WO2003033712A1 - Promoteurs inductibles chimiquement a partir d'orge et leur utilisation - Google Patents

Promoteurs inductibles chimiquement a partir d'orge et leur utilisation Download PDF

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Publication number
WO2003033712A1
WO2003033712A1 PCT/EP2002/011330 EP0211330W WO03033712A1 WO 2003033712 A1 WO2003033712 A1 WO 2003033712A1 EP 0211330 W EP0211330 W EP 0211330W WO 03033712 A1 WO03033712 A1 WO 03033712A1
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bci
promoter
seq
expression
nucleic acid
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PCT/EP2002/011330
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German (de)
English (en)
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Karl-Heinz Kogel
Ulrich Beckhove
Uta Geldermann
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Basf Plant Science Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline

Definitions

  • the invention relates to methods for the chemically inducible expression of nucleic acid sequences, preferably in plants. Also included are new chemically-inducible promoters, functional equivalents and functionally equivalent parts thereof, as well as expression cassettes and vectors which comprise these promoter sequences.
  • the invention further relates to transgenic plants transformed with these expression cassettes or vectors, cultures derived therefrom, parts or transgenic propagation material, and the use thereof for the production of foodstuffs, animal feed, seeds, pharmaceuticals or fine chemicals.
  • the invention further encompasses the use of these expression cassettes or vectors in methods for identifying substances which are able to induce pathogen resistance after exposure to plants.
  • the aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example, to 'increase agricultural productivity, to improve quality of human foods or for producing specific chemicals or pharmaceuticals.
  • Promoters are important tools in plant biotechnology to control the expression of a specific gene in a transgenic plant and thus to achieve certain essential characteristics of the plant. It is desirable to be able to use a wide variety of promoters with different properties in order to optimally implement the expression of a particular gene in a particular cell, tissue or plant species.
  • Various promoters with activity in plants are known to the person skilled in the art.
  • a promoter can be constitutive, tissue-specific, developmental or inducible. Constitutive promoters such as the 35S CaMV promoter are permanently active regardless of the development stage and tissue of a plant and cause a continuous expression of the genes controlled by them. Such constitutive expression can adversely affect plant quality.
  • constitutive expression can have different effects at different stages of development and thus also have an adverse effect on the plant. Similar adverse effects of constitutive expression can also occur with expression in tissue or cell cultures and with fermentations, where, for example, they can negatively influence the growth rates and thus reduce the yields. ' ,
  • Tissue- or development-dependent promoters are only active at defined times or in defined tissues of a plant.
  • control of the time, extent and / or location of expression is particularly desirable.
  • inducible promoters are those whose transcription activity is regulated by one or more internal or external inducers.
  • Inducers can be chemical compounds such as certain metabolites (e.g. sugar, alcohols), growth regulators, herbicides, plant hormones, phenolic compounds, but also factors such as light or also physiological stress (heat, salt, lack of oxygen, injury, toxic elements). Indirectly can also be the effect of a pathogen or one Illness (such as a virus or fungus) cause induction.
  • WO 93/21334 describes the alcA / alcR expression system, which can be induced by various alcohols (e.g. ethanol).
  • the system consists of two components: the promoter sequence and the alcR protein, which stimulates the promoter as an inducer protein in the presence of the alcohol.
  • SA Salicylic acid
  • SAR systemic acquired resistance
  • LAR local acquired resistance
  • Jasmonic acid (JA) and its methyl ester (MeJA; JM) are important signal substances in plant defense mechanisms and have an additional hormone function for controlling plant metabolism (Sticher L et al. (1997) Ann Rev Phytopath 35: 235-270; Creelman RA and Mullet JE (1997) Ann Rev Plant
  • Physiol Plant Mol Biol 48: 355-381 The biosynthesis is based on membrane lipids, mostly from linolenic acid under the action of a lipoxygenase or unspecifically by membrane peroxidation, for example during a hypersensitive reaction (Sticher L ef al. (1997) Ann Rev Phytopathol 35: 235-270). SA antagonizes the production of JA (Penninckx IAMA (1996) Plant: Cell 8: 2309-2323).
  • Abscisic acid is required for signal transduction from systemin to JA (Sticher L et al. (1997) Ann Rev Phytopathol 35: 235-270).
  • Ethylene can also modulate SAR, increase lignification and is presumably required for signal transduction between Systemin and Pinll (Sticher L et al. (1997) Ann Rev Phytopathol 35: 235-270).
  • induced resistance encompasses a number of plant defense mechanisms, in each of which an increased resistance to a subsequent secondary infection or stimulation is triggered by a primary infection or stimulation with a pathogen or other stress factor (Sticher L et al. (1997) Ann Rev Phytopathol 35: 235-270). Resistance can be local or systemic. The systemic resistance includes the accumulation of resistance proteins in areas that are further away from the site of primary infection or stimulation. Different classes of these proteins can be defined: hydrolases, especially the "pathogenesis related proteins” (PR proteins) (Stinzi A et al. (1993) Biochemie 75: 687-706), Defensine (Broekart WF et al.
  • PR proteins pathogenesis related proteins
  • SAR Systemic acquired resistance
  • SA salicylic acid
  • SAR reaction is mediated by endogenous messenger substances such as jasmonate (JA) or salicylic acid (SA). Exogenous application of these substances is capable of activating the corresponding PR genes and causing SAR (Ward et al. (1991) Plant Cell 3: 1085-1094; Uknes et al. (1992) Plant Cell 4 (6 ): 645-656). Similar effects can also be achieved by synthetic compounds such as 2, 6-dichloroisonicotinic acid (DCINA) (Vernooij et al. (1995) Mol Plant Microbe Interact 8: 228-234) or benzo [1,2,3] thiadiazole-7-thiocarboxylic acid.
  • DCINA 2, 6-dichloroisonicotinic acid
  • DCINA realizes this without increasing the SA level at the same time, i.e. it acts at the same location or below in the signal transduction chain (Vernooij B et al. (1995) Mol Plant Microbe Interact 8: 228-234).
  • SA salicylic acid
  • BION ® the active substance (benzo [1, 2,3] thiadiazole-7-thiocarboxylic acid S-methyl ester; BTH) of which SA is structurally similar and how it triggers SAR.
  • the promoters of the PR genes are generally also induced indirectly, for example by pathogen attack and / or stress, using endogenous messenger substances such as SA.
  • pathogen-inducible promoters are the PRP1 promoter from potato (Martini N et al. (1993) Mol Gen Genet 263: 179-186), the Fisl promoter (WO 96/34949), the Betv 1 promoter (Swoboda I et al. (1995) Plant Cell and Env 18: 865-874), the Vstl promoter (Fischer R (1994) dissertation, Univ. Hohen- heim; Schubert R et al. (1997) Plant Mol Biol 34: 417-426) , the sesquiterpene cyclase promoter (Yin S et al. (1997) Plant Physiol 115: 437-451) and the gstAl promoter (Mauch F and Dudler R (1993) Plant Physiol 2: 1193-1201).
  • promoters are only insufficiently suitable as chemically inducible promoters, since they have a not inconsiderable constitutive activity and can also be unwantedly induced by natural pathogens instead of the chemical inducer.
  • a disadvantage of using these promoters for the transgenic expression of anti-pathogenic proteins is the fact that the promoters are only reactive.
  • Delay phase until sufficient defense potential has been reached can result in damage to the plant which can impair its value and its suitability as food or feed.
  • the BCI-3 promoter is induced by DCINA, BTH and weaker by SA. After BTH induction, the accumulation of BCI-3 is only detectable in the mesophyll and weaker in the epidermis. Expression of BCI-3 was not affected by ethylene and ABA, but induced by JA application and wounding. Inoculation of barley with C. sativus, the non-host pathogen Bgt or aphids (S. avenae) had no effect on the transcript accumulation. In contrast, BCI-3 is even repressed by infection with a virulent and avirulent breed of Bgh.
  • BCI-4 gene expression can only be induced with the resistance inducers DCINA and BTH and - weaker and more transient - with SA. Weak expression of BCI-4 was detected locally and systemically after infiltration of the pseudomonad strain PG4180. All other investigated inducers, various biotic and abiotic stress factors, as well as fungicide applications or injuries had no influence on the expression of BCI-4. This shows that BCI-4 is not expressed in general stress reactions, but is specifically regulated. BCI-4 shows a very low constitutive expression activity. An increased accumulation of BCI-4 transcript and protein in barley plants occurred exclusively in leaves (with the exception of the flag leaf) and only after chemical induction (BTH casting treatment), the accumulation remaining limited to the mesophyll.
  • An advantage of the promoters according to the invention is that gene expression in plants - especially in the important, monocotyledonous plants - can be specifically activated by applying a chemical. An unwanted activation by pathogens can be largely excluded. There are many possible uses. For example, it is possible to prevent an impending pathogen attack in a quasi-preventive manner.
  • the cDNA of the BCI-3 and BCI-4 genes is partially known (WO 00/71748; Besser K et al. (2000) Molecular Plant Pathology 1 (5): 277-286; GenBank Acc.No .: AJ250282 and AJ250283) , An EST
  • expression tag (“expressed sequence tag”) was found by means of an expression analysis following treatment of barley with SAR ("systemic acquired resistance") triggering chemicals. In addition to the BCI-3 and BCI-4 EST, numerous other sequences were identified. Surprisingly, however, the promoters according to the invention show strong induction only when stimulated with exogenously applied chemicals such as DCINA or BTH, but not in the case of pathogen attack. This expression specificity is particularly advantageous because only a low background activity in the absence of the chemical inducer (triggered, for example, by environmental factors or pathogen infestation) can be expected.
  • a first subject of the invention relates to a method for chemically inducible transgenic expression of nucleic acid sequences, characterized in that
  • an expression cassette consisting of a nucleic acid sequence to be expressed transgenically in functional linkage with a chemically inducible promoter selected from the group of sequences consisting of
  • the expression of the nucleic acid sequence is induced by treating the organism or the tissue, organ, part, cell culture or cell thereof with a chemical inducer.
  • Plant means all plant organisms as described below by way of example. Plants that play a role in agriculture and as feed or food are particularly preferred. Most preferred are cereals such as barley, wheat, rye, oats, triticale, rice and corn.
  • the invention further relates to the barley BCI-3 promoter according to SEQ ID NO: 1, the sequence complementary thereto, and functional equivalents or parts thereof which are functionally equivalent.
  • Another object of the invention relates to the barley BCI-4 promoter according to SEQ ID NO: 2, the sequence complementary thereto, and functional equivalents or parts thereof which are functionally equivalent.
  • “Functionally equivalent parts” means partial sequences of the promoters described by SEQ ID NO: 1 or 2, which essentially have the same promoter activity as the BCI-3 promoter described by SEQ ID NO: 1 or the BCI-4 promoters according to SEQ ID NQ: 2 exhibit. Partial sequences can be composed of one or more parts of the promoters described by SEQ ID NO: 1 or 2.
  • Functionally equivalent parts of a BCI-3 promoter preferably comprise the sequences described by SEQ ID NO: 3, 4, 5, 6 or 7.
  • Functionally equivalent parts of a BCI-4 promoter preferably comprise the sequence described by SEQ ID NO: 8.
  • “Functional equivalents” means sequences which are derived from the BCI-3 promoter described by SEQ ID NO: 1 or the BCI-4 promoter according to SEQ ID NO: 2 and which have essentially the same promoter activity.
