Patent Grant
8178751
This application is a national stage application (under 35 U.S.C. §371) of PCT/EP2007/050062, filed Jan. 4, 2007, which claims benefit of European application 06100304.2, filed Jan. 12, 2006.
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequenece_Listing—13987—00094. The size of the text file is 82 KB, and the text file was created on Jun. 15, 2011.
The invention relates to a method of generating or increasing a pathogen resistance in plants by reducing the expression of at least one stomatin polypeptide or a functional equivalent thereof. The invention relates to novel nucleic acid sequences coding for a Hordeum vulgare stomatin (HvSTM1) polynucleotide and describes homologous sequences (STM1) thereof, and to their use in methods for obtaining a pathogen resistance in plants, and to nucleic acid constructs, expression cassettes and vectors which comprise these sequences and which are suitable for mediating a fungal resistance in plants. The invention furthermore relates to transgenic organisms, in particular plants, which are transformed with these expression cassettes or vectors, and to cultures, parts or transgenic propagation material derived therefrom.
There are only few approaches which confer a resistance to pathogens, mainly fungal pathogens, to plants. This shortcoming can partly be attributed to the complexity of the biological systems in question. Another fact which stands in the way of obtaining resistances to pathogens is that little is known about the interactions between pathogen and plant. The large number of different pathogens, the infection mechanisms developed by these organisms and the defence mechanisms developed by the plant phyla, families and species interact with one another in many different ways.
Fungal pathogens have developed essentially two infection strategies. Some fungi enter into the host tissue via the stomata (for example rusts, Septoria species, Fusarium species) and penetrate the mesophyll tissue, while others penetrate via the cuticles into the epidermal cells underneath (for example Blumeria species).
The infections caused by the fungal pathogens lead to the activation of the plant's defence mechanisms in the infected plants. Thus, it has been possible to demonstrate that defence reactions against epidermis-penetrating fungi frequently start with the formation of a penetration resistance (formation of papillae, strengthening of the cell wall with callose as the main constituent) underneath the fungal penetration hypha (Elliott et al. Mol Plant Microbe Interact. 15: 1069-77; 2002).
In some cases, however, the plant's defence mechanisms only confer an insufficient protection mechanism against the attack by pathogens.
The formation of a penetration resistance to pathogens whose infection mechanism comprises a penetration of the epidermal cells or of the mesophyll cells is of great importance both for monocotyledonous and for dicotyledonous plants. In contrast to described mlo-mediated resistance, it can probably make possible the development of a broad-spectrum resistance against obligatory biotrophic, hemibiotrophic and necrotrophic fungi.
The present invention was therefore based on the object of providing a method for generating a resistance of plants to penetrating pathogens.
The object is achieved by the embodiments characterized in the claims.
The invention therefore relates to a method of increasing the resistance to penetrating pathogens in a monocotyledonous or dicotyledonous plant, or a part of a plant, for example in an organ, tissue, a cell or a part of a plant cell, for example in an organelle, which comprises lessening or reducing the activity or amount of a stomatin protein (STM1) in the plant, or a part of the plant, for example in an organ, tissue, a cell or a part of a cell, for example in a cell compartment, for example in an organelle, in comparison with a control plant or a part of a control plant, for example its organ, tissue, cell or part of a cell, for example in a cell compartment, for example in an organelle.
Preferably, a race-unspecific resistance is obtained in the method according to the invention. Thus, for example, a broad-spectrum resistance against obligatorily biotrophic and/or hembiotrophic and/or necrotrophic fungi of plants, in particular against mesophyll-penetrating pathogens, can be obtained by the method according to the invention.
Surprisingly, it has been observed that the gene silencing via dsRNAi of a gene which codes for the stomatin protein HvSTM1 results in an increase in the resistance of monocotyledonous and dicotyledonous plants to fungal pathogens. Thus, this negative control function in the event of attack by fungal pathogens has been demonstrated for the stomatin protein HvSTM1 from barley (Hordeum vulgare) (HvSTM1), wheat (Triticum aestivum) and thale cress (Arabidopsis thaliana).
It has been found within the scope of a TIGS (=Transient Induced Gene Silencing) analysis in barley by the method of Schweizer et al. (2001) that a dsRNAi-mediated silencing of the gene HvSTM greatly increases the resistance to Blumeria graminis f. sp. hordei (synonym: Erysiphe graminis DC. f. sp. hordei). This effect has also been obtained in dicotyledonous species such as, for example, Arabidopsis thaliana by inducing the post-transcriptional gene silencing (PTGS). This emphasizes the universal importance of the loss-of-function of HvSTM1-homologous genes for the development of a broad-spectrum pathogen resistance of the plant.
Stomatin is an integral membrane protein which has first been identified in blood cells. In certain hereditary diseases, in which this protein is absent, a hemolytic anemia results. In this stomatocytosis, the blood cells suffer a pronounced passive diffusion of singly charged cations, which results in hyperhydration as the result of a high sodium concentration and a low potassium concentration. The name stomatin is derived from the mouth-like structure of these erythrocytes (Greek Stoma=mouth). Stomatin acts as a negative membrane permeability regulator for singly charged cations. Stomatin has a single membrane domain, while the remainder of the protein protrudes into the cytoplasm. The molecular mechanisms via which stomatin exerts its function is unclear. It is assumed that the cytoplasmic domain of the protein acts sterically as a sort of plug which closes ion channels and perhaps interacts with the cytoskeleton (Stewart G W, Argent A C, Dash B C J (1993) Biochim. Biophys. Acta 1225, 15-25; Stewart G W et al. (1992) Blood 79, 1593-1601).
A first potential plant stomatin was cloned in maize (Nadimpalli R et al. (2000) J. Biol. Chem. 275, 29579-29586). However, no functional data for this gene, Zm-stm1, were published. A potential role in pathogen defence has been assumed for other genes which were cloned in this work since transcripts of these genes were upregulated in the lesion Mimic-Mutante Les9. While such a context has not been demonstrated for Zm-stm1, the authors speculate about a role as a positive pathogen defence regulator, for example as promoters of the hypersensitive reaction (HR). In the same line as this argumentation, two other study groups have found that genes for proteins in Arabidopsis in the background of two mutants with an increased resistance are upregulated and that stomatin could therefore act as PR protein (Petersen M et al. (2000) Cell 103, 1111-1120; Brodersen P et al. (2002) Genes & Dev 16, 490-502).
The finding that a reduction in the expression of stomatin leads to a significant increase in the pathogen resistance in plants was all the more surprising.
In a further embodiment, the invention therefore relates to a method of generating a plant with an increased resistance to plant pathogens, preferably with a broad-spectrum resistance, in particular to fungal pathogens, for example from the classes Ascomycetes, Basidiomycetes, Chytridiomycetes or Oomycetes, for example of mildews of the family Erysiphaceae, genus Blumeria, by interfering with the cell wall structure, in particular by reducing the membrane permeability, for example for singly charged cations, in particular by modifying the ion concentration in the cell, preferably by increasing the concentration of singly charged cations, such as, for example, sodium, for example by mutation of an ion channel or of a protein which interacts with, or regulates, an ion channel.
In an embodiment, the invention therefore relates to a method of generating a plant with an increased resistance to plant pathogens, preferably with a broad-spectrum resistance, in particular to fungal pathogens, for example from the classes Ascomycetes, Basidiomycetes, Chytridiomycetes or Oomycetes, for example of mildews of the family Erysiphaceae, genus Blumeria, by reducing the expression or by mutation of a stomatin STM1 protein
In a further embodiment, the activity of a subtilisin-like polypeptide is reduced, for example blocked or eliminated, in the method according to the invention.
In a further embodiment, in the method according to the invention the activity of a polypeptide is reduced or eliminated, which is encoded by a polynucleotide comprising at least one nucleic acid molecule selected from the group consisting of:
In the method according to the invention, it is in particular the resistance to mesophyll-cell-penetrating pathogens which is preferably increased.
In one embodiment, the resistance is obtained by lessening, reducing or blocking the expression of a polypeptide, preferably of a polypeptide which is encoded by the above-described nucleic acid molecule, for example that of a stomatin STM1 from barley as shown herein in SEQ ID No. 2 and 3, or from
Rice:
On the other hand, it is also possible to reduce, lessen or block the endogenous activity of one of these polypeptides by methods known to the skilled worker, for example by mutating a genomically coding region for the active center, for binding sites, for localization signals, for domains, clusters and the like, such as, for example, of coding regions for coiled coil, HEAT, FBOX, LRR, IBIB, C2, WD40, beach, U-box or UND domains. The activity can be reduced in accordance with the invention by mutations which affect the secondary, tertiary or quaternary structure of the protein.
Mutations can be inserted for example by an EMS mutagenesis. Domains can be identified by suitable computer programs such as, for example, SMART or InterPRO, for example as described in Andersen P., The Journal of Biol. Chemistry, 279, 38, pp. 40053-40061, 2004 or Y. Mudgil, Plant Physiology, 134, 59-66, 2004, and literature cited therein. The suitable mutants can then be identified for example by tilling.
In one embodiment, the lessening of the polypeptide quantity, activity or function of a stomatin STM1 protein in a plant is combined with increasing the polypeptide quantity, activity or function of other resistance factors, preferably of a Bax inhibitor 1 protein (BI-1), preferably of the Bax inhibitor 1 protein from Hordeum vulgare (GenBank Acc.-No.: AJ290421), from Nicotiana tabacum (GenBank Acc.-No.: AF390556), rice (GenBank Acc.-No.: AB025926), Arabidopsis (GenBank Acc.-No.: AB025927) or tobacco and oilseed rape (GenBank Acc.-No.: AF390555, Bolduc N et al. (2003) Planta 216:377-386) or of ROR2 (for example from barley (GenBank Acc.-No.: AY246906), SnAP34 (for example from barley (GenBank Acc.-No.: AY247208) and/or of the lumenal binding protein BiP for example from rice (GenBank Acc.-No. AF006825). An increase can be achieved for example by mutagenesis or overexpression of a transgene, inter alia.
In one embodiment, the lowering of the polypeptide quantity, activity or function of a stomatin STM1 protein in a plant is combined with decreasing the protein quantity, activity or function of other resistance factors, preferably of the proteins RacB (for example from barley (GenBank Acc.-No.: AJ344223), CSL1 (for example from Arabidopsis (GenBank Acc.-No.: NM116593), HvNaOX (for example from barley (GenBank Acc.-No.: AJ251717), MLO (for example from barley (GenBank Acc.-No. Z83834), ARM1 (armadillo repeat protein; application number 05110468.5).
The activity or function of MLO, BI-1 and/or NaOX can be reduced or inhibited analogously to what has been described for MLO in WO 98/04586; WO 00/01722; WO 99/47552 and the further publications mentioned hereinbelow, whose content is herewith expressly incorporated by reference, in particular in order to describe the activity and inhibition of MLO. The description of the abovementioned publications describes processes, methods and especially preferred embodiments for lessening or inhibiting the activity or function of MLO; the examples indicate specifically how this can be realized.
The reduction of the activity or function, if appropriate of the expression of BI-1 is described in detail in WO 2003020939, which is herewith expressly incorporated into the present description. The description of the abovementioned publication describes processes and methods for lessening or inhibiting the activity or function of BI-1; the examples indicate specifically how this can be realized. The reduction or inhibition of the activity or function of BI-1 is especially preferably carried out in accordance with the embodiments especially preferred in WO 2003020939 and the examples and in the organisms shown therein as being especially preferred, in particular in a plant, for example constitutively, or a part thereof, for example in a tissue, but especially advantageously at least in the epidermis or in a considerable part of the epidermal cells. The reduction of the activity or function, if appropriate of the expression, of BI-1 is described extensively in WO 2003020939. The skilled worker finds in WO 2003020939 the sequences which code for BI-1 proteins and can also identify BI-1 with the method provided in WO 2003020939.
The reduction of the activity or function, if appropriate of the expression, of NaOX is described extensively in PCT/EP/03/07589, which is herewith expressly incorporated into the present description. The description of the abovementioned publication describes processes and methods for lessening or inhibiting the activity or function of NaOX, and the examples indicate specifically how this can be realized. The reduction or inhibition of the activity or function of NaOX is especially preferably carried out in accordance with the embodiments especially preferred in PCT/EP/03/07589 and the examples and in the organisms shown therein as being especially preferred, in particular in a plant, for example constitutively, or a part thereof, for example in a tissue, but especially advantageously at least in the epidermis or in a considerable part of the epidermal cells. The skilled worker finds in PCT/EP/03/07589 the sequences which code for NaOX proteins and can also identify NaOX with the method provided in PCT/EP/03/07589.
The terms “to lessen”, “to reduce” or “to repress” or their substantives are used synonymously in the present text.
