TISSUE TYPE: leaf (vii) IMMEDIATE SOURCE: (A) LIBRARY: sugar beet lampda-ZAP cDNA library (B) CLONE: beta-1,3-glucanase cDNA clone (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 34..1041 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AATITTGITT A1TCTTAGAG TTATITCTTC ACA ATG AGG CTA ATT AGC ACA ACT 54 Met Arg Leu lie Ser Ihr Ihr TCT GCA GIT GCT ACT TIG Leu CTG ITT Leu Phe 15 CIT CTA CTA AIT CTA CCT ACT ATT 102 Ser Ala Val 10 Ala Ihr Leu Val Val He Leu 20 Pro Ser lie CAA CTG ACA GAG GCA CAA AIT GGC CTA TCT AAC GGG AGA CTA GGC AAC 150 Gin Leu 25 Ihr Glu Ala Gin He Gly 30 Val Cys Asn Gly Arg 35 Leu Gly Asn AAC ΊΤΑ CCT TCC GAG GAA GAT GIT CTA AGC TIG TAC AAG TOG AGG GGA 198 Asn 40 Leu Pro Ser Glu Glu 45 Asp Val Val Ser Leu 50 Tyr Lys Ser Arg Gly 55 ATA ACG AGG ATG AGA ATC TAT GAC CCT AAC CAA CGG ACC CTC CAA GCG 246 He Ihr Arg Met Arg 60 He Tyr Asp Pro Asn 65 Gin Arg Ihr Leu Gin 70 Ala GIT AGA GGA TOG AAT ATA GGG CTA ATC CTC GAT GTC CCT AAG CCT GAC 294 Val Arg Gly Ser 75 Asn He Gly Leu He Val 80 Asp Val Pro Lys Arg Asp 85 CTA AGG TCA CTC GGC TCC GAT GCT GGG GCT GCG TCT CCT TGG GTC CAA 342 Leu Arg Ser 90 Leu Gly Ser Asp Ala 95 Gly Ala Ala Ser Arg 100 Trp Val Gin AAC AAT CTA GTC CCT TAC GCG TCT AAT AIT CGA TAC ATA GCA GIT GCT 390 Asn Asn 105 Val Val Pro Tyr Ala Ser 110 Asn He Arg Tyr He 115 Ala Val Gly AAT GAA ATA ATG CCT AAT GAT GCC GAG GCA GGG TCA ATT GTC COG GCC 438 Asn 120 Glu He Met Pro Asn 125 Asp Ala Glu Ala Gly 130 Ser He Val Pro Ala 135 ATG CAA AAT GTC CAA AAT GCC CIT GGA TCA GCT AAT TTA GCT GCT AGA 486 Met Gin Asn Val Gin 140 Asn Ala Leu Arg Ser 145 Ma Asn Leu Ala Gly 150 Arg ATT AAA GTC TCT ACC GOG ATA AAA ACT GAC CTC GIT GCT AAC TTC CCT 534 lie Lys Val Ser 155 Ihr Ala He Lys Ser Asp 160 Leu Val Ala Asn Phe 165 Pro CCC TCT AAA GCT GIT ITT ACT TCT TCA TCA TAC ATG AAT CCA ATT GIT 582 Pro Ser Lys 170 Gly Val Phe Ihr Ser 175 Ser Ser Tyr Met Asn 180 Pro He Val AAC TTC CIT AAA AAT AAC AAT TCA CCT TIG TTA GCC AAC ATT TAC CCT 630 Asn Phe Leu Lys Asn Asn Asn Ser Pro Leu Leu Ala Asn He Tyr Pro 185 190 195 TAC Tyr 200 ΤΊΤ TCT TTC ATT GGC ACC CCA ACT ATG GGT CTA GAT TAT GCA CTC 678 Phe Ser Phe He Gly 205 Thr Pro Ser Met Arg Leu Asp Tyr Ala Leu 210 215 TIT ACT TCA CCT AAT GCC CAA GTT AAT GAT AAT GCT TTA CAA TAC CAA 726 Phe Thr Ser Pro Asn 220 Ala Gin Val Asn Asp 225 Asn Gly Leu Gin Tyr Gin 230 AAT CTC ΊΊΤ GAT GCT TTA CTA GAC ACT CTG TAT GCG GCC TTA GCG AAG 774 Asn Val Phe Asp 235 Ala Leu Val Asp Thr 240 Val Tyr Ala Ala Leu Ala Lys 245 GCC GGT GCC CCC AAT GTC COG ATT GTT CTG TCC GAG ACT GGG TGG CCT 822 Ala Gly Ala Pro 250 Asn Val Pro He Val 255 Val Ser Glu Ser Gly Trp Pro 260 TOG GCT GGT GGT AAT GCT GCT ACT HT TCT AAC GCG GGG ACT TAT TAC 870 Ser Ala 265 Gly Gly Asn Ala Ala 270 Ser Phe Ser Asn Ala Gly Thr Tyr Tyr 275 AAG GGC TTA ATT GCT CAT CTA AAG CAA GGA ACT CCC CTG AAG AAA GGA 918 Lys 280 Gly Leu He Gly His 285 Val Lys Gin Gly Thr 290 Pro Leu Lys Lys Gly 295 CAA GCA ATT GAG GCA TAT TIG ΤΊΤ GCT ATG TIT GAT GAG AAC CAA AAG 966 Gin Ala He Glu Ala 300 Tyr Leu Phe Ala Met 305 Phe Asp Glu Asn Gin Lys 310 GCT GGA GGT ATT GAG AAC AAT ΊΊΤ GGA CTG TIT ACT CCC AAT AAA CAG 1014 Gly Gly Gly He 315 Glu Asn Asn Phe Gly 320 Leu Phe Thr Pro Asn Lys Gin 325 CCA AAA TAC CAA Pro Lys Tyr Gin CTC Leu AAT Asn TTC Phe AAT AAT Asn Asn TGAAACTACT TTAATIGCCT 1061 330 335 ACTATATATA TATATATGCT AATATGTTGT ATCTAGTTAT GTCATCTACA TATATAATAA 1121 GTGAAATCAA ACACCOGATC ATAGACTAAA ATIGTAATAA AAGATCCTCC TCTIGTAATA 1181 TTATCCTAGC TGCAATAATA TTTACTCTTA TATAGAGATC TTCTGAAAAA AAAAAAAAAA 1241 AAAAAAAA 1249 (2) INFORMATION FOR SBQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 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 1 5 10 15 Val Val lie Leu Pro Ser He Gin Leu