Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2442—Chitinase (3.2.1.14)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01014—Chitinase (3.2.1.14)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01039—Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01058—Glucan 1,3-beta-glucosidase (3.2.1.58)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a DNA sequence encoding the sugar beet chitinase referred to in the following as "the sugar beet chitinase 4" or an analogue of said DNA sequence encoding a polypeptide having the antifungal activity of sugar beet chitinase 4, as well as to a genetic construct useful for the construction of genetically transformed plants having an increased resistance to plant pathogens containing chitin, such as phytopathogenic fungi, as compared to untransformed plants.
• the genetic construct comprises and is capable of expressing the DNA sequence of the invention, preferably in combination with a DNA sequence encoding a second chitinase different from sugar beet chitinase 4 and a DNA sequence encoding a ⁇ -1,3-glucanase.
• the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet plant, from which a polypeptide having the antifungal activity of the sugar beet chitinase 4 is expressed in an increased amount as compared to the untransformed plant, preferably in combination with a polypeptide having chitinase activity and a polypeptide having ⁇ -1,3-glucanase activity so as to result in an increased resistance to chitin-containing plant pathogens.
• Most plants are susceptible to infection by pathogens such as micro-organisms and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently economical loss to farmers.
• the plants respond to infection with several defense mechanisms including phytoalexins, deposition of lignin-like material, accumulation of cell wall hydroxyproline-rich glycoproteins, pathogenesis related proteins (PR-proteins) and increase in the activity of several lytic enzymes such as chitinases and ⁇ -1,3-glucanases.
• PR-proteins pathogenesis related proteins
• Some of these responses can be induced not only directly by infection, but also by exposure of the plant to elicitors isolated from fungal cell walls, and in some cases by exposure to exogenous chemicals such as ethylene.
• the full capacity of the defense mechanism of the plant is, however, normally delaved in rela tion to the onset of infection, and thus, the plant may be severely injured before its defense mechanism reaches its maximum capacity. Also, the defense mechanism of the plant may not in itself be sufficiently strong to effectively combat the infectious organism. Therefore, a normal and necessary procedure is to treat infected plants or plants susceptible to infection with a chemical, e.g. a fungicide, either as a prophylactic treatment or shortly after infection.
• a chemical e.g. a fungicide
• 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-glucosamine and D-glucose.
• Chitinase and ⁇ - 1 ,3-glucanase activity has been observed in plant species such as tobacco, barley, potato, rice, maize, corn, bean, tomato, cucumber, wheat germv rape seed and pea and it has been shown that the chitinase activity increases in response to infection with most phytopathogenic fungi.
• Plant chitinases have been purified and characterized from crop plants such as tobacco, barley, corn, tomato, bean and pea, and cDNA and genomic clones have been obtained therefrom. Plant chitinases are reviewed by Bol and Linthorst, 1990 and Boiler, 1988.
• EP 0 292 435 relates basically to the regeneration of fertile Zea mays plants and mentions, inter alia, that a tobacco chitinase gene may be introduced in the plant in order to make it resistant to pathogens. Chitinase genes of other sources and other plants than Zea mays are not mentioned.
• EP 0 290 123, WO 88/00976 and US 4 940 840 disclose the use of chitinases of bacterial origin in the construction of transgenic plants; chitinase of plant origin is not mentioned or alternatively only mentioned in general terms.
• WO 90/07001 discloses DNA constructs comprising a high level promoter operably linked to a DNA sequence encoding a plant chitinase, which constructs are used in the transformation of plants so as to achieve overexpression of chitinase in the plant and thereby conferring resistance to plant pathogenic fungi.
• the only plant chitinase exemplified is a bean chitinase.
• EP 0 392 225 EP 0 307 841, EP 0 332 104, EP 0 440 304 and
• EP 0 418 695 disclose the construction of transgenic plants
• pathogenesis-related proteins e.g. chitinase and ⁇ -1,3-glucanase.
• Pathogenesis-related proteins from sugar beet plants or transgenic sugar beet plants are not mentioned.
• EP 0 448 511 also relates to transgenic plants comprising
• compositions comprising hydrolytic enzymes such as glucanase and chitinase for use for controlling plant pathogens. Chitinase or glucanase from sugar beet are not mentioned.
• WO 91/06312 discloses a composition for protecting a harvested crop comprising endoenzymes such as glucanase or chitinase. No particular source of chitinase or glucanase is mentioned. Rousseau-Limouzin M. and Fritig B. (1991) describe the production of basic and acidic PR-proteins in sugar beets infected with Cercospora beticola and the serological relation of these PR-proteins to the PR- proteins in tobacco. The described PR-proteins are found to be serological related to tobacco PR-proteins whereas the sugar beet chitinase 4 of the present invention does not show a serological relationship to any known chitinase, confer below. No information about the amino acid sequence or nucleotide sequence of any of the PR-proteins is given. In conclusion, none of the above cited publications disclose any sugar beet chitinase enzyme or the use thereof in the construction of transgenic plants.
• chitinase isoenzymes
• the chitinase isoenzymes were characterized by their molecular weight and kinetics of chitin hydrolysis. Chitinase preparations were indicated to be capable of hydrolyzing newly synthesized chitin in the cell wall of the growing fungi. No further characterization was reported and the chitinase enzymes were not separately discussed.
• the present invention relates to a DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in SEQ ID NO . : 1 or an analogue thereof, the analogue being a DNA sequence encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 as defined herein and i) being a characteristic part of the DNA sequence shown in SEQ ID NO.
• the chitinase 4 DNA sequence, SEQ ID NO.:1, shown in the Sequence Listing below was determined on the basis of a cDNA clone isolated from a sugar beet cDNA library prepared as described in the Material and Methods section below on the basis of hybridization with a very specific oligonucleotide probe.
• the oligonucleotide probe was prepared on the basis of a tryptic peptide produced from a substantially pure sugar beet chitinase 4 obtained as described in Materials and
• Example 4 The procedure used for isolating the chitinase 4 DNA sequence is outlined in Example 4 below.
• 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.
• the initial analysis of sugar beet chitinase 4, which revealed an enzyme with a small functional domain suggested that the chitinase enzyme had a low chitin affinity and thus low enzymatic activity. Thus the enzyme did not seem to be of any particular interest.
• the sugar beet chitinase 4 belongs to the plant chitinases of the hevein class in that it contains a leader sequence, a hevein domain and a functional (catalytic) domain.
• Hevein is a lectin which binds to chitin
• the hevein domain of the enzyme is the part of the enzyme which is expected to bind to chitin and chitin-containing structures, e.g. of phytopathogenic fungi.
• chitinase 4 By hydrophobic clustering analysis using the method according to Gaboriaud et al., 1987, the primary structure of chitinase 4 has been found to be more compact than the structures of other plant chitinases belonging to the sugar beet chitinase 2 class (as described in further detail below) . It is anticipated that the compact structure of chitinase 4 is an advantage in order to allow the enzyme to get access to chitin structures, e.g. in the cell walls of phytopathogenic fungi.
• chitinase 4 has been found to lack a C-terminal extension which means that the enzyme is translocated to the intercellular space, and thus not to the vacuole. The presence of the enzyme in the intercellular space has been experimentally verified.
• the sugar beet chitinase 4 has been found to have a surprisingly high antifungal activity and have shown a particularly good inhibiting effect on the growth of phytopathogenic fungi.
• the use of a combination of the sugar beet chitinase 4 enzyme a second different chitinase and a ⁇ -1,3-glucanase in the control of phytopathogenic "fungi has been found to result in an even more improved antifungal activity as compared to the use of the sugar beet chitinase 4 alone. This synergistic antifungal effect is reported for the first time in connection with this application.
• the present invention relates to a genetic construct comprising one or more copies of a DNA sequence .comprising the chitinase 4 DNA sequence shown in SEQ ID NO.:1 or an analogue thereof as defined above or a subsequence thereof (further defined below), one or more copies of a DNA sequence encoding a second chitinase different from the sugar beet chitinase 4, and one or more copies of a DNA sequence encoding a ⁇ - 1,3-glucanase, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.
• the constituents of the genetic construct and the synergistic effect are further explained below.
• the main use of the genetic construct of the invention is in the production of a genetically transformed plant having an increased resistance to chitin-containing plant pathogens such as phytopathogenic fungi as compared to plants which do not contain the construct such as untransformed or natural plants.
• the genetically transformed plants are advantageously prepared by use of a plant transformation vector harbouring the genetic construct of the invention.
• the chitinase 4 DNA sequence or an analogue thereof may also be used in the isolation of DNA sequences belonging to the chitinase 4 gene family as defined above.
• the chitinase 4 DNA sequence or an analogue thereof or a genetic construct of the invention may be used in a method of preparing a polypeptide, e.g. a recombinant sugar beet chitinase enzyme, or a polypeptide mixture having a potent antifungal activity.
• the polypeptide or polypeptide mixture may by prepared by use of recombinant DNA techniques and may be used in the antifungal treatment of various products, especially food products.
• the chitinase 4 DNA sequence, SEQ ID NO . : 1 encodes the basic sugar beet chitinase 4 enzyme, the amino acid sequence of which also appears from SEQ ID NO . : 2.
• chitinase 4" and “sugar beet chitinase 4" are used interchangeably.
• chitinase 4 DNA sequence of the invention and an analogue thereof 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.
• 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.
• chitinase activity denotes the enzyme's ability to decompose chitin and chitin-containing structures and the chitinase activity may be determined by 1) a biological assay and 2) a chemical assay.
• biological assay the effect of chitinase 4 on growing hyphae of pathogenic fungi, i.e. the ability of chitinase 4 to destroy the hyphae walls and thereby retard the growth of the hyphae. is directly observed.
• the chemical assay the decomposition of 3 H-chitin by chitinase 4 to result in mainly dimers of chitin is monitored.
• the biological assay may be carried out using any of the 3 different methods described in "Materials and Methods” herein under the heading "Antifungal activity”.
• Antifungal activity When a positive result is obtained in any of these methods, i.e. the observance of destruction of the hyphae walls and retardation of the growth of the fungal hyphae, it is taken as evidence of biological chitinase 4 activity.
• the chemical assay may be carried out as described in "Materials and Methods" under the heading "The radiochemical chitinase assay”.
• Chitinase 4 activity is shown by hydrolysis of 3 H-chitin and the resulting formation of mainly dimers of chitin in this assay.
• lysozyme activity denotes the enzyme's fungal cell wall lysing ability.
• the lysozyme activity is determined by carrying out the lysozyme assay described in "Materials and Methods" under the heading “Lysozyme assay”.
• the antifungal activity of the sugar beet chitinase 4 is a qualitative as well as a quantitative measure reflecting the ability of the polypeptide to destroy components e.g. chitin, of the hyphae walls of a phytopathogenic fungus thereby inhibiting or retarding the growth of the fungus.
• the analogue of the chitinase 4 DNA sequence is a DNA sequence having at least one of the properties i)-iv) listed above. The terms used to define the analogues of the invention are explained in further details below.
• characteristic part denotes a nucleotide sequence which is obtained from the nucleotide sequence of the chitinase 4 DNA sequence or which has a nucleotide sequence corresponding to a part of the chitinase 4 DNA sequence and which encodes a polypeptide having retained the antifungal activity of sugar beet chitinase 4.
• the characteristic part comprises a subsequence of the chitinase 4 DNA sequence, the subsequence being either a consecutive stretch of nucleotides of the chitinase 4 DNA sequence or being composed of one or more separate nucleotide sequences of the chitinase 4 DNA sequence.
• the part will normally be only a small number of nucleotides shorter than the chitinase 4 DNA sequence, e.g. 1-50, such as 1-25 nucleotides shorter.
• a typical example of a characteristic part of the chitinase 4 DNA sequence includes the nucleotides encoding the active site of chitinase 4.
• the analogue defined in ii) above is a DNA sequence which hybridizes with the chitinase 4 DNA sequence under the conditions specified in the "Materials and Methods" section below under the heading "Identification of DNA belonging to the chitinase 4 gene family".
• the conditions defined for the hybridization to take place are based on hybridization experiments carried out with a number of known plant chitinases and sugar beet chitinase 4 and is further described in Example 11 below.
• any DNA sequence hybridizing with the chitinase 4 DNA sequence under the hybridization conditions specified in the above cited part of "Material and Methods" is defined as belonging to the chitinase 4 gene family and is contemplated to encode a polypeptide having the structure and antifungal activity of the sugar beet chitinase 4. Furthermore, when the polypeptides produced from such DNA sequences react with .antibodies raised against sugar beet chitinase 4, it is a strong indication that the polypeptide encoded by the DNA sequence in question belongs to the sugar beet chitinase 4 serological class.
• Such DNA sequences constituting part of the present invention may either comprise sequences isolated from natural sources, e.g. plants, synthetically produced sequences or may be synthetically modified DNA sequences, e.g. as described below. In the following, DNA sequences belonging to the chitinase 4 gene family are also termed "chitinase 4 related DNA sequences".
• the analogue defined in iii) above is a DNA sequence which encodes a polypeptide comprising the amino acid sequence shown in SEO ID NO. : 2, i.e. the amino acid sequence of the mature chitinase 4 enzyme. It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia , to the preference of the organism in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the chitinase 4 DNA sequence of the invention may be exchanged by others which, when expressed, result in a polypeptide identical to or substantially identical to the polypeptide encoded by the chitinase 4 DNA sequence in question.
• the analogue defined in iv) above is a DNA sequence encoding a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4.
• the term "is recognized by” is used interchangeably with "binds to”.
• the sugar beet chitinase 4 enzyme belongs to a new serological class of basic chitinases hitherto not reported in the literature.
• the antibody to be used in determining the serological relationship between the polypeptide encoded by the chitinase 4 DNA sequence of the invention and a polypeptide encoded by a DNA sequence of another origin may be a monospecific polyclonal antibody or a monoclonal antibody.
• a particularly suitable antibody is a monoclonal or polyclonal antibody prepared against one or more characteristic epitopes encoded by the chitinase 4 DNA sequence. Such epitopes are explained in further detail below.
• the DNA sequences of the invention explained herein may comprise natural as well as synthetic DNA sequences, the natural sequence typically being derived directly from cDNA or genomic DNA, normally of plant origin, e.g. as described below.
• a synthetic sequence may be prepared by conventional methods for synthetically preparing DNA molecules, e.g. using the principles in solid or liquid phase DNA synthesis such as a DNA synthesizer 381 A (Applied Biosystems).
• the DNA sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic and mixed genomic and synthetic origin.
• composition of the chitinase 4 DNA sequence and each of the domains of the chitinase 4 enzyme encoded by the DNA sequence shown in SEQ ID NO. :1 and with the amino acid sequence shown in SEQ ID NO.: 2 are further described and compared to other plant chitinases.
• the chitinase 4 DNA SEQ ID NO . : 1 comprises a leader sequence
• nucleotides 71-174 encoding a hevein domain of 35 amino acid residues and a part (nucleotides 175-793) encoding a functional domain of 206 amino acid residues.
• the N-terminal part of the mature polypeptide chain is blocked and it has not been possible to determine the sequence by conventional amino acid sequencing methods.
• WGA-A wheat germ agglutinin
• potato chitinase based on an analysis by electrospray mass spectrometry (vide Example 4), the start codon of the chitinase 4 DNA sequence has been deduced.
• leader sequence from chitinase 4 DNA shows that the two first nucleotides in the leader sequence from chitinase 4 DNA (SEQ ID NO.:1) are missing.
• the leader sequence of the genomic chitinase 4 consists of 24 amino acid residues (SEQ ID NO.:4)
• the leader sequence from chitinase 4 consists of 24 amino acid residues although the almost full length chitinase form cDNA is missing the first amino acid Met (SEQ ID NO . : 2).
• Plant chitinases may be divided into 3 different groups, the hevein class, the non-hevein class and the cucumber class.
• Sugar beet chitinase 4 is a basic chitinase belonging to the hevein class. However, it is distinctly different from the other basic chitinases of this class. Whereas chitinases from bean, tobacco, tomato, potato, pea, poplar, barley (T and K) and sugar beet (chitinase 2) have molecular weights of 32-38 kDa (vide Example 10), chiti nase 4 is smaller with a molecular weight of about 26 kDa (as determined for the mature enzyme). In addition, since antibodies raised against chitinase 4 do not recognize the other basic
• chitinases described above (vide Example 10), it is evident that chitinase 4 also belong to a different serological class than all other basic plant chitinases from the hevein class.
• the primary structure of the mature chitinase 4 as determined on the basis of its amino acid sequence contains 2 different domains: the hevein domain and the functional domain. At the N-terminal part of the polypeptide chain, 12 out of 35 amino acid residues are conserved compared to the hevein structure.
• the functional domain contain 206 amino acid residues.
• the hevein domain consists of 43 amino acid residues and the functional domain contains 263 amino acid residues.
• hevein domain i.e the chitin binding domain
• chitinase 4 has a binding affinity which is of a similar magnitude as that of the other basic chitinases belonging to the hevein class.
• This class of chitinases does not contain the hevein domain, but only the functional domain.
• the homology between the functional domains of the hevein class and the non-hevein class is very high.
• polyclonal antibodies raised against the chitinases from the hevein-class recognize the chitinases from the non-hevein class.
• the specific activity of the non-hevein class, the acidic chitinase from tobacco and the basic chitinase C from barley are approximately 6-fold lower than that of the hevein class chitinases. Since the functional domain in chitinase 4 contains only 206 amino acid residues as compared to the 263 amino acid residues of the functional domain of the basic tobacco chitinase, a decrease in the specific activity was expected. Chitinase' 4, however, performs extremely well and was by the present inventors shown to be superior to chitinase T. K. and C from barley (results not shown) when ana lyzed by the radiochemical enzyme assay described in "Material and Methods" below.
• the most important parts of the chitinase 4 DNA sequence shown in SEQ ID NO . : 1 are the part encoding the hevein domain and especially the part encoding the functional domain of the enzyme. While the presence of a leader sequence in most cases is a prerequisite for allowing the polypeptide expressed from the DNA sequence to be transported out of the cell in which it is produced, the nature and origin of the particular leader sequence to be used may vary and need not be the leader sequence naturally associated with the chitinase 4 enzyme. Additionally, the leader sequence naturally associated with the chitinase 4 enzvme may be used in heterologous gene construct in transformation in plants, in particular sugar beet plants, when the encoded polypeptides are targeted to the extracellular space.
• a particularly interesting DNA sequence according to the present invention is a DNA sequence comprising nucleotides 71-793 of the chitinase 4 DNA sequence shown in SEQ ID NO.:1 and encoding the hevein domain and the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.
• analogue is referred to as a DNA sequence which either
• Ai is a characteristic part of said DNA sequence
• Aii) hybridizes with a DNA probe prepared from said DNA sequence, Aiii) encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or
• Aiv encodes a polypeptide which is recognized by an antibody raised against a polypeptide encoded by said DNA sequence.
• a still more interesting DNA sequence of the invention is a DNA sequence comprising nucleotides 175-793 of the chitinase 4 DNA se quence shown in SEQ ID NO.:1 encoding the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.