  • a promoter activity is said to be "essentially the same” if the transcription of a particular nucleic acid sequence to be expressed transgenically under the control of a promoter derived from SEQ ID NO: 1 or SEQ ID NO: 2 is not or only weakly by a chemical inducer can be activated by pathogens.
  • the maximum induction of expression by a chemical inducer is at least twice as high as the maximum induction by a pathogen, is preferred at least 5 times as high, very particularly preferably at least 10 times as high, most preferably at least 20 times as high.
  • Dichloroisonicotinic acid or benzo [1,2,3] thiadiazole-7-thiocarboxylic acid S-methyl ester (BTH) are preferably used as chemical inducers.
  • Barley powdery mildew (Blumeria [syn Erysiphe] graminis f.sp. hordei Speer, Bgh) is preferred as the pathogen; barley powdery mildew of breed A6 (BghA6) is particularly preferred.
  • Very particularly preferably, functionally equivalent promoters with essentially the same promoter activity are understood to be those which have a leaf-specific, particularly preferably a leaf-mesophyll-specific expression.
  • “specific" means that the expression in the conscious tissue is at least twice as high as in any other tissue. The expression is preferably at least 5 times as high, very particularly preferably at least 10 times as high.
  • the absolute level of expression after incubation with the chemical inducer can differ both downwards and upwards in comparison with the BCI-3 promoter according to SEQ ID NO: 1 or the BCI-4 promoter according to SEQ ID NO: 2.
  • the level of expression is measured in each case on the basis of the transcribed mRNA or the protein which has been translated as a result under otherwise unchanged conditions.
  • Preferred are promoter sequences whose expression level after induction receives a comparison value with the promoter described by SEQ ID NO: 1 or SEQ ID NO: 2 by no more than 50%, preferably no more than 25%, particularly preferably no more than 10% below.
  • Sequences are particularly preferred whose expression level after induction receives a comparison value with one of the promoters described by SEQ ID NO: 1 or SEQ ID NO: 2 by more than 50%, preferably 100%, particularly preferably 500%, very particularly preferably 1000% exceeds. Also particularly preferred are those sequences whose level of expression before induction does not receive a comparison value with a promoter described by SEQ ID NO: 1 or SEQ ID NO: 2 or does not exceed this by at least 10%, preferably at least 30%, particularly preferably at least 50%, most preferably falls below at least 90%. In general, induction is expected after 2 to 72 hours, preferably after 12 to 48 hours.
  • Preferred nucleic acid sequences to be expressed transgenically are those which allow easy quantification of the expression, ie in particular those whose transcription and translation results in so-called reporter proteins (Schenborn E, Groskreutz D (1999) Mol Biotechnol 13 (l): 29-44) such as beimés- as GFP ("green fluorescence protein"; Chui WL et al. (1996) Curr Biol 6: 325-330; Leffel SM et al. (1997) Biotechniques 23 (5): 912-8), chloramphenicol transferase, luciferase (Millar et al.
  • chemical inducer primarily includes compounds with a benzo-1, 2, 3-thiadiazole basic structure and includes, by way of example but not by way of limitation, benzo-1, 2, 3-thiadiazolecarboxylic acid, benzo-1, 2, 3-thiadiazolthiocarboxylic acid, cyanobenzo- 1,2,3-thia-diazole, benzo-1, 2, 3-thiadiazolecarboxamide, benzo-1, 2,3-thia-diazolecarboxylic acid hydrazide, benzo-1, 2, 3-thiadiazole-7 ⁇ -carboxylic acid, benzo-1, 2,3-thiadiazole-7-thiocarboxylic acid, 7-cyano-benzo-1,2,3-thiadiazole, benzo [1,2,3] thiadiazole-7-thiocarboxylic acid S-methyl ester, benzo-1,2 , 3-thiadiazole-7-carboxamide, benzo-1,2,3-thiouracil
  • inductors with a pyridinecarboxylic acid backbone such as isonicotinic acid and derivatives thereof; haloisonicotinic acids such as dichloroisonicotinic acid, 2,6-dichloroisonicotinic acid (DCINA) and derivatives thereof are particularly preferred.
  • haloisonicotinic acids such as dichloroisonicotinic acid, 2,6-dichloroisonicotinic acid (DCINA) and derivatives thereof are particularly preferred.
  • DCINA 2,6-dichloroisonicotinic acid
  • ben such as the lower alkyl esters (Cl to C6), especially the methyl ester.
  • inducers of PR genes or inducers of a SAR such as, for example, benzoic acid, salicylic acid (SA), jasmonic acid or jasmonic acid methyl ester, polyacrylic acid and substituted derivatives of the aforementioned inducers.
  • inducers are 2,6-dichloroisonicotinic acid (DCINA), benzo [1,2,3] thiadiazole-7-thiocarboxylic acid S-methyl ester, salicylic acid (SA), jasmonic acid (JA) and jasmonic acid methyl ester (JM).
  • DCINA 2,6-dichloroisonicotinic acid
  • SA benzo [1,2,3] thiadiazole-7-thiocarboxylic acid S-methyl ester
  • SA salicylic acid
  • JA jasmonic acid
  • JM jasmonic acid methyl ester
  • the application of the inductors can be used in pure form, in solution or suspension, as powder or dust or in any other form of formulation customary in agriculture.
  • Solid as well as liquid auxiliaries or carriers can be used with which the inductor is mixed, for example to facilitate application to the plant,
  • Silicates, clays, polymers, alcohols, ketones, aliphatic or aromatic hydrocarbons and the like can be used, for example, as auxiliaries or carriers.
  • the inductor is used in the form of a conventional "wettable powder" or as a concentrate of an aqueous suspension or emulsion, the formulation can contain one or more surface-active, ionic or non-ionic compounds ("surfactants"), "wetting" agents or emulsifiers.
  • surfactants surface-active, ionic or non-ionic compounds
  • the inductor can be used individually or in combination with other suitable inductors (for example a combination of DCINA and BTH).
  • the inductor can also be used in combination with other compounds such as adjuvants, herbicides, fungicides, insecticides or growth regulators or fertilizers.
  • the inductor can preferably be applied as a spray to leaves, stems and / or branches of the plant or else to the seeds before sowing.
  • the quantity and time period of the inductor are adapted to the quantity required for an optimal induction.
  • inductors When applied to whole plants, inductors are generally used in a concentration of 0.1 to 1000 mg of active compound per liter of soil volume as part of a soil treatment process or in concentrations of 0.1 to 100 mg / 1 active compound per liter of liquid for spray application. Lower concentrations can also be used in cell or tissue cultures. In general induction is expected after 2 to 72 h, preferably after 12 to 48 h.
  • a functional equivalent is understood to mean, in particular, natural or artificial mutations of the BCI-3 promoter according to SEQ ID NO: 1 or of the BCI-4 promoter according to SEQ ID NO: 2, as well as homologous sequences from other plant genera and species, which continue to be essentially the same Have promoter activity.
  • Mutations include substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which are obtained by modifying the BCI-3 promoter sequence according to SEQ ID NO: 1 or the BCI-4 promoter sequence according to SEQ ID NO: 2.
  • the aim of such a modification can e.g. further restricting the sequence contained therein to certain regulatory elements or e.g. also the insertion of further restriction enzyme interfaces, the removal of superfluous DNA or the addition of further sequences, for example further regulatory sequences.
  • the sequence can be limited to specific, essential regulatory regions - for example also for the production of functionally equivalent parts of the promoter sequences according to the invention - can also be carried out with the aid of a search routine for searching for promoter elements.
  • Certain promoter elements are often present in abundance in the regions relevant to promoter activity. This analysis can be carried out, for example, using computer programs such as the PLACE program ("Plant Cis-acting Regulatory DNA Elements") (Higo K et al. (1999) Nucl Acids Res 27 (1): 297-300).
  • the "PlantCARE” program which is available on the Internet, can be used in particular to find cis-acting regulatory elements (http: //sphinx.rüg. Ac .be: 8080 / PlantCARE / cgi / index.html).
  • Functional equivalents of the BCI-3 promoter derived from the BCI-3 promoter sequences according to the invention, for example by substitution, insertion or deletion of nucleotides, have a homology of at least 50%, preferably 60%, preferably at least 70%, particularly preferably at least 80%, entirely particularly preferably at least 90% of the promoter sequence according to SEQ ID NO: 1 or a functionally equivalent part thereof, preferably an equivalent part according to SEQ ID NO: 3, 4 5, 6 or 7 and are distinguished by essentially the same promoter activity in comparison the BCI-3 promoter sequence according to SEQ-ID NO: 1 or a functionally equivalent part thereof, preferably an equivalent part according to SEQ ID NO: 3, 4, 5, 6 or 7.
  • Functional equivalents of the BCI-4 promoter derived from the BCI-4 promoter sequences according to the invention, for example by substitution, insertion or deletion of nucleotides, have a homology of at least 50%, preferably 60%, preferably at least 70%, particularly preferably at least 80%, entirely particularly preferably at least 90% of the promoter sequence according to SEQ ID NO: 2 or a functionally equivalent part thereof, preferably an equivalent part according to SEQ ID NO: 8, and are characterized by essentially the same promoter activity compared to the BCI-4 promoter sequence according to SEQ-ID NO: 2 or a functionally equivalent part thereof, preferably an equivalent part according to SEQ ID NO: 8.
  • GAP Garnier ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • Gap Weight 12 Length Weight: 4
  • An example is a sequence that has a homology of at least 50% on a nucleic acid basis with the sequence
  • SEQ ID NO: 1 understood a sequence which, when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set, has a homology of at least 50%.
  • Further examples of the promoter sequences used in the expression cassettes or expression vectors according to the invention can easily be found, for example, from various organisms whose genomic sequence is known, for example from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthinum, by comparing homology from databases .
  • BCI-3 or BCI-4 gene or cDNA sequences for the production of probes for screening genomic libraries of other organisms.
  • homologous promoter sequences including functional equivalents, can also be found.
  • Functional equivalents furthermore means DNA sequences which, under standard conditions, have a nucleic acid sequence as described by SEQ ID NO: 1 or 2 or the functionally equivalent parts derived from them as described in SEQ ID NO: 3, 4, 5, 6, 7 or 8 or hybridize the complementary nucleic acid sequences and which essentially have the same promoter activities as the nucleic acid sequences described by SEQ ID NO: 1 or 2 or the functionally equivalent parts derived from them according to SEQ ID NO: 3, 4, 5, 6, Have 7 or 8.
  • Standard hybridization conditions are to be understood broadly and mean stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C. C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0).
  • the temperature during the washing step can be raised from low stringent conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can be kept constant and only the other can be varied. Denaturing agents such as formamide or SDS can also be used during hybridization. In counter If 50% formamide was used, the hybridization is preferably carried out at 42 ° C.
  • Ficoll 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium current at 42 ° C, or
  • Functional equivalents include - in addition to those which are "silent", ie which leave the promoter activity unchanged - also those promoter variants whose function is weakened or enhanced compared to the starting promoter.
  • a method for producing functional equivalents according to the invention preferably comprises the introduction of mutations into the BCI-3 promoter sequence according to SEQ ID NO: 1 or the BCI-4 promoter sequence according to SEQ ID NO: 2 or the functionally equivalent parts derived from these according to SEQ ID NO : 3, 4, 5, 6, 7 or 8.
  • Mutagenesis can be carried out in an undirected ("random") manner, the mutagenized sequences then being screened for their promoter activity according to a "trial-by-error" procedure.
  • partial sequences that are responsible for the promoter properties of the BCI-3 or BCI-4 promoter can be identified (e.g. using the above-mentioned computer algorithms).
