EP1257636A2

EP1257636A2 - Ozone and/or pathogen-inducible gene expression in plants

Info

Publication number: EP1257636A2
Application number: EP01903725A
Authority: EP
Inventors: Hélène CHIRON; Alain Drouet; Dieter Ernst; Heinrich Sandermann
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date: 2000-02-11
Filing date: 2001-02-09
Publication date: 2002-11-20
Also published as: WO2001059139A2; WO2001059139A3; AU2001231719A1; DE10006204A1

Abstract

The invention relates to recombinant nucleic acid molecules that comprise plant defense genes, as well as promoters that mediate a pathogen and/or ozone-inducible gene expression. The invention further relates to a method for producing new plants that contain said nucleic acid molecules or fragments thereof, to the corresponding new plants and to the use of the promoters for inducing pathogen and/or ozone-responsive gene expression in plants and plant cells.

Description

Ozone and / or pathogen inducible gene expression in plants

The invention relates to recombinant nucleic acid molecules which comprise plant defense genes and promoters which mediate gene expression which can be induced by pathogens and / or ozone. The invention further relates to methods for generating new plants which contain these nucleic acid molecules or fragments thereof, these new plants and the use of the promoters for generating pathogen and / or ozone-responsive gene expression in plants and plant cells.

Stilbenes were said to have an antibacterial and fungitoxic effect at an early stage. They are found in some unrelated plant families, but only rarely in important crop plants. In the pine (Pinus sylvestris), the constitutive stilbenes Pinosylvin (PS; 3,5-dihydroxystilbene) and Pinosylvinmonomethylether (PSM) occur exclusively in the heartwood (H. Kindl (1985), Biosynthesis of stilbenes in: Biosynthesis and Biodegradation of Wood Components ( Ed .: T. Higuchi), Academic Press, London, 349-377). The synthesis of these two compounds is, however, induced in the shoots, the phloem and the needles in response to wounding or attack by fungi (JH Hart (1981), Annu. Rev. Phytopathol. 19, 437- ^ 58; Kindl, supra; Lieutier et al. (1996), Eur. J. For. Path. 26, 145-158).

The stilbenes can generally be divided into constitutive compounds to protect wood degradation from microorganisms and into induced phytoalexins, which protect the phloem against bark beetles and other insects as well as against fungi symbiotically associated with beetles (Hart, supra; Lieutier, supra). The metabolism to methoxystilbenes starts from L-phenylalanine and includes the activities of the enzymes phenylalanine ammonium lyase (PAL), stilbene synthase (STS) and pinosylvin-3-O-methyltransferase (PMT). It has been shown that STS activity is the limiting factor in metabolism to PS and PSM (S. Schanz et al. (1992), FEBS 313, 71-74). Changes in the gene expression of STS and / or PMT are likely to have a decisive influence on the induced resistance of plants by stilbenoids acting as phytoalexins.

It is known that plants are protected from various stress factors by PSM much more efficiently than by PS. Examples of this are protection against pathogenic organisms such as, for example, fungi (Lieutier, supra) and protection against ozone (Rosemann et al. (1991), Plant Physiol. 97, 1280-1286), PSM being significantly more antimicrobially active than PS, the analog connection to resveratrol, another stilbene. Furthermore, it is known that pinosylvin-3-O-methyltransferase (PMT) can be induced much more strongly by ozone than stilbene synthase (STS) (Trost, dissertation LMU Munich, 1994). Thus the regulation of the activity of Pinosylvin-3-O-methyltransferase is of essential biological and economic importance.

Methyl transferases are known per se. They catalyze the transfer of methyl groups from one compound to another. Vegetable O-methyltransferases are also known which cause methylation of the oxygen atom. Examples of this are the O-methyltransferase from Pinus radiata (Acc. No. U70873), the caffeic acid-O-Methyltransferase from Pinus taeda (Acc. No. U39301), the catechol-O-methyltransferase from Nicotiana tabacum (Acc. No. X74452) and the lignin-bispecific acid / 5-hydroxyferulic acid-O-methyltransferase from Populus tremoides (Acc. No. X62096).

DNA sequences which code for enzymes which catalyze stilbene synthase enzymes, ie enzymes which catalyze the synthesis of stilbenes such as resveratrol or pinosylvin, are described, for example, in European patent specification EP 0 309 862, German patent application DE-A-41 07 396 and European patent application EP 0 464 461 and the US Pat. No. 5,500,367. These documents describe the isolation of stilbene synthase genes and their use in the generation of transgenic plants. The resulting transgenic plants have an increased resistance to various plant pests such as fungi, bacteria, insects, viruses and nematodes. For example, STS from grapevine was expressed in tobacco and rice and then led to increased resistance to pathogens (Hain et al. (1993), Nature 361, 153-156; Stark-Lorenzen et al. (1997), Plant Cell Rep. 16, 668-673). As mentioned above, since PSM protects plants from various stress factors much more effectively than PS, knowledge of the DNA sequence coding for the PMT catalyzing PSM synthesis would be extremely desirable.

