# Patent

## Title
Isolated oxide synthase and divinyl ether synthase and uses thereof

## Abstract

The present invention relates to enzymes from the cytochrome P450 family and to the nucleotide sequences encoding them, and to their use in a method for the generation of pathogen-resistant plants.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. 371) of PCT/EP02/07555 filed Jul. 6, 2002, which claims the benefit of German application 10133343.9 filed Jul. 12, 2001 and of German application 10220115.3 filed May 6, 2002.
FIELD OF THE INVENTION
The present invention relates to enzymes from the cytochrome P450 class, to the isolation of the corresponding coding nucleotide sequences and to their use in a method for generating pathogen-resistant plant cells and their progeny.
BACKGROUND OF THE INVENTION
The cytochrome P450 enzyme class includes the enzymes allene oxide synthase (AOS), hydroperoxide lyase (HPL) and divinyl ether synthase (DES). They form a separate subfamily referred to as CYP74.
Owing to the multiplicity of cytochrome P450 enzymes, a nomenclature was developed which assigns a specific family and subfamily to each protein of this class on the basis of its primary structure. Thus, all the AOSs form their own subfamily CYP74A, while CYP74B comprises the 13-HPLs, CYP74C the 9/13-HPLs and CYP74D the 9-DESs (Feussner et al.; 2001, Trends Plant Sci. 6, 268-273). Proteins from the same subfamily are numbered chronologically.
CYP74 enzymes are monooxygenases with a hemamolecule of prosthetic group. Although they also have a protoporphyrin IX group (hem b) attached as prosthetic group, they have very little affinity to CO (Matsui, 1998, Belgian Journal of Botany. 131, 50-62).
They constitute important enzymes in the metabolism of polyene fatty acids, which is known as the lipoxygenase (LOX) pathway (Feussner and Wasternack, 1998, Fett/Lipid. 100, 146-152).
LOXs are dioxygenases in which the iron in the catalytic center is bound to amino acid side chains (Brash, 1999, J. Biol. Chem. 274, 23679-23682). They catalyze the incorporation of molecular oxygen into the (1Z,4Z)-pentadiene system of polyunsaturated fatty acids. In plants, these are mainly linoleic and α-linolenic acid. Depending on the regioselectivity of the LOX employed, two different positional isomers of hydroperoxides, viz. (9S) isomers or (13S) isomers, may arise as products. For example, α-linolenic acid gives rise to (13S,9Z,11E,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13S-HPOTE) and linoleic acid gives rise to (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid (13S-HPODE).
In plants, these hydroperoxides are rapidly reacted further by a multiplicity of enzymes. Currently, seven different enzyme families which convert hydroperoxides and thus compete for LOX products are known in the plant kingdom: the allene oxide synthase (AOS) reaction, the hydroperoxide lyase (HPL) reaction, the divinyl ether synthase (DES) reaction, the reductase reaction, the peroxygenase reaction, the epoxyalcohol synthase (EAS) reaction and the LOX reaction itself (Feussner et al.; 2001, Trends Plant Sci. 6, 268-273). When 13-HPOTE is reacted in the presence of the enzyme allene oxide cyclase (AOC), a cyclization reaction takes place which gives 12-oxophytodienoic acid (12-oxo-PDA) (Ziegler et al.; 2000, J. Biol. Chem. 275, 19132-8), which, in turn, is the precursor of jasmonic acid, which is here considered a plant hormone.
AOS (EC4.2.1.92; CYP74A) was the first CYP74 enzyme to be described; the homogeneous protein was first isolated from flax (Song and Brash, 1991, Science. 253, 781-784). It catalyzes the reaction which gives an unstable allene oxide which can break down into the corresponding α- and γ-ketoles in the presence of water. AOS is involved in the biosynthesis of jasmonic acid (Vick and Zimmerman, 1983, Biochem. Biophys. Res. Commun. 111, 470-7). Jasmonic acid itself is involved in inducing the transcription of specific mRNA and regulating the translation of jasmonate-induced proteins (JIP), such as LOX, AOS and proteinase inhibitors. This makes jasmonic acid an important signal substance in plant stress response (Wasternack and Parthier, 1997, Trends Plant Sci. 2, 302-307). An involvement in processes of growth regulation and in the promotion of senescence is likewise described (Sembdner and Parthier, 1993, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 569-589).
A large number of AOSs have already been cloned and expressed functionally in E. coli, including the Arabidopsis thaliana, Lycopersicon esculentum, Linum usitatissimum and Hordeum vulgare AOSs. Apart from the barley AOSs, all of the AOSs cloned to date show substrate specificity for (13S)-hydroperoxide (Maucher et al.; 2000, Plant J. 21, 199-213).
HPL (CYP74B and C) cleaves the hydroperoxide into (3Z)-aldehydes and ω-oxoacids (Matsui, 1998, Belgian Journal of Botany. 131, 50-62). Even before the enzyme itself was discovered, the HPL reaction products were known as “leaf aldehydes”, which contribute to the characteristic odor of plants and fruits (Hatanaka, 1996, Food Rev. Int. 12, 303-350). In the case of 13-HPOTE as substrate, (3Z)-hexenal and (9Z)-12-oxo-9-dodecenoic acid are formed, the latter of which isomerizes to give (10E)-12-oxo-10-dodecenoic acid (traumatin), which is discussed as a wound hormone. A function as plant messenger substance is also discussed (Bate and Rothstein, 1998, Plant J. 16, 561-569). Traumatin can be oxidized further to give traumatic acid, which likewise appears to be involved in plant wound response (Zimmerman and Vick, 1970, Plant Physiol. 46, 445-453). HPLs which were cloned and expressed in E. coli as active protein were the A. thaliana, Cucumis sativus, Medicago sativus and L. esculentum HPLs, inter alia. Again, most of these enzymes show substrate specificity for (13S)-hydroperoxide. Only one HPL from cucumber and one from melon are without substrate specificity and are therefore referred to as 9/13-HPL (McIntyre et al.; 1999, J. Biol. Chem. 274, 25189-25192; Matsui et al.; 2000, FEBS Lett. 481, 183-188). A study of the relationship of both sequences with other members of the CYP74 family reveals a higher degree of homology with AOS than with the 13-HPLs. This is why these enzymes are classified as a separate subfamily CYP74C (Matsui et al.; 2000, FEBS Lett. 481, 183-188). It has been demonstrated that the Arabidopsis HPL is induced by wounding.
DES (CYP74D) catalyzes the formation of divinyl ethers which are fungicidally active (Weber et al. 1999). An involvement of the divinyl ethers in the defense against pathogenic fungi and bacteria, analogously to the aldehydes in the case of the HPL products, is also discussed (Weber et al.; 1999, Plant Cell. 11, 485-493; Göbel et al.; 2001, J. Biol. Chem. 276, 6267-6273). The first L. esculentum DES was cloned in 2001 by Itoh and Howe. It emerged that it has a high degree of homology on a sequence with AOS and HPL; it is therefore also thought as belonging to the cytochrome P450 class, subfamily CYP74D. Moreover, DES is unique in the CYP74 group in as far as it is the only enzyme which is highly specific for 9S-hydroperoxide (Itoh and Howe, 2001, J. Biol. Chem. 276, 3620-3627).
In nature, P450 enzymes are involved in many ways in the biosynthesis and the metabolism of a large number of endogenous substances. Their involvement in the detoxification of xenobiotics is of particular importance. Moreover, plant P450 enzymes are involved in the biosynthesis of wound signals (jasmonic acid, salicylic acid, traumatin) and hormones (gibberellins, brassinosteroids).
Substances which trigger wound signals in plants and subsequent signal transduction cascades come in many forms. They can be triggered by damage or injury to the plant, but can also be induced (artificially) by external chemical compounds.
In addition, however, it is in particular the incidence of plant diseases and mainly the promotion of plant's defense reactions against such pathogens which are agronomically of enormous relevance. The plant's response to a pathogen may involve quite a different pathway than is the case when the plant defends itself against stress by wounding. The incidence of plant diseases caused by, for example, viruses, bacteria or fungi, generally results in considerable damage or indeed death of the whole plant, in conjunction with a drastic quantitative or qualitative reduction of the crop.
If the yield losses are to be limited to an economically acceptable extent, it is imperative to carry out plant protection measures. In particular, it would be desirable to improve the plants' defense reactions against pathogens and/or pests without using chemicals, which constitute an additional pollutant for the soil, the groundwater and the user.
SUMMARY OF THE INVENTION
An object of the present invention was therefore to provide transgenic plant cells, plants and their progeny which display increased resistance to pathogens, and correspondingly improved methods for their generation.
This object is achieved by an allene oxide synthase with an amino acid sequence as shown in SEQ ID No. 2 or its isoenzymes and/or a divinylether synthase with an amino acid sequence as shown in SEQ ID No. 4 or its isoenzymes.
The two abovementioned enzymes belong to the CYP74 enzyme family. An increased specific activity of these CYP74 enzymes, alone or in combination, with regard to the endogenous specific activity of these enzymes in a plant cell leads to an increased resistance to pathogens in a plant cell.
Here, the specific CYP enzyme activity which is increased in accordance with the invention brings about a resistance of the plant cells or plants to pathogens which is increased by 20-90%, preferably by 30-80% and especially preferably by 40-70% over the specific endogenous activity in plant cells or plants. The resistance of the plants is determined by a drop in penetration frequency.
For the purpose of the invention, penetration frequency is understood as meaning the number of infection sites with successfully penetrated epidermal cell, divided by the total number of infection sites.