Thr 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 He Thr Arg Met Arg He Tyr Asp Pro 50 55 60 Asn Gin Arg Thr Leu Gin Ala Val Arg Gly Ser Asn lie Gly Leu He 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 He Arg Tyr He Ala Val Gly Asn Glu He Met Pro Asn Asp Ala Glu 115 120 125 Ala Gly Ser He Val Pro Ala Met Gin Asn Val Gin Asn Ala Leu Arg 130 135 140 Ser Ala Asn Leu Ala Gly Arg He Lys Val Ser Thr Ala He Lys Ser 145 150 155 160 Asp Leu Val Ala Asn Phe Pro Pro Ser Lys Gly Val Phe Thr Ser Ser 165 170 175 Ser Tyr Met Asn Pro He Val Asn Phe Leu Lys Asn Asn Asn Ser Pro 180 185 190 Leu Leu Ala Asn He Tyr Pro Tyr Phe Ser Phe He Gly Thr Pro Ser 195 200 205 Met 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 He 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 He Gly His Val Lys Gin 275 280 285 Gly Thr Pro Leu Lys Lys Gly Gin Ala He Glu Ala Tyr Leu Phe Ala 290 295 300 Met Phe Asp Glu Asn Gin Lys Gly Gly Gly He Glu Asn Asn Phe Gly 305 310 315 320 Leu Phe Ihr Pro Asn Lys Gin Pro Lys Tyr Gin Leu Asn Phe Asn Asn 325 330 335 ·* c '· (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6313 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: ENA (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) FEATURE: (A) NAME/KEY: unsure (B) LOCATION: 3214..4227 (D) OTHER INFORMATION: /note= Approximately 1000 base pairs (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join(1428..2169, 4619..4775, 5407..5824) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TCTAGAGAGA GAAAAAACAA OGCCATGTGA OGGTTGGGGT GGGACAAGTT CGACCCGCTA 60 ATATTAATGG GCGGATAIGG ACAACTCTTG AACCCCTCCC TGGCACCTAG AGGTGGGTAT 120 GGGCOGGCCC GGCATGATIT GGGACCGCTC GAGGCCOGAC CCGTTGTCCG CTAOGTGGCC 180 GCGGGTCTGT CATGGTCAAG ΑΑΑΑΤΊΤΑΊΤ GAAACIAAAT ΑΤΤΑΑΑΙΤΓΑ TTTAACCGGT 240 AAITAGTEAA CCTGATCATT TITTCCAAAA TACCTCAAAA TTATIGAAAC TAAATATTAA 300 AITTAAAITG AGAATGITIT TGTCAAGAGA ATCATAGTTA AAAGGAAAAT TTGGCAAAAA 360 ΑΤΤΠΊΤΠΤ ACGAGTTCAT TTTTGTGAAA AAAAACCTTA TAAAGCATTT TTTGCGAAAA 420 CAAACAAAAA TCAAA'iTlTT TCTGGGAAAA CAAACCAAAA TCAAAATTAT TTITGCAAGA 480 TGAAACCTAA CCAOCACTAC CATGCATCAA TCCCCAACCC CTCCCTCCAC CCCCTCGGGG 540 CACCCACACC CCCCITCGCC CCTACCCTAT CTCCACTCTA ITCTTCCCTC CITCACGCCT 600 CCTCTCTCTC CTCTCCCITC GTCCAACCCC CCCCCCCCCC AACOGTTGTG CCACTCAGAA 660 CGCACCCCAC CGCTGCGCCA CTCAAAACCC CCAACCGTTG CGCCACCCAG AAACAATGGT 720 titcgttctt ogaititiga tagtttccgt ttcattigtg GTGCTAGTGG ATAGTGGGCC 780 CTCTTTCGTG CTGGTGGTGG GGGTTTCATG GTGTTATGCT CATGTGATGG TGGGGGTATC 840 ATGTTGTTAT GGTCATGTGA TOGTGGCTTG GGGAGGGGAA GCTGOGGAGG GGGTGGGGTG 900 GACTGTGGTT GGGGGGGTGG ACAAAAGGAG AGGAGAGGGG GGAGGGAGCT AGGGAAGAAG 960 GGAGAGGAGA GAGAGTAGGG GTGAAGGGGG GIGGGGGACT ACOGCCTGAG AAGGTGTAGG 1020 CAGGGGTTGG GCTTGCTGCT GGGGGTGGGT GGGGGTTGGG CGCAACTCTT GGTAGTGGTG 1080 GCTAGAGGTG GTCAATGCOG GAAATTCTAT GCTCAACGCC AGAAAOGACA CTAGATTTCT 1140 TTTOGAAAAA AAATITGATC ITCATTTGTT TTTACAAAAA ATAATTTATA AGATi'ITl'lT 1200 CGCAAAAGTG AACTOGTAAA ΑΟΠΤΙΊΤΓΑ AAACAAATTT TCCTAGTTAA AATGATTCIT 1260 TTCACTl'lTT ACTATGTTAC ATACATAAAT TTIGATAGAT ATCTCTCTTA AATAAGCITG 1320 TCATTTCTCC CITATCCCAA AGACCCAAAG CCTCTATAAA TTGGCTACAA ACTCTCCTCA 1380 CAGTCACACA TGACACAAOG CAAGITTGAA AGGAAAAACA AGAGGTGATG AAAATAAAAA 1440 CCTCTCCTAG TTTTCTACTA GGATTAATAT GTTEAGCTCT AGTCCTCCTA CTAGGAGAAG 1500 GTCTACAATG TGGGCGGCAA TGCAACACAA COGATACTAA TTGTCTTTCC GGTTGCTCAG 1560 TOGGCCGCCC ATCACGTCOG ACACCTCCTC GTCCTCCCAC CCOGAGACCG CCACCTCCTC 1620 GTCCTCCCAC CCOGAGACCG CCACCTCCTC GTCCTCCTAC CCCGAGACCA CCACCACCTA 1680 CACCAAGACC AOCACCTCCT CGTCCTCCTA CCCOGAGACC ACCACCACCT CCTACACCAA 1740 GACCACCACC TCCTOGTOCT CCTACCCCAA GACOGCCACC ACCTCCTACA CCAAGACCAC 1800 CACCTCCTOC TACACCAAGA CCACCACCTC CTAGTCCTCC TACCCCTAGA CCACCACCAC 1860 CAOCACCTCC TAGTCCTCCT ACCCCAAGCC