There are now various indications that it is advantageous for the most efficient disease resistance based on the expression of plant defense genes such as STS or PMT in transgenic plants if the expression of the heterologous plant defense gene (or the heterologous plant defense genes) in the plant is stimulated only by the attack of the pathogens, ie only by the interaction of the plant and pathogen (Fischer and Hain (1994), Current Opinion in Biotechnology 5, 125-130; Fischer, optimization of the heterologous expression of stilbene synthase genes for crop protection, dissertation University of Hohenheim, 1994). For example, studies with transgenic tobacco plants in which STS genes were expressed under the constitutive 35S RNA promoter of Cauliflower Mosaic Virus showed that the disease resistance achieved in these plants is lower than in plants which have the same genes under the control of the pathogen. inducible homologous STS promoters after fungal infection (Fischer and Hain (1994) supra; Fischer (1994) supra.) Induction of pathogen defense in the latter system therefore occurs only at the required point in time and not permanently. It is therefore an important object of the present invention to provide DNA sequences which code for PMT. In particular, it is an essential object of the present invention to provide a PMT promoter which is inducible by pathogens and / or ozone in order to achieve increased disease resistance in plants in which the PMT genes or another plant defense gene are under the control of a pathogen and / or or ozone inducible promoter.

Furthermore, it is an essential object of the present invention to provide a method for producing transformed plant cells or plants which have increased pathogen and / or ozone-inducible disease resistance. Further objects of the invention will become apparent from the following description and the appended claims.

The features of the independent protection claims serve to solve these tasks.

Advantageous configurations are defined in the respective subclaims.

This object is achieved according to the invention by a recombinant nucleic acid molecule, the regulatory sequences of a promoter active in plant cells which mediates gene expression which can be induced by pathogens and / or ozone, and operatively linked thereto a DNA sequence which is essential for a protein with the enzymatic activity of a pinosylvin Encoded -3-O-methyltransferase (PMT). Recombinant nucleic acid molecules of this type, after introduction into plant cells, lead to an accumulation of PSM in the transgenic plant cells or plants and thus to effective protection against ozone and / or Pathogens like mushrooms. Since PSM has a significantly stronger antimicrobial activity than PS, the synthesis of which is catalyzed by the STS gene product described in the prior art, the nucleic acid molecule according to the invention can be used with above-average advantage, for example, in modern crop protection.

The recombinant nucleic acid molecules according to the invention comprise functional fragments of the DNA. “Functional” means fragments of the PMT sequence with specific functions, such as promoters, enhancers and other control sequences.

In a preferred embodiment of the present invention, the promoter and / or the DNA sequence of the recombinant nucleic acid molecule according to the invention is isolated from Pinus sylvestris (pine).

In particular, a genomic DNA sequence with the sequence SEQ No. 1 from Pinus sylvestris, which codes for a PMT protein. This genomic sequence includes a total of 1908 base pairs including exon, intron and flanking sequences. The gene that codes for a protein with the enzymatic activity of a PMT consists of two coding exon regions and an intron region. The intron is 102 base pairs in size, which is common for introns (introns usually consist of between 70 and thousands of nucleotides; GJ Goodall and W. Filipowicz (1991), EMBO J. 10, 2635-2644). The 5'-exon / intron and 3'-intron / exon limits confirm the well-known GT / AG donor / acceptor rule, which applies both in plants and in animals (JWS Brown (1986), Nucl. Acids Res. 14, 9549-9559). On closer inspection, the AG / GTA motif at the 5 'exon / intron splice point matches the AG / GTAAG consensus sequence. The intron also contains a CAG / motif at the 3'- Intron / exon splice site analogous to the TGCAG / G consensus sequence for plant genes (Goodall and Filipowicz, supra). The intron is AT-rich, which is essential for the splicing process (Goodall and Filipowicz, supra). The upstream sequences showed conserved prokaryotic elements such as the cap signal TCATTCGT at position -18, one identified as a TATA box

TGATAAAAGCA motif at position -46 and putative CAAT boxes at positions -80 and -210 (see SEQ ID No. 1).

In an essential object of the present invention, a promoter of the PMT gene or fragments thereof is provided which mediate gene expression in plant cells which can be induced by pathogen and / or ozone. Such an inducible promoter has the advantage over constitutive promoters that the pathogen defense by the products of the genes or coding sequences operatively linked to the promoter according to the invention (for example cDNA sequences) only takes place at the required point in time and not permanently, and thus a higher disease resistance in Plants that contain this chimeric gene construct can be obtained. The promoter according to the invention is preferably isolated from Pinus sylvestris.

Examples of fragments of the promoter of the PMT gene from Pinus sylvestris (jaw) according to the invention are W boxes in the range from -450 to -420, -280 to -240, -220 to -180 and -100 to -30, which are particularly suitable for defense against pathogens are important (Rushton and Somssich (1998), Curr. Opinion Plant Biol. 1, 311-315), as explained in detail below.

The Scottish pine PMT promoter according to the invention contains potential cis elements. yb-responsive elements (MRE) are located at positions - 121, -129, -194, -230, -310 and -358. The ACTTACCACCCT element in position - 358 corresponds in eight out of twelve positions to the consensus sequence T / A CT C / A ACCTA C / ACC / A in plant gene promoters of phenylpropanoid metabolism induced with UV light and with mushroom monitors (Lois et al. (1989) , EMBO J. 8, 1641-1648). At positions -121 and -230, the (A + C) -rich motifs CCAACCACCTCC and CCAACCACTC match with a second one

Consensus motif (CC A AJC CA / T AAC C / T CC) in ten or nine out of twelve positions (Lois et al., Supra). The element GTTG at position -194 is the inverse of the core motif CAAC. Another motif at position -129, TCCCATCTCC, corresponds in nine out of ten positions to the consensus sequence of the boxE (A / T CCC A / T ^' T / ACA / TA / TG / Q, which is evident in stress-inducible phenylpropanoid genes -Promotors is conserved (B. Grimmig and U. Matern (1997), Plant Mol. Biol. 33, 323-341).

