CA2048696A1 - Plant chitinase gene and use thereof - Google Patents

Abstract

ABSTRACT

A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in Sequence 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 hitherto unreported 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 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 transformed sugar beet plants, having an increased resistance to chitin-containing fungi as compared to untransformed plants.

Description

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FIELD OF THE INVENTION

The present invention relates to a DNA sequence encoding the sugar beet chitinase referred to in the followin~ as "the sugar beet chiti-nase 4" or an analogue of said DNA sequence encodlng 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 h~ving an increased resistance to plant pathogens containing chitin, such as phytopathogenic fungi, as compared to un-transformed plants. The genetic construct comprises and is capable of expressing the DNA sequence of the invention, preferably in combina-tion with a DNA sequence encoding second chitinase different from sugar beet chitinase 4 and a DNA sequence encoding a ~-1,3-glucanase.
In another aspect, the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet plant, from which a polypepti~e having the antifungal activity of the sugar beet chitinase 4 is expressed in an increased amount as com-pared to the untransformed plant, preferably in combination with a polypeptide having chitinase activity and a polypeptide having ~-1,3-glucanase activity so as to result in an increased resistance to ~ -chitin-containing plant pathogens.

BACKGROUND OF THE INVENTION

Most plants are susceptible to infection by pathogens such as micro-organisms and develop various undesirable disease sy~ptoms upon infection which cause retarded growth, reduced yiald and consequently 25 economical loss to farmers. The plants respond to infection with -several defense mechanisms including phytoalexins, deposition of - lignin-like material, accumulation of cell wall hydroxyproline-rich glycoproteins, pathogenesis related proteins (PR-proteins) and in-crease in the activity of several lytic enzymes such as chitinases .
- 30 and ~-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 cas0s by exposure to exogenous chçmicals such as ethylene. The full capacity of the de-fense mechanism of the plant is, however, normally delayed in rela-tion to the onset of infection, and thus, the plant may be severely . .
82945~001tl5/JKM/A12/1991 07 2~

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injured before its defense mechanism reaches its maximum capacity.
Also, the defense mechanism of the plant may not in itself be suffi-ciently strong to effectively combat the infectious organism. There-fore, a normal and necessary procedure is to treat infected plants or plants susceptible to infection with a chemical, e.g. a fungicide, either as a prophylactic treatment or shortly after infection.

However, the use of a chemical treatment is neither desirable from an ecological nor from an economic point of view and it would be desir-able to be able to enhance the defense of the host plant itself by 10 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 ~-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-glucos-amine and D-glucose.

Chitinase and ~-1,3-glucanase activity has been observed in plant species such as tobacco, barley, potato, corn, bean, tomato, cu-cumber, wheat germ and pea and it has been shown that the chitinase activity increases in response to iniection 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 Boller, 1988.

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

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EP 0 292 435 relates basically to the regenera~ion of fertile Zea ~ays 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 chiti-nases of bacterial origin in the construction of transgenic plants;
chitinase of plant origin is not mentioned or alternatively only mentioned in general terms.

WO 90/07011 discloses DNA constructs comprlsing a high level promoter operably linked to a DNA sequence encoding a plant chitinase, which constructs are ~sed 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 ex-emplified is a bean chitinase.

EP 0 392 225, EP 0 307 841 and EP 0 332 104 disclose the construction of transgenic plants harbouring DNA sequences encoding plant patho-genesis-related proteins (PRP), e.g. chitinase and ~-1,3-glucanase.
Pathogenssis-related proteins from sugar beet plants or transgenic ~ :
sugar beet plants are not mentioned.
:
In conclusion, none of the above cited publications disclose any ; sugar beet chitinase enzyme or the use thereof in the construction of transgenic plants.

At the Phytochemical Society of Europe International Symposium, Norwich,~ United Kingdom, Aprll 11-13, 1989: Biochemistry and Molecu-lar Biology of Plant: Pathogen Interactions, the present inventors disclosed the isolation and purification of 6 chitinase isoenzymes, including chitinase 4, from the leaves of sugar beet plants infected by Cercospor~ beticol~, a phytopathogenic chitin-containing fungi.
The chitinase isoenzymes were characterized by their molecular weight and kinetics of chitin hydrolysis. Chitinase preparations were indi-cated to be capable of hydrolyzing newly synthesized chitin in the 829450bi.GOI/LS/~llCM/A12/1991 07 29 (~) , ~ ,, ' ' -.' . ' ' ' ' ' ' . . ' ' ' . ".-.. '' '' . :. .... " .' , " .' . ' ' ' :' . ' , ' .. , ,., .'., ~" ', ','.', ' . ' ' ' '' .. ' ,', ; ',';.',''',.' ' . ,. '.' ' .' .'' ',., ,. ' ' ' ', cell wall of the growing fungi. No further characterization was reported and the chitinase enzymes were not separately discussed.

By the present invention a novel plant chitinase has been elucidated which, either alone or in combination with other pathogenesis-related proteins, shows promising results in the inhibition of chitin-con-taining fungi.

BRIEF DISCLOSURE OF THE INVENTION

In one aspect the present in~en~ion relates to a DNA sequence com-prising the sugar beet chitinase 4 DNA sequence shown in Sequence l or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the antifungal activity of the sugar beet chiti-nase 4 as defined herein and i) being a characteristic part of the DNA sequence shown in Sequence l, ', ':
ii) hybridizing with the DNA sequence shown in Sequence l at 55~C
under the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", iii) encoding a polypeptide having the amino acid sequence of the sugar beet chitinase 4 shown in Sequence l, or ~: :
iv) encoding a polypeptide being recognized by an antibody raised against sugar beet chitinase 4.
.
The chitinase 4 DNA sequence, Sequence l, shown in the Sequence Listing below was determined on the basis of a cDNA clone isolated from a sugar beet cDNA library prepared as described in the Material and Methods section below on ~he basis of hybridization with 2 very ; 30 speci~ie oligonucleotide probe. The oligonucleotide probe was prepar-ed on the basis of a tryptic peptide produced from a substantially pure sugar beet chitinase 4 obtained as described in Materials and ~ .
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Methods and i~ Example 1 below. The procedure used for isolatlng the chitinase 4 DNA sequence is outlined in Example 4 below.

Prior to the present invention, the amino acid sequence of the sugar beet chitinase 4 enzyme or the DNA sequence encoding sugar beet chitinase 4 had not been reported, and no indication had been given that it could be interesting to look for these sequences. In fact, the initial analysis of sugar beet chitinase 4, which revealed an enzyme with a small functional domain, suggested that the chitinase enzyme had a low chitin affinity and thus low enzymatic activity.
Thus the enzyme did not seem to be of any partlcular interest.

The elucidation of the amino acid sequence of the sugar beet chiti-nase 4 was an important step in the analysis of the enzyme. Thus, from the amino acid sequence it was clear that the sugar beet chiti- ~ -nase 4 belongs to the plant chitinases of the hevein class in that it contains a leader sequence, a hevein domain and a functional (cataly-tlc) 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 phytopatho-genic 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 j chitinases belonging to the sugar beet chitinase 2 class (as describ-ed in further detail below). It is anticipated that the compact structure of chitinase 4 is an advantage in order to allow the enzyme ::
,~ to get access to chitin structures, e,g. in the cell walls of phyto-' pathogenic fungi.
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~ urthermore, 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.

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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 S different chitinase and a ~-1,3-glucanase in the control of phyto-pathogenic fungi has been found to result in an even more lmproved antifungal activity as compared to the use of the sugar beet chiti-nase 4 alone. This synergistic antifungal effect is reported for the first time in connection with this application.

Accordingly, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence comprising the chitinase 4 DNA
sequence shown in Sequence 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 bset chitinase 4, and one or more copies oi a DNA sequence encoding a ~-1,3-glucanase, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA se-quences 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 phytopatho-genic 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.

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-The chitinase 4 DNA sequence or an analogue thereof, and in particu-lar a speciflc subsequence thereof (which will be further discussed below), may also be used in the isolation of DNA sequences be~onging 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 en7yme, or a polypeptide mixture having a potent antifungal activity. The polypeptide or polypeptide mixture may by prepared by use of recombinant DNA techniques and may be used in the antifungal treatment of various products, especially food products.

DETAILED DISCLOSURE OF THE INVENTION

The chitinase 4 DNA sequence Sequence l encodes the basic sugar beet chitinase 4 enzyme, the amino acid sequence of which also appears from Sequence 1. 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.
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i 25 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 decom-pose chitin and chitin-containing structures and the chitinase acti-,~ vity may be determined by l) a biological assay and 2) a chemical . : .

82~5~i.~1~5/nUM/A12/1991 ~ 29 . .

,-i assay. In the biological assay, the effect of chitinase 4 on growing hyphae of pathogenic fungi, i.e. the ability of chitinase 4 to de-stroy the hyphae walls and thereby retard the growth of the hyphae, is directly observed. In the chemical assay, the decomposition of 3H-chitin by chitinase 4 to result in mainly dimers of chitin is moni-tored.

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". Uhen a positive result is obtained in any of these methods, i.e. the observance of destruction of the hyphae walls and retardation of the growth of the fungal hyphae, it is taken as evidence of biological chitinase 4 activity.

The chemical assay may be carried out as described in "Materials and Methods" under the heading "The radiochemical chitinase assay".
Chitinase 4 activity is shown by hydrolysis of 3H-chitin and the resulting formation of mainly dimers of chitin in this assay.

The term "lysozyme activity" denotes the enzyme's fungal cell wall lysing ability. The lysozyme activity is determined by carrying out the lyso~yme assay described in ~Maéerials and Methods" under the heading "Lysozyme assay".

It wiIl be understood that the antifungal activity of the sugsr beet chitinase 4 is a qualitative as well as a quan~itative measure re-flecting the abili~y 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 DN~ sequence having at least one o~ the properties i)-iv) listed above. The terms used to define the analogues of the invention are explained in further de-tails below.

The term ~characteristic part" as used in connection with the ana-logue defined in i) above denotes a nucleotide sequence which is obtained from the nucleotide sequence of the chitinase 4 DNA sequence :
E~ i.~1/LS/nKM/A12/1s91 ~ 29 ($) ~ ' ':: . . ' 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. Typical-ly, 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 se-quence. In order to allow the polypeptide encoded by the characteris-tic part of the chitinase 4 DNA sequence to retain its characteristic antifungal activity, the part will normally be only a small number of nucleotides shorter than the chitinase 4 DNA sequence, e.g. 1-50, such as 1-25 nucleotides shorter.

A typical exa~ple of a characteristic part of the chitinase 4 DNA
sequence includes the nucleotides encoding the active site of chiti-nase 4.

The analogue defined in ii) above is a DNA sequence which hybridi7eswith the chitinase 4 DNA sequence under the conditions specified in the "Naterials and Methods" section below under the heading "Identi-fication of D~A belonging to ~he chitinase 4 gene family". The condi-tions defined for the hybridization ~o take place are based on hybri-dization experiments carried out with a number of known plant chiti-nases and sugar beet chitinase 4 and is further described in Example 11 below.
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In the present context, any DNA sequence hybridizing with the chiti-nase 4 DNA sequence under the hybridization conditions specified in the above cited part of "Material and Methods" is defined as belong-ing to the chltinase 4 gene family and is contemplated to encode a polypeptide having the structure and antifungal activity of the sugar beet chitinase 4. Furthermore, when the polypeptides produced from such DNA sequences react with antibodies raised against sugar beet chitinase 4, it is a strong indication that the polypeptide encoded by the DNA sequence in question belongs to the sugar beet chitinase 4 ; serological class. Such DNA sequences constituting part of the present invention may either comprise sequences isolated from natural sources, e.g. plants, synthetically produced sequences or may be , 8~ tLS/nKM/A12/1991 ~ 29 ~ -~ -.

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' ' ', . . , . ' ' ' , ~:, ' . ~ , . , ' . . ' ' synthetically modified DNA sequences, e.g. as described below. In the following, DNA sequences belonging to the chitinase 4 gene family are also termed ~chitinase 4 rel~ted DNA sequences".

The analogue defined in iii) above is a DNA sequence which encodes a polypeptide comprising the amino acid sequence shown in Sequence l, i.e. the amino acid sequence of the mature chitinase 4 enzyme. It is well known that the same amino acid may be encoded by various codons, the codon usage being related, i~ter alia, to the preference of the organism in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the chitinase 4 DNA sequence of the invention may be exchanged by others which, when expressed, result in a polypeptide identical to or substantiall~ identical to the polypep-tide encoded by the chitinase 4 DNA sequence in question.

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

ji 25 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 monospeciflc polyclonal antibody or a monoclonal -~ antibody. A particularly suitable a~tibody is a monoclonal or poly-clonal antibody prepared against one or more characteristic epitopes encoded by the chitinase 4 DNA sequence. Such epitopes are explained in further detail below.
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The DNA sequences of the invention explained herein may comprise natural as well as synthetic DNA sequences, the natural sequence ~: .
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ll 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 solid or liquid phase peptide synthesis 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 and with the amino acid sequence shown in Sequence 1 are further described and compared to other plant chitinases.
: -The chitinase 4 DNA sequence comprises a leader sequence (nucleotides -~ -1-70) encoding 24 amino acid residues, a part (nucleotides 71-175) encoding a hevein domain of 35 amino acid residues and a part (nu- ; ;
15 cleotides 176-793) encoding a functional domain of 206 amino acid residues. The N-terminal part of the sequence is blocked and it has not been poss~ble to determine the sequence by conventional amino acid or DNA sequencing methods. However, based on comparison with the DNA sequences of a wheat germ agglutinin (WGA-A) and a potato chiti-nase and based on an analysis by electrospray mass spectrometry (vide Example 4), the start codon of the rhitinase 4 DNA sequence has been deduced. -~; ,', ` Plant chitinases may be di~ided into 3 different groups, the he~ein class, the non-hevein class and the cucumber class. -Sugar beet chitinase 4 is a basic chitinase belonging to the hevein class. Howevar, 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 (chiti-' ~ nase 2) have molecular weights of 32-38 kDa (vide Exampl~ 10), chiti-nase 4 is smaller with a molecular waight of 26 kDa (as determined for the mature enzyme). In addition, since antibodies raised against chitinase 4 do not recognize the other basic chitinases described abo~e (vide Example 10), it is e~ident that chitinase 4 also belong : .
.; . ....
8~ /LS/nNM/AI2/l~l ~ ~ -to a different serological class than all other basic plant chiti-nases from the hevein class.

The primary structure of the mature chitinase 4 as determined on the basis of its amino acid sequence contains 2 different domains: the hevein domain and the functional domain. At the N-terminal part of the polypeptide chain, 12 out of 35 amino acid residues are conserved compared to the hevein structure. The functional domain contain 206 amino acid residues. In the basic chitinase from Nicotiana tabaccum (cv. Havanna) (Shinshi e~ al., 1989), the hevein domain consists of 43 amino acid residues and the func~ional 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 homo-logy between the functional domains of the hevein class and the non-hevein class is very high. In addition, polyclonal antibodies raised 20 against the chitinases from the hevein-class recognize the chitinases -from the non-hevein class.
, . .
In general, the specific activity of the non-hevein class, the acidic chitinase from tobacco and the basic chitinase C from barley (Kragh K. M., Thesis, 1990) are approximately 6-fold lower than that of the hevein class chitinases.

Since the functional domain in chitinase 4 contains only 206 amino acid residues as compared to the 263 amino acid residues af the functional domain of the basic tobacco chitinase; a decrease in the speciflc activity was expected. Chitinase 4, however, performs ex- -tremely well and was by the present inventors shown to be superior to chitinase T, K, and C from barley (results not shown) when ana-lyzed by the radiochemical enzyme assay described in "Material and Methods" below.
'' 829450bi.001/LS/,1KM/A12/lggl 07 2g rr , ~1 i . , ' . ~ , . , , , , . ' ' ' :' From the above explanation, it will be clear that the most important parts of the chitinase 4 DNA sequence shown in Sequence l are the part encoding the hevein domain and especially the part encoding the functional domain of the enzyme. While the presence of a leader sequence in most cases is a prerequisita 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.

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 Sequence l and encoding the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.
The term "analogue~' is referred to as a DNA sequence which either :":
Ai) is a characteristic part of said DNA sequence, :

Aii) hybridizes with a DNA probe prepared from said DNA se-.~ quence, Aiii) encodes a polypeptide having the same amino acid sequence ~, 20 as the polypeptide encoded by said DNA sequence, or " . :
;~ A~v) encodes a polypeptide which is recognized by an antibody ` raised against a polypeptide encoded by said DNA sequence.
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A still more interesting DNA sequence of the invention is a DNA
sequence comprising nucleotides 176~793 of the chitinase 4 DNA se-quence shown in Sequence 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, .
Bil) hybridizes with a DNA probe prepared from said DNA se- :
quence, .
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'' '''' ' ,,' . ''','"','." ', '' ,',', ' ~' ' ' " ' ' ' ' ' ' "''", ' " ' ', ' :, ''~; ''' ,' ' '' "' " ''' ', '' '; "' '' ' ' """, ""'' '', ' ' ' "'' ' '" "' ''" '"',"' "' ' "' ' " ' '' " ''''' .'.''"

., ,.. . .. . . , , , . .... ~ ' ' .'. .'. , , ' ~ , ' . '.,, ' 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 ~y the properties Ai)-Ai~) and Bi)-Biv) above are defined in a similar manner to the analogues of the chiti-nase 4 DNA sequence defined by the properties i)-iv) above, In a further aspect, the presen~ invention relates to a DNA sequence comprising a sugar beet chitinase 4 gene. In the present context, the term "gene" is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream se-quences) as well as intervening sequences, the so-called introns, which are pIaced between individual coding segments ~so-called exons) or in the 5'-upstream or 3'-downstream region. The 5'-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter. The 3'-downstream region comprises se-quences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcrlpt and the 3' un~ranslated region.

An example of a DNA sequence of the in~ention comprising a chitinase 4 gene is the genomic sugar beet DNA sequence harboured in the geno-- mic chitinase 4 clone (chit 4), the isolation of which is described in Example 4. The partial nucleotide sequence of the gene has been - 25 elucidated and is shown in Sequence 2, Based on comparison of the partial DNA sequence with the DNA sequence of th~ chitinasc 76 gene shown in Sequence 3 and further discussed below, and the nucleotide sequence of the chitinase 4 cDNA shown ip Sequence 1, (the compari-sons are shown in Sequence 6) it is contemplated that nucleotides ; 30 356-359 of the chitinase ~ gene sequence constitute the start codon of the chitinase 4 gene.
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8294~001/lS/3KM/A12/1991 07 29 '-~' . ' '. '` '~, ' '' ', '''' '' ,' ' ' . ~',: '" '' ' ,': ,' :, ' ', .' ,` '" "' ~ ' . . ,, , ' ~, . .:, ~: , Based on a comparison with the chitinase 76 sequence (comprising one intron) and the DNA sequence of chitinase 1 shown in Sequence 7 (comprising two introns), it is believed that the chitinase 4 gene comprises only one intron starting at nucleotide 298 downstream of the ATG start codon. The position of the intron is believed to cor-respond to a position between nucleotides 295 and 396 in the chiti-nase 4 cDNA sequence shown in Sequence 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 chiti-nase 4 enzyme has been revealed by comparison to the active site of other known enzymes catalyzing the hydrolysis of other oligosacchari-des 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 184 (Asp) and 190 (Glu).

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 ~he enzyme responsible fo~
its substrate specificity and substrate bindiDg may be envisaged or elucidated. Also, ths amino acid residues forming the epitopes of the enzyme may be elucidated. On the basis of the knowledge of such specific amino acids it is possible to specifically modify the enzyme so as to obtain a modified mode of action of the enzyme, e.g. with , ~29450bi.001/LS/JlCM/A12/1991 07 29 '~ ~) , , ...... .. .. . .... . ........ . ........................................... . .

''" ' ., '' ' , ' ' '' ,';' ' ' . ' ' . ' ' " " '" ' '' ' ' . ', ' '; '~ ' , ', ' '' ' . ' ~' ; ' :: . , "

respect to an increase~ catalytic activity, an improved, i.e.
broadened, substrate specificity, an improved substrate, e.g. chitin, binding or a modified epitope. Such modifications may be accomplished - -by use of well-known principles of protein engineering, such as site-directed mutagenesis, e.g. as described in Example 16 below.

As an exiEmple, the replacement of one or more of the Trp residues in position 170, 205 and 207 with Tyr residues is expected to change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity. Likewise, changes of the amino acid resi-dues constituting the active site or amino acid residues which formthe structure of the folded enzyme are expected to influence, e.g., the catalytic activity, substrate specificity and/or substrate bind-ing may be found to result in improved properties of the resulting modified enzyme. Of course, the nature of the modification to be carried oùt will depend on the desired result, i.e. the specific desired function of the resulting modified enzyme.

Corresponding to the chitinase 4 enzyme encoded by a DNA sequence of the invention, a DNA sequence encoding the modified chitinase 4 enzyme may either alone or in combination with a DNA sequences encod-ing other proteins, e.g. pathogenesis related proteins, such asthaumatin, osmothin and/or zeamatin (Viegers, 1991) or other enzymes such as chitinases and ~-1-3-glucanases be used in the construction of a genetically transformed plant, preferably a sugar beet plant, having a particularly high and advantageous antifungal activity.
Also, the modified chitinase 4 enzyme may prove to be a particular interesting co~ponent of an antifungal composition as described below.
.
; Within a gene family, a high degree of homology between coding re-~ gions of the genes is expected, whereas less homology is expected ;~ ~ 30 between non-coding regions. For different alleles of the same gene, the homology is expected to be extensive throughout the alleles. The term "homology~' is used here to denote the presence of the degree of complementarity between the amino acid seqùence of a glven polypep-tide and the amino acid sequence of a polypeptide being analyzed as determined by use of ~he com~uter program by Myers and Miller, ver-'`~ :.,' .' 82945Wi.001/15j~KM/A12/1991 07 29 . .

. : , . . , . . . . . : ~ , , , , , . . ~, ,,: ,::' , .
:. ' , '. . '. - - '' ' '. :~. .: ,,, . ': . ' .. .' .'. ' ' , ' .; . - - . . . . ... . . .. ..

, '`' ' . '' ' ', ,: ' '' ' ''' : ' '' '"' ' ' ''' ' ' '. . '' : -:, :, . , . ' , , :, ' ' : . ,' ',. .'' '~ ~ : .:, ~ 3~l ~
.~ _ sion 1.05, September 1990, using the comparison matrix: Genetic code, the Open Gap Cost 6 and the Unit Gap cost 1. See also Myers and :
Miller, 1988. The amino acid sequences may be deduced from a DNA
sequence or may be obtained by conventional amino acid sequencing methods. The degree of homology is preferably determined on the basis o~ mature proteins, i.e. without taking any leader sequance into account.

In accordance herewith, the present invention relates to a DNA se-quence encoding a chitinase isoenzyme which is at least 60% homo-logous with the sugar beet chitinase 4 enzyme encoded by the DNAsequence Sequence 1 and at the most 40% homologous with the sugar beet chitinase 1 encoded by the DNA sequence shown in Sequence 7.
The mini~um degree of homology of at least 60X has been determined on the basis of an analysis of a rape seed chitinase (based on the mature protein) which has been shown to belong to the sugar beet chitinase 4 serological class (see Example 11). The degree of homo-logy of 40Z with chitinase 1 (which does not belong to the chitinase 4 class) reflects the maximal degree which is expected to be accept- .
able for a polyyeptide belonging to the chitinase 4 class.

Of course, a higher degree of homology with the chitinase 4 enzyme and ior a lower degree of homology with the chitinase 1 enzyme re-flects an even higher similarity herewith and accordin~ly, the DNA
sequence described above preferably encodes a chitinase isoenzyme which is at least 65%, e.g. at least 70X homologous, such as at least : 25 75X or preferably 80X homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence Sequence 1 and/or at the most 38X
: such as at the most 35% homologous with the sugar beet chitinase 1 : . .
enzyme encoded by the DNA sequence Sequence 7.
. .
An example of a DNA sequence encoding a polypeptide being about 75X
homologous to the sugar beet chitinase 4 enzyme and at the most 40%
~ homologous to the sugar beet chitinase 1 enzyme is the genomic DNA
- sequence (chitinase 76 the sequence of which is shown in Sequence 3) contained in the genom~c clone chitinase 76 obtained as described in Example 5. ~ :
:

~ 829450bi.001/LS/JKM/A12/1991 07 29 : ' ($) ' ., ' ' : ' '' ' ' . . '. , ., ~ .', ' ' " ', ;, ' .; , ' .. , , ' . . ' :

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

In a further aspect, the present inven~ion relates to a modiiied DNA
sequence comprising a DNA sequence as defined above comprising the chitinase 4 DNA sequence or gene or an analogue thereof in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having retained the antifungal activity of the sugar beet chitinase 4 or having an increased antifungal acti~ity as compared to the sugar beet chitinase 4. The polypeptide encodin~ by the modified DNA sequence has normally an amino acid sequence which ,? iS different from the amino acid sequence of the sugar beet chitinase 4. It will be understood that a modified DNA sequence of the inven-.~ tion will be of importance in the preparation of novel polypeptides i 25 having an increased antifungal activity as compared to chitinase 4.
~. ' ' 1 :
When "substitution" is performed, one or more nucleotides in the full -nucleotide sequence are replaced with one or more different nucleo-tides, when "addition" is performed, one or more nucleotides are , added at either end of the full nucleotide sequence~ when t'insertion"
.~ 30 is performed one or more nucleotides within the full nucleotide sequence is inserted, and when "deletion" is performed one or more nucleotides are deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it.