CACCATCTCC TOCTAGCCCT GAACCACCAA 1920 CTCOGCCCGA ACCTAOGCCA CCAACTCCTA CACCAOCAAC TCATCTTACT GACATAATCT 1980 CTGAAGAAAT GTTTAATGAA TTCCTCTTCA ACCGCATTCA GCCACGTIGT CCTGCTAGAT 2040 GGTTCTACAC TTACCAGGCT TTCATTACIG CAGCTGAAAC CTTCCCTGAG TTTGCTAATA 2100 CTGGGAATGA TGAAATTAGA AAGAGAGAAA TTGCTGCTTT CTTTGGACAG ACCTCTCATC 2160 AAACCTCTGG TTCATTCTTC TACTTCTCAT TCTTCTTTAC TTCCGATCIG CTTCACTTTA 2220 2280 CTAACATGCA TCTTTTCATA CTATATCGTA TATITATAGA TGAACAAGTA GTACCTTATT ATTIGCTACT GCCTACTTCC TAGTGTTTAT TCTTTGTCAA TCTAATGATC TTTATACTTT 2340 ATGTTGGACA CAATAAATTA TATAATCTTA ACGTTAAGTT TAGACGATAT GAATTATTTC 2400 TITTTCAOGT ATCTACCCTA GTTATTATAG GTTGTTATTA CCAGTTITTT TACTTCTACT 2460 ΑΊΤΠΌΑΟΓΓ TTGACCATAT CGATTCITTA TGCATAAGAC ATATATAATA TATAAGATCT 2520 TGTIGTATTA TTTACITOGA TATATATATT TTGTTGGATT ATGCTCCAAA ATTAGTCAAG 2580 TTTTCCTAAT AGTTAIGATT AGTIGCTATT AATTAGTTAG IGGGTTAACA AGTAGTGGTC 2640 TAGATAATGG TCTAGCTAGT GGGATAGCAA GTAAGTAGTA GGCTACATAG TGGACATGTT 2700 GTTAGTAGTG CTITGTATGC CTATTTAAAG ATGGITTTGT TTATTCATAA TGTGTACATG 2760 AAAAATATAT AAAAAATGTA GTCTITCTAA TCTCITCAAG TTCTCTTCCT CCTCTAAAAA 2820 TTCTACATGG TATCAGAGCT CCAGGTTAGA TOOGGGAAAG GGAAAGAGAT GAAGCAAAAA 2880 AAAGAAAAGA AAAAAAAGAG AGAGATAGAG AAAGAAAGAG AGTATTAAAA ACAAAATIGA 2940 GTAAAAAGAA ACAAGCAATG CAGCTIGATT TCATTGITIG TGAGCAAITT TITGTTTGAG 3000 TGAAATHTT TGIGTITAAC CATGAAAGTA ATGGTIGGGT GTGATGTGTT GTTGGGCTCC 3060 CTTITAGCCC ATGAAAAIGG ATTITAATCC TGTGGAAAAA AGGGCATAAA AAATTGITIG 3120 AGTIGAAGAT CGAGTAATCT TTACGAGICT GIGGAAGTOG TACTCAACGC AGAGACGATT 3180 TCCAAAGTTG TACTAGTGGA IGTGGTCAAA ΤΠΝΝΝΝΝΝΝ 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 ΝΝΝΝΝΝΝΝΝΝ ΝΝΝΝΝΝΝΝΝΝ NNNNNNNTCT AGATTCACIT TCTAAGTTAA ACTTACCAAA 4260 TCITGATATA TAGTAATTTA CAAAGTATAT TATAAGCTAA GTOGTCATCT CICCCTCCAT 4320 CTiTiTTi'i'A CTACITGTTA CATl’ITiCi'i1 AAATGAATGT CTCATTITAC ACACTACATT 4380 GCTITATTTG CATTGGTAAC TATGACCCAT CAAAAAATAA CAATATTCCC CITTATTITT 4440 CTCATCITCT TAACTTTAAC TCTCAAATAT ΤΙϋΓΓΑΊΠΤ CAACAAAGTT AACTCTCAAA 4500 TATTTCTCAT AATAATCTCA AATCTTCGCT TCATAAAGTC TCCAAAACAA AATGGTGTCA 4560 TITAAAAAGA AAAATCAGAG GACATATATA CTAATGGATT TTIGAATIGA CAATATAGGA 4620 GAACCGACCG CACAACATGG ACCATITACA TGGGGGTATT GITTCATAGA AGAAATTGGA 4680 GCOGGCCCTC TCAGCCAATA TIGTGCACCC TCIGTAGAAT GGCCITGCAT TGGTGGGAGG 4740 TITTACTATG CTCGTCGACC AGTCCAACIT ACCTGCTAAG TACTCCCTCC GTTCCAAAAT 4800 ATAGTTCTCA TTTTCCITTT TTCACAGTAA HTATGCAAG TAGAATATAA GAGGCTAGCT 4860 AAAGATTHT ΊΤΠΤΑΊΤΓΑ AATAAATGIT GTATGGGAAA AGATCATTIT AGGAGAGAGA 4920 GTGGAGAATA ATTAGTGAAA GAGCATTAAT TCTAACATTT TCGTTCAATA AATAAAGGAA 4980 AAAACAAATT CAAGAAGCTA AAGTAATGAG GGCACAGGIT TTCTAGACAA ATTAOGGAAA 5040 AATGTGGAAC TAAATATGAA AATGGGAACT ATATOTGAG ACACNCAAAA TAAAAATGGG 5100 AACTATATIT TGGGAOGGAG GGAGTATTAT TATATTAGCT TACTCCTATT ACITGCATOG 5160 CATCTCCAAA TTHTATICT TCATAGAAAA GTCATTITCA AGAATHTGC TAITOGACCT 5220 CTTAAAATIT TTTACTAOGC TTTCTAAITA CATATTTTIA TAGTGTACIT ATITTATACC 5280 TTTCCAITTC TTCTCITl'lT CCITCCITTC CTTCACITAA GITITAACIT GATACATATA 5340 GCTAGCAAAA TTATCTTAGG TATHTAGCT ΑΑΙΤΓΑΑΑΑΤ ITTTGCTAAT GATAAATAAT 5400 ITGCAGGAAT TTCAACTATG GAAAGCAGGT GAAGCACTTA GGITTGGACC TCCTATTCAA 5460 COCAGACATA GTAGCACATG ACCCAGITAT ITCTTTOGAA ACTGCAATTT GGl'iTIGGAT 5520 GACTCCTGAA GGAAACAAGC CTTCTTOCCA TGAAGTCATA ACTGGGCAAT GGACACCAAC 5580 TCCTGCAGAC ATAGCTGGCA ACAGATTGCC TCGATATCGT TTAATCACAA ΑΤΑΊΤΠΤΑΑ 5640 TCGTGCITTA GAATGOGGCA CTCATCGACC AGATAATAGA GGGGAAAATC GAATTCAGTT 5700 TTACCAGAGA TACIGTGATC TTCTAGATCT TAGCTAIGGA GATAACCTIG ATTGCTACOG 5760 TCAAACTCCC ITTCATTOGG GTCITAAAAA ACTTCAGGGA GCTAGAGAAT CATGGTOGTC 5820 GAGCIAAAAT TATACGCATG CATCTAGTCT CTAAGTOCAT ACAHATICT CITCATGOGT 5880 GTATGATATT GACTAAGTTC CTATCTTCAA AAATATGTGG TCTCTCAAAA TATGCAAACA 5940 GAACCAGCAA TAAGTAATAA GCAAGGTTTA