The PMT promoter from Pinus sylvestris according to the invention also contains the following regulatory, ice-acting elements which are known for genes which synthesize flavonoids and stilbene: G-box-like motifs (GTGG on the

Positions -147 and -165, CACC at position -353; Faktor et al. (1996) Plant Mol.

Biol. 32, 849-859), the H-box-like motif CCATCC in inverse orientation

(GGTAGG at position -140), the GC box GGGGCAGAAT (consensus sequence G / T GGGCGG G / A G / A C / T) at position -72; Elicitor-responsive elements, i.e. W-

Boxing (ACTG at positions -55, -199 and -263, and TGAC at positions -440;

Rushton and Somssich, supra) and an SV40 enhancer core in inverse orientation

(TTACCAC at position -356).

The characterization of the promoter from Pinus sylvestris according to the invention enables the determination of potential cis-regulatory elements, which may be in relation to the rapid temporary accumulation of PMT mRNA by the treatment of jaws with ozone and fungal icitor stand. Genes encoding PAL, cinnamate 4-hydroxylase and 4-coumarate-CoA ligase (4CL) are known to be under strong control at the level of transcription and to respond to both developmental and environmental stimuli can be expressed in a coordinated manner in many plant species. The members of the My b family are involved in the regulation of these phenylpropanoid genes (Rushton and Somssich, supra).

MREs are located in the PMT promoter from Pinus sylvestris according to the invention at positions -121, -129, -194, -230, -310 and -358. The MREs at positions -358, -310 and -230 were in parsley as boxes L, A and P for almost all known PAL and 4 CL genes (Logemann et al. (1995), Proc. Natl. Acad. Sci. USA 92, 5905-5909) and also for CCoAOMT genes in parsley (Grimmig and Mattern, supra). However, these elements alone or a promoter region that contains all of these elements together cannot mediate the elicitor or light responsiveness of a reporter gene in transient expression assays. Thus, these elements are apparently necessary, but not sufficient for Elicitor- or light-mediated PAL and 4CL gene activation (Logemann et al, supra). Furthermore, apart from these genes, no other example of a gene involved in general phenylpropanoid metabolism is known, the promoter of which contains a complete set of all of these three boxes, which further emphasizes their functional importance in the coordinative regulation of these genes.

A G-box, which is located near the TATA box, is a widely used sequence motif in eukaryotic promoters. Such G boxes are present in the PMT promoter from Pinus sylvestris according to the invention at positions -147 and - 165 and in an inverse orientation at position -353. The functional analysis of plant promoters has shown that the G-Box plays a role in Promoter activation by various signals such as light, abscisic acid and UV light plays (Faktor et al., Supra).

The preservation of the G-Box and the H-Box between TATA and G-Boxes in different CHS promoters underlines their importance as regulatory motives (Faktor et al., Supra). An H box is located in the jaw promoter according to the invention in an inverse orientation at position -140 between the TATA and the G boxes. Both G and H boxes were found in the promoter-near region of a number of genes encoding phenylpropanoid biosynthesizing enzymes such as PAL, 4CL and CHS (Zhu et al. (1996), Current Oppinion in Genetics & Development 6, 624-630). The G and H boxes near the TATA box were both described as essential for floral expression (Faktor et al., Supra).

G boxes, triggered by development signals and pathogenic signals such as abscisic acid, light, UV radiation and wounding, are involved in the regulation of various genes. The H box is not as widespread, and is characteristic of phenylpropanoid biosynthesizing gene promoters (Zhu et al, supra). The G-box and the H-boxes are necessary together and obviously sufficient for feed-forward stimulation by 4-coumaric acid (Loake et al (1992), Proc. Natl. Acad. Sci. USA 89, 9230-9234). The H box is also present in parsley CHS and PAL promoters, and functional analysis has shown that this cis element is involved in UV induction (Loake et al., Supra).

Enhancer or activator elements dramatically increase the transcriptional activity of certain eukaryotic genes. A copy of the SV40 enhancer core sequence is found at position -356 in the PMT promoter. such Enhancer sequences have been reported in connection with the promoters of PAL genes from Phaseolus vulgaris (Cramer et al. (1989), Plant. Mol. Biol. 12, 367-383) and parsley CCoAOMT (Grimmig and Mattern, supra).

Elicitor-responsive elements (W-boxes) TTGACC were developed by Raventos et al. (Plant J. 7, 147-155 (1995)) and by Rushton et al. (supra). Such W boxes are found in the PMT promoter from Pinus sylvestris at position -440 and in inverse orientation at positions -55, -199 and -263. Sequences with such elicitor-responsive elements occur in promoters of various genes related to defense genes including PR1 from parsley (Rushton, Supra), in the CHS promoter from maize (Franken et al (1991) EMBO J. 10, 2605-2612) and in STS promoter from Wein on (Schubert et al. (1997), Plant Mol. Biol. 34, 417-426; Ernst et al. (1999) "Ozone induced genes mechanisms and biotechnological applications" in Plant Responses to environmental stress (ed .: MS Smallwood, CM. Calvert, DJ. Bowles), BIOS Scientific Publishers, Oxford, 33-41). Elicitor-responsive elements are generally responsible for the induction of plant defense mechanisms.

In the wine STS promoter, the ozone-responsive region (-430 to -280) differs from the pathogen-responsive region (-280 to -140) (Schubert et al., Supra; Ernst el al., Supra). In the PMT promoter according to the invention, the pathogen- or elicitor-responsive W boxes are present to a greater extent in the range between -263 and -50.