“Lessening”, “reduction” or “repression” or their verbs are understood as meaning, in accordance with the invention, that the activity in the plant is lower than in a control plant or is lower in a part of a plant than in the same part of a control plant, for example in an organ, an organelle, a tissue or a cell. In one embodiment, the activity of the abovementioned polypeptide is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more lower than in the control. In one embodiment, no expression of the abovementioned polypeptide takes place. As a consequence, these terms also comprise the complete inhibition or blocking of an activity, for example by the knock-out of a gene.
“Reduction”, “to reduce”, “lessening” or “to lessen”, “repression” or “to repress” comprise the partial or essentially complete inhibition or blocking of the functionality of a protein, based on a variety of cell-biological mechanisms.
Lessening within the purpose of the invention also comprises a quantitative reducing of a protein down to an essentially complete absence of the protein (i.e. lack of detectability of activity or function or lack of immunological detectability of the protein). Here, the expression of a certain protein or the activity or function in a cell or an organism is reduced by preferably more than 50%, especially preferably by more than 80%, very especially preferably by more than 90%.
For example, the expression of a nucleic acid molecule for a stomatin STM1 protein, for example in combination with a tissue-specific increase in the activity of a Bax inhibitor-1 protein may take place in the mesophyll tissue. The reduction of the stomatin STM1 protein quantity in a transgenic plant which for example overexpresses BI-1 in the mesophyll tissue offers the possibility of generating a complete and comprehensive fungal resistance in the plant.
In a further embodiment, the increase in the polypeptide quantity, activity or function of a Bax Inhibitor 1 protein from Hordeum vulgare (GenBank Acc.-No.: AJ290421), from Nicotiana tabacum (GenBank Acc.-No.: AF390556), rice (GenBank Acc.-No.: AB025926), Arabidopsis (GenBank Acc.-No.: AB025927) or tobacco and oilseed rape (GenBank Acc.-No.: AF390555, Bolduc N et al. (2003) Planta 216:377-386) or of ROR2 (for example from barley (GenBank Acc.-No.: AY246906), SnAP34 (for example from barley (GenBank Acc.-No.: AY247208) and/or of the lumenal binding protein BiP for example from rice (GenBank Acc.-No. AF006825) is effected in combination with the reduction in the protein quantity or activity or function of the proteins RacB (for example from barley (GenBank Acc.-No.: AJ344223), CSL1 (for example from Arabidopsis (GenBank Acc.-No.: NM116593), HvNaOX (for example from barley (GenBank Acc.-No.: AJ251717), and/or MLO (for example from barley (GenBank Acc.-No. Z83834). As a consequence, in one embodiment, at least one of the abovementioned genes which are suitable for overexpression or increased activity is activated or overexpressed and/or at least one of the abovementioned genes which is suitable for reduction is reduced.
An increase in the expression can be obtained as described herein. An increase in the expression or function is understood as meaning herein both the activation or enhancement of the expression or function of the endogenous protein, including a de novo expression, and an increase or enhancement by expression of a transgenic protein or factor.
For the purposes of the invention, “organism” means “nonhuman organisms” as long as the term relates to a viable multi-celled organism.
For the purposes of the invention, “plants” means all dicotyledonous or monocotyledonous plants. Preferred are plants which can be subsumed under the class of the Liliatae (Monocotyledoneae or monocotyledonous plants). The term includes the mature plants, seeds, shoots and seedlings, and parts, propagation material, plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures derived from the above, and all other types of associations of plant cells which give functional or structural units. Mature plants means plants at any developmental stage beyond the seedling stage. Seedling means a young, immature plant in an early developmental stage.
“Plant” also comprises annual and perennial dicotyledonous or monocotyledonous plants and includes by way of example, but not by limitation, those of the genera Bromus, Asparagus, Pennisetum, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum and Saccharum.
In a preferred embodiment, the method according to the invention is applied to monocotyledonous plants, for example from the family Poaceae, especially preferably to the genera Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum and Saccharum, very especially preferably to agriculturally important plants such as, for example, Hordeum vulgare (barley), Triticum aestivum (wheat), Triticum aestivum subsp. spelta (spelt), Triticale, Avena sativa (oats), Secale cereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharum officinarum (sugar cane) or Oryza sativa (rice). Thus, in a preferred embodiment, the expression or activity of the stomatin STM1 protein or polynucleotide is reduced in one of these plants.
“Epidermal tissue” or epidermis means the external tissue layers of the plants. It can be single layered or multiple layered; and there is epidermis-“enriched” gene expression, such as, for example, Cer3, which can act as marker, exists; Hannoufa, A. (1996) Plant J. 10 (3), 459-467.
By “epidermis”, the skilled worker preferably means the predominant dermal tissue of primary aerial plant parts, such as of the shoots, the leaves, flowers, fruits and seeds.
The epidermal cells excrete a water-repellent layer, the cuticle, towards the outside. The roots are surrounded by the rhizodermis, which resembles the epidermis in many ways, but also differs substantially therefrom. The epidermis develops from the outermost layer of the apical meristem. The origin of the rhizodermis, in contrast, is less clear. Phylogenetically speaking, it can be assigned either to the calyptra or to the primary bark, depending on the species. A large number of functions can be ascribed to the epidermis: it protects the plant from dehydration and regulates the transpiration rate. It protects the plant from a wide range of chemical and physical external factors and against feeding animals and attack by parasites. It is involved in the gas exchange, in the secretion of certain metabolites and in the absorption of water. It contains receptors for light and mechanical stimuli. It therefore acts as signal transformer between the environment and the plant. In accordance with the various functions, the epidermis comprises a number of differently differentiated cells. Other aspects are species having specific variants and different organization of the epidermides in the individual parts of a plant. Essentially, it consists of three categories of cells: the “actual” epidermal cells, the cells of the stomata and of the trichomes (Greek: trichoma, hair), which are epidermal appendages with different shapes, structures and functions.
The “actual”, i.e. the least specialized epidermal cells, account for most of the bulk of the cells of the epidermal tissue. In topview, they appear either polygonal (slab or plate shaped) or elongated. The walls between them are often wavy or sinuate. It is not known what induces this shape during development; existing hypotheses only offer unsatisfactory explanations herefor. Elongated epidermal cells can be found in organs or parts of organs that are elongated themselves, thus, for example, in stems, petioles, leaf veins and on the leaves of most monocots. The upper surface and undersurface of laminae can be covered in epidermides with different structures, it being possible for the shape of the cells, the wall thickness and the distribution and number of specialized cells (stomata and/or trichomes) per unit area to vary. A high degree of variation is also found within individual families, for example in the Crassulaceae. In most cases, the epidermis consists of a single layer, though multi-layered water-storing epidermides have been found among species from a plurality of families (Moraceae: most Ficus species; Piperaceae: Peperonia, Begoniaceae, Malvaceae and the like). Epidermal cells secrete a cuticle to the outside which covers all epidermal surfaces as an uninterrupted film. It may either be smooth or structured by bulges, rods, folds and furrows. However, the folding of the cuticle, which can be observed when viewing the surface, is not always caused by the formation of cuticular rods. Indeed, there are cases where cuticular folding is merely the expression of the underlying bulges of the cell wall. Epidermal appendages of various form, structure and function are referred to as trichomes and, in the present context, likewise come under the term “epidermis”. They occur in the form of protective hairs, supportive hairs and gland hairs in the form of scales, different papillae and, in the case of roots, as absorbent hairs. They are formed exclusively by epidermal cells. Frequently, a trichome is formed by only one such cell, however, occasionally, more than one cell is involved in its formation.
The term “epidermis” likewise comprises papillae. Papillae are bulges of the epidermal surface. The textbook example thereof is the papillae on flower surfaces of the pansy (Viola tricolor) and the leaf surfaces of many species from tropical rain forests. They impart a velvet-like consistency to the surface. Some epidermal cells can form water stores. A typical example is the water vesicles at the surfaces of many Mesembryanthemum species and other succulents. In some plants, for example in the case of campanula (Campanula persicifolia), the outer walls of the epidermis are thickened like a lens.
The main biomass of all tissues is the parenchyma. The parenchymatic tissues include the mesophyll which, in leaves, can be differentiated into palisade parenchyma and spongy parenchyma. Accordingly the skilled worker understands, by mesophyll, a parenchymatic tissue. Parenchymatic cells are always alive, in most cases isodiametric, rarely elongated. The pith of the shoots, the storage tissues of the fruits, seeds, the root and other underground organs are also to be considered as parenchymas, as is the mesophyll. “Mesophyll tissue” means the foliar tissue between the epidermal layers, and consists of palisade tissue, spongy tissue and the vascular bundles of the leaf.
In the leaves of most ferns and phanerogams, especially in the case of the dicots and many monocots, the mesophyll is subdivided into palisade parenchymas and spongy parenchymas. A “typical” leaf is of dorsiventral organization. In most cases, the palisade parenchyma is at the upper surface of the leaf immediately underneath the epidermis. The spongy parenchyma fills the underlying space. It is interspersed by a voluminous intercellular system whose gas space is in direct contact with the external space via the stomata.
The palisade parenchyma consists of elongated cylindrical cells. In some species, the cells are irregular, occasionally bifurcate (Y-shaped: arm palisade parenchyma). Such variants are found in ferns, conifers and a few angiosperms (for example in some Ranunculaceae and Caprifoliaceae species [example: elder]). Besides the widest-spread organization form which has just been described, the following variants have been found:
palisade parenchyma at the leaf undersurface. Particularly conspicuously in scaly leaves. (For example arbor vitae (thuja), and in the leaves of wild garlic (Allium ursinum).
Palisade parenchyma at both leaf surfaces (upper surface and undersurface). Frequently found in plants of dry habitats (xerophytes). Example: prickly lettuce (Lactuca serriola);
Ring-shaped closed palisade parenchyma: in cylindrically organized leaves and in needles from conifers.
The variability of the cells of the spongy parenchyma, and the organization of the spongy parenchyma itself, are even more varied than that of the palisade parenchyma. It is most frequently referred to as aerenchyma since it comprises a multiplicity of interconnected intercellular spaces.
The mesophyll may comprise what is known as the assimilation tissue, but the terms mesophyll and assimilation tissue are not to be used synonymously. There are chloroplast-free leaves whose organization differs only to a minor extent from comparable green leaves. As a consequence, they comprise mesophyll, but assimilation does not take place; conversely, assimilation also takes place in, for example, sections of the shoot. Further aids for characterizing epidermis and mesophyll can be found by the skilled worker for example in v. GUTTENBERG, H.: Lehrbuch der Allgemeinen Botanik [Textbook of general botany]. Berlin: Akademie-Verlag 1955 (5th Ed.), HABERLANDT, G.: Physiologische Pflanzenanatomie [Physiological plant anatomy]. Leipzig: W. Engelmann 1924 (6th Ed.); TROLL, W.: Morphologie der Pflanzen [Plant morphology]. Volume 1: Vegetationsorgane [Vegetation organs]. Berlin: Gebr. Borntraeger, 1937; TROLL, W.: Praktische Einführung in die Pflanzenmorphologie [Practical introduction to plant morphology]. Jena: VEB G. Thieme Verlag 1954/1957; TROLL, W., HÖHN, K.: Allgemeine Botanik [General botany]. Stuttgart: F. Enke Verlag, 1973 (4th Ed.)
As a consequence, epidermis or epidermal cells can be characterized in histological or biochemical, including molecular-biochemical, terms. In one embodiment, the epidermis is characterized in biochemical terms. In one embodiment, the epidermis can be characterized by the activity of one or more of the following promoters:
In another embodiment, the epidermis is characterized in that only some of the promoters are active, for example 2, 3, 5 or 7 or more, but at least one of the abovementioned promoters is active. In one embodiment, the epidermis is characterized in that all the abovementioned promoters are active in the tissue or the cell.
In one embodiment, the expression or activity of the stomatin STM1 protein or polynucleotide in the epidermis is reduced by the expression of an inhibitory molecule under the control of an epidermis-specific promoter, in particular under the control of one of the abovementioned promoters. Examples of inhibitory molecules are listed hereinbelow, for example RNAi, antisense-RNA, microRNA, cosuppression, antibodies and other methods which are known to the skilled worker. The epidermis-specific expression of an inhibitory molecule in the epidermis is particularly advantageous for increasing the resistance of a plant to mildew.
As a consequence, mesophyll or mesophyll cells can be characterized in biochemical, including molecular-biological, or histological terms. In one embodiment, the mesophyll is characterized in biochemical terms. In one embodiment, the mesophyll can be characterized by the activity of one or more of the following promoters:
OsPPDK, acc. AC099041.