• analogue refers to a DNA sequence which
• Bi is a characteristic part of said DNA sequence, Bii) hybridizes with a DNA probe prepared from said DNA sequence,
• Biii encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by said DNA sequence, or
• Biv encodes a polypeptide which is recognized by an antibody raised against a polypeptide encoded by said sequence.
• analogues as defined by the properties Ai)-Aiv) and Bi)-Biv) above are defined in a similar manner to the analogues of the chitinase 4 DNA sequence defined by the properties i)-iv) above.
• the present invention relates to a DNA sequence comprising a sugar beet chitinase 4 gene.
• the term "gene” is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5'-upstream or 3'-downstream region.
• the 5'-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter.
• the 3' -downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.
• DNA sequence of the invention comprising a chitinase 4 gene is the genomic sugar beet DNA sequence harboured in the genomic chitinase 4 clone (chit 4), the isolation of which is described in Example 4.
• the partial nucleotide sequence of the gene has been elucidated and is shown in SEQ ID NO.: 3.
• SEQ ID NO.:3 Based on comparison of the partial DNA sequence with the DNA sequence of the chitinase 76 gene shown in SEQ ID NO.:5 and further discussed below, and the nucleotide sequence of the chitinase 4 cDNA shown in SEQ ID NO.:1, (the comparisons are shown in Fig. 24) it is contemplated that nucleotides 356-358 of the chitinase 4 gene sequence constitute the start codon of the chitinase 4 gene.
• the chitinase 4 gene comprises only one intron starting at nucleotide 398 downstream of the ATG start codon.
• the position of the intron is believed to correspond to a position between nucleotides 395 and 396 in the chitinase 4 cDNA sequence shown in SEQ ID NO.:1.
• the knowledge of the amino acid sequence of the sugar beet chitinase 4 makes it possible to analyze the enzyme and elucidate the important parts of the enzyme, this being done, e.g., on the basis of a comparison with the amino acid sequence of other known chitinases.
• An especially interesting part of the enzyme is, for instance, a part comprising the active site of the enzyme, a part comprising epitopes of the enzyme and a part responsible for the enzyme's substrate specificity and/or binding properties.
• the contemplated position of the active site of the sugar beet chitinase 4 enzyme has been revealed by comparison to the active site of other known enzymes catalyzing the hydrolysis of other oligosaccharides such as explained in Example 16 below.
• the active site of the sugar beet chitinase 4 is constituted by amino acid residues 183 (Asp) and 189 (Glu) in SEQ ID NO.:2.
• amino acid residues 183 (Asp) and 189 (Glu) in SEQ ID NO.:2.
• the specific amino acids of the enzyme responsible for its substrate specificity and substrate binding may be envisaged or elucidated.
• the amino acid residues forming the epitopes of the enzyme may be elucidated.
• the replacement of one or more of the Trp residues in position 169, 204 and 206 with Tyr residues is expected to change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate specificity.
• changes of the amino acid residues constituting the active site or amino acid residues which form the structure of the folded enzyme are expected to influence, e.g., the catalytic activity, substrate specificity and/or substrate binding may be found to result in improved properties of the resulting modified enzyme.
• the nature of the modification to be carried out will depend on the desired result, i.e. the specific desired function of the resulting modified enzyme.
• a DNA sequence, encoding the modified chitinase 4 enzyme may either alone or in combination with DNA sequences encoding other proteins, e.g. pathogenesis related proteins, such as thaumatin, osmothin and/or zeamatin (Viegers, 1991) or thionin
• the modified chitinase 4 enzyme may prove to be a particular interesting component of an antifungal composition as described below.
• a high degree of homology between coding regions of the genes is expected, whereas less homology is expected between non-coding regions. Between different gene families, the homology may vary considerably.
• the term "homology” is used here to denote the presence of the degree of complementarity between the amino acid sequence of a given polypeptide and the amino acid sequence of another polypeptide being analyzed as determined by use of the computer program by Myers and Miller, version 1.05, September 1990, using the comparison matrix: Genetic code, the Open Gap Cost 6 and the Unit Gap cost 1. See also Myers and Miller, 1988.
• the degree of homology between different genes may thus be used to assess the degree of familarity between different genes.
• the amino acid sequences may be deduced from a DNA sequence or may be obtained by conventional amino acid sequencing methods.
• the degree of homology is preferably determined on the basis of mature proteins, i.e. without taking any leader sequence into account .
• the present invention relates to a DNA sequence encoding a chitinase isoenzyme which is at least 60% homologous with the sugar beet chitinase 4 enzyme encoded by the DNA sequence SEQ ID NO.:1 and at the most 40% homologous with the sugar beet chitinase 1 encoded by the DNA sequence shown in SEQ ID NO.: 11.
• the minimum degree of homology of at least 60% has been determined on the basis of an analysis of a rape seed chitinase (based on the mature protein) which has been shown to belong to the sugar beet chitinase 4 serological class. (see Example 11).
• the degree of homology of 40% with chitinase 1 (which does not belong to the chitinase 4 class) reflects the minimal degree which is expected to be acceptable for a polypeptide belonging to the chitinase 4 class.
• a higher degree of homology with the chitinase 4 enzyme and therefor a lower degree of homology with the chitinase 1 enzyme reflects an even higher similarity herewith and accordingly, the DNA sequence described above preferably encodes a chitinase isoenzyme which is at least 65%, e.g.
• DNA sequence encoding a polypeptide being about 75% homologous to the sugar beet chitinase 4 enzyme and at the most 40% homologous to the sugar beet chitinase 1 enzyme is the genomic DNA sequence (chitinase 76, the sequence of which is shown in SEQ ID NO.:5) contained in the genomic clone chitinase 76 obtained as described in Example 5.
• sugar beet chitinase 4 isolated from sugar beet leaves is recognized by an antibody raised against this sugar beet chitinase, but not by an antibody raised against the sugar beet chitinase 2. This is a very strong indication of the fact that the sugar beet chitinase 4 belongs to a different class of chitinases than the sugar beet chitinase 2 and thus that 2 different classes of sugar beet chitinases exist.
• the present invention further comprises a DNA sequence which encodes a polypeptide which is recognized by an antibody raised against sugar beet chitinase 4, but not by an antibody raised against sugar beet chitinase 2.
• the present invention relates to a modified DNA sequence
• a modified DNA sequence comprising a DNA sequence as defined above comprising the chitinase 4 DNA sequence or gene or an analogue thereof in which at least one nucleotide has been .deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having retained the antifungal activity of the sugar beet chitinase 4 or having an increased antifungal activity as compared to the sugar beet chitinase 4.
• the polypeptide encoding by the modified DNA sequence has normally an amino acid sequence which is different from the amino acid sequence of the sugar beet
• a modified DNA sequence of the invention will be of importance in the preparation of novel polypeptides having an increased antifungal activity as compared to chitinase 4.
• substitution one or more nucleotides in the full nucleotide sequence are replaced with one or more different nucleotides
• addition one or more nucleotides are added at either end of the full nucleotide sequence
• insertion one or more nucleotides within the full nucleotide sequence is inserted
• “deletion” 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.
• the present invention relates to a subsequence of the chitinase 4 DNA sequence of SEQ ID NO . : 1 encoding a
• subsequences of the chitinase 4 DNA sequence or of the genomic DNA sequence are subsequences comprising the nucleotide sequence defining the active site of the sugar beet chitinase 4 enzyme.
• An example of such a subsequence is a DNA sequence comprising the active site of the sugar beet chitinase 4 enzyme, e.g. the DNA sequence encoding the following peptide named peptide 4-22 (shown by use of the
• This sequence is the amino acid sequence of the tryptic peptide 4-22 obtained from the purified sugar beet chitinase 4 as described in
• a DNA sequence encoding this polypeptide may be of significant importance for carrying out modifications of the active site with the aim of improving the antifungal activity of the resulting polypeptide.
• the DNA sequence may be fused to a part of another DNA sequence encoding an enzyme different from the sugar beet chitinase 4 or substituted with a part of such enzyme encoding the active site thereof with the aim of obtaining a hybrid enzyme having the antifungal activity of sugar beet chitinase 4.
• the polypeptide chain of the hybrid enzyme should be able to fold in the correct manner so as to provide a useful conformation around the active site.
• a further interesting DNA sequence encoding a part of the chitinase 4 enzyme is a DNA sequence encoding the polypeptide having the following amino acid sequence consisting of the amino acids No's. 183-204 of SEQ ID NO. :6
• This polypeptide is deduced from the DNA sequence of the genomic chitinase 76 clone shown in SEQ ID NO.: 5 and corresponds almost to the DNA sequence of the peptide 4-22 given above, except for the most important fact that the bolded D is an N in peptide 4-22. It is believed that the chitinase 76 derived polypeptide may have the same or nearly the same interesting properties and uses as the peptide 4-22. Two further interesting DNA sequences are the sequence encoding the following peptide consisting of the amino acids No's. 163-169 of SEQ ID NO. :2
• G-P-L-Q-I-T-W which is the tryptic peptide 4.19.3 of chitinase 4 and the DNA sequence encoding the tryptic peptide 4-26 consisting of the amino acids No's. 201-224 of SEQ ID NO.: 2
• T-A-F-W-F-W-M-N-N-V-H-S-V-I-V-N-G-Q-G-F-G-A-S-I which sequences are described in Example 16 below.
• the peptides comprises one and two Trp-residues, respectively.
• the Trp-residues are contemplated to be involved in the active site and/or substrate specificity of the chitinase 4 enzyme, e.g. as further discussed in Example 16 below.
• Analogues of these above mentioned subsequences in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which still have the catalytic and/or binding activities as that of the three above-mentioned peptides encoded by the chitinase 4 DNA subsequences may be very interesting.
• an interesting subsequence is a subsequence of the chitinase 4 DNA sequence of SEQ ID NO. :1 encoding a polypeptide comprising the hevein domain of the sugar beet chitinase 4 enzyme, or an analogue of said subsequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which subsequence is encoding a polypeptide capable of binding to chitin as determined by affinity column chromatography on regenerated chitin prepared as described in "Materials and Methods" under the heading "Preparation of a chitin column”.
• chitinase 4 enzyme Due to the fact that the hevein domain of the chitinase 4 enzyme is compact and believed to be very efficient, i.e. capable of establishing an intimate binding to chitin, this domain may prove to be very useful in the modification of chitinases, such as other plant chitinases, containing either a weak or no hevein domain with the aim of conferring a stronger chitin-binding capability to such chitinases.
• Examples of chitinase which could advantageously be modified by insertion of the DNA sequence encoding the hevein domain of sugar beet chitinase 4 are chitinases of the non-hevein class or cucumber class (e.g. the sugar beet chitinase SE disclosed herein).
• a further interesting subsequence of the present invention is a subsequence of the chitinase 4 DNA sequence SEQ ID NO.:1 encoding the leader peptide of chitinase 4 or an analogue thereof in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted and which is capable of directing a passenger polypeptide to which it is fused out of the cell in which the fused leader and passenger polypeptide is produced to be deposited in the extracellular space.
• epitopes of the sugar beet chitinase 4 enzyme may be used to raise monospecific polyclonal and monoclonal antibodies which are useful in identifying chitinase 4 isoenzymes belonging to the chitinase 4 serological class and for epitope mapping. Suitable epitopes are expected to be found among the hydrophilic peptides of the chitinase 4 amino acid sequence SEQ ID NO.:2, because these peptides seem to be substantially different from peptide parts of other chitinases than sugar beet chitinase 4.
• Antibodies either monoclonai, monospecific or polyspecific may be prepared by use of conventional methods, e.g.
• epitopes are believed to be particularly suitable for the production of monospecific antibodies to sugar beet chitinase 4.
• Peptide 1 and Peptide 4 are believed to be the most suitable peptide sequences to be used in the production of monospecific antibodies to chitinase 4.
• a DNA sequence comprising a subsequence of the present invention in which one or more nucleotides have been modified, e.g. as explained above, and having substantially retained the function and/or characteristics of the subsequence should be understood as being within the scope of the present invention.
• bacterial as well as plant chitinases exist.
• the most interesting types of chitinases are believed to be plant chitinases, and accordingly it is preferred that the DNA sequence of the invention or an analogue or a subsequence thereof is of plant origin.
• Especially interesting plant chitinase DNA sequences are derived from a member of the family Chenopodiaceae, Solanaceae, Apiaceae, Brassicaceae, Cucurbitaceae or Fabaceae.
• sequences, subsequences and analogues are corn, alfalfa, oat, wheat, rye rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea.
• sequences, subsequences and analogues should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in isolated, purified, in vitro or recombinant form.
• the chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof as defined above and especially a single stranded DNA or RNA sequence which is substantially complementary to either strand of such a DNA sequence may be used to isolate corresponding sequences from other plants, whereupon they, if desirable, may be modified as described herein.
• chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may be fused to one or more second nucleotide sequences encoding a second polypeptide or part thereof under conditions which ensure that at least part of the DNA sequence of the invention is expressed in conjunction with the other nucleotide sequence(s), e.g. in the form of a fusion protein.
• a DNA sequence of the invention encoding a polypeptide having the antifungal activity of the sugar beet chitinase 4 enzyme may advantageously be fused to a C-terminal sequence encoding a signal peptide which gives rise to transport of the fusion protein expressed therefrom to specific organelles of the organism expressing the polypeptide.
• Signal peptides involving transport will be discussed in further detail below.
• interesting subsequences of the chitinase 4 DNA sequence such as those described above, e.g. a subsequence encoding the hevein domain and/or an epitope, may likewise be fused to DNA sequences encoding other proteins, such as enzymes, e.g.
• chitinases in order to confer to the proteins the desirable properties of the polypeptides encoded by the subsequences of the chitinase 4 DNA sequence.
• a polypeptide encoded by the chitinase 4 DNA sequence or an analogue or subsequence thereof as defined above preferably in a non-naturally occurring or recombinant form.
• the polypeptide of the invention has the advantage that it may be easily produced in large quantities by use of well known conventional recombinant productions techniques, e.g.
• polypeptide of the invention may be used as a constituent in an antifungal composition, e.g. as described below.
• the sugar beet chitinase 4 enzyme has been shown to have a number of advantageous properties including a surprisingly high antifungal activity as compared to other known chitinases such as other known sugar beet chitinases, probably due to its dual chitinase/lysozyme activity and its compact structure. Also, the strong hevein domain of the sugar beet chitinase 4 enzymes adds to its advantageous properties.
• the use of a DNA sequence encoding the sugar beet chitinase 4 or an analogue thereof encoding a polypeptide having the antifungal activity as defined above is expected to be very interesting in the construction of genetically modified plants having an increased resistance to phytopathogenic fungi as compared to untransformed plants. Accordingly, in another important aspect, the present invention relates to a genetic construct comprising
• the genetic construct may be used in the construction of a genetically modified plant in order to produce a plant showing an increased antifungal activity as determined by the procedure given in Example 2 and thus an increased resistance towards phytopathogenic fungi.
• the genetic construct may be used in increasing the chitin-degrading capability of a plant.
• An example of a genetic construct as defined above is given in Example 18 below.
• the present invention relates to a genetic construct comprising one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID NO. :1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding a polypeptide having the activity of a second chitinase different from the sugar beet chitinase 4, and/or one or more copies of a DNA sequence encoding a polypeptide having ⁇ -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.
• polypeptides with chitinase or ⁇ - 1,3-glucanase activity is preferably of plant origin.
• the chitinase and ⁇ - 1,3-glucanase activity may be determined as explained in the section "Materials and Methods" below.
• a genetic construct comprising one or more copies of a DNA sequence as defined above comprising the chitinase 4 DNA sequence shown in SEQ ID NO.:1 or an analogue or subsequence thereof, one or more copies of a DNA sequence encoding an acidic chitinase having a pI equal to or less than 4.0, and one or more copies of a DNA sequence encoding a basic ⁇ - 1,3-glucanase having a pI of at least 9.0, each of the DNA sequences being functionally connected to a promoter and a transcription terminator capable of expressing the DNA sequences into functional polypeptides.
• an "acidic chitinase” is defined as a chitinase having a pi of less than 4.0.
• the acidic chitinase is a chitinase which hydrolyses chitin into chitooligosaccharides of the hexamer type.
• the acidic chitinase is preferably of plant origin. Examples of such chitinases are cucumber lysozyme/chitinase and
• the term "basic ⁇ - 1 ,3-glucanase” means a ⁇ -1 ,3-glucanase having a pi of more than 9.0.
• the basic ⁇ -1 ,3-glucanase is one which is capable of hydrolyzing glucan into mainly dimers, e.g. as determined by the 3 H-laminarin assay described in the Materials and Methods section below.
• the basic ⁇ - 1,3-glucanase is preferably of 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 4, the DNA sequence of which is shown in SEQ ID NO.: 9 or an analogue thereof encoding a basic ⁇ -1,3-glucanase having a pi of at least 9.0 and preferably being capable of hydrolyzing 3 H-laminarin into mainly dimers of ⁇ -1,3-glucan.
• the basic sugar beet ⁇ -1,3-glucanase 4 is different from other plant ⁇ -1,3-glucanases in that it does not contain a C-terminal extension as appears from the amino acid sequence SEQ ID NO.: 10.
• the advantageous effect of using the basic sugar beet ⁇ -1,3-glucanase 4 may in part be due to this lacking C-terminal extension.
• sugar beet chitinase 1 which shows a very low homology with the sugar beet chitinase 4 of the present invention, confer above.
• the DNA sequence of the sugar beet chitinase 1 is shown in SEQ ID NO.: 11.
• the DNA sequence is about 6.3 kb long and encodes a polypeptide having 439 amino acid residues.
• the polypeptide shown in SEQ ID NO.: 12 contains a leader sequence of 26 amino acid residues, a hevein domain of 20 amino acid residues and a C-terminal extension of 23 amino acids. Additionally, the sequence contains a most interest proline rich domain of 238 amino acids which forms and interest aspect of the present invention.
• Example 2 The experiments reported in Example 2 below show that the combination of the sugar beet chitinase 4 enzyme, an acidic chitinase and a basic ⁇ -1,3-glucanase results in an increased antifungal activity as compared to the antifungal activity of each of the constituents.
• the increased antifungal activity observed when using this specific combination is partly believed to be due to the different mode of action of the acidic chitinase, basic ⁇ -1,3-glucanase and sugar beet chitinase 4, respectively.
• the acidic chitinase is one which hydrolyses chitinase primarily into hexamers (as compared to chitinase 4 which primarily hydrolyses chitin into dimers) and the basic ⁇ -1,3-glucanase is one which hydrolyses glucan primarily into dimers, it is believed that these different cleaving modes may be involved in the resulting advantageous total effect. Furthermore, the synergistic effect obtained when using a combination of the sugar beet chitinase 4, a polypeptide having the activity of a second chitinase different from chitinase 4, e.g.
• an acidic chitinase and a polypeptide having the activity of a ⁇ -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.
• genetic constructs as defined above may be designed and prepared. Without being an exhaustive list, elements of the genetic constructs which may be varied are the number of copies of each of the DNA sequences of the genetic construct, the specific nucleotide sequence of each of the DNA sequences, the type of promoter and terminator connected to each DNA sequence, and the type of any other associated sequences, e.g. a C-terminal or N-terminal sequence (described below). Thus, genetic constructs of the present invention may vary within wide limits.