CITGCACCAA ATCTGGATCT GTTCTAGTGA AATTGTTGTA TGTTCGTATT GTATGGTAAT GAATAAAGTT TCTGTTGTAT TTGCATTATC TGCACCTTAT TGATATTAAT TTITCATATT CAGGOGITTA CAATCATAAG GATTACItTTA GGACCATTGA ACATGCAGIT GAGTTACTCT TTAATATGGT GITCAAGAAG AGTATGGAAA ATAGAAATGA GGAATGAACG TACTCTATAT TATAAGAGAC TACTAGIGIT GHTAGTCAG TGCTATTGIT ACACCTAAAA AAGCTCTATG AGATTACATT TACATTATGG TCAAAAGGTC TTAATGTCTA COG (2) INFORMATION POR 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 6000 6060 6120 6180 6240 6300 6313 (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 Ihr Pro Pro Arg Pro Pro Thr Pro Arg Pro Pro Pro Pro 50 55 60 Arg Pro Pro Ihr Pro Arg Pro Pro Pro Pro Arg Pro Pro Ihr Pro Arg 70 75 80 Pro Pro Pro Pro Ihr Pro Arg Pro Pro Pro Pro Arg Pro Pro Ihr Pro 90 95 Arg Pro Pro Pro Pro Pro Ihr Pro Arg Pro Pro Pro Pro Arg Pro Pro 100 105 110 Ihr Pro Arg Pro Pro Pro Pro Pro Ihr Pro Arg Pro Pro Pro Pro Pro 115 120 125 Ihr Pro Arg Pro Pro Pro Pro Ser Pro Pro Ihr Pro Arg Pro Pro Pro 130 135 140 ? Pro Pro Pro Pro Ser Pro Pro 145 150 Pro Glu Pro Pro Ihr Pro Pro 165 Pro Thr His Leu Thr Asp He 180 Leu Leu Asn 195 Arg He Gin Pro Tyr Gin Ala 210 Phe He Thr Ala 215 Thr Gly Asn 225 Asp Glu He Arg 230 Gin Ihr Ser His Glu Thr Ser 245 Phe Ihr Trp Gly Tyr Cys Phe 260 Ser Gin Tyr Cys Ala Pro Ser 275 Phe Tyr Tyr Gly Arg Gly Pro 290 295 Gly Lys Gin 305 Val Lys His Leu 310 lie Val Ala His Asp Pro Val 325 Trp Met Ihr Pro Glu Gly Asn 340 Gly Gin Trp 355 Thr Pro Thr Pro Gly Tyr Gly 370 Leu He Thr Asn 375 Ihr His Gly 385 Pro Asp Asn Arg 390 Arg Tyr Cys Asp Leu Leu Asp 405 Thr Pro Ser Pro Pro Ser Pro Pro Ser 155 160 Glu Pro Ihr Pro Pro Thr Pro 170 Thr Pro 175 He Ser Glu Glu Met Phe Asn 185 190 Glu Phe Arg Cys Pro Gly Arg Trp Phe 200 205 Tyr Thr Ala Glu Thr Phe Pro Glu Phe 220 Gly Asn Lys Arg Glu He Ala Ala Phe 235 Phe Gly 240 Gly Glu Pro Ihr Ala Gin His 250 Gly Pro 255 He Glu Glu He Gly Ala Gly 265 270 Pro Leu Val Glu Trp Pro Cys He Arg Gly Arg 280 285 Val Gin Leu Thr Trp 300 Asn Phe Asn Tyr Gly Leu Asp Leu 315 Leu Fhe Asn Pro Asp 320 He Ser Phe 330 Glu Thr Ala He Trp Phe 335 Lys Pro 345 Ser Ser His Glu Val 350 He Thr Ala 360 Asp He Ala Arg Asn 365 Arg Leu Pro lie Fhe Asn Gly Ala 380 Leu Glu Cys Gly Gly Glu Asn Arg 395 He Gin Fhe Tyr Gin 400 Val Ser Tyr Gly Asp Asn Leu Asp Gly 410 415 Tyr Arg Gin Ihr Pro Phe Asp 420 Arg Glu Ser Trp Ser Ser Ser 435 (2) INPOEMATION FOR SEQ ID NO:13: Trp Gly Leu Lys Lys Leu Gin Gly Ala 425 430 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: C-terminal (Vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (xi) SEQUENCE DESCRIPTION: SBQ 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) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ 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) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide •Μ . ί r (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 Ihr Asn Ala Ihr 15 Ala Ser Leu Val Ser Glu Met (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SBQ 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: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Ala Cys Val Thr His Glu Thr Gly His Phe Cys Tyr lie Glu Glu He 15 10 15 Ala Lys (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (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) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Gly Pro Leu Gin lie Thr Trp 1 5 (2) INFORMATION FOR SBQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Ser He Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val Ala Asn Asn 15 10 15 Ala Val Ihr 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 __ I tl (iii) HYPOTHETICAL: NO (iv) ΑΝΠ-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Triticum aestivum (xi) SEQUENCE DESCRIPTION: SEQ 