In a further essential subject of the present invention, the invention relates to chimeric nucleic acid molecules into which the above-mentioned promoter according to the invention or fragments thereof, such as the aforementioned W-boxes, have been introduced. Preferably, the chimeric nucleic acid molecules according to the invention, in addition to the promoter mentioned above or fragments thereof, DNA sequences operatively linked to the promoter, which encode proteins which catalyze the synthesis of phytoalexins.

Phytoalexins in the context of the present invention are compounds which can be formed by plants due to an ozone load or due to an infection to ward off the pathogens, usually fungi.

Thus, the coding regions contained in the chimeric nucleic acid sequences according to the invention can be inductively expressed in plants by ozone and / or pathogens. These coding regions are particularly preferably defense genes, most preferably genes for the synthesis of phytoalexin-catalyzing proteins which are naturally not inducible by ozone and / or pathogens.

The nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or those produced by genetic engineering or chemical synthesis methods.

The present invention further relates to vectors which contain the abovementioned recombinant nucleic acid molecules, promoters or chimeric nucleic acid molecules. The present invention thus also includes vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above and which can be used for the transfer of the nucleic acid molecules according to the invention or for planting or plant cells. The invention also relates to transformed microorganisms, such as bacteria, viruses, fungi, which contain the nucleic acid sequences according to the invention.

By providing the nucleic acid molecules, promoters or vectors according to the invention it is now possible to use genetic engineering methods to modify plant cells to have an ozone and / or pathogen-inducible characteristic. In particular, there is now the possibility of changing plant cells by means of genetic engineering methods to the effect that they fuse one or more plant genes for the synthesis of proteins catalyzing phytoalexins, the expression of which is naturally not or not significantly induced by ozone and / or pathogens, by fusion of the promoter according to the invention or a fragment thereof with pathogen or ozone inducible with such plant genes.

Plant cells can also be modified by means of genetic engineering methods to the effect that by inserting the nucleic acid molecules according to the invention or at least one fragment thereof into plants or plant cells which do not naturally contain these DNA sequences, they have an ozone and / or pathogen-inducible characteristic ,

It is also an object of the invention to provide new plants and plant cells which are distinguished by an ozone and / or pathogen-inducible expression of genes which either are not naturally present in these plants or plant cells or which are naturally not above those according to the invention Promoter or fragments thereof. This object is achieved by the provision of the DNA sequence coding for the PMT and the provision of the promoter responsible for ozone and / or pathogen induction or fragments thereof which have a gene expression of the defense genes controlled by them which can be induced by ozone and / or pathogens enable in plants and plant cells.

The invention thus relates to transgenic plants which in the present case contain the recombinant nucleic acid molecules, promoters or chimeric nucleic acid molecules according to the invention integrated into the plant genome. In principle, these plants can be any plant. It is preferably a monocot or dicot crop. Examples of monocotyledonous plants are plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea (corn ) and the like. Dicotyledonous crops include cotton, legumes such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potatoes, ornamental plants and trees. Other useful plants can include fruit (in particular apples, pears, cherries, grapes, citrus, pineapples and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and, for medicinal plants, rauwolfia and digital. The cereals wheat, rye, oats, barley, rice, corn and millet, sugar beet, rapeseed, soybeans, tomato, potatoes and tobacco are particularly preferred.

The invention further relates to propagation material of plants according to the invention, for example seeds, fruits, cuttings, tubers, root pieces, etc., and parts of these plants, such as plant cells, protoplasts and calli.

The plant cells include differentiated and undifferentiated plant cells, including protoplasts, in which the recombinant according to the invention Nucleic acid molecules, promoters or chimeric nucleic acid molecules integrated into the plant genome or as autonomous molecules (including transient transformation).

In a further embodiment, the invention relates to host cells, in particular prokaryotic and eukaryotic cells, which have been transformed or infected with a nucleic acid molecule according to the invention, a promoter according to the invention or a vector according to the invention, and cells which originate from such host cells and contain the nucleic acid molecules or vectors described. The host cells can be bacteria or fungal cells as well as plant or animal cells.

The present invention is also based on the object of demonstrating methods for producing plants or plant cells which have increased pathogen and / or ozone-inducible disease resistance. This

The object is achieved by methods with the aid of which it is possible to produce new plants or plant cells in which there are naturally no plant defense genes against ozone and pathogens or the desired defense gene expression is not present to a sufficient extent and / or which have no or insufficient, naturally occurring ozone and / or pathogen inducible gene expression.

Various methods are available for producing such new plants or plant cells. On the one hand, plants or plant cells can be modified using conventional genetic engineering transformation methods in such a way that the DNA sequences or promoters according to the invention are integrated into the plant genome, so that stable transformants are produced. According to the invention, plants or plant cells with increased pathogen and / or ozone-inducible disease resistance are produced by a method in which one or more defense genes, preferably genes which encode proteins which catalyze the synthesis of phytoalexins, in the form of the recombinant nucleic acid molecules according to the invention comprise the promoter according to the invention and a sequence coding for PMT, or in the form of the chimeric nucleic acid molecules according to the invention which comprise the promoter according to the invention and one or more defense genes and preferably genes which code for proteins which catalyze the synthesis of phytoalexins.

Alternatively, plants or plant cells with increased pathogen and / or ozone-inducible disease resistance are produced by a process in which one or more defense genes, preferably genes which code for proteins which catalyze the synthesis of phytoalexins, are naturally present, but not by ozone and / or pathogens can be made inducible by introducing the promoter according to the invention or functional fragments thereof.

