Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

• the present invention relates to a method for the production of transgenic plants and / or plant cells having an increased pathogen resistance, wherein the transgenic plants or plant cells have a content that is altered in comparison to the wild type and / or an altered activity on at least one actin depolymerizing factor (ADF). exhibit.
• ADF actin depolymerizing factor
• the present invention also relates to the use of nucleic acids encoding at least one ADF for the production of transgenic plants or plant cells having an increased pathogen resistance. Furthermore, the present invention relates to nucleic acid sequences encoding an ADF.
• Plant diseases caused by various pathogens such as viruses, bacteria and fungi can lead to significant yield losses in the cultivation of crops.
• fungicides are widely used in agricultural production to control fungal diseases. Despite such controls, a significant portion of the potential yield due to disease is lost.
• crops with a natural resistance to important fungal pathogens in the context of integrated pest management.
• genetic engineering approaches play a major role, for example by introducing external resistance genes or by the manipulation of endogenous gene expression in plants targeted resistances in important crops should be introduced.
• non-host resistance describes the observation that an entire plant species is resistant to a specific pathogen. This hitherto unknown phenomenon is probably due to structural or chemical properties of the plant species. These may be, for example, the thickness of the coticle, the presence of inhibitory substances or the limited availability of nutrients.
• certain plant species or different cultivars of a species will be resistant or susceptible to a particular pathogen, depending on their genotype.
• the different resistance mechanisms that are responsible for the resistance or susceptibility of a plant species or their cultivars to certain pathogens are exemplified for the mildew ⁇ Blumeria graminis), which infects several different types of grasses.
• the powdery mildew fungus as a species comprises several formae speciales depending on whether the respective powdery mildew fungus e.g. Wheat or barley.
• the respective powdery mildew fungus e.g. Wheat or barley.
• the respective powdery mildew fungus e.g. Wheat or barley.
• Blumeria graminis f. sp. hordei In the case of infestation of barley is Blumeria graminis f. sp. hordei, while the infestation of wheat by Blumeria graminis f. sp. tritici acts.
• different races or pathotypes can be identified within the different formae speciales, to which different cultivars of the host species have a different resistance.
• Blumeria graminisf. sp. hordei exclusively affects the epidermis cell layer of barley leaves.
• the fungus penetrates into the plant cell mechanically and enzymatically through the cell wall by means of a penetration peg, which is conidia, i. Asexually formed spores act.
• the successful infestation of barley leaves is achieved when the haustorium, which is the fungal feeding organ, has formed.
• the first mechanism is based on the so-called "gene for gene” concept. In this mechanism, resistance is achieved by a dominant resistance gene rendering the plants resistant only to those fungal isolates carrying the corresponding avirulence gene. In most cases, this so-called race-specific resistance, in which a barley cultivar is resistant only to selected mildew isolates, is characterized by the hypersensitive response (HR), i. the host cells of the infection site die off (Heitefuss, R., vide supra).
• HR hypersensitive response
• the second mechanism confers broad-spectrum resistance to all known isolates of a formae specialis of powdery mildew fungi and is characterized by the absence of the so-called Mo wild-type gene.
• Mio is a presumably negative regulator of pathogen defense (Devoto, A. et al. (1999), J. Biol. Chem., 274, 34993-35004).
• the function of this mechanism also depends on at least two other genes, Rorl and Ror2 (Freialdenhoven, A. et al. (1996), Plant Cell, 8, 5-14).
• the resistance or Incompatibility, as mediated by the recessive / w / o resistance allele, is generally not characterized by the onset of HR.
• the papilla subcellular cell wall apposition
• the papilla which forms directly below the fungal penetration peg, the so-called appresorium.
• the penetration attempts of the fungus are inhibited at the stage of papule formation, ie there is no formation of a haustorium, which is essential for the establishment of an efficient infestation.
• transgenic plants which have increased resistance to have different plant pathogens. It is a further object of the present invention to provide plants or plant cells with a race non-specific resistance to various fungal pathogens such as mildew available. It is also an object of the present invention to provide transgenic barley plants having a race-nonspecific sequence against fungal pathogens such as the mildew pathogen. In addition, it is an object of the present invention to provide methods which enable the production of the abovementioned transgenic plants or plant cells having an increased (race-unspecific) resistance to plant pathogens, such as mildew.
• the stated objects of the present invention are essentially achieved by providing a process for producing transgenic plants with increased pathogen resistance, which is characterized in that the content and / or the activity of at least one actin depolymerizing factor (ADF ) is changed from the corresponding wild-type.
• ADF actin depolymerizing factor
• race-nonspecific resistance in barley and other gramineae is mediated by recessive m / o alleles. Therefore, efforts have been made for a long time to identify other genes that interact with the Mo gene. Using a mutagenesis screen, additional genes could be identified with Rorl and Ror2, which are genetically linked to the Mo gene interact (Freialdenhoven et al, vide supra).
• Rorl and Ror2 which are genetically linked to the Mo gene interact
• a general problem in identifying other genes that interact with the Mo gene, and thus could also be used by appropriate manipulation to produce race-unspecific resistance is that, depending on the screening method, evidence of modified infection types is provided. and in view of the barley's genomic redundancy, the mutagenesis screening methods used are not always sensitive enough to identify additional genes of the / rc / o mediated resistance mechanism.
• RNA interference RNA interference
• ADF3 actin depolymerizing factor 3
• transgenic plants or plant cells from barley in which the content and / or the activity of ADF3 is changed in comparison to the wild type, thus exhibit an increased race-unspecific resistance to the mildew pathogen.
• transgenic plants or plant cells of different plant species which are distinguished by an increased resistance to plant pathogens and in particular excel in pathogenic pathogens such as mildew. This should be especially true when the plant pathogens have to enter into a functionally relevant interaction with the actin cytoskeleton to establish an efficient infection (see below).
• the present invention therefore provides an isolated nucleic acid molecule which is suitable for the ADF3 of barley having the SEQ ID no. 1 coded.
• nucleic acid molecules which are suitable for functionally equivalent parts of the ADF3 of barley having the SEQ ID NO. 1 coding for mutants of ADF3 from barley with the SEQ ID NO. 1 or nucleic acid molecules which hybridize to the aforementioned nucleic acid molecules under stringent conditions.
• Another object of the present invention are proteins or protein fragments which are encoded by the aforementioned nucleic acid molecules.
• the present invention also provides a process for the production of transgenic plants or plant cells having an increased pathogen resistance compared to the wild type and an altered content and / or an altered activity of at least one ADF.
• the invention likewise provides a process for the production of transgenic plants or plant cells with increased pathogen resistance, in which the expression of at least one ADF by transfer of the abovementioned nucleic acid sequences or nucleic acid sequences corresponding to ADF3 from barley having SEQ ID NO. 1 are homologous, planting or plant cells is effected.
• Another object of the present invention are methods for the production of transgenic plants or plant cells with increased pathogen resistance, in which the content and / or activity of at least one endogenous ADF is up-regulated or down-regulated.
• Another object of the invention is a process for the preparation of transgenic plants or plant cells with increased pathogen resistance, in which the activity of at least one endogenous ADF by transfer of nucleic acid molecules for non-functional homologs or parts thereof of the ADF3 from barley with the SEQ ID no. 1, is degraded.
• Another object of the invention is a method for the production of transgenic plants or plant cells with increased pathogen resistance, in which antibodies that are specific for ADFs and possibly inhibit their function are expressed in the cell.
• Another object of the invention are methods for the production of transgenic plants or plant cells with increased pathogen resistance, in which the post-translational modification status of at least one overexpressed and / or endogenous ADF is changed.
• an object of the present invention are methods for the production of transgenic plants or plant cells with increased pathogen resistance, in which the expression of at least one ADF by methods such. antisense, post-transcriptional gene silencing (PTGS), virus-induced gene silencing (VIGS), RNA interference (RNAi), ribonuclease P constructs, hammerhead ribozyme constructs or homologous recombination.
• PTGS post-transcriptional gene silencing
• VIGS virus-induced gene silencing
• RNAi RNA interference
• ribonuclease P constructs hammerhead ribozyme constructs or homologous recombination.
• the invention also relates to transgenic plants or plant cells having an increased resistance to pathogens, which have a higher compared to the wild type altered content and / or altered activity of at least one ADF.
• the present invention also relates to transgenic plants or plant cells which have been produced by one of the methods according to the invention and have increased pathogen resistance in comparison with the wild type.
• Another object of the present invention is the use of nucleic acids which encode functional or non-functional ADFs or parts thereof from different organisms, for the production of transgenic plants or plant cells with increased pathogen resistance.
• nucleic acid sequences described in the invention for the described methods or for the production of said transgenic plants and plant cells is also an object of the present invention.
• Phathogen resistance means reducing or attenuating disease symptoms of a plant as a result of infestation by a pathogen.
• the symptoms may be of a variety of types, but preferably include those which directly or indirectly affect the quality of the plant, the quantity of yield, the suitability for use as feed or food, or else the sowing, growing, harvesting or processing of the plant Harvests difficult
• the term "increased pathogen resistance” is understood according to the invention to mean that the transgenic plants or plant cells according to the invention are affected less frequently and / or less frequently by plant pathogens.
• the term “increased pathogen resistance” also includes a so-called transient pathogen resistance, ie that the transgenic plants or plant cells according to the invention have an increased pathogen resistance compared to the corresponding wild type only for a certain period of time.
• the reduced frequency or the reduced extent of the pathogen infestation in the transgenic plants or plant cells according to the invention is determined in comparison with the corresponding wild type.
• an increase in resistance is preferred in the sense that the plant is infested with the pathogen by at least 5%, preferably at least 20%, likewise preferably at least 50%, 60% or 70%, particularly preferably at least 80%, 90 % or 100%, also more preferably by a factor of 5, more preferably by at least a factor of 10, also more preferably by at least a factor of 50, more preferably by at least a factor of 100 and most preferably by at least a factor of 1000 less or less strong compared to the wild type.
• plant pathogens is understood as meaning those plant pathogens which have to interact with the plant actin cytoskeleton in order to establish an efficient infection.
• plant pathogens includes fungal pathogens.
• Fungal pathogens or fungus-like pathogens are preferably selected from the group comprising Plasmodiophoramycota, Oomycota,
• Plasmodiophoromycota such as Plasmodiophora brassicae (cabbage hernia, clubroot of crucifer), Spongospora subterranea (powdery scab of potato tubers),
• Ascomycota such as Microdochium nivale (snow mold on rye and wheat), Fusarium graminearum, Fusarium culmorum (ear rot especially in wheat), Fusarium oxysporum (Fusarium wilt on tomato), Blumeria graminis (powdery mildew on barley (f.sp. hordei) and Wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew), Nectria galligena (fruit tree crayfish), Unicnula necator (grapevine powdery mildew), Pseudopeziza tracheiphila (red
• Burner of the grapevine Claviceps purpurea (ergot on eg rye and grasses), Gaeumannomyces graminis (blacklegs on wheat, rye grasses), Magnaporthe grisea (rice blast disease), Pyrenophora graminea (stripe disease on barley), Pyrenophora teres (net blotch on barley), Pyrenophora tritici-repentis (leaf blotch (leaf drought) on wheat), Venturia inaequalis (apple scab), Sclerotinia sclerotium (Thstengeltechnik, Rapskrebs), Pseudopeziza medicaginis (valve scab)
• Basidiomycetes such as Typhula incarnata (Typhula rot on barley, rye, wheat), Ustilago maydis (blubber on corn), Ustilago nuda (blaze of barley), Ustilago tritici (blaze of wheat, spelled), Ustilago avenae
• Puccinia striiformis yellow rust on wheat, barley, rye and numerous grasses
• Uromyces appendiculatus bean rust
• Sclerotium rolfsii root and stem reds of many plants.