  • Other related or unrelated nucleic acid sequences for example promoter sequences
  • non-essential sequences can be deleted without significantly impairing the promoter activity.
  • Methods for mutagenizing nucleic acid sequences include, for example, the use of oligonucleotides with one or more mutations in comparison to the region to be mutated (e.g. as part of a "site-specific mutagenesis").
  • primers with approximately 15 to approximately 75 nucleotides or more are used, preferably approximately 10 to approximately 25 or more nucleotide residues being located on both sides of the sequence to be changed.
  • the details and implementation of said mutagenesis methods are known to the person skilled in the art (Kunkel et al. (1987) Methods Enzy ol 154: 367-382; Tomic et al.
  • Mutagenesis can also be achieved by treating, for example, vectors which contain one of the nucleic acid sequences according to the invention with utagenizing agents such as hydroxylamine.
  • the invention further relates to transgenic expression cassettes which comprise one of the promoter sequences according to the invention. Expression cassettes for transgenic expression of nucleic acids are preferably claimed
  • a) or b) are functionally linked to a nucleic acid sequence to be expressed transgenically.
  • transgene means all such constructions which have been obtained by genetic engineering methods in which either a) the promoter of the BCI-3 gene according to SEQ ID No: 1 'or a functional equivalent or functional equivalent part of the same, preferably a functionally equivalent part according to SEQ ID NO: 3, 4, 5, 6 or 7, or the promoter of the BCI-4 gene according to SEQ ID No: 2 or a functional equivalent or part of the same, preferably a functionally equivalent part according to SEQ ID NO: 8, or
  • nucleotide residues are not in their natural, genetic environment (i.e. at their natural chromosomal locus) or have been modified by genetic engineering methods, the modification being, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • the expression of the nucleic acid sequence to be expressed can lead to the formation of sense RNA or anti-sense RNA.
  • the sense RNA can consequently be translated into certain polypeptides.
  • anti-sense RNA for example, the expression of certain genes can be down-regulated.
  • a functional link is understood to mean, for example, the sequential arrangement of one of the promoters according to the invention and a nucleic acid sequence to be expressed transgenically and, if appropriate, other regulatory elements such as a terminator in such a way that each of the regulatory elements can fulfill its function in the transgenic expression of the nucleic acid sequence, depending on the arrangement of the nucleic acid sequences to sense or anti-sense RNA. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other IJNA molecules.
  • nucleic acid sequence to be expressed transgenically is positioned behind (ie at the 3 'end) the promoter sequence according to the invention, so that both sequences are covalently linked to one another.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
  • the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector and inserted into a plant genome by, for example, transformation.
  • An expression cassette according to the invention is, however, also to be understood as such constructions in which one of the promoters according to the invention is introduced into a host genome, for example via a targeted homologous recombination or a random insertion, there takes over regulatory control over nucleic acid sequences then functionally linked with it and which controls transgenic expression thereof.
  • an expression cassette according to the invention is obtained which controls the expression of the specific polypeptide as a result of chemical induction.
  • the insertion of the promoter can also take place in such a way that antisense RNA is expressed to form the nucleic acid coding for a specific polypeptide.
  • the expression of the particular polypeptide is thus down-regulated or switched off as a result of chemical induction.
  • a nucleic acid sequence to be expressed transgenically - for example by means of a homologous recombination - can be placed behind an endogenous, natural promoter according to the invention (for example the BCI-3 or BCI-4 promoter), which likewise gives an expression cassette according to the invention.
  • Vectors according to the invention also contain the expression cassettes described above.
  • Vectors are recombinant DNA sequences into which the expression cassettes according to the invention can be inserted into a suitable host for the purpose of isolation, multiplication and transformation.
  • the vectors can preferably be propagated in a host such as E. coli and are suitable for the transformation of plant cells or of Agrobacterium.
  • Vectors can be, for example, plasmids, cosmids, phages, viruses or also Agrobacterium.
  • nucleic acid sequences contained in the expression cassettes or vectors according to the invention can be linked to further genetic control sequences in addition to a promoter according to the invention.
  • genetic control sequences is to be understood broadly and means all those sequences which have an influence on the formation or the function of the expression cassette according to the invention, the vector or a organism transformed with the aforementioned.
  • Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms.
  • the expression cassettes according to the invention preferably comprise 5 'upstream of the respective nucleic acid sequence to be expressed transgenically, one of the promoters according to the invention and 3' downstream a terminator sequence as an additional genetic control sequence and, if appropriate, further customary regulatory elements, in each case functionally linked to the transgene expressing nucleic acid sequence.
  • Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties.
  • Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH (1991) J Biol Chem 266 (26): 17131 -17135) and heat stress (Schöffl F et al. (1989) Mol Gen Genet 217 (2- 3): 246-53).
  • promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria.
  • Genetic control sequences also include the 5 'untranslated regions, introns or non-coding 3' regions of genes, preferably the barley BCI-3 or BCI-4 gene. Also Sequences from other plant species, such as the Actin-1 or Actin-2 intron. It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes.
  • the 5'-leader sequence from the tobacco mosaic virus may be mentioned as an example of such translation enhancers (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711).
  • Other 5 'untranslated sequences can promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440.).
  • the nucleic acid sequences given under SEQ ID N0: 1 and 2 each contain the 5 'untranslated region up to the ATG start codon of the BCI-3 or BCI-4 protein.
  • the expression cassette can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable an increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically.
  • the nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct.
  • Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome.
  • homologous recombination for example, the natural promoter of a specific gene can be exchanged for a promoter according to the invention.
  • Methods such as cre / lox technology allow tissue-specific, possibly inducible, removal of the expression cassette from the genome of the host organism (Sauer B. Methods. 1998; 14 (4): 381-92).
  • certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.
  • the promoter to be introduced can be placed by means of homologous recombination in front of the target gene to be expressed transgenically, in that the promoter is linked to DNA sequences which are, for example, homologous to endogenous sequences which are upstream of the reading frame of the target gene. Such sequences are to be understood as genetic control sequences.
  • the two homologous sequences can interact and thus place the promoter sequence at the desired location in front of the target gene, so that the promoter sequence is now functionally linked to the target gene stands and forms an expression cassette according to the invention.
  • the selection of the homologous sequences determines the insertion site of the promoter.
  • the expression cassette can be generated by homologous recombination using a single or a double reciprocal recombination.
  • simple reciprocal recombination only a single recombination sequence is used and the entire DNA introduced is inserted.
  • double-reciprocal recombination the DNA to be introduced is flanked by two homologous sequences and this is done
  • Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate. One way that happens randomly
  • Removing 20 integrated sequences and thus enriching cell clones with a correct homologous recombination consists in using a sequence-specific recombination system as described in US Pat. No. 6,110,736.
  • a variety of sequence specific recombination systems can be used, for example
  • the Cre / lox system 25 called the Cre / lox system, the FLP / FRT system, the gin recombinase, the pin recombinase from E. coli and the R / RS system of the pSRI plasmid.
  • the Pl Cre / lox and the FLP / FRT system are preferred.
  • the recombinase (Cre or FLP) interacts specifically with its respective recombination sequences (34 bp lox sequence or
  • Suitable polyadenylation signals as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J
  • terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.
  • a functional linkage or an expression cassette according to the invention can be produced by means of common recombination and cloning techniques, as described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) and in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) and in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience or Gelvin et al.
  • sequences can also be positioned between the promoter according to the invention and the nucleic acid sequence to be expressed, which, for example, have the function of a linker with certain restriction enzyme interfaces or a signal or transit peptide.
  • the insertion of sequences can also lead to the expression of fusion proteins.
  • An expression cassette according to the invention is produced, for example, by fusing a promoter according to the invention with a nucleotide sequence to be expressed, and a terminator or polyadenylation signal.
  • the expression cassettes according to the invention and the vectors derived from them can contain further functional elements.
  • the term functional element is to be understood broadly and means all those elements which have an influence on the production, reproduction or function of the expression cassettes, vectors or transgenic organisms according to the invention. Examples include, but are not limited to:
  • Selection markers which are resistant to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. to lend.
  • herbicides such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. to lend.
  • Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences which code for phosphinothricin acetyltransferases (PAT) and
  • Glyphosate ® N- (phosphonomethyl) glycine
  • the degrading enzyme for Glyphosat ® coding gox gene glyphosate oxidoreductase
  • the deh gene coding for a dehalogenase that inactivates dalapon
  • sulfonylurea imidazolinone inactivating acetolactate synthases (ALS) or acetohydroxy acid synthases (AHAS) as well as bxn genes that degrade nitrodynilase enzymes.
  • the selection marker allows one Selection of the transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84).
  • GUS ⁇ -glucuronidase
  • uidA uidA gene which encodes an enzyme for various chromogenic substrates
  • R-locus gene encodes a protein that inhibits the production of anthocyanin pigments (red color) in vegetable
  • the genes of the R gene complex from maize are particularly advantageous.
  • the R gene complex encodes a protein that regulates the production of anthocyanin pigments in most seeds and plant tissues.
  • Corn can have one to four R alleles that regulate pigmentation in a developmental and tissue dependent manner.
  • the transgenic expression of an R gene in a cell leads to the formation of a red pigment.
  • Corn lines that carry the dominant alleles of the genes of the anthocyanin biosynthetic pathways (C2, Al, A2, Bzl and Bz2), but have a recessive allele of the R locus, would result in red pigmentation when transformed and transgenically expressed.
  • the Wisconsin 22 line can be used, which contains the rg-Stadler allele and TR112, a K55 derivative which is r-g, b, Pl.
  • the alternative can be used, which contains the rg-Stadler allele and TR112, a K55 derivative which is r-g, b, Pl.
  • Genotype can be used as long as the Cl and R alleles are introduced together.
  • ⁇ -lactamase gene (Sutcliffe (1978) Proc Natl Acad Sei USA 75: 3737-3741): encodes an enzyme for various chromogenic substrates (e.g. PADAC, a chromogenic cephalosporin).
  • xylE gene (Zukowsky et al. (1983) Proc Natl Acad Sei USA 80: 1101-1105): encodes a catechol dioxygenase that can convert chromogenic catechols. 5.
  • Alpha amylase gene (Ikuta et al. (1990) Bio / technol. 8: 241-242).
  • Tyrosinase gene (Katz et al. (1983) J Gen Microbiol 129: 2703-2714): Coded for an enzyme that tyrosine too
  • ⁇ -galactosidase gene encoded for an enzyme for which various chromogenic substrates are available.
  • Luciferase (lux) gene (Ow et al. (1986) Science, 234: 856-859; Millar et al. (1992) Plant Mol Biol Rep 10: 324-414) allows bioluminescence detection. Luciferases such as the "firefly luciferase" enable quantification using X-ray films, scintillation counters, fluorescence spectrophometers, sensitive, photon-counting cameras or luminometers. Chromogenic substrates and other auxiliaries are used for this. The system can be used to screen
  • Aequoringen (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268): Can be used in calcium-sensitive bioluminescence detection.
  • GFP green fluorescent protein
  • GFP expression in a plant or cell can be visualized as a result of illumination with light of specific wavelengths.
  • Chloramphenicol acetyl transferase neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS) can also be used as marker proteins.
  • NPT neomycin phosphotransferase
  • NOS nopaline synthase
  • OCS octopine synthase
  • origins of replication which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli.