• variable elements of the genetic construct to be chosen will depend, e.g. on the desired strength of the antifungal effect to be obtained which may be determined as a function of gene dosage and specific nucleotide sequence of each of the DNA sequences, and the type and strength of the promoter and terminator used for each DNA sequence. Also, expression in specific parts of the plant with respect to organs and intracellular and extracellular location may be varied with different types of promoter and terminator.
• a genetic construct of the invention which is to be expressed in a given organism such as a plant
• the genetic construct of the invention is too large, it may be difficult to obtain a stable introduction thereof into the genome of the plant which may lead to excision of a part of or the entire genetic construct from the genome of the plant.
• the genetic construct should be adapted so that the expression products therefrom are generally acceptable to the host organism.
• the number of copies of the DNA sequences of the genetic construct of the invention together with the activity of the genes will determine the optimal number of copies of the DNA sequences of the genetic construct of the invention.
• a genetic construct 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.
• a significant antifungal effect is obtained from a protein encoded by the chitinase 4 DNA sequence of the invention or an analogue thereof. Accordingly, it is contemplated that a genetic construct of the invention, in which two copies of the chitinase 4 DNA sequence of the invention or an analogue thereof, and one copy of each of the DNA sequences encoding an acidic chitinase and a basic ⁇ -1,3-glucanase are present may show very potent antifungal effects when present in a genetically transformed plant of the invention. It is believed that such a genetic construct will not pose a too heavy burden on the plant in which it is harboured. Of course, also the choice of e.g. promoter used for each DNA sequence will influence the amount of protein expressed therefrom. This will be further explained below.
• the genetic construct of the invention as described above may be present on one or several DNA fragments.
• the size of the genetic construct to be introduced in an organism such as a plant in the case of a plant typically by means of a plant transformation vector, and the combination of promoters and transcription
• the DNA sequence of the invention When a polypeptide encoded by the DNA sequence of the invention is to be expressed in an organism, e.g. in a plant, it is desirable that the DNA sequence further comprises a nucleotide sequence encoding a leader sequence.
• the leader sequence may be the natural leader sequence, or a leader sequence derived from DNA encoding another protein. In any event, the leader sequence is to be functionally connected to the DNA sequence so that the polypeptide expressed from the resuiting nucleotide sequence serves to direct the polypeptide encoded by the DNA sequence out of the cell in which it is produced.
• polypeptide may be directed to specific locations of the organism in which it is produced, e.g. to lysosomes or vacuoles, or the passenger polypeptide may be excreted into the intracellular room.
• the leader sequence may be either N-terminally or C-terminally positioned.
• N-terminal sequence to be used will e.g. depend on the particular organism and the part thereof, e.g. the specific cell or tissue, in which the polypeptide encoded by the DNA sequence of the invention is to be produced and to which part of the same cell or another location in the organism the polypeptide is to be transported.
• a typical leader peptide has a core of hydrophobic amino acids and thus, a suitable leader sequence to be used in connection with the DNA sequence of the invention is a nucleotide sequence comprising a stretch of codons encoding hydrophobic amino acids.
• leader sequences examples are the following leader sequences which are also part of the invention. These leader sequences are the N-terminal leader sequence of the sugar beet chitinase 1 enzyme, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the N-terminal leader sequence of the genomic chitinase 76 clone, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 5 and SEQ ID NO : 6 , respectively; the N-terminal leader sequence of the acidic sugar beet chitinase SE, the nucleotide and amino acid sequence of which is shown in SEQ ID NO: 7 and SEQ ID NO: 8,
• N-terminal sequence of the ⁇ -1,3-glucanase 4 the nucleotide and amino acid sequence of which is shown in SEQ ID N0:9 and SEQ ID NO:10, respectively.
• Another interesting sequence is DNA subsequence from the sugar beet chitinase 1 encoding the proline rich domain of the chitinase 1 gene comprising 132 amino acids and shown in SEQ ID NO: 12 which may also be used in the direction of the polypeptide to specific locations of the organism.
• the abovementioned leader sequences are to be considered as non- limiting examples.
• leader sequences of the invention are all specific for sugar beet plants, these leader sequences may in another aspect of the invention be functionally connected to a DNA sequence different from the DNA sequences being part of the invention, and which DNA sequence is to be used in a transformation of a sugar beet plant.
• a DNA sequence may in particular be a DNA sequence which is not naturally present in sugar beet plant.
• the use of a leader sequence normally present in the sugar beet may be an advantage in a transformation of a sugar beet plant as such a leader sequence is known to function in a sugar beet.
• invention may thus serve to direct the polypeptide expressed from the nucleotide sequence to specific locations of the cell or organism in which it is produced.
• proline rich domain of the chitinase 1 shown in SEQ ID NO.:12 consisting of 132 amino acids. It is contemplated that the proline rich domain may be involved in the anchoring of the chitinase 1 protein to the cell wall after modification of the prolines to glycosylated hydroxyprolines, as in extensines. Thus, the subsequence containing the proline rich domain may be used when directing and obtaining a polypeptide at a desired location in the cell and/or organism in which the polypeptide is produced.
• At least one of the DNA sequences of the genetic construct of the invention further comprises a C-terminal sequence encoding a signal peptide capable of directing the polypeptide encoded by the DNA sequence to a part of an organism in which it is to be deposited, e.g. in the vacuole.
• the same DNA sequence may be present with and without a C-terminal sequence in the same genetic construct.
• the C-terminal sequence may be the C-terminal extension normally associated with the DNA sequence, if any, or may be derived from the host in which the genetic construct is to be expressed or may be of another origin.
• Non-limiting examples C-terminal sequences to be included in a genetic construct of the invention are C-terminal sequences selected from the following sequences; the C-terminal sequence of sugar beet chitinase 1, the amino acid of which is shown in SEQ ID NO.: 12, encoding the following polypeptide consisting of the amino acids No's 413-439
• N L R C * the C-terminal sequence of a bean chitinase (PHA) encoding the following polypeptide shown in SEQ ID NO.: 13
• a phytopathogenic fungus mainly present intracellularly it may be desirable that most of or all of the DNA sequences of the genetic construct are provided with a C-terminal sequence capable to transport the polypeptides expressed from the DNA sequences to the vacuole.
• each of the DNA sequences of the genetic construct of the invention or of a gene comprising such DNA sequences are accomplished by means of a regulatory sequence functionally connected to the DNA sequence or gene so as to obtain expression of said sequence or gene under the control of the inserted regulatory sequence.
• the regulatory sequence is a promoter which may be constitutive or regulatable.
• promoter is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is functionally connected, allowing transcription to take place.
• the promoter is usually situated upstream (5') of the coding sequence.
• promoter includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are. e.g. sequences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions,
• a "constitutive promoter” is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place is fulfilled.
• the constitutive promoter may be enhanced.
• a “regulatable promoter” is a promoter the function of which is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of each of the DNA sequences of the genetic construct of the invention. Other types of regulatory sequences are upstream and downstream sequences involved in control of termination of transcription (transcription terminators) and removal of introns, as well as sequences responsible for polyadenylation, and for initiation of translation. When the regulatory sequence is to function in a plant, it is preferably of plant origin.
• tissue specific regulation may be regulated by certain intrinsic factors which ensure that genes encoding proteins specific to a given tissue are expressed.
• tissue specific promoters are leaf specific promoters such as the chlorophyll a/b promoter and the AHAS promoter, and further root specific, stem specific, seed specific and petal specific promoters. Also factors such as pathogenic attack or certain biological factors have been shown to regulate promoters.
• heat-response promoters and promoters involved in the developmental regulation of plants may be found to be of interest.
• a suitable constitutive promoter is selected from the group consisting of plant promoters, fungal promoters, bacterial promoters, or plant virus promoters.
• a preferred group of plant virus promoters are promoters which may be derived from a cauliflower mosaic virus (CaMV) . Such promoters are normally strong constitutive promoters. Examples of a preferred CaMV promoter is a CaMV 19S promoter and a CaMV 35S promoter (Odell et al., 1985).
• promoters may be derived from the Ti-plasmid such as the octopine synthase promoter, the nopaline synthase promoter (HerreraEstrella et al., 1983), the mannopine synthase promoter, and promoters from other open reading frames in the T-DNA such as ORF7.
• the regulatory sequence may be a chitinase promoter, i.e. a promoter which is naturally found in connection with chitinase genes and involved in the transcription thereof.
• a chitinase promoter may be obtained from an isolated chitinase gene, e.g. an already known chitinase gene or a gene which may be identified and isolated e.g. by the methods disclosed herein.
• the chitinase promoter should be obtained from a plant which has been shown to have a fast response to pathogen challenge.
• fast responses have been observed in pea and barley and it is contemplated that chitinase promoters from these plants may be useful for the present purpose.
• An example of such promoters is the chitinase promoter of pea (K. Vad, 1991).
• An example of another promoter which is contemplated to be useful in the present context is the sugar beet chitinase 1 promoter (SEQ ID NO: 11) and the sugar beet acetohydroxyacid synthase promoter (AHAS) (P. Stougard and K.
• sugar beet promoters from the acidic chitinase SE, chitinase 1, chitinase 76 and chitinase 4 or ⁇ - 1 ,3-glucanase 4 may also be useful.
• the natural promoter may be modified for the purpose, e.g. by modifications of the promoter nucleotide se quence so as to obtain a promoter functioning in another manner than the natural promoter, preferably activating the transcription of the gene earlier after the challenge with a pathogen or being stronger.
• each of the coding DNA sequences of the genetic construct of the invention is functionally connected to a transcription terminator.
• the transcription terminator serves to terminate the transcription of the DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences and plant virus terminator sequences.
• Suitable transcription terminators are a NOS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium (Herrera-Estrella et al., 1983), a 35S transcription terminator sequence of the cauliflower mosaic virus (Paszkowski et al., 1984), a PADG4 transcription terminator to the DNA gene 4 (Wing et al., 1989), and a PADG7 transcription terminator to the T-DNA gene 7.
• One or more of the DNA sequences of the genetic construct of the invention may advantageously be functionally connected to an enhancer sequence which results in an increased transcription and expression of the DNA sequence(s).
• Suitable enhancer sequences and means for obtaining an increased transcription and expression are known in the art.
• the present invention relates to a vector which is capable of replicating in a host organism and which carries a DNA sequence of the invention comprising a chitinase 4 DNA sequence substantially as shown in SEQ ID NO:1 or an analogue or subsequence thereof, or a genetic construct of the invention.
• the vector may either be one which is capable of autonomous replication, such as a plasmid, or one which is replicated with the host chromosome, such as a bacteriophage or integrated into a plant genome via the border sequences of Ti vectors.
• the vector is an expression vector capable of expressing the DNA sequences in the organism chosen for the production.
• the expression vector is a vector which carries the regulatory sequences necessary for expression such as the promoter, an initiation signal and a termination signal, etc. These regulatory sequences may be the ones carried by the genetic construct of the invention.
• the vector may also be one used for identification and optionally isolation of chitinase genes or messengers from other organisms, e.g. other plants, for which purpose expression is not required. This may be done, e.g., as described below.
• the present invention relates to an organism which carries and which is capable of replicating or expressing an inserted DNA sequence as defined above, i.e. a chitinase 4 DNA sequence comprising a nucleotide sequence substantially as shown in SEQ ID NO:1 or an analogue thereof or a chitinase gene or pseudogene comprising said DNA sequence.
• inserted indicates that the DNA sequence (or subsequence or analogue, or gene or pseudo-gene) has been inserted into the organism or an ancestor thereof by means of genetic manipulation, in other words, the organism may be one which did not naturally or inherently contain such a DNA sequence in its genome, or it may be one which naturally or inherently contains such a DNA sequence, but in a lower number so that the organism with the inserted DNA sequence or the inserted genetic construct has a higher number of such sequences than its naturally occurring counterparts.
• the DNA sequence carried by the organism may be part of the genome of the organism, or may be carried on a vector as defined above which is harboured in the organism.
• the DNA sequence may be present in the genome or expression vector as defined above in frame with one or more second DNA sequences encoding a second polypeptide or part thereof so as to encode a fusion protein, e.g. as defined above.
• the organism may be a higher organism such as a plant, or a lower organism such as a microorganism.
• a lower organism such as a bacterium, e.g. a gram-negative bacterium such as a bacterium of the genus Escherichia, e.g. E. coli, or of the genus Pseudomonas, e.g. P. putida and P. fluorescens, or a gram-positive bacterium such as of the genus Bacillus, e.g. B. subtilis, or a yeast such as of the genus Saccharomyces or a fungus, e.g.
• a DNA sequence or a genetic construct according to the present invention may lead to a considerably increased chitinase and optionally ⁇ -1,3-glucanase expression and a correspondingly increased antifungal activity.
• the recombinant production may be performed by use of conventional techniques, e.g. as described by Sambrook et al., 1989.
• a microorganism producing chitinase may be used in combating soil plant pathogens, i.e. pathogens present in the soil and responsible for retarded growth or death of the plant.
• plant pathogens i.e. pathogens present in the soil and responsible for retarded growth or death of the plant.
• plant pathogens are soil fungi present in e.g. the rhizosphere.
• the organism may be a cell line, e.g. a plant cell line.
• the organism is a plant, i.e. a genetically modified plant such as will be discussed in further detail below.
• the genetic construct is preferably to be used in modifying a plant.
• the present invention also relates to a genetically transformed plant comprising in its genome a genetic construct as defined above.
• the genetically transformed plant has an increased antifungal activity compared to a plant which does not harbour a genetic construct of the invention, e.g. an untransformed or natural plant or a plant which has been genetically transformed, but not with a genetic construct of the invention.
• a constitutive expression of the polypeptides encoded by the genetic construct is desirable, but in certain cases it may be interesting to have the expression of the polypeptides encoded by the genetic construct regulated by various factors, for example by factors such as temperature, pathogens, and biological factors.
• Chitinase genes have been found in monocotyledonous as well as dicotyledonous plants and have there been found to be expressed into chitinase active in destroying the cell walls of phytopathogenic fungi.
• the plant to be transformed by the genetic construct of the invention may be a monocotyledonous as well as a dicotyledonous plant, since the genetic construct is expected to be active in such classes of plants.
• monocotyledonous plants which may be transformed are corn, oat, wheat, rye, rice, barley and sorghum.
• Non- limiting examples of dicotyledonous plants which may be genetically transformed are alfalfa, tobacco, cotton, sugar beet, fodder beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, cabbage, pepper, lettuce, bean and pea.
• the genetically transformed plant according to the invention has an increased resistance to chitin-containing plant pathogens such as phytopthogenic fungi and nematodes as compared to plants which have not been genetically transformed according to the invention or as compared to plants which do not harbour the genetic construct as defined above.
• phytopathogenic fungi The most important chitin-containing plant pathogens to be controlled according to the invention are represented by phytopathogenic fungi.
• Phytopathogenic fungi differ in the way which they interact with their host plant during infection. Some species invade the plant via natural openings or wounded tissue and grow in between the plant cells, in the intercellular space, during the entire infection cycle. The fungal hyphae excrete toxins or enzymes that weaken or destroy the plant cells and thereby provide the fungus with cell constituents leaking out of the plant cells. Other fungal pathogens immediately destroy the host cells by penetrating the cell wall of healthy host cells and disintegrate their protoplasts.
• Cercospora spp. is a fungus the growth of which is restricted to the intercellular space.
• Conidia i.e. spores
• spores germinate on the leaf surface and penetrate through the stomata of the leaves.
• the toxins cause the plasma membrane to degrade, whereby the cell content leaks out into the intercellular space. Later in the infection cycle the plant cells collapse and necrotic areas containing dead plant cells and fungal mycelia emerge.
• Verticillium alboatrum is a root pathogen which propagates in the intercellular space, but which penetrates through the openings made by the emergence of lateral roots, through mechanically injured areas or by direct penetration of hyphae through the tender root tissue in the regions of cell elongation or meristemic activity.
• the fungus destroys the parenchymatous cells and the tracery elements are mechanically plugged.
• Plant pathogenic fungi with an intercellular infection cycle include: Sclerotinia sclerotiorum, Rhizoctonia solani, Phytophtora megasperma and Helmintosporium spp.
• Colletotricum lindemuthianum causes "Bean anthracnose" .
• Conidia from this fungus germinate in a film of water in the infection court and the produced germ tube penetrates the cuticula and grows into the epidermal cells of bean leaves and pods.
• the fungus acts as a parasitic pathogen, penetrating living cells and causing disintegration of the protoplasts.
• Fusarium spp . is a typical soilborne fungus infecting the plants through the roots, where the hyphae penetrate the epidermal cells of young roots and invades the xylem of roots and stems. The vessels become plugged with granular material and surrounding cells of the outer phloem and cortex are destroyed.
• Puccinia graminis causes "Stem rust" of wheat.
• the growing mycelia produce haustoria that penetrate the walls of the host cells and invaginate their protoplasts.
• Ustilago maydis is a fungus with mainly intercellular growth, but occasionally penetrates the cell wall of host cells.
• the present invention relates to seeds, seedlings or plant parts obtained by growing the genetically transformed plant as described above. It will be understood that any plant part or cell derivable from the genetically transformed plant of the invention is to be considered within the scope of the present invention.
• the genetic information is introduced into the plant by use of a vector system or by direct introduction, e.g.
• the present invention relates to a transformation system comprising at least one vector which carries a genetic construct as defined above and which is capable of introducing the genetic construct into the genome of a plant such as a plant of the family Chienopodiaceae in particular of the genus Beta, especially Beta vulgaris.
• plant transformation systems are based on the use of plasmids or plasmid derivatives of the bacteria Agrobacterium.
• the two best known Agrobacteria are Agrobacterium tumefaciens and Agrobacterium rhizogenes (plasmids thereof are in the following termed pTi and pRi, respectively).
• the use of such plant transformation systems is based on the ability of the bacteria Agrobacterium to transfer a specific piece of DNA (T-DNA) to a plant cell in a wounded area.
• T-DNA is located between specific border DNA sequences on the pTi or pRi which further carries virulence genes necessary for the transfer of the T-DNA to the plant.
• the Agrobacterium transformation system mediates the transfer of any DNA sequence located between the "borders" and thus, it is possible to exchange the wild type Agrobacterium T-DNA with any desirable piece of DNA to be introduced into a plant.
• 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.
• the vector system with which the plant is transformed comprises one or two plasmids.
• 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.
• 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.
• a suitable plant transformation vector is pBI121 and derivatives thereof, e.g. as described by Jefferson 1987.
• the vector to be used is provided with suitable markers, eucaryotic as well as procaryotic, e.g. genes encoding antibiotic resistance or herbicide resistance or glucoronidase (GUS), e.g.
• hygromycin or other known markers, e.g. the markers disclosed by Lindsey, 1989 and Reynaerts et al., 1988.
• the marker is to be present so as to be able to determine whether the DNA insert has been inserted in the desired position in the plasmid and to be able to select plant cells transformed with the vector.
• the 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.
• a useful microorganism is E . coli , but other microorganisms having the above properties may be used.