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 Ihr Ser 35 40 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ΑΝΠ-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) LENGTH: 41 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (Vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Leu Cys Pro Gly Gly Asn 15 10 15 Cys Cys Ser Gin Phe Gly Trp Cys Gly Ser Ihr Ihr Asp Tyr Cys Gly 20 25 30 Pro Gly Cys Gin Ser Gin Cys Gly Gly 35 40 (2) INFORMATION FOR SEQ ID N0:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Glu Gin Cys Gly Ser Gin Ala Gly Gly Ala Arg Cys Ala Ser Gly Leu 1 5 10 15 cys Cys Ser Lys Phe Gly Trp Cys Gly Asn Thr Asn Glu Tyr Cys Gly 20 25 30 Pro Asp Asn cys Gin Ser Gin cys Pro Gly 35 40 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Glu Leu Cys Gly Asn 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 SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (KB-7), constructed freon beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GACTCTAGAA AYCCRCCRYG YCARTAYGAY AC (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (KB-9), constructed from Beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GGAGGATCCC ARRCNAAYCA RATHTT 26 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (270) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CCAAGCITGA ATTC1T1T1T ΤΤΤΤΤΠΤΤΤ ΤΠΤ 34 (2) INFORMATICS FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 292 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Cucumis sativus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Met 1 Ala Ala His Lys 5 He Thr Thr Thr Leu 10 Ser lie Phe Phe Leu 15 Leu Ser Ser lie Phe 20 Arg Ser Ser Asp Ala Ala Gly 25 lie Ala He 30 Tyr Trp Gly Gin Asn 35 Gly Asn Glu Gly Ser Leu Ala 40 Ser Thr Cys 45 Ala Thr Gly Asn Tyr 50 Glu Phe Val Asn He Ala Phe Leu 55 Ser Ser 60 Phe Gly Ser Gly Gin 65 Ala Pro Val Leu Asn 70 Leu Ala Gly His Cys 75 Asn Pro Asp Asn Asn 80 Gly Cys Ala Phe Leu 85 Ser Asp Glu He Asn 90 Ser Cys Lys Ser Gin 95 Asn Val Lys Val Leu Leu Ser He Gly Gly Gly Ala Gly Ser Tyr Ser Leu 100 105 110 Ser Ser Ala Asp Asp Ala Lys Gin Val Ala Asn 115 120 Phe He Trp Asn Ser 125 Tyr Leu Gly Gly 130 Gin Ser Asp Ser Arg Pro Leu 135 Gly Ala Ala Val Leu 140 Asp Gly Val Asp 145 Phe Asp He Glu Ser Gly Ser 150 155 Gly Gin Phe Trp Asp 160 Val Leu Ala Gin Glu Leu Lys Asn Phe Gly Gin 165 170 Val He Leu Ser Ala 175 Ala Pro Gin Cys 180 Pro He Pro Asp Ala His Leu 185 Asp Ala Ala lie Lys 190 Thr Gly Leu Phe 195 Asp Ser Val Trp Val Gin Phe 200 Tyr Asn Asn Pro Pro 205 Cys Met Phe Ala 210 Asp Asn Ala Asp Asn Leu Leu 215 Ser Ser Trp Asn Gin 220 Trp 225 Thr Ala Phe Pro Thr Ser Lys Leu Tyr Met 230 235 Gly Leu Pro Ala Ala 240 Arg Glu Ala Ala Pro Ser Gly Gly Phe He Pro 245 250 Ala Asp Val Leu He 255 Ser Gin Val Leu 260 Pro Thr He Lys Ala Ser Ser 265 Asn Tyr Gly Gly Val 270 Met Leu Trp Ser Lys Ala Phe Asp Asn Gly Tyr Ser Asp Ser He Lys 275 280 285 Gly Ser lie Gly 290 (2) INFORMATION FOR SBQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 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) SEQUENCE DESCRIFTICN: SEQ ID NO: 32: Met Thr Asn Met Thr Leu Arg Lys His Val He Tyr Phe Leu Phe Phe 15 Ile Ser Cys Ser Leu Ser 20 Lys Pro Ser Asp Ala Ser Arg Gly Gly Ile 25 30 Ala Ile Tyr Trp Gly Gin 35 Asn Gly Asn 40 Glu Gly Asn Leu Ser Ala 45 Ihr Cys Ala Ihr 50 Gly Arg Tyr Ala Tyr Val 55 Asn Val Ala Phe Leu Val 60 Lys Phe 65 Gly Asn Gly Gin Ihr 70 Pro Glu Leu Asn Leu Ala Gly His Cys 75 Asn 80 Pro Ala Ala Asn Ihr Cys 85 Ihr His Phe Gly Ser Gin Val Lys Asp 90 95 Cys Gin Ser Arg Gly Ile Lys 100 Val Met Leu 105 Ser Leu Gly Gly Gly Ile 110 Gly Asn Tyr Ser 115 Ile Gly Ser Arg Glu Asp 120 Ala Lys Val Ile Ala Asp Tyr 125 Leu Trp Asn 130 Asn Phe Leu Gly Gly Lys 135 Ser Ser Ser Arg Pro Leu 