A modified DNA sequence may be obtained by well-known methods, e.g., by use of site-directed mutagenesis.
.
82~s~i~l/LslnKMlAl2ll99~o729 ~ ... .

, .,., - . . , . : , ~ .. .: :
,... . . . . - . . .: . .. . . ,, : . .

;: : , . : ,.

::: . : :: .

f J 1' '`

In a further aspect, the present invention relates to a subsequence of the chitinase 4 DNA sequence of Sequence 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 5 chitinase 4 DNA sequence or of the genomic DNA sequence ~re subse- .
quences comprising the nucleotide sequence defining the active site of the sugar beet chitinase 4 enzyme. An example of such a subse-quence is a DNA sequence comprising the active site of the sugar beet chitinase 4 enzyme, e.g. the DNA sequence encoding the following peptide (shown by use of the conventional one-letter amino acid code):

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 result-ing polypeptide. Furthermore, the DNA sequence may be fused to a part :
of another DNA sequence encoding an en~yme different from the sugar beet chitinase 4 or substituted with a part of such en7yme encodingthe 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 old 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 follow-ing amino acid sequence S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-D-A-V-T-A-F-R

~; 30 This polypeptide is deduced from the DNA sequence of the genomic chitinase 76 clone shown in Sequence 3 and corresponds to the DNA
sequence of the peptide 4-22 given above, except for the fact that 8W50bi.001/LS/lKM/A12/1991 07 29 :

. ' . . . ' ". ,, ,~ ' r. .

J~ J ~,:

the bolded D is an N in peptide 4-22. It is believed that the chiti-nase 76 derived polypeptide may have the same or nearly the same interesting properties and uses as the peptide 4-22.

Two further interesting DNA sequence is the sequence encoding the following peptide G-P-L-Q-I-T-W

which is the tryptic peptide 4.19.3 of chitinase 4 and the DNA se-quence encoding the tryptic peptide 4-26 T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-~-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 in~olved in the active site and/or substrate `
specificity of the chitinase 4 enzyme, e.g. as further discussed in Example 16 below.

Another example of an interesting subsequence according to the inven-tion is a subsequence of the chitinase 4 DNA sequence of Sequence 1 encoding a polypeptide comprising the hevein domain of the sugar beet chitinase 4 enzyme, or an analogue of said subsequence which hybridi-zes to the chitinase 4 DNA sequence at 55C under the conditions specified in "Materials and Methods" under the heading "Identifica-tion of DNA belonging to the sugar beet chitinase 4 gene family" and -encoding a polypeptide capable of binding to chitln as determined by af~inity column chromatography on regenerated chitin prepared as described in "Materials and Methods" under the headin~ "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 cfficie~t, i.a. capable of establish-ing an intimate binding to chitin, this domain may prove to be very useful in the modification of chitinases, such as other plant chiti-nases, containing either a weak or no hevein dom~in with the 2im ofconferring a stronger chitin-binding capability to such chitinases.
Examples of chitinase which could advanta~eously be modified by ' 829~i.001/LS/JKM/A12/1991 07 29 ::

-:,: :: : : ~ , , , . , , . , .. , : , . :.
:., , . . ., , . , .: . . , : , : . . .,.. , :

-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 Sequence 1 encoding the leader peptide of chitinase 4 or an analogue thereof which hybridi-zes to the chLtinase 4 DNA sequence at 55C under the conditions specified in "Ma~erials and Methods" under the heading "Identiiica-tion of DNA belonging to the sugar beet chitinase 4 gene family" and which is capable of directing a passenger polypeptide to which it is fused out of the endoplasmatic reticulum of the cell in which the fused leader and passenger polypeptide is produced.
. ', 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 epltopes are expected to be found among the hydrophilic peptides of the chitinase 4 amino acid sequence Sequence 2, because these pep-tides seem to be substantially different from peptide parts of other chitinases than sugar beet chitinase 4. Antibodies (either monoclo-nal, monospecific or polyspecific) may be prepared by use of conven-tional methods, e.g. as descr~bed in the Materials and Methods sec-tion below on the basis of synthetically produced peptide parts of the sugar beet chitinase 4 enzyme. Based on a conventional computer 1 25 analysis of the chitinase 4 DNA and amino acid sequence, the follow-;~ ing possible epitopes of the sequence have been identified:
~.: ' '' Peptide 1: AGKRFYTRA
Peptide 2: NPSKQ
Peptide 3: GGNS
Peptide 4: TARVG m QYGQ
.
These epitopes are believed ~o 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 ' ' :'~
a~bi.OOltLS/JKM/A12/1991 01 29 , ,, , .. , ., ., ,., . ., ., .,, , " , ,, , . "~,~ .. ". ,, , . . j, : . .

';, ' .~.' ' : . ' . ' , ; .
i ' , ' ;' . ' " , ' ' ~ ' . ' . . ' , ,, ,; , ...' ~ ;. ' , ;' ', '' ',' ~ ' ' " ~

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 charac-teristics of the subsequence should be understood as being within the scope o 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 in 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 se- -quence 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, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and 20 pea. -The terms "sequence", "subsequence" and "analogue" as used herein with respect to sequences, subsequences and analogues according to the invention should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in iso-la~ed, purified, in vitro or recombinant form.
~ . .
The chitinase 4 DNA sequence of the invention or an analogue orsubsequence 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 motified as described herein.

From the abo~e explanation it will be clear that the chitinase 4 DNA
sequence of the invention or an analogue or subsequence thereof may .

E~ i.Oo1~5/JKM/A12/19910729 : - , . , , ,. . . :

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 5 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 encodin~ a signal peptide which gives rise to transport of the fusion protein expressed therefrom to specific organelles of the 10 organism expressing the polypeptide. Signal peptides involving trans-port will be discussed in further detail below. Interesting subse-quences of the chitinase 4 DNA sequence, such as those described above, e.g. a subsequence encoding the hevein domain and/or an epi-tope, may likewise be fused to DNA sequences encoding other proteins, 15 such as enzymes, e.g. chitinases, in order to confer to the proteins the desirable properties of the polypeptides encoded by the isubse- i~
~ quences of the chitinase 4 DNA sequence.

f Also wi~hin the invention is a polypeptide encoded by the chitinase 4 DNA sequence or an analogue or subsequence thereof as definsd above, 20 preferably in a non-naturally occurring or recombinant form. As compared to the naturally occurring chitinase 4 enzyme, the polypep-' tide of the in~ention has the advantage that it may be easily pro- -duced in large quantities by use of well known conventional recombi-i, nant productions techniques, e.g. as described in Sambrook et al., 25 1990, and that it may be obtained in a form which is free from im-purities normally associated with the naturally occurring sugar beet j chitinase 4. ~he polypeptide of the invention may be used as a con-stituent in an antifungal composition, e.g. as described below.

As it is expIained above and in the examples to follow, t~e sugar 30 beet chitinase 4 enzyme has been shown to have a number of advan-tageous properties including a surprisingly high antifungal activity i~ ~ 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 35 beet chitinase 4 enzymes adds to its advantageous properties. Thus, the use of a DNA sequence encoding the sugar beet chitinasa 4 or an .

,~ 829450bi.001/LS/JK~/A12/1991 07 29 (~) ': ' , ' ' , ' ' ' . j.'! ' " ',' : ... ' ., . , ~ , ' ' ;. ' - .. . , . , . : ' ' ' ' . ' . : ' ;'.: ~ ' ' ~ ' ' " . ' , ' ' " 'i' : ' ' ' ' ' ' ' " ' ' ' . ' ' ';" ' . : ' "' ' :
;' . .. ~ . ' ' '', ' , ' ' ' ' ' ' . 1: ' '''.
, ~ ' ' ' ' '~ " ' ' ' " ' ' ' . ' ' ~ , :
:~ ' , ' , ' " " ' ' ' ~ '' :

, ~ !". ~ 3 analogue thereof encoding a polypeptids having the antifungal ac-tivity 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 ana-logue 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 genet$cal-ly modified plant in order to produce a plant showing an increased antifungal activity as determined by the procedure given in Example 2 and thus an increased resistance towards phytopathogenic fungî.
Furthermore, it is contemplated that the genetic construct may be :
used in increasing the chitin-binding capability of a plant. An example of a genetic construct as defined above is given in Example 18 below.

Furthermore, experiments have revealed (vide Example 2) that when phytopathogenic fungi (C. beticola and T. viride) are treated with a composition comprising a polypeptide having the an~ifungal activity of the sugar beet chitinase 4 in admixture with an acidic chitinase and a basic ~-1,3-glucanase the growth rate of the fungal hyphae is drastically reduced and the number of germ~nating spores are decreas-ed. In this connection, it ig contemplated that the synergistic ~ effect will be obs0rved in general when the sugar beet chitinase 4 is ; used in combination with other chitinases and ~-1,3-glucanases, ~ 30 preferably of plant origin.

'. ~ .
: - :
8 ~ ~ nuMlAl2/lssl ~ 29 ; ~ , ~., .,, ., . - . . : - . . ., , : . . . . . . -~' ' ' ' ' ' ' .. , ' :. ' "' ''' .' '' .',' ." "' " ',. ' '. ' ' .' . . . . ' ' ' ' Thus, in another important aspect, the present invention relates to a genetic construct comprising one or more copies of a DNA sequence as defined abo~e comprising the chitinase 4 DNA sequence shown in Sequence 1 or an analogue or subse-quence thereof, one or more copies of a DNA sequence encoding a polypeptide havingthe activity of a second chitinase difierent from the sugar beet chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having ~-1,3-glucanase activity, each of the DNA sequences being functionally connected ~o a promoter and a transcription terminator capable of expressing the DNA se-quences into functional polypeptides.

The polypeptides with chitinase or ~-1,3-glucanase activity is pre-ferably of plànt origin. The chitinase and ~-1,3-glucanase activity may be determined as explained in the section "Materials and Methods"
below.

Of particular interest is a genetic construct comprising .

one or more copies of a DNA sequence as defined above comprising the chitinase 4 DN~ sequence shown in Sequence 1 or an analogue or subse-~uence thereof, one or more copies of a DNA sequence encoding an acidic chitinase having 8 pI eqoal to or less than 4.0, and .
one or more copies of a DNA sequence encoding a basic ~-1,3-glucanase `~ 25 having a pI of at least 9.0, each of the DNA sequences being functionally connected to a promoter and a transcrlption texminator capable of expressing the DNA se-quences into functional polypeptides.
:
82~b~.001/LS/JKM/A12/1991 07 29 , : . ' .: ' , ' , . ' , . , , , , ' ~ , . . . ' ,............................... ;'~ ~J~ f i ' f ~
.

In the present context, an ~acidic chitinase~ is defined as a chiti-nase having a pI of less than 4Ø 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 chi~inases are cuc~mber lysozyme/chitinase and Arabidopsis as well as the acidic sugar beet chitinase SE having the amino acid sequence shown in Sequence 8 or an analogue of said DNA
sequence encoding an acidic chitinase having a pI of at the most 4.0 and preferably capable of hydrolyzing 3H-chitin into mainly hexamers.

10 In the present context, the term "basic ~-1,3-glucanase" means a ~- :
1,3-glucanase having a pI of more than 9Ø Preferably, the basic ~- -1,3-glucanase is one which is capable of hydrol.yzing glucan into . mainly dimers, e.g. as determined by the 3H-laminarin assay described in the Materials and Methods section below. The basic ~-1,3-glucanase is preferably o plant origin. Examples of a suitable basic ~-1,3-glucanase are basic ~-1,3-glucanases derived from tobacco (Shinshi et -~ al., 1990), barley (Fincher et al., 1986) or sugar beet, e.g. the ` basic sugar beet ~-1,3-glucanase 3, the sequence of which i5 shown in i Sequence 9 or an analogue thereof encoding a basic ~-1,3-glucanase having a pI of at least 9.0 and preferably being capable of hydrolyz-`i ing 3H-laminarin into mainly dimers of ~'-1,3-glucan. The basic sugar beet ~-1,3-glucanase 3 is different from other plant ~'-1,3-glucanases I in that it does not contain a C-terminal extension. The advantageous effect of using the basic sugar beet ~-1,3-glucanase 3 may in part be due to this lacking C-terminal extension.

The experiments reported ln Example 2 below show that the combination of the sugar beet chitinase 4 enzyme, an acidic chitinase and a basic ~-1,3-glucanase results in an increased antifungal activity as com-pared to th0 antifungal activity o~ each of the constituents. The . ~ 3~ increased antifungal activity observed when using this specific combination is par~ly believed to be due to the different mode of action of the acidic chitinase, basic ~-1,3-glucanase and sugar beet chitinase 4, respectively. '.'~hen the acidic chitinase is one which hydrolysec. chitinase primarily into hexamers (as compared ~o chiti-..
. , .

8~bi.001/LS/JlCM/A12/1991 07 29 : .

nase 4 which primarily hydrolyses chitin into dimers) and the basic ~-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 efiect obtained when usin~ a combination of the sugar beet chitinase 4, a polypeptide having the activity of a second chitinase different from chitlnase ~, e.g. an acidic chiti-nase, and a polypeptide having the activity of a ~-1,3-glucanase, e.g. a basic ~-1,3-glucanase, is believed to be due to the fact that such combination will attack both the chitin and glucan constituents of the cell wall of phytopathogenic fungi and also parts of the cell wall in which the chitin and glucan constituents are intimately cross linked to one another. The ~-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 ~-1,3-glucanase may be obtained, e.g. from already known sources, or may be identified and isolated from natural sources, e.g. by use of the techniques disclosed herein.

~; It will be understood that a large number of diiferent genetic con-structs as defined above may be designed and prepared. Without being an exhaustive list, el~ments 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 ~he type of any other associated $equences, e.g. a C-terminal or N-terminal sequence (described below). Thus, genetic constructs of the present invention may vary wi~hin wide limits.
Normally, the combination of each of the above mentioned variable elements of the genetic construct to be chosen will depend, e.g.
the desired strength of the antifungal effect to be obtained which may be determined as a function of gene dosage and specific nucleo-~ .

8~ tLs/nKM/Al2/l99l ~ 29 ; ~ ~3 1 ' ' : ~ . . . . , . . . : , . .

,:, , , . ., . , . , . ~ : "
.. : , ',. ' ~ , . - , . ~, : ., tide sequenca of each of the DNA sPquences, and the type and strength of the promoter and terminator used for each DNA sequence.
However, in designing a genetic construct of the inventi~n 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 expressi~n 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 inven-tion is too large, it may be difficult to obtain a stable introduc-tion thereof into the genome of the plant which may lead to excision of a part of or the entire genetic construct from the geno~e of the plant. Thus, the genetic construct should be adapted so that the expression products therefrom are generally acceptable to the host organism.

It is believed that a number of copies of 3-4 of some or all of the DNA sequ~nces of the genetic construct of the in~ention will present a too large material to be introduced in the genome of the plant.
Expression of proteins from such a genetic construct may also prove to be too heavy a burden for the plant resulting in retarded growth and/or reduced yield. However, with the fast increasing knowledge within the field of plant genetic engineering, improved transforma-tion and biological containment techniques may be developed leading to the possibility of introducing larger foreign genetic fragments into a plant without causing retarded growth, yield or recombination-al events.
, At present, a genetic construct is preferred which contains only a few copies of the DNA sequence of the invention. Accordingly, it is preferred that each of the DNA sequences of the genetic construct of : `
the invention is present in only one copy. The construction of a genetic construct containing one copy of each of the DNA sequences is ; illustrated in the examples below.

As mentioned above, a significant antifungal effect is obtained from a protein encoded by the chitinase 4 DNA sequence of the invention or 35 an analogue thereof. Accordin~ly, it is contemplated that a genetic -: :"
829450bi.001/15/lKM/A12/1991 07 29 ~3 ' construct of the invention, in which two copies of the chitlnase 4 DNA sequence of the invention or an analogue thereof, and one copy of each of the DNA sequences encoding an acidic chitinase and a basic ~-1,3-glucanase are present may show very potent antifungal effects when present in a genetically transformed plant of the invention. It is believed that such a genetic construct will not pose a too heavy burden on the plant in which it i5 harboured. Of course, also the choice of e.g. promoter used for each DNA sequence will influence the amount of protein expressed therefrom. This will be further explained below.

The genetic construct of the invention as described above may be present on one or several DNA fragments. Depending on the size of the genetic construct to be introduced in an organism such as a plant, in the case of a plant typically by means of a plant transformation vector, 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. The use of more than one vector is dlscussed below. When the use of only one plant transformation vector is desirable, it is advantageous that the genetic construct is present on one DNA frag-ment.

j 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 se-quence, or a leader sequence derived from DNA encoding another prote-in. In Any event, the leader sequence is to be functionally connected to the DNA sequence so that the polypeptide expressed from the re-sulting nucleotide sequence serves to direct the poiypeptide encoded by the DNA sequence out of the endoplasmic reticulum 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 ls 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.
.~ .
8X~ /~ci/nuM/Al2/lssl ~ 29 . . . . . .
" ., . , .: , , The nature of the N-terminal sequence to be used will e.g. depend on the particular organism and the par~ 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 transport-ed. 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. Suitable non-~0 limitin~ examples of a leader sequence to be used in the presentcontext are the N-terminal leader sequence of the sugar beet chiti-nase 1 enzyme, the nucleotide and amino acid sequence of which is shown in Sequence 7, the N-terminal leader sequence of the genomic chitinase 76 clone, the amino acid sequence of which is shown in Sequence 3, and the N-terminal leader sequence of the acidic sugar beet chitinase SE, the amino acid and nucleotide sequence of which is shown in Sequence 8. ;

Furthermore, it may be advantageous that at least one of the DNA
sequences of the genetic construct of ~he invention further comprises a C-terminal sequence encoding a si~nal peptide capable of directing the polypeptide encoded by the DNA sequence to a part of an organism in which it is to be expressed, e.g. the vacuole. The C-terminal sequence may be the C-terminal extension normally associated with the ~
DNA sequence, if any, or may be derived from the host in which the ~ -genetic construct is to be expressed or may be of another origin.
This is especially relevant in connection with the chitinase 4 DNA
sequence and the DNA sequence of the basic sugar beet ~-1,3-glucanase 3 both 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 gene-tic construct of the invention are C-terminal sequences selected from the following sequences:

, 829450bi.001/LS/JKM/A12/1991 07 2~
~$) ".

: :
- . . .

'' .' ' .. ' ' . ' : ' ~ , , . . ., ,, ,:, . .,, , . , , : . , . ' . .' ,',; ,.

the C-terminal sequence of sugar beet chitinaqe 1 encoding the fol-lowing polypeptide N L D C Y R Q T P F D W G L K K L Q G A R E S W S S S *

The C-terminal end of the sugar beet chitinase 4 encoding the fol-lowing polypeptide N L R C *

the C-terminal sequence of a bean chitinase (PHA) encoding the following polypeptide .
. N L D C Y S Q T P F G N S L L L S D L V T S Q *

the C-terminal sequence of a basic tobacco chitinase encoding the . following sequence ,. . .
~ : N L D C G N Q R S F G N G L L V D T M *
~ .' ' .
the C-terminal sequence of an acidic tobacco chitinase encoding the following sequence lS N L D C Y N Q R N C P Q G *
.! . .
the C-terminal sequence of the barley chitinase CH26 encoding the ,~ following sequence .. .. .
N L D C Y S Q R P F A *, or ,; the C-terminal sequen~e of a basic ~-1,3-Glucanase from tobacco encoding the following sequence G V S G G V W D S S V E T N A T A S L V S E

The choice of whether a G-terminal sequence is to be added to one or ' more o~ the DNA sequences of the genetic construct will be determin-; ed, e.g. on the basis of to which plant compartment the polypeptide 1~:

8~ /Ls/nKM/Al2/l99l ~ 29 ~ (~) ` ' ~ :;' ' : ... ..

expressed fro~ 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-ter~inal sequence capable to transport the polypep-tides expressed from the DNA sequences to the vacuole.

As it will be apparent from the above explanation it is important to obtain a sufficient expression of the polypeptides encoded by the genetic construct in plants containing said construct in order to allow the polypeptides to exert their intended function, i.e. to exert their antifungal activity. One essential element in obtaining a sufficient expression is to provide a satisfactory regulation of the ~ :
transcription and expression of the DNA sequence or gene from which the polypeptide is expressed.
.: . ' The expression of each of the DNA sequences of the genetic construct of the invention or of a gene comprising such DNA sequences are accomplished by means of a regulatory sequence functionally connected to the DNA sequence or gene so as to obtain expression of said se-quence or gene under the control of the inserted regulatory sequence.
Typically, the regulatory sequence is a promoter which may be consti-tutive or regulatable.

The term "promotsr" is intended to mean a short DNA sequence to which RNA polym~rase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promo~er is function-ally connected, allowing transcription to take place. The promoter is -usually situatPd upstream (5') of ~he coding sequence. In its broader scope, the term "promoter" includes the RNA polymerase binding site as well as regulàtory sequence elements located within several hundreds oi base pairs, oceasionally even further away, from the transcription start site. Sueh regulatory sequences are, e.g. se-quenees whieh are invelved in the binding of protein factors whieh eontrol the effectiveness of transcription initiation in response to physiological conditions.
.
:, ' .
82~s~~ LslnKMlAl2ll99l ~ 29 . ~ .

A "constitutive promoter" is a promoter which is sub;ecte~ to sub-stantially no regulation such as induction or repression, but which allows for a steady and subs~antially 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.
:
A "regulatable promoter" i5 a promoter th~ 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 se-quence or may, alternatively, be such which suppress the expressionof the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated re-gulatory 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 Gi regulatory sequences are upstream and downstream sequences involved in control of termination of transcription (trans-cription terminators) and removal of introns, as well as sequences responsible for polyadenylation, and initiatlon 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 ~ivçn 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 æpecific, 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 o~ interest.
~ ' .
8294~.001/15/3RMtA12/1991 ~ 29 `~`: `'' .. ' - ~` .: . . ` ' ` . . ,, : , In the present context, a suitable constitutive promoter is iselected 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 5 derived from a cauliflower mosaic virus (CaMV). Su~h promoters are normally strong constitutive promoters. Examples of a preferred CaMV
promoter is a CaMV l9S promoter and a CaMV 35s promoter (Odell et al., 1985).

Other promoters may be derived from the Ti-plasmid such as the octo- ~ -pine synthase promoter, the nopaline synthase promoter (Herrera-; Estrella et al., 1983), the mannopine synthase promoter, and promo-ters from o~her open reading frames in the T-DNA such as ORF7.
Further examples of suitable promoters are MAS/35S (Janssen and ~- Gardner, 1989) I MAS dual Tr 1,2 (Velten et al., 1984) and a T-2 DNA
.J 15 gene 5 promoter (Koniz and Schell, 1986) . : ~:

The regulatory sequence may be a chitinase promoter, i.e. a promoter which is naturally found in connection with chitinase genes and ; involved in the transcription thereof. A chitinase promoter may beobtained from an isolated chitinase gene, e.g. an already known chitinase gene or a gene which may be identified and isolated e.g.
by the methods disclosed herein. Typically, the chitinase promoter ~ should be obtained from a plant which has been shown to have a fast ;, response to pathogen challenge. In this connection, fast responses have been observed in pea and barley and it is contempla-ted that ~, 25 chitinase promoters from these plants may be useful for the presentpurpose An example of su h promoters is the chitinase promoter of pea (K. Vad, 1991). An example of another promoter which is contem-plated to be useful in the present context is the sugar beet chiti-nase 1 promoter (Sequence 7) and the sugar beet acetohydroxyacid ,1 30 synthase promoter (AHAS) (P. Stougard and K. ~o~sen, Danisco A/S, ~ Denmark, personal communication).
I .
j~ Optionally, and if desired, the natural promoter may be modified for the purpose, e.g. by modifications of the promoter nucleotide se-i .

~ 8~ /Ls/nKM/Al2/l~l ~ 29 (~3 . , ~ .. .. . .. . . . . . . . . . . . . . .... .
.;..,., ..,,.. ~. ., ,, " , ., '''' ".' "., ' ' ''' ..'' ',". ''" '''.' ' ' ',' ''.. :'' '' ' ' ," ' ., '': ':'' ' quence so as to obtain a promoter functioning in another manner than the natural promoter, preferably being stronger.

As stated above, each of the coding DNA sequences of the genetic construct of ~he invention is functionally connected to a transcrip-tion terminator. The transcription terminator serves to terminate thetranscription of the DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bac-terial 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 PAD~7 transcription terminator to the T-DNA gene 7.

One or more o~ 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 speciflc terminators, respectively, to be connected with each of the DNA sequences of the genetic construct may be the sa~e 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 iurther 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 invent~on comprising a chitinase 4 DNA sequence substantially as shown in Sequence 1 or an analogue or subsequence thereof, or a genetic construct of the invention. The vector may . ' '", 82~ 5/nKM/Al2/lssl ~ 29 ~ ~: .
~ , , ;, ~ , , i , , . . -.~ . , . ~ . . . . - - .. : . : . :

. , - ,, . , ,: , . , S~ C ~ J '~

either be one which i5 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 sequenoes in the organism chosen for the production. Thus, the expression vector is a vector which carries the regulatory sequences necessary for ex-pression such as the promoter, an initiation signal and a termination signal, etc. These re~ulatory 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 irom 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 organ-ism 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 Sequence 1 or an analogue thereof or a chitinase gene or pseudogene comprising said DNA sequence.