  • origins of replication include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sabrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid sequence to be expressed transgenically numerous advantageous forms of use for the promoters, vectors, expression cassettes according to the invention or organisms transformed with them are possible. Numerous possibilities of use are known to the person skilled in the art. In addition to improving plant properties (e.g. nutrient content or composition, taste, yield, etc.), other advantageous properties such as male or female sterility, increased stress tolerance and / or resistance (against e.g. drought, heat, salt, cold, frost, increased) can also be used Moisture, oxidative stress etc.) can be achieved.
  • the applications and nucleic acid sequences mentioned as a result are therefore to be understood as exemplary and not restrictive.
  • a plant's natural defense mechanisms against pathogens are often inadequate.
  • the introduction of foreign genes from plants, animals or microbial sources can strengthen the immune system. Examples are protection against insect damage in tobacco by expression of the Bacillus thuringiensis endotoxin (Vaeck et al. (1987) Nature 328: 33-37) or protection of the tobacco against fungal attack by expression of a chitinase from the bean (Broglie et al. (1991 Science 254: 1194-1197).
  • Previous approaches have been based on constitutive overexpression of these genes. Desirably, however, these antibodies should only be expressed when the need arises as a result of a pathogen attack.
  • a constitutive overexpression - even when there is no need - can have an adverse effect on the growth, yield and other properties of the plant.
  • constitutive expression can increasingly switch off the transgene ("gene silencing").
  • Transgenic expression of certain polypeptides can lead to increased resistance to bacterial and fungal attack.
  • Preferred is the expression of so-called "peptide antibiotics", “pathogenesis related” (PR) proteins, toxin resistance, and of proteins which relate to the host-pathogen interaction.
  • Preferred peptide antibiotics count to the class of Cecropine or Magainine and inhibit the growth of different types of bacteria and fungi.
  • PR genes can also confer resistance to pests. These genes are naturally induced as a result of pest infestation and are divided into different classes (Bol et al. (1990) Annu Rev Phytopath 28: 113-138).
  • the PR proteins include ß-1, 3-glucanases, chitinases, osmotin and other proteins.
  • Resistance to, for example, fungi, insects, nematodes and diseases can be achieved through the targeted secretion or enrichment of certain metabolites or proteins.
  • examples include glucosinolates (defense against nematodes), lysozymes from non-plant sources such as, for example, T4 lysozyme or lysozyme from various mammals, arcelin, ⁇ -amylase inhibitor,
  • RNAsen or ribozymes as well as further nucleic acid sequences or proteins listed below.
  • Examples include:
  • Bt genes Bacillus thuringiensis crystal toxin genes (Bt genes; Watrud et al. (1985) In: Engineered Organisms and the Environment). Bt genes confer resistance to
  • BT genes Lepidopteran or Coleopteran parasites, such as the European corn borer (European Corn Borer (ECB)).
  • European corn borer European Corn Borer (ECB)
  • EBC European Corn Borer
  • Different BT genes coding for complete or truncated BT proteins are described.
  • BT genes are described in which the coding
  • protease inhibitors can also confer resistance to insect attack (Johnson et al. (1989) Proc Natl Acad Sei USA 86: 9871-9875).
  • the protease inhibitor II gene (pinII) from tomato or potato is particularly preferred. A combined one is very particularly preferred
  • Coexpression of a pinll gene with a Bt gene is also preferred.
  • cowpea trypsin inhibitor CpTI; Hilder VA et al. (1989) Plant Mol Biol 13 (6): 701-10.
  • genes which code for enzymes or cofactors which inhibit the digestive system of insects include its oryzacystatin and amylase inhibitors (e.g. from wheat or barley).
  • Lectins are multivalent carbohydrate-binding proteins with the ability to agglutinate red blood cells of different species. Lectins have insecticidal activity against a number of pests (Murdock et al. (1990) Phytochemistry 29: 85-89;
  • Lectingenes that can be used advantageously include phytohemagglutinin, snowdrop lectin, barley and wheat germ agglutinin (WGA), and rice agglutinins (Gatehouse et al. (1984) J Sei Food Agric 35: 373-380), with WGA being particularly preferred.
  • Genes that control the production of small or larger polypeptides with insecticidal activity are also included. Examples are juvenile hormone esterase (Hammock et al. (1990) Nature 344: 458-461) or compounds which impair the pupation of insects by inhibiting the expression or the activity of the ecdysteroid UDP-glucosyl transferase.
  • Genes coding for avermectin (avermectin and abamectin; Campbell WC (ed.), In: Avermectin and Aba ectin, 1989. Ikeda et al. (1987) J Bacteriol 169: 5615-5621), and genes coding for ribosome inactivating proteins (RIPs).
  • Enzymes that affect the integrity of the insect cover are also preferred. Examples include genes which code for chitinases (such as, for example, the chit42 endochitinase from Trichoderma harzianum; GenBank Acc.-No .: S78423) or glucanases, proteases or lipases, and also genes which code for the production of nikkomycin
  • genes are preferred which reduce the quality of the plant as insect food.
  • An insecticidal effect can be achieved by changing the sterol composition.
  • Lipoxygenase are also natural, plant-based enzymes, which can be assigned the property of reducing the suitability of a plant as an insect food.
  • Achieving insect defense or attraction for example by increasing the release of volatile fragrance or messenger substances by, for example, enzymes from terpene biosynthesis.
  • Expression of enzymes with a function in the biosynthesis of alkaloids such as is preferred
  • Benzophenanthridine alkaloids and indole alkaloids as well as of terpene biosynthesis enzymes such as monoterpenes and sesquiterpenes.
  • the overexpression of endoxyloglucan transferase may be mentioned as an example.
  • phenylammonylases and other enzymes of the phenylpropanoid pathway which are responsible for the biosynthesis of a large number of compounds based on the phenylpropane backbone, e.g. Lignin, phytoalexins, pterocarpones, furano-coumarins. Expression of is also preferred
  • Isoflavone-2-hydroxylases and of enzymes with a function in lignin polymerization such as anionic pe- roxidasen.
  • the expression of cell wall proteins such as glycine hydroxyproline-rich proteins (for example exlensins) is also preferred.
  • the transgenic expression of certain genes can provide resistance to virus attack.
  • virus infection can be prevented by expression of viral coat proteins (Cuozzo et al. (1988) Bio / Technology, 6: 549-553; Hemenway et al. (1988) EMBO J 7: 1273-1280; Abel et al. (1986) Science 232: 738-743).
  • antisense nucleic acid sequences of certain viral genes e.g. genes of the viral replication apparatus
  • ribozymes can also be used advantageously.
  • Other approaches include the use of satellite viruses.
  • An increase in the quality and / or quantity of the crop yield e.g. increased amount of fruit or seeds
  • influencing the rate of growth to faster or slower growth enabling growth outside the usual growth period
  • resistance to adverse weather conditions heat, dryness, wet, cold, Frost
  • Numerous methods, proteins etc. are known to the person skilled in the art, with the aid of which these properties can be achieved. Examples include:
  • Nucleic acids which code for the transcriptional activator CBF1 from Arabidopsis thaliana (GenBank Acc.-No .: U77378) or the "antifreeze protein" from Myoxocephalus octodecemspinosus (GenBank Acc.-No .: AF306348) or functional equivalents thereof are particularly preferred.
  • nucleic acid sequences that confer such resistance are described above.
  • the combination of these sequences with one of the promoters according to the invention is particularly advantageous, since the resistance can only be generated in a constitutive manner if necessary (after induction with a chemical inductor).
  • the constitutive expression has further potentially negative consequences when it is approved under consumer acceptance.
  • Expression of resistance is required from a practical point of view - in the laboratory - only when producing a transgenic plant (using the resistance as a selection marker) or - in agriculture - only when herbicides are used. Especially in the latter case, the combination of herbicide and chemical inducer in one formulation would be seen as an advantageous application.
  • nucleic acid sequences that can act as reporters or marker genes are described above.
  • the combination of these sequences with the promoters according to the invention is a particularly preferred embodiment, since it enables the identification of further chemical inducers, for example as part of the screening of chemical substance libraries. Due to the expression behavior of the BCI-3 and BCI-4 genes, it can be assumed that chemical inducers of these genes also induce SAR.
  • Such chemical compounds are extremely interesting compounds for use in agriculture. Similar to the BION ⁇ , they have the potential to induce resistance to pathogens after application to the plant.
  • An object of the invention therefore includes methods for the identification of pathogen resistance-inducing compounds, characterized in that
  • an expression cassette consisting of a reporter gene in functional linkage with a chemically inducible promoter selected from the group of sequences consisting of
  • the organism or the tissue, organ, part, cell culture or cell thereof is treated with a chemical compound selected from a library of chemical compounds, and
  • an "antisense” nucleic acid initially means a nucleic acid sequence which is completely or partially complementary to at least part of the "sense" strand of a target protein to be repressed. It is known to the person skilled in the art that he can use alternative cDNA or the corresponding gene as a starting template for corresponding antisense constructs.
  • the “antisense” nucleic acid is preferably complementary to the coding region of the target protein or a part thereof. However, the “antisense” nucleic acid can also be complementary to the non-coding region or a part thereof.
  • an antisense nucleic acid can be designed in the manner familiar to the person skilled in the art, taking into account the base pair rules of Watson and Crick.
  • the antisense nucleic acid comprises ⁇ -anomeric 0 nucleic acid molecules.
  • ⁇ -Anomeric nucleic acid molecules form special double-stranded hybrids with complementary RNA in which, in contrast to the normal ⁇ units, the strands run parallel to one another (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). 5
  • the antisense strategy can advantageously be coupled with a ribozyme method.
  • Ribozymes are catalytically active RNA sequences which, coupled to the antisense sequences, catalytically cleave the target sequences (Tanner NK (1999) FEMS 0 Microbiol Rev 23 (3): 257-75). This can increase the efficiency of an anti-sense strategy.
  • the expression of ribozymes to reduce certain proteins is known to the person skilled in the art (EP 291 533, EP 321 201, EP 360 257). Suitable target sequences and ribozymes can, for example, as described by Steinecke (Ribozymes, Methods in Cell Biology 50, Galbraith et al. Eds, Academic Press, Inc.
  • RNA By expression of a sense RNA that is homologous to that of an endogenous transcript, the expression of the corresponding endogenous protein can also be reduced (co-suppression). It has been demonstrated in tobacco, tomato and petunia that the expression of sense RNA to an endogenous gene can reduce or switch off its expression, similar to that described for antisense approaches (Goring et al. (1991) Proc Natl Acad Sei USA 88: 1770-1774; Smith et al. (1990) Mol ⁇ Gen Genet 224: 447-481; Napoli et al. (1990) Plant Cell 2: 279-289; Van der Krol et al. (1990) Plant Cell 2: 291-99). The introduced construct can represent the gene to be reduced in whole or in part. The possibility of translation is not necessary. Corresponding methods are described (EP 465 572; EP 647 715; US 5,283,184; US 5,231,020).
  • dsRNAi double-stranded RNA interference
  • double-stranded RNA intererence The method of gene regulation using double-stranded RNA (“double-stranded RNA intererence") is very particularly preferred.
  • Corresponding processes are known to the person skilled in the art and are described in detail (for example Matzke MA et al. (2000) Plant Mol Biol 43: 401-415; Fire A. et al (1998) Nature 391: 806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364).
  • the simultaneous introduction of strand and counter strand results in highly efficient suppression of native genes.
  • Another object of the invention relates to transgenic organisms, transformed with at least one expression cassette according to the invention or a vector according to the invention, as well as cells, cell cultures, tissues, parts - such as leaves, roots etc. in plant organisms - or well propagated from such organisms.
  • Organism, starting or host organisms are understood to mean prokaryotic or eukaryotic organisms, such as, for example, microorganisms or plant organisms.