140 Gly Asp 145 Ala Val Leu Asp Gly 150 Ile Asp Phe Asn Ile Glu Leu Gly Ser 155 Pro 160 Gin His Trp Asp Asp Leu 165 Ala Arg Ihr Leu Ser Lys Phe Ser His 170 175 Arg Gly Arg Lys Ile Tyr Leu 180 Ihr Gly Ala 185 Pro Gin Cys Pro Phe Pro 190 Asp Arg Leu Met 195 Gly Ser Ala Leu Asn Ihr 200 Lys Arg Phe Asp Tyr Val 205 Trp Ile Gin Phe 210 Tyr Asn Asn Pro Pro Cys 215 Ser Tyr Ser Ser Gly Asn 220 Ihr Gin 225 Asn Leu Phe Asp Ser 230 Trp Asn Lys Trp Thr Ihr Ser Ile Ala 235 Ala 240 Gin Lys Phe Phe Leu Gly 245 Leu Pro Ala Ala Pro Glu Ala Ala Asp 250 255 Ser Gly Tyr Ile Pro Pro Asp 260 Val Leu Ihr 265 Ser Gin Ile Leu Pro Ihr 270 Leu Lys Lys Ser 275 Arg Lys Tyr Gly Gly Val 280 Met Leu Trp Ser Lys Phe 285 Trp Asp Asp Lys Asn Gly Tyr Ser Ser Ser Ile Leu Ala Ser Val 290 295 300 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Trp Val Gin Asn Asn Val Val Pro Tyr 1 5 (2) INFORMATION POR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Ala Gly Ala Pro Asn Val Pro lie Val Val Ser Glu Ser Gly Trp Pro 15 10 15 Ser Ala Gly Gly 20 (2) INFORMATION POR 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 (2) INFORMATION FOR SBQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (TG-l), constructed from beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TGGGTNCARA AYAAYGT 17 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (TG-2), constructed from beta vulgaris (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: AAYGARATHA TGCCNAA 17 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) 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-3), constructed from N. tabacum and H. vulgare (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ? I -κ TCRTYRAACA TNGCRAA 17 (2) INFORMATION 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: SEQ ID NO: 39: Phe Ala Met The Asp Glu 1 5 (2) INFORMATION FOR SBQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Phe Ala Met Phe Asn Glu 1 5 (2) INFORMATICS FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pisum sativum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Glu Gin Cys Gly Arg Gin Ala Gly Gly Ala Ihr 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) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pisum sativum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Glu 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: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hordeum vulgare (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Glu 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) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hordeum vulgare (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: Xaa Gin Gin Gly Ser Gin Ala Gly Gly Ala Thr 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) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SBQ ID NO:46: Ala lie Gly Val Asp Leu Leu Asn Asn Pro Asp Leu Val Ala Thr Asp 15 10 15 Pro Val (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Gly Pro He Gin He 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 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (KB-3) , constructed from beta vulgaris (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: COGAAGCITA GATCTAAACA ACAACATGTC TTCTYTYGGA CC 42 (2) INFORMATION POR SEQ ID N0:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGIH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ΑΝΠ-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: primer (KB-4), constructed from beta vulgaris, chitinase 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GCACAOGTAG OGCTAGCTIG G 21 (2) INFORMATION POR SEQ 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 from beta vulgaris, chitinase 4 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CATCGGAGGA TCCACTACC