When the recombinant nucleic acid molecules according to the invention, comprising the promoter according to the invention and a sequence coding for PMT, are introduced, the method according to the invention comprises in particular the following steps:

(a) if necessary, the introduction of the DNA sequence coding for a stilbene synthase into a plant cell if there is no or insufficient STS gene activity in the plant cell (by the stilbene synthase

Pinosylvin provided, which serves as a substrate for the PMT, which is encoded by the DNA sequence of the recombinant nucleic acid molecules according to the invention), (b) the introduction of the recombinant nucleic acid molecule according to the invention into the plant cell, and optionally

(c) the regeneration of completely transformed plants from the transformed plant cells, and if appropriate

(d) obtaining the desired parts of the plants, including the seeds of the plants thus obtained.

The recombinant PMT enzyme is preferably expressed in such an amount that the methylation of pinosylvin provides at least partial protection against ozone and / or diseases of the plants caused by pathogenic organisms. It may be sufficient that only the functional parts of the recombinant protein are expressed to provide such protection.

In a further preferred embodiment, the recombinant protein or a functional part thereof is used for the methylation of Pinosylvin or a derivative of Pinosylvin, whereby this application can also extend to pharmaceutical fields of application or other fields in which a methylation of Pinosylvin of high specificity is required ,

Another object of the invention is to demonstrate advantageous uses of the nucleic acid molecules according to the invention or of the promoters according to the invention. The invention therefore includes uses of the nucleic acid molecules according to the invention or the promoters according to the invention for producing the plants and plant cells according to the invention described above, which differ from non-transgenic plants and plant cells due to the presence of the nucleic acid molecules according to the invention by an ozone and / or pathogen-induced characteristic expression ,

Furthermore, the invention comprises the use of the nucleic acid molecules according to the invention or the promoters according to the invention or of fragments thereof for the detection and identification of ozone and / or pathogen-responsive nucleic acid elements in plant genes.

The invention also relates to nucleic acid molecules or fragments thereof which hybridize with one of the DNA sequences according to the invention. In the context of this invention, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Nucleic acid molecules that hybridize with the molecules according to the invention can be isolated, for example, from genomic or from cDNA libraries.

Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, for example by means of hybridization according to standard methods (see for example Sambrook et al, supra).

The invention thus also relates to the use of a DNA sequence according to the invention or parts thereof for the identification and isolation of homologous sequences from plants or other organisms.

For example, nucleic acid molecules can be used as the hybridization probe which have the nucleic acid molecules according to the invention or parts of these sequences exactly or essentially. With the as

Fragments used hybridization probe can also be synthetic fragments, which were produced with the help of the usual synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention. If one has identified and isolated genes which hybridize with the nucleic acid sequences according to the invention, one is

Determination of the sequence and an analysis of the properties required. For this purpose, a number of standard molecular biological, biochemical and biotechnological methods are available to the person skilled in the art.

The molecules hybridizing with the nucleic acid molecules according to the invention also comprise fragments, derivatives and allelic variants of the DNA sequences described above which contain an ozone and / or pathogen-responsive sequence, in active or inactivated form. The term "derivative" in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions and have a high degree of homology to these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations from the nucleic acid molecules described above may have arisen through deletion, addition, substitution, insertion or recombination.

For the nucleic acid molecules that are homologous to those described above

Molecules are and represent derivatives of these molecules, there are usually variations of these molecules that represent modifications that perform the same biological function. This can involve both naturally occurring variations, for example sequences from other organisms, or mutations, it being possible for these modifications to have occurred naturally or to have been introduced by targeted mutagenesis. Furthermore, the variations can be synthetically produced sequences. The allelic variants can be both naturally occurring and synthetically produced variants or those produced by recombinant DNA techniques.

To prepare the introduction of foreign genes into higher plants or their cells, a large number of cloning vectors are available, which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be introduced into the vector at a suitable restriction site. The resulting plasmid is used for the transformation of E. co / z ^' cells. Transformed E. co / z ^' cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the Plasmid DNA is cleaved and DNA fragments obtained are linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.

A large number of known techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, the fusion of protoplasts, the direct gene transfer of isolated DNA to protoplasts, the electroporation of DNA, the introduction of DNA using the biolistic method and others Possibilities. Both stable and transient transformants can be generated.

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids such as pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker. Usual section markers are those which impart resistance to the transformed plant cells to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and the like.

Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. For example, for the transformation of If the plant cell uses the Ti or Ri plasmid, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti and Ri plasmid, must be connected as a flank region to the genes to be introduced.

If agrobacteria are used for the transformations, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by means of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. (1978), Molecular and General Genetics 163, 181-187). The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. T-DNA may also be present. The agrobacterium transformed in this way is used to transform plant cells.

The use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in EP 120 515.

For the transfer of the DNA into the plant cell, plant explants can expediently be used with Agrobacterium tumefaciens or Agrobacterium rhizogenes be cultivated. Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. The plants are regenerated using conventional regeneration methods using known nutrient media. The plants or plant cells obtained in this way can then be examined for the presence of the introduced DNA.

Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known to the person skilled in the art (cf. L. Willmitzer (1993) Transgenic Plants in: Biotechnology, A Multi-Volume Comprehensive Treatise (editor: HJ. Rehm et al. ), Volume 2, 627-659, VCH Weinheim, Germany).

While the transformation of dicotyledonous plants or their cells via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well established, recent work indicates that monocotyledonous plants or their cells are also very accessible to transformation by means of vectors based on Agrobacteria (see et al. Chan et al. (1993) Plant Mol. Biol. 22, 491-506).