  • Preferred microorganisms are bacteria, yeast, algae or fungi.
  • Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or cyano-bacteria, for example of the genus Synechocystis.
  • microorganisms which are capable of infecting plants and thus of transmitting the constructs according to the invention.
  • Preferred microorganisms are those from the genus Agrobacterium and in particular from the type Agrobacterium turnefaciens.
  • Preferred yeasts are Candida, Saccharo yces, Hansenula or Pichia.
  • Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Seetion B. Vol 37, No 1,2 (1995) on page 15, table 6 described mushrooms.
  • Plants are particularly preferred host or starting organisms as transgenic organisms. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Also included are the mature plants, seeds, sprouts and seedlings, as well as parts, propagation material and cultures derived from them, for example cell or callus cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
  • transgenic plants according to the invention are selected in particular from monocotyledonous crop plants, such as, for example, cereals of the genus Gramineae (such as rice, corn, wheat), in particular wheat, barley, millet, rye, triticale, corn, rice or oats and sugar cane.
  • the transgenic plants according to the invention are selected in particular from dicotyledonous crop plants, such as, for example
  • Brassicacae such as rapeseed, cress, Arabidopsis, turnip,
  • Leguminosae such as soy, alfalfa, pea, alfalfa, bean or peanut
  • Solanaceae such as potato, tobacco, tomato, or eggplant
  • Asteraceae such as sunflower, tagetes, lettuce or calendula, Cucurbitaceae such as melon, pumpkin or zucchini, Rubiaceae such as Coffea arabica or Coffea liberica (coffee bush)
  • Theaceae such as Camillia sinensis or Thea sinensis
  • Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae or cyanobacteria, and mosses.
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.
  • the production of a transformed organism or a transformed cell requires that the corresponding DNA (for example one of the expression cassettes or vectors according to the invention) is introduced into the corresponding host cell.
  • a large number of methods are available for this process, which is referred to as transformation (see also Keown et al. (1990) Methods in Enzymology 185: 527-537).
  • the DNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion.
  • the DNA can also be obtained by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes.
  • Electroporation is another suitable method for introducing DNA in which the cells are reversibly permeabilized by an electrical pulse.
  • Such and other methods are described, inter alia, by Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111-116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al. (1980) Science 208: 1265; Horsch et al. (1985) Science 227: 1229-1231; DeBlock et al.
  • Suitable methods are above all protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-containing solution and microinjection.
  • transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes (Horsch RB et al. (1985) Science 225: 1229; Marton L (1984) Cell Culture and Somatic Ceil Genetics of Plants 1: 514-521).
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes Horsch RB et al. (1985) Science 225: 1229; Marton L (1984) Cell Culture and Somatic Ceil Genetics of Plants 1: 514-521.
  • T-DNA transferred DNA
  • the Agrobacterium -mediated transformation is best suited for dicotyledonous, diploid plant cells, whereas the direct transformation techniques are suitable for every cell type.
  • the expression cassette is introduced by means of plasmid vectors.
  • Preferred vectors are those which enable stable integration of the expression cassette into the host genome.
  • plasmid In the case of injection or electroporation of DNA into plant cells, there are no special requirements for the plasmid used. Simple plasmids such as the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary that there is an additional selectable marker gene on the plasmid.
  • Transformation techniques are described for various monocot and dicotyledonous plant organisms. Furthermore, various possible plasmid vectors stand for the introduction of foreign ones Genes are available in plants, which usually contain an origin of replication for reproduction in E. coli and a marker gene for selection of transformed bacteria. Examples are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the expression cassette can be inserted into the vector via a suitable restriction site.
  • the resulting plasmid is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant plasmid obtained using methods familiar to the person skilled in the art. Restriction analysis and sequencing can be used to check the cloning step.
  • Transformed cells i.e. Those that contain the inserted DNA integrated into the DNA of the host cell can be selected by untransforced ones if a selectable marker is part of the inserted DNA. Any gene that can confer resistance to antibiotics or herbicides can act as a marker, for example. Transformed cells that express such a marker gene are able to survive in the presence of concentrations of a corresponding antibiotic or herbicide that kill an untransformed wild type. Examples are the bar gene that confers resistance to the herbicide phosphinotricin (Rathore KS et al.
  • the expression cassette must be integrated in special plasmids, either in an intermediate vector (English: shuttle or intermediate vector) or in a binary vector. If, for example, a Ti or Ri plasmid is to be used for the transformation, at least the right boundary, but mostly the right and the left boundary of the Ti or Ri plasmid T-DNA as flanking region, is connected to the expression cassette to be inserted.
  • Binary vectors are preferably used. Binary vectors can replicate in both E.coli and Agrobacterium.
  • the selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin.
  • a host Agrobacterium that is functioning should already contain a plasmid with the vir region. This is necessary for the transfer of T-DNA to the plant cell.
  • An Agrobacterium transformed in this way can be used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and described (EP 120516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V; Fraley RT et al. (1983) Proc Natl Acad Sei USA 80 (15): 4803-7. And An et al. (1985) EMBO J 4: 277-287).
  • Various binary vectors are known and some are commercially available, for example pBIN19 (Clontech Laboratories, Inc. USA).
  • plant explants are co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes Starting from infected plant material (e.g. parts of leaves, roots or stems, but also protoplasts or suspensions of plant cells), whole plants can be regenerated using a suitable medium that can contain, for example, antibiotics or biocides for the selection of transformed cells.
  • the plants obtained can then be screened for the presence of the introduced DNA, here the expression cassette according to the invention.
  • the corresponding genotype is generally stable and the corresponding insertion is also found in the subsequent generations.
  • the integrated expression cassette contains a selection marker which gives the transformed plant resistance to a biocide (for example a herbicide) or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc.
  • the selection marker allows the selection of transformed cells from untransforced (McCormick et al. (1986) Plant Cell Reports 5: 81-84).
  • the plants obtained can be in the usual
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens. Mieren, for example pBinl9 (Bevan et al. (1984) Nucl Acids Res 12: 8711).
  • a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.
  • the effectiveness of the expression of the transgenically expressed nucleic acids can be determined, for example, in vitro by sprout meristem propagation using one of the selection methods described above.
  • transgenic organisms described above cell cultures, parts - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits.
  • Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as food or feed, for example directly or after preparation known per se.
  • Another object of the invention relates to the use of the transgenic organisms according to the invention described above and the cells, cell cultures, parts derived therefrom - such as roots, leaves etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits for the production of food or feed, pharmaceuticals or fine chemicals.
  • This process is widely applicable to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavors, aromas and colors.
  • the production of vitamins and vitamin precursors is particularly preferred, in particular tocopherols and tocotrienols and caro- tinoiden.
  • the transformed host organisms and the isolation from the host organisms or from the growth medium are cultivated using methods known to those skilled in the art.
  • the production of pharmaceuticals, such as antibodies or vaccines is described in Hood EE and Jilka JM (1999) Curr Opin Biotechnol 10 (4): 382-6; Ma JK Vine ND (1999) Curr Top Microbiol Immunol 236 275-92.
  • SEQ ID NO: 1 promoter and 5 'untranslated region of the barley BCI-3 gene (1850 bp)
  • SEQ ID NO: 2 promoter and 5 'untranslated region of the BCI-4 gene from barley
  • SEQ ID NO: 5 "Prom900" deletion variant of the BCI-3 promoter from barley (898 bp)
  • SEQ ID NO: 6 "Prom1200" deletion variant of the BCI-3 promoter from barley (1173 bp)
  • SEQ ID NO: 7 "Proml700” variant of the BCI-3 promoter from barley (1772 bp)
  • SEQ ID NO: 8 "Prom900" variant of the BCI-4 promoter from barley (904 bp)
  • SEQ ID NO: 9 oligonucleotide primers M13-fwd 5 '-GTAAAACGACGGCCAGTG-3'
  • SEQ ID NO: 10 oligonucleotide primers Ml3-Rev 5 '-GGAAACAGCTATGACCATG-3'
  • SEQ ID NO: 11 oligonucleotide primers PPl-5 5'-CTC GCA GTT GGC CGG CAC CGT-3 '
  • SEQ ID NO: 12 oligonucleotide primers PPl-3 5 '-TGA CCC CAT GTA CTA CAT CGCCT-3'
  • SEQ ID NO: 13 oligonucleotide primers PP2-5 5'-CTT CCA GTC CCG CAC GTT GTA C-3 '
  • SEQ ID NO: 14 oligonucleotide primer PP2-3 5 '-GTC GGT TGC GGC TAT TTG ATT GC-3'
  • SEQ ID NO: 15 oligonucleotide primers PP3-5 5 '-AGC CAT CAT CCC TGC GGA TCC A-3' 16.
  • SEQ ID NO: 16 oligonucleotide primers PP3-3 5 '-GAG CGT CCG GGC GCG GCC TT-3'
  • SEQ ID NO: 17 oligonucleotide primer PP4-5 5'-GGA TCC AAG CGA GAT TTG AAC GGA-3 '
  • SEQ ID NO: 18 oligonucleotide primers PP4-3 5 '-GCG GCC TTG CCG GAC GCG GT-3'
  • SEQ ID NO: 19 oligonucleotide primer PP5-5 5 '-GTA TGC CGC TCC ATG TTA GAA GAT-3'
  • SEQ ID NO: 20 oligonucleotide primers PP5-3 5 '-ACA TTA CAG GCA GCG TCC GAC AC-3'
  • SEQ ID NO: 21 oligonucleotide primer PP6-5 5'-ACT CAT GCC GGT GCA GAT CTT CG-3 '
  • SEQ ID NO: 22 oligonucleotide primer PP6-3. - 5 '-ATG CGG GGA TGA GGA GGA CAT G-3'
  • SEQ ID NO: 23 oligonucleotide primer BCI3-5 5 '-aagcttcaccaactcccttcaaggtctaa-3'
  • SEQ ID NO: 24 oligonucleotide primer BCI3-3 5 '-ctgcagtgtgtgtgcttgctgtgatgc-3'
  • SEQ ID NO: 25 oligonucleotide primers P300-5
  • SEQ ID NO: 26 oligonucleotide primer P600-5 5 '-aag £ tttagctccgttcccatgatct-3'
  • SEQ ID NO: 27 oligonucleotide primer P900-5 5 '-aagcttggctaatatgcgcgtaaaa-3'
  • SEQ ID NO: 28 oligonucleotide primer P1200-5 5 '-aagcttcgtccaattctaggatgatgatgc-3' BCI3-3
  • SEQ ID NO: 29 oligonucleotide primer OPNl 5 '-TGA ATG GTC ATG GCT GCT TA-3'
  • SEQ ID NO: 30 oligonucleotide primers OPN2 5 '-GGC TCT GAA TCG CAC ATT CT-3' 31.
  • SEQ ID NO: 31 oligonucleotide primer OPN3 5 '-GTA CCT CTC CTC CCC GCT CAC T-3'
  • SEQ ID NO: 32 oligonucleotide primer OPN4 5 '-GCC TCG TTG TAA CGG GTA CT-3'
  • SEQ ID NO: 33 oligonucleotide primer OPN5 5 '-TCATGCATATCTATGTCTTTCTCTTC-3'
  • SEQ ID NO: 34 oligonucleotide primer OPN6 5'-AAA CGA TTC GCT GCA AAA AT-3 '
  • SEQ ID NO: 35 oligonucleotide primer OPN7 5 '-GTA CCT CTC CCC GCT CAC T-3'
  • SEQ ID NO: 36 oligonucleotide primer BCI4-5 5 '-agcgaatcgtttttcccttt-3'
  • SEQ ID NO: 37 oligonucleotide primer BCI4-3 5 '-cctgagtgtacggctctgagatg-3'
  • Fig. 1 Overview of the BCI-3 promoter-reporter constructs that were used for the transient assay.