• Deuteromycetes such as Septoria nodorum (teal tubers) on wheat (Septoria tritici), Pseudocercosporella herpotrichoides (broken wheat on cereals, barley, rye), Rynchosporium secalis (leaf spot disease on rye and barley), Alternaria solani (drought on potato, tomato), Phoma betae (root burn on beta beet), Cercospora beticola (Cercospora leaf spot disease on beta
• Phytophthora infestans (late blight, late blight in tomato, etc.), Microdochium nivale (formerly Fusarium nivale, snow mold on rye and wheat), Fusarium graminearum, Fusarium culmorum (wheat spike), Fusarium oxysporum (Fusarium wilt Tomato), Blumeria grammis (powdery mildew on barley (formerly hordei) and wheat (for sp.
• Tritici Tritici
• Magnaporthe grisea rice blast disease
• Sclerotinia sclerotium white stem, rape
• Septoria nodorum and Septoria tritici Tuber spot on wheat
• Alternaria brassicae oval seed rape on canola, cabbage and other cruciferous vegetables
• Phoma Hungary turnip disease, blackleg on cabbage, root-neck or stem rot on rape).
• bacterial pathogens include the pathogens listed in Table 2 and the diseases associated with them.
• transgenic plants produced according to the invention are particularly preferably resistant to the following pathogenic bacteria:
• Corynebacterium sepedonicum (bacterial ring rot on potato), Erwinia carotovora (blackleg on potato), Erwinia amylovora (fire blight on pear, apple, quince), Streptomyces scabies (potato scab), Pseudomonas syringae pv. Tabaci (wildfire on tobacco), Pseudomonas syringae pv. Phaseolicola (Fatty stain disease on bushbeam), Pseudomonas syringae pv. Tomato ("bacterial speck” on tomato), Xanthomonas campestris pv. Malvacearum (leaf spot disease on cotton) and Xanthomonas campestris pv. Oryzae (bacterial blight on rice and other grasses).
• viral pathogens includes all plant viruses such as tobacco or or cucumber-mosaic virus, ringpot virus, Necrosis virus, corn dwarf-mosaic virus etc.
• viral pathogens include the pathogens listed in Table 3 and the diseases associated with them.
• insects and nematodes can also be resistant to animal pests such as insects and nematodes.
• insects such as beetles, caterpillars, lice or mites may be mentioned.
• the plants according to the invention are preferably resistant to insects of the genera Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera. Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc.
• insects of the genera Coleoptera and Lepidoptera such as the European corn borer (ECB), Diabrotica barberi (“Northern com rootworm”), Diabrotica undecimpunctata (“Southern com rootworm”), Diabrotica virgifera (“Western com rootworm”), Agrotis ipsilon (“black cutworm”), Crymodes devastator (“glassy cutworm”), Feltia **ns (“dingy cutworm”), Agrotis gladiaria (“claybacked cutworm “), Melanotus spp., Aeolus mellillus (“ wireworm “), Aeolus mancus (“ wheat wireworm “), Horistonotus uhlerii (“ sand wireworm “), Sphenophorus maidis (“ maize billbug “), Sphenophorus zeae (“ timothy billbug ").
• EBCB European corn borer
• Diabrotica barberi Northern com rootworm
• Diabrotica undecimpunctata
• Sphenophorus parvulus bluegrass billbug
• Sphenophorus callosus southem com billbug
• Phyllogphaga spp. White grubs
• Anuraphis maidiradicis com root aphid
• Delia platura seedcom maggot
• Colaspis brunnea grain colaspis
• Stenolophus lecontei seedcom beetle
• Clivinia impressifrons (“lender seedcom beetle”).
• Femer are the cereal cockerel (Oulema melanopus), the frit fly (Oscinella frit), wireworm (Agrotis lineatus) and aphids (such as oat aphid Rhopalosiphum padi, large cereal aphid Sitobion avenae).
• nematode pests include the pathogens listed in Table 4 and the diseases associated with them.
• the transgenic plants according to the invention are resistant to Globodera rostochiensis and G. pallida (cysts on potatoes, tomato and other nightshade plants), Heterodera schachtii (beet cysts on sugar beets and fodder beet, oilseed rape, cabbage, etc.), Heterodera avenae (oat cysts on oats cereals), Ditylenchus dipsaci (stem or small beetle, turnip head on rye, oats, corn, clover, tobacco, turnip), Anguina tritici (wheat, wheel disease on wheat (spelled, rye), Meloidogyne hapla (Wurzelgallenälchen to carrot, cucumber, Salad, tomato, potato, Sugar beet, alfalfa).
• the plants according to the invention are preferably resistant to the following pathogens:
• Canola against the fungal, bacterial or viral pathogens Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata.
• Puccinia graminis f.sp. tritici Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.
• Zignosellus lesser cornstalk borer; Oulema melanopus (cereal leaf beetle); Hypera punctata (clover leaf weevil); Diabrotica undecimpunctata howardi (southern com rootworm); Russian wheat aphid; Schizaphis graminum (greenbug); Macrosiphum avenae (English grain aphid); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Melanoplus sanguinipes
• Agromyza parvicornis (com blot leafininer); Anaphothrips obscrurus (grass thrips); Solenopsis milesta (thief ant); Tetranychus urticae (twospotted spider mite).
• Diatraea saccharalis (sugarcane borer); Spodoptera frugiperda (fall armyworm); Helicoverpa zea (com earworm); Colaspis brunnea (grape colaspis); Lissorhoptrus oryzophilus (rice water weevil); Sitophilus oryzae (rice weevil); Nephotettix nigropictus (rice leafhopper); Blissus leucopterus leucopterus (chinch bug); Acrosternum hilare (green stink bug);
• plant pathogen includes pathogens of the group Blumeria graminisf. sp. hordei, tritici, avenae, secalis, lycopersici, vitis, cucumis, cucurbitae, pi $ i, pruni, solani, rosae, fragariae, rhododendri, mali and nicotianae.
• ADF3 barley actin depolymerizing factor 3
• ADFs actin-depolymerizing factors
• ADF3 is discussed below, the ADF3 of barley with SEQ ID no. 1, whereas by use of the term “ADFs” is meant the ADF3 from barley and / or those proteins having significant or substantial homology to the ADF3 from barley.
• content of ADF3 from barley or ADFs in general means according to the invention the amount of ADF3 or a specific ADF, as can be determined for the wild type of a plant or plant cell.
• ADF3 or ADFs in general is their ability to interact with globular actin (G-actin) or filamentous actin (F-actin) or other physiological binding partners.
• a “content altered with respect to the type of WT" of ADFs is therefore understood according to the invention to be an increased or reduced amount of ADFs compared to the wild type.
• Increasing the level of ADFs can be achieved by increasing the amount of endogenous ADFs or by adding an additional amount of exogenous ADFs.
• the lowering of the amount of ADFs in the transgenic plants or plant cells according to the invention is generally achieved by a lowering of the content of endogenous ADFs.
• a wild-type is understood to mean the corresponding non-genetically modified starting organism.
• the increase in activity on ADFs can be achieved by increasing the activity of the endogenous ADFs and / or by adding an additional amount of functional ADFs. Decreasing activity on ADFs can be achieved by lowering the activity of the endogenous ADFs.
• a decrease in the activity of ADFs is understood to mean that the activity on endogenous ADF3 or endogenous ADFs remains unchanged, but the interaction of the ADFs with their physiological binding partners is significantly inhibited by, for example, the expression of non-functional forms of the ADFs or antibodies.
• the increase in the content and / or the activity of ADFs in a transgenic plant or plant cell caused by a method according to the invention is at least 5%, preferably at least 20%, also preferably at least 50%, particularly preferably at least 100%, also particularly preferred at least the factor 5, particularly preferably at least the factor 10, also particularly preferred at least the factor 50, more preferably at least the factor 100 and most preferably at least the factor 1000.
• the transgenic plants according to the invention have comparable increases in content and / or activity of ADFs in a transgenic plant or plant cell.
• the reduction of the content and / or the activity of ADFs in a transgenic plant cell or plant caused by a method according to the invention is at least 5%, preferably at least 10%, particularly preferably at least 20%, also particularly preferably at least 40%, also particularly preferred at least 60%, more preferably at least 80%, also more preferably at least 90% and most preferably at least 98%.
• an object of the present invention relates to an isolated nucleic acid molecule which is suitable for the ADF3 of barley having the SEQ ID no. 1 coded.
• Another object of the present invention relates to nucleic acid molecules suitable for functionally equivalent parts of the ADF3 from barley having the SEQ ID NO. 1 encode.
• ADF3 ADF3 fragments of the nucleic acid sequences as described for the ADF3 of SEQ ID no. 1, whose expression still leads to proteins with the binding properties and structural properties of ADF3.
• functionally equivalent parts is to be understood when referring to protein fragments in general. These are particularly preferably nucleic acid sequences which lead to ADF3 fragments which have deletions of several amino acids at the N- and / or C-terminus, without there being any change in the structural properties or the
• binding properties of the ADF3 comes.
• binding properties of ADF3 is meant in particular the binding behavior of ADF3 to G-actin and / or F-actin.
• mutants of ADF3 are nucleic acid molecules encoding mutants of ADF3.
• mutants of ADF3 is meant both functional and non-functional mutants of ADF3.
• Functional mutants are forms of ADF3 that have point mutation (s), insertion (s) and / or deletion (s), without any significant loss of the structural or binding properties of ADF3. ⁇
• ADF3 The binding properties of ADF3 from Zea mays as well as its structural properties have been described (Jiang et al., (1997), Proc. Natl. Acad., See, USA, 94, 9973-9978).
• ADF3 is an ADF which is essentially homologous to ADF3 from barley for the purposes of the present invention, the findings described in said publication regarding the binding behavior of ADFs to G-actin and F-actin in the production of functional and non-functional mutants of the barley ADF3 according to the invention (see below).
• An example of a conservative amino acid exchange is an exchange of a valine for an alanine.
• the skilled person will note whether the conservative amino acid exchange is located in a region of ADF3 that is essential for its binding behavior to F-actin or G-actin. Evidence of whether a specific region is essential for the binding behavior of ADF3 can be obtained through a so-called sequence alignment with already known ADFs for which the binding properties and structural properties have already been determined (see Jiang et al., Vide supra).
• the mutated amino acid is located at position 6 of the amino acid sequence, with the wild type serine (S) being changed to an alanine (A) (Smertenko, A.P. et al. (1998) Plant J 14, 187-193).
• an object of the invention are also nucleic acid molecules which code for non-functional mutants of ADF3 from barley.
• Such non-functional mutants of ADF3 are forms of ADF3 that are no longer or at least very limited in their ability to interact with G-actin and / or F-actin or other physiological binding partners of ADF3.
• Such non-functional mutants of ADF3 may in turn comprise point mutation (s), insertion (s) and / or deletion (s).
• Such non-functional mutants of ADF3 are e.g. useful in the production of transgenic plants or cells in which the content of endogenous ADF3 in the barley is not altered, but the activity on endogenous ADF3 is blocked by overexpression of said non-functional mutants.
• non-functional mutants of ADF3 have substantially the same nucleic acid or amino acid sequences as functional mutants of ADF3. However, they have in some places point mutation (s), insertion (s) or deletion (s) of nucleotides or amino acids, which cause, in contrast to functional mutants of ADF3, that the non-functional mutants of ADF3 not or only very limited in are able to associate with F-actin, G-actin and / or other physiological binding partners to interact.