• the plasmid harboring the genetic construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A . tumefaciens , so as to obtain an Agrobacterium cell harboring the genetic construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified.
• a suitable Agrobacterium strain e.g. A . tumefaciens
• This transformation may be performed in a number of ways, e.g. as described in An et al. (1988). Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher et al. (1980). Typically, a plant to be infected is wounded, e.g.
• Example 15 by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive or brushing the plant with a steel brush (e.g. as described in Example 15).
• the wound is then inoculated with the Agrobacterium, e.g. in a suspension.
• the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
• the inoculated plant or plant part is then subjected to selection and regeneration and grown on a suitable culture medium and allowed to develop into mature plants. This is accomplished by use of methods known in the art.
• tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
• Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
• a genetically transformed plant may be performed as a double transformation event (introducing the genetic construct in two transformation cycles) or may be associated with use of conventional breeding techniques.
• two genetically modified plants according to the present invention may be cross breeded in order to obtain a plant which contains the genetic construct of each of its parent plants.
• the chitinase 4 DNA sequence of the present invention or an analogue thereof may be used for diagnostic purposes, which will be further explained in the following.
• Various types of diagnosis may be performed by use of the chitinase 4 DNA sequence of the invention.
• chitinase messenger RNA's transcribed from a gene belonging to the chitinase 4 gene family may be qualitatively as well as quantitatively determined by hybridization to the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof under conditions suitable for said hybridization.
• genes belonging to the chitinase 4 gene family and present in an organism such as a plant may be identified and isolated by use of the DNA sequence of the invention, e.g. by screening a gene library of such an organism.
• 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.
• 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.
• DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof may be used in a method of isolating a gene or messenger belonging to or derived from the chitinase 4 gene family from an organism, e.g.
• a plant in particular a dicotyledon
• the method comprising hybridizing a nucleic acid containing sample obtained from a gene library or cDNA library from the organism with the DNA sequence of the invention comprising the chitinase 4 DNA sequence or an analogue or subsequence thereof, optionally in a labelled form, in a denatured form or an RNA copy thereof under conditions favorable to hybridization between the DNA sequence or RNA copy and the nucleic acid of the sample, and recovering the hybridized clone so as to obtain a gene or cDNA belonging to the chitinase 4 gene family of the organism.
• the identification and isolation of a gene or cDNA clone in a sample belonging to the chitinase 4 gene family by use of the chitinase 4 DNA sequence of the invention or an analogue thereof, in particular a subsequence thereof, may be based on standard procedures, e.g. as described by Sambrook et al., 1989. For instance, to characterize chitinase 4 related genes in other plants, it is preferred to employ standard Southern techniques.
• the chitinase 4 DNA sequence of the invention or an analogue or subsequence thereof may also be used in a method of quantifying the amount of a chitinase 4 related messenger present in different tissues in an organism, e.g.
• a plant comprising hybridizing a nucleic acid containing sample obtained from the organism with the chitinase 4 DNA sequence of the invention comprising a nucleotide sequence substantially as shown SEQ ID NO : 1 or an analogue thereof, especially a subsequence thereof, optionally in labelled form, in denatured form or an RNA copy thereof under conditions favorable to hybridization between the denatured DNA sequence or RNA copy and the RNA of the sample and determining the amount of hybridized nucleic acid (Barkardottir et al., 1987).
• the hybridization should be carried out in accordance with conventional hybridization methods under suitable conditions with respect to e.g.
• the choice of conditions will, inter alia, depend on the degree of complementarity between the fragments to be hybridized, i.e. a high degree of complementarity requires more stringent conditions such as low salt concentrations, low ionic strength of the buffer and higher temperatures, whereas a low degree of complementarity requires less stringent conditions, e.g. higher salt concentration, higher ionic strength of the buffer or lower temperatures, for the hybridization to take place.
• the support to which DNA or RNA fragments of the sample to be analyzed are bound in denatured form is preferably a solid support and may be any of the supports conventionally used in DNA and RNA analysis.
• the DNA sequence used for detecting the presence of the chitinase 4 related gene is preferably labelled, e.g. as explained above, and the presence of hybridized DNA is determined by autoradiography, scintillation counting, luminescence, or chemical reaction.
• Another approach for detecting the presence of a specific chitinase 4 related gene is to employ the principles of the well-known polymerase chain reaction, e.g. as described in the "Materials and Methods" section below.
• 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 are increasingly used to follow specific alleles of genes in various organisms.
• the alleles are either themselves followed or they are used as markers (unlinked or linked) in crosses involving other characteristics, e.g. pathogen resistance and morphological characteristics such as tuber colour. So far, the method has primarily been employed in humans, but it has also been employed in plants. It is contemplated that the chitinase 4 DNA sequence of the invention or a analogue thereof may be useful in RFLP-analysis of chitinase 4 related genes, especially in sugar beet.
• the present invention relates to an antifungal composition
• the present invention relates to an antifungal composition comprising a microorganism capable of expressing a poly peptide encoded by the DNA sequence comprising the chitinase 4 DNA sequence shown in SEQ ID NO : 1 or an analogue or subsequence thereof as defined above, or by a genetic construct of the invention defined above and a suitable vehicle.
• Microorganisms suitable as constituents in an antifungal composition are mentioned above.
• the antifungal composition according to the present invention may be prepared by a method comprising culturing a microorganism harbouring and being capable of expressing a DNA sequence of the invention comprising the chitinase 4 DNA sequence shown in SEQ ID NO:1 or an analogue or subsequence thereof or a genetic construct of the invention in an appropriate medium and under conditions which result in the expression of one or more antifungal polypeptides encoded by the DNA sequences, optionally rupturing the microorganisms so as to release their content of expressed antifungal polypeptide(s) into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the polypeptide(s) to freeze-drying or spray-drying thereby obtaining an antifungal composition comprising the antifungal polypeptide(s).
• the antifungal proteins may be excreted to the medium, and optionally after removal of the microorganisms by conventional methods or after purification of the proteins by conventional methods or after purification of the prolines by conventional methods, the medium may be used directly or after freeze drying.
• the antifungal composition according to the invention may be used in combating or inhibiting the germination and/or growth of a phytopathogenic fungus in or on a plant or in any other material in which the presence of fungi is undesirable. This will be further discussed below.
• antifungal composition of the invention shall, of course, be adapted to its intended purpose, both with respect to the vehicle to be used and with respect to the form, in which the antifungal agent is present.
• antifungal agent is meant the active constituent of the antifungal composition responsible for or involved in providing the antifungal activity.
• antifungal polypeptide is meant a polypeptide encoded by the chitinase 4 DNA se quence of the invention or an analogue thereof or a genetic construct of the invention having antifungal activity, i.e. chitinase activity and optionally ⁇ - 1 ,3-glucanase activity as defined above.
• the antifungal composition may in addition to the polypeptide encoded by the chitinase 4 DNA sequence of the invention or an analogue thereof or a genetic construct of the invention having antifungal activity, i.e. chitinase activity and optionally ⁇ - 1,3-glucanase activity as defined above, contain one or several chemicals, e.g. fungicides, conventionally used in the combatting of fungi either therapeutically or prophylactically.
• fungicides conventionally used in the combatting of fungi either therapeutically or prophylactically.
• the antifungal agent is in itself a microorganism or will be prepared by a microorganism. In most cases, the most easy and inexpensive way of preparing the antifungal composition will be to use the microorganism as such or the medium in which it is grown as the antifungal agent.
• the antifungal polypeptide(s) expressed from the microorganisms may be secreted into the medium, e.g. as a consequence of the action of a suitable signal peptide capable of directing the polypeptide out into the medium, or may be released from the microorganism by well known mechanical or chemical means. Before use, it may be advantageous to remove the microorganisms or any cell debris from the medium.
• the medium may, in principle, serve as the vehicle for the antifungal agent, but it is preferred to add a further vehicle suited for the particular intended use.
• a culture of the microorganisms expressing the antifungal polypeptide(s) may be obtained as described above using methods known in the art. As mentioned above, it may be necessary or advantageous to subject the microorganism culture to a further treatment so as to release the content of the antifungal polypeptide(s) into the medium or to increase the amount released by secretion,
• the medium comprising a substantial amount of the antifungal polypeptide(s) may be directly applied to the soil in which the plants are present or in which the plants are to be grown, or to the plants or plant parts or to the irrigation water.
• seeds may be treated with the medium, optionally in combination with a conventional seed coating composition.
• the microorganisms expressing the antifungal polypeptide(s) can be applied in various formulations containing agronomically acceptable vehicles, i.e. adjuvants or carriers, in dosages and concentrations chosen to maximize the beneficial effect of the microorganism.
• agronomically acceptable vehicles i.e. adjuvants or carriers
• the microorganisms may also be distributed as such under circumstances allowing the microorganisms to establish themselves in the material to be treated.
• the microorganism is a microorganism conventionally found in the soil, e.g. a rhizobacterium, it will generally be desirable that the transformed microorganism establishes itself in the soil so that it continuously may secrete the antifungal polypeptide(s) out into the soil surrounding the plant.
• microorganisms or the medium comprising the antifungal polypeptide(s) may be added to pre-mixes, e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question.
• pre-mixes e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question.
• the microorganisms or the medium is in a solid form, e.g. in a powdery form or in the form of a granule.
• the powdery form may be obtained by conventional means, e.g. by applying the microorganism on a particulate carrier by spray-drying or an equivalent method.
• the microorganism expressing the antifungal polypeptide(s) 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.
• the present invention further relates to a method of inhibiting the germination and/or growth of a chitin containing plant pathogen, such as phytopathogenic fungus, in or on a plant, which method comprises 1) transforming the plant or a part thereof with a genetic construct as defined above and regenerating the resulting transformed plant or plant part into a genetically transformed plant, and/or
• While genetic transformation of plants is for most purposes are the preferred method, it may be an advantage to combine transformation with treatment of the plant with an antifungal composition of the invention. Since the genetic transformation is a time-consuming and in certain aspects difficult process, it may be an advantage to use a biologically based composition instead of or in addition to the conventionally used and from an environmental point of view undesirable chemical fungicides. In most cases the material to be treated with the antifungal composition of the invention is a plant. However, a number of chitin containing fungi exist which infect other materials than plants, e.g. food products such as bread or bread products, milk products cheese, meat, vegetables, cereals, in which the presence and growth of fungi are undesirable.
• Fig. 1 describes the purification of sugar beet chitinase 2, 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elution of the proteins was performed with a linear gradient of NaCl. The absorbance was recorded at 280 ⁇ m.
• Fig. 2 describes the polypeptide pattern of sugar beet chitinase 2, 3 and 4 after purification on a Mono-S FPLC column. Lanes contain 50 ⁇ 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 3 H-chitin by chitinase 4.
• -"H-chitin was incubated with 4 ⁇ g chitinase 4 at 37°C for 0.25, 0.5, 3 and 24 hours.
• 3 H-chitin was incubated without enzyme at 37°C for 24 hours.
• the chitooligosaccharides released were separated by TLC and identified by comparing their migration with that of N-acetylglucosamine (monomer) (Fig. 3A), chitobiose (dimer) (Fig. 3B), chitotriose (trimer) (Fig. 3C) and chitotetraose (tetramer) (Fig. 3D) standards.
• the chitooligosaccharides released were separated by TLC and identified by comparing their migration with that of N-acetylglucosamine (monomer) (Fig. 3A), chitobio
• 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.
• Fig. 5 shows the inhibition of the growth of Cercospora by a combination of chitinase 4, acidic chitinase SE and ⁇ -1,3-glucanase 4 using the microscope slide bioassay. After 48 hours of incubation the cultures were stained with Calcofluor White and investigated under fluorescent light.
• Fig. 5A shows the growth of the fungus when 20 ⁇ g of each of the enzymes chitinase 4, acidic chitinase SE and ⁇ -1,3-glucanase 4 were added to the culture at time 0
• Fig. 5B shows the growth of a control culture where no antifungal proteins have been added.
• Fig. 6 shows the inhibition of growth of Cercospora by chitinases using the microtiter plate bioassay.
• the time course curves (absorbance at 620 nm) describe the growth of the fungus during the first 92 hours of incubation. The absorbance (an indication of the growth) was measured at 8 to 16 hours time intervals and each measurement is an average of 5 replicates, Curve A is a control curve showing the growth of Cercospora when no growth inhibitors were added to the culture.
• Curve B shows the growth of the fungus when 20 ⁇ l of a chitinase containing fraction from the chitin-column was added at time 0.
• curve C 20 ⁇ g of purified chitinase 4 was added to the culture at time 0.
• Fig. 7 is an autoradiography showing the effect of chitinase 4 on chitin in the apex of Cercospora hyphae.
• Incorporation of 3 H- labelled N-acetylglucosamine into the hyphae of Cercospora beticola was performed by growing the fungus for 20 minutes on growth medium containing radioactive monomer of chitin.
• Incorporation of N-acetylglucoseamine into the cell wall in the apex of the fungal hyphae is seen as black dots.
• Fig. 7A shows the hyphae before treatment with purified chitinase 4.
• Fig. 7B shows the hyphae after the radioactive incorporation followed by treatment with purified chitinase 4 for 24 hours.
• Fig. 8 shows the separation of tryptic peptides of chitinase 4 by reverse phase HPLC on a Vydac RP-18 column.
• the peptides were eluted with a linear gradient from 10% to 45% acetonitrile from 25 to 75 minutes.
• Buffer A was water, whereas B was acetonitrile. Both solvents contained 0.1% trifluoroacetic acid.
• the flow rate was
• Fig. 9 shows the separation of three acidic SE chitinase isozymes on an anion exchange column (Mono P) by the FPLC system.
• the proteins were eluted with a linear sodium chloride gradient in a 25 mM Bis-Tris buffer at pH 7.0.
• Fig. 10 describes the two different serological classes of sugar beet, the chitinase 2 and chitinase 4 class. 5 ⁇ g of both chitinase 2 (32 kD) and 4 (26 kD) were blotted on to the nitrocellulose membrane before reaction with antibody to sugar beet chitinase 2 (Fig. 10A) or antibody to sugar beet chitinase 4 (Fig. 10B).
• Fig. 11 Hybridization of different chitinase genes with a chitinase 4 cDNA probe under specific hybridization conditions.
• the different chitinase genes were spotted on Hybond N-nylon membranes as 1 ⁇ l probes of a plasmid preparation containing the chitinase sequences.
• the hybridization was carried out over night at 55°C in the following hybridization buffer: 2 ⁇ SSC, 0.1% SDS. 10 ⁇ Denhardt's. 50 ⁇ g/ml Salmon sperm DNA and a chitinase 4 cDNA sequence as probe and under washing conditions of 55°C. 2 ⁇ SSC, 0.1% SDS in two times 15 minutes followed bv two times 15 minutes 1 ⁇ SSC. 0.1% SDS, 55°C.
• Fig. 12 describes the induction of chitinase and ⁇ - 1,3-glucanase in sugar beet leaves after infection with Cercospora beticola . Plants were inoculated with a suspension of fungal spores.
• Fig. 13 describes the immuno-detection of sugar beet chitinase 2 and 4 and ⁇ - 1 ,3-glucanase 3 in protein extracts of Cercospora infected sugar beet leaves.
• Lanes I and c contain protein extracts from infected and control plants, respectively.
• Antibodies raised against chitinase 2 (left), chitinase 4 (centre) and ⁇ - 1,3-glucanase 3 (right) were employed.
• Fig. 14 Site directed mutagenesis of amino acids contemplated to form part of the active site of the chitinase 4 enzyme by the use of the PCR technique described in "Materials and Methods".
• SDO is used as 5' primers for all the suggested PCR-reactions. The sequence is indicated by the arrow and is chosen 5' to the unique BamHI site, The sequences for the SD1, SD2 , SD3 , SD4 and SD5 primers are indicated by arrows. For these 3' primers the complementary sequence with the indicated substitutions are used.
• the primers can be used for the following substitutions with reference to the genomic chitinase DNA sequence (SEQ ID NO.: 3) encoding the amino acid sequence shown in SEQ ID NO.:4. Numbers in brackets denote the number of the corresponding amino acid encoded from the chitinase 4 cDNA (SEQ ID NO.:2)
• PCR products are digested with the relevant restriction enzymes and exchanged with the corresponding sequence in the chitinase 4 gene.
• Fig. 14A and Fig. 14B should be considered as one figure.
• Fig. 15 Construction of a hybrid ⁇ -1,3-glucanase gene construct with a C-terminal extension from tobacco Fig. 15A.
• PCR primers which can be used to change the stop codon and to introduce a part of the C-terminal extension, a DraI site is created at the 3' end.
• the arrows indicate the PCR primers; for the 5' primer the sequence underneath the arrow is used, for the 3' primer the
• FIG. 15B and Fig. 15C should be considered as one figure.
• Fig. 15D Four annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a stop codon, a SmaI site and an BglII site.
• the fused gene product can be made by digesting the glucanase gene with XbaI and EcoRI and ligating it with the PCR product digested with XbaI and Dral and the annealed synthetic oligonucleotides digested with Smal and BglII.
• Fig. 16 Construction of a hybrid chitinase 4 gene construct with a C-terminal extension
• Fig. 16A Chitinase 4 with an underlined tobacco C-terminal extension.
• Fig. 16B and Fig. 16C PCR primers which can be used to introduce a SmaI site near the stop codon in the chitinase 4 gene.
• the arrows indicate the PCR primers; for the 5' primer the sequence underneath the arrow is used, for the 3' primer the complementary sequence with the indicated substitutions is used.
• Fig. 16B and Fig. 16C should be considered as one figure.
• Fig. 16D Four annealed synthetic oligonucleotides containing the sequence for the C - terminal extens ion , a changed stop codon, a Smal site and an EcoRI site.
• the fused gene product can be made by digesting the chitinase 4 gene with BamHI and EcoRI and ligating it with the PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI.
• Fig. 17 Construction of the plant transformation vector pBKL4K4 containing the chitinase 4 DNA sequence shown in SEQ ID NO: 1.
• the boxed sequences indicate the B15 chitinase 4 cDNA, the enhanced 35S promoter and the 35S terminator sequences used for the construct.
• ⁇ B15K4.1 is pBluescript carrying the 966 bp EcoRI fragment encoding the chitinase 4.
• the hatched boxes indicate the coding regions contained in the final product.
• Plasmid pPS48 carries a conventional 35S enhanced promoter and a conventional 35S terminator separated by a polylinker containing unique cloning sites.
• the plant transformation vector pBKL4 (a modification of pBin 19 Bevon, 1984) carries a right and a left T-DNA border sequence from the Agrobacterium Ti plasmid pTiT37, a GUS gene with a 35S promoter and a conventional NOS terminator, a conventional NPTII gene with a 35S promoter and a conventional OCS terminator.
• a polylinker containing several unique cloning sites is situated between the GUS and the NPTII genes.
• Fig. 17A and Fig. 17B should be considered as one figure.
• Fig. 18 Construction of the plant transformation vector pBKL4K4KSE1 containing the DNA sequences encoding chitinase 4 and acidic
• chitinase SE respectively shown in SEQ ID NO: 1 and SEQ ID NO: 8.
• the boxed sequences indicate the acidic chitinase SE cDNA.