T~e term "inserted" indicates tha~ 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 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 urganism. The DNA sequence may be present in the ' ~enome or expression vector as defined above in frame with one or more second ~NA sequences encoding a second polypeptide or part thereof so as to encode a fusion protein, e.g. as defined above.
. . . .
' , .
82~ 5/nKM/Al2/l~l ~ 29 -Normally, plant transformation systems are based on the use of plasmids or plasmid derivatives of the bacteria Agrobacterium. The two best known Agrobacte~ia are Agrobacterium tumefaciens and A~r~-bacterium rhizogenes (plasmids thereof are in the following termed S pTi and pRi, respectively). The use of such plant transformation systems is based on the ability of the bacteria Agrobacterium to transfer a specific piece of DNA (T-DNA) to a plant cell in a wounded area. In nature, the T-DNA is located between specific border DNA
sequences on the pTi or pRi which further carries virulence genes necessary for the transfer of the T-DNA to the plant. The Agr~bac-terium transformation system mediates the transfer of any DNA se-quence lvcated 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 Agro-bacterium of a small plasmid containing the DNA to be transferred between pTi or pRi identical borders and a suitable origin of repli-! cation, results in a vector system where the virulence funotions 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 transforma~ion vector is pBI121 and : derivatives thereof, e.g. as described by Jefferson 19~7. :~

Suitably, the vector to be used i5 provided with suitable mar~ers, eucaryotic as well as procaryotic, e.g. genes encoding antibiotic resistance or herbicide resistance or glucoronidase (GUS), e.g.
.~ .
.~.

`~ 82!~450bi.001/LS/JKM/A12/1991 07 29 : ~
, . , . , . ~, , , , , . , ~ . . .

hygromycin or Dther 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 insert-ed in the desired position in the plasmid and to be able to select plant cells transformed with the vector.

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

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

.
The plasmid harboring the genetic construct of the invention is thus preferably transferred into a suitable Agrobac~erium strain, e.g. A.
~umefaciens, so as to obtain an Agxobacterium cell harboring the genetic construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified. This transformation m y be performed in a number of ways, e.g. as described in (An et al., 1988). :
i ' .' Direct iniection of plant tissues by Agrob~cterium is a simple tech-nique which has been widely employed and which is described in (But- -cher et al., 1980). Typically, a plant to be infected is wounded, e.g. ~y cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasiva or brushing the plant with a steel brush (e.g. as described in Exa~ple 15). The wound is then inoculated with the Agrob~cterium, 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 .~.
8294~ /Ls/nuM/Al2/lssl ~ 29 ,., ~ ., , , , , . , ;, . , ~ , -another part of the plant. The inoculated plant or plant part is then sub;ected to selection and regeneration and grown on a suitable culture medium and allowed to develop into mature plants. This is accomplished by use of methods known in the art.

Other very suitable methods for transforming the plant is by use of sonication, electroporation ~Joersbo, l990~ or particle gun methods, e.g. as described by Klein et al., 1989.

When genetically transformed plant cells are produced these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable cul-ture 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 ~issue cultures, for example by selecting transformed shoots using an anti-biotic 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 gene-tic construct in two transformation cycles) or may be associated w1th 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 con-struct of each of its parent plants.

As will be understood from the introductory part of the pre~ent :
speclfication, the chitinase 4 DNA sequence of the present invention or an analogue thereof may be used for diagnostic purposes, which will be furrher explained in the following.
`,; ':
;:

,: -- ., 8~i.~1/Ls/nKM/A~2/1s91 ~ 29 ... -: ~: :,: . :
: . :- . : : : , . ,. . . , . . . .: . :

Various types of diagnosis ~ay be performed by use of the chitinase 4 DNA sequence of the invention. In a given example, chitinase mes- -senger 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 be-longing 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 parti-cular 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 recover-~ ing the hybridLzed 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 lnstance, to characterize . ~'.

,: .
8294~ s/nKM/Al2ll99l ~ 29 :

.. .: . , . ~, , -, ,: . :. . . , - , ~ .
:, ~ ., .,-,.: :, ",j, , ., ~.. . ; .,. . . : . .
: ~: : : : ,,., ,: :, ,: ,.:: ,.
, . . .: . , . . . , . . .. : ~ .
~ . . - , , . . : .
;, ,. . - .: . ;.. .... .. - , .. .:,, ,., :~ . : ..
', : . . :

-chitinase 4 related genes in other plants, it is preferred to employ standard Southern techniques.

The chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may also be used in a method of quantifying the amount of a chitinase 4 related messenger present in differ~nt tissues in an organism, e.g. a plant, the method comprising hybridiz-ing 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 Sequence 1 or an analogue thereof, especially a subsequence thereof, optionally in labelled form, in denatured form or an RNA copy thereof under conditions fa~orable 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 carrîed out in accordance with conven-tional hybridization methods under suitable conditions with respect .
to e.g. strineency, incubation time, temperature, the ratio between the DNA sequence of the invention comprising ~he chitinase 4 DNA
sequence or an analogue or subsequence thereof to be used for the 20 identification and the sample to be analyzed, buffer and salt concen- -tration or other conditions of importance for the hybridization. The .:
choice of conditions will, inter alia, depend on the degree of com-plementarity between thP fragmsnts 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 temperatur~s, whereas a low degree oi 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 ta which DNA or RNA fragments of the sample to be analyz-ed 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 DN~ sequence used for detecting the presence of the chitinsse 4 ~ related gene is preferably labelled, e.g. as sxplained ahove, and the ;
- 8294501~i.001/LS/JKM/A12/1991 07 29 ,. - ' ', ' ~ : i, - ., .' ,-'- . : ' . ' ' , ' ~ ' ,; ,, , :' .

` -presence of hybridized DNA is determined by autoradiography, scin-tillation counting, luminescence, or chemical reaction.

Another approach for detecting the presence of a specific chitinase 4 related gene, e.g introduced by the genetic methods described pre-viously, or a part thereof in an organism, e.g. a plant, in particu-lar 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 ~ample to be analy~ed for the presence of a chitinase 4 relatsd 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 prinoiples 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 ~-1,3-glucanase activity.
~ :, '.
Restriction fragment length polymorphisms (RFLP) are increasingly used to follow specific alleles of genes in various organisms. The -alleles are either themselves followed or they are used as markers .20 (unlinked or linked) in crosses involving other characteristics, e.g.
pathogen resistance and morphological characteristics such as tuber colour. So far, the method has primarily been employed in humans, but .it has also been employed in plants. It is contemplated that the chitinase 4 DNA sequence of the invention or a analogue thereof may `25 be useful in RFLP-analysis of chitinase 4 related genes, especially in sugar beet.
' `~In a further aspect the present invention relates to an antifungal composition comprising a polypeptide encoded by a DNA sequence com-prising the chitlnasa 4 DNa sequence shown in Sequence 1 or an ana-~;30 logue or subsequence thereof as defined above, or by a genetic con-struct of the invention as defined above and a suitable vehicle. In another embodiment, the present invention relates to an antifungal composition comprising a microorganism capable of expressing a poly-'' 8W ~i.~l/LS/nKM/AI2/lssl ~ 29 .. , : , ,, .: .,. ., :: : . . , .:. . . , ; , . :
: . .~ . ~ . " , , :. , , , . , , . ~ i . , :

.

peptide encoded by the DNA sequence comprising the chitinase 4 DNA
sequence shown in Sequence 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 Sequence 1 or an analogue or subsequence thereof or a genetic construct of the inven-tion in an appropriate medium and under conditions which result in the expression of one or more antifungal polypeptides encoded by the DNA sequences, op~ionally rupturing the ~icroorganisms so as to release their content of expressed antifungal polypeptide(s) into the - ;
medium, removing cell debris from the medium, and optionally subject-ing the medium containing the polypeptide(s) to freeze-drying or spray-drying thereby obtaining an antifungal composition comprising the antifungal polypeptide(s).

The antifungal composition according to the invention may be used in combating or inhibiting the germination and/or growth of a phytopa-thogenic 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 25 adapted to its intended purpose, both with respect to the v~hicle 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 con-stituent of the antifungal composition responsible fox or involved in providing the antifungal activity. By the term "antifungal poly-peptide" is meant a polypeptide encoded by the chitinase 4 DNA se-quence of the invention or an analogue thereof or a g~netic construct ; of the invention having antifungal activity, i.e. chitinase activity ; and optionally ~-1,3-glucanase activity as defined above. ~ ;
, . .

, .
82~ /Ls/nK~/At2/l99lo729 : .

.... .. .. , . . :, ... . . .. . . .

:

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 i5 grown as the antifungal agent. The antifungal polypeptide(s) expressed from the microorganisms may be secreted into the medium, e.g. as a conse-quence of the action of a suitable signal peptide capable of direct-ing 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 mediu~ 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 polypep-tide(s) may be obtained as described above using methods known in the art. As mentioned above, it may be necessary or advantageous to sub~ect 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 polypep-tide(s) may be directly applied to the soil in which the plants are present or in which the plants are to be grown, or to the plants or plant parts or to the irrigation watsr. Alternatively, seeds may be treated with the medium, optionally in combination with a convention-al sesd coating composition.

The microorganisms expressing the antiiungal polypeptide(s) can be ; applied in various formulations containing agronomically acceptable vshicles, i.e. ad~uvants or carriers, in dosages and concentrations chosen to maximize the beneficial effect of the microorganism. How-ever, the microorganisms may also be distributed as such under cir-cumstances allowing the microorganisms to establish themselves in .
~ the material to be treated. When the microorganlsm is a microorganism ,~ conventionally found in the 90il, e.g. a rhizobacterium, it will .~ .
8X~i.~l/LS/nKM/AI2/l~l ~ 29 . ' ' .' : ' ~. ' '' ' . . . ' , . I . ,i ,. ' ' ' . , "' , , ::

," ' ' ' ' ,, ' ,:, . ', , ' ' . ''' ' '. ~ ' ' ", ' ~ :
': . - ': " ' ' . ' .~ .' , ' .: ' . . , , ' ':

generally be desirable that the transformed microorganism establishes itself in the soil so that it continuously may secrete the antlfungal polypeptide(s) out into the soil surrounding the plant.

It may be advantageous to add the microorganisms or the medium com-prising 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 i~ is convenient that the microorgan-isms 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 conven-tional means, e.g. by applying the microorganism on a particulatecarrier 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 ordex to induce the chitinase activity of the transformed microor-ganism it may be advantageous to add a small amount of chitin to the medium in which the transformed microorganism is present. - -.
In accordance with the above, the present invention further relates -to a method of inhibiting or the germination and/or growth of a chitin containing plant pathogen, such as phytopathogenic fungus, in or on a plant, which method comprises ; 1) transforming the plant or a part thereof with a genetic construct as defined above and regenerating the resulting transformed plant or plant part into a genetically transformed plant, and/or 2) treating the plant or a part thereof, a seedling or se~d from which the plant is to be propagated, or the medium on which it is grown with an antifungal composition as defined above.
~: ~ '., ' While ~enetic transformation of plants is for most purpose~ 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 82~ 5/nKM/Al2/l~l ~ 29 -.: . . .. ,,: : . ,, . .. . : . . .. . ..

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in certain aspects difficult process, it may be an ad~antage to use a biologically based composition instead of or in addition to the conventionally used and from an environmental point of view undesir-able chemical fungicides.

In most cases the material to be treated with the antifungal composi-tion of the invention is a plant. However, a number of chitin con-taining 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 par~ 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 se-quences, 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 perfoxmed with a linear gradient of NaCl. The absorbance was recorded at 280 ~m.
, Fig. 2 describes the polypeptide pattern of sugar beet chitinase 2, 3 and 4 after puriflcation on a Mono-S FPLC column. Lanes contain 50 ~g of the following proteins. Lanes a and b, chitinase 4; lanes d and e, chitinase 3; lanes f and g, chitinase 2; and lanes c and h, molecular weight markers. The proteins were stained with silver.
' ~'" '. '.
Fig. 3 shows the analysis of the water-soluble products released from 3H-chitin by chitinase 4. 3H-chitin was incubated with 4 ~g 30 chitinase 4 at 37C for 0.25, 0.5, 3 and 24 hours. As a control 3H- ~-' ~ ~ :
8~5~i.~1/L8/JKM/A12/1991 ~ 29 , ' .

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chitin was incubated without enzyme at 37C for 24 hours. The chito-oligosaccharides released were separated by TLC and identified by comparing their migration with that of N-acetylglucosamine (monomer), chitobiose (dimer), chitotriose (trimer) and chitotetraose (tetra~er) 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 ~g of the enzyme was incubated with cell walls from Micrococcus lysodeikticus and the decrease in absorbance at 450 nm was recorded at specified time intervals. 1 ~g of SE ("Sure Ellen") was used as a control, and (50 ng and 5~g) lysozyme (lys) was used as standards.

Fi8. 5 shows the inhibition of the growth of Cercospora by a combina-tion 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 ~g of each of the enzymes chit 4, SE and glucanase 4 were added to the culture at time 0.

Fig. 5B shows the growth of a control culture where no anti-fungal proteins have been added.
:.

Fig. 6 shows the inhibition of growth of Cercospora by chitinases using the microtiter plate bioassay. The time course curves (absor-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 ~1 of a : ~ :
chitinase containing fraction from the chitin-column was added at ~: .
8WSObi.OOl/LS/JKM/A12/1991 07 29 ~ , :

time 0. In curve C 20 ~g of purified chitinase 4 was added to the culture at ti~e 0.

Fig. 7. is an autoradiography showing the effect of chitinase 4 on chitin in the apex of Cercospora hyphae. Incorporation of 3H-labelled N-acetylglucosamine into the hyphae of Cercospora heticola was per-formed by growing the fungus for 20 minutes on growth medium con-taining radioactive monomer of chitin. Incorporation of N-acetylglu-coseamine 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 chiti-nase 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 ~P-18 column. The peptides were eluted with a linear gradient f~om lO~ to 45X acetonitrile from 25 to 75 minutes. Buffer A was water, whereas B was acetonitrile. Both sol-vents contained 0.1% trifluoroacetic acid. The flow rate was 0.7 ml/minute.
':
Fig. 9 shows the separation of three acidic SE chitinase isozymes on an anion exchange column (Mono P3 by the FPLC system. The proteins were eluted with a linear sodium chloride gradient in a 25 mM Bis-Tris buffer at pH 7Ø

Fig. lO describes the two different serological classes of sugar beet, the chitinase 2 and chitinase 4 class. S ~g 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 (left) or antibody to sugar beet chitinase 4 (right) 8~ i.~1/Ls/JKM/A12/19s1 ~ ~9 ,~ .

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~./ s7 ;~ f Fig. 11. Hybridization of different chltinase genes with a chitinase - -4 cDNA probe under specific hybridization conditions. The difierent chitinase genes were spotted on Hybond N-nylon membranes as 1 ~l probes of a plasmid preparation containing the chitinase sequences.

1 a chitinase 1 clone from sugar beet 2 a chitinase 4 clone form sugar beet 3 a chitinase 76 clone form sugar beet 4 a chitinase clone from pea a "SE" clone from sugar beet 6 a chitinase clone 1 from tobacco 7 a chitinase clone 2 from tobacco 8 a chitinase clone 3 from tobacco 9 a chitinase clone from bean a chitinase 4 like clone from rape seed.

The hybridization was carried out at 55C in the following hybridiza-tion buffer: 2 x SSC, 0.1% SDS, 10 x Denhardt's, 50 ~g/ml Salmon sperm D~A and a chitinase 4 cDNA sequence as probe.
- :

. . ' Fig. 12 describes the induction of chitinase and ~-1,3-glucanase in sugar best 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 and ~-1,3-glucanase were measured using the radiotracer assays with 3H-chitin and 3H-laminarin as the substrate, respectively.

: , ' Fig. 13 describes the immunodetection of sugar beet chitinase 2 and 4 and ~-1,3-glucanase 3 in protein extracts of Cercospora Lnfected sugar beet leaves. L~nes I and c contain protein sxtracts from in-fected and control plants, respectively. Antibodies raised against chitinase 2 (left), chltinase 4 (centre) and ~1,3~glucanase 3 (right) ~ere employed.

' ' ' . ' 82g450bi001/LS/J}CM/A12/1991 07 29 , .~

Fig. 14. Site directed mutagenesis of amino acids contemplated to form part of the active site of ~he chitinase 4 enzyme by the use of the PCR technique described in "Materials and Methods". SD0 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:

10 SDl: Trpl70~Tyr TGG~TAC
SD2: Glul90~Gln GAA~C M
SD3: Aspl84~Asn GAT~M T
SD4: Trp207~Tyr TGG~TAC
SD5: Trp205~Tyr TGG~TAC .
, ',~
The PCR products are digested with the relevant restriction enzymes and exchanged with the corresponding sequence in the chitinase 4 gene.

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

j~ Fig. 15B. PCR primers which cAn be used to change the stop codon and to introduce a part of the C-terminal extension, a DraI site i9 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 ~he complementary sequence with the indicated -sùbstitutions is used.
1~ .' Fig. 15C. Four annealed synthetic oligonucleotides containing ,~ the last part of the C-terminal extension, a stop codon, a SmaI
.~ 30 site and an BglII site.
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5/~gM/A12/1~1 ~ 29 .

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The fused gene product can be made by digesting the glucanase gene with XbaI and EcoRI and ligating it with the PCR product digested with XbaI and DraI and the annealed synthetic oligonucleotides di-gested with SmaI and BglII.

Fig. 16. Construction of a hybrid chitinase 4 gene construct with a C-terminal extension Fig. 16A. Chitinase 4 with an underlined tobacco C-terminal extension.

Fig. 16B. PCR primers which can be used to introduce a SmaI site near the stop codon in the chitinase 4 gene. The arrows indicate the PCR primers; for the 5' primer the sequence underneath the arrow is used, for the 3' primer the complementary sequence with the indicated substitutions is used.
', -~ - ' Fig. 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 with B~mHI and EcoRI and ligating it with the PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with Smal and EcoRI.

~ " ' Fig. 17. Construction of the plant transformation vector pBKL4K4 containing the chitinase 4 DNA sequence shown in Sequence 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 encodlng ths chitinase 4. The hatched boxes indicate the coding regions con-tained in the final product. Kb3 (-KB3) and Kb4 (-KB4) are synthetic oligonucleotides acting as primers in the polymerase chain reaction (PCR) using pBlSK4.1 DNA as template. The DNA sequ~nces of KB3 ~nd KB4, respectively, are given in Example 18. Plasmid pPS48 carries a , .~ ' 829450bi.001/LS/ KM/A12/1991 07 29 ~;, .,. 1',,;'',~!,:

conventional 35S enhanced promoter and a conventional 35S terminator separated by a polylinker csntaining unique cloning sites. The plant transformation vector pBKL4 carries a right and a left T-DNA border sequence from the Agrobacterium Ti plasmid pBI121 (Bevan et al, 1984), a GUS ~ene with a 35S promoter and a conventional NOS termi-nator, a conventional NPTII gene with a 3SS promoter and a conven-tional 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 pBKLhK4KSEl containing the DNA sequences encoding chitinase 4 and SE, respective-ly shown in Sequence 1 and Sequence 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 is likewise pBluescript carrying almost the entire "SE" cDNA. The hatched boxes indicate the coding regions contained in the final product. 5 AGCTGTAC3 is an adaptor used for the KpnI-HindIII liga-tion. pPS48 is mentioned in connection with Fig. 17. The construction of the plant transformation vector harboring the chitinase 4 se~uence (p8KL4K4) 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 Sequence 3. The boxed sequences indicate the chitinase 76 ~; gene, the enhanced 35S promoter and the 35S terminator sequences.
pK76.1 is pUCl9 carrying the HindIII-EcoRI fragment encoding chiti-nase 76 in the HindIlI/EcoRI site of the pUCl9 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 ,~ 30 DNA sequences of KB3 and 340, respectively, are shown in Example 18.
Plasmid pPS48 was used in connection with Fig. 17. The plant transformation vector pBKL4 is described in Fig. 17.

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829450bi.001/LS/n~M/A12/1991 07 29 .
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'`''' ~ ' ' ' : ' ' ' ' ' ' , ' ' ~ . ,; ' " .
'.: ~ . , :, ,. , :. j . , : : ~ : , ' ' , . .

-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). Ampli~ication 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' pri-mer. 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.

Fig. 21 describes the separation of sugar beet ~-l,3-glucanases l, 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elu-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 se~uences encoding chitinase 4, SE
and ~-1,3-glucanase, respectively, and shown in Sequence l, Sequence 8 and Sequence 9. The boxed sequences indicate the ~-1,3-glucanase cDNA, the enhanced 35S promoter and the 35S terminator. pGluc l is pBluescript carrying the 1249 bp EcoRI fragment encoding the ~-1,3-glucanase. The hatched box indicates the coding region. Plasmid -~
pPS48M is the same as pPS48 described in connection with the con-struct shown in Fig. 17, except that the plasmid ~s supplemented with two additional restriction sites (EcoRI and KpnI) at each site of the E35S-35St box. The construction of the plant transformation vector harboring the chitinase 4 and SE sequences is described in Fig. 17 and Fig. 18.
, :. .
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i.~1/LS/~UM/A12/1ss1 ~ 29 . .' - . ' . '. : .' . ' . ' '. ~ ' ; ' . ' . ' , ,': ,' . ', ' : . ... :, ' ,,, . .' '': ' ,' : "' " ' '.' ' ' ' " . ' ' ':, ' . . " . ,' ,,. ' ' . ' .' ~ ;' ' .' ' . " ' ' '' REFERENCES

An G. et al., 1986, Plant Physiol., Vol. 81, pp. 301-305 An G. et al., 1988, Plant ~olecular Biology Manual A3, 1-19, Kluwer Academic Publishers, Dordrecht 5 Barkardottir et al., 1987, Developmental Genetics, Vol. 8, pp. 495-Barkholt et al., 1989, Anal. Biochem., Vol. 117, pp. 318-322 Benton et al., 1977, Science, Vol. 196, p. 180 Bevan, 1984, Nuc. Acid. Res. 12, p. 8711 Bol J. F. et al., Plant pathogenesis-related proteins induced by virus infection, 1990, Annu. Rev. Phytopathol., Vol. 28, pp. 113-38 ;' ' Boller T., Ethylene and the regulation of antifungal hydrolases in plants, 1988, Oxford Survey of Plant Molecular & Cell Biology, Vol.
5, pp. 145-17b Butcher D. N. et al., Tissue Culture Methods for Plant Pathologi.sts, 1980, eds.; D. S. Ingrams and J. P. Helgeson, pp. 203-208 Chiirgwin et al., 1978, Biochemistry, Vol. 18, pp. 5294-5299 Feistner et al., Charting of rat pituitary peptides by plasma desorp-tion and electrospray mass spectrometryj Proceedings of the Nato Advanced Research workshop on Methods and Mechanisms for Producing Ions from Large Molecules, Minaki, June 1990 ~ ' Fincher G. B. et al., Primary structure of the (1~3,1~4~-~-D-glucan 4-glucohydrolase from barley aleurone, 1986, Proc. Natl. Acad. Sci.
- USA, Vol. 83, pp. 2081-2085 i.00l/LS/nUM/AI2/lssl0729 :~:

-Fraley R. T. ~t al., Expression of bacterial genes in plant cells, August 1983, Proc. Natl. Acad. Sci. USA, Genetics, Vol 80, pp.

Fritsch E. F., Molecular Cloning, A laboratory manual, 1989, 2nd edition, Cold Sprin~ Harbor Laboratory Press Herrera-Estrella L. et al., Expression of chimaeric genes transferred 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 Biolo~y Manual Bl, 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, Leai` disc transformation, Plant Molecular Biology Manual A9, 1-16 : . .
Jacobsen S. et al,, 1990, Physiol. Plantarum, Vol. 79, p. 554 .

Janssen B.-J. et al., Localized transient e~pression of GUS in leaf discs following cocultivation with Agrobacteriu~, 1989, Plant Molecu-- lar Biology, Vol. 14, pp. 61-72 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 ; ~ 25 Joersbo M. et al., Direct gene transfer to plant protoplasts by .
8294~i.~1/LStnKM/A12/lssl ~ 29 .:: .. , .. .. . : . . : . .. :, , .: .: . . . : ...... . . .. . . . .. .. .

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.

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 TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector, 1986, Mol Gen Genet, Vol. 204, pp.

15 Kragh K. M. et al., 1990, Plant Science, Vol. 71, p. 55 Kragh K. M., 1991, Chitinases and ~-1,3-glucanases in barley (Hordeum vulgax L.), Plant Biology Section, Ris0 National Laboratory, Ros-kilde, Denmark ' ' .
Kyhse-Andersen, 1984, J. Biochem. Biophys. Methods 10, pp. 203-209 , 20 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, 1991, The Journal o~ Biological Chemistry, Vol. 266, No. 3, pp. 1564-1573 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 : ' 8~bi.~l/LS/nCM/AI2/l99l ~ 29 ..

t` ~

van Loon L. C. et al., Identification, purification. and characteriz-ation of pathogenesis-related proteins from virus-iniected Samsun NN
tobacco leaves, 1987, Plant Molecular Biology, Vol. 9, pp . 593-609 Marcussen J. et al., Manuscript submitted, Anal.Biochem.