• Such functional or non-functional mutants of ADF3 according to the invention can be identified by a person skilled in the art in a simple manner.
• a variety of techniques are available to those skilled in the art with which it is possible to insert point mutation (s), insertion (s) or deletion (s) into the nucleic acid sequences encoding functional or non-functional mutants of ADF3 (Sambrook (2001), Molecular Cloning: A Laboratory Manual, 3rd Edition, Coldspring Harbor Laboratory Press). After introduction of the point mutation / insertion and / or deletion, which are also generally referred to as mutation, the skilled worker can determine by appropriate binding tests, as shown in the examples or known from the prior art, whether the mutagenized ADF3 still on their have normal binding properties with respect to G-actin, F-actin and / or other physiological binding partners.
• Non-functional mutants of ADF3 have a decreased binding specificity compared to the non-mutagenized ADF3 or the functional mutant of ADF3, preferably towards G-actin and / or F-actin.
• a non-functional mutant of ADF3 has from 1 to 90%, preferably from 1 to 70%, more preferably from 1 to 50%, also more preferably from 1 to 30%, most preferably from 1 to 15% and most preferably Over 1 to 10% of the binding efficiency of ADF3 or the corresponding functional mutants of ADF3 to the respective pathogenic and / or physiological binding partner, in this case preferably to G-actin and / or F-actin.
• Non-functional mutants of ADF3 are also referred to as inactivated or inactive ADF3 in the invention.
• the functional and / or non-functional mutants of ADF3 according to the invention which carry the abovementioned point mutation (s), insertion (s) and / or deletion (s) or the functionally equivalent parts are distinguished by a substantial sequence homology to ADF3 ,
• Essential sequence homology is generally understood according to the invention as meaning that the nucleic acid or amino acid sequence of a DNA molecule or a protein is at least 40%, preferably at least 50%, more preferably at least 60%, also preferably at least 70%, 80% or 85%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% to the Nukleinklare ⁇ or amino acid sequences of ADF3 or its functionally equivalent parts is identical.
• the homology is determined over the entire sequence length of ADF3.
• identity between two proteins is meant the identity of the amino acids over a particular protein region, preferably the total protein length, especially the identity, as determined by comparison with the laser gene software of DNA Star Inc., Madison, Wisconsin (USA) Application of the CLUSTAL method (Higgins et al., 1989), Comput. Appl. Biosci., 5 (2), 151). The homology is thus preferably calculated over the entire amino acid or nucleic acid sequence range.
• other programs are available to the person skilled in the art for the comparison of different sequences, which are based on different algorithms. The algorithms of Needleman and Wunsch or Smith and Waterman provide particularly reliable results. For example, the program PiIe Aupa can also be used for the sequence comparisons (J.
• the Clustal-W program was used, as it can be accessed via http://www.ebi.ac.uk/clustalw.
• the parameters of the mentioned start page remained unchanged for the alignments.
• nucleic acid molecules which hybridize with the nucleic acid molecules encoding ADF3, functionally equivalent parts thereof or functional or non-functional mutants of ADF3 under stringent conditions or are substantially complementary to these.
• complementarity is meant the ability of one nucleic acid molecule to hybridize to another nucleic acid molecule due to hydrogen bonding between complementary bases. The person skilled in the art knows that two nucleic acid molecules do not have to have 100% complementarity in order to be able to hybridize with one another.
• nucleic acid sequence which is intended to hybridize with another nucleic acid sequence, to this at least 40%, at least 50%, at least 60%, preferably at least 70%, particularly preferably at least 80%, also more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% or 100% complementary.
• homology, complementarity and identity levels are to be determined over the entire protein or nucleic acid length.
• Nucleic acid molecules are identical if they have identical nucleotides in the same 5 '-3'
• stringent conditions thus refers to conditions under which a nucleic acid sequence preferentially binds to a target sequence, but not or at least substantially reduced to other sequences.
• Stringent conditions depend on the circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are chosen so that the hybridization temperature is about 5 ° C below the melting point (Tm) for the specific sequence at a defined ionic strength and a defined pH. Tm is the temperature (at a defined pH, a defined ionic strength and a defined nucleic acid concentration) at which 50% of the molecules that are complementary to a target sequence hybridize to this target sequence.
• stringent conditions include salt concentrations between 0.01 and 1.0 M sodium ions (or another salt) and a pH between 7.0 and 8.3. The temperature is at least 30 ° C for short molecules (eg, those that range from 10 to 50 nucleotides).
• stringent conditions may include the addition of destabilizing agents such as formamide.
• Typical hybridization and washing buffers have the following composition.
• Hybridization solution prehybridization solution.
• nucleic acid sequences encoding barley ADF3, functionally equivalent portions thereof, or functional or non-functional mutants thereof can be used to target transgenic plants having altered levels and / or altered activity To produce ADF3, which has the consequence that these plants have an increased pathogen resistance to the mildew fungus Blumeria graminisf. sp. hordei grouting.
• the present invention is not directed to methods of producing transgenic barley cells that exhibit increased resistance to Blumeria graminisf due to altered content and / or altered activity of barley ADF3. sp. hordei show limited.
• the present invention thus generally relates to methods for the production of transgenic plants or plant cells with an increased resistance to pathogens, in which the content and / or the activity of at least one ADF is changed compared to the wild type.
• the methods described below for producing transgenic plants with increased pathogen resistance and changing the content and / or the activity of at least one ADF it is therefore also possible to use, in addition to the abovementioned nucleic acid sequences, those nucleic acid sequences which code for ADFs which are substantially homologous to the ADF3 with the SEQ ID no. 1 are from barley.
• nucleic acid or amino acid sequence of such ADF to at least 40%, preferably at least 50%, more preferably at least 60%, also preferably at least 70%, 80% or 85%, especially preferably at least 90%, more preferably at least 95%, and most preferably at least 98% to the nucleic acid or amino acid sequences of the ADF3 from barley.
• sequence homology can be determined by the methods mentioned above.
• nucleic acid sequences which code for ADFs which are substantially homologous to barley ADF3 the methods according to the invention may also use those nucleic acids which code for functionally equivalent parts of the ADFs or functional or non-functional mutants of the ADFs provided that they are ADFs that are substantially homologous to the ADF3 of barley.
• the ones described above Definitions for functionally equivalent parts or functional or nonfunctional mutants also apply to the ADFs in general.
• ADFs which can be used for the methods according to the invention are the Arabidopsis thaliana, Zea mays, Hordeum vulgare, Oryza sativa and Triticum aestivum ADFs given in Table 5.
• Suitable nucleotide and amino acid sequences for the ADFs can be deduced by the person skilled in the art both from the database entries given in the table and from the sequence listing.
• GenBank database can be accessed at http://www.ncbi.nlm.nih.gov/entrez.
• the TIGR database can be accessed at http://www.tigr.org.
• DNA sequences with a high homology, ie a high similarity or identity are bona f ⁇ de candidate for DNA sequences corresponding to the DNA sequences of the invention, ie ADF3.
• These gene sequences can be isolated by standard methods such as PCR and hybridization, and their function determined by appropriate enzyme activity assays and other experiments by those skilled in the art.
• Homology comparisons with DNA sequences can also be used according to the invention to design PCR primers by first identifying the regions that exist between the DNA sequences of different organisms are most conserved. Such PCR primers can then be used to isolate, in a first step, DNA fragments that are components of DNA sequences that are homologous to the DNA sequences of the invention.
• search engines that can be used for such homology comparisons. These search engines include e.g. the CLUSTAL program group of the BLAST program provided by the NCBI.
• the ADF3 from barley and the ADFs generally have a so-called consensus region.
• consensus region there is a so-called sequence alignment of various ADF sequences from A. thaliana with ADF3 from barley.
• Consensus Sequence I comprises the following sequence:
• X 1 may comprise any desired amino acid, preferably L or I.
• X 2 may comprise any amino acid.
• X 3 may comprise any amino acid, preferably N or D.
• X 4 may comprise any amino acid, preferably D or E.
• X 5 may comprise any amino acid, preferably Y or F,
• X 6 may be any amino acid, preferably A or C include.
• X 7 may comprise any amino acid, preferably V or I.
• Xg can include any amino acid.
• X 9 may include any amino acid.
• X 10 may include any amino acid, preferably D or E.
• X 11 may comprise any amino acid, preferably F or Y. The amino acids are given after the usual one-letter code.
• ADFs that can be used in the methods of the invention are characterized by the presence of a second consensus sequence.
• This second consensus sequence II comprises the following sequence:
• Y 1 can be any amino acid, preferably K or R.
• Y 2 can be any amino acid, preferably K or R.
• Y 3 may be any amino acid, preferably F or Y.
• Y 4 may be any amino acid, preferably F, I or V.
• Y 5 can be any amino acid.
• Y 6 may be any amino acid, preferably S or C.
• Y 7 may be any amino acid, preferably S, E or D.
• Y 8 can be any amino acid.
• Y 9 may be any amino acid, preferably S or A.
• Y 10 may be any amino acid.
• Y 11 may be any amino acid, preferably I, V or M.
• Y 12 can be any amino acid.
• Y 13 may be any amino acid, preferably I, V or M.
• Y 14 can be any amino acid.
• Y 15 may be any amino acid, preferably S or A. Again, the amino acids are given in single-letter code.
• ADFs suitable for use in any of the methods of the invention are characterized by the following consensus sequence.
• Consensus Sequence III comprises the following sequence: RZ 1 Z 2 Z 3 GZ 4 Z 5 Z 6 EZ 7 Z 8 ATDZ 9 Z 1 OZ 11 Z 12 (SEQ ID NO. 91)
• Z 1 may be any amino acid, preferably E, V or T.
• Z 2 may be any amino acid, preferably L or M.
• Z 3 may be any amino acid, preferably Q, E or D.
• Z 4 may be any amino acid, preferably I or V.
• Z 5 may be any amino acid, preferably H or Q.
• Z 6 can be any amino acid.
• Z 7 may be any amino acid, preferably I, L, M or F.
• Z 8 may be any amino acid, preferably Q or H.
• Z 9 can be any amino acid.
• Z 10 may be any amino acid, preferably T or S.
• Z 11 may be any amino acid, preferably E or D.
• Z 12 may be any amino acid, preferably V 5 M or I. The amino acids are again indicated in single letter code.
• the ADF sequences according to the invention or the ADFs that can be used for the methods according to the invention can also contain the aforementioned three consensus sequences I, II and III in combination.
• the present invention thus also relates to nucleic acid sequences which i.a. for the consensus sequence shown above with the SEQ ID no. 89, 90 and / or 91 and their use in the inventive method for the production of transgenic plants with an increased pathogen resistance by changing the content and / or the activity of at least one ADF.
• ADFs the change in the content and / or the activity of ADFs or ADF3 can take place in various ways.
• this always includes the ADF3 from barley.
• the increase in the ADF activity and the ADF content for example, by switching off inhibitory regulatory mechanisms at the transcriptional, translational and protein level or by increasing the gene expression of a nucleic acid encoding at least one ADF or functional homologs, parts or mutants thereof to the wild-type respectively. This can be done, for example, by inducing the respective endogenous ADF gene (s) or by introducing nucleic acids coding for ADFs or functional homologs, parts or mutants thereof.
• the increase in the ADF activity or the ADF content compared to the wild type by increasing the gene expression of a nucleic acid encoding an ADF.