In another embodiment, the mesophyll is characterized in that only some of the promoters are active, for example 2, 3, 5 or 7 or more, but at least one of the abovementioned promoters is active. In one embodiment, the mesophyll is characterized in that all the abovementioned promoters are active in the tissue or the cell.
In one embodiment, all of the abovementioned promoters are active in the epidermis of a plant which is used or generated in accordance with the invention or of a plant according to the invention in the epidermis and in the mesophyll. In one embodiment, only some of the abovementioned promoters are active, for example 2, 5, 7 or more, but at least one of the promoters enumerated above is in each case active.
In one embodiment, the expression or activity of the stomatin STM1 protein or polynucleotide in the mesophyll is reduced by the expression of an inhibitory molecule under the control of a mesophyll-specific promoter, in particular under the control of one of the abovementioned promoters. Examples of inhibitory molecules are listed hereinbelow, for example RNAi, antisense-RNA, microRNA, cosuppression, antibodies and other methods which are known to the skilled worker. The epidermis-specific expression of an inhibitory molecule in the mesophyll is particularly advantageous for increasing the resistance of a plant to Septoria and/or rusts.
In one embodiment, the expression or activity of the stomatin STM1 protein or polynucleotide in the mesophyll and in the epidermis is reduced by the expression of inhibitory molecules under the control of mesophyll- and/or epidermis-specific promoters, in particular under the control of the abovementioned promoters. Examples of inhibitory molecules are mentioned hereinbelow, for example RNAi, antisense-RNA, antibodies and others.
“Nucleic acids” means biopolymers of nucleotides which are linked with one another via phosphodiester bonds (polynucleotides, polynucleic acids). Depending on the type of sugar in the nucleotides (ribose or deoxyribose), one distinguishes the two classes of the ribonucleic acids (RNA) and the deoxyribonucleic acids (DNA).
The term “crop” means all plant parts obtained by growing plants agriculturally and collected within the harvesting process.
“Resistance” means the preventing, the repressing, the reducing or the weakening of disease symptoms of a plant as the result of infection by a pathogen. The symptoms can be manifold, but preferably comprise those which directly or indirectly lead to an adversely affect on the quality of the plant, on the quantity of the yield, on the suitability for use as feed or foodstuff, or else which make sowing, growing, harvesting or processing of the crop more difficult.
In a preferred embodiment, the following disease symptoms are weakened, reduced or prevented: formation of pustules and hymenia on the surfaces of the affected tissues, maceration of the tissues, spreading necroses of the tissue, accumulation of mycotoxins, for example from Fusarium graminearum or F. culmorum.
“Conferring”, “existing”, “generating” or “increasing” a pathogen resistance means that the defence mechanisms of a certain plant or in a part of a plant, for example in an organ, a tissue, a cell or an organelle, have an increased resistance to one or more pathogens as the result of using the method according to the invention in comparison with a suitable control, for example the wildtype of the plant (“control plant”, “starting plant”), to which the method according to the invention has not been applied, under otherwise identical conditions (such as, for example, climatic conditions, growing conditions, type of pathogen and the like). Preferably, at least the epidermis and/or mesophyll tissue in a plant, or the organs which have an epidermis and/or mesophyll tissue, have an increased resistance to the pathogens. For example, the resistance in the leaves is increased. In one embodiment, the resistance in lemma, palea and/or glume (anther primordium) is increased.
In one embodiment, the activity of the protein according to the invention, stomatin STM1, is therefore reduced in the abovementioned organs and tissues.
In this context, the increased resistance preferably manifests itself in a reduced manifestation of the disease symptoms, where disease symptoms—in addition to the abovementioned adverse effects—also comprises for example the penetration efficiency of a pathogen into the plant or the plant cell, or the proliferation efficiency in or on the same. In this context, the disease symptoms are preferably reduced by at least 10% or at least 20%, especially preferably by at least 40% or 60%, very especially preferably by at least 70% or 80%, most preferably by at least 90% or 95%.
For the purposes of the invention, “pathogen” means organisms whose interactions with a plant lead to the above-described disease symptoms; in particular, pathogens means organisms from the Kingdom Fungi. Preferably, pathogen is understood as meaning a pathogen which penetrates epidermis or mesophyll cells, especially preferably pathogens which penetrate plants via stomata and subsequently penetrate mesophyll cells. Organisms which are preferably mentioned in this context are those from the phyla Ascomycota and Basidiomycota. Especially preferred in this context are the families Blumeriaceae, Pucciniaceae, Mycosphaerellaceae and Hypocreaceae.
Especially preferred are organisms of these families which belong to the genera Blumeria, Puccinia, Fusarium or Mycosphaerella.
Very especially preferred are the species Blumeria graminis, Puccinia triticina, Puccinia striiformis, Mycosphaerella graminicola, Stagonospora nodorum, Fusarium graminearum, Fusarium culmorum, Fusarium avenaceum, Fusarium poae and Microdochium nivale.
However, it is to be assumed that the reduction in the expression of Hvstomatin STM1, its activity or function also brings about a resistance to further pathogens. Changes in the cell wall structure may constitute a basic mechanism of pathogen resistance, as shown, for example, in Jacobs A K et al. (2003) Plant Cell, 15(11):2503-13.
Especially preferred are Ascomycota such as, for example, Fusarium oxysporum (fusarium wilt on tomato), Septoria nodorum and Septoria tritici (glume blotch on wheat), Basidiomycetes such as, for example, Puccinia graminis (stem rust on wheat, barley, rye, oats), Puccinia recondita (leaf rust on wheat), Puccinia dispersa (leaf rust on rye), Puccinia hordei (leaf rust on barley), Puccinia coronata (crown rust on oats).
In one embodiment, the method according to the invention leads to a resistance in
In a preferred embodiment, the invention relates to a stomatin STM1 polypeptide which has the activity shown in the examples.
In one embodiment, a stomatin STM1 protein is understood as meaning a protein with a homology to one of the amino acid sequences shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 15, 16 or 17 or in the figures, for example a stomatin STM1 polypeptide from maize (ZmStomatin STM1) as in SEQ ID NO: 5 and/or from rice (Oryza sativa) as shown in SEQ ID NO: 3ar from A. thaliana as shown in SEQ ID NO: 7, 9, 11 and/or 13, or according to one of the consensus sequences as shown in SEQ ID NO.: 15, 16 or 17, or a functional fragment thereof. In one embodiment, the invention relates to functional equivalents of the abovementioned polypeptide sequences.
“Polypeptide quantity” means for example the number of molecules, or moles, of stomatin STM1 polypeptide molecules in an organism, a tissue, a cell or a cell compartment. “Reducing” the polypeptide quantity means the molar reduction in the number of stomatin STM1 polypeptides, in particular of those shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 15, 16 or 17, in an organism, a tissue, a cell or a cell compartment—for example by one of the methods described hereinbelow—in comparison with a suitable control, for example the wildtype (control plant) of the same genus and species to which this method has not been applied, under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like). The reduction in this context amounts to at least 5%, preferably at least 10% or at least 20%, especially preferably at least 40% or 60%, very especially preferably at least 70% or 80%, most preferably at least 90% or 99%.
The present invention furthermore relates to the generation of a pathogen resistance by reducing the function or activity of a TOMATIN polypeptide comprising the sequences shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 15, 16 or 17 or of a homolog thereof and/or a polypeptide which has a homology of at least 40% with the above, and/or of a functional equivalent of the abovementioned polypeptides.
Homology between two nucleic acid sequences is understood as meaning the identity of the nucleic acid sequence over in each case the entire sequence length, in a preferred embodiment over the entire expressed sequence length, preferably cDNA, yet more preferably over the coding sequence, preferably CDS, which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389ff), setting the following parameters:
For example, a sequence which has at least 80% homology with the sequence SEQ ID NO: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 80% homology.
“Identity between two proteins” is understood as meaning the identity of the amino acids over a specific protein region, preferably the entire length of the protein, in particular the identity which is calculated by comparison with the aid of software, for example the Lasergene software from DNA Star Inc., Madison, Wis. (USA) using the CLUSTAL methods (Higgins et al., 1989), Comput. Appl. Biosci., 5 (2), 151). Homologies can also be calculated with the aid of the Lasergene software from DNA Star Inc., Madison, Wis. (USA) using the CLUSTAL method (Higgins et al., 1989), Comput. Appl. Biosci., 5 (2), 151).
For the alignments shown herein (for example as in
Homology between two polypeptides is preferably understood as meaning the identity of the amino acid sequence over the indicated entire sequence length which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
For example, a sequence which has at least 80% homology at the polypeptide level with the sequence SEQ ID NO: 2 is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set has at least 80% homology.
In a preferred embodiment of the present invention, the stomatin STM1 protein activity, function or polypeptide quantity is reduced in the plant or in a part of the plant, for example in a plant organ, plant tissue, a plant cell or a part of a plant cell, for example a plant-specific organelle.
For example, the stomatin STM1 protein is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
Preferably, the activity of the abovementioned polypeptides is reduced in the epidermal and/or mesophyll cells of a plant as detailed above.
In one embodiment, the activity of stomatin STM1 is reduced in lemma, palea and/or glume.
“Epitope” is understood as meaning the regions of an antigen which determine the specificity of the antibodies (the antigenic determinant). Accordingly, an epitope is the portion of an antigen which actually comes into contact with the antibody. Such antigenic determinants are those regions of an antigen to which the T-cell receptors react and, as a consequence, produce antibodies which specifically bind the antigenic determinant/epitope of an antigen. Accordingly, antigens, or their epitopes, are capable of inducing the immune response of an organism with the consequence of the formation of specific antibodies which are directed against the epitope. Epitopes consist for example of linear sequences of amino acids in the primary structure of proteins, or of complex secondary or tertiary protein structures. A hapten is understood as meaning a epitope which is dissociated from the context of the antigen environment. Although haptens have by definition an antibody directed against them, haptens are, under certain circumstances, not capable of inducing an immune response in an organism, for example after an injection. To this end, haptens are coupled with carrier molecules. An example which may be mentioned is dinitrophenol (DNP), which, after coupling to BSA (bovine serum albumine), has been used for generating antibodies which are directed against DNP. (Bohn, A., König, W. 1982).
Haptens are therefore substances (frequently small molecules) which, while they themselves do not trigger immune response, will indeed trigger such a response when coupled to a large molecular carrier.
The antibodies generated thus also include those which can bind to the hapten as such.
In one embodiment, the present invention relates to an antibody against a polypeptide characterized herein, in particular to a monoclonal antibody which binds a polypeptide which comprises an AA sequence or consists thereof, as shown in the sequences shown in SEQ ID No: 15, 16 or 17.
Antibodies within the scope of the present invention can be used for identifying and isolating polypeptides disclosed in accordance with the invention from organisms, preferably plants, especially preferably monocotyledonous plants. The antibodies can either be monoclonal, polyclonal or synthetic in nature or else consist of antibody fragments such as Fab, Fv or scFv fragments, which are formed by proteolytic degradation. “Single chain” Fv (scFv) fragments are single-chain fragments which, linked via a flexible linker sequence only comprise the variable regions of the heavy and light antibody chains. Such scFv fragments can also be produced as recombinant antibody derivatives. A presentation of such antibody fragments on the surface of filamentous phages makes possible the direct selection, from combinatory phage libraries, of scFv molecules which bind with high affinity.
Monoclonal antibodies can be obtained in accordance with the method described by Köhler and Milstein (Nature 256 (1975), 495).
“Functional equivalents” of a stomatin STM1 protein preferably means those polypeptides which have at least 40% homology with the polypeptides described by the sequences SEQ ID No: 15, 16 or 17 and which have essentially the same properties or function. Preferably, the homology amounts to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more.
The functional equivalence can be determined for example by comparing the phenotypes of test organisms after expression of the polypeptides in question, under the most identical conditions possible, or after reduction of the expression or activity of the polypeptides to be compared, in the source organisms in question.
“Essentially identical properties” of a functional equivalent means above all imparting a pathogen-resistant phenotype or imparting or increasing the pathogen resistance to at least one pathogen when reducing the polypeptide quantity, activity or function of said functional stomatin STM1 protein equivalent in a plant, organ, tissue, part or cells, in particular in epidermal or mesophyll cells of same, preferably measured by the penetration efficiency of a pathogen, as shown in the examples.
“Analogous conditions” means that all basic conditions such as, for example, culture or growth conditions, assay conditions (such as buffers, temperature, substrates, pathogen concentration and the like) between the experiments to be compared are kept identical and that the set-ups only differ by the sequence of the stomatin STM1 polypeptides to be compared, by their source organism and, if appropriate, by the pathogen.