• the enhanced 35S promoter and the 35S terminator sequences also used in connection with the construct shown in Fig. 17.
• pSurl is pBluescript carrying the 5' end of the SE gene
• pSE22 is likewise pBluescript carrying almost the entire SE cDNA.
• the hatched boxes indicate the coding regions contained in the final product.
• 5' AGCTGTAC 3' is an adaptor used for the KpnI-HindIII ligation.
• pPS48 is mentioned in connection with Fig. 17.
• the construction of the plant transformation vector harboring the chitinase 4 sequence (pBKL4K4) is described in Fig. 17.
• Fig. 18A and Fig. 18B should be considered as one figure.
• Fig. 19 Construction of the plant transformation vector pBKL4K76 containing the genomic chitinase 76 gene, the sequence of which is shown in SEQ ID NO : 5.
• the boxed sequences indicate the chitinase 76 gene, the enhanced 35S promoter and the 35S terminator sequences.
• pK76.1 is pUC19 carrying the HindIII-EcoRI fragment encoding chitinase 76 in the HindIII/EcoRI site of the pUC19 polylinker.
• the hatched boxes indicate the coding regions contained in the final product.
• KB3 and 340 are synthetic oligonucleotides acting as primers in the polymerase chain reaction (PCR) using pK76.1 as template.
• Plasmid pPS48 was used in connection with Fig. 17.
• the plant transformation vector pBKL4 is described in Fig. 17.
• Fig. 19A and Fig. 19B should be considered as one figure.
• Fig. 20 PCR amplification. of a part of the acidic chitinase SE cDNA using mRNA as a template. mRNA was reverse transcribed using a primer consisting of oligo-dT linked to two restriction sites (270) (see Example 7). Amplification was carried out using a gene specific mixed oligonucleotide linked to a restriction site (XbaI-KB7) as the 5' primer and 270 as the 3' primer. A second round of amplification was then carried out using another gene specific mixed oligonucleotide linked to a restriction site (XbaI-KB7) as the 5' primer and 270 as the 3' primer. A second round of amplification was then carried out using another gene specific mixed oligonucleotide linked to a restriction site (XbaI-KB7) as the 5' primer and 270 as the 3' primer. A second round of amplification was then carried out using another gene specific mixed oligonucleotide linked to a restriction site (
• oligonucleotide linked to a restriction site (BamHI-KB9) as the 5' primer and 270 as the 3' primer.
• the DNA sequence of 270 is shown in Example 7 and SEQ ID NO: 30.
• Fig. 21 describes the separation of sugar beet ⁇ - 1,3-glucanases 1, 2. 3 and 4 by Mono-S cation exchange chromatography at pH 4.5. Elution was performed with a linear gradient of NaCl. The absorbance was measured at 280 run.
• Fig. 22 describes the construction of the plant transformation vector pBKL4K4KSE1G1 containing the DNA sequences encoding chitinase 4, acidic chitinase SE and ⁇ - 1 ,3-glucanase, respectively, and shown in SEQ ID NO:1, SEQ ID NO: 7 and SEQ ID NO: 9.
• the boxed sequences indicate the ⁇ - 1 ,3-glucanase cDNA, the enhanced 35S promoter and the 35S terminator.
• pGluc 1 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 construct shown in Fig. 17, except that the plasmid is supplemented with two additional restriction sites (EcoRI and KpnI) at each site of the E35S-35St box.
• EcoRI and KpnI additional restriction sites
• K76 The genomic chitinase (see Fig. 19).
• K4+SE Chitinase 4 and the acidic chitinase (SE) (see Fig. 18).
• Std. 10 pg of purified sugar beet chitinase 4.
• Fig. 24 A comparison between the DNA sequence of the chitinase 4 cDNA sequence shown in SEQ ID N0.:1 and the genomic clone chitinase 76 shown in SEQ ID NO.:5. The position of the chitinase 76 intron is easily seen at position 875 to 1262. The homology of the sequences is about 73%.
• the figures Fig. 24A, Fig. 24B, Fig. 24C, Fig. 24D, Fig. 24E and Fig, 24F should be considered as one figure.
• Fig. 25 A comparison between the amino acid sequence of the chitinase 4 cDNA sequence shown in SEQ ID NO .:2 and chitinase 76 shown in SEQ ID NO. :6. A homology of about 80% is seen. The extra 3 amino acids in chitinase 76 are the amino acids (Ser, Thr, Pro) in position 62-64.
• Fig. 25A and Fig. 25B should be considered as one figure.
• Fig. 26 A comparison between the non-coding 5' sequences of the chitinase 4 and chitinase 76 genomic sequences shown in SEQ ID NO . : 3 and SEQ ID NO.:5, respectively. 8 boxes of strong homology is observed in the non-coding 5' sequence. It is contemplated that some of these boxes may be of regulatory importance.
• Fig. 26A and Fig. 26B should be considered as one figure.
• SEQ ID NO.:1 the chitinase cDNA sequence (harbored in the cDNA sugar beet chitinase 4 clone B15)
• SEQ ID NO.:2 the chitinase 4 amino acid sequence (harbored in the cDNA sugar beet chitinase 4 clone B15)
• SEQ ID NO.: 3 the partial DNA sequence of the genomic chitinase 4 clone
• SEQ ID NO.:4 the partial amino acid sequence of the genomic
• chitinase 4 clone SEQ ID NO.: 5 the DNA sequence of the genomic clone chitinase 76
• SEQ ID NO .:6 the deduced amino acid sequence of the genomic clone chitinase 76
• SEQ ID NO.:7 the cDNA sequence of the acidic sugar beet chitinase
• SEQ ID NO.:9 the cDNA sequence of the basic sugar beet ⁇ -1,3- glucanase
• SEQ ID NO.:10 the deduced amino acid sequence of the basic sugar beet ⁇ - 1 ,3-glucana ⁇ e
• SEQ ID NO.:11 The DNA sequence of the entire sugar beet chitinase 1 gene including introns, promoter and leader sequence, and the amino acid sequence deduced from the coding region of the chitinase 1 gene.
• SEQ ID NO.:12 The amino acid sequence deduced from the coding region of the chitinase 1 gene.
• SEQ ID NO.:13 C-terminal amino acid sequence of a bean chitinase (PHA).
• SEQ ID NO. :14 C-terminal amino acid sequence of a basic tobacco chitinase.
• SEQ ID NO. :15 C-terminal amino acid sequence of an acidic tobacco chitinase.
• SEQ ID NO.:16 C-terminal amino acid sequence of the barley
• SEQ ID NO.:17 C-terminal amino acid sequence of a basic ⁇ -1,3 glucanase from tobacco.
• SEQ ID NO. :18 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.: 19 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.:19 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.: 20 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.: 21 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.: 22 Amino acid sequence of a tryptic peptide of a
• SEQ ID NO.:23 N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from WGA-A (Triticum aestivum).
• SEQ ID NO.:24 N-terminal amino acid sequence of an amino acid sequence of a chitin binding protein from hevein (Hevea brasi liensis ) .
• SEQ ID NO.:25 N-terminal amino acid sequence of the amino acid
• SEQ ID NO.:26 N-terminal amino acid sequence of the amino acid
• SEQ ID NO.: 28 DNA primer named KB-7 constructed partly from the
• SEQ ID NO.:9 polypeptide sequence of the acidic chitinase from sugar beet
• SEQ ID NO.:29 Complementary DNA primer named KB-9 constructed partly from the polypeptide sequence of the acidic chitinase from sugar beet (SEQ ID NO.:9).
• SEQ ID NO.:30 Complementary DNA primer named Oligo-dT constructed from the general knowledge of polyA mRNA's
• SEQ ID NO.: 31 Amino acid sequence of a lysozyme/chitinase from
• cucumber (Cucumis sativus ) .
• SEQ ID NO.:32 Amino acid sequence of a lysozyme/chitinase from
• SEQ ID NO.:33 Amino acid subsequence 3-15 of the amino acid sequence of a ⁇ -1,3-glucanase from sugar beet.
• SEQ ID NO.:34 The amino acid subsequence 3-17 of the amino acid
• SEQ ID NO. :35 The amino acid subsequence 3-16 of the amino acid sequence of a ⁇ -1,3-glucanase from sugar beet
• SEQ ID NO.:36 DNA 5'primer named Oligo TG-1 constructed from the amino acid sequence of a ⁇ -1,3-glucanase from sugar beet.
• SEQ ID NO.:37 DNA 5'primer named Oligo TG-2 constructed from the amino acid sequence of a ⁇ -1,3-glucanase from sugar beet.
• SEQ ID NO.:38 DNA 3'primer named Oligo TG-3 constructed from the amino acid sequences of a glucanase from tobacco and barley.
• SEQ ID NO.:39 Amino acid subsequence from the amino acid sequence of a glucanase from barley used to construct the primer Oligo TG-3.
• SEQ ID NO.:40 Amino acid subsequence from the amino acid sequence of a glucanase from tobacco used to construct the primer Oligo TG-3.
• SEQ ID N0.:41 N-terminal amino acid sequence of the amino acid
• SEQ ID NO.: 43 N-terminal amino acid sequence of the amino acid
• SEQ ID NO.: 44 N-terminal amino acid sequence of the amino acid
• SEQ ID NO. :46 Amino acid subsequence of the active site of the amino acid sequence from a chitinase from tobacco.
• SEQ ID NO.:47 Amino acid subsequence of the active site of the
• polypeptide from a chitinase from tobacco SEQ ID NO.:48: Amino acid sequence of the active site of the
• polypeptide from a chitinase from tobacco is a polypeptide from a chitinase from tobacco.
• SEQ ID NO.:49 DNA 5'primer named KB-3 constructed partly from
• SEQ ID NO.:50 Complementary DNA 3'primer named KB-4 constructed from nucleotides No's. 261-241 of SEQ ID NO.:1.
• SEQ ID NO.: 51 Complementary DNA primer named 340 constructed partly from the nucleotide numbers 341-323 of SEQ ID NO.:1.
• the sequence is 966 bp long and encodes a protein having 264 amino acid residues in the polypeptide chain
• the leader sequence consists of 23 amino acid residues followed by a hevein domain of 35 amino acid residues and a functional domain of 206 ammo acid residues.
• the cDNA 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.
• the chitinase 4 gene the partial nucleotide sequence of which is shown in SEQ ID NO 3, encodes a protein having 265 amino acid residues shown in SEQ ID NO 4
• the leader sequence encoded by the gene consists of 24 amino acid residues
• the SEQ ID NO: 1 are missing the nucleotide A and T and the first amino acid Met is not present in the polypeptide sequence encoded by the chitinase 4 cDNA
• SEQ ID NO: 3 and SEQ ID NO:4 The partial DNA sequence (SEQ ID NO 3) and deduced ammo acid sequence (SEQ ID NO 4) of a genomic clone encoding the chitinase 4 gene isolated from a sugar beet EMBL3 genomic library
• the sequence is 691 bp long and encodes the first 112 of the 265 amino acids of the chitinase 4 polypeptide chain
• the leader sequence consists of 24 amino acid residues followed by a hevein domain of 35 amino acids
• the partially sequenced clone has a 5' non-coding region of 355 bp with a TATA-box sequence (TATAAA) located at position 285. which is 70 bp upstream of the ATG start codon SEQ ID NO : 5 and SEQ ID NO : 6
• the DNA sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO: 6) of a genomic clone encoding the chitinase 76 gene isolated from a sugar beet EMBL3 genomic library The sequence is 1838 bp long and encodes a protein having 268 amino acid residues in the polypeptide chain.
• the leader sequence consists of 24 amino acid residues followed by a hevein domain of 35 and a functional domain of 209 amino acid residues.
• the gene contains one intron which is located in position 875 to 1262. The exact location of this intron is based on an alignment with the B15 chit 4 cDNA
• TATAAA TATA-box sequence
• AATAAA putative poly-A signal
• the sequence is 1106 bp long and encodes a protein having 293 amino acid residues in the polypeptide chain.
• the leader sequence consists of 25 amino acid residues and the functional domain of 268 amino acid residues.
• the cDNA clone has a 5' non-coding region of 17 bp and a 3' flanking region of 202 bp .
• SEQ ID NO: 9 and SEQ ID NO: 10 The DNA sequence (SEQ ID NO.:9) and the deduced amino acid sequence (SEQ ID NO.:10) of a ⁇ - 1 ,3-glucanase 4 cDNA clone isolated from a sugar beet ⁇ ZAP cDNA library
• the sequence is 1249 bp long and encodes a protein having 336 amino acid residues in the polypeptide chain.
• the cDNA clone has a 5' non-coding region of 33 bp and a 182 bp 3' flanking region containing a putative polyadenylation signal at position 1157 and a poly A tail.
• the sequence is about 6.3 kb lang and encodes a protein having 439 amino acid residues in the polypeptide chain.
• the leader sequence is deduced to consist of 26 amino acid residues followed by a hevein domain of 20, a proline rich domain of 132 and a functional domain of 238 amino acid residues.
• the protein has a C-terminal extension of 23 amino acid residues which probably direct the protein to the vacuole.
• the sequence contains two introns at position 2170-4618 and 4776-5406.
• the intron borders contain the consensus GT/AG sequences.
• a TATA box sequence (TATAAA) is located at position 1355-1360 which is about 70 bp upstream of the ATG start codon.
• a putative poly A signal (AATAAA) is located at position 6032.
• T L -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.
• Shinshi H. et al. Agric. Biol. Chem. 47, 1455-1460, 1983 Shinshi H. et al., Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro- ⁇ -1,3-glucanase, 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, pp.
• Beta vulgaris, cv. "Monova” Seeds of Beta vulgaris, cv. "Monova”, were sown in clay mixed peat ("Cycas") and placed in growth chamber with 11/13 hours day/night cycles, 25/18°C (day/night) and 70% rh. Light intensity was approximately 25000 lux ("Osram HQI-T", 400 W/DH).
• Osram HQI-T 400 W/DH.
• Nicotiana tabacum and N . benthamiana plants were obtained as described above. Fungi
• the fungus was grown on solid growth medium in Petri dishes, Sterile "Potato Dextrose Agar” ("Difco", 39 g/l) was used as growth medium. A plug of mycelia was placed in the center of the Petri dish and the culture was incubated at room temperature for 4 weeks. Mycelia for spore induction was "harvested” by cutting off the whole mycelia "mat” including some agar. Sporulation of Cercospora speci es
• Mycelia was mixed with distilled water (1:2) in a 50 ml sterile glass tube and homogenized using a "Ultra Turrax T25" mixer operated at 8000 rpm for 2 minutes. 1 ml of the homogenate was transferred to a Petri dish containing solid sporulation medium. "V-8" was used as medium. It contained 200 ml “V-8” juice (Cambells, Italy), 800 ml water, 3 g CaCO 3 and 20 g agar.
• the suspension was allowed to settle for 1 hour. After airdrying the culture (approximately 1 hour) the Petri dish was closed, sealed and placed in an incubation chamber at 13oC and 24 hours light (cool white).
• the spores were harvested by pouring 10 ml distilled water onto the Petri dish and firmly brushing the surface of the culture with a sterile brush.
• the resulting spore suspension contained approximately 100,000 spores/ml.
• An inoculum of R . solani was prepared on barley grains soaked twice in 1% of potato dextrose broth and autoclaved. The grains were inoculated with agar disks of a growing culture of the fungus and incubated for two weeks, after which they were airdried.
• disks of R . solani growing on potato dextrose agar can be used directly as inoculum.
• the inoculum was mixed into potting soil in different concentrations, and the transgenic plantlets which had been rooted for 14 days, were transplanted into the infected soil.
• the percentage of surviving plants may be recorded after 1, 2 and 3 weeks, respectively, and after 3 weeks the surviving" plants are assessed for root damage.
• the small scale purification was carried out as follows. 1 g of leaf material was homogenized by a Ultra-Turrax homo-genizer in citrate buffer (0.1 M, pH 5, 2 ml/g tissue), containing 1 mM of both benzamidine, dithiothreitol and phenylmethylsuphonyl fluoride. Particulate matters were removed by centrifugation at 15,000 ⁇ g for 15 minutes. The supernatant comprising the enzymes was transferred to another test tube before the centrifugation was repeated.
• citrate buffer 0.1 M, pH 5, 2 ml/g tissue
• the gel pieces were transferred to a Warring blender, covered with methanol, and homogenized for 2 minutes at full power. Methanol, acetic acid and unreacted acetic anhydride were removed by filtration in a Buchner funnel using Whatman No. 1 filter paper. The filtrate was transferred to a beaker, 1 1 of 1 M Na 2 CO 3 was added and the pH was adjusted to 9 with 6 N NaOH . 50 ml of acetic anhydride was slowly added and the pH adjusted to 9. The reaction was allowed to take place for 1 hour before the final product was collected by filtering on a Buchner funnel. After extensive washing with water, the product was equilibrated in a 10 mM Tris buffer at pH 8.0 before storing at 4°C. The yield was 700 ml of regenerated chitin. A chitin column was prepared from the regenerated chitin by use of the conventional procedure according to Pharmacia.
• Divinylsulfone activated agarose (Mini-leak high, KEM-EN-TEC, Denmark) was employed to immobilize laminarin ( ⁇ - 1 ,3-glucan) (from Laminaria digi tata , Sigma). 50 g Mini-leak High was dispensed in 200 ml 1 M potassium phosphate (K-P) at pH 11, and 750 mg laminarin dissolved in 5 ml H 2 O was added. The reaction was allowed to proceed for 16 hours at 25oC on a shaking table. Unreacted divinylsulfone groups were blocked by incubation with a solution of 5% mercaptoethanol in 1 M K-P-buffer at pH 9.5. The reaction time was 16 hours at 25°C.
• K-P potassium phosphate
• Laminarin was labelled with radioactivity by reduction with 3 H-labelled NaB 3 H 4 .
• 500 mg laminarin (from Laminaria digi tata , Sigma) was dissolved in 2 ml H 2 O, and purified by precipitation by addition of 800 ⁇ l NaCl (0.2 g/ml) followed by 8 ml absolute ethanol. The precipitate was collected by centrifugation for 5 min. at 15.000 g. The supernatant was discarded and the pellet containing the laminarin was dissolved in 4 ml of 0.1 N NaOH. This solution was transferred to a reaction wessel containing 5 mCi of NaB 3 H 4 . After stirring for 90 min. at 25°C, 600 ⁇ l of 1 M HCl was added to destroy unreacted
• the reaction mixture was divided into 500 ⁇ l aliquots and 200 ⁇ l of NaCl and 2 ml of absolute ethanol was added to each test tube. After storage for 10 min. at 0oC, the precipitate was collected by centrifugation for 5 min. at 15.000 ⁇ g.
• the H-labelled laminarin was dissolved in 500 ⁇ l of H 2 O and the precipitation was repeated until the background level in the supernatant was less than 100 cpm per 20 ⁇ l.
• the labelled solution of laminarin was stored at -20°C. Before use in the ⁇ -1,3-glucanase -assay, the solution was diluted 20-fold with water.
• Chitinase activity was determined radiochemically with ⁇ H-chitin as a substrate.