5 Métraux J.-P. et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86, pp.

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, Intexlaken, Swit~erland, 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 , .
Paszkowski J. et al., Direct gene transfer to plants, 1984, The EMBO
20 Journal, Vol. 3, No. 12, pp. 2717-2722 Reynaerts A. et al., 1988, Selectable and screcnable markers, Plant Nolecular Biology Manual A9, 1-16 Rogers S.G. et al., Use of cointegrating Ti plasmid vector~, 1988, Plant Molecular Biology Manual A2, 1-12 25 Samac et al., 1990, Plant Physiol., Vol. 93, pp. 907-914 '' ' ' .
8~i.~1tLS/nKM/A12/1991 ~ 29 ~ , , ... , . ' . , ' ,. ' .', ' .' ' '; '', ,; ." '. '; ' "' ; '.' ~ '" . "' ' Sambrook J., Molecular Cloning, A laboratory manual, 1990, 2nd edi-tion, Cold Sp~ing 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 i Shinshi H. et al., Agric. Biol. Chem. 47, 1455-1460, 1983 :
Shinshi H. et al., Evidence for N- and C-terminal proc~ssing of a plant de~ense-related enzyme: Primary structure of tobacco prepro-~-1,3-glucanase, 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, pp.

Shinshi H. et al., Structure of a tobacco endochitinass gene:
evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain, 1990, Plant Molecular Biolo~y, Vol. 14, pp. 357-368 `` Sierks M. R. et al., Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Aspl76, Glu179 and Glu180 in the enzyme from Aspergillus awamori, 1990, Protein Engineering, Vol. 3, No. 3, pp. 193-198 .
,~ 20 Stougaard J. et al., 1986, Nature, Vol. 321, pp.669-674 Tomes D.T. et al.j Direct DNA transfer into intact plant cells and recov~ry of transgenic plants via micropro~ectile 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 Agrobact~rium tumefaciens, 1984, The EMB0 Journal, Vol. 3, No. 12, pp. 2723-2730 . '.
i.OOl~/nuM/Al2/l~lo729 ~ ';

, . . . . - . .,, . :: ., ,, , . . .~: .: .. . .. . . .. .. .

,'~ .' ` ' ' ' ' ' ' '' . ' " ' . ` ,' . ' '~ . ' , ' ' , " '"', ' " " ` ' ", ',. . ,", , " ', ' ' 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. S, pp. 103-108 Wing D. et. al., Conser~ed function in Nicotiana tabacum of a single 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 i .
i 8W ~ /Ls/JKM/Al2/lssl ~ 29 '::

,' :' . ~,.~ ~ . . ' ', . ., . ' SEQUENCE LISTING

BRIEF EXPLANATION

Sequence 1 the chitinase 4 cDNA sequence and the chitinase 4 amino acid sequence (harboured in the cDNA sugar beet chitinase 4 clone B15) Sequence 2 the partial DNA sequence and amino acid sequence of the genomic chitinase 4 clone Sequence 3 the DNA sequence and deduced amino acid sequence of the genomic ~lone chitinase 76 Sequence 4 A compa~ison between the DNA sequence of the chitinase 4 cDNA sequence shown in Sequence 1 and the genomic clone chitinase 76 shown in Sequence 3 Sequence 5 A compa~ison between the amino acid sequence of the chitinase 4 cDNA sequence shown in Sequence 1 and chitinase 76 shown in Sequence 3 . .
Sequence 6 A comparison between the non-coding 5' sequences of the chitinase 4 and chitinase 76 genomic sequences shown in Sequence 2 and Sequence 3, respectively ' : . Sequence 7 The DNA sequence of the entire sugar beet chitinase 1 :
: 20 gene including introns, promoter and leader sequence, ~ .
and the amino acid sequence deduced from the coding region of tbe chitinase 1 gene : , ' .
~: ~ Sequence 8 the cDNA sequence and the deduced amino acid sequence ~:
of the acidic sugar beet chitinase SE
.
Sequence 9 the cDNA sequence and the deduced amino acid sequence ~: of the basic sugar beet ~-1,3-glucanase ....
.: :
8X~ StnKM/AI2/l991 ~ 29 :
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. .

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DETAILED EXPLANATION

Sequence 1 The DNA and deduced amino acid sequence of the B15 chitinase 4 cDNA
clone isolated from a sugar beet ~ZAP cDNA library The sequence is 966 bp long and encodes a protein having 264 amino ; acid residues in the polypeptide chain. The leader sequence consistsof 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 Sequence 2, encodes a protein having 265 amino ; acid residues. The signal sequence encoded by ~he gene consists of 24 ~.
`~ amino acid residues.
'` : : Sequence 2 The partial DNA and deduced amino acid sequence of a genomic clone ` encoding the chitinase 4 gene isolated from a sugar beet EMBL3 geno-., mic library '. :.
The sequence is 691 bp long and encodes the first 112 of ths 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 (TATMA) located at posLtion 287, which is 70 bp upstream of the ATG start codon.

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, : . :: . : . , Seguence 3 The DNA and deduced amino acid sequence of a genomic clone encoding the chitinase 76 gene isolated from a sugar beet EMBL3 genomic libra-ry The sequence is 1838 bp long and encodes a protein ha~ing 268 amino acid residues in the polypeptide chain. The leader se~uence consists of 24 amino acid residues followed by a hevein domain of 35 and a functional domain of 209 amino acid residues. The gene contains one intron which is located in position 875 to 1262~ The exact location 10 of this intron is based on an alignment with the B15 chit 4 cDNA .
(Sequence 4). The intron borders con~ain the consensus GT/AG se-quences. 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 Sequence 4 .. :
A comparison between the DNA sequence of the B15 chit 4 cDNA clone and the genomic chitinase 76 clone The position of the chitinase 76 intron is easily seen at position 875 to 1262~ The homology of the sequences is about 73X~

indicates identical nucleotides.

Sequence 5 ' A comparison of the amino acid sequences of the genomic chitinase 76 clone and the B15 chitinase 4 cDNA clone A homology of about 80X is seen. The extra 3 amino acids in chitinase 76 are the amino acids (Ser, Thr, Pro) ln position 62 64~
' indicates identical amino acids.
:
;

829450bi.001/LS/.lKM/A12/lYY1 07 2Y
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;.' ' , `, , . , . , . ' 'I ' . , . , ' ' ' . . ., ". . ' . . . ,' . ' ." ' . , " ; ~ ' Sequence 6 A comparison of the n~n-coding 5' sequences of the chitinase 4 and 5' sequences of the chitinase 4 and chitinase 76 genomic sequences 8 boxes of strong homology is observed. It is contemplated that some of these boxes may be of regulatory importance.

: indicates identical nucleotides.

The ATG start codons are underlined.
.. :
Sequence 7 DNA sequence of the CHl gene The amino acids of the CHl protein are shown below the corresponding ~ codons. The following are underlined: TATA-box (TATAAA), methionine ; start codon (ATG), intron borders (AG and GT), stop codon for trans-, lation (TAA), and polyadenylation signal ( M TAAA).
,~ ,. .
Sequence 8 ,` ~ '.
.~ 15 The DNA and deduced amino acid sequence of the "SE" cDNA clone iso-,~ lated from a sugar beet ~ZAP cDNA library The sequence is 1106 bp long and encodes a protein ha~ing 293 amino acid residues in the polypeptide chain. The leader sequence consists , of 25 amino acid residues and the functional domaln of 268 amino acid residues. The cDNA clone has a 5' non-coding region of 17 bp and a 3' flan~ing region of 202 bp.

Sequence 9 ,~ The DNA and the deduced amino acid sequence of a ~-1,3-glucanase cDNA
~ clone isolated from a su~ar beet ~ZAP cDNA library i .

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The sequence is 1249 bp long and encodes a protein having 336 amino acid residues in the polypeptide chain. The cDNA clone has a 5' non-coding region of 33 bp and a 205 bp 3' flanking region containing a putative polyadenylation signal at position 1157 and a poly A tail. : -'', " ' .

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MATERIALS AND NETHODS

Biological material Plants .:
Seeds of Beta vulgaris, cv. "Monova'l, were sown in clay mixed peat ("Cycas'~) and placed in growth chamber with 11/13 hours day/night cycles, 25/18C (day/night) and 70X rh. Light intensity was approxi- :
mately 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. Twic~ a day the plants were supplied with weter containing 0.1X fertilizer: "St~erne" uni~ersal iertilizer, 4:1:4 (N:P:K). Six weeks after sowing ~he plants were ready for infection experiments with Cercospora beticola.

Nicotiana tabacum and N. benthamiana plants were obtained as describ-ed above.

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

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

Growth of Cercospor~ species .
Yhe fungus was grown on solid growth medium in Petri dishes. Sterlle "Potato Dextrose Agar" ("Difco", 39 g/l) was used as growth medium.
A plug o~ mycelia was placed in the center of the Petri dish and the culturs was incubated at room temperature for 4 weeks. Mycelia for spore induction was "harvested" by cutting of the whole mycelia "mat"
including some agar.

829450e~001/lS/~RM/A12/1991 07 29 t5:55 : . , . - . . . .............. : ;

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Sporulation of Cercospo~a species Mycelia was mixed with distilled water (1:2) in a 50 ml sterile glass tube and homogenized using a "Ul~ra Turrax T25" mixer operated at 8000 rpm for 2 minutes.

S 1 ml of the homogenate was transferred to a Petri dish containing solid sporulation medium. "V-8" was used as medium. It contained 200 ml "V-8" juice (Cambells, Italy), 800 ml water, 3 g CaC03 and 20 g agar.

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

After 7 days of incubation the spores were harvested by pouring 10 ml distilled water onto the Petri dish and firmly brushing the surface . 15 of the culture with a sterile brush.
. . .
- The resulting spore suspension contained approximately 100,000 spores/ml.

`. Infection with Cercospora species ,~ .
For inoculation, 12.500 spores were suspended in 1 ml of water con-taining 20 ~g of Tween-20. Using a chroma~ographic atomizer the suspension was applied to the upper leaf surface of six-week old ~ sugar beet or Nicoti~na plants until "run off". Immediately after inoculation the plants were placed in a "mist chamber" kept at 30~C, ; lOOX rh and 24 hours light (cool white). After 5 days of incubation the plants were moved to a growth chamber kept at 30C, 80X 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 - 8~XkL~l~/n~M/Al2/lssl ~ ~9 IS~S
; ., - :. :
., ., ,, . " ,,~., ,, .,, .. ,. ,.~ .. ,.. . ,, ., ,,, .. ., . ~.. : -i i ~: ,_ ; j, -i~ small scale purification of chitinase 4, the acidic chitinase ~SE" and ~-1,3-glucanase, and ii) a time course study to determine the expression level of total enzyme activity using radiochemical assays and immunoblotting.
iii) determination of the expression level of each of the enzymes in transgenic plants using the above (ii) techniques.
vi) isolation of mRNA for use in the construction of a cDNA library.

Infection of Nicotiana plants with the root pathogen Rhîzoctonia solani An isolate of R. solani was obtained from Dr. K. Tæavella-Klonavi (Saloniki, Greece).

An inoculum of R. solani was prepared on barley grains soaked twice in lX of potato dextrose broth and autoclaved. The grains were inocu-lated with agar disks of a growing culture of the fungus and incubat-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 concentratio~s, and the transgenic plantlets which had been rooted for 14 days, were j 20 transplanted into the infected soil. The percentage of surviving plants may be recorded after 1, 2 and 3 weeks, respectively, and after 3 weeks the surviving plants are assessed for root damage.
Alternatively, seeds from transgenic plants were sown d~rectly in the infected soil.

Extr~ct{on of protein from 1 g of sug~r beet leaf material More specifically, the small scale purification was carried out as follows. 1 g of leaf material was homo~enized by a Ultra-Turrax homo-genizer in citrate buffer (0.1 M, pH 5, 2 ml/g tissue), containing 1 mM of both ben~amidine, dithiothreitol and phenylmethylsuphonyl fluo-ride. Particulate matters were removed by centrifugation at 15,000 .

8~4xkx~ /nuM/Al2/l99l ~ 29 15:55 ,: ,' ' ': ~ . ' '.:., " ;'' ' '," ' ' ~ ' ',:' . . ,. ,,. ' .: '' , : ' ' ' :

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g for 15 minutes. The supernatant comprising the enzymes was trans-ferxed to another test tube before the centrifugation was repeated.
.
Large scale extraction of proteins from sugar beet leaf material To determine the antifungal potential and the amino acid sequence of the enzymes, large quantities of pure enzymes arP required. To obtain sufficient quantities, i.e. mg quantities, a large scale purification of chitinase 4, the acidic chitinase "SE" and ~-1,3-glucanase was carried out from 2 kg of leaf material from naturally infected sugar beet plan~sj cv. "Monova". Naturally infected leaves carrying 50 or more necrotic lesions were picked in the field at a breeding station in Italy (Maribo-Italy, Bologna) and stored at 4C until the extrac-tion of chitinase 4 was carried out.

Preparation of a chitin column - 30 g of chitosan (from Protan; Sea Cure P, No. 709, Norway) was dis-solved in 600 ml of 10X 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 trans-ferred to a beaker on a magnetic stirrer, and 40 ml of acetic anhy- `~
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, co-vered with methanol, and homogenized for 2 minutes at full power.
Methanol, acetic acid and unreacted acetic anhydride were removed by ` filtration in a Buchner funnel using Whatman No. 1 filter paper. The ; filtrate was transferred to a beaker, 1 l of 1 M Na2C03 was added andthe pH was adjusted to 9 with 6 N NaOH. 50 ml of acetic anhydride was slowly added and the pH ad~usted to 9. The reaction was allowed to , ~ 30 take place for 1 hour before the final product was collected by fil-tering 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 ~'~ ' '.
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829450cx.001/LS/JKM/A12/1991 0'7 29 15:55 . ~ .

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column was prepared from the regenerated chitin by use of the conven-tional procedure according to Pharmacia.

Preparation of radioaceive colloidal chitin 2 g of chitosan was acetylated with 3H-labelled acetic anhydride as S described for the synthesis of unlabelled chitin (see above). After e~tensive washing of the 3H-labelled chitin on a Buchner funnel, it was transferred to a beaker. 50 ml of concentrated ice-cold HCl was added, and the chitin was dissolved by stirrin~ for 5 minutes at 0C.
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 sedi-mented by centrifugation and resuspended in water several times to remove excess acid and ethanol. Finally the colloidal chitin was suspended in 200 ml of water and sonicated for 5 x 1 minute at full power. The 3H-labelled chitin was stored at 4C before use.

Preparation of a Laminarin column .
7`: Divinylsulfone activated agarose (Mini-leak high, KEM-EN-TEC, Den-~' mark) was employed to immobilize laminarin (~-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 H20 was added. The reaction was allowed to proceed for 16 hours at 25C on a shaking table. Unreacted divinylsulfone groups were blocked by incubation with a solution of 5X mercaptoe--, thanol in 1 M K-P-buffer at pH 9.5. The r~action time was 16 hours at 25C. Residual mercaptoethanol was removed by excessive washing of the gel on a Buchner funnel. The Laminarin-Agarose was suspended in 20 mM Tris-bu~fer at pH 8.0, and stored at 4~C. A laminarin column was prepared from the Laminarin-Agarose using the conventional proce-dure accordin~ to Pharmacia.

,~ ~ 30 Synthesis of 3H-labelled laminarin i,~ - ,, i Laminarin was labelled with radioactivity by reduction with 3H-label-,~ led NaB3H4. 500 mg laminarin (from Laminaria digitata, Sigma) was ~ 82~kx~ool~s/nEM/Al2/l~lo729 15:55 ,:: . . . : .. . - . :: , . .~ ~, ,, , , . . : . , , ,.,, ~ . , , :

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dissolved in 2 ml H20, and purified by precipitation by addition of 800 ~1 NaCl (0.2 g/ml) followed by 8 ml absolute ethanol. The preci-pitate 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 O.1 N NaOH. This solution was transferred to a reaction wessel containing 5 mCi of NaB3H4. After stirring for 90 min at 25C, 600 ~l of 1 M HCl was added to destroy unreacted NaB H4. The reaction mixture was divided into 500 ~1 aliquots and 200 ~l of NaCl and 2 ml of absolute ethanol was added to each test tube.
After storage for 10 min. at O~C, the precipitate was collected by centrifugation for 5 min. at 15.000 x g. The 3H-labelled laminarin was dissolved in 500 ~l of H20 and the precipitation was repeated until the background le~el in the supernatant was less than 100 cpm per 20 ~1. The labelled solution of laminarin was stored at -20C.
Before use in the ~-1,3-glucanase-assay, the solution was diluted 20-fold with water.

Reverse Phase-~PLC

A Kontron AG (Zurich, Switzerland) instrument consisting of 2 model 420 pumps snd a solvent mixer was used. Gradient control and data 20 acquisition was performed by a Kontron model 450-MT Data system ~
according to the manufacturers instructions. Proteins eluted from the ~:
Mono S column (see below) were subjected to RP-HPLC on either VYDAC
RP4 (0.46 x 15 cm; 10 ~m particle size; The Separa~ions Group, He-speria, California) column or a Poly F (Du Pont de Nemours) column.
The mobile system used for RP-HPLC was buffer A: 0.1% TFA in water and buffer B: O.lX TFA in acetonitrile.

SDS-PAG~

SDS-PAGE of crude plants extracts or partly purified chitinases were performed on an Easy-4 apparatus (Kem-En-Tec, Denmar~) using the Tricine SDS-PAGE system described by Schagger and ~on Jagow (1987). A
total of 25 ~g of protein was applied to each lane. Pure chitinase isoenzymes were analyzed on the Phast-System (Pharmacia) in accor-dance with the manufacturers instructions. ;~
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En~yme assays The radiochemical chitinase assay Chitinase activity was determined radiochemically with 3H-chitin as a substrate.

The specific activity of the 3H-chitin was 460 cpm/nmol N-acetyl-glucosamine (GlcNAc) equivalent (or 2,3 x 106 cpm/mg 3H-chitin). It was determined by scintillation counting and colorimetric determina-tion of GlcNAc after total hydrolysis of 3H-chitin by crude chitinase preparations from sugar beet leaves and e~ochitinase from serratia mlrcescens or St~eptomyces griseus.
:
The assay mixture contained in a total volume of 200 yl of enzyme solution, 50 ~1 of 3H-chitin suspension (containing 100.000 cpm) and 10 ~mol of sodium citrate (pH 5,0). After mixing, the enzymatic hydrolysis of 3H-chitin was allowed to take place at 40C for 15 min.
before addition of 300 ~1 of 10 % (w/v) TCA. In order to decrease the background reading, 100 ~1 of bovine serum albumin (lO mg/ml) were added before the insoluble 3H-chitin was removed by centrifugation at 15.000 x g for 5 min. The radioactivity in 300 ~1 supernatant was determined by scintillation counting.

20 The radiochemlcal ~-1,3-glucan~se assay ,- ~
1,3-glucanase activity was determined radiochemically with 3H-labelled laminarin as substrate.
;~ ~ . ., ~ ~ The assay mixture consisted of 50 ~l of enzyme extract, 50 ~il of 0,1 ,~ M Na-citrate pH 5,0 and 10 ~1 of 3H-labelled laminarin (192.000 cpm).
Incubation was carried out for 15 min. at 40C. To terminate the reaction, 1000 ~l of abs. Ethanol and 50 ~il of a saturated NaCl-~ solution was added. After 10 min. at 0C, unreacted laminarin was `~ ~ removed by centrifugation at 10.000 x g for 5 min. An aliquot of 400 ~l of supernatant was transferred to a scintillation vial. 5 ~l of PICO-FLUOR-40 were added and the amount of radioacti~ity was deter-mined by a liquid scintillation counting.

.
` ~829450cx.001/15/JRM/A12/1991 07 29 15:5S
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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.

~-glucuronidase (GUS)-Ass2y When GUS is employed as a reporter gene in connection with the con-struction of the genetically transformed plants ~ccording to the present invention, the success of the transformation may be deter-mined by use of the following GUS-assay described by Jefferson, 1987.
15 Leaf tips were sliced into thin sections (<0.5 mm) and incubated in a :r 2 mN solution of x-gluc. (5-bromo-4-chloro-3-indolyl-B-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 trea-ted for 2-4 hours at 37C, rinsed with water and the staining inten-20~ sity recorded by visual inspection by microscopy.

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829450ex.001/LS/1RM/A12/1991 07 29 15:55 -Purification of ch~tinase 2, 3 and 4, acidic chit~nase "SE" and ~-1,3-glucanase isoenzymes Acidic and basic chitinase isoenzymes were purified together with ~-l,3-glucanases from sugar beet leaves as shown in the following flow diagram.

2 kg of sugar beet leaves O.l M Na-citrate 1 ~M DTT Homogenization l mM BAM, pH 5.3 Centrifugation~ ~ .
Heat treatme It, 50C, 20 min.
90X (NH4)2so4 Dialysis lO ~ Tris, ~ pH 8.0 FF-Sepharose Q ---Chitin column 1, 1 ~ ..
Ro~ff FPLCi Mono-S FF S~ ~pharose Q
FF-Sepharose S RP-HPLC Chromato-focusing Laminarin-Agarose chltinase 2,3, and 4 FPLCi Mono P
acidic chitin~se .
FPLC, Mono-S
RP-HPLC
~-l,3-glucanase 3 and 4 : :
The sugar beet leaves were obtained in Italy (large scale, see "Bio-logical Material"). In the following each of the purii`ication steps outlined below will be explained, The equipment and procedure used ; ~ for each step are carried out as described below.

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82~s~l/Ls/nKM/Al2/l99l ~ 29 ~:55 .

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E~traction of protein from Cercospor~ beticola infected su~ar beet leaves All steps were performed at 4C. Centrifugation was carried ~ut at -20000 x g for ~0 minutes in a Centrikon model H-401B centrifuge, throughout the purification procedure.

Preparation of cellfree-extracts 2 kg of Cercorspora infected leaves were homogenized in 4 1 Na-ci-trate buffer pH 5.0 containing 1 mM DTT (Dithiothreitol), 1 mM BAN
(Benzamidine) (starting buffer) and 200 g Dowex lx2 (100 ~m/mesh size. The homogenate was squeezed through a double layer of 3i ~m mesh nylon gau7e, before centrifugation.

` P~ecipitation with heat and Ammoniu~sulfate ~ . :
~. :
The supernatant fraction obtained after the centrifugation was heated :
at 50C for 2n minutes and after cooling to 4C, the precipitate was collected by centrifugation. Solid ammoniumsulfate was added to the supernatant until a 90% saturation was achieved. After centrifuga- -! tion, the precipitated proteins were dissolved in starting buffer; 1 ~ ml of buffer/10 g of starting matérial.

j Purification of chitinase 2, 3 and 4, acidic chit~nase "SE" and ~- -1,3-glucanase by col~mn chromatography Chitlnase and ~-1,3-glucanase isoenzymes were purified from the am-monium sulfate precipitated protein fraction. After solubilization, the protein solution was dialyzed against 10 mM Tris pH 8.0 contain-ing 1 mM DTT and 1 mM BAM. Denatured proteins were removed by centri-fugation and the supernatant was loaded on the above outlined two ; columns e.g. i) a 50 ml Fast Flow Sepharose Q (Pharm~cia) and ii~ a 100 ml Chitin column (prepared RS described above), the columns being connected in series. The columns were equilibrated with the Tris :
`~ buifer, before 281 ml of the sample were loaded. Unbound proteins in-cluding ~-1,3-glucanase were removed by extensive washing with the starting buffer. After disconnecting ~he Fast Flow Sepharose Q co-.

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lumnl 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.

Puri~ication of ~-1,3-glucanase Separation of ~-7,3-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-acstate buffer at pH 4.2 containing 1 mN 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 ma;or peaks A, B and C of ~-1,3-glucanase activity were ob-served. Peak B was further fractionated by affinity col~n chromatog-raphy on Laminarin-Agarose. Peaks A and C were not further purified.

; Pu~ification of ~-1,3-glucanase on Laminarin-Agarose ; 20 A 28 ml column of Laminarin-Agarose was equilibrated wi.th 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 presqure dialysis to 15 ml and dialyzed against the Tris buffer. After loading of the ~ -sample on the Laminarin-Agarose column, the flow through the colu~n was stopped for 20 minutes to allow the ~-1,3-glucanase to bind to the af~inity ligand. Unabsorbed protein was removed by w~shing with Tris buffer. ~-1,3-glucanase was eluted with 1 N NaCl in Tris buffer.
~ .
Purification o~ 4 ~-1,3-glucanase isoenzymes by FPLC

Fractions from the Lamlnarin-Agarose column with ~-1,3-glucanase actiYity were combined, concentrated and dialyzed overnight against a B29450cx,001/15/JKM/A12/1991 07 29 15:55 ,: ., . . . , i . . ~ , , ,., . : : ~ . ~ , : .

20 mM acetate buffer pH 4.5. The proteins were separated on a cation exchange column (Mono S) (Pharmacia) on the FPLC system usin~ a linear NaCl gradient. Four major protein peaks were observed (see Fig. 21). They all four hydrolyzed the 3H-labelled laminarin substra-te in the radiochemical assay for ~-1,3-glucanase (see above).

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

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

After this desalting/purification step, peak 3 and 4 were ready for i) N-terminal amino acid sequencing, ;i) amino acid composition i5 analysis (see Example 8), iii) tryptic digestion to achieve peptides and iv) injecting into rabbits to produce polyclonal antibodies.

Puriiication of chi~nase 2, 3 and 4 . .

Elution of the chitin column with 20 mN 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 mN Na-acetate buffer at pH 4.5.

2 ml aliquots were loaded onto the above mentioned cation ~xchange column (Mono-S) by the FPLC system (Pharmacia). Non-adsorbed mate-rials were removed by washing with the acetate buffer. Elution of the chitinase isoenzymes was achieved with a linear gradient from 0 to 1 M NaCl in the acetate buffer. The elution profile is shown in Fig.
1. For further purification, the reverse phase VYDAC RP4 HPLC column was employed. The conditions were similar to those described above in connection witb the purification of ~-1,3-glucanase.
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82945~x.~l/LS/nUM/AI2/l9sl ~ 29 15:55 . .