“Functional equivalents” also means natural or artificial mutation variants of the stomatin STM1 polypeptides as shown in SEQ ID No: 15, 16 or 17 and homologous polypeptides from other monocotyledonous and dicotyledonous plants which furthermore have essentially identical properties. Preferred are homologous polypeptides from preferred plants described herein. The sequences from other plants, which sequences are homologous to the stomatin STM1 protein sequences disclosed within the scope of the present invention, can be found readily for example by database search or by screening gene libraries using the stomatin STM1 protein sequences as search sequence or probe.
Functional equivalents can also be derived for example from one of the polypeptides according to the invention as shown in SEQ ID No: 15, or 17 by substitution, insertion or deletion and can have at least 60%, preferably at least 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98% homology with these polypeptides and are distinguished by essentially identical properties to the polypeptides as shown in SEQ ID No: 15, 16 or 17.
Functional equivalents are also nucleic acid molecules which are derived from the nucleic acid sequences according to the invention as shown in SEQ ID No: 1, 3, 5, 7, 9, 11 or 13, 1, 3, 5, 7, 9, 11 or 13 by substitution, insertion or deletion and have at least 60%, preferably 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98% homology with one of the polynucleotides according to the invention as shown in SEQ ID No: or, 3, 5, 7, 9, 11 or 13 and code for polypeptides with essentially identical properties to polypeptides as shown in SEQ ID No: 15, 16 or 17.
Examples of the functional equivalents of the stomatin STM1 proteins as shown in SEQ ID No: 15, or 17 which are to be reduced in the method according to the invention can be found by homology comparisons from databases, from organisms whose genomic sequence is known.
Screening cDNA libraries or genomic libraries of other organisms, preferably of the plant species mentioned further below, which are suitable as transformation hosts, using the nucleic acid sequence described in SEQ ID No: or, 3, 5, 7, 9, 11 or 13 or parts of the same as probe is also a method known to the skilled worker for identifying homologs in other species. In this context, the probes derived from the nucleic acid sequence as shown in SEQ ID No: or 1, 3, 51 or 1 have a length of at least 20 bp, preferably at least 50 bp, especially preferably at least 100 bp, very especially preferably at least 200 bp, most preferably at least 400 bp. The probe can also be one or more kilobases in length, for example 1 kb, 1.5 kb or 3 kb. A DNA strand which is complementary to the sequences described in SEQ ID No: or, or a fragment of same strand with a length of between 20 bp and several kilobases may also be employed for screening the libraries.
In the method according to the invention, those DNA molecules which hybridize under standard conditions with the nucleic acid molecules described by SEQ ID No: or 13 and which code for stomatin STM1 proteins, with the nucleic acid molecules which are complementary to the above or with parts of the above and which, as complete sequences, code for polypeptides which have identical properties to the polypeptides described in SEQ ID No: 15, 16 or 17, 101115, 16 or 17, may also be used.
“Standard hybridization conditions” is to be understood in the broad sense and means, depending on the application, stringent or else less stringent hybridization conditions. Such hybridization conditions are described, inter alia, in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
The skilled worker would choose hybridization conditions which allow him to differentiate between specific and unspecific hybridizations.
For example, the conditions during the wash step can be selected from among low-stringency conditions (with approximately 2×SSC at 50° C.) and high-stringency conditions (with approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Moreover, the temperature during the wash step can be raised from low-stringency conditions at room temperature, approximately 22° C., to higher-stringency conditions at approximately 65° C. The two parameters, salt concentration and temperature can be varied simultaneously or else singly, keeping in each case the other parameter constant. During the hybridization, it is also possible to employ denaturant agents such as, for example, formamide or SDS. In the presence of 50% formamide, the hybridization is preferably carried out at 42° C. Some examples of conditions for hybridization and wash step are detailed hereinbelow:
In one embodiment, the hybridization conditions are selected as follows:
A hybridization buffer comprising formamide, NaCl and PEG 6000 is chosen. The presence of formamide in the hybridization buffer destabilizes double-strand nucleic acid molecules, whereby the hybridization temperature can be lowered to 42° C. without thereby reducing the stringency. The use of salt in the hybridization buffer increases the renaturation rate of a duplex, in other words the hybridization efficiency. Although PEG increases the viscosity of the solution, which has a negative effect on the renaturation rates, the presence of the polymer in the solution increases the concentration of the probe in the remaining medium, which increases the hybridization rate. The composition of the buffer is as follows:
The hybridizations are carried out overnight at 42° C. On the following morning, the filters are washed 3× with 2×SSC+0.1% SDS for in each case approximately 10 minutes.
In a further preferred embodiment of the present invention, an increase in the resistance in the method according to the invention is achieved by
“Gene expression” and “expression” are to be understood as being synomymous and mean the realization of the information which is stored in a nucleic acid molecule. Reducing the expression of a gene therefore comprises the reduction of the polypeptide quantity of the encoded protein, for example of the stomatin STM1 polypeptide or of the stomatin STM1 protein function. The reduction of the gene expression of a stomatin STM1 protein gene can be realized in many different ways, for example by one of the methods listed hereinbelow.
“Reduction”, “reducing” or “to reduce” in the context of a stomatin STM1 protein or stomatin STM1 protein function is to be interpreted in the broad sense and comprises the partial or essentially complete inhibition or blockage of the functionality of a stomatin STM1 polypeptide in a plant or a part, tissue, organ, cells or seeds derived therefrom, based on different cell-biological mechanisms.
Reducing within the meaning of the invention also comprises a quantitive reduction of a stomatin STM1 polypeptide down to an essentially complete absence of the stomatin STM1 polypeptide (i.e. lack of detectability of stomatin STM1 protein function or lack of immunological detectability of the stomatic STM1 protein). Here, the expression of a certain stomatin STM1 polypeptide or the stomatin STM1 protein function in a cell or an organism is preferably reduced by more than 50%, especially preferably by more than 80%, very especially preferably by more than 90%, in comparison with a suitable control, i.e. to the wildtype of the same type, for example of the same genus, species, variety, cultivar and the like (“control plants”), to which this method has not been applied, under otherwise identical conditions (such as, for example, culture conditions, age of the plants and the like).
In accordance with the invention, there are described various strategies for reducing the expression of a stomatin STM1 protein or a stomatin STM1 protein function. The skilled worker recognizes that a series of further methods is available for influencing the expression of a stomatin STM1 polypeptide or of the stomatin STM1 protein function in the desired manner.
In one embodiment, a reduction in the stomatin STM1 protein function is achieved in the method according to the invention by applying at least one method selected from the group consisting of:
These and modified methods, and further methods, are known to the skilled worker and extensively described, for example also in PCT/EP2005/003297, which is explicitly included herein by reference with regard to these methods.
Each one of these methods can bring about a reduction in the stomatin STM1 protein expression or stomatin STM1 protein function for the purposes of the invention. A combined use is also feasible. Further methods are known to the skilled worker and can comprise the hindering or prevention of the processing of the stomatin STM1 polypeptide, of the transport of the stomatin STM1 polypeptide or its mRNA, inhibition of the ribosome attachment, inhibition of the RNA splicing, induction of a stomatin-STM1-protein-RNA-degrading enzyme and/or inhibition of the translational elongation or termination.
A reduction in the stomatin STM1 protein function or stomatin STM1 polypeptide quantity is preferably achieved by a reduced expression of an endogenous stomatin STM1 protein gene.
The individual preferred processes shall be described briefly hereinbelow:
The method of regulating genes by means of double-stranded RNA (“double-stranded RNA interference”; dsRNAi) has been described many times for animal and plant organisms (e.g. Matzke M A et al. (2000) Plant Mol Biol 43:401-415; Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). Efficient gene suppression can also be demonstrated in the case of transient expression, or following the transient transformation, for example as the result of a biolistic transformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903). dsRNAi processes are based on the phenomenon that simultaneously introducing the complementary strand and counterstrand of a gene transcript suppresses the expression of the corresponding gene in a highly efficient manner. The phenotype caused is very similar to that of a corresponding knock-out mutant (Waterhouse P M et al. (1998) Proc Natl Acad Sci USA 95:13959-64).
The dsRNAi method has proved to be particularly efficient and advantageous when reducing the stomatin STM1 protein expression (WO 99/32619).
With regard to the double-stranded RNA molecules, stomatin STM1 protein nucleic acid sequence preferably means one of the sequences as shown in SEQ ID No: 1, 3, 5, 7, 9, 11 or 13, or coding for a consensus sequence as shown in SEQ ID NO.: 15, 16 or 17, or sequences which are essentially identical to those, preferably which have at least 50%, 60%, 70%, 80% or 90% or more identity to these, for example approximately 95%, 96%, 97%, 98%, 99% or more identity to these, or fragments of these with a length of at least 17 base pairs. “Essentially identical” means here that the dsRNA sequence may also have insertions, deletions and individual point mutations in comparison with the stomatin STM1 protein target sequence while still bringing about an efficient reduction in the expression. In one embodiment, the homology as defined above is at least 50%, for example approximately 80%, or approximately 90%, or approximately 100%, between the “sense” strand of an inhibitory dsRNA and a subsection of a stomatin STM1 protein nucleic acid sequence (or between the “antisense” strand and the complementary strand of a stomatin STM1 protein nucleic acid sequence). The length of the subsection is approximately 17 bases or more, for example approximately 25 bases, or approximately 50 bases, approximately 100 bases, approximately 200 bases or approximately 300 bases. Alternatively, an “essentially identical” dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing under stringent conditions with a part of a stomatin STM1 protein gene transcript.
The “antisense” RNA strand, too, can have insertions, deletions and individual point mutations in comparison with the complement of the “sense” RNA strand. The homology is preferably at least 80%, for example approximately 90%, or approximately 95%, or approximately 100%, between the “antisense” RNA strand and the complement of the “sense” RNA strand.
“Subsection of the “sense” RNA transcript” of a nucleic acid molecule coding for a stomatin STM1 polypeptide or a functional equivalent thereof means fragments of an RNA or mRNA transcribed by a nucleic acid molecule coding for a stomatin STM1 polypeptide or a functional equivalent thereof, preferably by a stomatin STM1 protein gene. In this context, the fragments preferably have a sequence length of approximately 20 bases or more, for example approximately 50 bases, or approximately 100 bases, or approximately 200 bases, or approximately 500 bases. Also comprised is the complete transcribed RNA or mRNA.
The dsRNA can consist of one or more strands of polymerized ribonucleotides. Modifications both of the sugar-phosphate backbone and of the nucleosides may also be present. For example, the phosphodiester bonds of the natural RNA can be modified in such a way that they comprise at least one nitrogen or sulfur heteroatom. Bases can be modified in such a way that the activity of, for example, adenosin deaminase is restricted. Such and further modifications are described hereinbelow in the methods of stabilizing antisense RNA.
To achieve the same purpose, it is, of course, also possible to introduce, into the cell or the organism, a plurality of individual dsRNA molecules, each of which comprises one of the above-defined ribonucleotide sequence segments.
The dsRNA can be prepared enzymatically or fully or partially by chemical synthesis.
If the two strands of the dsRNA are to be combined in one cell or plant, this can be accomplished in various ways:
The formation of the RNA duplex can be initiated either externally or internally of the cell. As described in WO 99/53050, the dsRNA can also comprise a hairpin structure, by linking “sense” and “antisense” strand by means of a “linker” (for example an intron). The autocomplementary dsRNA structures are preferred since they only require the expression of a construct and always comprise the complementary strands in an equimolar ratio.
The expression cassettes coding for the “antisense” or “sense” strand of a dsRNA or for the autocomplementary strand of the dsRNA are preferably inserted into a vector and stably (for example using selection markers) inserted into the genome of a plant using the methods described hereinbelow in order to ensure permanent expression of the dsRNA.
The dsRNA can be introduced using a quantity which makes possible at least one copy per cell. Higher quantities (for example at least 5, 10, 100, 500 or 1000 copies per cell) can make, if appropriate, a more efficient reduction.
In order to bring about an efficient reduction in the stomatin STM1 protein expression, 100% sequence identity between dsRNA and a stomatin STM1 protein gene transcript or the gene transcript of a functionally equivalent gene is not necessarily required. Accordingly, there is the advantage that the method tolerates sequence deviations as they can exist as the result of genetic mutations, polymorphisms or evolutionary divergences. The large number of highly conserved amino acid residues between different stomatin STM1 protein sequences of different plants, as shown in the figures with reference to the consensus sequences, allows the conclusion that this polypeptide is highly conserved within plants, so that the expression of a dsRNA derived from one of the disclosed stomatin STM1 protein sequences as shown in SEQ ID No: or should also have an advantageous effect in other plant species.