• the gene expression of a nucleic acid coding for an ADF is increased by introducing nucleic acids encoding at least one ADF into the respective plant or plant cell.
• the ADF genes of a wide variety of organisms ie any nucleic acid coding for an ADF with substantial homology to ADF3 from barley or functional homologues, parts or mutants thereof, can be used for this purpose.
• nucleic acid sequences from eukaryotic sources containing introns in the event that the host organism is unable or unable to splice the corresponding ADF sequences, preferably already processed nucleic acid sequences such as corresponding cDNAs to use. All nucleic acids mentioned in the description can e.g. an RNA, DNA or cDNA sequence.
• a nucleic acid sequence which codes for at least one ADF is transferred to a plant or plant cell.
• This transfer leads to an increase in the expression or the activity of ADF compared to the wild type and, correspondingly, to an increase in the pathogen resistance in the transgenic plants or plant cells.
• Such a method can be used to increase the expression of DNA sequences encoding ADFs or their functionally equivalent homologues, parts or functional mutants, and thus also increase the pathogen resistance in the transgenic plants or plant cells.
• vectors which comprises these sequences and regulatory sequences, such as promoter and termination sequences, is known to the person skilled in the art.
• Such a method typically comprises the following steps according to the invention:
• step a) The person skilled in the art knows how a vector from step a) can be transferred to plant cells and what features a vector must have in order to be integrated into the plant genome.
• An example of the overexpression of a functional mutant of an ADF is the overexpression of HvADF3-S 6 A (SEQ ID No. 2, see examples). Due to the amino acid exchange of serine for alanine, the ADF3 can no longer be phosphorylated in position 6. This leads to a constitutively active form of ADF3. Thus, overexpression of this mutant leads both to an increase in the content of ADF3 in the transgenic plants and to an increased activity on ADF3.
• the ADF content in transgenic plants or plant cells is increased by transferring a nucleic acid which codes for an ADF from another organism such as Dictyostelium discoideum, then it is recommended that the amino acid sequence encoded by the nucleic acid sequence, for example from Dictyostelium discoideum, by backtranslating the Polypeptide sequence according to the genetic code in a nucleic acid sequence to carry over, which mainly comprises those codons, which are used more frequently due to the organism-specific codon Usage.
• the codon usage can be easily determined by computer evaluations of other known genes of the organisms concerned.
• the manipulation of the expression of the organism-specific and, in particular, the plant-specific endogenous ADFs is understood according to the invention. This can be achieved, for example, by altering the promoter DNA sequence for ADF-encoding genes. Such a change, which results in an altered, preferably increased expression rate of at least one endogenous ADF gene, can be effected by deletion or insertion of DNA sequences.
• a change in the promoter sequence of endogenous ADF genes usually leads to a change in the expressed amount of the ADF gene and thus also to a change in the cell or plant detectable ADF activity.
• an altered or increased expression of at least one endogenous ADF gene can be achieved in that a regulatory protein that does not occur in the untransformed organism interacts with the promoter of these genes.
• a regulatory protein that does not occur in the untransformed organism interacts with the promoter of these genes.
• Such a regulator may be a chimeric protein consisting of a DNA binding domain and a transcriptional activator domain, as described, for example, in WO 96/06166.
• Another way to increase the activity and content of endogenous ADFs is to up-regulate transcription factors involved in the transcription of the endogenous ADF genes, eg, by overexpression.
• the measures for the overexpression of transcription factors are known in the art and are also disclosed in the context of the present invention for ADFs.
• a change in the activity of endogenous ADFs can be achieved by targeted mutagenesis of endogenous gene copies.
• Altering the endogenous ADFs can also be achieved by influencing the post-translational modifications of ADFs. This can e.g. This can be done by regulating the activity of enzymes such as kinases or phosphatases involved in the post-translational modification of ADFs by appropriate means such as overexpression or gene silencing.
• endogenous ADFs can also be regulated via the expression of aptamers that bind specifically to the promoter sequences of ADFs. Depending on whether the aptamers bind to stimulating or repressing promoter regions, the amount and, in this case, the activity of endogenous ADF is increased.
• the reduction of the content and / or the activity of at least one ADF can be achieved by different strategies.
• expression of at least one ADF in transgenic plants can be lowered by "silencing".
• a nucleic acid which codes for and / or is complementary to at least one ADF or parts thereof is transferred to the plant.
• the nucleic acid to be transferred is usually transferred to the plant by a vector, such as a plasmid, which is able to stably replicate within the plant cell or the to integrate transferred nucleic acid into the plant genome.
• the RNAi method can be used for the silencing of ADFs.
• a vector to the plant cell comprising, in the 5 '-3' direction, the following elements: a plant-functional promoter; operatively linked thereto, a DNA sequence comprising the antisense sequence of the sequence encoding the ADF or portions thereof and having 3 'exon sequences recognizable at its 3' end by the spliceosome; an intron; a DNA sequence comprising the sense sequence of the DNA sequence encoding the ADF or portions thereof and having 5'-exon sequences recognizable at its 5'-end from the spliceosome; and a termination sequence.
• a vector is shown in Figure 2.
• the position of the antisense and sense sequences can be reversed. It is clear to the person skilled in the art that the respective 5 'and 3' splice sites must be adapted accordingly.
• RNA molecules comprising a first exon comprising the antisense sequence of the sequence encoding the ADF or parts thereof, an intron and a second exon containing the sense sequence for the ADF or parts thereof consists of coding DNA sequence. Since the intron is removed by the splicing process, a continuous RNA molecule is formed with regions that are complementary to one another. Such an RNA molecule will form a double-stranded structure (Smith et al, 2000, Nature, 407: 319-320).
• Such double-stranded RNA molecules are capable of specifically silencing the mRNA of ADFs by induction of the PTGS system, resulting in ADFs no longer be expressed. Which ADFs are no longer expressed, can be determined by the appropriate choice of antisense and sense sequences.
• the finding of protein-characteristic sequences belongs to the usual knowledge of a person skilled in the art. The skilled worker knows that a multiplicity of ADFs can also be achieved by the multiple use of characteristic sequences in each case.
• Such a method may e.g. following steps include:
• a) Preparation of a vector comprising the following nucleic acid sequences in 5'-3 'orientation: a plant-functional promoter sequence operatively linked to the identical or homologous antisense sequence of the sequence coding for the at least one ADF or parts thereof, the sequence at its 3 'end of the spliceosome recognizable 3' exon sequences
• vectors for the RNAi method or PTGS can also be used.
• Such vectors may be constructed, for example, such that the sense and antisense sequences each transcribed from a U6 promoter, hybridize in the cell and induce the PTGS system (Tuschl, 2002, Nat. Biotechnol., 20, 446-448; Miyagishi et al, 2002, Nat. Biotechnol., 20, 497- 500, Lee et al., 2002, Nat. Biotechnol., 20, 500-505).
• the sense and antisense sequences are linked by a "loop" sequence and are transcribed from a human RNAse P RNA H1 promoter.
• the sense and antisense sequences can hybridize, form double-stranded RNA, and induce the PTGS system (Tuschl, 2002, vide supra; Paul et al., 2002, Nat. Biotechnol., 20, 505-508 Paddison et al., 2002, Genes Dev., 16, in press, Brummelkamp et al., 2002, Science, 296, 550-553).
• PTGS system Tuschl, 2002, vide supra; Paul et al., 2002, Nat. Biotechnol., 20, 505-508 Paddison et al., 2002, Genes Dev., 16, in press, Brummelkamp et al., 2002, Science, 296, 550-553.
• RNAi method no vectors are used, but pre-synthesized double-stranded RNA molecules which possess the above-described sense or antisense sequences, e.g. biolistic methods are introduced directly into the cell to be transfected.
• the vectors used to transfer the nucleic acids comprise a promoter in 5'-3 'orientation, operably linked thereto, a DNA sequence comprising the sequence encoding ADFs or portions thereof and complementary to itself Contains sections, as well as a termination sequence.
• the transcription of these vectors into the plant cell produces RNA molecules that have sequence regions that can hybridize to themselves.
• double-stranded RNA molecules can be found in the cell that induce the PTGS system, which then causes the mRNA to be specifically degraded by ADFs.
• This process also referred to as co-suppression, for silencing of plant proteins requires that the mRNA of the ADF (s) to be suppressed has sections which are complementary to one another.
• Such portions may be identified by one skilled in the art by simple visual inspection of the DNA sequence encoding the particular protein, or by appropriate sequence programs such as DNAStar from DNASTAR Inc., Madison, USA.
• Such a method may include, for example, the following steps:
• Termination sequence b) transferring the vector from a) to plant cells and optionally integration into the plant genome.
• the vectors used to transfer the nucleic acids in 5'-3 'orientation comprise a promoter, operably linked thereto, a DNA sequence comprising the antisense sequence of the sequence encoding ADFs or portions thereof, as well as a termination sequence.
• the transcription of such vectors into plant cells produces an RNA molecule whose sequence is complementary to the mRNA sequence coding for ADFs or parts thereof.
• Such a method may e.g. the following steps include:
• promoter-functional promoter sequence operatively linked to the identical or homologous antisense sequence of the sequence encoding the endogenous ADF (s) or parts thereof;
• vectors are used to transfer the nucleic acids to the plant cells, in the 5'- 3 'orientation a promoter which is functional in plants, operatively attached thereto a DNA sequence which codes for a ribozyme, the specifically recognize the mRNA of at least one ADF, and include a termination sequence.
• a promoter which is functional in plants, operatively attached thereto a DNA sequence which codes for a ribozyme, the specifically recognize the mRNA of at least one ADF, and include a termination sequence.
• ribozymes also refers to those RNA sequences which, in addition to the actual ribozyme, also comprise guide sequences which are complementary to the mRNA of the ADFs or parts thereof and thus more specifically target the mRNA-specific ribozyme to the mRNA. Substrate of the ribozyme drove.
• Such a method includes e.g. the following steps:
• RNA molecules are produced in the cell which have a leader sequence (the antisense sequence) which directs the RNAse P to the mRNA of the ADFs, causing cleavage of the mRNA by RNAse P (US Pat 5,168,053).
• the leader sequence comprises 10 to 15 nucleotides complementary to the DNA sequence of the ADFs and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
• the transcripts of the external leader sequence bind to the target mRNA through the formation of base pairs, allowing cleavage of the mRNA by the RNAase P at nucleotide 5 'of the paired region. Such a cleaved mRNA can not be translated into a functional protein.
• Such a method may e.g. the following steps include:
• Termination sequence b) transferring the vector from a) to plant cells and optionally integration into the plant genome.
• vectors which contain a DNA sequence which has the following components in the 5'-3 'orientation: A DNA sequence which corresponds to the 5' region the DNA sequence encoding an ADF, a DNA sequence for resistance genes, and a DNA sequence corresponding to the 3 'region of the ADF coding sequence.
• Such vectors can be used to induce specific gene knock-out of the ADF of interest via homologous recombination.
• the sequence encoding the resistance gene is inserted into the DNA encoding the ADF so that functional mRNA of the ADF can no longer be produced in the cell.
• Antibiotic resistance genes are typically used as resistance genes. Of course, other resistance genes that allow for selection of the cells in which the recombination has taken place can also be used.
• Such a method may e.g. following steps include:
• Termination sequence b) transfer of the vector from a) to plant cells and integration into the plant genome.