DESCRIPTION OF THE FIGURES
FIG. 1. Affinity purification of clone Pp291: (A) Elution with pH gradient, and (B) elution gradient with imidazole gradient.
FIG. 2. Photometric activity assay.
FIG. 3. Elution profile of HPLC analysis of hydrazones as derivatives of volatile aldehydes.
FIG. 4. Elution profile of HPLC analysis of catalysis products.
FIG. 5. Elution profile of HPLC analysis of nonvolatile products.
FIG. 6. Elution profile of HPLC analysis of nonvolatile products.
FIG. 7. BC/MS chromatogram of the product formed by catalysis of the CYP74 enzyme.
FIG. 8. Relative enzyme activity of the enzyme encoded by clone Pp291.
FIG. 9. Relative conversion rates of various substrates by the CYP74 enzyme.



DESCRIPTION OF THE INVENTION
The CYP74 enzymes according to the invention, alone or in combination, advantageously bring about an increased resistance in plants to pathogens such as, for example, biotrophy fungi. An increased concentration (increased specific activity) in CYP74 protein(s) according to the invention preferably brings about increased resistance to powdery mildews, especially preferably Blumeria graminis f. sp. hordei or f. sp. tritici. However, this does not preclude an increased resistance to other plant pathogens.
Further examples of such plant pathogens are Pythium spec., Albugo spec., Rhizoctonia solani, Peronospora parasitica, Erysiphe crucifearum, E. cichoreacearum, Alternaria brassicicola, Botrytis cinerea, Sclerotium rolfsii, Sclerotinia sclerotium, Fusarium oxysporum, F. culmorum, F. graminearum, F. nivale, phytophtora infestans or Pseudomonas syringae. 
It must be noted that the abovementioned CYP74 enzymes according to the invention display a much broader substrate spectrum than previously known CYP enzymes from this class. The CYP74 enzymes according to the invention are capable of converting not only 9-HPOD/TE, but also 13-HPOD/TE as substrate.
In accordance with the invention, the present CYP74 enzymes originate from moss or higher plants. Preferably, the CYP74 enzymes according to the invention originate from Physcomitrella patens. 
The present invention also relates to an isolated nucleotide sequence encoding an allene oxide synthase of the abovementioned type which is involved in the biosynthesis of polyunsaturated fatty acids for increasing the resistance of plant cells or plants to pathogens, selected amongst

    
    
        a) a nucleotide sequence as shown in SEQ ID No. 1,
        b) a nucleotide sequence with at least 70% identity with the nucleotide sequence shown in SEQ ID No. 1,
        c) a nucleotide sequence which is complementary to a) or b).
    