Alternative systems for the transformation of monocotyledonous plants or their cells are transformation using the biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104, 37-48; Vasil et al. (1993) Bio / Technology 11, 1553-1558; Ritala et al. (1994) Plant Mol. Biol. 24, 317-325; Spencer et al. (1990), Theor. Appl. Genet. 79, 625-631), protoplast transformation, electroporation of partially permeabilized cells and the introduction of DNA using glass fibers. The transformed cells grow within the plant in the usual way (see also McCormick et al. (1986), Plant Cell Reports 5, 81-84). The resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties.

Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.

Likewise, transgenic lines can be determined by conventional methods, which are homozygous for the new nucleic acid molecules and which investigate their phenotypic behavior with regard to an existing or non-existing ozone and / or pathogen responsiveness and compare it with that of hemizygotic lines.

Of course, plant cells containing the nucleic acid molecules according to the invention can also be cultivated further as plant cells (including protoplasts, calli, suspension cultures and the like).

The present invention furthermore relates to the use of the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules for the identification and isolation of homologous molecules which comprise ozone and / pathogen-responsive elements from plants or other organisms. The definition of the term "homology" is given above. The following examples serve to illustrate the present invention without restricting it in any way.

Examples:

Example 1: Plant and mushroom material

Seven-year-old pine seedlings were obtained from the Bauchery Nursery, Crouy-sur-Cosson, Loir et Cher, France and grown in a greenhouse for another five months. After the experimental treatments, the trees were brought back to the greenhouse, and after a year, they were removed

Survival rate determined. The fungus Leptographium wingfieldii comes from the INRA collection (Orleans, France) and was originally isolated from the bark beetle Tomicus piniperda. It was further purified by a monospur culture and grown on malt agar in the dark at 22 ° C (Lieutier et. Al. (1989), Ann. Sei. For. 46, 201-216). Cultures were kept at 4 ° C with an annual run on Scottish pine tree trunks at 22 ° C to maintain their activity.

Example 2: Ozone treatment

The seedlings were acclimatized in climatic chambers for six days (Thiel et al. (1996), J. Plant Physiol. 148, 456-463). The light duration was 14 hours per day (PAR: 1300 μE m ^"2 s ^"1; UV-A: 22.5 Wm ^'2 ; UV-B: 0.45 MEDh ^"1 ); the day / night temperatures were 22 ° C and 16 ° C and the relative humidity during the day and at night were 70% and 85%. The seedlings were treated with ozone (0.15 or 0.3 μl l ^"1 ) for ten hours a day for a period of two Days. In all RT-PCR experiments, only individual bands at 1.15 kb for PMT, 1.17 kb for STS and 0.8 kb for cab were detected. RT-PCR control experiments carried out with cab primers showed a constant level of cab transcripts in non-ozone-treated tissues and a decrease in these transcripts in tissues which were exposed to 0.15 or 0.3 .mu.l ^"1" ozone.

In control trees that were exposed to ozone-free air, STS or PMT transcripts in the needles and the phloem were almost undetectable. Two ten-hour periods of ozone fumigation were sufficient to dramatically increase the STS and PMT transcript levels in needles from Scottish pine trees. Fumigation with 0.15 ul 1 ^"1 ozone resulted in a first peak of the STS transcripts after six hours of exposure, followed by a further increase 48 hours after the start of treatment, while the PMT transcripts initially up to 24 hours after the start fumigation remained at the control level and only then began to accumulate. Exposure to 0.3 μl l ^"1 ozone led to a further increase in the two transcript levels and remained at a high level in the needles. In contrast, the amounts of STS and PMT mRNA in the phloem were not significantly affected by ozone.

Example 3: Inoculations

48 hours after the start of ozone fumigation, two sterile inoculations and two fungal inoculations were performed on each tree in each room. According to the method described by Wright (Phytopathol. 23, 487-588 (1933)), calibrated agar discs (diameter 3 mm) of a three-week-old, sporulating Leptographium wingfieldii culture were introduced into the tree at the level of the cambium. Mock vaccinations without fungus were carried out with sterile malt agar discs (diameter 3 mm). The inoculations were carried out at two different heights of the trunk with a distance of at least 20 cm. A 4 cm (horizontal) x 7 cm (vertical) rectangle around the respective infection site of the phloem tissue was removed and used for the RNA analysis after a 1 cm x 2 cm rectangle of the phloem tissue that was directly around the Infection point was removed.

The sham inoculation led to an accumulation of both transcripts on day 5 (three days after the inoculation). The SJS mRNA showed a progressive accumulation up to day 9 (120 ng ng ^"1 cDNA), then slowly decreased and was still at a high level after 16 days. The P T mRNA progressively increased after day 5 (50 ng ng ^"1 cDNA) to return to the control level at the end of the experiment.

The two kinetics in response to sham inoculation were strongly affected by previous ozone fumigation. 0.15 μl 1 ^_1 ozone led to a dramatic decrease in the STS mRNA reaction pattern. The R TmRNA response curve was also lowered. Pretreatment with 0.3 μl 1 ^"1 ozone led to a peak in SJS transcripts on the same 9th day as with sham inoculation alone, but extended the accumulation up to the 16th day. The RT transcripts showed a progressive accumulation up to at day 16 (75ng ng ^"1 cDNA).