• nucleic acid sequences which code for ADFs or parts thereof this means both the complete coding DNA sequence of the ADFs and the complete mRNA sequence or the respective subregions. Since some of the above-mentioned methods of producing transgenic plants in which the expression of ADFs is significantly reduced rely on specific hybridization between the endogenous mRNA of ADFs and the sequences resulting from the transcription of the above-mentioned vectors (such as the antisense strategy), the person skilled in the art will understand that the transferred nucleic acids need not always contain all of the coding sequence for the ADFs, whether it be the sense or antisense sequence. Rather, for a specific hybridization, even relatively short regions of the ADF-encoding sequences may be sufficient for efficient silencing.
• sequences corresponding to the sequence regions of the mRNA of ADFs ultimately lead to double-stranded RNA molecules with approximately 25 nucleotides, preferably 21, 22 or 23 nucleotides in length.
• the sequences transferred in the antisense strategy generally comprise between 20-1000 nucleotides, preferably between 20-750 nucleotides, more preferably around 400-800 and 500-750 nucleotides. However, sequences may also be used which comprise between 20-500 nucleotides, between 20-300 nucleotides, between 20-150 nucleotides and between 20-100 or 20-50 nucleotides.
• RNAi or PTGS the sense and antisense RNAs used for the formation of double-stranded RNA molecules can also comprise around 21, 22 or 23 nucleotides with a characteristic 3 'overhang (Tuschl, 2002, Nat Biotechnol., 20, 446-448).
• nucleic acids When nucleic acids are transferred to the plant cells whose transcription in the cell results in sequences complementary to the mRNA of ADFs (as in the antisense strategy, for example), then these sequences need not be one hundred percent complementary to the mRNA. Rather, it is sufficient if these sequences are at least 50%, preferably at least 60%, more preferably at least 70%, further particularly preferably at least 80%, more preferably at least 90% and most preferably at least 95% complementary , The deviations can be caused by deletion, substitution and / or insertion. Of course, it will be clear to those skilled in the art that with decreasing complementarity, the likelihood of multiple ADFs increasing will increase.
• the length of complementary sequences is preferably between 20-1000 nucleotides, also preferably between 20-750 nucleotides, more preferably between 20-500 nucleotides, also more preferably between 20-300 nucleotides, particularly preferably between 20-150 nucleotides, also particularly preferred between 20-75 nucleotides, and most preferably about 20-50 nucleotides. Under certain circumstances, the sequences may also comprise only about 20 or 25 nucleotides.
• Some of the above-mentioned methods can also be performed with sequences that are not part of or complementary to the coding part of the mRNA of ADFs. It can e.g. be sufficient if they are sequences from the 5 'or 3' untranslated region, provided that these regulatory sequences are characteristic of the mRNA of the respective ADF's.
• mRNA encompasses not only the coding components of the mRNA of ADFs, but also all regulatory sequences which occur in the pre-mRNA or mature mRNA and which are characteristic of the mRNA of the ADFs. This also applies to the DNA sequence. This may be e.g. 5'- and 3'-untranslated regions to act promoter sequences, upstream activating sequences, introns etc.
• the leader sequence When using vectors whose transcription results in RNA molecules consisting of a leader sequence and RNAse P, then the leader sequence must be sufficiently complementary to specifically recognize the ADF. Which region of the mRNA of the ADF through the leader sequence can be selected according to the respective requirements. Preferably, such leader sequences comprise around 20 nucleotides, but they should not be significantly shorter than 15 nucleotides. With 100% complementarity of the leader sequence, 12 nucleotides should suffice. Of course, the leader sequences may comprise up to 100 nucleotides or more, as this only increases their specificity for the particular mRNA.
• sequences which correspond to the coding strand of the genes for ADFs or comprise parts thereof are meant those sequences which correspond to the coding strand of the genes for ADFs or comprise parts thereof.
• sequences need not be 100% identical to the sequences encoding the ADFs of interest. It suffices if the sequences are sufficiently similar to the ADF-encoding sequences that their expression in plant cells results in efficient and specific silencing of the ADFs in the cell, e.g. by RNA interference or co-suppression.
• sequences are at least 50% identical, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, more preferably at least 90% and most preferably at least 95% identical are.
• sequences are homologous to one another or have homology (see above).
• the deviations from the sequences coding for the ADFs or parts thereof may be due to deletion, addition, substitution and / or insertion.
• the skilled person is of course clear that with decreasing identity the
• sequences which correspond to the noncoding DNA strand of the genes of the ADFs of interest are not sufficiently specific and unsuitable for the methods of the invention. Accordingly, when referring to antisense sequences, it is meant according to the invention those sequences which correspond to the noncoding DNA strand of the genes of the ADFs of interest. Of course, these sequences, too, need not be one hundred percent identical to the sequence of the non-coding DNA strand of the genes of the respective ADFs of interest, but may have the abovementioned degrees of homology. This fact is also expressed in the fact that antisense sequences, which by definition are complementary to the mRNA of a gene, need not be 100% complementary to this mRNA.
• the antisense sequences may also be at least 50% complementary, preferably at least 60% complementary, more preferably at least 70% complementary, still more preferably at least 80% complementary, more preferably at least 90% complementary and most preferably at least 95%, 98% and / or 100% complementary.
• the antisense sequences are capable of specifically hybridizing to the particular mRNA of interest of ADFs. Hybridization can occur either in vivo under cellular conditions or in vitro.
• Hybridization of an antisense sequence with an endogenous ⁇ iRNA sequence typically occurs in vivo under cellular conditions or in vitro.
• antisense sequences which, for example, can not specifically hybridize in vivo and / or in vitro with coding sense sequences of ADFs, ie also hybridize with the coding sense sequences of other protein classes, can not be used.
• the antisense strategy can be coupled with a ribozyme method. Ribozymes are catalytically active RNA sequences which, coupled to the antisense sequences, catalytically cleave the target sequences (Tanner et al., (1999) FEMS Microbiol Rev. 23 (3), 257-75). This can increase the efficiency of an antisense strategy.
• ADFs genes for reducing the expression of ADFs, particularly in plants as organisms, include overexpression of homologous ADF nucleic acid sequences leading to cosuppression (Jorgensen et al., (1996) Plant Mol. Biol. 31 (5), 957-973) or the induction of specific RNA degradation by the plant using a viral expression system (amplicon) (Angell et al., (1999) Plant J. 20 (3), 357-362). These methods are also referred to as "post-transcriptional gene silencing" (PTGS) (see above).
• PTGS post-transcriptional gene silencing
• gene repression is also possible with specific DNA-binding factors, for example factors of the type of the zinc finger transcription factors.
• factors can be introduced into a cell that inhibit the target protein itself.
• the protein binding factors may be eg aptamers (Famulok et al., (1999) Curr Top Microbiol Immunol., 243, 123-36). The reduction can also be done by aptamers. Aptamers can also be designed to bind specifically to the ADF proteins and reduce the activity of the ADFs by, for example, binding to the catalytic center of the ADFs.
• aptamers The expression of aptamers is usually done by vector-based overexpression and is well known to those skilled in the art as well as the design and selection of aptamers (Famulok et al., (1999) Curr Top Microbiol Immunol., 243, 123-36).
• ADF-specific antibodies Other protein-binding factors whose expression in plants causes a reduction in the content and / or activity of ADFs are ADF-specific antibodies.
• the production of monoclonal, polyclonal or recombinant ADF-specific antibodies follows standard protocols (Guide to Protein Purification, Meth. Enzymol. 182, pp. 663-679 (1990), M.P. Deutscher, ed.).
• the expression of antibodies is also known from the literature (Fiedler et al., (1997) Immunotechnology 3, 205-216, Maynard and Georgiou (2000) Annu Rev. Biomed, Eng 2, 339-76). This approach is detailed below.
• a further method according to the invention for the production of transgenic plants with increased pathogen resistance provides that the activity of endogenous ADFs is reduced by non-functional mutants of ADFs being expressed in the plant or in the plant cells.
• non-functional mutants which are preferably dominant-negative mutants
• the interaction of the endogenous ADFs with their cellular binding partners is inhibited.
• non-functional mutations into the endogenous ADFs it is likewise possible according to the invention for plants and plants Plant cells are produced, which have an increased pathogen resistance.
• non-functional mutants are understood to be forms of ADFs which possess mutations which prevent the ADFs from interacting with G-actin, F-actin, components of the pathogen and / or with other physiological binding partners.
• Such dominant negative mutants When such dominant negative mutants are expressed or overexpressed in the transgenic cell or plant, they are capable of interfering with the interaction of the pathogen components with wild-type ADFs and the interaction of wild-type ADFs with the other physiological factors, e.g. Actin, so that the pathogen has no opportunity for propagation.
• the pathogen Surprising in this method is that it can produce transgenic plants which have increased pathogen resistance and at the same time have a substantially normal phenotype, although such dominant-negative mutants should influence the endogenous cytoskeleton of the plant cell.
• transgenic plants and plant cells according to the invention can be produced not only by expression or overexpression of dominant negative mutants of ADFs from plants such as ADF3, but of course also by expression or overexpression of dominant-negative mutants of ADFs other organisms.
• ADFs from eukaryotes such as yeasts (for example S. cerevisiae), C. elegans or higher mammals such as mice, rats and humans are suitable.
• yeasts for example S. cerevisiae
• C. elegans or higher mammals such as mice, rats and humans are suitable.
• the prerequisite is that the expression of these dominant-negative mutants a Konipetition the endogenous plant ADFs with pathogen components and / or their cellular
• the ADFs from other organisms can be identified by the database analyzes and homology comparisons described above. The prerequisite is that they have a region or regions which are homologous to the consensus sequences mentioned above.
• Transgenic plants expressing dominant-negative mutants of ADFs can be made by transferring a corresponding expression vector to plant cells.
• Such a method may e.g. the following steps include:
• Non-functional mutations that are dominant-negative mutants of ADFs can be identified by one skilled in the art by simple routine experimentation.
• a number of mutations e.g. from the ADF3 of maize that inhibit interaction with F-actin and G-actin, respectively.
• These are mutations in which the tyrosine residues at positions 67 and 70 of ADF3 from maize are replaced by phenylalanine.
• sequence alignments By means of so-called sequence alignments, the positions equivalent to tyrosines 67 and 70, e.g. in ADF3 from barley or other ADFs, thus producing similar mutations.
• the barley ADF3 is e.g. around the positions phenylalanine-66 and phenylalanine-69. These may e.g. be replaced by alanine.
• Mutations preventing the interaction of ADFs with G-actin, F-actin, other physiological binding partners and / or pathogen components, can be easily determined by preparing recombinant ADF proteins carrying different mutation and / or deletion and testing these recombinant proteins in binding assays with the aforementioned components.
• mutant-negative mutations all types of mutations, i. Insertion, deletion and point mutation capable of inhibiting the interaction of ADFs with G-actin, F-actin, other cellular binding partners and / or pathogen components.
• point mutation s
• insertion mutation s
• deletion mutation s
• Point mutations are preferred ("PCR technology: Principle and Applications for DNA Amplification", H. Ehrlich, Id, Stockton Press). Examples of the introduction of point mutations into DNA sequences coding for ADF3 can also be found in the exemplary embodiments.
• Transgenic plants or plant cells, which have an increased pathogen resistance can also be prepared according to the invention such that, for example, a recombinant antibody specifically the interaction of ADFs (and preferably ADF3 from barley) with G-actin, F-actin, other cellular Binding partners and / or pathogen components blocked or competitively expressed in the plants.
• a specific domain of ADFs can be isolated and identified, as known to those skilled in the art and can be found in the literature (Harlow et al., 1999, Using antibodies: a laboratory nianual, ColD Spring Harbor Laboratory Press).