    


The invention also encompasses nucleotide sequences which hybridize with a) or b).
Likewise, the invention encompasses an isolated nucleotide sequence encoding a divinyl ether synthase of the abovementioned type which is involved in the biosynthesis of polyunsaturated fatty acids for increasing the resistance of plant cells or plants to pathogens, selected amongst

    
    
        a) a nucleotide sequence as shown in SEQ ID No. 3,
        b) a nucleotide sequence with at least 70% identity with a nucleotide sequence shown in SEQ ID No. 3,
        c) a nucleotide sequence which is complementary to a) or b).
    
    


The invention also encompasses nucleotide sequences which hybridize with a) or b).
In accordance with the invention, an isolated nucleic acid or an isolated nucleic acid fragment is understood as meaning an RNA or DNA polymer which can be single-stranded or double-stranded and which can optionally comprise natural, chemically synthetized, modified or artificial nucleotides. In this context, the term DNA polymer also includes genomic DNA, cDNA or mixtures of these.
Hybridizing nucleotide sequences for the purpose of the invention are understood as meaning oligo- or polynucleotides which bind under standard hybridization conditions with the corresponding nucleotide sequence according to the invention which encodes CYP74 enzymes. The term standard hybridization conditions is to be understood in the broad sense and refers to stringent and less stringent hybridization conditions. Such conditions are described, inter alia, by Sambrook et al. (1989, Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press). In accordance with the invention, the term hybridizing sequences includes substantially similar nucleotide sequences from the DNA or RNA group which undergo a specific interaction (binding) with the nucleotide sequences according to the invention under standard hybridization conditions known per se. These also include short nucleotide sequences with a length of, for example, 10 to 30, preferably 12 to 15, nucleotides. In accordance with the invention, this also encompasses what are known as primers or probes, inter alia.
In accordance with the invention, complementary nucleotide sequences are understood as meaning DNA or RNA (mRNA) sequences which constitutes a transcription of the starting sequence in question in accordance with the base pairing rules.
In accordance with the invention, allenes are understood as being functionally equivalent nucleotide sequences, i.e. nucleotide sequences with essentially the same type of action. Functional equivalent sequences are those sequences which retain the desired functions despite a deviating nucleotide sequence, for example owing to the degeneracy of the genetic code. Thus, functional equivalents encompass naturally occurring variants of the sequences described herein as well as artificial nucleotide sequences, for example nucleotide sequences which have been obtained by chemical synthesis and, if appropriate, adapted to the codon usage of the host organism. In addition, functionally equivalent sequences encompass those with a modified nucleotide sequence which impart for example a desensitivity or resistance to inhibitors to the protein.
A functional equivalent is in particular also understood as meaning natural or artificial mutations of an originally isolated sequence which retain the desired function. Mutations encompass substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
Also included here are what are known as sense mutations which at the protein level may, for example, lead to the substitution of conserved amino acids, but which do not lead to a fundamental change in functionality of the protein, i.e. which are functionally neutral. This also includes modifications of the nucleotide sequence which, at the protein level, relate to the N or C terminus of a protein without, however, adversely affecting the function of the protein to a substantial degree. Indeed, these modifications can have a stabilizing influence on the protein structure. Functional equivalents are also those variants whose activity is weakened or enhanced in comparison with the original gene or gene fragment.
Artificial DNA sequences are also subject matter of the present invention, as long as they confer the desired properties, as described above. Such artificial DNA sequences can be determined for example by back translating proteins generated by means of molecular modeling, or by in-vitro selection. DNA sequences which are especially suitable are coding DNA sequences which have been obtained by back translating a polypeptide sequence in accordance with the host-organism-specific codon usage. The specific codon usage can be determined readily by the skilled worker who is familiar with molecular-genetic methods by means of computer evaluations of other, known genes of the organism to be transformed.
The term “functional equivalent” also refers to the protein encoded by the nucleotide sequence in question. In this case, the term “functional equivalent” describes a protein whose amino acid sequence shows a certain percentage of homology with that of the reference protein (in this case the CYP74 enzymes). This percentage is at least 75%, preferably 80%, especially preferably 90-95% and in particular 99.9%.
Furthermore, the present invention also encompasses for example those nucleotide sequences which are obtained by modifying the nucleotide sequence, which results in corresponding derivatives. The aim of such a modification can be for example the further delimitation of the coding sequence present therein or else for example the insertion of further restriction enzyme cleavage sites.
The nucleotide sequences according to the invention are furthermore distinguished by the fact that they originates from moss or higher plants. Preferably, they originate from Physcomitrella patens. 
By way of clarification of the terminology, it should be pointed out that the protein encoded by SEQ ID No. 1 is a class CYP74A cytochrome P450 enzyme (allene oxide synthase; AOS). Owing to its substrate specificity, inter alia, the enzyme encoded by SEQ ID No. 3 is assigned to the CYP74E class (divinyl ether synthase; DES).
The present invention furthermore relates to a gene construct comprising a nucleotide sequence as shown in SEQ ID No. 1 and/or a nucleotide sequence as shown in SEQ ID No. 3 and regulatory nucleotide sequences which are operatively linked thereto.
The gene constructs according to the invention also include those comprising derivatives, alleles or parts of the nucleotide sequence as shown in SEQ ID No. 1 and/or SEQ ID No. 3. In this context, the coding regions, alone or in combination (i.e. jointly), are under the control of the same regulatory sequences or of a plurality of separate regulatory sequences.
Operative linkage is understood as meaning the sequential arrangement of, for example, promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its intended function when the coding sequence is expressed. These regulatory nucleotide sequences can be of natural origin or be obtained by chemical synthesis. A suitable promoter is, in principle, any promoter which is capable of regulating the gene expression in the host organism in question. According to the invention, such a promoter may also take the form of a natural or synthetically generated chemically inducible promoter by which the expression of the genes governed by it can be controlled in the host cell at a specific point in time. These also include tissue-specific promoters. A gene structure is generated by fusing a suitable promoter to at least one nucleotide sequence according to the invention employing customary recombination and cloning techniques as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). In order to connect the DNA fragments with one another, adapters or linkers may be added to the fragments.
Also encompassed in accordance with the invention are the sequence regions which precede the coding regions (structural genes; 5′ or upstream sequence regions) and/or which follow them (3′ or downstream sequence regions). Also included are, in particular, sequence regions which have a regulatory function. They are capable of influencing transcription, RNA stability or RNA processing, and translation. Examples of regulatory sequences are, inter alia, promoters, enhancers, operators, terminators or translation enhancers.
Also encompassed by the present invention is a vector comprising an isolated nucleotide sequence as shown in SEQ ID No. 1, alleles, derivatives or parts thereof and/or an isolated nucleotide sequence as shown in SEQ ID No. 3, alleles, derivatives or parts thereof and/or a gene construct of the abovementioned type, and additional nucleotide sequences for selection and/or replication in a host cell and/or for integration into the genome of a host cell.