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-Purificat~on of acidic chitinase "SE"

Purification of the acidic chitinase "SE" on anion-exchange chroma- -tography The acidic chitinase "SE" was eluted from the above described Fast Flow Sepharose Q column with the Tris buffer containing 0.5 M NaCl as shown in the purification scheme. The proteins were dialyzed against 10 mM Tris-HCl, pH 8.0, and loaded onto a 40 ml "Fast Flow Sepharose Q" column equilibrated with the same buffer. The proteins were eluted with a 800 ml linear sodium chloride gradient from 0 to 0,5 mM NaCl.
Fractions containing chitinase activity as determined by the radio-chemical 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 ~he same buffer and 50 ml of the sample was loaded Unabsorbed proteins were removed by washing with tha 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".
, .
Purific~tion of acidic chitin~se "SE" by FPLC
':
Protsin fraction with high chitinase activity as determined by the ; 25 r~diochemical chitinase assay described above were pooled and dialy-zed against 25 mM Bis-Tris at pH 7Ø 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 isoen- :
zymes of acidic chitinase "SE" was separated using a linear salt gradient from 0 to 0.3 M NaCl (Fig. 3).
,, ': ' .:

8W ~x~ 5/n~M/Al2/lssl ~ 29 l5:55 ~, " ,, "", ,1 ",' ",, "" , , . ",".",,,, ,, ,,, ;,, ,,, ,,'~ "" . ", ~"", .. ",,,, ., ,," ", ,, , ~", ~., ,,, "

Analysis of the enzymatic cleavage pattern of sugar beet chitinase 4 40 ~l of 3H-labelled chitin (50,000 cpm) was incubated with 7 ~g of chitinase 4 (purified as described above) in a 0.1 M citrate buffer at pH 6.5. The total volume was 300 ~l. After a specified time (15 min., 30 min., 1 hour and 24 hours) the reaction was stopped by the addition of 300 ~1 of 10X (w/v) TCA. The unreacted polymer of 3H-labelled chitin was removed, and an aliquot (300 ~l) of the super-natant was applied to a thin layer chromatography (TLC) plate (Silica 10 gel 60 H, Merck). The mobile phase was n-propanol/H20/~H3 (70/30/1;
v/v/v ) . , , '.
, ..-:
A standard of N-acetylglucosamine-derived oligosaccharides was pro-duced by acid hydrolysis of chitin (Rupley, 1964). This standard was used to localize the products from the enzymatic cleavage on the TLC
plate. Zones of interest on the TLC plate were removed by scraping with a razor blade, and the silica gel containing the 3H-labelled oligosaccharides was transferr0d to a scintillation vial. 10 ml of scintillation liquid Dimilume (Packard Instruments) were added and the radioactivity was determined by a liquid scintillation spectro-photometer.
':
Ant~fungal nctivity An inhibitory efiect of sugar beet chitinase 4 has been observed onthe growth of both Cercospora beticola and Trichoderma reesei either alone or in combination with the acidic chitinase "SE" and the basic ~-1,3-glucanase 3. Germination of spores and/or growth of hyphae from phytopathogenic fungi, e.g. Cercospora, in the presence of antifungal proteins may be analyzed with three different methods.

M0thod I is carried out on microscope slides covered with a thin film of medium ~nd incubated with either buffcr (control) or ~g quantities of the antifungal proteins. Germination of spores or growth of the mycelium is followed by staining with Calcofluor ~hite before analy-sis by a fluorescent microscope. ~
:
. :
.
. .
82945oex~ool/Ls/lKM/Al2/l99l 07 29 15:55 Method II is carried out in microtiter plates containing growth media, 10 or 100 spores from Cercospora, buffer (control) or the antifungal proteins. The plates are incubated at 25°C before the optical density (at 620 nm) is determined at specified time inter-vals.
In method III, radiotracer techniques in combination with autora-diography are used to demonstrate that chine and .beta.-1,3-glucan are important cell wall components in Cercospora and the 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 dex-trose agar (PDA) and stored for 6 hours on moistened filter paper in petri dishes. 10 µl of a spore suspension (10,000 spores/ml) was placed in the center of the slide. 10 µl of a 10 mM Tris-buffer, pH
8.0 or 10 µl of a preparation containing 20 µg of the antifungal protein to be tested was applied to the spore suspension. The an-tifungal protein was dissolved in the Tris-buffer and filtered through a 0,22 µm filter before mixing with the spore suspension. The petridish was sealed with tape and incubated for 24-48 hours at 30°C
and full light. At the time for evaluation, the culture was stained with the fluorescent dye Calcofluor White (0.05% (w/v) in water) for 10 min. Calcofluor White binds primarily to cell walls containing nascent structures of chitin, and the fluorescent dye may therefore serve as a marker for differentiation and growth of the hyphae cell wall.
Method II: Microtiter Plate Bioassay 100 µl of potato dextrose broth (PDB) liquid growth medium was placed in each wall 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 1:1000 and 1:1000 with sterile water, before aliquots of 100 µl was transferred to the microtiter walls. The antifungal proteins were dissolved in the same buffer and treated as described above for method I. The bioassays were carried out with 5 repeats for each dilution of the fungal spores. The microtiter plate was sealed with tape to avoid evaporation and contamination. After incubation at room temperature on an agitator operated with 50 rpm, the tape was removed and twice a day, the absorbance was measured at 620 nm. The germina-tion 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-ace-tylglucosamine was distributed uniformly over a one day old culture.
After incubation for 20 min. (pulse labelling), the reaction (growth-/incorporation) was stopped by dipping the microscope slide in 6~
(w/v) of TCA. The preparation was dPhydrated in an ethanol gradient t70-lO0%) and dried.

After the pulse labelling, 50-lO0 ~l of a fraction containing chiti-nase ~ in lO mM Tris-buffer at p~ g.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 petri-dish, the preparation was incubated at 30C for 20 hours. The enzyme treatment was stopped by dipping the slide in 6% TCA and the prepara-tion 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 ~n the dark for 7 days at 7C and low relative humidity for exposure. The emulsion was developed by placing the slide in Kodak D-l9 developer for 10 minutes followed by fixation for 2 minutes and washed in running water for lO minutes. After ~; 30 drying the preparation was ready for a microscope analysis of the :
; hyphae of the fungus.

'~.

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,~ : :, , . .. . : ,. . .
',~' : , : . ' '' ''',':' ' : ' - . ' .

,,'~! ;, .~il ! .;

Production of antibodies for use in serological analysis Production o~ 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 ~,0) and diluted 1:1 with Freunds incomplete adjuvant. Polyclonal antibodles were raised in rabbits according to conventional methods by Dakopatts (Denmark).

Production o~ monospecific polyclonal antibodies to chitinase 4 pep~ides The procedure was carried out as described in detail for the produc-tion of monospecific antibodies to AHAS peptides (Narcussen and Poulsen, submitted). Based on computer analysis of the amino acid sequence for chitinase 4, four peptides were selected on the criteria of hydrophilicity and variability between chitinase 4 and other basic chitinases. Peptides were custom synthesized by Cambridge Research 15 Biochemicals (UK). The structures were verified by mass spectroscopy -and amino acid analysis to estimate purity.
: .
Bei~ore immunization the peptides were conjugated to diphtheria toxo-id. The carrier, diphtheria toxoid was converted to the toxoid-sul- -fosuccinyl-ester derivative by reaction wi~h 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. Im-munization in rabbits werc performed as described above for the production of polyclonal ant~bodies to chitinase 2 and ~.

SDS-PAGE and i~munoblotting For immunoblotting, proteins were transferred by seml-dry blotting onto a 0.45 ~m nitrocellulose membrane (Schleicher and Schuell, FGR) after separation by SDS-PAGE. The antigens ~ere probed with primary polyclonal rabbit antibodies raised against chitinase 2 and 4 (see above) and subsequently ~isualized using alcaline phosphatase con-829450e~(.001/LS/ll~M/A12/1991 07 29 15:55 . , - : . : ~, . . , . ~" : . .: . . : .:

"' .,, ,' ' ' , . ' . ' :~ : , ' ' . , . , ' ~' '' . ' ~' . , . ' , ' ' . ' ' ' ', ' . .
'` ', . . '' '; ' ' ' ' , . ' , , . , ' , ' .
.~':' ".'''' " ' '' "' ",',''.,' ~''' ,. ' ' ' " ~ , ' '.. '. ' ' . " ' ''.' ' ' J ', ~ ~

jugated secondary antibodies (Dakopatts, Denmark) according to Kyhse-Andersen (1984).

Analysis of the amino acid composition of the purified chitinase iso-enzymes 2,3 and 4 and ~-1,3-glucanase 3 and 4 After freeze-drying, the purified chitinase isoenzymes 2,3 and 4 and ~-1,3-glucanase 3 and 4 were subjected to amino acid analysis as described by Barkholt and Jensen (1989). An aliquot (4.2 ~g) of each of the chitinase isoenzymes and the ~-1,3-glucanase respectivPly 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 bees chitlnase 3 and 4, "SE" and ~-1,3-glucanase 3 and 4 : ':
Tryptic digestion .:
.
After RP-HPLC as described above and freeze-drying, 100 ~g of pro-teins were redissolved in 200 ~1 of 0.2 M Tris-HCl (pH 8.4) contain-ing 7 M guanidine hydrochloride. 20 mM dithiothreitol was added and the proteln was reduced at 37C for 4 hours under nitrogen. 30 mM
, .
iodoacetamide was added and the reaction was allowed to proceed in the dark for 40 minutes at 25C under nitrogen. Unreacted iodoacet-amide was inactiva~ed by addition of 5 ~1 of ~-mercaptoethanol fol-lowed by incubation for lS minutes at 25C in the dark. The protein solution was dialyzed against 0.2 M ammonium carbonate (pH 8.0) for 24 hours at 4C 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 ~1 of ammonium carbonate (pH 8.0) at 40C for 30 minutes. Finally the supernatant and 6 ~g of TPCK-treated trypsin were added. The digestion was al-lowed to take place at 40C for 4 hours and stopped by addition of 20 ~: :
82945~ /nRM/A12/l99l ~ 29 15:55 ,: ; : ~ -,: :.. :.:-. . : ., . . . .. ,, . .. .,, ;, : . .:........... . .

:, : .,. . ::, -; : : :.~ , . , . :: ,:: : : :

' ~1 of TFA. The peptide solution was subjected to RP-HPLC on a VYDAC
C18 column (0.46 ~ 15 cm; 10 ~m 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 Cl~ column (0.4 x 10 cm; 5 ym particle size; Dr. O Schou, Novo-Nordisk, Denmark) using the mobile system described above. Selected peptides were sub~ected to amino acid sequence analysis.

Amino acid sequencing :

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

Bacter~al strains and enzymes Restriction enzymes, Klenow polymerase and T4 DNA ligase were suppli-ed by Boehringer Mannhe;m and used in accordance with the manufac-turers instructions.

pBluescript was supplied by Stratagene (USA), pUC19 was supplied by Boehringer Mannheim.

For subcloning in E. coli, transfer of DNA was carried out using DH5 E. coli cells (from BRL) according to the manufacturers instructions.

Isolation of R~A from sugar beet leaves . ' , ' .. ' Isolation of RNA was carried out as described by Stougaard et al. : ~:
(1986).

Only sterile solutions and glassware baked overnight at 180C were used in order to avoid contamination with RNase. 2 g of Cercospora infected sugar beet leaves (cv. Monova) obtained as described above were ground in liquid N2 ant transferred to a 50 ml Falcon tube. 20 ~ , .
829450ex~001/15/JKM/AlV19~1 07 29 15:55 ~ .

.: : . : - :, , .,: .. " , ., . .,- , . :...... ", . .. .. .. .. ........

~8 ml of extraction buffer (100 mM Tris pH 7.5, 50 mM ~DTA, 500 mM NaCl) and 2 ml of 10% SDS, 0.7 g of N-laurylsarcosine, 0.05 g of aurintri-carboxylic acid and 1 ml of (14 M) ~-mercaptoethanol were added and the resulting mixture was incubated for 15 minutes at 65~C. The incubated mixture was spun at 10,000 x g for 20 minutes at room temperature in a Corex tubP. The supernatant was transferred to a Falcon tube and 2.7 g of CsCl was dissolved therein. The mixture was spun for 5 minutes at 10,000 x g and room temperature in a Corex tube, and the supernatant was transferred to 6 SW 55 tubes each containing 1.4 ml of CsCl cushion. (The CsCl cushion was prepared by dissolving 9.6 g of CsCl in 5 ml of 0.2 M EDTA and adding H20 to a total volume of 10 ml).

The RNA of the supernatant was pelleted through the cushion by cen-trifugation for 18 hours at 37,000 x g and 18C in a Beckman SW 55 rotor. The green supernatant was removed, and the CsCl cushion was carefully washed twice with H20. The CsCl cushion was removed and the RNA pellets were dissolved in a total of 2 ml of H20. The RNA was precipitated with 7 ml of 96% ethanol for 30 minutes at -20C, subse-quently spun and redissolved in 2 ml of H20, ad~usted to 100 mM NaCl and precipitated with 2.5 volumes of 96X ethanol. The resulting RNA
pellet was spun and dissolved in 1 ml of H20. The RNA content was measured by determination of the D260 (0D26o - 1.0 ~ 40 yg of RNA/ml), and the RNA was stored at -20C. Yield: about 1 mg of total RNA. i Purification of poly-A RNA
.
Purification was carried out according to Chirgwin e~ al. (197~).
The RNA with a poly-A tail was purified by affinity chromatography through an ol~go-dT cellulose column. 0.5 g of oligo-dT cellulose was mixed in 5 ml of O.S M NaOH or 5 minutes (1 g of oligo-dT cellulose binds 1.2 mg of poly-A RNAj. The resulting mixture was neutralized with 10 mM Tris pH 7.5 until pH reached 7.5. An 1 cm column with a diameter of 1 cm was made and equilibrated with 20 ml of column buffer (500 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA). The RNA was denatured at 65~C for 5 minutes, and 5 volumes of column buffer were added to the RNA before chromatography through the column. The "run-829~50ex.001/LS/JKM/A12/1991 07 29 15:55 :

,' .''';. : ',;'.,' :',.;, ,'' " .`',''" '' ,.. ,, .. ,~ ~ . '" ' ,' ' , .: ' ., / . . . ,: .
. " ' ' ''. " '' ', : ' '-' ., ' ' .

-through'~ was collected and sub;ected to chromatography again. The column was washed with column buffer until OD260 reached 0.01 or le~s. The poly-A RNA was eluted with TE buffer in 1 ml fractions, and the RNA concentration for each of the fractions was determined at OD260. The poly~A RNA~containing fractions were pooled and adjusted to 100 mM NaCl and the RNA was precipitated overnight with 2.5 vo-lumes of 96% ethanol at -20C. The poly-A RNA was spun and dissolved in H20 at a concentration of 1 ~g/~l and stored at -20C. ~he yield was about 1-2X of total RNA applied to the column.

Isolation of genomic DNA from sugar beet leaves Genomic DNA was isolated form sugar beet leaves (60.159.83i~-131-4) ~Dellaporta et al., 1983).

2 g of Cercospora infected sugar beet leavss 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 pHi 8.0, 50 mM EDTA and 500 ~M NaCl) were added together with 1 ml of 20X SDS and after mixing, the mix-ture was incubated at 65C 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 4C, 17,000 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~. After another centrifugation at 15,000 x g for 15 min. at 4~C, the pellet was washed with 70% ethanol and thereafter dried briefly before being resuspended in 0.7 ml of X-TE buffer (50 mM Tris, pH 8.0 and 10 mM
EDTA). The suspension was transferred to an eppendorf tube and cen-trifugated for 5 min. The supernatant was extracted twice with phe-nol/chloroform. The DNA was precipitated by adding 75 ~1 of 3 M Na-acetate and 500 ~1 of iso-propanol, mixing and spinning for 30 se-conds. Afterwards, the pellet was dissolved in 400 ~l of H20, and the suspension was ad~usted to 100 mM NaCl and precipitated with 1 ml of 96X ethanol. The suspension was centrifugated for 5 min. and the supernatant removed. The pellet was dried briefly, and the DNA dis-solved in 200 ~1 of TE buffer. The DNA concentration was determinated 8~so~x~l/Lsi/nKM/Al2/lsslo72s 15:55 : .
,, . , ... .. , . , . , - .. : , .
- ~ - , ,, " , . . ,, : : : : :
.: : , " ~
. : . ., .. - - . . . :, . . . :. ~ . . . . :
. , . , , , . ,, - .. : ... . .. . . .

:: . - . : . ~ . , . ., , , : . : .

:~: , : .. ,.... . , : , ~ ,.. . :
.. .. ..
.. ,: : ,, . . . : : : :
, : , . . . : . , , :, . :

-using the absorbance at OD260~ where oD260-1-5 ~g DNA~ml. The DNA
was stored at -20C until use. ;

Construction of a sugar beet cDRA library On the basis of sugar beet mRNA isolated as described above a ~ZAP
5 cDNA library was constructed by Stratagene Cloning Systems. ~

Construction of a sugar beet genomic D~A libr~ry :

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. Tha 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 106 phages were used for each screening. For each 24.5 x 24.5 mm plates, ; 15 2.5 x 105 phages were mixed with 3 ml of the E. coli strain XL l-Blue (in case of a sugar beet ~ZAP cDNA library) or LE392 (in case of the sugar beet genomic library (EMBL3)) and grown in LB medium with 10 mM
MgSO4 and 0.2X maltose to an OD600-1. The mixture was allowed to stand at 37"C for 20-30 minutes.

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

Transfer of plaques to nltrocellulose filtar ln situ The screening of ~ZAP recombinant clones by hybridization to single plaques in sltu was done as follows.

After growth overnight at 37C, the plates wers cooled for about 15 minutes at 5C. Phages and DNA were transferred to a hybond~N nylon ' ~ .
./nKMtAl2/l~l ~ 29 15:55 ~: .
, .. , ,. - . . . . , . . : ; , . -: . . : - . . . . . , , : , ,: ,:' ' ' ;', ', " ,'' ,'' ' ;' ' ' ', :" .

: . . . . . .
. , . , : , . . . . .

~ - \

membrane (Amersham) by placing the dry filter on the lawn of cells.
Phages were allowed to adsorb to the filter for l 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 What-man 3M~ filter paper sheets soaked with 0.5 M NaOH, 1.5 N NaCl for 30 seconds. They were then washed for 30 seconds in each of the follow-ing solutions: l) 0.5 M NaOH, 1.5 M NaCl, 2) 0.5 M Tris, p~ 7.5, 1.5 M NaCl, 3) 2 x SSC (modified Benton, 1977). The filters were air dried and illuminated with W for 3 minutes with the phage side upwards.

Preparation of radioactive probes for use in screening for sugar beet chi~inase 4 in sugar bee~ cDNA librarias Relevant oligonucleotides were labelled by phosphorylation with bac-teriophage T4 polynucleotide kinase according to the me~hod described in Sambrook et al. (1990). More specifically, the oligonuclsotides were synthesized without a phosphate group at their 5' termini and were labelled with ~ 32p from [~-32P]ATP using the enzyme bacterio-phage T4 polynucleotide kinase.
. , Pur~fication o~ radiolabelled oligonucleotides by precipitation with ; ethanol After inactivation of the bacteriophage T4 polynucleotide kinase by heat, 40 ~l of H20 was added to the tube, the content of which was sub~ected to thorough mLxing. Then 240 ~l of a 5 M solution of am-monium acetate and l ~g of herring sperm DNA were added. The result-ing mixture was mixed well, and 750 ~l of ~ce-cold ethanol were added. Again, thorough mixing was performed, and the resulting mix-ture was stored for 30 ~inutes at 0C.

The radiolabelled oligonucleotide was recovered by centrifugation at12,000 x g for 20 minutes at 4C Ln a microfuge. Using an au~omatic ~ .. . . .
'~ ' 8~5~l~5/nuM/Al2/l99l ~ 29 15:55 ,: : : . . . , ~ . , . ,.. , , . :. ,, - : . .~ . . . :.. :. .. :.... .
:'', " ', ~ ' ' ' . ' . .: ~ . :' " " , . ':

j,J '"`r '-," ~
, . . ~

pipette device equipped with a disposable tip, all of the supernatant fluid (which contained most of the unincorporated [~-32P]ATP) and any free 32p generated during the phosphorylation reaction were carefully removed The resulting residue was redissolved in 100 ~1 of H20 and 10 ~1 of 3M sodium acetate and thereaf~er 250 ~1 of 96% ethanol were added. The mixture was subjected to centrifugation for 20 minutes at 4C, dried and redissolved in 200 ~1 of H20.

Oligonucleotide hybridization of chitinase 4 DNA by filter hybridiza-tion The oligonucleotide hybridization procedure used eliminates the preferential melting of A-T versus G-C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. The hybridization was carried out essentially as described by UoDd e~ ~1. (1985~. The nitrocellulose filters obtained as described above were wetted on the surface with 2xSSC and subse-quently prehybridized in hybridization buffer ~6xSSC, lX BSA, lX
Ficoll 4000, 1~ PVP, 50 ~g/ml of heat denaturated salmon sperm DNA, 50 mM sodium phospha~e, pH 6.8). The hybridization was performed at 37C for 4 hours in a plastic bag with shaking. The filter was hybri-dized overnight in the same solution plus the radioactive oligonucle-otide probe ~the 23-mer chit 4 probe) at 37C with shaking. A lx106 cpm/ml solution of the hybridization buffer was used. The filter was rinsed three times in 6xSSC at 4C and thereafter washed twice for 30 ~in. at 4C in 6xSSC. Further, the filter was washed ~hree times in 2S TMAC-buffer (3 M Tetramethylammonium chloride, 50 mM Tris pH 8,0, 2 mM EDTA, 0,1% SDS) for 5 min. at 37C. ~The tetramethylammonium chloride is made in a 5 M stock solution. Since TMAC is hydroscopic, the actual molar concentration (c) must determined from the refra-ctive index (n) by the formula c-~n-1,331)/0,018). The filter was ~; 30 then washed twice for 20 min. in TMAC-buffer at 55C.

The filters were dried in the air at room temperature. Inkmarks on the filters servin~ to align the autoradiographs with the filters and the agar plates were marked with an autoradio~raphy marker ~Ultermit, Du Pont de Nemours). The filters were covered with Saran Wrap and an :::
35 X-ray film ~AGFA CURIX RP2) were exposed to the filters for 16-70 ~ .
/nEM/A12/1~1 ~ 29 1S:SS

hours at -70C 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 ~l 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 l-2 hours at room temperature so as to allow the phage particles to diffuse out of the agar. About lO6-107 phages per plaque were ob-tained.

The p~ages were then diluted in SM phage buffer and mixed with 200 ~il of XLl blue cells (OD600 ~ l). The mixture was allowed to stand ~or 15 20 minutes at 37C and 2.5 ml of top agarose (48C) 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 (l990) using several steps of replating and rescreening.

A phage stock was prepared according to the method of Sambrook et al.
(1990) . . :, ':
In vivo excision 25 In vivo excision of plaques was performed as described in "In vivo -.
; excision Protocol" in the Instruction Manual (CAT# 236201, August 30, 1988) for undigested Lambda ZAP II Cloning Kit, Stratagene Clon-ing Systems.
~ .

8~5~*~l~5/nKM/Al2/l99l ~ 29 15:55 ; ~ . . ~ , : ': . .

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 (DH5~ 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 cen-trifugation for 10 minutes at 3000 x g. The pellet was resuspended in 200 ~1 of Solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM
EDTA) in 1.5 ml tubes. 400 ~1 of Solution II (0.2 N NaOH, lX SDS) was added, the mixture subjected to gentle mixing and placed on ice for 5 minutes. 300 ~1 of 5 M KOAc pH 4.8 was added and sub~ected to tho-rou~h mixing. The resulting mixture was placed on ice for 10 minutes and subsequently sub;ected to centrifugation at 15,000 x g for 10 minutes at 4C.

15 The supernatant (900 ~1) was ~ransferred to new tubes and 0.6 volume (540 ~l) of icecold isopropanol was added. The resulting mixture was allowed to stand for 15 minutes at room temperature. The mixture was .
again sub~ected to centrifugation at 15,000 x g and 4C for 10 minu-tes and the supernatant was removed.

20 The pellet was dissolved in lOO ~l vf TE and 100 ~1 of 5 M LiCl was added. The mixture was allowed to stand on ice for 5 minutes and subjected to cen~rifugation at 15,000 x g and 4C for 10 minutes.
~ . .
The supernatant was transferred to new tubes and 500 ~l of 96% etha-nol was added. The tubes were centrifugated at 15,000 x g and 4C for 30 minutes and the supernatant was removed. The pellet was washed with 70X ethanol (about 100 ~l) and dried. The dried pellet was ~ redissolved in 50 ~l of TE.
'~ :
DNA sequencing . ~ ,' , .
The plasmid DNA (double-stranded template) to be sequenced was puri-fied by the above described method. Sequencing was performed as fol-lows:
. .
~s~l/l~/~KM/AI2/199l ~ 29 15:55 : :

,,.:. -: .. , :. : .

: . .