As the result of the high number of conserved residues and of the homology between the individual stomatin STM1 polypeptides and their functional equivalents, it may also be possible to suppress the expression of further homologous stomatin STM1 polypeptides and/or their functional equivalents of the same organism, or else the expression of stomatin STM1 polypeptides in other, related species, using a single dsRNA sequence which has been generated starting from a specific stomatin STM1 protein sequence of an organism. For this purpose, the dsRNA preferably comprises sequence regions of stomatin STM1 protein gene transcripts which correspond to conserved regions. Said conserved regions can be derived readily from sequence alignments, for example as shown in the figures. It is preferred to derive dsRNA sequences from the conserved regions of the consensus sequence which are shown in the figures.
A dsRNA can be synthesized chemically or enzymatically. To this end, it is possible to use cellular RNA polymerases or bacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNA polymerase). Suitable methods for the in vitro expression of RNA are described (WO 97/32016; U.S. Pat. No. 5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S. Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). A dsRNA which has been synthetized chemically or enzymatically in vitro can be purified from the reaction mixture fully or in part, for example by extraction, precipitation, electrophoresis, chromatography or combinations of these methods, before it is introduced into a cell, tissue or organism. The dsRNA can be introduced into the cell directly or else applied extracellularly (for example into the interstitial space).
However, it is preferred to transform the plant stably with an expression construct which realizes the expression of the dsRNA. Suitable methods are described hereinbelow.
Methods of suppressing a certain polypeptide by preventing the accumulation of its mRNA by means of the “antisense” technology have been described many times, including in plants (Sheehy et al. (1988) Proc Natl Acad Sci USA 85: 8805-8809; U.S. Pat. No. 4,801,340; Mol J N et al. (1990) FEBS Lett 268(2):427-430). The antisense nucleic acid molecule hybridizes with, or binds to, the cellular mRNA and/or genomic DNA coding for the callose synthase target polypeptide to be suppressed. The transcription and/or translation of the target polypeptide is thereby suppressed. The hybridization can be accomplished in a traditional manner via the formation of a stable duplex or, in the case of genomic DNA, by binding the antisense nucleic acid molecule to the duplex of the genomic DNA as the result of specific interaction in the large groove of the DNA helix.
An antisense nucleic acid molecule suitable for reducing a stomatin STM1 polypeptide can be derived using the nucleic acid sequence which codes for this polypeptide, for example the nucleic acid molecule according to the invention as shown in SEQ ID No: oror a nucleic acid molecule coding for a functional equivalent thereof following Watson's and Crick's base-pairing rules. The antisense nucleic acid molecule can be complementary to all of the transcribed mRNA of the said polypeptide, be limited to the coding region or else only consist of an oligonucleotide which is complementary to part of the coding or noncoding sequence of the mRNA. Thus, for example, the oligonucleotide can be complementary to the region which comprises the translation start for said polypeptide. Antisense nucleic acid molecules can have a length of, for example, 20, 25, 30, 35, 40, 45 or 50 nucleotides, but they may also be longer and comprise 100, 200, 500, 1000, 2000 or 5000 nucleotides. Antisense nucleic acid molecules can be expressed recombinantly or synthesized chemically or enzymatically, using methods known to the skilled worker. In the case of chemical synthesis, natural or modified nucleotides can be used. Modified nucleotides can impart an increased biochemical stability to the antisense nucleic acid molecule and lead to an increased physical stability of the duplex formed of antisense nucleic acid sequence and sense target sequence. Examples which can be used are phosphoro-thioate derivatives and acridine-substituted nucleotides such as 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylamino-methyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil and 2,6-diaminopurine.
In a further preferred embodiment, the expression of a stomatin STM1 polypeptide can be inhibited by nucleic acid molecules which are complementary to a conserved region (for example a region which has been conserved as described above) or to a regulatory region of a stomatin STM1 protein gene (for example a stomatin STM1 protein promoter and/or enhancer) and which form triple-helical structures with the DNA double helix therein, so that the transcription of the stomatin STM1 protein gene is reduced. Suitable methods have been described (Helene C (1991) Anticancer Drug Res 6(6):569-84; Helene C et al. (1992) Ann NY Acad Sci 660:27-36; Maher L J (1992) Bioassays 14(12):807-815).
In a further embodiment, the antisense nucleic acid molecule can be an α-anomeric nucleic acid. Such α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which—as opposed to the conventional β-nucleic acids—the two strands run in parallel with one another (Gautier C et al. (1987) Nucleic Acids Res 15:6625-6641). The antisense nucleic acid molecule can furthermore also comprise 2′-O-methylribonucleotides (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett 215:327-330).
Catalytic RNA molecules or ribozymes can be adapted to any target RNA and cleave the phosphodiester backbone at specific positions, whereby the target RNA is functionally deactivated (Tanner N K (1999) FEMS Microbiol Rev 23(3):257-275). As the result, the ribozyme is not modified itself, but is capable of cleaving further target RNA molecules in an analogous manner, whereby it obtains the characteristics of an enzyme.
In this manner, it is possible to use ribozymes (for example hammerhead ribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591) in order to cleave the mRNA of an enzyme to be suppressed, for example callose synthases, and to prevent translation. Methods of expressing ribozymes for reducing certain polypeptides are described in (EP 0 291 533, EP 0 321 201, EP 0 360 257). A ribozyme expression has also been described in plant cells (Steinecke P et al. (1992) EMBO J. 11 (4):1525-1530; de Feyter R et al. (1996) Mol Gen Genet 250(3):329-338). Ribozymes can be identified from a library of various ribozymes via a selection process (Bartel D and Szostak J W (1993) Science 261:1411-1418). Preferably, the binding regions of the ribozyme hybridize with the conserved regions of the stomatin STM1 protein as described above.
The above-described antisense strategy can advantageously be coupled with a ribozyme method. The incorporation of ribozyme sequences into “antisense” RNAs imparts this enzyme-like, RNA-cleaving characteristic to precisely these antisense RNAs and thus increases their efficiency in the inactivation of the target RNA. The preparation and use of suitable ribozyme “antisense” RNA molecules is described, for example, in Haselhoff et al. (1988) Nature 334: 585-591.
The ribozyme technology can increase the efficiency of an antisense strategy. Suitable target sequences and ribozymes can be determined for example as described in “Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds, Academic Press, Inc. (1995), p. 449-460”, by calculating the secondary structure of ribozyme RNA and target RNA and by their interaction (Bayley C C et al. (1992) Plant Mol. Biol. 18(2):353-361; Lloyd A M and Davis R W et al. (1994) Mol Gen Genet 242(6):653-657). For example, it is possible to construct derivatives of the Tetrahymena L-19 IVS RNA which derivatives have complementary regions to the mRNA of the stomatin STM1 protein to be suppressed (see also U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742).
The expression of a stomatin STM1 protein nucleic acid sequence in sense orientation can lead to a cosuppression of the corresponding homologous, endogenous gene. The expression of sense RNA with homology to an endogenous gene can reduce or cancel the expression of the former, similar to what has been described for antisense approaches (Jorgensen et al. (1996) Plant Mol Biol 31(5):957-973; Goring et al. (1991) Proc Natl Acad Sci USA 88:1770-1774; Smith et al. (1990) Mol Gen Genet 224:447-481; Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol et al. (1990) Plant Cell 2:291-99). Here, the construct introduced can represent the homologous gene to be reduced either fully or only in part. The possibility of translation is not required. The application of this technology to plants is described for example in Napoli et al. (1990) The Plant Cell 2: 279-289 and in U.S. Pat. No. 5,034,323.
The cosuppression is preferably realized using a sequence which is essentially identical to at least part of the nucleic acid sequence coding for a stomatin STM1 protein or a functional equivalent thereof, for example of the nucleic acid molecule according to the invention, for example of the nucleic acid sequence as shown in SEQ ID No: or 13, or of the nucleic acid sequence coding for a functional equivalent thereof.
The activity of a stomatin STM1 protein can probably also be realized by expression of a dominant-negative variant of this stomatin STM1 protein. Methods of reducing the function or activity of a polypeptide by means of coexpression of its dominant-negative form are known to the skilled worker (Lagna G and Hemmati-Brivanlou A (1998) Current Topics in Developmental Biology 36:75-98; Perlmutter R M und Alberola-IIa J (1996) Current Opinion in Immunology 8(2):285-90; Sheppard D (1994) American Journal of Respiratory Cell & Molecular Biology. 11 (1):1-6; Herskowitz 1 (1987) Nature 329(6136):219-22).
A dominant-negative stomatin STM1 protein variant can be accomplished for example by altering amino acid residues which are part of the stomatin STM1 and, as the result of their mutation, the polypeptide loses its function. Amino acid residues which are preferably to be mutated are those which are conserved in the stomatin STM1 proteins of different organisms. Such conserved regions can be determined for example by means of computer-aided comparison (“alignment”). These mutations for obtaining a dominant-negative stomatin STM1 protein variant are preferably carried out at the level of the nucleic acid sequence coding for stomatin STM1 proteins. A suitable mutation can be realized for example by PCR-mediated in vitro mutagenesis using suitable oligonucleotide primers, by means of which the desired mutation is introduced. Methods which are known to the skilled worker are used for this purpose. For example, the “LA PCR in vitro Mutagenesis Kit” (Takara Shuzo, Kyoto) can be used for this purpose.
A reduction of a stomatin STM1 protein gene expression is also possible using specific DNA-binding factors, for example using factors of the zinc finger transcription factor type. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about a repression of the endogenous gene. The use of such a method makes possible the reduction of the expression of an endogenous stomatin STM1 protein gene without it being necessary to recombinantly manipulate the sequence of the latter. Suitable methods for the preparation of suitable factors are described (Dreier B et al. (2001) J Biol Chem 276(31):29466-78; Dreier B et al. (2000) J Mol Biol 303(4):489-502; Beerli R R et al. (2000) Proc Natl Acad Sci USA 97 (4):1495-1500; Beerli R R et al. (2000) J Biol Chem 275(42):32617-32627; Segal D J and Barbas C F 3rd. (2000) Curr Opin Chem Biol 4(1):34-39; Kang J S and Kim J S (2000) J Biol Chem 275(12):8742-8748; Beerli R R et al. (1998) Proc Natl Acad Sci USA 95(25):14628-14633; Kim J S et al. (1997) Proc Natl Acad Sci USA 94(8):3616-3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai S Y et al. (1998) Adv Drug Deliv Rev 30(1-3):23-31; Mapp A K et al. (2000) Proc Natl Acad Sci USA 97(8):3930-3935; Sharrocks A D et al. (1997) mnt J Biochem Cell Biol 29(12):1371-1387; Zhang L et al. (2000) J Biol Chem 275(43):33850-33860).
The selection of these factors can be accomplished using a suitable portion of a stomatin STM1 protein gene. This segment is preferably located in the region of the promoter region. However, for the purpose of suppressing a gene, it may also be located in the region of the coding exons or introns. The corresponding segments are obtainable for the skilled worker by means of database search from the gene library or, starting from a stomatin STM1 protein cDNA whose gene is not present in the gene library, by screening a genomic library for corresponding genomic clones. The methods required for this purpose are known to the skilled worker.
Furthermore, it is possible to introduce, into a cell, factors which themselves inhibit the stomatin STM1 protein target polypeptide. The polypeptide-binding factors can be, for example, aptamers (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibodies or antibody fragments. The preparation of these factors is described and known to the skilled worker. For example, a cytoplasmic scFv antibody has been employed for modulating the activity of the phytochrome A protein in recombinantly modified tobacco plants (Owen M et al. (1992) Biotechnology (N Y) 10(7):790-794; Franken E et al. (1997) Curr Opin Biotechnol 8(4):411-416; Whitelam (1996) Trend Plant Sci 1:286-272).
Gene expression can also be suppressesd by customized, low-molecular-weight synthetic compounds, for example of the polyamide type (Dervan P B and Burli R W (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld J M et al. (2000) Gene Expr 9(1-2):77-91). These oligomers consist of the units 3-(dimethylamino)propylamine, N-methyl-3-hydroxypyrrole, N-methylimidazole and N-methylpyrrole and can be adapted to each segment of double-stranded DNA in such a way that they bind into the major group in a sequence-specific fashion and block the expression of the gene sequences therein. Suitable methods are described (see, inter alia, Bremer R E et al. (2001) Bioorg Med. Chem. 9(8):2093-103; Ansari A Z et al. (2001) Chem. Biol. 8(6):583-92; Gottesfeld J M et al. (2001) J Mol. Biol. 309(3):615-29; Wurtz N R et al. (2001) Org Lett 3(8):1201-3; Wang C C et al. (2001) Bioorg Med Chem 9(3):653-7; Urbach A R and Dervan P B (2001) Proc Natl Acad Sci USA 98(8):4343-8; Chiang S Y et al. (2000) J Biol. Chem. 275(32):24246-54).