In general, host cells which are suitable in accordance with the invention can be cells of higher plants. Preferred cells are cells of useful plants, preferably monocotyledoneous useful plants, especially preferably cereals and in particular barley and/or wheat. Preferred cells of dicotyledoneous useful plants are those from the Solanaceae family.
Thus, the present invention also encompasses at least one transgenic plant cell, intact plant and/or their progeny, comprising, in replicable form, an isolated nucleotide sequence as shown in SEQ ID No. 1, alleles, derivatives or parts thereof and/or an isolated nucleotide sequence as shown in SEQ ID No. 3, alleles, derivatives or parts thereof and/or a gene construct of the abovementioned type and/or a vector of the abovementioned type, the transgenic plant cell, intact plant and/or their progeny showing enhanced expression of the nucleotide sequence encoding an allene oxide synthase and/or of the nucleotide sequence encoding a divinyl ether synthase in comparison with the endogenous gene expression (i.e. the natural gene expression as is found, for example, in an untransformed plant or plant cell), which brings about an increased resistance of plants to pathogens.
The reason for enhanced gene expression may be an increased copy number of the nucleotide sequence in question. Alternatively, it is based for example on the fact that the coding region of a nucleotide sequence is linked operatively to one or more regulatory sequences which bring about an enhanced initiation of gene expression. This can be brought about for example by a strong and/or inducible promoter and/or enhancer and/or other regulatory sequences.
As regards the copy number, one or both of the abovementioned nucleotide sequences may be present as 2-100, preferably 5-50 and especially preferably 2-15 copies in a variant of the present invention.
In the abovementioned transgenic plant cells, plants and/or their progeny according to the invention, the nucleotide sequences according to the invention (alone or in combination), alleles, derivatives or parts thereof and/or gene constructs and/or vectors of the abovementioned type may be present extrachromosomally and/or integrated stably into the plant genome.
Suitable procedures and aids, such as gene constructs or vectors, and suitable helper organisms for integrating the nucleotide sequence into the plant genome are known to the skilled worker and will not be detailed further.
The transgenic plant cell, intact plant and/or their progeny which has an allene oxide synthase as shown in SEQ ID No. 2 and/or a divinyl ether synthase as shown in SEQ ID No. 4 and/or corresponding isoenzymes, derivatives and/or parts thereof, the relevant specific activity of the enzymes in the transgenic host systems being increased in comparison with the relevant endogenous enzyme activity (for example in the wild-type cells), is likewise encompassed by the present invention.
Isoenzymes are understood as meaning enzymes with the same or a similar substrate specificity and specificity of action, but with a different primary structure.
In accordance with the invention, derivatives are understood as meaning enzymes with modifications in the sequence, for example at the N and/or C terminus of the polypeptide, or in the conserved amino acid region, without, however, adversely affecting the function of the enzyme. These modifications can be carried out in the form of amino acid substitutions by methods known per se.
A particular embodiment of the present invention encompasses variants of the enzymes according to the invention whose activity, in comparison with the starting protein in question, is reduced or increased owing to amino acid substitutions. The same applies to the stability of the enzymes according to the invention in those cells which are more or less sensitive to, for example, degradation by proteases.
Furthermore, the present invention relates to enzymes with the function of an allene oxide synthase or divinyl ether synthase whose amino acid sequence has been modified in such a way that they are desensitive to regulatory compounds, for example the catabolites which regulate their activity (feedback desensitive).
In accordance with the invention, the abovementioned transgenic plant cell, intact plant and/or their progeny takes the form of a useful plant or its cells, preferably from the Solanaceae family or the cereal family, especially preferably potato, barley or wheat.
The present invention also encompasses a method for increasing the resistance of plant cells or plants to pathogens, wherein a nucleotide sequence as shown in SEQ ID No. 1 and/or a nucleotide sequence as shown in SEQ ID No. 3 and/or a gene construct and/or a vector of the above-detailed type is transferred, in replicable form, into plant cells and intact plants are regenerated from the plant cells thus transformed.
Methods for the generation of such transgenic plants according to the invention are standard laboratory practice. In an advantageous variant of the present invention, the nucleotide sequence and/or gene construct and/or vectors are transferred into the plant or plant parts or cells by what is known as “particle bombardment” and/or by agrobacteria-mediated transformation.
A particular advantage of the transgenic plant and its progeny obtained in accordance with the invention is that an increased copy number of at least one of the nucleotide sequences according to the invention or, analogously, the presence of an increased concentration of at least one correspondingly encoded protein, leads to a substantially increased disease resistance. This means that the gene(s) according to the invention and/or the encoded protein(s) is/are causative for the development of the resistance to pathogenic pests. Examples of pathogenic pests are, inter alia, Blumeria graminis f. sp. hordei or f. sp. tritici, Pythium spec., Albugo spec., Rhizoctonia solani, Peronospora parasitica, Erysiphe cruciferarum, E. cichoracearum, Alternaria brassicicola, Botrytis cinerea, Sclerotium rolfsii, Sclerotinia sclerotium, Fusarium oxysporum, F. culmorum, F. graminearum, F. nivale, phytophtora infestans or Pseudomonas syringae. 
In a preferred variant of the method according to the invention, the plant cells employed are cells of useful plants, preferably from the Solanaceae family or the cereal family, especially preferably potato, barley or wheat.
The present invention furthermore relates to the use of a nucleotide sequence as shown in SEQ ID No. 1 and/or a nucleotide sequence as shown in SEQ ID No. 3 and/or their alleles, derivatives and/or parts thereof for increasing the resistance of transgenic plant cells, intact plants and/or their progeny to pathogens.
The present invention also relates to the use of at least one polypeptide as shown in SEQ ID No. 2 and/or a polypeptide as shown in SEQ ID No. 3 and/or isoenzymes and/or derivatives thereof for increasing the resistance of transgenic plant cells, intact plants and/or their progeny to pathogens.
Also encompassed in accordance with the invention is the use of at least one of the abovementioned enzymes, where an increased specific activity of at least one enzyme in comparison with the corresponding endogenous specific enzyme activity brings about an increase in the resistance of the plant cells or plants to pathogens by 20-90%, preferably by 30-80% and especially preferably by 40-70%.
In this context, the use of at least one of the enzymes according to the invention brings about, for example, an increased resistance to powdery mildews, preferably Blumeria graminis f. sp. hordei or f. sp. tritici and/or Phytophtera infestans. 
The examples which follow are intended to illustrate the invention and are not limiting.
General Methods:
General DNA and cloning techniques, gel electrophoreses, sequencing, PCR, Northern blots, expression and purification of recombinant proteins, Western blots, HPLC and GC analyses and the cultivation of microorganisms are conventional laboratory methods and described, inter alia, in Sambrook et al. (Molecular cloning. A laboratory manual (1989) Cold Spring Harbor Laboratory Press).
The treatment of moss and plant material is likewise customary laboratory practice and described, inter alia, in Gelvin et al. (Plant Molecular Biology Manual, 1995, Dovdrecht/Holland, Kluwer Academic Publ.).
Cloning the Genes Encoding CYP74 Enzymes Via PCR
Two Physcomitrella patens lambda ZapII cDNA libraries, one from Protonematal and one from Gametophyte tissue, were available for the RACE-PCR. The T7-lang primer (5′-GTA ATA CGA CTC ACT ATA GGG CGA ATT GGG-3′) acted as vector primer for the 5′-RACE-PCR. Standard methods were used.
For the nested RACE-PCR, a first PCR was carried out using M13rev primer (5′-GGA AAC AGC TAT GAC CAT G-3′) and a RACE primer. This mixture acted as template for a second PCR with T7-lang primer and a (gene-specific) nested-RACE primer selected from amongst:









PP291AOS5R:
5′-TCA CCT CAT CCG ATA CGC TAG TC-3′



 


PP364AOS5R:
5′-GTC GAT GTC GTC TCA ATG TTC C-3′


 


PP364AOS5R2:
5′-CCA TTC GTG ATT GCC AGA ACT GC-3′






To clone the complete fragments, the latter were amplified with the aid of the ExpandTM PCR system. In addition to the Taq polymerase, this system contains a Pwo polymerase with a proof-reading activity. The purpose of this second polymerase is to keep down the reading error rate in order to obtain as few mutations as possible in the DNA to be cloned. The following cDNA libraries were used as templates: Protonematal P. patens cDNA lambda ZapII and Gametophytic P. patens cDNA lambda ZapII. The following primers were employed:








PP291/5′SphI



5′-AAA GCA TGC ATG GCA GTC CCT TCA TCC AAG C-3′


 


PP291/3′PstI


5′-AAA CTG CAG TCA CTT TTT GAG ATC GGA AAA GAA AAC


CTT GGT CGC-3′


 


PP364/5′BamHI


5′-GGA TCC CGT ACG GTT GTA GCC AGT CTT GGG-3′


 


PP364/3′HindIII


5′-AAG CTT TCA ATC TGA TCG CGG CGT CAG TG-3′






To check the clones during the cloning of RACE and complete cDNAs, a process known as “colony” PCR with Tfl polymerase was carried out since the error rate was of no importance in this context. The primers were retained and the program was carried out using standard settings.
Isolation of the CYP74 Enzymes
Two genes encoding CYP74 enzymes were isolated from two Physcomitrella patens lambda ZapII cDNA libraries.
Clone Pp291
The clone Pp291 which was isolated had an open reading frame for 322 amino acids. This protein had 48% identity with a Parthenium argentatum AOS (Pan et al.; 1995, J. Biol. Chem. 270, 8487-8494). However, a comparison of its nucleotide sequence with other known AOSs revealed that approximately 500 to 650 bp were still missing at the 5′ terminus. This is why a 5′-RACE-PCR was carried out with a lambda ZapII cDNA library from Protonematal tissue and with a lambda ZapII cDNA library from Gametophyte tissue. Using the RACE primer PP291AOS5R (see above) and an annealing temperature of 60° C., three fragments of different lengths were successfully amplified from the Protonematal library. An estimation of the fragment length was possible by comparison with the size standard also included in the agarose gel. The fragment lengths were 600 bp, 700 bp and 800 bp. The two longer fragments were cloned into the vector pGEM-T and sequenced. Both of the resulting sequences constituted an extension of the 5′ terminus of the starting clone. A stop codon was located 15 bp upstream of the first start codon (ATG) in both of the sequences. To clone the complete cDNA, the expression primers (PP291/5′SphI and PP291/3′PstI, see above) with restriction cleavage sites for SphI and PstI were derived from the cDNA sequence of the RACE-PCR. The complete cDNA sequence obtained is shown in SEQ ID No. 1 and encodes a protein with 475 amino acids (SEQ ID No. 2).
Clone Pp364
The second cDNA clone contained an open reading frame which encoded a protein with 489 amino acids. This protein had 42% identity with the Arabidopsis thaliana AOS (Laudert et al.; 1996, Plant Mol. Biol. 31, 323-335) and 41% identity with the L. usitatissimum AOS, both at the amino acid level. Despite its length, this clone contained no start codon. The expression primers PP364/5′BamHI and PP364/3′HindIII with the restriction cleavage sites for BamHI and HindIII were derived from the known clone sequence. The complete cDNA sequence was obtained by RACE-PCR and inverted PCR. The complete cDNA sequence is shown in SEQ ID No. 3 and encodes a protein with 532 amino acids (SEQ ID No. 4).
Expression and Purification of Recombinant Proteins
In order to express the isolated cDNAs, they had to be ligated into an expression vector. The vector pQE30 (Qiagen, Hilden) was used in order to express proteins with N-terminal His6-Tag. The ligation between pre-cut pQE30 and donor DNA (approximate ratio 3:1) was carried out with T4-DNA ligase.
The recombinant proteins were expressed in the E. coli strain SG13009 (Gottesmann et al.; 1981, J. Bacteriol. 148, 265-73). First, the expression clones were incubated at 37° C. in LB medium (with carbenicillin and kanamycin) until an OD600 of 0.6-0.8 was reached. After induction with IPTG (final concentration 1 mM), the bacteria were grown for a further 2-3 days at 10° C.
As far as possible, the purification was carried out on ice, while the centrifugation steps were carried out at 4° C. The cells were sedimented by centrifugation for 15 minutes at 4000×g.
This cell sediment was resuspended completely in 50 mM sodium phosphate, pH 8, and sonicated (Sonopuls GM 70, Bandelin, Berlin) for 5×1 minute at 50% intensity and 50% pulse. The cell debris was removed by centrifugation in 15 minutes at 4000×g, and the cell membranes were sedimented from the supernatant by a further centrifugation (1 hour at 100 000×g).
In the case of both clones, Pp291 and Pp364, the recombinantly produced proteins were purified via affinity chromatography. This was followed by elution using a pH gradient (FIG. 1A) and, as an alternative, an imidazole gradient (FIG. 1B). Identical volumes (1 ml) were precipitated in all wash and elution steps and applied to the gel.
As can be seen from FIG. 1A, pure Pp291 protein was eluted from the column at a pH of 4. In the case of the imidazole gradient, the protein eluted in relatively pure form at an imidazole concentration of 100 mM and above. In comparison of the two methods, no difference was observed. This is why hereinbelow the protein was purified using the pH gradient only.
Activity Assays
Since sequence alignments do not allow conclusions regarding the catalytic activity, the products formed during the catalysis of the enzymes were analyzed by means of a photometric activity assay and by HPLC and GC analyses, all of which were carried out by standard methods.