• Recombinant antibodies according to the invention are understood to be the known different forms of recombinant antibodies, as used e.g. in Skerra et al. (Curr Opin Immunol (1993) 2, 250-262).
• the recombinant antibodies according to the invention comprise the so-called Fab fragments, Fv fragments, scFv antibodies, scFv homodimers which are linked to one another via disulfide bridges and so-called VH chains.
• the Fab fragments consist of assembled full light and truncated heavy chains, whereas Fv fragments consist of non-covalently linked VH and VL chains.
• scFv antibodies are preferably used according to the invention. These consist of the variable portion of the light chain and the variable portion of the heavy chain, which are fused together via a flexible linker peptide.
• the production of such scFv antibodies has been extensively described in the prior art (see, inter alia Conrad et al., Vide supra; Breitling et al. (1999) Recombinant Antibodies, John Wiley & Sons, New York).
• the scFv antibodies have the same antigenic specificity and activity as normal antibodies, but unlike other natural or recombinant antibodies, do not have to be assembled from single chains in vivo. They are therefore particularly suitable for the inventive method.
• phage display libraries Another way of producing recombinant antibodies known to those skilled in the art is the screening of recombinant antibody libraries (so-called phage display libraries, see also Hoogenboom et al., (2000) Immunology Today 21, 371-378; Winter et al Ann., Rev. Immunol., 12, 433-455, De Wildt et al., (2000) Nat. Biotechnol., 18, 989-994).
• recombinant antibodies to a given antigen can be enriched, selected and isolated in this method.
• a method of expressing antibodies to ADFs may be e.g. the following steps include:
• a) production of a vector comprising the following nucleic acid sequences in the 5'-3 'orientation: a plant-functional promoter sequence, operatively linked thereto a DNA sequence encoding a recombinant antibody specific for the endogenous ADF (s) and / or blocking interactions with physiological binding partners, preferably G-actin and / or F-actin , operatively linked to a plant functional
• a plant-functional promoter sequence operatively linked thereto a DNA sequence encoding a recombinant antibody specific for the endogenous ADF (s) and / or blocking interactions with physiological binding partners, preferably G-actin and / or F-actin , operatively linked to a plant functional
• Termination sequence b) transferring the vector from a) to the plant cells and optionally integration into the plant genome.
• Another object of the present invention relates to plant cells and plants in which the endogenous genes of ADFs mutations, i. Substitutions, insertions and / or deletions have the result that the expressed endogenous ADFs are no longer or only partially able to interact with pathogen factors and / or their endogenous cellular binding partners.
• Plants or plant cells having endogenous gene copies for ADFs having such mutations will, like the transgenic plants and plant cells described above, be characterized by increased transient or permanent pathogen resistance to the above-mentioned virus groups and strains.
• Such plants and plant cells, which are not transgenic in contrast to the above-mentioned plants and plant cells can be prepared by classical mutagenesis.
• non-transgenic plants or plant cells must have the abovementioned mutation types in the genes coding for endogenous ADFs, which lead to a modulation of the expression of the endogenous ADFs and / or the binding behavior of the endogenous ADFs.
• Modulation of the expression of the endogenous ADFs can mean, for example, that the expression of the endogenous ADFs is downregulated by mutations in regulatory DNA elements of the genes of the endogenous ADFs, such as promoters, enhancers or generally so-called "upstream activating sequences".
• the modulation of the binding behavior of ADFs in the context of the present invention means that the abovementioned mutation types lead to a change in the binding behavior of the endogenous ADFs in relation to the pathogenic factors and / or the normal cellular binding partners. Preference is given to a modulation of the binding behavior of the endogenous ADFs, which means that they no longer or only to a limited extent interact with pathogen factors and / or their cellular partners. A combination of the modulation of the expression and the binding behavior of the endogenous ADFs is also conceivable.
• plants or plant cells may have mutations in the gene sequences for endogenous ADFs that result in the reduction of expression of these proteins.
• Other plants or plant cells have mutations that lead to the dominant-negative mutants described above. In both cases, plants with increased pathogen resistance are obtained.
• mutagenesis for example, plants or plant cells can be produced which have a reduction in the expression of these proteins due to mutations in enhancer and / or promoter sequences of the genes for endogenous ADFs and at the same time mutations in the coding
• Have areas of genes encoding endogenous ADFs that cause the remaining expressed ADFs can no longer or only limitedly interact with the pathogenic and / or other cellular binding partners.
• the non-transgenic plants and plant cells according to the invention which are distinguished by a modulation of the expression and / or the binding behavior of the endogenous ADFs and have a permanent or transient pathogen resistance, can preferably be produced by the so-called "TILLING" approach (Targeting Induced Local Lesion in US Pat Genomes).
• TILLING Targeting Induced Local Lesion in US Pat Genomes. This method is described in detail in Colbert et al. (2001, Plant Physiology, 126, 480-484), McCallum et al. (2000, Nat. Biotechnol., 18, 455-457) and McCallum et al. (2000, Plant
• the TILLING procedure is a so-called reverse genetics strategy that allows the production of high levels of point mutations in mutagenized plant collections, e.g. by chemical mutagenesis with ethyl methanesulphonate (EMS), combined with rapid systematic identification of mutations in target sequences.
• EMS ethyl methanesulphonate
• the target sequence is amplified by PCR in DNA pools of mutagenized M2 populations.
• Denaturing and annealing reactions of the heteroallelic PCR products allow the formation of heteroduplexes in which one strand of DNA originates from the mutated and the other from the "wild-type" PCR product, at the point of point mutation, a so-called mismatch, either denaturing HPLC (DHPLC, McCallum et al., 2000, Plant Physiol., 123, 439-442) or with the CeZI mismatch detection system
• the vectors which are used for the expression or silencing of ADFs comprise, in addition to the nucleic acid sequence to be transferred, further regulatory elements. Which specific regulatory elements these vectors must contain depends in each case on the method that should be carried out with these vectors. Which regulatory elements and other elements need to have these vectors is known to those skilled in the art familiar with the various methods mentioned above for producing transgenic plants in which the expression of a protein is inhibited.
• operably linked means that the sequences that link the different nucleic acid sequences used are selected so as to preserve the function of the respective linked nucleic acid segment. If, for example, If the coding sequences of ADF3 are expressed in a cell, care must be taken that there are no sequences between the sequence for the promoter and the coding sequence for ADF3 which would lead to termination of the transcription.
• the regulatory elements that contain the vectors are those that ensure transcription and, if desired, translation in the plant cell.
• such elements can also bring about targeted localization of the proteins in specific cell types or cell organelles. This can be done, for example, by using epidermis cell-specific promoters.
• the nucleic acid sequences to be transferred may be under the control of promoters functional in plants.
• These promoters may be constitutive, but also inducible or tissue- or development-specific promoters. In addition, it may also be fungus-specific promoters.
• constitutive 35S promoter typically, one will use the constitutive 35S promoter as promoter for vectors.
• other promoters can be used, which are derived from different sources such. are obtained from plants or plant viruses and are suitable for the expression of genes in plants. The selection of the promoter as well as other regulatory sequences determine the spatial and temporal expression pattern and thus also the expresson or the silencing of the ADFs in transgenic plants.
• Promoters of the phosphoenolpyruvate carboxylase from maize Hudspeth et al., 1989, Plant Mol. Biol., 12: 579) or potato fructose-1,6-bisphosphatase (WO 98/18940), which mediate leaf-specific expression, are suitable . It is also possible to use wound, light or pathogen induced as well as other developmentally dependent promoters or control sequences (Xu et al., 1993, Plant Mol. Biol.
• Suitable promoters also include promoters which guarantee expression only in photosynthetically active tissues, such as the ST-LS1 promoter (Stockhaus et al., (1987) Proc Natl Acad., See, USA 84: 7943-7947, Stockhaus et al (1989) EMBO J. 8: 2445-2451). Also useful may be promoters that are active during plant transformation, plant regeneration or certain stages of these processes, such as cell division specific promoters such as the histone H3 promoter (Kapros et al., (1993) In Vitro Cell Cev. Biol. Plant 29: 27- 32) or the chemically inducible Tet repressor system (Gatz et al. (1991) Mol. Gen.
• ST-LS1 promoter Stockhaus et al., (1987) Proc Natl Acad., See, USA 84: 7943-7947, Stockhaus et al (1989) EMBO J. 8: 2445-2451.
• inducible promoters include virus-inducible promoters, such as the ACMV virion-sense promoter (Hong et al., 1996, Virology, 220: 119-227), which is induced by the gene product AC2.
• virus-inducible promoters such as the ACMV virion-sense promoter (Hong et al., 1996, Virology, 220: 119-227), which is induced by the gene product AC2.
• all promoters of such proteins that are induced in virus-infested tissue such as e.g. Phenylalanine, ammonium lyase, chalcone synthase, hydroxyproline-rich glycoprotein, extensin, pathogenesis-related proteins (e.g., PR-Ia) and wound-inducible protease inhibitors (US 6,013,864).
• cDNA clones which are based on poly (A) + RNA molecules from a non-storage organ tissue, from the first bank identified by hybridization those clones whose corresponding poly (A) + RNA molecules accumulate only in the tissue of the storage organ.
• promoters which have storage-organ-specific regulatory elements are isolated.
• the skilled person is also available further PCR-based methods for the isolation of suitable storage organ-specific promoters.
• the potato class I patatin gene B33 promoter is the potato class I patatin gene B33 promoter.
• Further preferred promoters are those which are active in particular in fruits. These include, for example, the promoter of a polygalacturonase gene, e.g. from tomato which expresses expression during the ripening of tomato fruits (Nicholass et al. (1995) Plant Mol. Biol. 28: 423-435; this prior art describes the analysis of promoter / GUS fusion constructs), the promoter of an ACC- Oxidase, eg from apple, which confers maturity and fruit specificity in transgenic tomatoes (Atkinson et al. (1998) Plant Mol. Biol.
• inducible promoters allows the production of plants and plant cells which express the sequences according to the invention only transiently or transiently.
• transient expression allows the production of plants that are only transient Show pathogen resistance.
• Such a transient resistance may be desirable, for example, when the danger of pathogen contamination threatens and the plants therefore only have to be resistant to the pathogen for a certain period of time.
• Other situations in which transient resistance is desirable are known to those skilled in the art. It is also known to those skilled in the art that they can achieve transient or transient silencing and transient resistance by using unstable replicating vectors in plant cells carrying the appropriate sequences for expression for silencing ADFs.
• Enhancer elements may also contain resistance genes, replication signals and other DNA regions which may cause propagation of the vectors in bacteria such as e.g. Allow E. coli.
• the regulatory elements also include sequences that cause stabilization of the vectors in the host cells.
• regulatory elements include sequences that enable stable integration of the vector into the host genome of the plant or autonomous replication of the vector in the plant cells.
• Such regulatory elements are known to the person skilled in the art.
• termination sequences are sequences that ensure that transcription or translation is properly terminated. If the transferred nucleic acids are to be translated, they are typically stop codons and corresponding regulatory sequences; If the transferred nucleic acids are only to be transcribed, they are generally poly A sequences.
• vectors are plasmids, cosmids, viruses and other vectors commonly used in genetic engineering, with which nucleic acid molecules can be transferred to plants or plant cells.
• cloning vectors which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
• examples of such vectors are pBR322, pUC series, M13mp series, pACYC184, etc.
• the desired sequence can be introduced into the vector at an appropriate restriction site.
• the resulting plasmid is used for the transformation of E. coli cells.
• Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered.
• the analytical method used to characterize the plasmid DNA obtained is generally restriction analyzes, gel electrophoresis and other biochemical molecular biological methods.