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

The mixture was neutralized by adding 1 ~l of 5 M NH4AC and then precipitated by adding 20 ml of 96X eth~nol. The resulting mixture was spun for 30 minutes at 4~C and resuspended in 6 ~1 of H2O. 1.5 ~1 of 5 x conc. sequenase buffer was added. The mixture was placed at 37C for 5 minutes.

4 ~1 of sequetide (Biotechnology Systems NEN~ Research Products, Du Pont de Nemours) and 2 ~1 of sequenase (United States Biochemical) were added, resulting in a total volume of the mixture of 13.5 ~l.
The mixture was placed at room temperature for 5 minutes.
. : .
3.1 ~l of the labelling reac~ion was transferred to each termination tube (G, A, T and C) containing 2.5 ~1 of the dNTP terminating mix-ture. The mixtures in each of the tubes were allowed to react for 5minutes at 37C and 4 ~1 of stop solution was added. The ~ixtures were then heated to 85C and 2 ~1 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 ~f 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 ~ore specifically, the following procedure was used: ~ -::
A sample comprising 25 ng tl-23 ~l) of the DNA tempIate to be labe-led, 0-22 ~1 of H2O and 10 ~l of random oligonucleotide primers (con-stituting a totsl volume of 33 ~1) were added to the bottom of a clean microcentrifuge tube. Tha reaction tubes were heated to 95-100C 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 '.: .' ~ : .
82~soex~ool/Ls/nKM/Al2ll59lo729 l5:55 . .

:: `
`:~ , ' . . : .

, ' . ~.` , . , , ', . ,, ,. : ':;. , , '' :. , .~ . . : . ... .. .

D~A sample in LMT agarose was placed at 37C and the following re-agents were added to the reaction tubes:

10 ~1 of 5X primer buffer containing dATP, dGTP and dTTP.

5 ~1 of labeled nucleotide ~-32PdCTP (3000 Ci/mM) (Amersham), and 2 ~1 of diluted T7 DNA Polymerase. The T7 DNA Polymerase was diluted in ice cold Enzyme Dilution Buffer immediately before use ~o a final concentration of 1 U/~l. The reaction components were mixed with the tip of a pipette.

The tubss were incubated at 37-40C for between 2 and 10 minutes and subsequently, the reaction was stopped by the addition of 2 ~l of Stop Mix. The probes with the 32P-labeled DNA were further purified ~
using the Elutip~-D column system (Schleicher & Schuell). .

Then, the probe DNA was made ready for hybridization by mixing the :
requisite amount of radioactive probe with 200 ~1 of 10 mg/ml salmon : -~ 15 sperm DNA. The mixture was heated to 95-100C in a boiling water bath ; for 5 minutes and cooled on ice. The resulting probe was stored at -20 C for up to one week and heated to 95-100C 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 ~-ZAP cDNA library were subjected to ;
prehybridization for 2 hours at 67C under conventional prehybridi-zation conditions using a prehybridizing solution comprising 2 x SSC, 10 x Denhardt's, 0.1~ SDS and 50 ~g/ml salmon sperm DNA.

Hybridization was carried out overnight using a hybridization solu-tion identical to the prehybridization solution except for the fact ;~ ~ that a radioactive DNA probe prepared as described ~bove had been added.

. ~
' 8~skx~ 5/nK~/Al2/l99l ~ 29 l5:55 ,~ ', .

;: - ,- , ,:, ~,:,: , . . , , . ,, :: . . . . . .

:, - - , ,, ~ . . . . . .

.. . . . . .
,. ., ,, . :
: ,. , ~, , .

After hybridi~ation, a washing procedure was carried out in accor-dance with the following scheme:

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

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

Identification of DNA belonging to the chitinase 4 gene family To identify DNA belonging to the chitinase 4 gene fa~ily, hybridiza-tion of the DNA in question with a chitinase 4 probe was carried out ~ ~-I0 using the hybridiza~ion procedure disclosed in "hybridization of "SE"-DNA" except for the fact that the hybridization is carried out at a temperature of 55C.

E~cision of DNA from agaros~ gels DNA fragments to be used, e.g. in the construction of genetic con-15 structs according to the invention were isolated as follows. ~ ~ ~
: `' The DNA was run on L~T (Low Meltin~ Temperature) a~arose (Sea Plaque-O 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 68DC ', ',' 20 for 10 min. and re-equilibrated to 37C. Subsequently, 2U/100 ~1 of ; ~ agarose (free of DNase, from Calbiochem) was added. The mixture was allowed to stand o~srnight at 37DC and was subsequently extracted twice with phenol and twice with chloroform, sub~ected to EtOH preci-pitation and finally resolubilized in H2O.

PCR used ~or the ampllfication of cDNA encodlng "SE", ~-1,3-glucanaisa and ch~t 76 on the basis of sug~r beet mRNA
; :
;~ The preparation of a partial cDNA molecule was done by use of the Gene Amp~ RNA Amplification Reagent Kit (Perkin Elmer Cetus, USA).
The PCR was performed in accordance with the manufacturer's instruc-, .
~ 8W~k~VLS/nUM/A~2/l99l ~ 29 ~:55 ,.,.:, ... . , . , : , :.:, .. , ~ ,.i,, ,. , , . .,; , .. ...

tion with a few modifications. The re~erse transcription protocol was followed using the concentrations in the scheme below.

Component volume Final conc.
.
5 MgC12 solution 4 ~il 20 mN
10 x PCR buffer II 2 ~1 10 mMi dGTP 2 ~il 10 mM
dATP 2 ~il 10 mM
dTTP 2 yl 10 mM :
10 dCTP 2 ~il 10 mM :~
RNase Inhibitor 1 ~il 5 U/~il Reverse Transcriptase 1 ~il 5 U/~il primer 270 0.4 ~1 0,25 mM/100 ~1 :
mRNA 3.6 ~il Total volume per sample 20 , , .

In the step cycle the following procedure was used. -:

Segment 1: 42C for 2 hours Segment 2: 99C for 5 minutes :: .
Segment 3: 5C 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:
:: ~ : . :. .
:
~ .

~ ' ::
X~b~ /nRM/A12/1991 ~ 29 IS:SS
.
~.. . i . - , .. . .. . ., ~-.. - , . . . .. . .

~ ,. . .. ., , .; . . . - , : , , . :

., ~' ' .,'., .. ',., . "", . .' '. .'' ','.'. ,' "'. ', ' ' ,' ' "' . , '' ' . .. '.. ' ' .' ','. ' ' ." ' ' ; " i ' ,; '; ' , " '" " " ' ' ,' " ' , " ' ", ' ' , ' ~ " ~ ' ' ' ' ' ', ' . ' ' ~ ' ' ' ," ' . ,' . :. ' ., . : ' .

~: ` ~. , ,` ! .;
" ~ .

PCR cycles:

no. of cycles DC time (min.) addition of Taq polymerase and oil 37 2 :~:

37 2 ~ -72 10 .

42 2 ~ ~
72 5 :

. ' PCR used in th& construction o~ genetic con~tructs of the inven~ion ~ and in site-directsd mutagenesis on the basis o~ cloned D~A templa~es ,: . .' : ~.
The preparation of the relevant DNA molecule was done by use of the Gene Amp~ DNA Amplification Reagent Kit (Perkin Elmer Cetus, ~SA) :
and in accordance with the manufactures instructions except for the temperature cycling. Here the following procedure was used:

PC~ cycles : : :
~ 25no. of cycles C time (min.) ~ . .

~ 60 1 l/2 i~ 72 4 ~ 30 1 72 7 ,~ ~
50~00l~5/nKM/At2/l99l0729 l5:55 .~ ,; " ... ,, , ,.. , ,; ,, , .,.. ,., ,,,. ,.. ,- ..... .. . .

PURIFICATION ~ND 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 chi~in-ase 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 ~he 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-Syst~m as described in Materials and Methods". After separation on the Mono S FPLC column (Fig. 1) only a single silver stained band for each chitinase iso-; zymes 2,3 and 4 could be observed on the SDS-gel (Fig. 2). Further analysis by reverse phase ~PLC on a VYDAC RP4 column gave only one ~30 protein peak for each of the isozy~es. This is further evidence for a homogeneous protein preparation for each of the basic chitlnase isozymes.

:
8WSOCKOOl/LSi/JKM/A12/1991 07 29 15:55 ": ' .

,., , , : .,. ,. , , . : ,.. . , . : . . ~',i :: ,:: ' , : :

',,',':,' .' i" ' " ' " "" "'''; ' '', ' ' : '',', ', ' , ~ ' ,' "' ' '. ' ,. '' ~ ; ' ., '' ' ~.~ I,i ~, ,' ' The molecular weights determined by SDS-PAGE for chitinase 2, 3 and 4 are 32, 27 and 27 kDa, respectively (Fig. 2). By isoelectric focus-ing, the isoelectric points for chitinase 2, 3 and 4 were determined to 8.3, 9.0 and 9.l, 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 produc-ing chitooligosaccharides or an exochitinase liberating only N-ace-tylglucosamine from the non-reducing end of chitin or chitooligosac-charides, the pattern of reaction products liberated by chitinase 4 from 3H-chitin was analyzed by TLC (Fig. 3). Irrespecti~e of dura-tion of incubation, N-acetylglucosamine was only a very minor reac-tion product, whsreas chitobiose, chitotriose and chitotetraose werethe major product. This strongly implies that chitinase 4 is an endochitinase.

In addition to the catalytic activity exerted on 3H-chitin, chiti-nase 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 bi-functional enzyme having boeh chitinase and lysozyme activity.

ANTIFUNGAL ACTIVITY OF PURIFIED CHITINASE AND ~-1,3-GLUCANASE ISOEN-ZYMES FROM SUGAR BEET LEAVES

Three different bioassays were conducted to ascertain the in vitro antifungal activity of chitinase and ~-1,3-glucanase isoenzymes on the germination and growth of Cercospor~ bct~col~. In the same manner the antifungal activity of cbitinases and ~-1,3-glucanases from other sources or othar isozymes from sugar beets may be determinated using either purified enzymes or extracts containing the enzymes. Also, the , .
':

8X~s~ 5/nUM/A12/1ss1 ~ 29 15:55 . .

assays may be used to deter~ine 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 ~ilm 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 llght. The number of hyphae with fluorescent tips and the exten-sion of the staining at the individual tip reflect the growth ac-ti~ity 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 com~ination of chitinase 4, "SE" and ~-1,3-glucanase 3 is applied ~o the culture. 60 ~1 of protein solution containing 20 ~g of each an-tifungal proteins were spplied to each microscope slide. When chiti-nase 4 was used slone or in combination with either ~-1,3-glucanase 3 or "SE" alone, the inhibitory effect was less pronounced. Neither ~-1,3-glucanase 3 nor "SE" had any significant inhibitory effect alone or when combined. However, as seen from Fig. 5 when all 3 enzymes were used together, a very strong inhibitory effect was seen indicat-ing a synergistic effect between chitinase 4 "SE" and ~-1,3-gluca-nase.
..
; Method II - Mlcrotiter pla~e Bio~ssay - .
, .
The germination of spores and growth of the mycelium can be followed in a microtiter plate by measuring the absorbance ~620 nm) at Sp2Ci- :.:
fied time intervals. In the control experiments, the growth of Cer-30 cospora is initiated after an approx. 40 hours lag period and in-; creases almost line~rly for the next 40-50 hours (curve A in Fig.
6). When pure chitinase 4 (5 ~g per well) is included, the inial lag period is increased to 75 hours and the growth rate is decreased BS
. .
~ 82~5~ LslnKM/Al2/l99l ~ 29 l5:55 .~ .
. . , , , ., ,, ., . , . .. , . , .,: .. . : :

-, .. ' ,;, :, ', ' ' , : ", ' ,. .. , . . : . , ', compared to the control ~curve C in Fig. 6). The eluate fro~ the chitin column is shown as a comparison.

Method III - Autoradiography In the third bioassay, the chitin in the hyphae cell wall was label-led with 3~-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 ~-1,3-glucanase was added afeer 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 Cercosp~ra is specifically to hydrolyze the chitin fibers in the hyphae tip and ~hereby 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 ~-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 plsnts.

:

~s~x~ool/Ls/ncM/Al2/l99lo729 15:55 AMINO ACID COMPOSITION AND PARTIAL A~INO ACID SEQUENCES OF THE PURI-FIED CHITINASE ISOENZYMES 2, 3 AND 4 After freeze-drying, the amino acid composition of pure sugar beet chitinases 2, 3 and 4 were determined (see ~Materials and Methods").

The results are shown in Table I. For comparison, the amino acid composition of chitinase from barley, wheat and bean (Leah et al., 19~7) are included in the Table. The amino acid composition of chiti- -nase 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, chltinase 3 and 4 have a significant different amino acid composition than any of the other chitinases.

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

.: ' `: .

~ 8W5~ 5/DK~/A12/1991 ~ 29 15:55 , . - . . . ,: , , , .. . . - : .. - ~.. ,, .. . : , ., . : , : : . :. : : . , , . , ::, . , :

:: . - , , . , . , :. . . . .
:: :: : : : -: ~ ., , . . -, . ,, . , ,: , :~::: ' . ': : ' ' ' :1 ,, ' . ' ' : . ::. . . . .. .

TABLE I

Amino acid composition of barley, wheat, bean and sugar beet chitinases 2, 3 and 4 5 Amino acid Barley Wheat Bean S.B.2 S.B.3 S.B.4 cDNA

Aspartic acid 23 28 29 34.4 24.7 24.4 22 Threonine13.8 22 22 16.2 13.0 12.8 12 Serine 17.7 24 26 21.0 24.8 24.8 24 10 Glutamic acid 18 20 22 24.9 22.1 21.0 18 Proline 17 15 20 17.1 10.3 10.2 9 Glycine 30.7 52 37 39.7 30.6 30.4 27 :.
Alanine 37.3 27 26 28.0 28.2 28.5 26 Cysteine 7.2 12 16 16.9 16.8 16.9 15 15 Valine 12.5 14 10 8.6 14.4 14.3 14 Methionine 1.6 3 2 1.8 1.1 1.1 Isoleucine 10.8 9 11 11.9 10.9 11.0 11 : Leucine 11.3 13 17 16.2 9.0 9.0 8 Tyrosine11.9 14 15 17.3 12.7 12.7 12 i 20 Phenylalanine12.7 14 13 11.5 18.3 18.1 17 Histidine4.9 4 3 4.4 4.7 5.4 4 Lysine 6.9 8 8 8.7 4.3 3.1 3 ~ Arginine15.2 14 16 11.3 14.2 16.1 15 7 : 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.

1'` . :

j; .
. 8~Xkx.~l/LS/~KM/A12/l~l ~ 29 15:55 ,: :

.. ~ :,, . . ., . , , , ...... , ; . , ... , ,, , . :

,: . ,., ., . : ' , , . :,. ' , i .

Tryptic digestion of sugar beet chitinase 3 and 4 Analysis of the pure chitinase 4 enzyme has revealed that the N-terminal part of the enzyme is blocked. Thus, by analysis of the mature chitinase 4 it was not directly possi~le 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 charac-terize the DNA by which it is encoded, it was chosen to subject the mature enzyme to tryptic digestion in order to obtain protein frag-ments (peptides) susceptible to amino acid sequencing.

The tryptic digestion of the purified chitinase enzymes was carried out as descri~ed in "Materials and Methods~' above. The tryptic pep-tides were separated by reverse phase-HPLC on the Vydac RP-18 column -mentioned above under the conditions specified in ~Materials and Me-thods" see (Fig. 8). Peptides representing large peaks at an absor- :
15 bance of 214 nm and displaying a high retention time (indicating long -polypeptide chains) were selected for further purification on a Develosil ~P-18 column. :

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

When comparing the aMino acid sequences of each of the peptides withthe 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 oligo-nucleotide probe would require only few codon choices. Thus, this peptide was chosen to form the basis of the construction of an oligo-nucleotide probe to be used in the isolation of DNA encoding chiti-nase 4 (see Example 4 below).

8Ws~001~5/nKM/AI2/1s910729 15:55 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-~-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 .

15 4-24 S-P-S-S-G-G-G-S-V-S-S-L-V-T-D-A-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 .

:
: .

ISOLATION AND CHARACTERIZATION OF THE cDNA GENE FOR CHITINASE 4 FrOm the amino acid sequance obtained for peptide 4.26 (see Table II
in E~ample 3), the following very specific oligonucleotide gene probe was synthesi7ed using a DNA syntheziser 381 A (Applied Biosystems).

Peptide 4.26 T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-g-I

.
- F W F W M N N ---Phe Trp Phe Trp Met Asn Asn TTT TGG TTT TGG ATG M T AAT
C C ~ C C

~:
,~ .
829450ex,001/LS/JKM/A12/1991 07 29 ,~ .

,:: . . ,:::, ,. ;, , . . , , , ~ ;:. ; : , . ,, ,; ..

Using this gene probe, the expression cDNA library ~ZAP was screened using the procedure given in ~Materials and Methods" above. 8 cDNA
clones were obtained from ~ZAP, and one of the clones was fully sequenced while the others were only partly sequenced. The sequencing was performed as described in ~Materials and Methods" above. An almost full length cDNA clone, chit 4-B15, was obtained from the ~ZAP
library and the DNA sequence thereof is shown in Sequence 1.

On the basis of the cDNA sequence, a deduced amino acid sequence of chitinase 4 was obtained Sequence 1. The deduced amino acid sequence was aligned with the partial sequence obtained from the chitinase 4 protein (as described in Example 3 above) and an almost 100% identity was observed. This demonstrates that the isolated cDNA clone codes for the chitinase 4 polypeptides purified by the chromatographic procedure described above. The chit 4-B15 cDNA clone is 966 bp long and encodes a protein having 264 amino acid residues in the polypep-tide chain out of the 265 amino acids predicted for the chltinase 4 .
genomic DNA. The~leader sequence consist probably of 23 amino acid residues (out of 24 amino acid residues as determined for the genomic chitinase 4 DNA, see below), followed by a hevein domain of 35 and a functional domain of 206 amino acid residues. Af~er the stop codonthe cDNA has a 158 bp 3' noncoding region.
'~'~.' .
As mentioned in Example 3 above, it has not been possible to sequence the N-terminal amino acid sequence of chitinase 4 directly, because the N-terminal is blocked. However comparison with wheat germ ag-glutinin (UGA-A) and potato chitinase lead to the guess of the first amino acid being Gln. Hereafter the rest of the amino acid sequence of the chltinase 4 N-terminal was deduced from the DNA sequence to be Gln-Asn-Cys-Gly-Cys.......

The N-terminal sequence of chitinase 4 was further examined by de-termining the molecular weight (MU) of the mature chi~inase 4 by electrospray mass spectrometry as described by G.J. Feistner et al., 1990. A MW of 25393.6 ~/- 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 oi' 14 protons) and that the first amino acid resldue Gln is converted to the pyroglutamyl deriva-829450ex.001/LS/JKM/A12/1991 0'7 29 15:55 : :.
., ' : ~ , . ...... . . . : ........... . ,. . . : . , ::

...... . . . . . . .. ... . .

tive (loss of - NH~ - 15 MW), the calculated MW of the mature chiti-nase 4 is 25894. This is in agreement with the data observed by the electrospray mass spectrometric analysis and confirms the deduced N-terminal amino acid sequence given above for the mature chitinase 4.
.
The N-terminal amino acid sequence could be determined for chitinase 2 and the following terminal amino acid sequence was found in chiti-nase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala.
: .
' .

Table III

Comparison of the N-terminal amino acid ssquence between different chitin binding proteins:
.
UGA-A: QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCG~G--CQNGACWTS
Hevein: EQ**R*AGGKL*********W-*W**STDE**SPDHN**SN-*KD
Chit. Bean: EQ**R*AGGAL**GGN****F-*W**STT****P*--**SQ-*GG
Chit. Tob.: EQ**S*AGGAR*ASG****KF-*W**NTN****P*-N**SQ-*PG
Chit. SB 2: EL**N*AGGAL*I*G******-*W**NTNP***N
Chit. SB 4: *N**C-A**LC*SRFGF*GSTDA***E*CREGP----CRS-----.
* - amino acid residues identical to WGA-A

ISOLATION AND CHARACTERIZATION OF TH~ SUGAR BEET GENOMIC CLONES CHIT
76 AND CHIT ~

Screening of 500,000 clones from the amplified EMBL3 librsLy contain-ing genomic sugar beet inserts from a partial Sau3A digestion, resul-ted in the isolation of three clones wi~h the CH4 cDNA as probe.
~ .
The three hybridizing clones were characterized by restriction frag-ment analysis and sequencing. These analysis showed, that one of the clones contained a chitinase gene, now called Chit 76, the sequence ' ~5~ KM/A12/l991 ~ 29 15:55 .. , . .. , ~ ~ , .. . .
.: , . ~ ; . . ~ .: . .: :

,:, . : ::, .- . ", . ., . ,. . . . , : ,:, :

: . . . . . :: ::
,., ., ., ;. . . , ': ' , ' . : ' ' .

-of which is shown in Sequence 3. Sequencing of this gene was initiat-ed with the primer used for screening of the ~ZAP library (see Ex-ample 4), and continued with other primers complementary to sequences inside the chit 76 gene.

S The chit 76 gene codes for a 268 amino acid long chitinase which has 80X homology to the CH4 amlno acid sequence (vide Sequence 1) but only 34Z homology to the entire CH1 protein (vide Sequence 7). 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 CH4 cDNA Sequence 4. The intron borders contain the consensus GT/AG
sequences. The chit 76 intron is located exactly at the same position as the second intron in the C~l gene, when the amino acid sequences of CHl and chit 76 are aligned.

A TATA-box sequence (TATAAA) is located at position 378, which ~s 90 bp upstream for the ATG start codon for translation. A poly-A signal (AATA M ) is located at positicn 1725 in Sequence 3.

In a similar way a genomic clone encoding chitinase 4 was isolated.
The DNA has bean partially sequenced and about 350 nucleotides of the 5' noncoding region has been elucidated. About 340 nucleotides of the coding region haq been sequenced. Ihe sequence appear from the Sequence 2.

Alignment of the 5' noncoding regions from the two genomic genes show b~xes of homology (e.g. chitinase 4 nucleotides 14-49, 60~122, 123-135, 159-173, 174-207 and 277-328, (Sequence 6).
.
Basad on knowledge o~ the chit 4 B15 cDNA sequence and the partially sequenced genomic ch$tinase 4 gene, the rest of the. gene can easily be sequenced. It $s contemplated that the chitinase 4 gene comprises at least 1 intron, probably only 1 corresponding to the chitinase 76 sequence.

8X~bx.~l/~/nKMtA12/l~l ~ 29 15:55 ~: ' . , : -. . . . . . .. . . . . .. . .
:;:' ' , : ~ , ., '. ,' .. ~ .' ' ' , :
: . , ; . . . : ' : : , :' , - . :
lll CHARACTERIZATION OF THE ACIDIC CHITINASE ISOENZYME "SE" AND DETER-MINATION OF PARTIAL AMINO ACID SEQUENCE

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

After the final purification on the Nono P FPLC column three isozymes of "SE" could be resolved (see Fig. 9). By analysis on SDS-PAGE only a single protein band for each of the isozymes could be demonstrated.
The same molecular weight of 29 kD was determined by SDS-PAGE. Analy-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 3H-labelled chitin. The major product of the enzymatic hydrolysis was the hexamers oi 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 sub~ected 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 sub;ected to further puriflcation 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-tcrminal amino acid sequence wac also determined aq shown in the Table IV.

8~k~l~s/nKM/Al2/l99l ~ 29 l5:55 ~ :-: : , . , : ., . . , , , , . : .
, ;.::: , ' , , , . ' ; ~ , , , . " : . .
', ' . : ., ., ~ ,, :' . ' ' '. ' . , , . :, :.: , .. , : .
.. . . . . ..

TABLE IV

N-terminal: S-Q-I-V-I-Y-W-G-Q~N-G-D-~-G-S-L-A-D-T-C-N
SE 22.5: V-L-L-S-I-G-G-G-A-G-G-Y
SE 23.0 A-D-Y-L-W-N-T-Y
SE 25.1 N-N-P-P-C-Q-Y-D-T-S-A-D-N-L-L-S-S
SE 26.1 Y-G-G-V-M-L-V
SE 30.4 S-L-S-S-T-D-D-H-H-T-F-H-D-Y-L-W-N-T
SE 31.1 T-T-V-Q-A-N-Q-I-F-L-L-G-P-A-S-T-D-A-A-G-S-G-F-I

EXAMPLE 7 ~ .