In the photometric activity assay, the drop in absorption is measured at 234 nm. This drop is based on the breakdown of the conjugated diene system of the hydroperoxides during the reaction with Cyp74 enzymes. To this end, the respective substrate was dissolved (for the purposes of pure activity assay, generally to a final concentration of 20 μM 13-HPOTE or 9-HPODE) in phosphate buffer (the pH depending on the pH optimum of the enzyme) and the reaction was started by addition of the enzyme. The substrate concentration was monitored photometrically via the absorption at 234 nm. The molar absorption coefficient (λ234 nm) is 25 000 M−1 cm−1.
Both substrates (13-HPOTE and 9-HPODE) were converted both by the enzyme encoded by clone Pp291 and by the enzyme encoded by clone Pp364. A graphic representation is shown in FIG. 2.
The reaction of hydroperoxides with DES gives rise to divinyl ethers which hydrolyze in an acidic environment to give aldehydes. These aldehydes are volatile. In order to identify these aldehydes which are formed, they can be derivatized with DNPH (Kohlmann et al.; 1999, Eur. J. Biochem. 260, 885-895). The hydrazones thus formed are no longer volatile and can be identified with the aid of an HPLC analysis via their retention time. As FIG. 3 shows, divinyl ethers, when treated with the derivatizing reagent, can also breakdown into aldehydes, which can then be detected in the form of DNPH derivatives.
Using this method, it is possible to show that aldehydes are formed in the reaction mixture of the enzyme encoded by clone Pp364 and the substrates 13-HPOTE and 9-HPODE (FIG. 4). These aldehydes were identified by their retention times as (2E)-hexenal and (2E)-nonenal. They also revealed a UV spectrum which is typical of aldehyde-DNPH derivatives. No aldehydes were detected in the case of Pp291 (data not shown).
In the HPLC analysis of the non-volatile compounds, 1-14C-labeled hydroperoxides are reacted. Labeled products which can be visualized with the aid of a radio detector instead of a UV detector are formed. In this manner, even substances which have no typical UV maximum can be identified via their retention time. A further advantage is the fact that this type of detection is more sensitive than UV detection. If [1-14C-13-HPOTE is employed as substrate in the enzyme reaction, the chromatogram shown in FIG. 5 reveals the same elution profile for Pp291 as for barley 9/13-AOS1 (Maucher et al.; 2000, Plant J. 21, 199-213), which was used as reference. It has already been demonstrated for other AOSs that the main reaction product in the absence of allene oxide cyclase is the α-ketol.
The chromatogram of the expression and the subsequent conversion of Pp364 showed a signal at approximately 30 minutes (FIG. 6). This signal was found neither in cucumber 9/13-HPL nor in barley 9/13-AOS1. Pp364 additionally showed other signals with the same retention time as the products of the cucumber 9/13-HPL.
Since, in radio-HPLC, liquid scintillator is added to the column eluate, the same HPLC run was carried out with unlabeled substrates in order to collect the products and to analyze them further. Since α-ketols have no typical UV maximum, the detection was carried out at 210 nm in this case (Gardner, 1997, Advances in Lipid Methodology—four (Christie, W. W., ed.) pp. 1-43, The Oily Press, Dundee).
In the case of Pp291, a substance with the same retention time as the standard for the α-ketol formed from 13-HPOTE was collected, derivatized and studied with the aid of GC/MS. The chromatogram shown in (FIG. 7) resulted. The mass spectrum is identical with that of the corresponding α-ketol standard.
Determination of Enzyme Characteristics
pH Optimum
The pH optimum for clone Pp291 was determined photometrically. To this end, 13-HPOTE was used as substrate (approx. 25 μM in 50 mM sodium phosphate with the appropriate pH). The highest activity is shown by the enzyme in a pH range of between 5.0 and 6.0. This is shown in FIG. 8.
Enzyme-Kinetic Parameters
Pp291 was studied with regard to its substrate specificity in particular regarding hydroperoxides of arachidonic acid (HPETE) since this is the predominant fatty acid of this organism (Girke et al.; 1998, Plant J. 15, 39-48). The ratio of the conversion rates shown in FIG. 9 were found. Among the hydroperoxides studied in the present context, 8-HPETE emerges as the substrate which is converted most rapidly. It is followed by 13-HPOTE, 9-HPODE and 11-HPETE in the ratio 8-HPETE:13-HPOTE:9-HPODE:11-HPETE, 100:70:60:57.
Overexpression for Generating Increased Pathogen Resistance
For cloning the Pp291 and Pp364 cDNAs into binary vectors, they were restricted with BamHI and NotI and transferred into a BamHI- and NotI-cut pCRScript vector. They are subsequently excised from this vector using SalI and ligated into a suitably SalI-cut pBinAR vector. Those clones in which the genes are in sense orientation are determined by control excision with BamHI and sequencing.
Transformation into Arabidopsis thaliana: 
Using these pBinAR vectors containing in each case a Pp291 and Pp364 cDNA in sense orientation, Agrobacterium tumefaciens (C58C1 pMP90) is transformed. Thereafter, wild-type Arabidopsis thaliana plants (cv. Columbia) are transformed with the respective transformed Agrobacterium tumefaciens strain on the basis of a modified method of the vacuum infiltration method of Clough and Bent (Clough S. and Bent A., Plant J 1998, 16(6):735-43) and Bechtold, et al. (Bechtold, N. et al., CRAcad Sci Paris 1993, 1144(2):204-212).
Seeds of the primary transformants are screened on the basis of the kanamycin resistance by planting seed on kanamycin-containing MS plates (MS medium (Sigma) supplemented with 40 mg/l kanamycin, 10 mg/l benomyl and 100 mg/l timentin). After 2 weeks, kanamycin-resistant seedlings are transferred into soil; when they have grown into fully developed plants they are used for phenotypic and molecular analysis.
Among the transgenic plants which have been transformed with Pp291 and Pp364 in sense orientation, those in which no cosuppression effect was observed were selected. To this end, total RNA is isolated from the plants, and the presence of the messenger is detected by means of real-time PCR (Perkin-Elmer). The transgenic plants identified thus were infected with various pathogens. These included Blumeria grammis f.sp. hordei and f.sp. tritici, Pythium spec., Albugo spec., Rhizoctonia solani, Peronospora parasitica, Erysiphe crucifearum, E. cichoracearum, Alternaria brassicicola, Botrytis cinerea, Sclerotium rolfsii, Sclerotinia sclerotium, Fusarium oxysporum, F. culmorum, F. graminearum, F. nivale or Pseudomonas syringae. 