The stomatin STM1 protein expression can also be realized efficiently by induction of the specific stomatin STM1 protein RNA degradation by the plant with the aid of a viral expression system (Amplikon) (Angell, S M et al. (1999) Plant J. 20(3):357-362). These systems—also referred to as “VIGS” (viral-induced gene silencing)—introduce, by means of viral vectors, nucleic acid sequences with homology to the transcripts to be suppressed into the plant. Transcription is then cancelled, probably mediated by plant defence mechanisms against viruses. Suitable techniques and methods are described (Ratcliff F et al. (2001) Plant J 25(2):237-45; Fagard M and Vaucheret H (2000) Plant Mol Biol 43(2-3):285-93; Anandalakshmi R et al. (1998) Proc Natl Acad Sci USA 95(22):13079-84; Ruiz M T (1998) Plant Cell 10(6): 937-46).
The methods of the dsRNAi, of cosuppression by means of sense RNA and of “VIGS” (“virus-induced gene silencing”) are also referred to as “post-transcriptional gene silencing” (PTGS). PTGS methods are particularly advantageous because the demands for the homology between the endogenous gene to be suppressed and the recombinantly expressed sense or dsRNA nucleic acid sequence are less stringent than, for example, in a traditional antisense approach. Suitable homology criteria are mentioned in the description of the dsRNAI method and can generally be applied to PTGS methods or dominant-negative approaches. As the result of the high degree of homology between the stomatin STM1 proteins from maize, wheat, rice and barley, it can be concluded that this polypeptide is highly conserved in plants. Thus, it is probably also possible, using the stomatin STM1 protein nucleic acid molecules as they are shown herein, in particular by means of the nucleic acid molecules which are derived from the consensus sequences, or else for example from the nucleic acid molecules from Arabidopsis, barley, maize or rice, also efficiently to suppress the expression of homologous stomatin STM1 polypeptides in other species without the isolation and structure elucidation of the stomatin STM1 protein homologs found in these species being compulsory. This substantially simplifies the labor required.
To generate a homologously-recombinant organism with reduced stomatin STM1 protein function, one uses for example a nucleic acid construct which comprises at least part of an endogenous stomatin STM1 protein gene which is modified by a deletion, addition or substitution of at least one nucleotide, for example in the conserved regions, in such a way that the functionality is reduced or entirely nullified.
For example, the primary, secondary, tertiary or quaternary structure can be disrupted, for example in such a manner that the binding ability, or regulatory ability, of the cytoplasmic protein domain or the integration of the protein into the membrane no longer exists or is disrupted, in particular reduced. Such a disruption can be accomplished for example by the mutation of one or more residues which are indicated in the consensus sequence as being conserved or highly conserved.
The modification can also relate to the regulatory elements (for example the promoter) of the gene, so that the coding sequence remains unaltered, but that expression (transcription and/or translation) does not take place and is reduced.
In the case of conventional homologous recombination, the modified region is flanked at its 5′ and 3′ terminus by further nucleic acid sequences which must be of sufficient length for making possible the recombination. As a rule, the length is in the range of from several hundred or more bases up to several kilobases (Thomas K R and Capecchi M R (1987) Cell 51:503; Strepp et al. (1998) Proc Natl Acad Sci USA 95(8):4368-4373). To carry out the homologous recombination, the host organism—for example a plant—is transformed with the recombination construct using the methods described hereinbelow, and clones which have undergone successful recombination are selected using for example a resistance to antibiotics or herbicides.
Further suitable methods for reducing the stomatin STM1 protein function are the introduction of nonsense mutations into endogenous stomatin STM1 protein genes, for example by means of generation of knockout mutants with the aid of, for example, T-DNA mutagenesis (Koncz et al. (1992) Plant Mol Biol 20(5):963-976), ENU (N-ethyl-N-nitrosourea)-mutagenesis or homologous recombination (Hohn B and Puchta (1999) H Proc Natl Acad Sci USA 96:8321-8323.) or EMS mutagenesis (Birchler J A, Schwartz D. Biochem Genet 1979 December; 17(11-12):1173-80; Hoffmann G R. Mutat Res. 1980 January; 75(1):63-129). Point mutations can also be generated by means of DNA-RNA hybrid oligonucleotides, which are also known as “chimeraplasty” (Zhu et al. (2000) Nat Biotechnol 18(5):555-558, Cole-Strauss et al. (1999) Nucl Acids Res 27(5):1323-1330; Kmiec (1999) Gene therapy American Scientist 87(3):240-247).
The cell- or tissue-specific reduction in the activity of a stomatin STM1 can be effected for example by expressing a suitable construct, which, for example, an abovementioned nucleic acid molecule, for example the antisense RNA, dsRNA, RNAi, ribozymes, with a suitable tissue-specific promoter, for example a promoter as described herein as being specific for epidermis or mesophyll.
For the purposes of the present invention, “mutations” means the modification of the nucleic acid sequence of a gene variant in a plasmid or in the genome of an organism. Mutations can arise for example as the result of errors in the replication, or they can be caused by mutagens. While the spontaneous mutation rate in the cell genome of organisms is very low, the skilled worker is familiar with a multiplicity of biological, chemical or physical mutagens.
Mutations comprise substitutions, additions, deletions of one or more nucleic acid residues. Substitutions are understood as meaning the exchange of individual nucleic acid bases; one distinguishes between transitions (substitution of a purine base for a purine base, or of a pyrimidine base for a pyrimidine base) and transversions (substitution of a pyrimidine base for a purine base (or vice versa)).
Additions or insertions are understood as meaning the incorporation of additional nucleic acid residues into the DNA, it being possible to result in reading-frame shifts. In the case of such reading-frame shifts, one distinguishes between “in-frame” insertions/additions and “out-of-frame” insertions. In the case of the “in-frame” insertions/additions, the reading frame is retained, and a polypeptide which is enlarged by the number of the amino acids encoded by the inserted nucleic acids results. In the case of “out-of-frame” insertions/additions, the original reading frame is lost, and the formation of a complete and functional polypeptide is no longer possible.
Deletions describe the loss of one or more base pairs, which likewise lead to “in-frame” or “out-of-frame” reading-frame shifts and the consequences which this entails regarding the formation of an intact protein.
The mutagenic agents (mutagens) which can be used for generating random or site-specific mutations, and the methods and techniques which can be applied, are known to the skilled worker. Such methods and mutagens are described for example in A. M. van Harten [(1998), “Mutation breeding: theory and practical applications”, Cambridge University Press, Cambridge, UK], E Friedberg, G Walker, W Siede [(1995), “DNA Repair and Mutagenesis”, Blackwell Publishing], or K. Sankaranarayanan, J. M. Gentile, L. R. Ferguson [(2000) “Protocols in Mutagenesis”, Elsevier Health Sciences].
Usual molecular-biological methods and processes, such as the in vitro mutagenesis kit, LA PCR in vitro Mutagenesis Kit (Takara Shuzo, Kyoto), or PCR mutageneses using suitable primers may be employed for introducing site-specific mutations.
As has already been mentioned above, a multiplicity of chemical, physical and biological mutagens exists.
Those mentioned hereinbelow are given by way of example, but not by limitation.
Chemical mutagens can be distinguished by their mechanism of action. Thus, there are base analogs (for example 5-bromouracil, 2-aminopurine), mono- and bifunctional alkylating agents (for example monofunctional agents such as ethylmethylsulfonate, dimethyl sulfate, or bifunctional agents such as dichloroethyl sulfite, mitomycin, nitrosoguanidine-dialkylnitrosamine, N-nitrosoguanidine derivatives) or intercalating substances (for example acridine, ethidium bromide).
Physical mutagens are, for example, ionizing radiation. Ionizing radiation is electromagnetic waves or particle radiation capable of ionizing molecules, i.e. of removing electrons from the latter. The remaining ions are highly reactive in most cases, so that, if they are generated in live tissue, are capable of causing great damage, for example to the DNA, and (at low intensity) thereby inducing mutations. Ionizing radiation is, for example, gamma-radiation (photo energy of approximately one megaelectron volt MeV), X-rays (photo energy of a plurality of or many kiloelectron volts keV) or else ultraviolet light (UV light, photon energy of above 3.1 eV). UV light causes the formation of dimers between bases; with thymidine dimers, which give rise to mutations, being the most frequent here.
The traditional generation of mutants by treating the seeds with mutagenic agents such as, for example, ethylmethylsulfonate (EMS) (Birchler J A, Schwartz D. Biochem Genet 1979 December; 17(11-12):1173-80; Hoffmann G R. Mutat Res. 1980 January; 75(1):63-129) or ionizing radiation has been joined by the use of biological mutagens, for example transposons (for example Tn5, Tn903, Tn916, Tn1000, Balcells et al., 1991, May B P et al. (2003) Proc Natl Acad Sci USA. September 30; 100(20):11541-6.) or molecular-biological methods such as the mutagenesis by means of T-DNA insertion (Feldman, K. A. Plant J. 1:71-82.1991, Koncz et al. (1992) Plant Mol Biol 20(5):963-976).
The use of chemical or biological mutagens is preferred for the generation of mutated gene variants. In the case of chemical agents, the generation of mutants by application of EMS (ethylmethylsulfonate) mutagenesis is mentioned by particular preference. In the case of the generation of mutants using biological mutagenesis, the T-DNA mutagenesis or transposon mutagenesis may be mentioned by preference.
Thus, it is also possible to employ those polypeptides for the method according to the invention which are obtained as the result of a mutation of a polypeptide according to the invention, for example as shown in SEQ ID No: 15, 16 or 17.
All substances and compounds which directly or indirectly bring about a reduction in the polypeptide quantity, RNA quantity, gene activity or polypeptide activity of a stomatin STM1 protein will hereinbelow be summarized under the term “anti-stomatin STM1 protein compounds”. The term “anti-stomatin STM1 protein compound” explicitly includes the nucleic acid sequences, peptides, proteins or other factors which are employed in the above-described methods.
In a further preferred embodiment of the present invention, an increase in the resistance to pathogens from the families Blumeriaceae, Pucciniaceae, Mycosphaerellaceae and Hypocreaceae in a monocotyledonous or dicotyledonous plant or an organ, tissue or a cell thereof, is obtained by:
For example, regarding a nucleic acid sequence, an expression cassette or a vector comprising said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, “transgenic” means all those constructs or organisms which are the result of recombinant methods and in which either
For the purposes of the invention, “introduction” comprises all those methods which are suitable for introducing an “anti-stomatin STM1 protein compound” directly or indirectly into a plant or into a cell, compartment, tissue, organ or seeds thereof, or for generating such a compound therein. It comprises direct and indirect methods. The introduction can lead to a transient presence of one “anti-stomatin STM1 protein compound” (for example of a dsRNA) or else to a stable presence.
As the result of the differing nature of the above-described approaches, the “anti-stomatin STM1 protein compound” can exert its function directly (for example by insertion into an endogenous stomatin STM1 protein gene). However, the function can also be exerted indirectly after transcription into an RNA (for example in the case of antisense approaches) or after transcription and translation into a protein (for example in the case of binding factors). Both direct and indirectly acting “anti callose synthase compounds” are comprised in accordance with the invention.
“Introduction” comprises for example methods such as transfection, transduction or transformation.
Thus, “anti-stomatin STM1 compound” also comprises for example recombinant expression constructs which bring about an expression (i.e. transcription and, if appropriate, translation) of, for example, a stomatin STM1 protein dsRNA or a stomatin STM1 protein “antisense” RNA, preferably in a plant or in a part, tissue, organ or seed thereof.
In said expression constructs/expression cassettes, a nucleic acid molecule whose expression (transcription and, if appropriate, translation) generates an “anti-stomatin STM1 protein compound” is preferably in operable linkage with at least one genetic control element (for example a promoter) which ensures an expression in plants. If the expression construct is to be introduced directly into the plant and the “anti-stomatin STM1 protein compound” (for example the stomatin STM1 protein dsRNA) is to be generated therein in planta, plant-specific genetic control elements (for example promoters) are preferred. However, the “anti-stomatin STM1 protein compound” can also be generated in other organisms or in vitro and then be introduced into the plant. Here, all procaryotic or eucaryotic genetic control elements (for example promoters) which permit the expression in the respective plant which has been chosen for the generation are preferred.