10 ISOLATION AND CHARACTERIZATION OF THE cDNA FOR THE ACIDIC CHITINASE ~ - ISOENZYME "SE"

On the basis of the amino acid sequence of the tryptic peptides listed in Table IV two subsequences from the peptides SE 25.1 and SE
31.1 (Table IV) were selected for the synthesis of mixed oligonucleo-tides as ~hey had the best codons. The PCR primers KB 7 (SE 25.1), ~ KB-9 (SE 31.1) and the oligo-dT primer (270) were prepared ln the : same manner as described in Example 4 in relation to chitinase 4. The nucleotide sequence of the gene probes are shown in Table V.
: .
TABLE V

N P P C Q Y D T ~ -KB-7. 5'-GACTCTAGAG M cTCC8CCgTGcTCAGATATc5AcTAC-3' Q A N Q I F
KB-9. 5'-GGAGGATCCCC2GCGAATcCAAATATT-3' : :
. A C
T T

: ,:: :
270. 5'-CC M GCTTG M TTCTTT~TTTTTTTTTTTTTTTT-3' ~: .:
:- .
829450~?x.001/LS/r~M/A12/1991 07 29 ', :. , .. . ~ . .. ,: ~ : ... . . . :. .. ,:, . ... ., I :: ,., ,::.: ,.: ., ., :.: . :

, , ~, , . ! .. , .. ' ., , . . , ; , ~ ,, . . ' ~ ~,' M~

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

The DNA sequence obtained for the partial cDNA molecule constituted 10 by nucleotides 711-962 of the DNA sequence shown in Sequence 8. This cDNA was used to screen the ~-ZAP cDNA library described in "Materi-als 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 15 constituted by nucleotides 37-1106 of the DNA sequence shown in Sequence 8. As normally observed in connection with the isolation of cDNA, the entire cDNA was found to be difficult to isolate. Rescreen- -ing of the ~ZAP library with a 122 bp EcoRI-KpnI from the 5' end of the longest cDNA clone (SE22), gave a sequence containing the entire 5' end The clones were ligated using the KpnI site. The structural gene has a 5' noncoding region of 17 bp, a leader sequence of 25 amino acid residues, a functional domain of 268 amino acid residues and a 3' noncoding region of 20~ bp after the stop codon. The cDNA
sequence and the amino acid sequence are shown in Sequence 8.
~:
When the amino acid sequence obtained from the N-terminal and the 6 tryptic peptides (107 residues) were compared an almost lGOX agree-ment 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" ls apparent from Table IV.

.
.
. ..
~g~ /nKM/Al2/l~l ~ 29 15:55 ~ ~
.

' ' ' ' ,, ', , . ', ' ; , . , . , , " ' ' . . . .
`'.~ ' . . ~' ', '.' ' ', ' ' ., " ' .'' '' ,; '' ' ' ' ' ' ' ' ~ ~ . . .: ' ' '. ' ' . . I ' ' "

TABLE VI

SE22S~ML n~AKIVS--VLFLlSLLIF~SFESSHGS--OIVXYWGQNGDEGSLADTCN 46 CUCUMBER MAAHKIT--TTLSIFFLLSSIF~SSDAA--GIAIYWGONGNEGSLASTCA 46 AR~IDOPS ~TN~TLRKHVIYFLFFISCSLSKPSDASRGGIAIYWGONGNEGNLSATCA 50 .. . . . .. . . ~... ~, 0~ 01r~0, ~. 9.. ~
SE22S~L SGNYGTV~LAFVATFGNGOTPALNLAGHCDP~TN-CNSLSSDIKTCOQ~G 95 CUCUMBER TGNYEFVNIAFLSSFGSGO~PVLNLAGHCNPDNNGC~FLSDEIffSC~SQN 96 ~RA~IDOPS TGRYAYVNV~FLVKFGNGOTPELNL~GHC~P~ANTCT~FGSQVKDC~SRG 100 SE225A~L IKYLLSIGGGAGRYSLSSTDDANTF~DYLWNTYLGGOSSTRPLGDAVLDG 145 CUCU~BER VKVLLSIGGGAGSY5LSSADDAKOVANFIW~SYLGGQSDSRPLGAAVLDG 146 AR~8IDOPS IKV~LSLGGGIGNYSIGSREDAKVlADYLWNNFLGGKSSSRPLGDAVLDG 150 ~ P.~ .r~ r..~ o~*.~
SE22S~L IDFDIESGDGRFWDDLARAL~GHN~GQKTVYLSAAPOCPLPDASLSTAIA 195 ` .
CUCUnBER VDFD~ESGSGQFWDVLAQELKNFGQ~ VILSAAPQCPIPD~HLDAAIK 192 ~RA3~DOP5 IDFNIELGSPOHWD~LARTLSKFSHRGRKIYLTGAPOCPFPDRLMGSALN 200 SE22S~ML TGLFDYVWVQFYNNPPCQYDT-SADNLLSSWNQWTT-V~ANOIFLG W AS 243 CUCU~aER TGLFDSVWVOFYN~PPCMFAD-NADNLLSSWNQWTA-FPTSXLY~GLP~ 240 ~R~IDOPS TXRFDYVWIQFYNNPPCSYSSGNTONLFDSWNKWTTSIAAQXFFLGLP~ 250 SE225A~L TDA~-GSGFIP~D~LTSQVLPTIKGSAKYGGV~LWSK~YD--SGYS5~IK 290 CUCU~BER RE~PSGGFIP~DVLISOV W TIKASSNYGGV~LWS~FD--NGYSDSIK 2BB
ARA~IDOPS PEAA-DSGYIP~DVLTSQILPTLKKSRKYGGY~LWSKFWDDKN~YSSSIL 299 . ~ , . * . o r . o . o . ~ ~ ~, o r . ~ , . * ~ - ~
sE225AnL SSV~ 293 Consenl3us l~ngth: 304 CUCUM8ER GSIG 292 Iden~lty : 137 ~ 45.1'X.) ARABIDOPS ~SV~ 302 51mll~rlty: 106 ~ 34.9'X.) : .' .
Table VI shows an alignmen~ of the amino acid sequence corresponding ~ :
to the structural gene for the acidic chitinase nSE" and the am~no acid sequence oi a cucumber lysozyme/chitinase (EP 0 392 225 and 5 ~étraux, et al, 1989) and an Arabidopsis lysozyme/chitinase (Samac et :~
~1., 1990). It appears from ~his that there is a homology o~ about ~:
45X when all tree segment are comparsd. ~hsn ~SEn is compared with :.
ths cucumber lysozyme/chitln~se ~ homology of about 60Y wa~ abserved. ~

~.
, 829450CLOOI/LS/nCM/AI~/1991 07 29 IS:SS

-. " : ~. :. :,. :. . , . . ,,: , . . .. . .
'.... , , , ' ' : , : ''' .' , : . ''' ' ' , . ,,, ~ . . : , .
.. . .
~, CHARACTERIZATION AND DETERMINATION OF THE PARTIAL AMINO ACID SEQUENCE
FOR THE SUGAR BEET ~-1,3-GLUCANASES 3 AND 4 The sugar beet ~-1,3-glucanases 3 and 4 were isolated from Cercospora infected sugar beet leaves as described in the above "Materials and Methods". They are basic proteins having a strong affinity for ~-1,3-glucan. The amino acid composition of the sugar beet ~-1,3-glucanase 3 and 4 isoenzymes are similar to the one given for ~-1,3-glucanases from tobacco and barley as shown in Table VII.

8~Xx.~1/LS/nKM/AI2/199l ~ 29 15:55 "~
'" ' ': ': ' . ~ . ' ' , , ~ .~ ' ' .

' : .. ~ ' '. '' ' ' ' ''""
.": . ' ' " ' ~ ' ' TABLE VII

Amino acid composition of tobacco and sugar beet ~-1,3-glucanases Amino acid Tob2ccoa) Sugar beet 3 Sugar be~t 4 Barley Gsb) Aspartic A. 35 46.4 53.4 39 Threonine10 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 ~,: -:25 b) From Kragh e~ al., 1991 : . , ~ ~ . ' ' . ' : .
'' ' .,, . .:
8~5~ /nKM/A1211991 ~ 29 15:55 .. .
. : . . . , . - . . .

:~ ~ .. .. . . . ... .. . . . .... .. .

-SDS-PAGE of ~-1,3-glucanase The apparent molecular weight of ~-1,3-glucanase 3 and 4 determined on a 10-15X 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 ~-1,3-glucanase isoenzymes 3 and 4 were the dimer, laminari-biose. This strongly suggests that the ~-1,3-glucanase 3 and 4 iso-zymes are endoglucanases.

Amino acid sequencing of ~-1,3-gl~canase 3 and 4 The purified ~-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 connestion with the selection of the tryptic peptides of chitinase 4 (see E~ample 3). The amino acid sequence of the peptides ar- shown in Ta~le Vlll.

:; ~

~5~x.~l/LS/~KM/AI2/l99l ~ 29 15:55 ,~ , . .
~: ' ' ~ , . :,:,,, . , ;
,- ~ .. . . . . . .
', '1 , ' , ': . .' - .

TABLE VIII

Amino acid sequences for ~-1,3-glucanase 3 and 4 isolated from sugar beet leaves Peptide 3.15. W-V-Q-N-N-V-V-P-Y

Peptide 3.17. (A)-G-A-P-N-V-P-I-V-V-S-E-S-G-W-P-S-A-G-G

Peptide 3.16. L-Q-G-K-V-S

Peptide 4.25.1. L-G-N-N-L-P-S-E-E-D-V-V-S-L-Y

Peptide 4.26.3 L-D-Y-A-L-F

Peptide 4.27.1. Y-I-A-V-G-N-E-I-M-P-N-D-A-E-A-G-S-I-V-P-A-M-Q-N-V ~ .
(~) (Q) (A) (P)-(R) Peptide 4.28.2. W-V-Q-N-N-V-V-P-Y

~ :

ISOLATION AND CHARACTERIZATION OF THE cDNA FOR ~-1,3-GLUCANASE 3 AND -`I 15 In the same manner as described ~bove in connection with "SE", oligo-nucleotide probes corresponding to pep~ides from the ~-1,3-glucanase 3 and 4 polypeptides were synthesized. As 5'primer was used the fol-lowing two sequences in the first round of PCR for isolation of ~- .
1,3-glucanase 4:

,~ . .

. ' .

/Ls/nKM/A~2/lr~9lo72s l5:ss Pep. 3.15: W, V, Q, N, N, (V)...

Oligoseq. TG-l: 5' TGGGT~C~ AA ~ ~GT 3' In the second round of PCR the following sequence was used as 5'pri-mer:
Pep. 4.27,1: ...... N, E, I, M, P, N
Oligoseq. TG-2: 5' M ~GAAT M TGCC~ M

By comparing the amino acid sequences from ~-1,3-glucanases in barley (Fincher, 1986) and tobacco (Shinshi et al. ,1988), a consensus se-quence was selected and used for construction of a 3'primer with the following consensus sequence:
~ , Pep. seq: F,A,~,F,D/N,E.
G
Oligoseq. TG- 3: 5'TC~TT~ M CAT~GC~ M

C
This sequence was used in the second PCR round whereas the 270 primer used for cioning of "SE" was used in the first round. To isolate a ~-1,3-glucanase 3 clone, the TG-l primer was used since peptide 4.28.2 - peptide 3.15 (see Table VII in ExampIe 8). This primer was used as the 5' primer for both the PCR reactions. As the 3' primer, the TG-3 ; 25 and 270 oligonucleotides were used for the first and second round of PCR, respectively.

The resulting PCR products were employed to screen the above descri-bed sugar beet cDNA ~-ZAP library to isolate clones harboring cDNA
encoding ~-1,3-glucanases 3 and 4, re~pectively. One of the cDNA se-quences and the deduced amino acid sequence are shown in Sequence 9.

~ ~ , ~: :
~ 8W xk~ 5lnuM/Al2/l99l ~ 29 l5:55 ... .. . . . .. .. . . . . .. . . . . . .

EXAMPLE lO

SEROLOGICAL CHARACTERIZATION OF SUGAR BEET CHITI~ASES 2 AND ~

The serological relationship between chitinase 2 and 4 was analyzedby immunoblotting. WhPn a protein sample containing both chitinase 2 (MW 32 kDa) and 4 (MW 27 kDa) was separated by SDS-PAGE before im-munoblotting the following results were observed (see Fig. lO).
Chitinase 4 antibodies detect only a 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 chitlnase 4, whereas antibodies directed against chitinase 2 only recognize chitinase 2 and no cross-reactivity at all was observed.
The above results suggest that sugar beet contain two different classes of basic chi~inases. 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 chiti-nases 2 and 4. The difference indicates that the genes coding for chitinase 2 and 4 constitute two distinct gene families.

As far as the present inventors are aware, the fact that two dif-fcrent classes of basic sugar beet chitinases exist has hltherto not been reported in the literature.

~efinltion of sugar beet chi~inase 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., l99l~, 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 subs~antiated by serological cross reactivity carried out with an-~.

; 8~50~x.001~/nKM/A12/19910729 15:55 '," . " "~''' ' '.''', ', ,' : "" . "' ' ' ," ' '' '.'' '' , ', '," """,' ~ '', . " ' , ,' .' . ' '; ~ ', ' ,'' ' ',", ' ' '' , ' ,' : ' .,. , ', '' "', . ' ' ,'", , , '' "' ' ," ',, , ' ' . ' ', ' ~ ' " . ',' ' ' "" " ' ' ' ' r,~ ' , , . , . , ' ' ' ; ' ~ ' " -, , ' : , ; ~

tibodies raised against sugar beet chitinase 2. This antibody recog-nized not only sugar beet chitinase 2, but in addition also chitinase P (27.5 kD), Q (28.5 kD), Ch. 32 and Ch. 34 from tobacco (Bol and Linthorst, 1990), chitinases T, K and C from barley and chitinase Al, A2 and B from Pea. ~hsn antibodies raised against barley chitinase K
or wheat germ chitinase were employed, similar serological cross reactivities were observed. Therefore the chitinases described above : :
were defined as belonging to a chitinase class serologically related to sugar beet chitinases 2, e.g. a sugar beet chitinase 2 class chitinase.
'~
TABLE IX

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

Chitinase 2 ELCGNQAGGALCINGLCCSQYGWCGNTNPYCGN
15 Bean E~CGRQAGGALCPGGNCCSQFGWCGSTTDYCGP
Tobacco EQCGSQAGGARCASGLCCSKFGW
Pea B EQCGRQAGGATCPNNLCCSQYGY
Pea Al EQCGNQAGGXVPPNG
Pea A2 EQCGTQAGGALCPGGL
20 Barley K EQCGSQAGGATCPNXLCCSRFG
` Barley T XQQGSQAGGATCPNXLCCSXFGW
':
Definit~on o~ a sugar beet ch~tinzse 4 class When antibodies raised against sugar beet chitinase 4 was employed, none of the chitinases from the chitinase 2 class described above could be recognized. Chitinase 4 from sugar beets thus belongs to a new chitinase class so far not detected in other plant species than sugar beets. However, recent studies have indicated that chitinases belonging to the same new class exist in rape seed. Thus, protein extracts of rape seed obtained by a method similar to the one out-lined above for sugar beet chitinases were shown to react wi~h the above mentioned polyclonal antibodies directed against chitinase 4 : :
from sugar beets. . .

' 8~5~~ nKM/Al2ll99l ~ 29 15:55 ;: , :

i' . ' '.. ', '",,',"', ' ,,' ,'."',, ' ,""' '. ,.' ' . ' ~', . : ~' ': .

EXAMPLE ll EXAMINATION OF THE HOMOLOGY BETWEEN THE CHITINASE 4 cDNA AND OTHERCHITINASES ~SING THE HYBRIDIZATION TECHNIQUE

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

It appaars from Fig. ll that there is a very low degree of homology examined at 55C 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 chiti- -nase l and "SE" enzymes. These results therefore further indicate that ths chitinase 4 enzyme belon~,s to a new class of chitinases.

The high de~,ree 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 lO showing a high degree of serological homology between the mature enzymes from the two plants.
, . .

TRANSFORMATION OF BACTERIA CELLS
':
25 Agrobacterium tumefaciens JDM ???? was transformed with the plant transformation vector, pBKLhK4, the preparation of which is described in Example 18. ????? using a freeze/thaw method essentially as de-scribed by An et al, (1988). For the freeze/thaw method the bacteria to be transformed were cultivated in I~Rt-medium~ pH 7.4, overnight at 30 28C, 280 rpm, The next day the bacteria were subcultivated in 50 ml ' . :
~5~x~l/Ls/nKM/Al2/l99l ~ 29 15:55 ,~'' ''''""'. ''' " ',' , ''' , ", ' ' ''''' "' ' ;'''.''" ' '',' '''~ ` ;'.'" " . '""'''' , ''. .. . '. . . ' , ' ' .,. ' " .,,, '.' , " . . .

of LB-medium, pH 7.4, and grown for about 4 hours until OD 600 (OD600) was 0.5-l.O. The culture was cooled on ice and centrifuged for 5 minutes at lO.OOO x g at 4C. The supernatant was removed and the bacteria were carefully suspended in l ml of icecold 20 m~ CaCl2.
O.l ml of the bacteria suspension was pipetted off in icecold cryo tubes and the bacteria were frozen in liquid nitro8en and maintained at -80C.

For transformation of the bacteria l ~g of plasmid DNA was first added to a cryo tube with the frozen bacteria. The bacteria were incubated in a 37C 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 agitat$on (agitation table, lOO
x rpm). The cryotube was centrifuged for 30 sec. at lO.OOO x g, 4C.
The supernatant was removed and the bacteria were resuspended in O.l ml of LB-medium, pH 7.4. The bacteria were plated on to a YMB-dish with 50 ~g/l ~anamycin and incubated for 2 to 4 days at 28C until colonies appeared. The presence of a proper plasmids in the bacteria are verified be 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 wlth other genetic constructs of the invention may be performed, e.g. as shown in Figs.
18, l9, and 22 and explained in Example 18.

EXAMPLE l3 P~EPARATION OF GENETICALLY TRANSFORMED TOBACCO (Nicotiana benthamiana and N. tabacum) PLANTS
. .
~ Plant material .~ .
Lesves from plants to be genetically transformed were obtained from plants grown ln vltro or in vivo. In the latter case, the leaves were sterilized prior to transformation. Sterilization was performed by ` 30 placing the leaves for 20 min. in a solution of 5X Ca-hypochlorite containing O.l ml Tween 80 per l followed by washing 5 times in 82~s~ slnKM/Al2/l~l ~ 29 15:55 ' .

.: . .. : : .. : ' ': -, . ' : . : .-; .'. ' : . ' .: . . ..

.. ~i . . , . . . . . : : . .

sterile water In vitro plants were grown in containers on 1/2 shoot inducing medium (1/2 NS) (Murashige & Skoog, 1962).

The leaves were placed one at a time in a 14 cm Petri dish. They were then cut into squares of about 1 cm2, all 4 sides consisting of tissue which had been cut. Any cut tissue which had been bleached by hypochlorite sterilization was removed.

Cultivation of bacteria 24 hours before transformation a suspension of Agrobac~eria transfor-med as described above was started by inoculating 2-3 ml media with appropriate antibiotics with the transformed Agrobacteria. The bac-teria are grown at 28C with agitation (300 x rpm).

Transformation Transformation of the plant was done essentially as described by R.B. Horsch et al. (1986). The bacteria culture was diluted 50x with 15 1/10 US 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-cul tivation : ' .
The day before transformation co-cultivation Petri dishes containing 1/10 MS medium were coated with acetosyringone (200 ~M). 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 dippad in the bacteria suspension were placed upside down on the filter paper.
The leaf piecas were incubated in a growth chamber in weak light, e.g. 12 hours of light and 12 hours of darkness for 2-3 days.

~ ~''' ' 8y~5~ LslnuMlAl2ll~l ~ 29 15:55 :, , , ,' ,. . , , ~ . ~ .. '- .. . .

! . .' ~ . . " ~ J ; ~ ~
-Selection/regeneration The leaf pieces were transferred to Petri dishes containing shoot-inducing MS-medium with 300 mg/l of kanamycin and 800 mg/l of carbe-nicillin and sub-cultivated every 4 wPeks 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 sub-cultivated when needed. After approximately 2 weeks, the expression of the ~-glucoronidase activity using the G~S-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 compos~. They were then covered with plastic bags and grown for about 1 week, after which the two corners of the plastic bags were cut off. After another week the plastic bags were removed.

~' ' ' "' PR~PARATION OF GENETICALLY TRANS~ORMED S~GAR BEETS PLANTS BY MEANS OF
TRANSFORMATION WITH BACTERIA

Transformation was carried out using cotyledonary explants as de- -20 scribed below. Seeds were germinated for 4 days in darkness on a ~
substrate containing 0,7 g/l of agarose and 2 g/l of sucrose. The -~eedlings 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 trans-ferred to a MS substrate supplemented with 0,25 mg/l of BAP, 400 mg/l of kanamycin, 800 mg/l of carbenicillin and 500 mg/ml of cefo-. . .

~ s~x.~ltLs/J~M/Al2tl99l ~ 29 l5:55 : .. : , : ::: -, ,: :: :: :. ~ .
:: : . . : ~ . . . ,, . ::
,: : :: . . . . . . . .

. - , ' ~ ' : ' ' ' ' "' ' , .~ '' ' ' . : . ' . ;' ` ' : ' ' . - : : . , ::
.'. : . .. . . :, taxime and the explants were incubated for 14 days on this substrate.
The regenerated shoots were then transferred to containers with MS
containing 0.25 mg/l of BAP, 400 mg/l of kanamycin, and 800 mg/i of ..
carbenicillin as the substrate. The isolated shoots were transferred to fresh substrates with 4 weeks intervals for selection and multi-plication. Selected shoots were rooted on l/2 MS substrate containing l mg/l IBA.

.
Tissue from sugar beet and tobacco have been transformed with a .
genetics constructs containing either chi~inase l, chitinase 4 or chitinase 76 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 l5 .,, , ' ' .
~ 15 ANALYSIS OF CHITINASE AND ~-1,3-GLUCANASE IN TRANSGENIC PLANTS
. .
i The expression levels for chitinase and ~-1,3-glucanase isoenzymes can be evaluated either by measuring the total enzyme activity by the .
i two radiochemical assays, by measuring the antifungal activity using ' the biological methods I-III or by measuring the level of the dif-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 infec-tion system described in "Materials and Methods".

~,- 25 Using the biological methods I-III, the antifungal activity of the ~ -~ enzymes in the genetically transformed plants can be determined. A
,~ retarded growth of ths fungi hyphae shows that the transformation .~ has resulted in a plant having an improved tolerance i.e. an increa-sed antifungal activity to the phytopatogenic fungi compared to a non-transformed plant.
:', ~ .:
~,` -'.,', ~,:

;~ 82~xk~ool/Ls/nKMlAl2/l99lo729 15:55 , .

", , ' "-', , '',, " ' , ..... ' , .' ' ' .~ i ,. ,' .,, , :, -, . -,, : , "

~. ~ . "

.-:; ' ' :~. . . '''' ' , , ' ,:

In the radiochemical assays, 3H-chitin or 3H-laminarin are used as substrates for either chitinase or ~-1,3-glucanase, respectively.
Usin~ standard curves of product formation vs. enzyme amount, the activity for both chitinase and ~-1,3-glucanase in crude plant ex-tracts can be determined. This is illustrated further in a timecourse experiment where the level of either chitinase (Fig. 12a~ or ~-1,3-glucanase (Fig. 12b) is quantified in sugar bset leaves at specified time intervals after infection with C. beticola. Although the enzyme level of both the chitinase and the ~-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 en-hanced production of both enzymes was first observed 8-9 days after the infection with the fungal pathogen.

With these techniques, the constitutive level of chitinase as well as 15 ~-1,3-glucanase in transgenic plants can easily be recorded. -~

These techniques, however, do not differentiate between the various chitinase and ~-1,3-glucanase isozymes. Only the total enzyme ac-tivities for àll the chitinase or all the ~-1,3-glucanase isoenzymes are determined. ~owever, the presence of the various chitinase and ~-1,3-glucanase isoenzymes can easily be detected separately by analyz-ing the crude protein extracts by immunoblotting after separation by SDS-PAGE.

The antibody to ~-1,3-glucanase 3 recognized only one single protein in the Cercospora infected leaf material (Fig. i3~. 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 ior ~-1,3-glucanase using the radiochemical assay. When antibodies raised against either chitinase 2 or 4 were employed, two ma~or protein bands were induced in the infected leaf tissues. Chitinase 2 antibodies detect a 26 and a 32 kDa band, whereas two proteins havin~
molecular wei~hts 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 antibod-ies were chitinase 1 (26 kDa) and chitinase 2 (32 kDa), respectively.
Similarly, the antibody to chitinase 4 detected the authentic chitin-8X~5~l/Ls/nUM/AI2tl991 ~ 29 l5:55 .

: , . .. .. : - . . : .. . , ~ ;. ~ , " . . : , ,~ . " ;
~. : : . i . .,, ., , . , , , . :, .... : :

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 Sequence 1 and Sequence 8). The "3-D~' structure of chitinase 4 and "SE" on the nitrocellulose membrane may create sufficient epitope recognition to allow the antigen-antibody interaction between the "SE" antigen and the chiti-nase 4 antibody. The reaction between the "SE" antigen and the chiti-nase 4 antibody was only pronounced when the antibody solution is diluted 1:100 or 1:200. A much weaker reaction was observed when the 10 antibody is diluted 1:5000 or 1:10,000. :

Transgenic tobacco plants containing either chitinase 4 or chitinase 76 have been produced. In addition to the GUS positive reaction, the plants also expressed the chitinase 4 and or chitinase 76 when ana-lyzed by immunoblotting using the chitinase 4 antibody. Two protein bands (27 ~D and 21 kD) were present on the nitrocellulose membrane.
No positive reaction was observed wi~h the control plants. The rea-sons for the double band is not known at present, but may indicate that chitinase 4 may be processed at two sites in tobacco during the translocation process when the leader sequence is removed. In addi-tion to the normal processing site at the Leu Val Val Ala - Gln Asn Cys in chitinase 4 (amino acid position 23-24 in Sequence 1), a second putative tobacco processing site is localized at the amino acid sequence Ala Ser Ala Ser - Cys Ala tposition 85-86 in Sequence 1). A cleavage at thls site will give rise to the 21 kD polypeptide observed in the immunoblotting analysis. Southern analysis carried out as described in Materials and Methods above confirms that the transgene tobacco plants contain chitinase 4 and chitinase 76, respectively.
.