The result, i.e. the macroscopic degree of resistance of the plant to the pathogenic fungus or bacterium, was analyzed with the aid of fluorescence and light microscopy, inter alia. It emerged that the Pp291- and Pp364-transgenic Arabidopsis plants showed an increased resistance to the abovementioned pathogens in comparison with the wild type.
Transformation into Barley:
Barley cv. Pallas leaf segments were transformed with a cDNA of Pp291 or Pp364 which were present in the GFP expression vector (green fluorescence protein). The leaves were subsequently inoculated with the pathogenic fungus Blumeria graminis f.sp. hordei (powdery mildew of barley), and the result was analyzed after 48 hours by means of light microscopy and fluorescence microscopy. The penetration into GFP-expressing cells was assessed by detecting haustoria in live cells and by assessing the fungal development in precisely those cells. In all six experiments, bombardment of barley cv. Pallas with Pp291 or Pp364 cDNA resulted in a reduced number of cells which were successfully penetrated by Blumeria graminis f.sp. hordei (powdery mildew of barley) in comparison with cells which had been bombarded with a foreign control cDNA (human thyroid hormone receptor dsRNA, TR). The resistance-inducing effect of the Pp291 and Pp364 cDNA resulted in a reduced penetration efficiency by Blumeria graminis f.sp. hordei by 44% on average.
The transient transformation of barley was carried out with a method which had already been described for the biloistic introduction of cDNA into epidermal cells of barley leaves (Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54; Schweizer P et al. (2000) Plant J 2000 24: 895-903). Tungsten particles with a diameter of 1.1 mm (particle density 25 mg/ml) were coated with cDNA together with plasmid DNA of the vector PGFP (GFP under the control of the CaMV 35S promoter) as transformation marker. To this end, the following amount of cDNA and reporter plasmid were used for coating purposes for each bombardment: 1 mg of pGFP and 2 mg of cDNA. To prepare the microcarriers, 55 mg of tungsten particles (M 17, diameter 1.1 mm; Bio-Rad, Munich) were washed twice with 1 ml of autoclave distilled water and once with 1 ml of absolute ethanol, dried and taken up in 1 ml of 50% strength glycerol (approximately 50 mg/ml stock solution). The solution was diluted with 50% strength glycerol to 25 mg/ml, mixed thoroughly before use and suspended in an ultrasonic bath. To coat the microcarriers, 1 mg of plasmid, 2 mg of cDNA (1 mL), 12.5 ml of tungsten particle suspension (25 mg/ml), 12.5 ml of 1 M Ca(NO3)2 solution (pH 10) per bombardment were combined dropwise with constant mixing, left to stand for 10 minutes at RT and centrifuged briefly, and 20 ml of supernatant were removed. The remainder, which contained the tungsten particles, is resuspended (ultrasonic bath) and employed in the experiment.
Barley primary leaf segments approximately 4 cm in length were used. The tissue was placed on 0.5% Phytagar (GibcoBRLt Life Technologiest, Karlsruhe) supplemented with 20 mg/ml benzimidazole in Petri dishes (diameter 6.5 cm); immediately prior to the particle bombardment, the edges were covered with a stencil with a rectangular slot 2.2 cm×2.3 cm in size. One after the other, the dishes were placed on the bottom of the vacuum chamber (Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54) above which a nylon mesh (mesh size 0.2 mm, Millipore, Eschborn) had been inserted on a perforated board (5 cm above the bottom, 11 cm underneath the macrocarrier, see hereinbelow) to act as diffuser in order to diffuse particle aggregations and to slow down the particle stream. For each bombardment, the macrocarrier (plastic sterile filter holder 13 mm, Gelman Sciences, Swinney, UK) which had been attached at the top of the chamber was loaded with 5.8 ml of DNA-coated tungsten particles (microcarrier, see hereinbelow). The pressure in the chamber was reduced by 0.9 bar using a membrane vacuum pump (Vacuubrand, Wertheim), and the surface of the plant tissue was bombarded with the tungsten particles at a helium gas pressure of 9 bar. The chamber was aerated immediately thereafter.
To label transformed cells, the leaves were bombarded with the plasmid (pGFP; pUC18-based vector, CaMV 35S promoter/terminator cassette with inserted GFP gene; Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54; provided by Dr P. Schweizer, Schweizer P, Institute of Plant Genetics IPK, Gatersleben, Germany). Before carrying out the bombardment with another plasmid, the macrocarrier was cleaned thoroughly with water in each case. After the leaves had been incubated for four hours post-bombardment at RT in daylight, with the Petri dishes being left slightly open, they were inoculated with 100 conidia/mm of the powdery mildew of barley fungus (race A6) and incubated for a further 40 to 48 hours under identical conditions.
Leaf segments were bombarded with the coated particles using a particle inflow gun. For each bombardment, 312 mg of tungsten particles were applied. 4 hours after the bombardment, the leaves were inoculated with Blumeria graminis mildew (race A6), and, after a further 40 hours, evaluated for infection symptoms. The result (for example the penetration efficiency, defined as percentage of attacked cells which form a mature haustorium and a secondary elongating hyphae; calculation: see hereinbelow) was analyzed by means of fluorescence microscopy and light microscopy. Inoculation with 100 conidia/mm2 results in an attack frequency of approximately 50% of the transformed cells. A minimum of 100 interaction sites was evaluated for each individual experiment. Transformed (GFP-expressing) cells were identified under excitation with blue light. Three different categories of transformed cells were distinguished:

    1. penetrated cells containing a readily recognizable haustorium. A cell with more than one haustorium was rated as one cell.
    2. Cells which, while attacked by a fungal appressorium, contain no haustorium. A cell which had been attacked repeatedly by Blumeria graminis ssp. hordeum, but which contains no haustorium, was rated as one cell.
    3. Cells not attacked by Blumeria graminis ssp. hordeum.


Stomatol cells and subsidiary cells were not included in the evaluation. Surface structures of Blumeria graminis ssp. hordeum were analyzed by light microscopy and fluorescence staining of the fungus with 0.1% Calcofluor (w/v in water) for 30 seconds. The fungal development can be evaluated readily by fluorescence microscopy after staining with Calcofluor. While the fungus develops a primary and an appressorial germ tube in Pp291- or Pp364-dsRNA-transformed cells, no haustorial growth takes place. Haustorial development is a condition for the development of a secondary hypha.
The relative penetration efficiency (RPE) is calculated as the difference between the penetration efficiency in transformed cells (transformation with Pp291 or Pp364 cDNA) and the penetration efficiency in untransformed cells (here: average penetration efficiency 57%). The RPE in percent (% RPE) is calculated by subtracting 1 from the RPE and multiplying the result by 100.

    RPE=[PE in transformed cells]
    
        [PE in untransformed cells]

% RPE=100×(RPE−1)

    
    


The % RPE value (deviation from the average penetration efficiency of the control) serves to determine the susceptibility of cells transformed with pp291 or Pp364 cDNA.
In five independent experiments, the control cDNA revealed no difference between the transfection with the control cDNA and water with regard to the penetration efficiency of Blumeria graminis ssp. hordeum. 
To rule out an effect of the cDNA on the transformation rate or survival rate of the attacked cells, the number of GFP-expressing cells between control and Pp291, or Pp364, cDNA experiments was compared. The Pp291 and Pp364 cDNA had no effect on the total number or the number of attacked GFP-expressing cells.
The transfection rate with Pp291 and Pp364 resulted in a drastic drop in the penetration frequency of Blumeria graminis f.sp. hordei (average % RPE value=−30%).

## Claims

1. An isolated allene oxide synthase with the amino acid sequence of SEQ ID NO: 2.

2. An isolated divinyl ether synthase with the amino acid sequence of SEQ ID NO: 4.

3. The isolated allene oxide synthase of claim 1 which converts both 9-HPOD/TE and 13-HPOD/TE as substrates.

4. A method for increasing resistance of a plant cell, plant or progeny thereof to a pathogen, the method comprising administering the isolated allene oxide synthase of claim 1 to said plant cell, plant or progeny thereof.

5. The method of claim 4, wherein the administering of the synthase results in an increase in resistance to the pathogen by 20-90% when compared to the pathogen resistance shown in a corresponding untreated plant cell, plant, or progeny thereof.

6. The method of claim 4, wherein the administering of the synthase results in an increased resistance to mildew.

7. The isolated divinyl ether synthase of claim 2, which converts both 9-HPOD/TE and 13-HPOD/TE as substrates.

8. A method for increasing resistance of a plant cell, plant or progeny thereof to a pathogen, the method comprising administering the isolated divinyl ether synthase of claim 2 to said plant cell, plant or progeny thereof.

9. The method of claim 8, wherein the administering of the synthase results in an increase in resistance to the pathogen by 20-90% when compared to the pathogen resistance shown in a corresponding untreated plant cell, plant, or progeny thereof.

10. The method of claim 5, wherein the increase in resistance to the pathogen is 30-80%.

11. The method of claim 9, wherein the increase in resistance to the pathogen is 30-80%.

12. The method of claim 5, wherein the increase in resistance to the pathogen is 40-70%.

13. The method of claim 9, wherein the increase in resistance to the pathogen is 40-70%.

14. The method of claim 8, wherein the administering of the synthase results in an increased resistance to mildew.

15. The method of claim 6, wherein the mildew is caused by Blumeria graminis, Blumeria hordei, Blumeria tritici or Phytophtora infestans.

16. The method of claim 14, wherein the mildew is caused by Blumeria graminis, Blumeria hordei, Blumeria tritici or Phytophtora infestans.