An “operable” linkage is understood as meaning for example the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example an “antistomatin STM1 protein compound”) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regulatory elements is capable of fulfilling its function in the transgenic expression of the nucleic acid sequence, depending on the arrangement of the nucleic acid sequences to sense or antisense RNA. A direct linkage in the chemical sense is not necessarily required for this purpose. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further removed or else from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence which acts as promoter, so that the two sequences are bonded covalently with one another. In this context, the distance between the promoter sequence and nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
The preparation of a functional linkage and the preparation of an expression cassette can be accomplished by means of customary recombination and cloning techniques as are described for example in Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy T J, Berman M L and Enquist L W (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel F M et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) in: Plant Molecular Biology Manual. However, it is also possible to position further sequences which, for example, act as a linker with specific restriction enzyme cleavage sites or as a signal peptide between the two sequences. Moreover, the insertion of sequences can lead to the expression of fusion proteins. Preferably, the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed can be present in vector-integrated form and can be inserted into a plant genome by, for example, transformation.
However an expression cassette is also understood as meaning those constructs in which a promoter is placed behind an endogenous stomatin STM1 protein gene, for example by means of a homologous recombination, and where the expression of an antisense stomatin STM1 protein RNA brings about the reduction according to the invention of a stomatin STM1 protein. Analogously, an “anti-stomatin STM1 protein compound” (for example a nucleic acid sequence coding for a stomatin STM1 protein dsRNA or a stomatin STM1 protein antisense RNA) can be placed behind an endogenous promoter in such a way that the same effect occurs. Both approaches result in expression cassettes for the purposes of the invention.
Plant-specific promoters means in principle any promoter which is capable of controlling the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues, plant cultures. Here, the expression can be for example constitutional, inducible or development-dependent.
The following are preferred:
a) Constitutive Promoters
Preferred vectors are those which make possible a constitutive expression in plants (Benfey et al. (1989) EMBO J. 8:2195-2202). “Constitutive” promoter means those promoters which ensure expression in numerous, preferably all, tissues over a relatively large period of plant development, preferably at all times during plant development. In particular, a plant promoter or a promoter derived from a plant virus is preferably used. The promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21:285-294; Odell et al. (1985) Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288; Gardner et al. (1986) Plant Mol Biol 6:221-228) or the 19S CaMV Promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J. 8:2195-2202) is particularly preferred. A further suitable constitutive promoter is the rubisco small subunit (SSU) promoter (U.S. Pat. No. 4,962,028), the promoter of agrobacterium nopaline synthase, the TR double promoter, the agrobacterium OCS (octopine synthase) promoter, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promoter, the cinnamyl-alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the promoters of vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991), and further promoters of genes whose constitutive expression in plants is known to the skilled worker. Especially preferred as constitutive promoter is the promoter of nitrilase-1 (nit1) gene from A. thaliana (GenBank Acc.-No.: Y07648.2, Nukleotide 2456-4340, Hillebrand et al. (1996) Gene 170:197-200).
b) Tissue-specific Promoters
One embodiment employs promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds.
Seed-specific promoters such as, for example, the promoter of phaseolin (U.S. Pat. No. 5,504,200; Bustos M M et al. (1989) Plant Cell 1(9):839-53), of the 2S albumin gene (Joseffson L G et al. (1987) J Biol Chem 262:12196-12201), of legumin (Shirsat A et al. (1989) Mol Gen Genet 215(2): 326-331), of the USP (unknown seed protein; Bäumlein H et al. (1991) Mol Gen Genet 225(3):459-67), of the napin gene (U.S. Pat. No. 5,608,152; Stalberg K et al. (1996) L Planta 199:515-519), of sucrose binding protein (WO 00/26388) or the legumin B4 promoter (LeB4; Bäumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baeumlein et al. (1992) Plant Journal 2(2):233-9; Fiedler U et al. (1995) Biotechnology (NY) 13(10):1090f), the oleosin promoter from arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). Further suitable seed-specific promoters are those of the genes coding for the high molecular weight glutenin (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase. Further preferred promoters are those allowing seed-specific expression in monocotyledons such as maize, barley, wheat, rye, rice etc. It is possible and advantageous to employ the promoter of the lpt2 or lpt1 gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, of the glutelin gene, of the oryzin gene, of the prolamin gene, of the gliadin gene, of the zein gene, of the kasirin gene or of the secalin gene).
Tuber-, storage root- or root-specific promoters, for example the patatin class I promoter (B33) or the promoter of the potato cathepsin D inhibitor.
Leaf-specific promoters, for example for example the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of the rubisco (ribulose-1,5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J. 8:2445-2451). Epidermis-specific promoters, for example the promoter of the OXLP gene (“oxalate oxidase like protein”; Wei et al. (1998) Plant Mol. Biol. 36:101-112).
Examples of Other Tissue-Specific Promoters are:
In one embodiment, the activity of STM1, in particular of HvSTM1, in particular of the STM1 as described herein, is lowered, blocked or prevented in the epidermis, in particular for increasing the resistance to mildew, for example by means of gene silencing, for example by means of an RNAi, antisense, cosuppression or microRNA approach as can be carried out by the skilled worker on the basis of the methods and sequences disclosed herein.
In one embodiment, the activity of STM1, in particular of HvSTM1, in particular of STM1 as described herein, is lowered, blocked or prevented in the mesophyll, in particular for increasing the resistance to Septoria and rusts, for example by means of gene silencing, for example by means of an RNAi, antisense, cosuppression or microRNA approach as can be carried out by the skilled worker on the basis of the methods and sequences disclosed herein.
c) Chemically Inducible Promoters
The expression cassettes may also comprise a chemically inducible promoter (review article: Gatz et al. (1997) Annu. Rev. Plant Physiol Plant Mol Biol 48:89-108) through which expression of the exogenous gene in the plant can be controlled at a particular point in time. Promoters of this type, such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J 2:397-404), an abscisic acid-inducible promoter (EP 0 335 528) and an ethanol- or cyclohexanone-inducible promoter (WO 93/21334) can likewise be used. Thus, for example, the expression of a molecule which reduces or inhibits the stomatin STM1 protein function, such as, for example, the dsRNA, ribozymes, antisense nucleic acid molecules and the like which have been listed above can be induced at suitable points in time.
d) Stress- or Pathogen-inducible Promoters
Very especially advantageous is the use of inducible promoters for expressing the RNAi constructs employed for reducing the callose synthase polypeptide quantity, activity or function, which, for example, when pathogen-inducible promoters are used, makes possible an expression only when required, i.e. in the case of attack by pathogens).
In one embodiment, the method according to the invention therefore uses promoters which are active in plants which are pathogen-inducible promoters.
Pathogen-inducible promoters comprise the promoters of genes which are induced as a result of pathogen attack, such as, for example, genes of PR proteins, SAR proteins, β-1,3-glucanase, chitinase etc. (for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al. (1992) Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2:325-342; Somssich et al. (1986) Proc Natl Acad Sci USA 83:2427-2430; Somssich et al. (1988) Mol Gen Genetics 2:93-98; Chen et al. (1996) Plant J 10:955-966; Zhang and Sing (1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, et al. (1993) Plant J 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968) (1989).
Also comprised are wound-inducible promoters such as that of the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 gene (U.S. Pat. No. 5,428,148), of the win1 and win2 gene (Stanford et al. (1989) Mol Gen Genet 215:200-208), of the systemin gene (McGurl et al. (1992) Science 225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76), of the MPI gene (Corderok et al. (1994) Plant J 6(2):141-150) and the like.
A source of further pathogen-inducible promoters is the PR gene family. A series of elements in these promoters have proved advantageous. Thus, the region −364 to −288 in the promoter of PR-2d mediates salicylate specificity (Buchel et al. (1996) Plant Mol Biol 30, 493-504). The sequence 5′-TCATCTTCTT-3′ (SEQ ID NO: 20) occurs repeatedly in the promoter of the barley β-1,3-glucanase and in more than 30 other stress-induced genes. In tobacco, this region binds a nuclear protein whose abundance is increased by salicylate. The PR-1 promoters from tobacco and Arabidopsis (EP-A 0 332 104, WO 98/03536) are also suitable as pathogen-inducible promoters. Preferred, since particularly specifically induced by pathogens, are the “acidic PR-5”-(aPR5) promoters from barley (Schweizer et al. (1997) Plant Physiol 114:79-88) and wheat (Rebmann et al. (1991) Plant Mol Biol 16:329-331). aPR5 proteins accumulate within approximately 4 to 6 hours after attack by pathogens and only show very little background expression (WO 99/66057). One approach for obtaining an increased pathogen-induced specificity is the generation of synthetic promoters from combinations of known pathogen-responsive elements (Rushton et al. (2002) Plant Cell 14, 749-762; WO 00/01830; WO 99/66057). Other pathogen-inducible promoters from different species are known to the skilled worker (EP-A 1 165 794; EP-A 1 062 356; EP-A 1 041 148; EP-A 1 032 684).
Further pathogen-inducible promoters comprise the Flachs Fis1 promoter (WO 96/34949), the Vst1 promoter (Schubert et al. (1997) Plant Mol Biol 34:417-426) and the tobacco EAS4 sesquiterpene cyclase promoter (U.S. Pat. No. 6,100,451).
Other preferred promoters are those which are induced by biotic or abiotic stress, such as, for example, the pathogen-inducible promoter of the PRP1 gene (or gst1 promoter), for example from potato (WO 96/28561; Ward et al. (1993) Plant Mol Biol 22:361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (U.S. Pat. No. 5,187,267), the chill-inducible alpha-amylase promoter from potato (WO 96/12814), the light-inducible PPDK promoter or the wounding-inducible pinII promoter (EP-A 0 375 091).
e) Mesophyll-tissue-specific Promoters
In one embodiment, the method according to the invention employs mesophyll-tissue-specific promoters such as, for example, the promoter of the wheat germin 9f-3.8 gene (GenBank Acc.-No.: M63224) or the barley GerA promoter (WO 02/057412). Said promoters are particularly advantageous since they are both mesophyll-tissue-specific and pathogen-inducible. Also suitable is the mesophyll-tissue-specific Arabidopsis CAB-2 promoter (GenBank Acc.-No.: X15222), and the Zea mays PPCZm1 promoter (GenBank Acc.-No.: X63869) or homologs thereof. Mesophyll-tissue-specific means that the transcription of a gene is limited to as few as possible plant tissues which comprise the mesophyll tissue as the result of the specific interaction of cis elements present in the promoter sequence and transcription factors binding to these elements; preferably, it means a transcription which is limited to the mesophyll tissue.
As regards further promoters which are expressed essentially in the mesophyll or in the epidermis, see the enumeration inserted further above.
f) Development-dependent Promoters
Examples of further suitable promoters are fruit ripening-specific promoters such as, for example, the fruit ripening-specific promoter from tomato (WO 94/21794, EP 409 625). Development-dependent promoters include some of the tissue-specific promoters because the development of individual tissues naturally takes place in a development-dependent manner.
Constitutive, and leaf- and/or stem-specific, pathogen-inducible, root-specific, mesophyll-tissue-specific promoters are particularly preferred, with constitutive, pathogen-inducible, mesophyll-tissue-specific and root-specific promoters being most preferred.
A further possibility is for further promoters which make expression possible in further plant tissues or in other organisms such as, for example, E. coli bacteria to be operably linked to the nucleic acid sequence to be expressed. All the promoters described above are in principle suitable as plant promoters.
Other promoters which are suitable for expression in plants are described (Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl et al. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA 86:8402-8406).
The nucleic acid sequences present in the expression cassettes or vectors of the invention may be operably linked to further genetic control sequences besides a promoter. The term genetic control sequences has a wide meaning and means all sequences which have an influence on the coming into existence or the function of the expression cassette of the invention. For example, genetic control sequences modify transcription and translation in prokaryotic or eukaryotic organisms. The expression cassettes of the invention preferably comprise a promoter with an above-mentioned specificity 5′-upstream from the particular nucleic acid sequence which is to be expressed transgenically, and a terminator sequence as additional genetic control sequence 3′-downstream, and if appropriate further conventional regulatory elements, in each case operably linked to the nucleic acid sequence to be expressed transgenically.
Genetic control sequences also comprise further promoters, promoter elements or minimal promoters capable of modifying the expression-controlling properties. It is thus possible for example through genetic control sequences for tissue-specific expression to take place additionally dependent on particular stress factors. Corresponding elements are described for example for water stress, abscisic acid (Lam E and Chua N H, J Biol Chem 1991; 266(26): 17131-17135) and heat stress (Schoffl F et al., Molecular & General Genetics 217(2-3):246-53, 1989).
It is possible in principle for all natural promoters with their regulatory sequences like those mentioned above to be used for the method of the invention. It is additionally possible also for synthetic promoters to be used advantageously.