  • Fig. 2 Induction of BCI3 promoter activity by BTH (BION). Eight leaf segments per experiment were evaluated.
  • the number of GFP-positive cells [GFP] is indicated on the y-axis.
  • the entire BCI3 promoter (BCI3-Prom 1700) and the different deletion variants (BCl3-Prom 300; BCI3-Prom 600; BCI3-Prom 900; BCl3-Prom 1200) are listed on the y axis.
  • the results are given in each case for leaves treated with BTH (white bars) or untreated sheets (black bars). Transformation with the pGFP blank vector did not result in GFP positive cells.
  • the high background activity in untreated cells is probably due to the injury to the cells due to the bombardment with the tungsten particles (see below).
  • Fig. 3 Induction of BCI3 promoter activity by jasmonate (JA). Eight leaf segments per experiment were evaluated. The number of GFP-positive cells [GFP] is on the y-axis. given. The y-axis shows the entire BCI3 promoter (BCI3-Prom 1700) and the various deletion variants (BCI3-Prom 300; BCI3-Prom 600; BCI3-Prom 900; BCI3-Prom 1200). The results are given in each case for sheets treated with JA (white bars) or untreated sheets (black bars). Transformation with the pGFP blank vector did not result in GFP positive cells. The high background activity in untreated cells is probably due to the injury to the cells due to the bombardment with the tungsten particles (see below).
  • Fig. 4 Repression of BCI3 promoter activity by sorbitol. Eight leaf segments per experiment were evaluated. The number of GFP-positive cells [GFP] is indicated on the y-axis. The entire BCI-3 promoter (BCI3-Prom 1700) and the various deletion variants (BCI3-Prom 300; BCl3-Prom 600; BCl3-Prom 900; BCI3-Prom 1200) are listed on the y axis. The results are given for leaves treated with sorbitol (white bars) and untreated leaves (black bars). Transformation with the pGFP blank vector did not result in GFP positive cells. The high background activity in untreated cells is probably due to the injury to the cells due to the bombardment with the tungsten particles (see below).
  • Fig. 5 Analysis of the BCI-3 and BCI-4 promoter properties by Northern blot analyzes.
  • RNA 100 mg / 1 SA based on the volume of the soil or poured with water (H0). Primary leaves of the barley plants were harvested at the times indicated on the x-axis (hours after induction) and total RNA was extracted. After gel electrophoretic separation (9 to 10 ⁇ g RNA per lane) and preparation of Northern blots, the mRNA was hybridized with probes from BCI-3 and BCI-4 under stringent conditions. The uniform RNA loading of the gel was checked using ethidium bromide-mediated fluorescence of the ribosomal RNA under UV light.
  • RNA separation (10 ⁇ g RNA per lane), Northern blots were hybridized with BCI-3 and BCI-4 transcript or DNA probes under stringent conditions. The uniform RNA loading of the gel was checked on the basis of ethidium bromide-mediated fluorescence of the ribosomal RNA under UV light. BCI-4 showed no differential expression after sorbitol treatment.
  • RNA loading of the gel was determined using ethidium bromide mean fluorescence of the ribosomal RNA checked under UV light.
  • Fig. 7 Analysis of the BCI-3 and BCI-4 promoter properties in wheat by Northern blot analysis.
  • the transcript accumulation of the BCI genes after BTH application or real wheat powdery mildew (Bgt) inoculation of wheat plants was examined.
  • Fourteen day old wheat plants cv. Registrars were sprayed with 100 mg / 1 BTH (BTH) or, as a control, with the empty formulation WP (K), or inoculated with powdery mildew (Bgt). Secondary leaves were harvested at the indicated times (dpt / dpi) and total RNA extracted. After gel electrophoretic separation (approx. 10 ⁇ g RNA per lane) and preparation of Northern blots, the mRNA was hybridized with probes from BCI-3 and BCI-4 under less stringent (60 ° C.) conditions than barley samples.
  • a watering treatment with 20 mg / 1 BTH ("+”) based on the soil volume was carried out three days before the harvest, or no treatment ("-").
  • Intercellular washing liquid (IWF) was two days after the application of 20 mg / 1 BTH ("+"), or the equivalent amount of the empty formulation (WP, "-") based on the soil volume from seven-day-old barley plants cv. Ingrid won.
  • oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the cloning steps carried out in the context of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria Multiplication of phages and sequence analysis of recombinant DNA are carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6.
  • the barley variety Ingrid (Hordeum vulgäre L. cv Ingrid) comes from James McKey, University of Uppsala, Sweden.
  • the Golden Promise barley variety was provided by Paul Schulze-Lefert, Max Planck Institute for Plant Breeding Research, Cologne.
  • the seeds which had been pre-germinated in the dark on moist filter paper for 12 to 36 hours, were, unless otherwise stated, placed on the edge of a square pot (8x8cm) in Fruhstorfer earth of type P, 5 grains, covered with earth and regularly watered with tap water. All plants were grown in climate cabinets or chambers at 16 to 18 ° C, 50 to 60% relative humidity and a 16-hour light / 8-hour dark cycle with 3000 or 5000 lux (50 or 60 ⁇ ols - ⁇ - 2 photon flux density ) Cultivated for 5 to 8 days and used in the seedling stage in the experiments. In experiments in which applications were carried out on primary leaves, these were fully developed. Plants that were to be cultivated until the development of spikes were potted individually in Fruhstorfer Erde LD 80, watered regularly, repotted into larger pots if necessary and additionally illuminated in the greenhouse.
  • the epidermis of the abaxial leaf blade was removed from barley primary leaves at different harvest times and separated from the rest of the leaf tissue (mesophyll and epidermis of the adaxial leaf blade) in liquid nitrogen and stored at -70 ° C.
  • Conidiospores of Blumeria [syn Erysiphe] graminis f.sp. were used for the inoculation of barley plants with the real barley powdery mildew. Hordei spear of breed A6 (BghA ⁇ ) used (Wiberg A (1974) Hereditas 77: 89-148). This was provided by Jörn Pons-Kühne- mann, Institute for Biometry, JLU G devisen. The inoculum was grown in climatic chambers under the same conditions as described for the plants above, by transferring the conidia from infected plant material to regularly grown, 7-day-old barley plants cv. Golden Promise at a density of 100 Konidia / mm 2 .
  • BghA6 was inoculated using 7-day-old seedlings by shaking off the conidia of plants already infected in an inoculation tower with about 1 to 8 conidia / mm (unless stated otherwise).
  • Conidia from Blumeria graminis f.sp. were used to inoculate with the powdery mildew of wheat.
  • tritici spear (Bgt) used.
  • Bgt was a field isolate from Aachen, which was isolated from wheat in 1995 by Uli Beckhove, Institute for Phytopathology and Applied 5 Zoology (IPAZ) G collecten, and made available to the institute. In climatic chambers or cabinets, the isolate was exposed to wheat cv under the conditions mentioned above. Chancellor (seed breeding Engelen-Büchling oHG, Büchlingen / Oberschneiding).
  • Bacillus subtilis came from the culture collection of the Institute for Phytopathology and Applied Zoology (IPAZ), G corden, Germany.
  • the Pseudomonas strains were made available by Ina Budde and Dr Ullrich, Max Planck Institute for Terrestrial Microbiology, Marburg
  • RNA Extraction Buffer AChinsky RNA Extraction Buffer
  • the central primary leaf segment of 5 cm in length was harvested and homogenized in liquid nitrogen in mortars.
  • the homogenate was stored at -70 ° C. until the RNA extraction.
  • the frozen leaf material was extracted using an RNA
  • RNA was then dissolved in 50 ⁇ L DEPC water on ice, mixed and centrifuged for 5 min (see above). 40 ⁇ l of the supernatant was transferred as an RNA solution into a new microcentrifuge tube and stored at -70 ° C.
  • the concentration of the RNA was determined photometrically.
  • the concentrations of the RNA solutions were then adjusted to 1 ⁇ g / ⁇ L with DEPC water and checked in the agarose gel.
  • RNA concentrations in the horizontal agarose gel 1% agarose in 1 x MOPS buffer with 0.2 ⁇ g / mL ethidium bromide
  • 1 ⁇ L RNA solution with 1 ⁇ L 10 ⁇ MOPS, 1 ⁇ L color marker and 7 ⁇ L DEPC- Water added, separated according to their size at 120 V voltage in the gel in 1 x MOPS running buffer for 1.5 h and photographed under UV light. Any differences in concentration of the RNA extracts were compensated with DEPC water and the adjustment was checked again in the gel.
  • DEPC water bidistilled water, 0.1% [w / v] DEPC (diethyl pyrocarbonate), stir for at least 2 h, incubate overnight at 37 ° C, then autoclave.
  • RNA was separated in the agarose gel under denaturing conditions. A portion of RNA solution (corresponding to 6 to 15 ⁇ g RNA) was mixed with the same volume of sample buffer (with ethidium bromide), denatured for 5 min at 94 ° C., placed on ice for 5 min, briefly centrifuged and applied to the gel. The 1 x MOPS gel (1.5% agarose, ul tra pure) contained 5 volume percent concentrated formaldehyde solution (36.5% [v / v]). The RNA was separated at 100 V for 2 h and then blotted.
  • Northern blotting was carried out as an upward-directed RNA transfer in the capillary stream.
  • the gel was first swung in 25 mM sodium hydrogen / dihydrogenphosphate buffer (pH 6.5) and cut to size. Whatman paper was prepared so that it rested on a horizontal plate and protruded on two sides in a tub with 25 mM sodium hydrogen / dihydrogen phosphate buffer (pH 6.5). The gel was placed on this paper, leaving uncovered parts of the Whatman paper with a plastic film were covered. The gel was then covered with a positively charged nylon membrane (Boehringer-Mannheim) free of air bubbles, after which the membrane was again covered with absorbent paper in several layers about 5 cm high.
  • a positively charged nylon membrane Boehringer-Mannheim
  • the absorbent paper was weighed down with a glass plate and a 100 g weight. The blotting was carried out overnight at room temperature. The membrane was briefly distilled in A. swung and irradiated for RNA fixation with a light energy of 125 m in the crosslinker (biorad) UV light. The uniform RNA transfer to the membrane was checked on the UV light bench.
  • RNA from each sample were separated on an agarose gel and blotted onto a positively charged nylon membrane by capillary transfer. The detection was carried out with the DIG systems.
  • mRNA probes based on the BCI-3 (GenBank Acc.-No: AJ250282) and BCI-4 (GenBank Acc. -No.: AJ250283) cDNA sequences were prepared by first using the in the vector The cDNA fragments contained in pGEMT were amplified by the plasmid in such a way that a polymerase binding site was present at the 3 'end of the sequence, from which the antisense strand was then synthesized by in vitro transcription using a T7 or SP6 RNA polymerase with labeled UTPs could be. M13reverse and M13universal primers were used as primers in the PCR reaction. The probe was labeled with digogygenin or fluorescein or radioactive.
  • the insert of the individual vectors was amplified by PCR with flanking standard primers (M13 fwd and rev). The reaction proceeded with the following final concentrations in a total volume of 50 ⁇ L PCR buffer (Silverstar):
  • the amplificate was cleaned up using the High Pure PCR Product Purification Kit (Boehringer, Mannheim) according to the manufacturer's instructions and 4 ⁇ L were used as templates for the reaction with the corresponding DNA-dependent RNA polymerase (SP6 or T7 polymerase).