Fungal inoculation resulted in transient peaks in STS and RT transcripts after 5 (200 ng ng ^"1 cDNA) and 9 (130 ng ng ^{" 1} cDNA) days after the start of the experiment. Due to the gassing of ozone with 0.15 μl l ^"1 , the peak of the STS transcript accumulation decreased slightly and delayed the peak of RΛJT transcripts until day 16 (110 ng ng ^{" 1} cDNA). Ozone fumigation with 0.3 μl l ^"1 extended the Peak of S7S to a stable level of 150 ng ^"1 cDNA, while the amount of RT transcripts increased to 170 ng ^{" 1} cDNA by 16 days after the start of the experiment. The mushrooms approximately doubled the amount of S7S and RT transcript that occurred during inoculation, occurring four days earlier for STS. The S S and P T transcript sets showed similar responses to ozone. A significant decrease occurred at 0.15 μl l ^"1 , while at 0.3 μl l ^{" 1} the peak height was lengthened or increased slightly.

The dose-dependent accumulation after ozone induction of STS and PMT transcripts in needles agrees well with previously found increases

Stilbene, SJS and RT enzyme activities and STS transcripts in

Scottish pine seedlings (Rosemann et al, supra; Zinser et al.

(1998), Planta 204, 169-176). The comparison of these data suggests that the

Stilbene metabolites can be regulated at the level of transcription.

Ozone does not induce STS and P T mRNA in phloem, which indicates that there is no systematic ozone effect.

Wounds resulted in temporary induction of the SDS and PMT transcripts, which increased due to fungi during the first week after vaccination. The stilbenes PS and PSM were only detected in the reaction area in phloem, which was wounded or inoculated by a fungus related to the bark beetle, which corresponds to the proposed involvement of stilbenes in tree resistance (Lieutier et al. Supra; Bois and Lieutier (1997), Plant Physiol. Biochem. 35, 819-825). The ozone treatment leads to a temporary increase in STS and PMT mRNA in the needles, which shows a similarity between the ozone and pathogen-induced increase in transcripts. Example 4: Total RNA extraction

Lyophilized needles (70 to 90 mg) discarding the current annual flower and lyophilized phloem tissue (100 mg) were ground to a fine powder and the total RNA was isolated in a conventional manner.

Example 5: Extraction of the genomic DNA

The genomic DNA was extracted from the needles of adult Scottish pine according to protocol 1 in Csaikl el. Al (Plant Mol. Biol. Rep. 16, 69-86 (1998)).

Example 6: Isolation of the PMT genomic clone

The promoter sequence was obtained in the gene-specific reverse primer from the PMT cDNA sequence (German patent application DE-A-198 36 774; Ernst et al. (1999) Forest Biotechnology, University of Oxford, July 11-16, 1999 ; Ernst et al. (1999) IUFRO Conference, University of Munich, 12-17 September 1999) according to the method described by Cormack and Somssich (Gene 194, 273 to 276 (1997)). 1.5 μg of the genomic DNA of the Scottish pine were completely digested with 20 units of EcoRI. The DNA was precipitated on ice with two volumes of isopropanol for five minutes, washed with 70% ethanol and resuspended in 15 ul HO. After a five minute incubation at 90 ° C, the DNA was polyadenylated at 37 ° C with 0.5 mM dATP and 1.5 mM CoCl ₂ in buffer for 1.5 hours. 20 μl of terminal transferase (TdT) buffer (Röche) with 50 units of TdT served as the buffer. The reaction was terminated by heating the sample at 72 ° C for five minutes.

The first polymerase chain reaction (PCR) was carried out with a tenth of the volume of the polyadenylated DNA (150 ng), 100 pmol of the gene-specific primer 1 (5'-TCCCCGAGTTCCATGCCCAGAA-3 '), 100 pmol of the universal T17 primer (5 '- GTAAAACGACGGCCAGTCGACTTTTTTTTTTTTTTTTT-3'), 200 μM dNTPs and 5 units of AGS-Gold ™ DNA polymerase (AGS) in 100 μl 1 x AGSGold ™ - Buffer performed with the heat cycle conditions an initial denaturation step at 94 ° C for 1 minute, followed by 35 cycles of 1 minute denaturation at 94 ° C, primer attachment at 60 ° C for 1 minute, and 3 minutes extension at 72 ° C with a final ten minute extension step at 72 ° C.

The second PCR was performed using 1 µl of the first PCR product under the same conditions as the first PCR, except that 100 pmol of the gene-specific primer 2 (5'-GCCGATCCCATTTCGAATCC-3 ') and 100 pmol of the universal primer (5'-GTAAAACGACGGCCAGT-3 ') were used. The final PCR product was purified and cloned into the pGEM-T vector (Promega) according to the manufacturer's instructions. The plasmids were sequenced commercially (MWG).

Example 7: RT-PCR analysis

The total RNA (5 μg) from pine needles or phloem tissue was digested with DNasel and reverse transcribed by 200 units of Superscript II RT (Live Technologies) for 1 hour at 42 ° C., with a corresponding buffer, 10 mM DTT, 0, 4 mM of each dNTP's, 100 nM oligo-dTι _2-18 primer (Life Technologies) and 10 units of RNase inhibitor (Lfve Technologies) were added.