• the specific activity of the 3 H-chitin was 460 cpm/nmol N-acetylglucosamine (GlcNAc) equivalent (or 2,3 ⁇ 10 6 cpm/mg 3 H-chitin). It was determined by scintillation counting and colorimetric determination of GlcNAc after total hydrolysis of 3 H-chitin by crude chitinase preparations from sugar beet leaves and exochitinase from serratia marcescens or Streptomyces griseus .
• the assay mixture contained in a total volume of 200 ⁇ l of enzyme solution, 50 ⁇ l of 3 H-chitin suspension (containing 100,000 cpm) and 10 ⁇ mol of sodium citrate (pH 5,0). After mixing, the enzymatic hydrolysis of 3 H-chitin was allowed to take place at 40°C for 15 min. before addition of 300 ⁇ l of 10 % (w/v) TCA. In order to decrease the background reading, 100 ⁇ l of bovine serum albumin (10 mg/ml) were added before the insoluble 3 H-chitin was removed by centrifugation at 15.000 ⁇ g for 5 min. The radioactivity in 300 ⁇ l supernatant was determined by scintillation counting. The radiochemical ⁇ - 1 , 3 -glucanase assay ⁇ - 1,3-glucanase activity was determined radiochemically with 3 H-labelled laminarin as substrate.
• the assay mixture consisted of 50 ⁇ l of enzyme extract, 50 ⁇ l of 0,1 M Na-citrate pH 5,0 and 10 ⁇ l of 3 H- labelled laminarin (192.000 cpm). Incubation was carried out for 15 min. at 40°C. To terminate the reaction, 1000 ⁇ l of abs. Ethanol and 50 ⁇ l of a saturated NaCl-solution was added. After 10 min. at 0°C, unreacted laminarin was removed by centrifugation at 10.000 ⁇ g for 5 min. An aliquot of 400 ⁇ l of supernatant was transferred to a scintillation vial. 5 ml of PICO-FLUOR-40 were added and the amount of radioactivity was determined by a liquid scintillation counting. Lysozyme assay
• the lysozyme activity of chitinase 4 was determined by the method described by Selstes et al . (1980). More specifically, lysozyme activity was measured in microtiter plates. Each well contains cell walls from Micrococcus lysodeikticus suspended in a 20 mM sodium phosphate buffer, pH 7.4, containing 1 mg/ml of BSA. The initial absorbency at 450 nm was adjusted to 0.6 before addition of egg-white lysozyme or plant chitinase 4. The reaction was followed by measuring the decrease in absorbance at 5 min. intervals for 50 min. ⁇ -glucuronidase (GUS) -Assay
• the success of the transformation may be determined by use of the following GUS-assay described by Jefferson, 1987.
• Leaf tips were sliced into thin sections ( ⁇ 0.5 mm) and incubated in a 2 mM solution of x-gluc. (5-bromo-4-chloro-3-indolyl- ⁇ -glucuronide) dissolved in 0.1 M sodium phosphate buffer pH 7.0 containing 0.5 mM potassium ferri cyanide and 10 mM EDTA.
• the leaf sections were treated for 2-4 hours at 37°C, rinsed with water and the staining intensity recorded by visual inspection by microscopy.
• Acidic and basic chitinase isoenzymes were purified together with ⁇ - 1,3-glucanases from sugar beet leaves as shown in the following flow diagram.
• the supernatant fraction obtained after the centrifugation was heated at 50°C for 20 minutes and after cooling to 4°C, the precipitate was collected by centrifugation. Solid ammoniumsulfate was added to the supernatant until a 90% saturation was achieved. After centrifugation, the precipitated proteins were dissolved in starting buffer; 1 ml of buffer/10 g of starting material.
• Chitinase and ⁇ - 1 , 3-glucanase isoenzymes were purified from the ammonium sulfate precipitated protein fraction. After solubilization, the protein solution was dialyzed against 10 mM Tris pH 8.0 containing 1 mM DTT and 1 mM BAM. Denatured proteins were removed by centrifugation and the supernatant was loaded on the above outlined two columns e.g. i) a 50 ml Fast Flow Sepharose Q (Pharmacia) and ii) a 100 ml Chitin column (prepared as described above), the columns being connected in series. The columns were equilibrated with the Tris buffer, before 281 ml of the sample were loaded.
• Unbound proteins ineluding ⁇ - 1,3-glucanase were removed by extensive washing with the starting buffer. After disconnecting the Fast Flow Sepharose Q co- lumn, the chitinase was eluted from the chitin column with 20 mM acetic acid, pH 3.2 containing 1 mM DTT. The acidic chitinase SE was eluted from the Fast Flow Sepharose Q column with the Tris-buffer containing 0,5 M NaCl. Purification of ⁇ - 1 ,3-glucanase
• Proteins which were not adsorbed on either the Fast Flow Sepharose Q nor the chitin column were collected, and concentrated to 60 ml by pressure dialysis with an Amicon PM-10 filter (Danver, MA, U.S.A.). After dialysis overnight against 20 mM Na-acetate buffer at pll 4.2 containing 1 mM DTT and 1 mM BAM, the protein solution was loaded on to a 50 ml Fast Flow Sepharose S column (Pharmacia) equilibrated in the dialysis buffer, Unadsorbed proteins were removed by washing with the equilibration buffer. Bound proteins were eluted with a 600 ml linear gradient from 0 to 0.5 M NaCl in the starting buffer.
• Peak B was further fractionated by affinity column chromatography on Laminarin-Agarose. Peaks A and C were not further purified.
• the protein fractions from peak B was combined, concentrated by pressure dialysis to 15 ml and dialyzed against the Tris buffer. After loading of the sample on the Laminarin-Agarose column, the flow through the column was stopped for 20 minutes to allow the ⁇ - 1 ,3-glucanase to bind to the affinity ligand. Unabsorbed protein was removed by washing with Tris buffer, ⁇ - 1,3-glucanase was eluted with 1 M NaCl in Tris buffer.
• 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 70%.
• peak 3 and 4 were ready for i) N-terminal amino acid sequencing, ii) amino acid composition analysis (see Example 8), iii) tryptic digestion to achieve peptides and iv) injecting into rabbits to produce polyclonal antibodies.
• the acidic chitinase SE was eluted from the above described Fast Flow Sepharose Q column with the Tris buffer containing 0.5 M NaCl as shown in the purification scheme.
• the proteins were dialyzed against 10 mM Tris-HCl, pH 8.0, and loaded onto a 40 ml "Fast Flow Sepharose Q" column equilibrated with the same buffer.
• the proteins were eluted with a 800 ml linear sodium chloride gradient from 0 to 0,5 mM NaCl. Fractions containing chitinase activity as determined by the radiochemical chitinase assay described above were pooled.
• the protein fractions were dialyzed against 25 mM Bis-Tris, adjusted to pH 7.0 with iminodiacetic acid.
• a 15 ml "polybuffer Exchanger” column (Pharmacia; PBE 74) was equilibrated with the same buffer and 50 ml of the sample was loaded. Unabsorbed proteins were removed by washing with the Bis-Tris buffer.
• Protein fraction with high chitinase activity as determined by the radiochemical chitinase assay described above were pooled and dialyzed against 25 mM Bis-Tris at pH 7.0, The proteins were resolved on a Mono-P FPLC column (Pharmacia) equilibrated with the Bis-Tris buffer. After an initial wash with the starting buffer, three isoenzymes of acidic chitinase SE was separated using a linear salt gradient from 0 to 0.3 M NaCl (Fig. 3). Analysis of the enzymatic cleavage pattern of sugar beet chitinase 4
• a standard of N-acetylglucosamine-derived oligosaccharides was produced by acid hydrolysis of chitin (Rupley, 1964). This standard was used to localize the products from the enzymatic cleavage on the TLC plate. Zones of interest on the TLC plate were removed by scraping with a razor blade, and the silica gel containing the 3 H-labelled oligosaccharides was transferred to a scintillation vial. 10 ml of scintillation liquid Dimilume (Packard Instruments) were added and the radioactivity was determined by a liquid scintillation spectrophotometer.
• Method I is carried out on microscope slides covered with a thin film of medium and incubated with either buffer (control) or ⁇ g quantities of the antifungal proteins. Germination of spores or growth of the mycelium is followed by staining with Calcofluor White before analysis by a fluorescent microscope. Method II is carried out in microtiter plates containing growth media, 10 or 100 spores from Cercospora, buffer (control) or the antifungal proteins. The plates are incubated at 25°C before the optical density (at 620 nm) is determined at specified time intervals.
• microscopy slides were covered with a thin layer of potato dextrose agar (PDA) and stored for 6 hours on moistened filter paper in petri dishes.
• 10 ⁇ l of a spore suspension (10.000 spores/ml) was placed in the center of the slide.
• 10 ⁇ l of a 10 mM Tris-buffer, pH 8.0 or 10 ⁇ l of a preparation containing 20 ⁇ g of the antifungal protein to be tested was applied to the spore suspension.
• the antifungal protein was dissolved in the Tris-buffer and filtered through a 0,22 ⁇ m 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.
• 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.
• the tape was removed and twice a day, the absorbance was measured at 620 nm. The germination and growth of the fungus was followed for 4 days by measuring the absorbance. For each combination of antifungal protein and spore dilution the absorbance vs. time was plotted.
• Method III Autoradiography Cercospora cultures were grown on a microscope slide as described in method I.
• Liquid growth medium (PDB) containing 3 H-labelled N-acetylglucosamine was distributed uniformly over a one day old culture. After incubation for 20 min. (pulse labelling), the reaction (growth/incorporation) was stopped by dipping the microscope slide in 6% (w/v) of TCA. The preparation was dehydrated in an ethanol gradient (70-100%) and dried.
• the microscope slide was coated with an autoradiographic emulsion (Ilford K 5). After drying the emulsion extensively with a "fan dryer” the slide was placed in the dark for 7 days at 7°C and low relative humidity for exposure. The emulsion was developed by placing the slide in Kodak D-19 developer for 10 minutes followed by fixation for 2 minutes and washed in running water for 10 minutes. After drying the preparation was ready for a microscope analysis of the hyphae of the fungus. Production of antibodies for use in serological analysis
• Freezedried purified chitinase 2, 3 and 4 (obtained as described above) were dissolved in Tris buffer (10 mM, pH 8,0) and diluted 1:1 with Freunds incomplete adjuvant. Polyclonal antibodies were raised in rabbits according to conventional methods by Dakopatts (Denmark).
• diphtheria toxoid Before immunization the peptides were conjugated to diphtheria toxoid.
• the carrier, diphtheria toxoid was converted to the toxoid-sulfosuccinyl-ester derivative by reaction with carbodiimide (EDC) followed by N-hydroxy sulfosuccinimide.
• EDC carbodiimide
• N-hydroxy sulfosuccinimide N-hydroxy sulfosuccinimide.
• the four different peptide-diphtheria toxoid conjugates were purified by gel filtration on a Sephacryl S-300 column. The high molecular weight fractions were collected, freeze-dried and dissolved in water. Immunization in rabbits were performed as described above for the production of polyclonal antibodies to chitinase 2 and 4.
• proteins were transferred by semi-dry blotting onto a 0.45 ⁇ m nitrocellulose membrane (Schleicher and Schuell, FGR) after separation by SDS-PAGE.
• the antigens were probed with primary polyclonal rabbit antibodies raised against chitinase 2 and 4 (see above) and subsequently visualized using alcaline phosphatase con jugated secondary antibodies (Dakopatts. Denmark; according to Kyhse-Andersen (1984).
• the ECL enhanced chemiluminescence
• 1 ⁇ g protein was applied to each lane of the SDS-PAGE gel.
• the nitrocellulose membrane was treated according to Amershams protocol. In brief, the nitrocellulose membrane was initially treated with 10% BSA, before the primary antibodies to sugar beet chitinase 4 diluted 1:1000 was added. The antigen was detected with horse-radish peroxidase conjugated secondary antibodies. Detection reagent was added and after 2 minutes the protein bands are visualized on a Hyperfilm-ECL.
• the protein solution was dialyzed against 0.2 M ammonium carbonate (pH 8.0) for 24 hours at 4°C in the dark using Eppendorf test tubes with dialysis tubing (10 kDa cut off; Servapore, Serva, FRG) inserted underneath a punctured lid. Thereafter, precipitated protein was pelleted by centrifugation for 5 minutes at 10,000 ⁇ g and the supernatant was transferred to another test tube. The protein pellet was partially solubilized by addition of a few particles of guanidine hydrochloride and incubated with 4 ⁇ g TPCK- treated trypsin in 20 ⁇ l of ammonium carbonate (pH 8.0) at 40°C for 30 minutes.
• TPCK- treated trypsin were added.
• the digestion was allowed to take place at 40°C for 4 hours and stopped by addition of 20 ⁇ l 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 C 18 column (0.4 ⁇ 10 cm; 5 ⁇ m particle size; Dr. O Schou, Novo-Nordisk, Denmark) using the mobile system described above.
• Selected peptides were subjected to amino acid sequence analysis. Amino acid sequencing
• RNA with a poly-A tail was purified by affinity chromatography through an oligo-dT cellulose column.
• 0.5 g of oligo-dT cellulose was mixed in 5 ml of 0.5 M NaOH for 5 minutes (1 g of oligo-dT cellulose binds 1.2 mg of poly-A RNA).
• the resulting mixture was neutralized with 10 mM Tris pH 7.5 until pH reached 7.5.
• An 1 cm column with a diameter of 1 cm was made and equilibrated with 20 ml of column buffer (500 mM NaCl, 10 mM Tris pH 7.5 , 1 mM EDTA).
• RNA was denatured at 65°C for 5 minutes, and 5 volumes of column buffer were added to the RNA before chromatography through the column. The "runthrough" was collected and subjected to chromatography again. The column was washed with column buffer until OD 260 reached 0.01 or less.
• the poly-A RNA was eluted with TE buffer in 1 ml fractions, and the RNA concentration for each of the fractions was determined at OD 260 .
• the poly-A RNA-containing fractions were pooled and adjusted to 100 mM NaCl and the RNA was precipitated overnight with 2.5 volumes of 96% ethanol at -20°C.
• the poly-A RNA was spun and dissolved in H2O at a concentration of 1 ⁇ g/ ⁇ l and stored at -20°C. The yield was about 1-2% of total RNA applied to the column. Isolation of genomic DNA from sugar beet leaves
• Genomic DNA was isolated from sugar beet leaves (of the variety 60.159.838-131-4) (Dellaporta et al . , 1983).
• the supernatant was filtered through 1-2 layers of miracloth into a new centrifuge tube, 15 ml iso-propanol was added and the mixture was incubated for 30 min. at -20°C. After another centrifugation at 20,000 ⁇ g for 15 min. at 4°C, the pellet was washed with 70% ethanol and thereafter dried briefly before being resuspended in 0.7 ml of X-TE buffer (50 mM Tris, pH 8.0 and 10 mM EDTA). The suspension was transferred to an eppendorf tube and centrifugated for 5 rain. The supernatant was extracted twice with phenol/chloroform.
• genomic sugar beet library was constructed by cloning the genomic DNA in the BamHI site of the vector EMBL3.
• the library was constructed by Clontech. Plating libraries for screening for relevant DNA sequences
• the titer of the library was determined according to Sambrook et al . (1989), and about 10 6 phages were used for each screening.
• top agar (0.7% agarose in LB medium with 10 mM MgSO 4 and 0.2% maltose) (48°C) were added and the resulting mixture plated onto 24.5 ⁇ 24.5 mm plates containing 200 ml of LB agar and allowed to grow overnight at 37°C.
• Phages were allowed to adsorb to the filter for 1 to 5 minutes.
• the filters were then placed with the "plaque side" upwards on Whatman 3MM filter paper sheets soaked with 0.5 M NaOH, 1.5 M NaCl for 30 seconds. They were then washed for 30 seconds in each of the following solutions: 1) 0.5 M NaOH, 1.5 M NaCl, 2) 0.5 M Tris, pH 7.5,
• oligonucleotides were labelled by phosphorylation with bacteriophage T4 polynucleotide kinase according to the method described in Sambrook et al. (1989). More specifically, the oligonucleotides were synthesized without a phosphate group at their 5' termini ends and were labelled with ⁇ - 32 P from [ ⁇ - 32 P]ATP using the enzyme bacteriophage T4 polynucleotide kinase.
• the radiolabelled oligonucleotide was recovered by centrifugation at 12,000 ⁇ g for 20 minutes at 4°C in a microfuge. Using an automatic pipette device equipped with a disposable tip, all of the supernatant fluid (which contained most of the unincorporated [ ⁇ - 32 P]ATP) and any free 32 P generated during the phosphorylation reaction were carefully removed. The resulting residue was redissolved in 100 ⁇ l of H 2 O and 10 ⁇ l of 3M sodium acetate and thereafter 250 ⁇ l of 96% ethanol were added. The mixture was subjected to centrifugation for 20 minutes at 4°C, dried and redissolved in 200 ⁇ l of H 2 O.
• the oligonucleotide hybridization procedure used eliminates the preferential melting of A-T versus G-C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only.
• the hybridization was carried out essentially as described by Wood et al . (1985).
• the nitrocellulose filters obtained as described above were wetted on the surface with 2xSSC and subsequently prehybridized in hybridization buffer (6 ⁇ SSC, 1% BSA . 1% Ficoll 4000, 1% PVP, 50 ⁇ g/ml of heat denaturated salmon sperm DNA, 50 mM sodium phosphate, pH 6.8).
• the hybridization was performed at 37°C for 4 hours in a plastic bag with shaking.
• the filter was hybridized overnight in the same solution plus the radioactive oligonucleotide probe (the 23-mer chit 4 probe) at 37°C with shaking. A 1x10° cpm/ml solution of the hybridization buffer was used. The filter was rinsed three times in 6xSSC at 4°C and thereafter washed twice for 30 min. at 4°C in 6 ⁇ SSC. Further, the filter was washed three times in TMAC-buffer (3 M Tetramethylammonium chloride, 50 mM Tris pH 8, 0, 2 mM EDTA, 0,1% SDS) for 5 min. at 37°C. (The tetramethylammonium chloride is made in a 5 M stock solution.
• the filters were dried in the air at room temperature. Inkmarks on the filters serving to align the autoradiographs with the filters and the agar plates were marked with an autoradiography marker (Ultermit, Du Pont de Nemours).
• the filters were covered with Saran Wrap and an X-ray film (AGFA CURIX RP2) were exposed to the filters for 16-70 hours at -70°C with an intensifying screen.
• the films were developed and positive plagues were identified by aligning the dots on the film with those on the agar plates.
• SM phagebuffer Standardbrook et al . , 1989
• chloroform contained in an eppendorf tube.
• the eppendorf tubes were allowed to stand for
• a single well-isolated positive plague useful for making a phage stock to be used in the in vivo excision was picked from the agar plates according to the method described by Sambrook et al . (1989) using several steps of replating and rescreening.
• a phage stock was prepared according to the method of Sambrook et al .