• the plasmid DNA can be cleaved and recovered DNA fragments can be linked to other DNA sequences.
• Each plasmid DNA sequence can be cloned in the same or different plasmids. Cloning standard methods can be found in Sambrook et al., 2001 (Molecular cloning: A laboratory manual, 3rd edition, CoId Spring Harbor Laboratory Press).
• plasmids By injection and electroporation of DNA into plant cells per se, no special requirements are imposed on the plasmids used. The same applies to direct gene transfer. Simple plasmids such as pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The skilled worker knows the usual selection markers and it is no problem for him to select a suitable marker. Common seclution markers are those which confer resistance to a biocide or an antibiotic on the transformed plant cells, such as kanamycin, G418, ampicillin, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin,
• the Ti or Ri plasmid are used for the transformation of the plant cell, then at least the right border, but frequently the right and left borders of the T-DNA contained in the Ti and Ri plasmid, must be connected as the flank region with the genes to be introduced ,
• the DNA to be introduced must be cloned into specific plasmids, either into an intermediate or a binary vector.
• the intermediate vectors can be integrated by homologous recombination into the Ti or Ri plasmid of the Agrobacterium due to sequences homologous to sequences in the T-DNA. This also contains the necessary for the transfer of T-DNA viral Region. Intermediate vectors can not replicate in agrobacteria. By means of a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and Agrobacteria.
• the host cell serving Agrobacterium is to contain a plasmid carrying a vir region.
• the vir region is necessary for the transfer of T-DNA into the plant cell.
• T-DNA may be present.
• the thus transformed Agrobacterium is used to transform plant cells.
• T-DNA for plant cell transformation has been extensively studied and sufficiently described in EP 120,515.
• plant explants may conveniently be cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
• Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium which may contain antibiotics or biocides for the selection of transformed cells.
• the regeneration of the plants is carried out by conventional regeneration methods using known nutrient media.
• the plants or plant cells thus obtained can then be examined for the presence of the introduced DNA.
• Cells are the transformation by the biolistic approach (Wan and Lemaux (1994) Plant Physiol 104, 37-48, Vasil et al (1993) Bio / Technology 11, 1553-1558, Ritala et al (1994) Plant Mol. Biol., 24, 317-325, Spencer et al., (1990), Theor. Appl. Genet., 79, 625-631), protoplast transformation, electroporation of partially permeabilized cells, and introduction of DNA by means of glass fibers.
• the transformed cells grow within the plant in the usual way (see also McCormick et al., (1986) Plant Cell Reports 5, 81-84).
• the resulting plants can be grown normally and crossed with plants having the same transformed genetic material or other genetic material.
• the resulting hybrid individuals have the corresponding phenotypic properties.
• Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the corresponding phenotype or other peculiarities have been preserved.
• transgenic lines that are homozygous for the new nucleic acid molecules and their phenotypic behavior can be determined by conventional methods examined for existing or non-existent pathogen responsiveness and compared with that of hemizygous lines.
• plant cells containing the nucleic acid molecules according to the invention may also be used as plant cells (including
• the vectors shown above can be transferred to plant cells in a variety of ways. Whether the vectors must be in linear or circular form depends on the particular application. The skilled person knows whether and when he can use corresponding linearized vectors or not. For example, one skilled in the art will appreciate that to produce specific knock-outs of genes for ADFs by homologous recombination, it may be sufficient to linearize the corresponding vectors and to inject them into transgenic plants.
• transgenic plant encompasses both the plant in its entirety and all plant parts in which the expression and / or activity of ADFs according to the invention is altered.
• plant parts may be plant cells, plant seeds, leaves, flowers and pollen.
• Propagating material of transgenic plants according to the invention such as e.g. Seeds, fruits, cuttings, tubers, root pieces, etc., said propagation material optionally containing transgenic plant cells described above, as well as parts of these plants such as protoplasts, plant cells and calli.
• transgenic plants In the production of transgenic plants, various methods and possibilities are available, as has already been stated above.
• plants or plant cells can be modified by conventional genetic engineering transformation methods such that the new nucleic acid molecules are integrated into the plant genome, ie that stable Transformants are generated and the transferred nucleic acid molecules are replicated with the plant genome.
• the vector system used it is also possible according to the invention to produce transgenic plants in which the nucleic acids to be transferred are contained in the plant cell or plant as a self-replicating system.
• the vectors used to transfer the plants must then have correspondingly DNA sequences enabling the replication of plasmids used for transfer within the cell.
• the plants used for the process according to the invention may, in principle, be any plant. Preferably, it is a monocot or dicotyle useful, food or forage plant.
• monocotyledonous plants are plants belonging to the genera Avena (oats), Triticum (wheat), sea ale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea (maize ) and the like.
• Dicotyledonous crops include, but are not limited to, cotton, legumes such as legumes, and especially alfalfa, soybean, rapeseed, tomato, sugarbeet, potato, ornamentals, and trees.
• Other crops may include fruits (especially apples, pears, cherries, grapes, citrus, pineapple and bananas), squash, cucumber, wine, oil palms, tea, cocoa and coffee bushes, tobacco, sisal and medicinal plants Rauwolfia and Digitalis.
• the cereals are particularly preferably wheat, rye, oats, barley, rice, corn and millet, sugar beet, rapeseed, soy, tomato, potato and tobacco.
• Other crops can be found in US Pat. No. 6,137,030.
• Preferred plants are cereals, alfalfa, oats, barley, rye, wheat, triticale, millet, rice, alfalfa, flax, cotton, hemp and Brassicacaeen such as rape or canola.
• Such transgenic plants, their propagation material, and their plant cells, tissue or parts are a further subject of the present invention.
• the invention thus also relates to harvest products and propagation material of transgenic plants, which were prepared by a method according to the invention and have an increased pathogen resistance.
• the harvested products and the propagation material are, in particular, fruits, seeds, flowers, tubers, rhizomes, seedlings, cuttings, etc. These may also be parts of these plants, such as plant cells, protoplasts and calli.
• the present invention also relates to the use of the abovementioned nucleic acid sequences for the production of transgenic plants or plant cells having an increased pathogen resistance in the context of the present invention.
• the present invention has for the first time succeeded in identifying, in addition to Rorl and Ror2 with ADF3, another gene in the barley which is involved in the mutant-mediated resistance.
• ADF3 another gene in the barley which is involved in the mutant-mediated resistance.
• transgenic plants have the advantage over other resistant plants that they are resistant not only to some specific mildew isolates, but to a variety of said mildew isolates, and this resistance is not limited to individual barley cultivars.
• a particularly preferred embodiment of the present invention relates to transgenic barley plants or cells having increased resistance opposite Blumeria graminisf. sp. hordei in which the content and / or the activity of ADF3 from barley having the SEQ ID no. 1 is changed from the wild type.
• particularly preferred embodiments of the present invention relate to methods for producing transgenic barley plants or the corresponding cells which have an increased resistance to Blumeria graminisf. sp. hordei in which the content and / or the activity of ADF3 with SEQ ID NO. 1 is changed from the wild type.
• a particularly preferred embodiment of the present invention relates to the isolated nucleic acid sequence encoding ADF3 from barley of SEQ ID NO. 1 encoded as well as functionally equivalent parts and functional or non-functional
• nucleic acid sequences which are essentially complementary to the particularly preferred last-mentioned nucleic acid sequences and hybridize to them under stringent conditions.
• the Mo gene has been identified in all investigated land plants and thus in other organisms than barley such as ⁇ rabidopsis thaliana and in other Gramineae such as wheat, oats, corn, rye, rice, Panicum, Pennisetum, Setaria, sorghum, Maize and the like, it can be considered that the barley ADF3 acts as a prototype for the corresponding homologous ADFs from other plants in the production of transgenic plants having increased pathogen resistance.
• the respective pathogen resistance may preferably be a resistance to formae speciales of Blumeria graminis, since this parasitism also occurs, for example, in wheat, oats and rye.
• transgenic plants and plant cells according to the invention can have a permanent or transient pathogen resistance.
• the type of resistant depends on the vectors used and the selection mechanisms used.
• transgenic plants having increased pathogen resistance selected from the group consisting of wheat, barley, oats, rice, panicum, pennisetum, setaria, sorghum, corn and the like.
• the above plants are resistant to the various formae speciales of the mildew pathogen Blumeria graminis, e.g. the isolates Blumeria graminis f. sp. Hordei, Blumeria graminis f. sp. tritici, Blumeria graminis f. sp. avenae.
• the identification of factors involved in the / w / o-mediated race-nonspecific resistance mechanism in barley has usually been carried out by mutation screening methods (Freialdenhoven et al., vide supra). This is based on barley cultivars that have mlo alleles and are resistant to isolates of Blumeria graminisf. sp. hordei are.
• the identification of factors that interact with the Mo locus is accomplished by selecting for plants that are mutagenic despite a mlo genotype for infection with isolates of Blumeria graminisf. sp. hordei are sensitive.
• RNA interference-based screening method was used to identify genes that influence broad-band resistance as mediated by recessive loss-of-function mloallele.
• Tissue Epidermis was withdrawn from 7-day old plants inoculated with Blumeria graminis hordei or tritici 6 and 24 hours after inoculation
• the cDNA library was constructed using a Stratagene kit (pBluescript II XR cDNA Library Construction Kit, Catalog No. 200455). The selection of the transformed cells was carried out with ampicillin.
• the thus-isolated cDNA fragments were then cloned using the Gateway ® technology Invitrogen into the vector pUAMBN.
• the use of the Gateway ® technology is described in detail in Walhout et al. (Walhout et al., (2000) "GATEWAY recombinational cloning: Application of the cloning of large frames of ORFeoms", Applications of Chimeric Genes and Hybrid Proteins, San Diego: ACADEMIC PRESS Inc., pp 575-592 ).
• the cDNA fragments were amplified by PCR through Gateway®- compatible oligonucleotides having the following sequences:
• the underlined sequence is specific for the cDNA library.
• the mirotiter plates were incubated at RT for 24 h; the complete batches were transformed into 50 ⁇ l chemocompetent E. coli (DH5 ⁇ ) (1 hr 4 ° C, 2 min 42 ° C); the complete transformations were plated on LB-Kan 3. then colonies were picked and grown in microtiter plates and Millipore 96 min minipreparation made. The DNA was taken up in 50 ⁇ l
• the mirotiter plates were incubated at RT for 24 h; the complete batches were transformed into 50 ⁇ l chemocompetent E. coli (DH5 ⁇ ) (1 hr 4 ° C, 2 min 42 ° C); the complete transformations were plated on LB-Amp
• the pU AMBN vector has a corn polyubiquitin promoter followed by two Gateway ® recombination cassettes in opposite orientation separated by the third intron of the barley mai resistance gene (see Fig.
• This arrangement of the vector ensures that the fragments amplified by PCR are in the sense and antisense direction in the vector.
• PCR fragment once in the sense and antisense direction in the vector is cloned, created after transfection of the vector into the plant cell and expression of the sequences, a double-stranded oligonucleotide molecule, which is able to trigger an RNAi response in the plants.
• the Particle Delivery System ® Biolistic PDS- 1000 / He BioRad
• gold particles were coated with the corresponding DNA.
• To coat the gold particles typically 5 ⁇ l of DNA (1 ⁇ g / ⁇ l), 50 ⁇ l of 2.5 M CaCl 2 and 20 ⁇ l of 0.1 M spermidine were added to pre-prepared gold particles.