The cDNA was carried out according to the method described by Kiefer et. al. (supra) described method, and 10 ng were for the PCR with 2.5 units of 7α ^ polymerase (Amersham Pharmacia Biotech), a corresponding buffer, 0.2 mM of each dNTP and 0.2 μ 3'-and 5 'Primers used. The amplification was performed during 35 cycles of 1 minute denaturation at 94 ° C, 1 minute primer attachment at 58 ° C (R T and cαb primer) or 62 ° C (STS primer) and 2 minutes extension 72 ° C carried out. The reaction products were analyzed by electrophoresis using a 1% agarose gel, visualized under UV light after staining with ethidium bromide and quantified using the Picogreen method (Molecure Probes). The authenticity of the PCR products was checked by two directed partial sequencing using the Thermo Sequenase kits (Amersham Pharmacia Biotech).

pictures:

Figure 1 (= SEQ ID No. 1)

Nucleotide sequence of the PMT gene from Pinus sylvestris and the protein sequence derived from it: The predicted transcription start site is designated as +1. Conserved eukaryotic promoter elements (CAP signal, TATA, CATA, CAAT and GC boxes) and potential plant regulatory elements (G and H boxes) are underlined and Myb-responsive elements (MREs) are considered. Elicitor regulatory elements (W boxes) are printed in bold and the SV40 enhancer is printed in italics.

Claims

PATENT CLAIMS

1. Recombinant nucleic acid molecule, comprising a) regulatory sequences of a promoter active in plants which mediates gene expression which can be induced by pathogens and / or ozone; and b) operatively linked to it a DNA sequence that encodes a protein with the enzymatic activity of a Pinosylvin-3-O-methyltransferase (PMT).

2. Recombinant nucleic acid molecule according to claim 1, wherein the promoter and / or the DNA sequence from the jaw (Pinus sylvestris) is isolated.

3. Recombinant nucleic acid molecule comprising the sequence SEQ ID No. 1 indicated promoter sequence or fragments thereof, which mediate pathogen and / or ozone-responsive expression of a coding region operatively connected thereto.

4. Isolated promoter of a Pinosylvin-3-O-methyltransferase (PMT) gene or fragments thereof which mediate pathogen and / or ozone inducible gene expression in plant cells.

5. Promoter according to claim 4, isolated from pine (Pinus sylvestris).

6. Promoter as in SEQ ID No. 1 specified.

7. Chimeric nucleic acid molecule comprising the promoter according to any one of claims 4 to 6 or fragments thereof.

8. The chimeric nucleic acid molecule according to claim 7, which comprises at least one DNA sequence operatively linked to the promoter, which codes for a defense gene, in particular for a protein catalyzing the synthesis of phytoalexins.

9. The chimeric nucleic acid molecule according to claim 8, wherein the DNA sequence codes for a protein which is naturally not pathogen and / or ozone inducible.

10. Vectors containing a promoter or a nucleic acid molecule according to any one of the preceding claims.

11. Transgenic plants which contain a promoter, a nucleic acid molecule or a vector according to one of the preceding claims, and parts of these plants and their propagation material, such as protoplasts, plant cells, calli, seeds, tubers or cuttings, etc., and the offspring of these plants.

12. Dicotyledonous plants according to claim 11, in particular useful plants, such as soybean, rapeseed, tomato, sugar beet, potato, cotton, tobacco and ornamental plants or trees.

13. Monocot plants according to claim 11, in particular cereals such as oats, wheat, rye, barley, rice, millet or corn.

14. Transgenic plant cells, including protoplasts, which have a

Contain a promoter, a nucleic acid molecule or a vector according to any one of claims 1 to 10.

15. Plant cells according to claim 14, in which due to the introduction of the nucleic acid molecule according to one of claims 1 to 3, a promoter according to one of claims 4 to 6 or a chimeric nucleic acid molecule according to one of claims 7 to 10 or at least one fragment thereof an ozone and / or pathogen-inducible expression of a gene which does not naturally have the corresponding DNA sequence.

16. A process for producing transgenic plant cells or transgenic plants with increased pathogen and / or ozone-inducible disease resistance, in which a defense gene, in particular a gene for the synthesis of

Phytoalexin-catalyzing protein, in the form of the nucleic acid molecules according to one of claims 1 to 3 or in the form of the nucleic acid molecules according to claim 8 or 9 is introduced.

17. Process for the production of transgenic plant cells or transgenic

Plants with increased pathogen and / or ozone-inducible disease resistance, in which a defense gene, in particular a gene for a protein catalyzing the synthesis of phytoalexins, under the control of a promoter according to any one of claims 4 to 6 or fragments thereof which are caused by ozone and / or mediate inducible expression.

18. The method according to claim 16 or 17, wherein the protein encoded by the nucleic acid molecule according to one of claims 1 to 3 is expressed in such an amount that the methylation of pinosylvin provides at least partial protection against diseases caused by pathogenic organisms and / or ozone of the plants.

19. A method for producing ozone and / or pathogen-inducible characteristics in transgenic plants or plant cells by transferring the nucleic acid molecule according to one of claims 1 to 3, a promoter according to one of claims 4 to 6 or a chimeric nucleic acid molecule according to one of the claims Plant 7 to 9 or at least one fragment thereof or plant cells.

20. Use of the nucleic acid molecule according to one of claims 1 to 3 or 7 to 10 or a fragment thereof for generating increased pathogen and / or ozone-inducible disease resistance in transgenic plants.

21. Use of the promoter according to any one of claims 4 to 6 or a fragment thereof for the generation of ozone and / or pathogen-inducible characteristics in transgenic plants or plant cells.

22. Use of the promoter according to one of claims 4 to 6 or a fragment thereof for the expression of heterologous plant defense genes.

23. Use of the nucleic acid molecule according to one of claims 1 to 3 or 7 to 10 or a fragment thereof and / or the promoter according to one of the

Claims 4 to 6 or a fragment thereof for the detection and identification of ozone and / or pathogen-responsive nucleic acid elements in plant genes.