EXA`MPLE 16 MODIFICATION OF THE SUGAR BEET C~IITINASE 4 BY SITE DIRECTED MUTAGENE-SIS
.
Site directed ~utagenesis on a DNA sequence encoding the sugar beet chitinase 4, e.g. the chitinase 4 gene, may be carried out by use of 8~5~K~1~5/nKM/A12/1991 ~ 29 15:55 :. ;, . , : , , ~ . .. . .
. -, . : ,, , . , ., . ,. , , ~ ................... : , , , . :, ,: ~ .

,. , ~ ., . , ., , ,, ,. ,,., . . :

~ 3 PCR reactions (described in ~Materials and Methods" under the heading "PCR used in the construction of genetic constructs of the in~ention and in site-directed mutagenesis on the basis of cloned DNA templa-tes") using specific 3' and 5' primers for each site directed mutage-nesis. The choice of the specific 3' and 5' primers to be used dependon the position in the DNA sequence in which the modification is to be carried out.

Typically, suitable amino acids to be modified, either by substitu-tion, deletion or insertion are selected on the basis of an analysis of the amino acid sequence of the mature chitinase 4 enzyme, op-tionally in combination with an analysis of the enzyme's 3-D struc-ture. 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 sug2r beet chitinase 4 :

The position of the essential amino acid residues included in the active site of chitinase 4 have been tentatively ident~fied by the folldwing observations. Firstly, recent investigations with barley chitinase C demonstrated that chemical modification with carbodiimide and N-bromosuccinimide (NBS) completely inhibits the enzymatic acti-vity (results not shown). Similar experiments carried out with gluco-amylase from Aspergillus niger (Sierks et al. ,1990) have elucidated the mode of action by which carbodiimide and NBS inactivates this enzyme. Carbodiimide is covalently linked to the three essential acidic groups (glutamic and aspartic acid residues) constituting the catalytic site of glucoamylase. NBS oxidizes ~rp residues important in either stabilizing the transition state intermediate of the cata-`~ lysis or Trp residues involved in substrate binding at a distance from the catalytic site. The experiments with chitinase C indicatqthat three acidic and two Trp-residues are vqry i~portant constitu-ents of the active site. Secondly, by comparison to the active sites of other enzymes which hydrolyze oligosacch~ride chains including the ;~ glucoamylase described above, the active site of chitinase 4 is contemplated to be constituted by amino acid residue 184 ~Asp) and .
:
~b~l/L~lnKM/Al2/l99l ~ 29 15:55 ; -,, ~' .

,. : : .... . : . :
:,: . - , . . .. , . , , : :,: : . : . . :. : :

~ ' ' ,, , , , ~ , : ~ .
... , ., ., , , .,.. .. . . , : .,,: ., . : : :
, .. .: :. , . ., . : :.............. . .

190 (Glu). ~n contrast, chitinase C from barley and all other plant chitinases of the same serological class (the sugar beet chitinase 2 class) have three aspartic acid residues ("corresponding" to amino acid residues 184, 190 and 195 of chitinase 4) in the active site.
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 chiti-nase 4 , The two acidic residu~s 184 Asp and 190 ~lu forming the active site of chitinase 4 is contained in the peptide 4.22: SIGFDGLNAPETVANNAV.
Important Trp-residues of the active sites may be contained in pep-tide 4.19.3: GPLQITW and peptide 4.26: TAFWFWMNNVHS.

The active site of the chitinase 4 differs from the active sites of other plant chitinases, e.g. tobacco, which has the following corre-sponding amino acid sequences AIGVDLLNNPDLVATDPV, GPIQISH and SALWFU-MT~QSP, 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 informa-tion about the active site and possibly identify suitable modifica-tions resulting in improved properties of the modified en yme. 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 190 substituted with aspargine and/or the aspartic acid in position 184 are substituted with glutamine. Changing the carboxyl groups Asp 184 to Asn and for Glu 190 to Gln in chitinase are in itself expected to have ~ negative influence on the enzymatic activity, but is contemplated to result in further knowledge of the mode of action of the chitinase 4 enzyme.

The substitution oi Trp in positions 170, 205 and 207 to Tyr may change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity. The scheduled substitution given above is only shown as examples, and numerous changes is in-B2g~50ex.001/LS/JKM/A12/lg91 07 2g 15:55 . , . : .. . . . . .
. - :. . . .
: . .: . - . . . .
.. .... . . . . .

::, . , . . - . . ... :

~ H

ferred to achieve a more potent antifungal chitinase. This may be accomplished by site-directed mutagenesis e.g. usin~ the method outlined below.

Site directed mutagenesis For all the PCR reactions suggested here primers are chosen either themselves containing restriction sites or being located near re-striction sites in a manner creatlng the possibility of exchanging the PCR product with a corresponding sequence in the gene by restric-tion enzyme digestion followed by ligation of the relevant fragments.

The 5' primer to be used in the following examples is termed SD 0 (see Fig. 14).

When Trpl70 of the chitinase 4 amino acid sequence is to be substi-tuted by the amino acid Tyr, the following procedure may be carried out: -~ ' . ' For the PCR reaction the 3' primer SDl is used (see Fig. 14).

The resulting PCR product (from bp 301 to 538) is digest~d with BamHI
and PvuII and interchanged with the corresponding fragment of the chitinase 4 gene by conventional methods (Sambrook et al, 1990).
:
When Glul90 is-to be substituted with the amino acid Gln, the 3' primer SD2 is used (Fig. 14).
.
When Aspl84 is to be substituted with the amino acid Asn, the 3' primer SD3 is used (Fig 14).
, The PCR products are digested with BamHI and BspMII and interchanged with the BamHI-BspMII fragment of ~he chitinase 4 gene in a similar 25 manner as described abovs for exchange of Trpl70.
.: :
When Trp207 is to be substituted with the amino acid Tyr, the 3' : primer SD4 is used (Fig. 14).

82~5~1/LS/~KM/A12/1991 ~ 29 15:55 ;

.. . . .. . . . .. . . . .. . .. . . . .

~': ~ ' . ' . '' . .. ' ' ' ' ' . .' . . .. :. . . ' . ' ' ' . ' ' ' ' : ., : . :
'; , ' ' :'. :' . ' ',,' ~,,: ' ',: . , '' , ,., : , , : . , ' :' . ' , ' ': , '-; - ' " ' . ' ' ' ' ' -' "'' .' .''. . " . '. . . ' . " ' , : '. ', ' " ' ' ,, . ' ~.: ' , : ` . , ' ': ~ . ' . ' . , . , , . , , .,. ' , ,, . ,., :.: '' , . : : ' :

' ,' ' ',, '.,', '" ,' ',"' : ' , ' , ,"" ' " , '' ' ' ~ '. ', '", ' '' ": :, ', ' " ' ' .:'.

When Trp205 is to be substituted with the amino acid Tyr, the 3' primer SD5 is used (Fig. 14).

PCR products are digested wi~h BamHI and BalI and interchanged with the BamHI-BalI fragment in the chitinase 4 gene as described above.

5 In a similar manner, other desirable modifications may be carried :
out.
' CONSTRUCTIONS OF GENETIC CONSTRVCTS WITH SUITABLE C-TERMINAL EXTEN- ~ .
SION
.
C-terminal amino acid sequences found in connection with various plant chitinases and glucanases are exemplified in the speciiication and are believed to prove useful in modification of one or more of the antifungal en~ymes encoded by the genetic constructs accordin~
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 seq~ences encoding one or more of the antifungal proteins of the invention by any suitable technique such as PCR.
~` ,, , ~; Fig. 15a illustrates the su~ar beet ~-1,3-glucanase cDNA with atobacco C-terminal extension which is underlined in the figure.

Fig. 15b illustrates PCR primers which can be used to chan~e the stop codon and to introduce a part of the C-terminal extension, a :
DraI site is created at the 3' end.

Fig. lSc illustrates 4 annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a ~top codon, a SmaI s1te and an EcoRI site.

, 8294~0ex.001/LS/JKM/A12/1991 07 29 IS:SS

.,:, . , , , , ~ - . . " , ., , , . , , , , . , . , ~ , .
, , , . ,, ' : . ,, , : ; . ' ' ' ' The C-terminal extension can be introduced by exchanging the XbaI-EcoRI fragment in the ~-1,3-glucanase gene with the PCR product digested with XbaI and DraI and the annealed synthetic oligonucleo-tides digested with SmaI and EcoRI using conventional methods (Sam- -brook et ai, 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 SmaI
site near the stop codon in the chitinase 4 gene.
' ,:' Fig 16c illustrates four annealed synthetic oligonucleotides contain-ing the sequence for the C-terminal extension, a changed stop codon, a SmaI site and a EcoRI site.

The C-terminal extension can be introduced by exchanging the Bam~lI-EcoRI fragment with thP PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI
li~ewise 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 ~-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 Sequence 7. Other interesting N-terminal sequences may be the ones shown in Table IX.

::::
Genetic constructs The excised recombinant pBluescript containing the chitinase 4 cDNA
; gene (B15 chitinase 4) was subcloned in order to supply tha gene with :
~; an enhanced 35S promoter and a 35S terminator. This construct was .

.
82945~00l/LS/nKM/Al2/l99l0729 l5:55 ~:

transferred to the plant transformation vector pBKL4 containing the NPTII and the GUS genes.

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

The oligonucleotide KB3:
(5 CCGAAGCTTAGATCTAAAC M CAACATGTCTTCT(~)T(TC)GGACC3 ) containing the two restriction sites, a ribosome binding site, the ATG (underlined) and the first 15 nucleotides of the B15 chit 4 clone (nucleotide 8 and 10 were mixed (~) nucleotides due to the fact that the KB3 primer was used for the chit 76 clone as well) was used as the 5' PCR primer and the oligonucleotide KB4 The oligonucleotide KB4:
t5 GCACACGTAGCG~TAGC~TGG3 ) ', I **
261 NheI 241 1~ was used as the 3' PCR primer (nucleotide 255 and 256 was interchan~
l~ 20 ged in order to destroy the second NheI site).

The PCR product was extracted twice with phenol and twice with chlo-roform and EtOH precipitated. After resuspension in H20 the DNA was digested with HindIlI and NheI. The HindIII-NheI fragment from pB15 chit 4 was exchanged with the HindIII-NheI PCR fragment (Fig. 17).
, , 25 The construct was sequenced with the T7 sequencing primer (correspon-' ding to the pBluescript T7 promoter) and primer 340 ~ ~ (CATCGGAGGATCCACTACC) i. ' I I
~ 341 323 .1~ ' .
and it was confirmed that the entire exchanged sequence was correct.
Furthermore, in the 5' sequence the original nucleotide 8 was a T and '. ' .:
'' ' .: .
8~k~ nuulAl2llssl ~ 29 l5:55 . .; . r nucleotide lO was a C as in the pBl5 chit 4 clone and both the NheI
sites at posi~ion 245 and 251 were still present.
:
The construct was digested with EcoRI and a fill in reaction was performed with Klenow enzyme in the presence of dATP and TTP, the construct was further digested with BglII after removal of the Klenow enzyme. The DNA fragment BglII-EcoRI containing the entire chitinase 4 sequence was cloned into the vector pPS48 containing an enhanced 35S promoter and a 35S terminator. The chitinase 4 gene was inserted in the correct orientation by digesting the pPS48 vector with BamHI-SmaI (Fig. 17). The chitinase 4 gene with the enhanced 35S promoterand 35S terminator was transferred to the plant transformation vector pBKL4 (Fig. 17) as a HindIII fragment (Fig. 17). The resulting vec-tor, pBKL4K4, harboured in an E. coli DH5~ has been desposited with the Deutsche Sam~lung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300 Braunschweig (DSM) on 30 July, l99l under the provisions of the Budapest Treaty under the provisional accession No. ................................................................................ ~
:, The "SE" gene was then introduced into the construct pBKL4K4 (Fig.
17). A full length "SE" gene was constructed by combining the 5' end of the gene fr~m the pSurl clone (EcoRI-KpnI) with the rest of the gene from pSE22 (KpnI-HindlII) in the cloning vector pUCl9 (Fig.
18). The "SE" gene was subcloned in the SmaI site of pPS48 as a EcoRI-HindIII fragment filled in with Klenow pol~merase in the pre-sence of all four nucleotides. The orientation of the gene with respect to the enhanced 35S promo~er and 35S terminator was examined by restriction enzyme analysis and further confirmed by sequence i analysis.
:' : .
The "SE" gene with the enhanced 35S promoter and 35S terminator was cloned in the KpnI site o~ pBKL4R4 as a HindIII fragment in the 30 presence of a HindIII-KpnI ad2pter (Fig. 18). The HindIII fragment -w~s furthermore cloned in the HindIlI site of pBKL4.

Similarly to the chitinase 4, the chitinase 76 gene was clonsd in pBKL4 (Fig. l9).
: ,.
;~ .,' ' .
~ 8~5~001/LS/~UM/A12/19910729 15:55 ' i ' , ~ i' , . , , ; ,, ,~ ' ' ' . ' ',:
:: ` ' , .
~s:: .. - , , . :

'~ , , . ' ' . . . . .

In a similar manner, the glucanase gene can be introduced into the construct pBKL4, pBKLhK4, pBKL4KSE, or pBKLKK4KSE (Fi~. 22).

The full length cDNA clone (Sequence 9) was digested with EcoRI and BglII, the sticky ends were filled in with Klenow polymerase in the presence of all four dNTP's. The glucanase gene is then subcloned in the SmaI site of pPS48Mod. The orientation of the gene with respect to the enhanced 35S promoter and the 35S terminator, respectively, may be examined by restriction enzyme analysis and further confirmed by sequence analysis.
<
The glucanase gene with the enhanced 35S promoter and the 35S termi-nator is cloned in the EcoRI site of pBKL4, pBKL4K4, pBKL4KSE, pBKL4K4KSE.

..

~ .
:
~ ' ~45~x.~l/LS/~KM/AI2/l99l ~ 29 l5:55 , ............. . ......................... . .
"'',~ ',' ' ~''''; , ," ', '. . ' .
;"~.' ' ' . . ". ' ' ' . : ~ , " 1 . ' '' ' . ' .' . ' ' ' ' '' ~ '~ " ,' ' . , 'i

Claims (65)

1. A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in Sequence 1 or an analogue thereof, the analogue being a DNA
sequence encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 as defined herein and i) being a characteristic subsequence of the DNA sequence shown in Sequence 1, ii) hybridizing with the DNA sequence shown in Sequence 1 at 55°C
under the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", iii) encoding a polypeptide having the amino acid sequence of the sugar beet chitinase 4 shown in Sequence 1, 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 Sequence 1 and encoding the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

3. A DNA sequence according to claim 1, comprising nucleotides 176-793 of the chitinase 4 DNA sequence shown in Sequence 1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

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

5. A DNA sequence encoding a chitinase isoenzyma which is at least 60% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence Sequence 1 and at the most 40% homologous with the sugar beet chitinase 1 encoded by the DNA sequence shown in Sequence 7.

6. A DNA sequence according to claim 5 which encodes a chitinase isoenzyme which is at least 65% homologous, e.g. at least 70%
homologous, such as at least 75% or preferably at least 80%
homologous with the sugar beet chitinase 4 enzyme encoded by the DNA
sequence Sequence 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 Sequence 7.

7. A DNA sequence according to claim 5 or 6 comprising the genomic chitinase 76 sequence shown in Sequence 3.

8. A DNA sequence according to any of claims 1-7 encoding a polypeptide which reacts with an antibody raised against sugar beet chitinase 4, but not with an antibody raised against sugar beet chitinase 2.

9. A modified DNA sequence comprising a DNA sequence as defined in any of claims 1-8 in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having retained the antifungal activity of the sugar beet chitinase 4 or having an increased antifungal activity as compared to the sugar best chitinase 4.

10. A subsequence of the chitinase 4 DNA sequence of Sequence 1 comprising a DNA sequence encoding a polypeptide comprising the active site of the sugar beet chitinase 4 enzyme, e.g. a DNA sequence encoding the following peptide S-I-G-F-D-G-L-N-A-P-E-T-V-A-N-N-A-V-T-A-F-R

or a polypeptide comprising a part of the sugar beet chitinase 4 enzyme which is involved in the active site of the sugar beet chitinase 4, e.g. a DNA sequence encoding the peptide G-P-L-Q-I-T-W

or the peptide T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-S-I

or an analogue thereof hybridizing to the chitinase 4 DNA sequence at 55°C under the conditions specified in "Materials and Methods" under the heading "Identification of DNA belonging to the chitinase 4 gene family".

11. A subsequence of the chitinase 4 DNA sequence of Sequence 1 encoding a polypeptide comprising the hevein domain of the sugar beet chitinase 4 enzyme or an analogue of said subsequence which hybridi-zes to the chitinase 4 DNA sequence at 55°C under the conditions specified in "Materials and Methods" under the heading "Identification of DNA belonging to the sugar beet chitinase 4 gene family" and encoding a polypeptide capable of binding to chitin as determined by affinity column chromatography on regenerated chitin prepared as described in "Materials and Methods" under the heading "Preparation of a chitin column".

12. A subsequence of the chitinase 4 DNA sequence Sequence 1 encoding the leader peptide of chitinase 4 or an analogue thereof which hybridizes to the chitinase 4 DNA sequence at 55°C under the conditions specified in "Materials and Methods" under the heading "Identification of DNA belonging to the sugar beet chitinase 4 gene family" and which is capable of directing a passenger polypeptide to which it is fused out of the endoplasmic reticulum of the cell in which the fused leader and passenger polypeptide is produced.

13. A subsequence of the chitinase 4 DNA sequence Sequence 1 encoding one or more of the following epitopes of the sugar beet chitinase 4 enzyme Peptide 1: AGKRFYTRA
Peptide 2: NPSRQ
Peptide 3: GGNS
Peptide 4: TARVGYYTQYCQ

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

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

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

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

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

19. A genetic construct comprising one or more copies of a DNA sequence as defined in any of claims 1-16 comprising the chitinase 4 DNA sequence shown in Sequence 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 .beta.-1,3-glucanase activity, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA
sequences into functional polypeptides.

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

21. A genetic construct according to any of claims 19 or 20, in which the second chitinase and the .beta.-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 Sequence 8 encoding an acidic sugar beet chitinase SE having the amino acid sequence shown in Sequence 8 or an analogue of said DNA
sequence encoding an acidic chitinase having a pI of at the most 4.0 and being capable of hydrolysing 3H-chitin into mainly hexamers, and/or the DNA sequence encoding a basic .beta.-1,3-glucanase is the DNA
sequence encoding the basic sugar beet .beta.-1,3-glucanase 3 having the amino acid sequence shown in Sequence 9 or an analogue thereof encoding 8 basic .beta.-1,3-glucanase having a pI of at least 9.0 and being capable of hydrolysing 3H-laminarin into mainly dimers of .beta.-1,3-glucans.

23. A genetic construct according to any of claims 18-22 in which the promoter is a constitutive or regulatable promoter.

24. A genetic construct according to claim 23 wherein the constitutive promoter is selected from the group consisting of plant promoters, bacterial promoters or plant virus promoters.

25. A genetic construct according to claim 24, in which the promoter is selected from the group consisting of the sugar beet Acetohydroxyacid synthase promoter (AHAS) or the sugar beet Chitinase 1 promoter, the sequence of which appears from Sequence 7.

26. A genetic construct according to claim 24, in which the promoter is selected from the group consisting of a NOS promoter and an OCS
promoter of the opine synthase genes of Agrobacterium.

27. A genetic construct according to claim 24, in which the promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) promoter such as a CaMV 19S promoter or a CaMV 35S promoter, a MAS/35S, MAS dual Tr 1,2 and a T-2 DNA gene 5 promoter.

28. A genetic construct according to claim 23 wherein the regulatable promoter is regulatable by at least one factor selected from the group consisting of a growth factor, a chemical factor, a biological factor, and a physical factor.

29. A genetic construct according to claim 23 in which the promoter is a tissue specific promoter.

30. A genetic construct according to any of claims 18-29 wherein the transcription terminator is selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences, fungal transcription terminators and plant virus terminator sequences.

31. A genetic construct according to claim 30, in which the transcription terminator is selected from the group consisting of a NOS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium, a CaMV 35S transcription terminator sequence, a PADG4 transcription terminator to the DNA gene 4, a PADG7 transcription terminator to the T-DNA gene 7.

32. A genetic construct according to any of claims 18-31 in which at least one of the DNA sequences of the construct is functionally connected to an enhancer sequence which results in an increased transcription and expression of the DNA sequence(s).

33. A vector which is capable of replicating in a host organism and which carries a DNA sequence as defined in any of claims 1-16, or a genetic construct as defined in any of claims 18-32.

34. A host organism harboring a vector as defined in claim 33.

35. A host organism according to claim 34, which is capable of replicating or expressing the DNA sequence as defined in any of claims 1-16 or the genetic construct as defined in any of claims 18-32.

36. 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 18-32 and which is capable of replicating or expressing the DNA sequence or the genetic construct.

37. A host organism according to any of claims 34-36 which is a microorganism such as bacteria or yeast.

38. A host organism according to any of claims 34-36 which is a plant cell or a protoplast.

39. A genetically transformed plant comprising in its genome a genetic construct according to any of claims 18-32.

40. A genetically transformed plant according to claim 39 which is selected from the group of monocotyledonous plants consisting of corn, oat, wheat, rice, barley, rye and sorghum.

41. A genetically transformed plant according to claim 40 which is selected from the group of dicotyledonous plants consisting of alfalfa, tobacco, cotton, sugar beet, sunflower, carrot, chenille, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce and pea.

42. A genetically transformed plant according to any of claims 39-41 having an increased resistance to a chitin containing plant pathogen as compared to a plant which does not harbour the genetic construct as defined in any of claims 18-32.

43. A genetically transformed plant according to claim 42, having increased resistance to a phytopathogenic fungus or a nematode.

44. A genetically transformed plant according to claim 42 having increased resistance to phytopathogenic fungi of the genus Cercospora, Rhizoctonia, Fusarium, Cladosporium, Phytophthora, Phoma, Sclerotonia, Ascochyta, Pyrenophora, Helmithosporium, Ustilago, Puccinia, Ramularia, Botrytis or Verticillium.

45. A genetically transformed plant according to claim 44 having increased resistance to phytopathogenic fungi selected from the group consisting of Rhizoctonia solani, Cercospora beticola, Cercospora nicotianae, Cladosporium herbarium, Phytophthora megasperma, Sclerotonia sclerotiorum, Ramularia beticola, Botrytis cinerea and Phoma lingam.

46. Seeds, seedlings or plant parts obtained by growing the genetically transformed plant according to any of claims 39-45.

47. A transformation system comprising at least one vector which carries a genetic construct according to any of claims 18-32 and which is capable of introducing the genetic construct into the genome of a plant.

48. The transformation system according to claim 47 which comprises a binary or a co-integrate vector system.

49. The transformation system according to claim 47 or 48, 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.

50. The transformation system according to any of claims 47-49, which comprises an Agrobacterium tumefaciens Ti or an Agrobacterium rhizogenes Ri plasmid or a derivative thereof.

51. A microorganism capable of infecting a plant and harboring a transformation system according to any of claims 47-50.

52. The microorganism according to claim 51 which is an Agro-bacterium spp.

53. A method of producing a genetically transformed plant having increased resistance to chitin containing plant pathogens such as phytopathogenic fungi as compared to a natural plant, comprising transferring a genetic construct according to any of claims 18-32 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.

54. The method according to claim 53 in which the genetic construct is transferred into the plant by means of a microorganism according to claim 51 or 52.

55. The method according to claim 53, in which the genetic construct is transferred into the plant or into a part thereof by direct introduction of naked DNA by injection, sonication or electroporation.

56. 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 18-32 and a suitable vehicle.

57. A method of preparing an antifungal composition comprising culturing a microorganism according to claim 34-37 in an appropriate medium and under conditions which result in the expression of one or more polypeptides encoded by the DNA sequence according to any of claims 1-16 or the genetic construct according to any of claims 18-32, optionally rupturing the microorganisms so as to release their content of expressed polypeptide(s) into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide(s) to freeze-drying or spray-drying thereby obtaining an antifungal composition comprising the polypeptide(s) encoded by said DNA sequence or said genetic construct.

58. A method of inhibiting the germination and/or growth of a chitin containing plant pathogen such as a phytopathogenic fungus in a plant which method comprises 1) transforming the plant or a part thereof with a genetic construct as defined in any of claims 18-32 and regenerating the resulting transformed plant or plant part into a genetically transformed plant and/or 2) treating the plant or a part thereof, a seedling or seed from which the plant is to be propagated, or the medium on which the plant is grown with a composition as defined in claim 56.

59. A method according to claim 58 wherein the composition according to claim 56 or prepared by the method according to claim 57 has been added to water or a nutrient composition supplied to the plant.

60. A method of biologically controlling the germination and/or growth of a chitin containing plant pathogen such as a phytopathogenic fungus present on a material comprising treating the material with a culture of microorganisms as defined in claim 34-37 under conditions allowing the culture of microorganism to establish itself on the material to be treated.

61. A method according to claim 60 wherein the microorganism is a Pseudomonas spp., or a Streptomyces spp. or another microorganism conventionally used for biological pest control.

62. A method according to claim 60 or 61, wherein the material is a plant.

63. A method of inhibiting the germination and/or growth of undesirable fungi on a material, comprising treating the material with an antifungal composition according to claim 56 or prepared by the method according to claim 57.

64. A method according to claim 63, 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.

65. The plant transformation vector pBKL4K4 harbored in the E. coli strain DH5.alpha. deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) on 30 July, 1991 under the provisional accession number.