• the particles were then pelleted by means of a bench top centrifuge and washed with 140 ⁇ l 70% ethanol and 140 ⁇ l 100% ethanol. After re-centrifugation, the coated gold particles were resuspended in 48 to 60 ⁇ l of 100% ethanol. Subsequently, the epidermis cells were bombarded according to the manufacturer's instructions with the coated particles.
• the so-called single-cell transient expression assay as described by Shirasu et al. (Shirasu et al. (1999), Plant J., 17, 293-299).
• the reporter plasmid containing the GUS gene was coated on gold particles. The coating and bombardment of the leaves was done as shown above. The bombarded leaves were transferred to 1% agar plates supplemented with 8% mM benzimidazole and incubated at 18 ° C. for 4 hours. Subsequently, the leaves were stained for GUS activity and examined microscopically.
• the GUS construct is described in Nielsen et al. (Nielsen et al., (1999) Physiol. Mol. Plant Pathol., 54, 1-12).
• dsRNAi constructs each containing a barley gene in the form of inverted repeats in pUAMBN as described above, were used together with a plasmid expressing the constitutive expression of the reporter protein ⁇ -glucuronidase (GUS). mediated transfected into the epidermis cells of individual barley leaves.
• GUS reporter protein ⁇ -glucuronidase
• the transformed samples with Blumeria graminisf. sp. hordei (Bgh) 96 hours after ballistic transfection. Forty-eight hours after inoculation, the leaves were stained for GUS activity, and individual transformed epidermal cells inoculated with IgA spores were examined microscopically for the fungal penetration success.
• the barley cultivars transfected with the two named vectors were both the wild-type Mio and powdery mildew resistant m / o genotypes, as well as genotypes conferring race-specific resistance to certain mildew isolates. Mal, Mla6, Mlal2, MIg).
• Cultivar Golden Promise (Mio): Max Planck Institute, Cologne Cultivar 110 (Mlal2): near isogenic to cv Ingrid cultivar BCPallasM ⁇ i cultivar BCPallasM ⁇ tf cultivar BCPallasMg cultivar BCIngridm / o5 cultivar BCIngridw / o5
• a gene whose expression is expressed by the dsRNAi construct may be considered to be part of the Mo race non-specific resistance mechanism if it causes increased sensitivity to the Bgh pathway in the resistant m / o genotype, but does not affect breed-specific resistance in the Mal, Mla6, Mal 2, Mg genotypes.
• the penetration success is examined microscopically, e.g. the extent of haustoria formation is observed.
• ADF3 which is referred to below as ADF3 or HvADF3
• HvADF3 HvADF3
• This barley exhibited high amino acid sequence similarity to the previously described Arabidopsis thaliana and Zea mays actin depolymerizing factors.
• the barley ADF3 has the sequence with SEQ ID no. 1.
• a sequence alignment with other Arabidopsis thaliana ADFs is shown in FIG.
• BCIngrid / M / o5 Penetration rate 15% ⁇ 3% Control: 0% ⁇ 0%
• HvADF3 Variant of HvADF3 carrying an S 6 A-amino acid substitution that renders the protein inaccessible for N-terminal phosphorylation.
• a corresponding mutant has been described for maize ADF3 (Smertenko et al., Plant J 14, 187-193.)
• HvADF3-CA-F (has HmdlII site and mutation Ser6 ⁇ Ala6): 5'-TTT AAG CTT GCC ACC ATG GCA AAC GCT TCA GCA GGT GCT GGG-3 1
• the serine ⁇ against alanine exchange was inserted into the wild-type gene of HvADF3 by design of the corresponding primers.
• the PCR product was cut with the above-mentioned restriction enzymes and ligated into the overexpression vector pUbi-MCS-Nos previously cleaved with the same restriction enzymes.
• the resulting overexpression vector (pHvADF3-CA, Fig. 3) has a maize polyubiquitin promoter (pUbi), the mutant HvADF3 gene (HvADF3-CA), and a nopaline synthase transcription / termination sequence (NOS).
• HvADF3 SEQ ID No. 45
• Gateway primers SEQ ID No. 45
• Primer HvADF3 gate F (has attB 1 region):
• Primer HvADF3-Gate-R (has attB2 region):
• HvADF3 specific sequences are underlined.
• the dsRED vector (pUbi-RFP-Nos, Fig. 4) has a maize polyubiquitin
• PTS peroxisome target sequence
• Primer GFP-F (has Hm ⁇ II site and binds to GFP sequence): 5'-GCG AAG CTT GCC ACC ATG GTG AGC AAG GGC GAG-3 '
• Primer GFP-PTS-R (has additional PTS and Mwl site, binds in GFP sequence): 5'-AAG ACG CGT TTA GAG GCG GGA CTT GTA CAG CTC G-3 '
• the PCR was carried out with a GFP sequence as template.
• the PCR product was cut with the above-mentioned restriction enzymes and ligated into the overexpression vector pUbi-MCS-Nos previously cleaved with the same restriction enzymes.
• the GFP peroxisome target sequence vector (pGFPTS, Fig. 6) has a maize polyubiquitin promoter (pUbi), the green fluorescent protein coding gene including the peroxisome target sequence (GFPTS) and a nopaline synthase transcription termination sequence (NOS). ,
• HvADFS are changed from the wild type
• HvADFS intracellular transport mechanisms as a consequence of overexpression or silencing of HvADFS
• transport-dependent defense mechanisms e.g.
• the accumulation of vesicles at sites of infection may be the reason for an increase in the number of experiments that led to the identification of HvADFS
• the fungus can form only very short hyphae in cells in which HvADFS is overexpressed, which prevents the establishment of a sustainable infection.
• the fungal structures are fully formed in stomata cells that have not been transfected (see Fig. 8 c).
• the fungal pathogen initially benefits from the host's degraded actin cytoskeleton in successful cell wall penetration, it can not establish a successful infection because apparently intact actin filaments are necessary for maintaining a compatible interaction.
• Degradation of the content and / or activity of ADF3 in barley may result in race-nonspecific resistance to various Bgh isolates.
• the SEQ ID NO. 11 specified nucleic acid sequence for ADF 3 from Ar ⁇ bidopsis th ⁇ li ⁇ n ⁇ can be amplified by the following primers:
• the resulting fragment is cloned into a binary vector.
• a subcloning into the vector pCR®2.1 TOPO (Invitrogen, Düsseldorf), from which the gene with the enzymes EcoRV and HindUI can be excised again (see Fig. 9).
• the overhanging ends are filled in with the help of the Klenow enzyme.
• the fragment prepared above is ligated into the Smal-opened, dephosphorylated binary vector pSUN2 (see Figure 10).
• AtADF3 In order to enable the pathogen-inducible expression of AtADF3 as well, it is excised from pSUN2 with BgHI and ⁇ l and ligated into the vector Lo215.
• This vector already contains the Arabidopsis thaliana Thi2.1 promoter (Acc. L41244, EppleJP., Apel, K. and Bohlmann, H. (1995)
• An Arabidopsis thaliana thionin gene is inducible via a signal transduction pathway different from that for pathogenesis Plant Physiol. 109 (3), 813-820), which is induced by pathogen attack, which has already been demonstrated via a downstream GUS gene.
• AtADF3 is cloned in place of the existing GUS gene by excising it with SacI and Smal from the vector, dephosphorylating and filling the vector and ligating the AtADF3 fragment into the vector. Via a homologous recombination (Gateway® reaction, Invitrogen, Düsseldorf) the promoter gene construct was subsequently recloned into the binary vector Lol23 (see FIG. 11).
• the transformation is carried out according to the floral-dip method (modified according to Clough and Bent, 1998).
• the seeds are sterilized overnight after harvesting with chlorine gas and then laid out on selection plates.
• the antibiotics are added depending on the plant resistance marker. at BASTA is added to pSUN2, kanamycin is added to Lo 123.
• the seeds are placed on the selection plates after sterilization and stored for stratification for 2 days at 4 0 C in the cold room. Thereafter, they will continue to be observed under short-day conditions.
• the first selection of the plants can be carried out. Non-transgenic plants bleach during selection, while the transgenic plants that possess the corresponding resistance gene remain green.
• the plants that remain green after the first selection are selected a second time under the same conditions.
• the plants that do not bleach during the second selection can then be transplanted to soil.
• the plants are selfed and the resulting T2 seed populations subjected to phytopathological analysis.
• Refrigerator at about 16 ° C with a plastic bag covered dark and kept moist.
• the plastic bag After one day, the plastic bag is opened slightly and later completely removed.
• the biotrophic mildew fungus is cultured on Arabidopsis thaliana PüanzQn.
• Arabidopsis thaliana PüanzQn To infect the 4 week old transgenic Ar ⁇ bidopsis PüaDzen conidia carriers are removed with a fine brush on the surface of the leaves and painted on the leaves of the transgenic plants. The plants are incubated for 7 days at 20 0 C. Seven days after inoculation, the conidiophores become visible on the leaves and chlorosis and necrosis appear in the following days. These symptoms are quantified and tested for significance.
• transgenic Arabidopsis plants expressing vltADF3 constitutively or pathogen-inducible show significantly increased resistance to both Peronospor ⁇ p ⁇ r ⁇ sitic ⁇ and Erysiphe cichoracearum compared to non-transgenic wild-type plants.
• Fig. 1 shows a sequence alignment of HvADFS and various ADFs from Arabidopsis thaliana (see also Table 7)
• Figure 2 shows the vector pUAMBN used for dsRNAi-based silencing in barley epidermal cells.
• Ubi corn polyubiquitin promoter; attRl and attR2, Gateway recombination sites; ccdB, negative selection marker; nos, Agrobacterium tumefaciens nopaline synthase transcriptional terminator.
• Fig. 3 shows the vector pHvADF3-CA.
• Fig. 4 shows the vector pUbi-RFP-nos.
• FIG. 5 shows the visualization of the actin cytoskeleton in transfected individual epidermis leaf cells of oats by phalloidin staining.
• the cells were transfected with a plasmid expressing dsRED (RFP) to label bombarded cells.
• RFP plasmid expressing dsRED
• FIG. 5 shows the visualization of the actin cytoskeleton in transfected individual epidermis leaf cells of oats by phalloidin staining.
• the cells were transfected with a plasmid expressing dsRED (RFP) to label bombarded cells. When no additional gene was expressed (control, A), stained actin fibers were detectable within the labeled cells as well as in adjacent cells.
• RFP plasmid expressing dsRED
• Fig. 6 shows the vector pGFPTS.
• Fig. 7 shows the movement of GFP-labeled peroxisomes in individual leaf epidermis cells of barley.
• a plasmid encoding a GFP variant with a C-terminal peroxisomal targeting sequence was either isolated (A, control) or together with a plasmid carrying a "constitutively active" variant of HvADF3 carrying an S A-amino acid substitution (B ), expressed. While in the control transfections (A) GFP-labeled peroxisomes were constantly moving within the bombarded cells, the co-expressing peroxisomes of the constitutively active variant of HvADF3 (B) slowed down and finally aggregated.
• Fig. 8 shows that overexpression of HvADF3 inhibits the development of the fungus.
• Barley leaf dermis cells were transfected with GUS ( ⁇ -glucuronidase) reporter plasmids and a plasmid expressing the ectopic expression of a constitutively active variant of HvADF3 (which has an S 6 A-
• FIG. 9 shows the vector ⁇ CR2.1 TOPO with inserted -4tADF3 gene.
• Fig. 10 shows the vector pSUN2 with inserted -4tADF3 gene.
• Fig. 11 shows the vector Lol23 with inserted AtAOF3 gene.

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