  • 4 ⁇ l of the purified PCR product were mixed with 2 ⁇ L of transcription buffer, 2 ⁇ l of NTP labeling mix, 2 ⁇ l of NTP mix and 10 ⁇ l of DEPC water. 2 ⁇ L of the T7 RNA polymerase solution were then pipetted in.
  • the reaction was then carried out at 37 ° C. for 2 h and then made up to 100 ⁇ L with DEPC water.
  • the RNA probe was detected in the ethidium bromide gel and stored at -20 ° C.
  • the reaction mixture (20 ⁇ L) for non-radioactive probes was mixed with 1 ⁇ digoxigenin or fluorescein-RNA labeling mix (Boehringer, Mannheim), that for radioactive probes with 1 mM ATP, GTP and CTP, as well as 25 ⁇ Ci a 32 P UTP , each with 40 u RNase inhibitor (Boehringer, Mannheim) and 40 u RNA polymerase in 1 x transcription buffer (Boehringer, Mannheim) incubated at 37 ° C for 2 h.
  • the reaction for the non-radioactive labeling was stopped by adding 80 ⁇ L of DEPC water and the result was checked in the agarose gel.
  • the approach for the radioactive labeling was mixed with 30 ⁇ L DEPC water, the probe was cleaned over MicroSpin G-25 columns (Amersham Pharmacia Biotech, Freiburg), and the number of radioactive decays measured in an aliquot (cpm, multipurpose Scintillation Counter, Coulter LS6500, Beckman, Kunststoff).
  • RNA polymerization, hybridization and immunodetection were largely carried out according to the manufacturer of the kit for non-radioactive RNA detection (BIG system User's Guide, DIG-Luminescence detection Kit, Boehringer-Mannheim, Kogel et al. (1994) Plant Physiol 106: 1264-1277).
  • the membranes were first swung for 5 3 ⁇ 20 min at 68 ° C. in 2 ⁇ SSC (salt, sodium cations), 0.1% SDS buffer (sodium dodecyl sulfate). The membranes were then placed on the inner wall of 68 ° C. preheated hybridization tubes (Hybaid, Heidelberg) and 30 min with 10 mL Dig-üasy hybridization buffer in the preheated hybridization
  • RNA-RNA hybrids were washed strictly twice for 20 min each in 0.1% (w / v) SDS, 0.1 x SSC at 20 68 ° C.
  • the blots were first swirled twice for 5 min at RT in 2 x SSC, 0.1% SDS. This was followed by 2 stringent washing steps at 68 ° C in 0.1 x SSC, 0.1% SDS for
  • DEPC water Distilled water is treated overnight at 37 ° C with diethyl pyrocarbonate (DEPC, 0.1%, w / v) and then autoclaved.
  • DEPC diethyl pyrocarbonate
  • 10 x MOPS buffer 0.2 M MOPS (morpholine-3-propanesulfonic acid), 0.05 M sodium acetate, 0.01 M EDTA, pH adjusted to pH 7.0 with 10 M NaOH, 1% [v / v] DEPC, autoclave. A small amount of concentrated MOPS buffer was correspondingly diluted with autoclaved DEPC water from the stock solution.
  • RNA sample buffer 760 ⁇ L formamide, 260 ⁇ L formaldehyde, 100 ⁇ L ethidium bromide (10 mg / mL in DEPC water), 80 ⁇ L glycerol, 80 ⁇ L bromophenol blue (saturated), 160 ⁇ L 10 x MOPS, 100 ⁇ L water
  • 10 x wash buffer without tween 1.0 M maleic acid, 1.5 M NaCl; without DEPC, adjust to pH 7.5 with NaOH (solid, approx. 77 g) and 10 M NaOH.
  • Substrate buffer adjust 100 mM Tris (trishydroxymethylamino-methane), 150 mM NaCl with 4 M HCl to pH 9.5.
  • 10 x color markers 50% glycerol (v / v), 1.0 mM EDTA pH 8.0, 0.25% bromophenol blue (w / v), 0.25% xylenecanol (w / v).
  • the membrane was prehybridized for at least 30 min in 10 mL lx hybridization buffer.
  • a volume of the probe corresponding to approx. 300000-400000 cpm / mL was used in a hybridization tube after denaturation in hybridization buffer (5 min at 90 ° C).
  • the mixture was then washed twice for 5 min with 2x SSC, 0.1% SDS in the tube and then with 0.1 x SSC, 0.1% SDS in a water bath at hybridization temperature until no radioactive background could be detected with the hand monitor.
  • the membranes were wrapped in cling film or sealed in plastic bags and exposed to an X-ray film (Kodak X-OMAT AR, Röntgen Bender, Baden-Baden) or a phosphor screen (Kodak Imaging Screen-K, Bio-Rad, Kunststoff).
  • the X-ray films were developed after a variable exposure time (see above), the phosphor screens were read out in the phosphor imager (Molecular Imager FX, Bio-Rad, Kunststoff) and with the Quantity One-4.1.1 program (Bio-Rad, Kunststoff) in a resolution of 200 ⁇ m documented.
  • 5x hybridization buffer 1% BSA (bovine serum albumin) (w / v), 1% polyvinylpyrrolidone 10 to 40 kDa (w / v), 1% Ficoll 1400000 (w / v), 250 mM TrisCl pH 7.5 (0, 8 M stock solution, autoclaved), 0.5% tetra-sodium diphosphate decahydrate (w / v), 5% SDS (sodium dodecyl sulfate) (w / v), sterile filtered (0.45 ⁇ m), with autoclave. Fill up water, lx hybridization buffer is diluted from 5x hybridization buffer with DEPC water.
  • Membranes that had already been hybridized with one probe could be "stripped" again for hybridization with another probe.
  • Digoxigenin or fluorescein labeled probes were washed by washing in A. bidest. (1 min at RT) and subsequent incubation of the membranes with boiled 0.1% SDS (w / v) (10 min at RT) removed.
  • Radioactively labeled probes were completely detached from the membranes by washing in 1% SDS (w / v), 50% formamide (v / v) for 20 to 40 min at 80 ° C.
  • the promoter of the BCI-3 gene from barley was isolated by means of the inverse polymerase chain reaction (iPCR) starting from genomic DNA from barley (variety Ingrid).
  • iPCR inverse polymerase chain reaction
  • 400 to 500 mg of leaf material cv. Ingrig WT
  • AXG 500 nucleobond columns Macherey & Nagel
  • 750 ng of genomic DNA were cut with the restriction endonucleases EcoRV, Ncol, Rsal, Eco91I or BamHI (MBI Fermentas, St. Leon-Rot, Germany).
  • the DNA was incubated in a total volume of 5 100 ⁇ l with 30 units of enzyme in the buffer for 4 h at 37 ° C. according to the manufacturer's instructions. This was followed by precipitation of the DNA with 0.1 volume of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of ethanol overnight at -20 ° C.
  • the ligation was followed by precipitation with 0.1 volume of sodium acetate buffer (pH 5.2) and 2.5 volume of ethanol. After centrifugation (30 min, 14000 rpm (20,000 g), 4 ° C.), the pellet was washed with ethanol (70%), dried and suspended in 80 ⁇ l H 2 0 resus 0. 3 ⁇ l of this was used as a template for the iPCR.
  • the iPCR was carried out as a standard PCR.
  • a PCR was carried out using the primer pair 1 (SEQ ID NO: 3 and 4) with Pfu polymerase (Promega).
  • Pfu polymerase Promega
  • 5 the amplificate was re-amplified with the Taq polymerase using the nested primer pair 2 (SEQ ID NO: 5 and 6).
  • the PCR product was then cloned into pGEM-T (Promega) and sequenced. Primer pairs 3 and 40 were derived from the sequence information.
  • ligated genomic DNA - the primer pairs 3 and 4 were used as described above and the PCR product was cloned into pGEM-T (Promega) and sequenced. Primer pairs 5 and 6 were derived from the sequence information.
  • ligated genomic DNA - the primer pairs 5 and 6 were used as described above.
  • the PCR product was isolated from an agarose gel and cloned into the pGEM-T vector (Promega, Mannheim, Germany) by means of T-overhang ligation.
  • the cDNAs were sequenced from the plasmid DNA using the "Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit" (Amersham, Freiburg, Germany).
  • Primer pair 1 PP1-5: 5 '-CTC GCA GTT GGC CGG CAC CGT-3' SEQ ID NO: 11 PP1-3: 5 '-TGA CCC CAT GTA CTA CAT CGCCT-3' SEQ ID NO: 12
  • Primer pair 2 PP1-5: 5 '-CTC GCA GTT GGC CGG CAC CGT-3' SEQ ID NO: 11 PP1-3: 5 '-TGA CCC CAT GTA CTA CAT CGCCT-3' SEQ ID NO: 12
  • Primer pair 2 Primer pair 2:
  • PP3-5 5'-AGC CAT CAT CCC TGC GGA TCC A-3 'SEQ ID NO: 15
  • PP3-3 5' -GAG CGT CCG GGC GCG GCC TT-3 'SEQ ID NO: 16
  • Primer pair 4 PP4-5: 5 '-GGA TCC AAG CGA GAT TTG AAC GGA-3' SEQ ID NO: 17 PP4-3: 5 '-GCG GCC TTG CCG GAC GCG GT-3' SEQ ID NO: 18
  • PP5-5 5 '-GTA TGC CGC TCC ATG TTA GAA GAT-3' SEQ ID NO: 19
  • PP5-3 5 '-ACA TTA CAG GCA GCG TCC GAC AC-3' SEQ ID NO: 20
  • composition of the PCR mix (25 ⁇ l):
  • the sequence of the BCI-3 promoter was composed using the computer program Genedoc align from the sequences obtained in each case and is reproduced under SEQ ID NO: 1.
  • the entire promoter sequence was amplified using the PCR conditions below. The following primers were used:
  • BCI3-5 (SEQ ID NO: 23): 5 '-aagcttcaccaactcccttcaaggtctaa-3'
  • BCI3-3 (SEQ ID NO: 24): 5 '-ctgcagtgtgtgtgcttgctgtgatgc-3' Composition of the PCR mixture: 3.00 ⁇ l template 2.50 ⁇ l 10 ⁇ buffer 2.50 ⁇ l dNTP s [2mM of each] 0.75 ⁇ l MgCl 2
  • the PCR product was cloned into TG overhang ligation in pGEM-T [Promega).
  • BCI3-3 (SEQ ID NO: 24: 5 '-ctacagtgtgtgtgcttgctgctgtgatgc-3'
  • the sequence of the BCI-4 promoter was elucidated using a BCI4-BAC (BAC No. 266D20; provided by IPK, Gatersleben, Germany).
  • the BAC clone was identified using the BCI-4 cDNA as a probe in a manner familiar to those skilled in the barley genomic BAC library.
  • the sequence of the BCI-4 promoter which is shown in SEQ ID NO: 2, was finally determined using the method of primer walking.
  • OPN1 5 '-TGA ATG GTC ATG GCT GCT TA-3' (SEQ ID NO: 29)
  • OPN2 5 '-GGC TCT GAA TCG CAC ATT CT-3' (SEQ ID NO: 30)
  • OPN3 5 '-GTA CCT CTC CTC CCC GCT CAC T-3 (SEQ ID NO: 31)
  • OPN5 5 '-TCATGCATATCTATGTCTTTCTCTTC-3' (SEQ ID NO: 33)
  • the following primer (e.g. 0PN2) was derived from the sequence information obtained using one primer (e.g. OPN1).