Claims (80)

1. A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO:.l or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the 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:.l 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 the 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 NO:.1 and encoding 20 the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

3. A DNA sequence according to claim 1, comprising nucleotides 175793 of the chitinase 4 DNA sequence shown in SEQ ID NO:.1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an 25 analogue of said DNA sequence.

4. A DNA sequence comprising a sugar beet chitinase 4 gene.

5. A DNA sequence encoding a chitinase isoenzyme which is at least 60% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO:.l and at the most 40% homologous with the 30 sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO:.11. 829746CL.001/MKA/SPK/A36/1992 04 02

6. A DNA sequence according to claim 5 which encodes a chitinase isoenzyme which is at least 65% homologous, e.g. at least 70% homologous, such as at least 75% or preferably at least 80% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO:.1 and/or at the most 38% such as at the most 35% homologous with the sugar beet chitinase 1 enzyme encoded by the DNA sequence SEQ ID NO:.11.

7. A DNA sequence according to claim 5 or 6 comprising the genomic chitinase 76 sequence shown in SEQ ID NO:.5. 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 ' 9 or the peptide T-A-F-W-F-W-M-N-N-V-H-S-V-1-V-N-G-Q-G-F-G-A-S-1 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 NO: .1 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 NO:.1 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 NO:.1 encoding one or more of the following epitopes of the sugar beet chitinase 4 enzyme Peptide 1: AGKRFYTRA 30 Peptide 2: CNPSKQYY Peptide 3: IECNGGNS Peptide 4: TARVGYYTQYCQ 829746CL.001/MKA/SPK/A36/1992 04 02

14. A DNA sequence according to any of the preceding claims which is of plant origin.

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

16. A DNA sequence according to claim 15 which is derived from a corn, alfalfa, oat, wheat, rye, rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, bean, chenille, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce and 10 pea.

17. A polypeptide encoded by a DNA sequence according to any of the preceding claims.

18. A genetic construct comprising i) a promoter functionally connected to 15 2) a DNA sequence as defined in any of claims 1-16 comprising a chitinase 4 DNA sequence or an analogue or a subsequence thereof and 3) a transcription terminator functionally connected to the DNA sequence .