• Bacterial strains (DH5 ⁇ and XL1-Blue) harboring the plasmids were grown overnight in 5 ml of LB medium supplied with the relevant antibiotics and 5 ml of the overnight culture was harvested by centrifugation for 10 minutes at 3000 ⁇ g. The pellet was resuspended in 200 ⁇ l of Solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM
• EDTA in 1.5 ml tubes. 400 ⁇ l of Solution II (0,2 N NaOH, 1% SDS) was added, the mixture subjected to gentle mixing and placed on ice for 5 minutes. 300 ⁇ l of 5 M KOAc pH 4.8 was added and subjected to thorough mixing. The resulting mixture was placed on ice for 10 minutes and subsequently subjected to centrifugation at 15,000 ⁇ g for 10 minutes at 4°C. The supernatant (900 ⁇ l) was transferred 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 subjected to centrifugation at 15,000 ⁇ g and 4°C for 10 minutes and the supernatant was removed.
• Solution II 0.,2 N NaOH, 1% SDS
• the pellet was dissolved in 100 ⁇ l of TE and 100 ⁇ l of 5 M LiCl was added. The mixture was allowed to stand on ice for 5 minutes and subjected to centrifugation at 15,000 ⁇ g and 4°C for 10 minutes.
• the supernatant was transferred to new tubes and 500 ⁇ l of 96% ethanol was added.
• the tubes were centrifugated at 15,000 ⁇ g and 4°C for 30 minutes and the supernatant was removed.
• the pellet was washed with 70% ethanol (about 100 ⁇ 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 purified by the above described method. Sequencing was performed as follows :
• a mixture comprising about 2 ⁇ g of the relevant plasmid. 1 ⁇ l of 2 M NaOH, 2 mM EDTA, 1 ⁇ l of primer (100 ⁇ g/ml) and H 2 O up to 10 ⁇ l was incubated at 85 °C for 5 minutes and subsequently put on ice.
• the mixture was neutralized by adding 1 ⁇ l of 5 M NH 4 Ac and then precipitated by adding 20 ml of 96% ethanol. The resulting mixture was spun for 30 minutes at 4°C and resuspended in 6 ⁇ l of H 2 O. 1.5 ⁇ l of 5 ⁇ cone, sequenase buffer was added. The mixture was placed at 37°C for 5 minutes.
• DNA probes to be used in the isolation of the sugar beet acidic chitinase SE was labelled by use of the Stratagene oligolabelling kit prime IT, (Random Primer Kit) according to the manufacturers instructions. More specifically, the following procedure was used:
• a sample comprising 25 ng (1-23 ⁇ l) of the DNA template to be labeled, 0-22 ⁇ l of H 2 O and 10 ⁇ l of random oligonucleotide primers (constituting a total volume of 33 ⁇ l) were added to the bottom of a clean microcentrifuge tube.
• the reaction tubes were heated to 95-100°C in a boiling water bath for 5 minutes and then centrifuged briefly at room temperature to collect the liquid, which may have condensed on the cap of the tubes.
• the reaction tube containing the DNA sample in LMT agarose was placed at 37°C and the following reagents were added to the reaction tubes: 10 ⁇ l of 5X primer buffer containing dATP, dGTP and dTTP.
• T7 DNA Polymerase 2 ⁇ l of diluted T7 DNA Polymerase.
• the T7 DNA Polymerase was diluted in ice cold Enzyme Dilution Buffer immediately before use to a final concentration of 1 U/ ⁇ l.
• the reaction components were mixed with the tip of a pipette.
• the tubes were incubated at 37-40°C for between 2 and 10 minutes and subsequently, the reaction was stopped by the addition of 2 ⁇ l of Stop Mix.
• the probes with the 32 P-labeled DNA were further purified using the ElutipTM-D column system (Schleicher & Schuell). Then, the probe DNA was made ready for hybridization by mixing the proper amount of radioactive probe with 200 ⁇ l of 10 mg/ml salmon sperm DNA. The mixture was heated to 95-100°C in a boiling water bath for 5 minutes and cooled on ice. The resulting probe was stored at 20 °C for up to one week and heated to 95-100oC in a boiling water bath for 5 minutes and cooled on ice before use.
• Filter prints obtained as described above under "Oligonucleotide hybridization" of the sugar beet ⁇ -ZAP cDNA library were subjected to prehybridization for 2 hours at 67°C under conventional prehybridization conditions using a prehybridizing solution comprising 2 ⁇ SSC, 10 x Denhardt's, 0.1% SDS and 50 ⁇ g/ml salmon sperm DNA.
• Hybridization was carried out overnight using a hybridization solution identical to the prehybridization solution except for the fact that a radioactive DNA probe prepared as described above had been added.
• the positive plaques were identified as described under "Oligonucleotide hybridization of chitinase 4 DNA in filter hybridization".
• hybridization of the DNA in question with a chitinase 4 probe was carried out using the hybridization procedure disclosed in "hybridization of SE- DNA" except for the fact that the hybridization is carried out at a temperature of 55 °C.
• the chitinase 4 probe may be the chitinase 4 DNA sequence shown in SEQ ID NO.:1. It is contemplated that a probe prepared on the basis of a characteristic part or a specific subsequence of the chitinase 4 DNA sequence as disclosed herein, e.g. a probe prepared on the basis of the peptide 4-26 may also be useful.
• the nucleotide probe is advantageously prepared on the basis of the amino acid sequence of said specific part or a subsequence thereof.
• DNA fragments to be used e.g. in the construction of genetic constructs according to the invention were isolated as follows.
• the DNA was run on LMT (Low Melting Temperature) agarose (Sea Plaque ® GTG, FMC) in TAE (0.04 M Tris-acetate, 0.002 M EDTA) buffer.
• the DNA band was excised with a Pasteur pipette. To the excised DNA, 1 vol 200 mM NaCl, 10 mM EDTA was added. The gel was melted at 68°C for 10 min. and re-equilibrated to 37°C. Subsequently, 2U/100 ⁇ l of agarase (free of DNase, from Calbiochem) was added. The mixture was allowed to stand overnight at 37°C and was subsequently extracted twice with phenol and twice with chloroform, subjected to EtOH precipitation and finally resolubilized in H 2 O,
• PCR used for the amplification of cDNA encoding SE, ⁇ - 1 ,3-glucanase and chit 76 on the basis of sugar beet mRNA
• the preparation of a partial cDNA molecule was done by use of the Gene Amp ® RNA Amplification Reagent Kit (Perkin Elmer Cetus, USA). The PCR was performed in accordance with the manufacturer's instruc tion with a few modifications. The reverse transcription protocol was followed using the concentrations in the scheme below.
• the PCR protocol was followed except that the Taq polymerase was added later (see PCR cycles) 'and the temperature cycling was changed to the following:
• PCR used in the construction of genetic constructs of the invention and in site-directed mutagenesis on the basis of cloned DNA templates
• the preparation of the relevant DNA molecule was done by use of the Gene AmpTM DNA Amplification Reagent Kit (Perkin Elmer Cetus, USA) and in accordance with the manufactures instructions except for the temperature cycling. Here the following procedure was used:
• the method used for the synthesis of regenerated chitin has been specifically developed in order to make it possible to obtain a high yield of active chitinase 4.
• a high yield of active and pure chitinase is required in order to have sufficient protein material for i) determining the antifungal potential,
• the isolation and characterization of the DNA encoding the chitinase is necessary when the DNA is to be used for the construction of genetically modified plants having an increased chitinase activity. Also, a high amount of pure chitinase is required to make it possible to elucidate and characterize the important parts of the enzyme such as the active site.
• the regenerated chitin was obtained by acetylating the free amino groups at low as well as at high pH as described above (as compared to the conventional method in which this synthesis is performed only at low pH).
• the new combined method was easier, faster and gave a much higher yield and a more stable product than the conventional method in which acetylation is carried out only at low pH.
• the molecular weights determined by SDS-PAGE for chitinase 2, 3 and 4 are 32, 27 and 26 kDa, respectively (Fig. 2).
• the isoelectric points for chitinase 2, 3 and 4 were determined to 8.3, 9.0 and 9.1, respectively.
• 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.
• chitinase 4 is an endochitinase producing chitooligosaccharides or an exochitinase liberating only N-acetylglucosamine from the non-reducing end of chitin or chitooligosaccharides
• the pattern of reaction products liberated by chitinase 4 from 3 H-chitin was analyzed by TLC (Fig. 3). Irrespective of duration of incubation, N-acetylglucosamine was only a very minor reaction product, whereas chitobiose, chitotriose and chitotetraose were the major product. This strongly implies that chitinase 4 is an endochitinase.
• chitinase 4 was also capable of hydrolyzing the cell walls of Micrococus lysodeicticus using the lysozyme assay described in "Materials and Methods" (see Fig. 4). This demonstrate, that chitinase 4 is a bifunctional enzyme having both chitinase and lysozyme activity.
• Fig. 5 is shown the results when a combination of chitinase 4, acidic chitinase SE and ⁇ -1,3-glucanase 3 is applied to the culture. 60 ⁇ l of protein solution containing 20 ⁇ g of each antifungal proteins were applied to each microscope slide. When chitinase 4 was used alone 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 indicating a synergistic effect between chitinase 4 SE and ⁇ -1,3-glucanase.
• the germination of spores and growth of the mycelium can be followed in a microtiter plate by measuring the absorbance (620 nm) at specified time intervals.
• the growth of Cercospora is initiated after an approx. 40 hours lag period and increases almost linearly for the next 40-50 hours (curve A in Fig. 6).
• the inial lag period is increased to 75 hours and the growth rate is decreased as compared to the control (curve C in Fig. 6).
• the eluate from the chitin column is shown as a comparison.
• the chitin in the hyphae cell wall was labelled with 3 H-labelled N-acetylglucosamine. After a short pulse, the radioactivity was deposited in the tip of the fungal hyphae (see Fig. 7). When chitinase 4 alone or in combination with SE and ⁇ -1,3-glucanase was added after the pulse labeling, the radioactivity deposited in the hyphae tip wasa effectively removed.
• the amounts of enzymes is similar to that described in Method I (see above). This strongly indicates that the mode of action of chitinase 4 on the cell wall of Cercospora is specifically to hydrolyze the chitin fibers in the hyphae tip and thereby inhibit cell wall synthesis.
• the inhibition is primarily seen as a lag time for germination, the length of which depends of the strength and concentration of the growth inhibitor.
• S.B.2 sugar beet chitinase 2
• S.B.3 sugar beet chitinase 3
• cDNA amino acid composition derived from the cDNA sequence encoding the mature protein, chitinase 4
• the tryptic peptides were separated by reverse phase-HPLC on the Vydac RP-18 column mentioned above under the conditions specified in "Materials and Methods" see (Fig. 8). Peptides representing large peaks at an absorbance of 214 nm and displaying a high retention time (indicating long polypeptide chains) were selected for further purification on a Develosil RP-18 column.
• the purified peptides were subjected to amino acid sequence analysis as described above in "Materials and Methods" and the amino acid sequence of each of the peptides is shown below in Table II.
• tryptic peptides proved to be very advantageous to form the basis for the construction of an oligonucleotide probe
• amino acid sequence of the tryptic peptide 4-26 it was found that use of this sequence in the construction of an oligonucleotide probe would require only few codon choices
• this peptide was chosen to form the basis of the construction of an oligonucleotide probe to be used in the isolation of DNA encoding chitinase 4 (see Example 4 below) TABLE II
• a deduced amino acid sequence of chitinase 4 was obtained SEQ ID NO.:2.
• the deduced amino acid sequence was aligned with the partial sequence obtained from the chitinase 4 protein (as described in Example 3 above) and an almost 100% identity was observed. This demonstrates that the isolated cDNA clone codes for the chitinase 4 polypeptides purified by the
• the chit 4-B15 cDNA clone is 966 bp long and encodes a protein having 264 amino acid residues in the polypeptide chain out of the 265 amino acids predicted for the chitinase 4 genomic DNA.
• the leader sequence consist probably of 23 amino acid residues (out of 24 amino acid residues as determined for the genomic chitinase 4 DNA, see below), followed by a hevein domain of 35 and a functional domain of 206 amino acid residues.
• the cDNA After the stop codon the cDNA has a 158 bp 3' noncoding region.
• the N-terminal sequence of chitinase 4 was further examined by determining the molecular weight (MW) of the mature chitinase 4 by electrospray mass spectrometry as described by G.J. Feistner et al., 1990. A MW of 25893.6 +/- 10 was observed. On the basis of the amino acid sequence, a MW of 25923 can be calculated. Given that the mature chitinase 4 contains 7 S-S-bridges (loss of 14 protons) and that the first amino acid residue Gln is converted to the pyroglutamyl derivative (loss of - NH2 - 15 MW), the calculated MW of the mature chitinase 4 is 25894. This is in agreement with the data observed by the electrospray mass spectrometric analysis and confirms the deduced N-terminal amino acid sequence given above for the mature chitinase 4.
• N-terminal amino acid sequence could be determined for chitinase 2 and the following terminal amino acid sequence was found in chitinase 2: Glu-Leu-Cys-Gly-Asn-Gln-Ala.
• WGA-A QRCGEQGSNMECPNNLCCSQY-GYCGMGGDYCGKG--CQNGACWTS
• WGA-A shown in SEQ ID NO.:23
• chitinase 76 the sequence of which is shown in SEQ ID NO . : 5. Sequencing of this gene was initiated with the primer used for screening of the ⁇ ZAP library (see Example 4), and continued with other primers complementary to sequences inside the chit 76 gene.
• the chit 76 gene codes for a 268 amino acid long chitinase which has 80% homology to the chitinase 4 amino acid sequence (vide SEQ ID NO.:1) but only 34% homology to the entire chitinase 1 protein ( vide SEQ ID NO.:11).
• the gene contains one intron which is located in position 875 to 1262. The exact location of this intron is based on an alignment with the chitinase 4 cDNA SEQ ID NO.:1 (Fig. 24).
• the intron borders contain the consensus GT/AG sequences.
• the chit 76 intron is located exactly at the same position as the second intron in the chitinase 1 gene, when the amino acid sequences of chitinase 1 and chit 76 are aligned.
• TATAAA TATA-box sequence
• a poly-A signal AATAAA is located at position 1725 in SEQ ID NO.:5.
• Alignment of the 5' noncoding regions from the two genomic genes show boxes of homology (e.g. chitinase 4 nucleotides 14-49, 60-122, 123-135, 159-173, 174-207 and 277-328, (Fig. 26).
• the rest of the gene can easily be sequenced. It is contemplated that the chitinase 4 gene comprises at least 1 intron, probably only 1 corresponding to that given in the same position as that of the chitinase 76 sequence.
• the acidic chitinase SE was purified as described in "Materials and Methods" above.
• 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 3 H-labelled chitin.
• the major product of the enzymatic hydrolysis was the hexamers of chitin or higher homologous of chitin oligo saccharides. Since the major product for chitinase 4 was the dimer, a different mode of action for SE is inferred. No lysozyme activity could be determined for SE at pH 4-9.
• the purified enzyme was subjected to tryptic digestion as described in "Materials and Methods" and in Example 3 above for chitinase 4 and 6 peptides were selected.
• the peptides were subjected to further purification in the same manner as the tryptic peptides of chitinase 4 described in Example 3 above and the amino acid sequence of the 6 peptides were determined.
• the peptides were selected using the same criteria as the ones used in connection with chitinase 4.
• the amino acid sequence of these peptides are shown in Table IV.
• the N-terminal amino acid sequence was also determined as shown in the Table IV.
• N-terminal consisting of amino acids No's 26-46 of SEQ ID NO.:8
• SE 22.5 consisting of amino acids No's 98-109 of SEQ ID NO.:8
• SE 23.0 consisting of amino acids No's 121-128 of SEQ ID NO.:8
• SE 25.1 consisting of amino acids No's 208-224 of SEQ ID NO.:8
• SE 26.1 consisting of amino acids No's 271-277 of SEQ ID NO.:8
• SE 30.4 consisting of amino acids No's 110-128 of SEQ ID NO.:8
• SE 31.1 consisting of amino acids No's 229-252 of SEQ ID NO.:8
• a partial cDNA molecule was prepared in two steps using the PCR- technique and mRNA, the first step using the above mentioned primers KB7 and 270.
• the PCR- technique was performed as described above in "Materials and Methods”.
• the cDNA synthesized was isolated on LTM agarose gel and the agarose was removed with agarase.
• the primers KB9 and 270 were used. The method is illustrated in Fig. 20.
• the product from the second PCR reaction was cloned in pUC 19 (Boehringer Mannheim) and sequenced.
• the DNA sequence obtained for the partial cDNA molecule constituted by nucleotides 711-962 of the DNA sequence shown in SEQ ID NO.:7. This cDNA was used to screen the ⁇ -ZAP cDNA library described in
• the structural gene has a 5' noncoding region of 17 bp, a leader sequence of 25 amino acid residues, a functional domain of 268 amino acid residues and a 3' noncoding region of 202 bp after the stop codon.
• the cDNA sequence and the amino acid sequence are shown in SEQ ID NO . : 7 and SEQ ID NO.:8, respectively.
• Table VI shows an alignment of the amino acid sequence corresponding to the structural gene for the acidic chitinase SE and the amino acid sequence of a cucumber lysozyme/chitinase (EP 0 392 225 and Metraux, et al , 1989) and an Arabidopsis lysozyme/chitinase (Samac et al . , 1990). It appears from this that there is a homology of about 45% when all tree segment ire compared. When SE is compared with the cucumber lysozyme/chitinase a homology of about 60% was observed.
• 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.
• 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 connection with the selection of the tryptic peptides of chitinase 4 (see Example 3).
• the amino acid sequence of the peptides are shown in Table VIII.
• 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
• Pep. 4-25.1 consisting of amino acids No's 37-51 of SEQ ID NO.:10
• Pep. 4-26.3 consisting of amino acids No's 211-216 of SEQ ID NO.:10
• Pep. 4-27.1 consisting of amino acids No's 115-139 of SEQ ID NO.:10
• Pep. 4-28.2 consisting of amino acids No's 101-109 of SEQ ID NO.:10
• Pep. 4-40.1 consisting of amino acids No's 249-272 of SEQ ID NO.:10 EXAMPLE 9
• oligonucleotide probes corresponding to peptides from the ⁇ - 1 ,3-glucanase 3 and 4 polypeptides were synthesized.
• 5'primer was used the following two sequences in the first round of PCR for isolation of ⁇ - 1 ,3-glucanase 4:
• the resulting PCR products were employed to screen the above described sugar beet cDNA ⁇ -ZAP library to isolate clones harboring cDNA encoding ⁇ - 1 ,3-glucanases 3 and 4, respectively.
• the cDNA sequences and the deduced amino acid sequence of ⁇ - 1 ,3-glucanase 4 are shown in SEQ ID NO.: 9 and SEQ ID NO.: 10, respectively.
• Chitinase 4 antibodies detect only an approximately 26 kDa protein (chitinase 4), but not the 32 kDa protein (chitinase 2 isozyme) although it is also present on the same nitrocellulose membrane. In contrast chitinase 2 antibody recognizes only a 32 kDa protein
• sugar beet chitinase 2 class When the N-terminal amino acid sequence of sugar beet chitinase 2 was aligned with the following chitinases from bean, tobacco, pea A1, pea A2, pea B (Vad et al ., 1991), barley T (Jacobsen et al . , 1990), and barley K (Kragh et al., 1990), a strong homology between these basic chitinases were observed (see Table IX). This suggests that these chitinases belong to the same chitinase class. This was further substantiated by serological cross reactivity carried out with antibodies raised against sugar beet chitinase 2.