  • the sequence of the BCI-4 promoter was composed using the computer program Genedoc align from the sequences obtained in each case and is reproduced under SEQ ID NO: 2.
  • the entire promoter sequence was amplified using the PCR conditions below. The following primers were used:
  • BCI4-5 (SEQ ID NO: 36): 5 '-agcgaatcgtttttcccttt-3'
  • BCI4-3 (SEQ ID NO: 37): 5 '-cctgagtgtacggctctgagatg-3'
  • Composition of the PCR approach :
  • the PCR product was isolated from an agarose gel and cloned into the pGEM-T vector (Promega, Mannheim, Germany) by means of T-overhang ligation.
  • the cDNAs were sequenced from the plasmid DNA using the "Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit” (Amersham, Freiburg, Germany).
  • the PCR product was cloned and sequenced in pGEM-T (Promega) via T overhang ligation.
  • pGYl-GFP vector based on pUC18, 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, Institute of Plant Genetics IPK, Gatersleben, Germany
  • the GFP gene was cut out with the restriction enzyme Kpnl and ligated into the vector pUC18.
  • the desired orientation of the reporter gene was determined using restriction digests.
  • the vector was named pGFP.
  • the BCI-4 promoter was cut out of pGEM-T as an Ncol / PstI fragment. It was first digested with Ncol and the Ncol site was filled in using the Klenow fragment. The following batch was pipetted together: 16 ul DNA (eluted from the gel), 2 ul T4 ligation buffer, 1 ul dNTP's [2 mM], 1 ul Klenow fragment. This approach was incubated at 37 ° C for 30 min. The reaction was then stopped at 65 ° C. for 10 minutes. subse- Send was digested with PstI and the fragment thus obtained was ligated into the pGFP (see below) digested with PstI and Smal.
  • the various BCI-3 promoter sequences were cut out of the pGEMT vectors with the BCI-3 promoter or the BCI-3 promoter deletion variants with HindiII and PstI, purified using a gel and inserted into the HindIII / PstI digested pGFP vector.
  • the various constructs are shown schematically in FIG. 1.
  • the induction of the BCI-3 promoter, the deletion variants and the BCI-4 promoter was examined using different stimuli.
  • the induction was either realized by means of Northern blot analysis with detection of the endogenous BCI-3 or BCI-4 mRNA, or was shown using the Pr ⁇ motor reporter constructs described above.
  • DCINA induction 2, 6-dichlorisonicotinic acid (DCINA, CGA41396, Syngenta, Basel, Switzerland) was formulated as a 25% active substance with wettable powder (WP) or after pre-dissolving the pure substance with prostitute hylformamide (DMF) in aqueous solution (1% DMF) adjusted to pH 7 with NaOH and applied in concentrations of 5 and 10 mg / 1 (corresponds to 0.02 or 0.04 M) based on the soil volume. Control plants were watered with WP or 1% DMF solution.
  • WP wettable powder
  • DMF prostitute hylformamide
  • BTH Benzo (1, 2, 3) thiadiazole-7-carbothionic acid S-methyl ester
  • BTH also Azibenzolar-S-methyl, CGA245704, Bion®, Syngenta, Basel, Switzerland
  • Soil application was carried out in concentrations of 20 and 50 mg / 1 (corresponds to 0.095 and 0.24 mM, respectively) based on the volume of the soil.
  • Salicylic acid induction Salicylic acid (SA) was dissolved directly in water or after pre-dissolving in DMF in aqueous solution
  • Ethylene induction primary leaves from seven-day-old plants were placed on moistened filter paper in gastight Erlenmeyer flasks with ethylene gas in concentrations of 0.001, 1, 10 and 100 ⁇ L / 1 (corresponds to 0.028, 28, 280 and 2800 ⁇ M) in a climatic chamber incubated (see above). Control sheets were gassed with air.
  • Abscisic acid (ABA) induction ABA was applied by spraying an aqueous solution of 50 ⁇ M to seven-day-old seedlings until they were evenly covered by fine droplets. Control plants were sprayed with water. Treated plants were incubated in climatic chambers (see above).
  • JA induction Induction with the phytohormone jasmonate (JA) (Sigma) was carried out by "floating" leaf segments on a solution of 45 ⁇ M JA in climatic chambers. Control sheets were “floated” on water. Treatment with jasmonic acid methyl ester (JM) was carried out in glass petri dishes by "floating" primary leaves of seven-day-old plants on a solution of 45 ⁇ M JM in climatic chambers (see above). Control sheets were "floated” on water.
  • JM jasmonic acid methyl ester
  • Sorbitol induction was carried out by "floating" leaf segments or primary leaves of seven-day-old seedlings for 4 h on a 1 M sorbitol solution (Fluka). The leaves were then “floated” on water overnight. Control sheets were “floated” on water.
  • Bacillus subtilis came from the culture collection of the Institute for Phytopathology and Applied Zoology (IPAZ), Giessen, Germany, the various Pseudomonas strains were provided by Ina Budde and Matthias Ullrich, Max Planck Institute for Terrestrial Microbiology, Marburg.
  • a wound was achieved by perforating the primary leaves of seven day old plants with 15 needles / cm 2 . Control plants remained uninjured and were further cultivated together with the wounded in the climate cabinet (see above).
  • the individual constructs were shot into chemically induced barley leaves (cv. Ingrid WT) with the aid of particle bombardment. 24 hours later, GFP cells were counted in the leaf segments. Tungsten particles with a diameter of 1.1 ⁇ m (particle density 25 mg / ml) were coated with plasmid DNA. For this purpose, 1 ⁇ g reporter plasmid was used for coating per shot (method according to Schweizer P et al. (1999) Mol Plant Microbe Interact
  • microcarrier preparation 55 mg of tungsten particles (M 17, diameter 1.1 ⁇ ; Bio-Rad, Kunststoff) were washed twice with 1 ml of autoclaved distilled water and once with 1 ml of absolute ethanol, dried and in 1 ml of 50% strength Glycerin taken up (approx. 50 mg / ml stock solution). The solution was diluted to 25 mg / ml with 50% glycerol, mixed well before use and suspended in an ultrasonic bath.
  • plasmid 12.5 ⁇ l of tungsten particle suspension (25 mg / ml), 12.5 ⁇ l of 1 M Ca (N0 3 ) 2 solution (pH 10) were added dropwise with continuous mixing, 10 Leave at RT for min, centrifuge briefly and remove 20 ⁇ l from the supernatant. The rest with the tungsten particles is resuspended (ultrasonic bath) and used in the experiment.
  • the pressure in the chamber was reduced by 0.9 bar and the tungsten particles were shot at 9 bar helium gas pressure on the surface of the plant tissue.
  • the chamber was immediately ventilated.
  • the macro carrier was thoroughly cleaned with water. 24 h after the bombardment, GFP cells were counted in the leaf segments.
  • the empty vector ⁇ UC18 with GFP cloned in was shot into induced barley leaves as controls and a search for GFP cells was carried out under the microscope.
  • Example 11 Inducibility of the BCI-3 and BCI-4 promoter based on Northern blot experiments
  • the BCI-3 promoter is induced by DCINA, BTH and weaker by SA (Fig. 5, A, B, C).
  • the accumulation of BCI-3 was only detectable in mesophyll after BTH induction in Northern blot analyzes, however further investigations with the more sensitive detection method of RT-PCR showed an inducibility also in the epidermis.
  • Expression of BCI-3 was not influenced by ethylene and ABA, but induced by JA application (FIG. 5, D) and wounding (FIG. 6, B). In contrast to induction by JA application, BCI-3 was repressed with an increased endogenous JA content after sorbitol treatment (FIG. 6, A).
  • BCI-3 was affected by a virulent and avirulent race repressed by Bgh. As with sorbitol treatment (see above), this repression could be an expression of a changed metabolism in the interaction with the biotrophic pathogen, but this is probably not related to resistance, since the expression of BCI-3 was also repressed in the compatible interaction.
  • Gene expression after infiltration with bacteria was only slightly activated in one of two experiments carried out and therefore does not appear to be causally related to bacteria (FIG. 6, D).
  • the gene expression of BCI-4 can only be induced with the resistance inducers SA, DCINA and BTH ((FIG.
  • BCI-4 functions not in general Stressreak ⁇ is expressed, but s is regulated specifically.
  • BCI-4 shows a very low constitutive expression activity. Stenhold increased accumulation of BCI-4 transcript and protein in Ger ⁇ occurred exclusively in leaves (with the exception of the flag leaf), and only after chemical induction (BTH-G thinkbe- treatment) (s. Fig. 8), the accumulation of the mesophyll remained limited as Northern and Western analyzes show might ⁇ th. activation of the expression in compatible or inkompa ⁇ tiblen interactions with the barley powdery mildew fungus could (Northern analysis, RT-PCR) on the transcript can not be detected.

Abstract

L'invention concerne des procédés d'expression inductibles chimiquement de séquences d'acides nucléiques, de préférence dans des plantes. Cette invention concerne également de nouveaux promoteurs inductibles chimiquement, des équivalents fonctionnels et des fragments desdits promoteurs fonctionnellement équivalents ainsi que des vecteurs et cassettes d'expression, qui contiennent ces séquences de promoteurs. Ladite invention concerne, en outre, des plantes transgéniques transformées à l'aide de ces vecteurs et cassettes d'expression, des cultures, des fragments ou un produit de reproduction transgénique dérivés desdites plantes ainsi que l'utilisation de ces derniers dans la fabrication de produits alimentaires, d'aliments pour animaux, de semences, de produits pharmaceutiques ou produits chimiques fins. Cette invention concerne, par ailleurs, l'utilisation de ces vecteurs ou cassettes d'expression dans des procédés d'identification de substances qui sont en mesure d'induire une résistance aux agents pathogènes dans des plantes.
PCT/EP2002/011330 2001-10-17 2002-10-10 Promoteurs inductibles chimiquement a partir d'orge et leur utilisation WO2003033712A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071748A2 (fr) * 1999-05-21 2000-11-30 Basf Aktiengesellschaft Genes ers, procede de criblage de composes chimiques capables d'induire un etat de resistance accru dans les plantes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071748A2 (fr) * 1999-05-21 2000-11-30 Basf Aktiengesellschaft Genes ers, procede de criblage de composes chimiques capables d'induire un etat de resistance accru dans les plantes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BESSER K ET AL: "EXPRESSION ANALYSIS OF GENES INDUCED IN BARLEY AFTER CHEMICAL ACTIVATION REVEALS DISTINCT DISEASE RESISTANCE PATHWAYS", MOLECULAR PLANT PATHOLOGY, XX, XX, vol. 1, no. 5, September 2000 (2000-09-01), pages 277 - 286, XP001097217 *
GATZ CHRISTIANE: "Chemically inducible promoters in transgenic plants.", CURRENT OPINION IN BIOTECHNOLOGY, vol. 7, no. 2, 1996, pages 168 - 172, XP001135217, ISSN: 0958-1669 *
JEPSON I ET AL: "CHEMICAL-INDUCIBLE GENE EXPRESSION SYSTEMS FOR PLANTS - A REVIEW", PESTICIDE SCIENCE, ELSEVIER APPLIED SCIENCE PUBLISHER. BARKING, GB, vol. 54, no. 4, December 1998 (1998-12-01), pages 360 - 367, XP000804296, ISSN: 0031-613X *

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