19. A genetic construct comprising 20 one or more copies of a DNA sequence as defined in any of claims 116 comprising the chitinase 4 DNA sequence shown in SEQ ID NO:.1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide having the activity of a second chitinase different from the sugar beet 25 chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having β1,3-glucanase activity, 829746CL.001/MKA/SPK/A36/1992 04 02 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 J H-chitin into mainly chito hexamers, and/or the DNA sequence encoding the β-1,3-glucanase encodes a basic /9-1,3glucanase having a pi of at least 9.0 and preferably being capable of 10 cleaving H-laminarin into mainly 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 /9-1,3-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 /9-1,ΟΣΟ glucanase is the DNA sequence encoding the basic sugar beet /3-1,3glucanase 4 having the amino acid sequence shown in SEQ ID NO:.10 or an analogue thereof encoding a basic β-1,3-glucanase having a pi of at least 9.0 and being capable of hydrolysing ^H-laminarin into mainly dimers of /3-1,3-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 02 191

25. A genetic construct according to olalm 24, in which the promoter Is selected froa the group consisting of the sugar beet Acetohydroxyacid synthase promoter (ABAS) , the sugar beat chitinase 1 promoter, the sequence of which appears from SBQ ID 80:.11 5

26. A genetic construct according to &ny of claims 18-25, in which the N-terminal leader sequence is selected from the group consisting of the coding regiona of the sugar beet chitinase 1, the sequence of which appears froa SBQ ID 80:.11, the sugar beet chitinase 4, the sequence of which appears fro· SBQ ID 80:.1, the sugar beet fl-t,310 glucanase. the sequence of which appears from SBQ XD 80:.9, the sugar beet chitinase 76, the sequence of which ie shown in SBQ XD MO . -. 5, end the acidic chitinase SE froa sugar beet, the sequence of which appeare from SEQ ID 80:.7.

27. A genetic construct according to any of claims 18-26 which IS contains the DHA subsequence from the sugar beet chitinase 1 encoding Che proline rich region.

28. A genetic cone truce according te claim 24, in which the promoter ia selected froa the group consisting of a BOS promoter and an OCS promoter of the opine synthase genes of dgrobacterium. 20

29. A genetic construct according to claia 24, in which the promoter is selected froa the group consisting of a cauliflower mosaic virus (CaMV) promoter such as « CaMV 19$ promoter or a CaMV 356 promoter, a MAS/35S, MAS dual Tr 1,2 and a T-2 DKA gene 5 promoter.

30. A genetic construct according to claim 23 wherein th* 25 regulatable promoter is regulatable by At least one factor selected from the group consisting of a growth factor, a chemical factor, s biological factor, and a physical factor.

31. A genetic construct according to claim 23 in which the promoter is a tissue specific proooter. 30 32. A genetic construct according to any of claims 18-31 wherein the transcription terminator ia selected froa the group consisting of

U»7«Ch00VMKA/SrK/A34/t992 M 06 192 plane trenacrlptlon terminator saquancea, bacterial transcription terminator sequences, fungal transcription terminators and plant virus terminator sequences.

33. A genetic construct according to claim 32, in which the S transcription terminator is selected from tha group consisting of a

BOS snd OCS transcription terminator sequence of the opine synthase genes of Agrobactarlua, a CeMV 35S transcription terminator sequence, a PAD04 transcription terminator to the DMA. gene 4, a ?ADC7 transcription terminator to the T-DNA gene 7. 10 34. A genetic construct according to any of claims 13-33 in which at least one of the DMA sequences of the construct is functionally connected to an enhancer sequence which results in an Increased transcription and expression of the SNA sequence (a).

35. A genetic construct comprising a ERA sequence encoding a 15 polypeptide, which DNA sequence is linked to a DMA subsequence encoding a N-terminal leader sequence selected from the group consisting of the sugar beet chitinase 4 N-terminal sequence shown in SBQ ID NO:.l, the sugar beet /)-1,3-glucanase N-terminal sequence shown in SEQ ID NO:.9, tha sugar beet acidic chitinase SE N20 terminal sequence shown in SEQ ID MO:.7 and the sugar beet chitinase 1 N-terminal sequence shown in SBQ ID MO:.11.

36. A genetie cone cruet according co claim 35, fur use 1» Llie transformation of a plant, in particular a sugar beet plant.

37. A genetic construct according to any of claims 18-36 which 25 contains the DNA subsequence from the sugar beet chitinase 1 encoding the proline rich domain.

38. A vector which is capable of replicating in a host organism and which carries a MIA sequence as defined in any of claims 1-16, or a genetic construct as defined in any of claims 18-37. 30 39. A host organism harboring a vector aa defined in claim 38. »29746CU»VMKA/BPK/A36/19B 04 06

Q ·>

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 1837 . 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. 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

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 1 ingam.

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. 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. 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

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. 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 1837, 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 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. 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 02 197

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. 5

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 10 strain DH5e 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.

73. A DNA sequence according to any one of claims 1, 5 or 9, substantially as hereinbefore described with reference to the accompanying drawings.

74. A subsequence according to any one of claims 10-13, substantially as hereinbefore described with reference to the accompanying drawings.

75. A polypeptide according to claim 17, substantially as hereinbefore described.

76. A genetic construct according to any one of claim 18, 19 or 35, substantially as hereinbefore described with reference to the accompanying drawings.

77. A vector according to claim 38, substantially as hereinbefore described with reference to the accompanying drawings.

78. A host organism according to claim 39 or 41, substantially as hereinbefore described.

79. A genetically transformed plant according to claim 44, substantially as hereinbefore described and exemplified.

80. A seed, seedling or plant part according to claim 51, substantially as hereinbefore described.