• This antibody recognized not only sugar beet chitinase 2, but in addition also chitinase P (27.5 kD), Q (28.5 kD), Ch. 32 and Ch, 34 from tobacco (Bol and Linthorst, 1990), chitinases T, K and C from barley and chitinase A1, A2 and B from Pea.
• 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.
• Tobacco consisting of the amino acids No's 1-23 of SEQ ID NO.: 26
• Pea A1 shown in SEQ ID NO. :42
• Barley K shown in SEQ ID NO. :44
• the chitinase 4 enzyme belongs to a new class of chitinases.
• the high degree of homology between the cDNA encoding the chitinase 4 enzyme and the DNA encoding the chitinase from rape seed chitinase shown by the high degree of DNA hybridization further indicates that the genes encoding chitinase 4 in sugar beets and the genes encoding the chitinases in rape seed are significantly homologous and thus belong to the same gene class. This is supported by the results disclosed in Example 10 showing a high degree of serological homology between the mature enzymes from the two plants.
• TRANSFORMATION OF BACTERIA CELLS Agrobacterium tumefaciens (the strain LBA 4404, Ooms et al., 1982) was transformed with the plant transformation vector, pBKL4K4, the preparation of which is described in Example 18, using a freeze/thaw method essentially as described by An et al , (1988).
• the bacteria to be transformed were cultivated in LB- medium, pH 7.4, overnight at 28°C, 280 rpm.
• the next day the bacteria were subcultivated in 50 ml of LB-medium, pH 7.4, and grown for about 4 hours until OD 600 (OD 600 ) was 0-5-1.0.
• the culture was cooled on ice and centrifuged for 5 minutes at 10.000 ⁇ g at 4°C. The supernatant was removed and the bacteria were carefully suspended in 1 ml of icecold 20 mM CaCl 2 . 0.1 ml of the bacteria suspension was pipetted off in icecold cryo tubes and the bacteria were frozen in liquid nitrogen and maintained at -80°C.
• plasmid DNA For transformation of the bacteria 1 ⁇ g of plasmid DNA was first added to a cryo tube with the frozen bacteria. The bacteria were incubated in a 37°C water bath for 5 minutes, 1 ml of LB-medium, pH 7.4, was added to the cryo tube, and the mixture was incubated for 4 hours at room temperature using mild agitation (agitation table, 100 ⁇ rpm). The cryo tube was centrifuged for 30 sec. at 10.000 ⁇ g, 4°C. The supernatant was removed and the bacteria were resuspended in 0.1 ml of LB-medium, pH 7.4.
• the bacteria were plated on to a YMB-dish with 50 mg/l kanamycin and incubated for 2 to 4 days at 28oC until colonies appeared.
• the presence of a proper plasmids in the bacteria are verified by restriction analysis of the extracted plasmid prior to the use of the bacteria in the transformation of the plants.
• bacterial transformation with other genetic constructs of the invention may be performed, e.g. as shown in Figs. 17, 18, 19, and 22 and explained in Example 18.
• Leaves from plants to be genetically transformed were obtained from plants grown in vi tro or in vivo . In the latter case, the leaves were sterilized prior to transformation. Sterilization was performed by placing the leaves for 20 min. in a solution of 5% Ca-hypochlorite containing 0.1 ml Tween 80 per 1 followed by washing 5 times in sterile water. In vi tro plants were grown in containers on 1/2 shoot inducing medium (1/2 MS) (Murashige & Skoog, 1962).
• the leaves were placed one at a time in a 14 cm Petri dish. They were then cut into squares of about 1 cm , all 4 sides consisting of tissue which had been cut. Any cut tissue which had been bleached by hypochlorite sterilization was removed.
• Transformation of the plant was done essentially as described by R.B. Horsch et al . (1985).
• the bacteria culture was diluted 50x with 1/10 MS immediately before transformation.
• Approximately 10 ml of the diluted bacteria suspension was poured into a 9 cm Petri dish, and the leaf pieces were dipped in this suspension for about 15 min. The leaf pieces were then removed and excess bacteria suspension was removed with sterile filter paper.
• the leaf pieces were transferred to Petri dishes containing shootinducing MS-medium with 300 mg/1 of kanamycin and 800 mg/l of carbenicillin and sub-cultivated every 4 weeks to the same medium.
• Shoots which appear on shoot- inducing MS-medium 300 k/c dishes were transferred to containers with 1/2 MSO 300 k/c.
• the shoots were subcultivated when needed.
• the expression of the ⁇ -glucoronidase activity using the GUS-assay was performed on the leaf tips of green shoots. Planting out
• the transformed explant were transferred to a MS substrate supplemented with 0,25 mg/l of BAP, 400 mg/l of kanamvcin. 800 mg/l of carbenicillin and 500 mg/ml of cefo taxime and the explants were incubated for 14 days on this substrate.
• the regenerated shoots were then transferred to containers with MS containing 0.25 mg/l of BAP, 400 mg/l of kanamycin, and 800 mg/l of carbenicillin as the substrate.
• the isolated shoots were transferred to fresh substrates with 4 weeks intervals for selection and multiplication. Selected shoots were rooted on 1/2 MS substrate containing 1 mg/l IBA.
• Tissue from tobacco have been transformed with a genetic construct containing either chitinase 1, chitinase 4, chitinase 76 and acidic chitinase SE and the selective markers, NPT-II and GUS. Selection of the callus and shoots on kanamycin has proved that the obtained tissue expresses the GUS marker and thus that the transformation has occurred.
• the expression levels for chitinase and ⁇ - 1,3-glucanase isoenzymes can be evaluated either by measuring the total enzyme activity by the two radiochemical assays, by measuring the antifungal activity using the biological methods I-III or by measuring the level of the different isoenzymes by immunoblotting using specific antibodies, all of the methods being described above in "Materials and Methods”.
• the final test of the resulting transgenic plants is the analysis of their degree of resistance to phytopathogenic fungi using the infection system described in "Materials and Methods”.
• the antifungal activity of the enzymes in the genetically transformed plants can be determined.
• a retarded growth of the fungi hyphae shows that the transformation has resulted in a plant having an improved tolerance i.e. an increased antifungal activity to the phytopatogenic fungi compared to a non-transformed plant.
• H-chitin or 3 H-laminarin are used as substrates for either chitinase or ⁇ -1,3-glucanase, respectively.
• the activity for both chitinase and ⁇ -1,3-glucanase in crude plant extracts can be determined. This is illustrated further in a time course experiment where the level of either chitinase (Fig. 12a upper part) or ⁇ -1,3-glucanase (Fig. 12b lower part) is quantified in sugar beet leaves at specified time intervals after infection with C.
• the antibody to ⁇ -1,3-glucanase 3 recognized only one single protein in the Cercospora infected leaf material (Fig. 13). In contrast, no antigen was detected in the control leaves. This is in agreement with the low constitutive level of expression observed in control plants for ⁇ -1,3-glucanase using the radiochemical assay.
• antibodies raised against either chitinase 2 or 4 were employed, two major protein bands were induced in the infected leaf tissues. Chitinase 2 antibodies detect a 26 and a 32 kDa band, whereas two proteins having molecular weights of 29 and 26 kDa were observed with the chitinase 4 antibody.
• the "3-D" structure of chitinase 4 and SE on the nitrocellulose membrane may create sufficient epitope recognition to allow the antigen-antibody interaction between the SE antigen and the chitinase 4 antibody.
• the reaction between the SE antigen and the chitinase 4 antibody was only pronounced when the antibody solution is diluted 1:100 or 1:200. A much weaker reaction was observed when the antibody is diluted 1:5000 or 1:10,000.
• Transgenic tobacco plants (Nicotiana tabacum and/or N . benthamiana ) were transformed with either chitinase 4, chitinase 76, the acidic chitinase or chitinase 4 + the acidic chitinase.
• the transformed plants were examined with respect of i) GUS activity, II) expression of chitinase genes, and iii) degree of resistance against C. nicotiana or R . solani .
• the transgenic plants expressed GUS-activity in variable amounts. Only plants with high GUS-activity were subjected to further analysis. The expression of the chitinase gene products were analysed by
• lanes SE, K76, K4, K4 + SE respectively.
• lane Std in Fig. 23 A broad protein band was observed in extracts from transgenic plants with the chitinase 4 or chitinase 76 gene constructs.
• This cleavage site may give rise to the 25 kD polypeptide band.
• the translocation of chitinase 4 was inhibited.
• this basic chitinase 4 is deposited in the
• Site directed mutagenesis on a DNA sequence encoding the sugar beet chitinase 4, e.g. the chitinase 4 gene may be carried out by use of PCR reactions (described in "Materials and Methods" under the heading "PCR used in the construction of genetic constructs of the invention and in site-directed mutagenesis on the basis of cloned DNA templates") using specific 3' and 5' primers for each site directed mutagenesis.
• the choice of the specific 3' and 5' primers to be used depend on the position in the DNA sequence in which the modification is to be carried out.
• suitable amino acids to be modified are selected on the basis of an analysis of the amino acid sequence of the mature chitinase 4 enzyme, optionally in combination with an analysis of the enzyme's 3-D structure.
• 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.
• Carbodiimide is covalently linked to the three essential acidic groups (glutamic and aspartic acid residues) constituting the catalytic site of glucoamylase.
• NBS oxidizes Trp residues important in either stabilizing the transition state intermediate of the catalysis or Trp residues involved in substrate binding at a distance from the catalytic site.
• the experiments with chitinase C indicate that three acidic and two Trp-residues are very important constituents of the active site.
• the active site of chitinase 4 is contemplated to be constituted by amino acid residue 183 (Asp) and 189 (Glu) in SEQ ID NO.:1 (corresponding to amino acid residue 184 and 190 in the amino acid sequence encoded by the genomic chitinase 4 amino acid sequence.
• the number given below in brackets denotes the number of the amino acid from the corresponding amino acid sequence encoded by the genomic chitinase 4).
• chitinase C from barley and all other plant chitinases of the same serological class have three aspartic acid residues ("corresponding to amino acid residues 183, 189 and 194 of chitinase 4 (SEQ ID NO: 2)) in the active site (184, 190 and 195. respectively).
• the position of the two important Trp residues involved in the active site of chitinase C have not been elucidated. Since chitinase 4 only contain three Trp residues in contrast to the 6 present in chitinase C, the important Trp residues may be more easily identified in chitinase 4.
• the two acidic residues 183 Asp and 189 Glu of SEQ ID NO: 2 (184 and 190, respectively) forming the active site of chitinase 4 is
• peptide 4-22 SIGFDGLNAPETVANNAVTAFR. Important Trp-residues of the active sites may be contained in peptide 4-19.3:
• GPLQITW and peptide 4-26 TAFWFWMNNVHSVIVNGQGFGASI.
• the active site of the chitinase 4 differs from the active sites of other plant chitinases, e.g. tobacco, which has the following corresponding amino acid sequences AIGVDLLNNPDLVATDPV shown in SEQ ID NO.: 46, GPIQISH shown in SEQ ID NO.: 47 and SALWFWMTPQSP shown in SEQ ID NO.
• an interesting modification is one in which the glutamic acid in position 189 (190) is substituted with aspargine and/or the aspartic acid in position 183 (184) are substituted with glutamine.
• Changing the carboxyl groups Asp 183 (184) to Asn and for Glu 189 (190) to Gln in chitinase 4 are in itself expected to have a
• Trp in positions 169. 204 and 206 (170, 205 and 207, respectively) to Tyr may change the binding of the substrate (chitin) to the catalytic site and perhaps the substrate
• primers are chosen either themselves containing restriction sites or being located near restriction sites in a manner creating the possibility of exchanging the PCR product with a corresponding sequence in the gene by restriction enzyme digestion followed by ligation of the relevant fragments.
• the 5' primer to be used in the following examples is termed SD 0 (see Fig. 14).
• the number in brackets denotes the number of the corresponding amino acid residue encoded by the genomic chitinase 4 DNA sequence.
• Trp169(170) of the chitinase 4 amino acid sequence is to be substituted by the amino acid Tyr, the following procedure may be carried out:
• the resulting PCR product (from bp 301 to 538) is digested with BamHI and PvuII and interchanged with the corresponding fragment of the chitinase 4 gene by conventional methods (Sambrook et al . , 1989).
• Glu189(190) is to be substituted with the amino acid Gln
• the 3'primer SD2 is used (Fig. 14).
• the 3' primer SD3 is used (Fig 14).
• the PCR products are digested with BamHI and BspMII and interchanged with the BamHI -BspMII fragment of the chitinase 4 gene in a similar manner as described above for exchange of Trp169(170).
• PCR products are digested with BamHI and Ball and interchanged with the BamHI -BalI fragment in the chitinase 4 gene as described above. In a similar manner, other desirable modifications may be carried out.
• the C-terminal extension may be introduced in the DNA sequences encoding one or more of the antifungal proteins of the invention by any suitable technique such as PCR.
• Fig. 15a illustrates the sugar beet ⁇ -1,3-glucanase cDNA with a tobacco C-terminal extension which is underlined in the figure.
• Fig. 15b illustrates PCR primers which can be used to change the stop codon and to introduce a part of the C-terminal extension, a Dral site is created at the 3' end.
• Fig. 15c illustrates 4 annealed synthetic oligonucleotides containing the last part of the C-terminal extension, a stop codon. a SmaI site and an EcoRI site.
• the C-terminal extension can be introduced by exchanging the XbaI- EcoRI fragment in the ⁇ - 1 ,3-glucanase gene with the PCR product digested with XbaI and DraI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI using conventional methods (Sambrook et al ., 1989).
• Fig 16a illustrates the chitinase 4 gene with a tobacco C-terminal extension (the underlined sequence in the figure).
• Fig 16b illustrates PCR primers which can be used to introduce a SmaI site near the stop codon in the chitinase 4 gene.
• Fig 16c illustrates four annealed synthetic oligonucleotides containing the sequence for the C-terminal extension, a changed stop codon, a SmaI site and a EcoRI site.
• the C-terminal extension can be introduced by exchanging the BamHI-EcoRI fragment with the PCR product digested with BamHI and SmaI and the annealed synthetic oligonucleotides digested with SmaI and EcoRI likewise using conventional methods.
• chitinase 76 chitinase 4
• SE ⁇ -1,3-glucanase sequences.
• the N-terminal sequence may in a similar manner be exchanged with other N-terminal sequences.
• Of particular interest may be the N-terminal sequence of chitinase 1 shown in the SEQ ID NO.: 12, the N-terminal sequence of the acidic chitinase SE shown in SEQ ID NO.:8, the N-terminal sequence of chitinase 4 shown in SEQ ID NO.:2, the N-terminal sequence of chitinase 76 shown in SEQ ID N0.:6.
• N-terminal sequence of ⁇ - 1,3-glucanase shown in SEQ ID NO.:10.
• Other interesting N-terminal sequences of the mature protein may be the ones shown in Table III, or in Table IX, or the proline rich region from the sugar beet chitinase 1 shown in SEQ ID NO . : 1.
• the excised recombinant pBluescript containing the chitinase 4 cDNA gene (B15 chitinase 4) was subcloned in order to supply the gene with an enhanced 35S promoter and a 35S terminator.
• This construct was transferred to the plant transformation vector pBKL4 containing a NPTII and a GUS gene.
• pBKL4 is a derivative of the A. tumefaciens Ti-plasmid pBI121 (Bevan et al., 1984), in which the genes between the left and right borders have been replaced with the following genes: 1) ⁇ -glucoronidase (GUS) from E.
• NPT Neomycin phosphotransferase
• PCR amplification reaction was performed in order to introduce the ATG site, a ribosome binding site and two restriction sites (HindIII and BglII) 5' to the cDNA sequence.
• the oligonucleotide KB3 (shown in SEQ ID NO.:49): containing the two restriction sites, a ribosome binding site, the
• the PCR product was extracted twice with phenol and twice with chloroform and EtOH precipitated. After resuspension in H 2 O the DNA was digested with HindIII and Nhel. The HindIII-NheI fragment from pB15 chit 4 was exchanged with the HindIII-NheI PCR fragment (Fig. 17).
• the construct was sequenced with the T7 sequencing primer (corresponding to the pBluescript T7 promoter) and primer 340 (shown in SEQ ID NO. :51) :
• nucleotide 8 was a T and nucleotide 10 was a C as in the pB15 chit 4 clone and both the NheI sites at position 245 and 251 were still present.
• the construct was digested with EcoRI and a fill in reaction was performed with Klenow enzyme in the presence of dATP and TTP, the construct was further digested with BglII after removal of the Klenow enzyme.
• the DNA fragment BglII-EcoRI containing the entire chitinase 4 sequence was cloned into the vector pPS48 containing an enhanced
• the chitinase 4 gene was inserted in the correct orientation by digesting the pPS48 vector with BamHI -SmaI (Fig. 17).
• the chitinase 4 gene with the enhanced 35S promoter and 35S terminator was transferred to the plant transformation vector pBKL4 (Fig. 17) as a HindIII fragment (Fig. 17).
• the resulting vector, pBKL4K4, harboured in an E. coli DH5 ⁇ has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb. D-3300 Braunschweig (DSM) on 30 July. 1991 under the provisions of the Budapest Treaty under the accession number DSM 6635.
• the SE gene was then introduced into the construct pBKL4K4 (Fig. 17).
• a full length SE gene was constructed by combining the 5' end of the gene from the pSur1 clone (EcoRI-KpnI) with the rest of the gene from pSE22 (KpnI-HindIII) in the cloning vector pUC19 (Fig.
• the SE gene was subcloned in the Smal site of pPS48 as a EcoRI-HindIII fragment filled in with Klenow polymerase in the presence of all four nucleotides.
• the orientation of the gene with respect to the enhanced 35S promoter and 35S terminator was examined by restriction enzyme analysis and further confirmed by sequence analysis.
• the SE gene with the enhanced 35S promoter and 35S terminator was cloned in the KpnI site of pBKL4K4 as a HindIII fragment in the presence of a HindIII-KpnI adapter (Fig. 18).
• the HindIII fragment was furthermore cloned in the HindIII site of pBKL4.
• the chitinase 76 gene was cloned in pBKL4 (Fig. 19).
• the glucanase gene can be introduced into the construct pBKL4 , pBKL4K4 , pBKL4KSE, or pBKLKK4KSE (Fig. 22).
• the full length cDNA clone (SEQ ID NO.:9) was digested with EcoRI and BglII, the sticky ends were filled in with Klenow polymerase in the presence of all four dNTP's.
• the glucanase gene is then subcloned in the SmaI site of pPS48Mod.
• the orientation of the gene with respect to the enhanced 35S promoter and the 35S terminator, respectively, may be examined by restriction enzyme analysis and further confirmed by sequence analysis.
• the glucanase gene with the enhanced 35S promoter and the 35S terminator is cloned in the EcoRI site of pBKL4 , pBKL4K4 , pBKL4KSE.

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