Genetic control sequences further comprise also the 5′-untranslated regions, introns or noncoding 3′ region of genes such as, for example, the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (generally: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994)). It has been shown that these may play a significant function in the regulation of gene expression. It has thus been shown that 5′-untranslated sequences are capable of enhancing transient expression of heterologous genes. An example of a translation enhancer which may be mentioned is the 5′ leader sequence from the tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. They may in addition promote tissue specificity (Rouster J et al. (1998) Plant J 15:435-440).
The expression cassette may advantageously comprise one or more so-called enhancer sequences in operable linkage with the promoter, which make increased transgenic expression of the nucleic acid sequence possible. Additional advantageous sequences such as further regulatory elements or terminators can also be inserted at the 3′ end of the nucleic acid sequences to be expressed recombinantly. The nucleic acid sequences to be expressed recombinantly may be present in one or more copies in the gene construct.
Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular to gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J. 3:835 ff) or functional equivalents thereof. Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator.
Control sequences additionally mean those which make homologous recombination or insertion into the genome of a host organism possible or allow deletion from the genome. In homologous recombination, for example, the natural promoter of a particular gene can be specifically replaced by a promoter with specificity for the embryonal epidermis and/or the flower.
An expression cassette and/or the vectors derived from it may comprise further functional elements. The term functional element has a wide meaning and means all elements which have an influence on the production, replication or function of the expression cassettes, the vectors or the transgenic organisms of the invention. Non-restrictive examples which may be mentioned are:
To select successfully transformed cells, it is generally required additionally to introduce a selectable marker which confers to the successfully transformed cells a resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglucose 6-phosphate (WO 98/45456) or an antibiotic. The selection marker permits the selection of the transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84).
The introduction of an expression cassette according to the invention into an organism or into cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissues, organs, parts or seeds) can advantageously be accomplished using vectors in which the expression cassettes are present. The expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site. The resulting plasmid is first introduced into E. coli. Correctly transformed E. coli are selected, cultured, and the recombinant plasmid is obtained using methods known to the skilled worker. Restriction analysis and sequencing can be used for verifying the cloning step.
Examples of vectors can be plasmids, cosmids, phages, viruses or else agrobacteria. In an advantageous embodiment, the introduction of the expression cassette is accomplished by means of plasmid vectors. Preferred vectors are those which make possible a stable integration of the expression cassette into the host genome.
The generation of a transformed organism (or a transformed cell) requires the introduction of suitable DNA molecules, and thus of the RNA molecules or proteins formed as the result of their gene expression, into the host cell in question.
A multiplicity of methods (Keown et al. (1990) Methods in Enzymology 185:527-537) is available for this procedure, which is referred to as transformation (or transduction or transfection). Thus, DNA or RNA can be introduced for example directly by means of microinjection or by bombardment with DNA-coated microparticles. Also, it is possible to permeabilize the cell chemically, for example with polyethylene glycol, so that the DNA can enter the cell by diffusion. Alternatively, the DNA can be introduced by protoplast fusion with other DNA-comprising units such as minicells, cells, lysosomes or liposomes. Another suitable method for introducing DNA is electroporation, where the cells are reversibly permeabilized by means of an electrical pulse. Suitable methods are described (for example in Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104-112; Guerche et al. (1987) Plant Science 52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987) Nature 327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al. (1985) Science 227:1229-1231; DeBlock et al. (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).
In plants, the described methods for the transformation and regeneration of plants from plant tissues or plant cells for the transient or stable transformation are used. Suitable methods are mainly the transformation of protoplasts by means of polyethylene-glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-comprising solution, and Microinjection.
In addition to these “direct” transformation techniques, a transformation can also be carried out by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The methods are described for example in Horsch R B et al. (1985) Science 225: 1229f).
If agrobacteria are used, the expression cassette is to be integrated into specific plasmids, either into a shuttle or intermediate vector or into a binary vector. If a Ti or Ri plasmid is used for the transformation, at least the right border, but in most cases the right and the left border, of the Ti or Ri plasmid T-DNA is linked as flanking region with the expression cassette to be introduced.
It is preferred to use binary vectors. Binary vectors are capable of replicating both in E. coli and in Agrobacterium. As a rule, they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187). The selection marker gene permits a selection of transformed agrobacteria and is, for example, the nptII gene, which confers a resistance to kanamycin. The agrobacterium which acts as host organism in this case should already comprise a plasmid with the vir region. This is required for transferring the T-DNA to the plant cell. An agrobacterium thus transformed can be used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has been studied and described extensively (EP 120 516; Hoekema, in: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; An et al. (1985) EMBO J. 4:277-287). Various binary vectors are known and in some cases commercially available, such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).
In the case of the injection or electroporation of DNA or RNA into plant cells, the plasmid used need not meet any particular requirements. Simple plasmids such as those from the pUC series can be used. If intact plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be located on the plasmid.
Stably transformed cells, i.e. those which comprise the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed cells when a selectable marker is a component of the introduced DNA. For example, any gene which is capable of conferring a resistance to antibiotics or herbicides (such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin and the like) can act as marker (see hereinabove). Transformed cells which express such a marker gene are capable of surviving in the presence of concentrations of a suitable antibiotic or herbicide which kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene, which confers resistance to the herbicide phosphinothricin (Rathore K S et al. (1993) Plant Mol Biol 21(5):871-884), the nptII gene, which confers resistance to kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP gene, which confers resistance to the herbicide glyphosate. The selection marker permits the selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84). The plants obtained can be bred and hybridized in the customary manner. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary.
The abovementioned methods are described for example in Jenes B et al. (1993)
Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S D Kung and R Wu, Academic Press, p. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res 12:8711f).
As soon as a transformed plant cell has been generated, an intact plant can be obtained using methods known to the skilled worker. Here, the starting material is, for example, callus cultures. The development of shoot and root can be induced in the known manner from these as yet undifferentiated cell lumps. The plantlets obtained can be potted on and bred.
The skilled worker is also familiar with methods of regenerating plant parts and intact plants from plant cells. For example, methods described by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533 are used for this purpose.
The method according to the invention can advantageously be combined with other methods which bring about a pathogen resistance (for example to insects, fungi, bacteria, nematodes and the like), stress resistance or another improvement of the plant's characteristics. Examples are mentioned inter alia in Dunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000; 51 Spec No; pages 487-96.
In a preferred embodiment, the reduction of the function of a stomatin STM1 protein in a plant is accomplished in combination with an increase in the activity of a Bax inhibitor 1 protein. This can be effected for example by expressing a nucleic acid sequence which codes for a Bax inhibitor 1 protein, for example in the mesophyll tissue and/or root tissue.
In the method according to the invention, the Bax inhibitor 1 proteins from Hordeum vulgare or Nicotiana tabacum are especially preferred.
Another subject matter of the invention relates to nucleic acid molecules which comprise nucleic acid molecules coding for stomatin STM1 proteins from barley as shown by the polynucleotides SEQ. ID No: 1, and to the nucleic acid sequences which are complementary thereto, and to the sequences derived as the result of the degeneracy (degeneration) of the genetic code and to the nucleic acid molecules which code for functional equivalents of the polypeptides as shown in SEQ. ID No: 1, the nucleic acid molecules not consisting of the SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.
Another subject matter of the invention relates to the stomatin STM1 protein from barley as shown in SEQ. ID No.: 2 or to one which comprises these sequences, and to functional equivalents thereof, which do not consist of the SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42 or 44.
Another subject matter of the invention relates to doublestranded RNA nucleic acid molecules (dsRNA molecule) which, when introduced into a plant (or into a cell, tissue, organ or seed thereof), bring about the reduction of a stomatin STM1 protein, where the sense strand of said dsRNA molecule has at least 30%, preferably at least 40%, 50%, 60%, 70% or 80%, especially preferably at least 90%, very especially preferably 100%, homology with a nucleic acid molecule as shown in SEQ ID No: or 13, or to a fragment of at least 17 base pairs, preferably at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs, especially preferably at least 40, 50, 60, 70, 80 or 90 base pairs, very especially preferably at least 100, 200, 300 or 400 base pairs, most preferably at least 500, 600, 700, 800, 900, at least 1000, base pairs and which has at least 50%, 60%, 70% or 80%, especially preferably at least 90%, very especially preferably 100%, homology with a nucleic acid molecule as shown in SEQ ID No: 1, 3, 5, 7, 9, 11 or 13 but do not correspond to SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.
The double-stranded structure can be formed starting from a single, autocomplementary strand or starting from two complementary strands. In an especially preferred embodiment, sense and antisense sequence are linked by a linking sequence (linker) and can form for example a hairpin structure. The linking sequence can very especially preferably be an intron, which is spliced out after the dsRNA has been synthesized.
The nucleic acid sequence coding for a dsRNA can comprise further elements, such as, for example, transcription termination signals or polyadenylation signals.
A further subject matter of the invention relates to transgenic expression cassettes which comprise one of the nucleic acid sequences according to the invention. In the transgenic expression cassettes according to the invention, the nucleic acid sequence coding for the stomatin STM1 proteins from barley, wheat and maize is linked with at least one genetic control element as defined above in such a manner that the expression (transcription and, if appropriate, translation) can be accomplished in a desired organism, preferably monocotyledonous plants. Genetic control elements which are suitable for this purpose are described above. The transgenic expression cassettes can also comprise further functional elements as defined above.
Such expression cassettes comprise for example a nucleic acid sequence according to the invention, for example one which is essentially identical to a nucleic acid molecule SEQ ID No: or 13, or a fragment thereof according to the invention, where said nucleic acid sequence is preferably arranged in sense orientation or in antisense orientation relative to a promoter and can therefore lead to the expression of sense or antisense RNA, where said promoter is a promoter which is active in plants, preferably a promoter which is inducible by pathogen attack. Also comprised according to the invention are transgenic vectors which comprise said transgenic expression cassettes.
Another subject matter of the invention relates to plants which, as the result of natural processes or of artificial induction, comprise one or more mutations in a nucleic acid molecule which comprises the nucleic acid sequence as shown in SEQ ID No: or 13, where said mutation brings about a reduction in the activity, function or polypeptide quantity of a polypeptide encoded by the nucleic acid molecules as shown in SEQ ID No: or. For example a mutation prepared and identified by tilling.
Preferred in this context are plants which belong to the family Poaceae, especially preferred are plants selected among the plant genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea, Saccharum and Oryza, very especially preferably plants selected from the species Hordeum vulgare (barley), Triticum aestivum (wheat), Triticum aestivum subsp. spelta (spelt), Triticale, Avena sativa (oats), Secale cereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharum officinarum (sugar cane) and Oryza sativa (rice).
One embodiment of the invention therefore relates to a monocotyledonous organism comprising a nucleic acid sequence according to the invention which comprises a mutation which brings about, in the organisms or parts thereof, a reduction in the activity of one of the proteins encoded by the nucleic acid molecules according to the invention. For example, the mutation relates to one or more amino acid residues which are identified as being conserved or highly conserved in the consensus sequence shown in the figures.
In accordance with the invention, stomatin STM1 of different organisms, in particular of plants, especially preferably of useful plants, in particular of stomatins of the plant genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea, Saccharum and Oryza and also Arabidopsis generally have a so-called consensus region.
The consensus sequence derived therefrom which can be assumed to be decisive for the physiological function of the different stomatin STM1s reads:
EXIPIXXXXAITKDNVXIXIXXXXYVKIXDPXXASYGVXXPIXAVXQLAQ
TTMRSELGKITLDKTFEERDXLNXXIVXXINXAAXXWGLXCXXYEIRDIX
PPXGXXXAMEMQAXAERKKRAQILESEXXXXXXXXXXXXXXXXXXXXXXX
AXXXXXNRAXGXAEAILAXXXATAXGXXXXSXXXXXXGXXXAAXLXXAEQ
YXXAFXXXAXXXXXXLLPXXXXXPXXXXAQXX
where X can be one or more of any amino acids, with X preferably being any 1 to 3 amino acids, with X more preferably being any one amino acid (SEQ ID No.: 15) The underlined amino acids were identified as being conserved in all compared sequences.
The consensus sequence preferably reads:
P
PXNWGIRIVPERKAFVIERFGKYXTTLPSGIHFLXPFVDRIAYVHSLKE
E
A
IPI
PNQT
AITKDNV
S
I
H
I
DGVL
YVKI
V
DP
KL
ASYGV
EN
PI
Y
AVXQLAQ
TTMRSELGKITLDKTFEERD
T
LN
EK
IV
EA
IN
V
AA
KD
WGL
Q
C
LR
YEIRDI
M
PPXGVRXAMEMQAEAERKKRAQILESEGERQXHINXADGKKSSVILXSEA
A
MMDQV
NRA
Q
G
E
AEAILA
RAQ
ATA
K
G
LXLV
S
QSLKEXGGXEAASLRVAEQ
Y
IXAFGNIAKEGTTMLLPSXAXNPASMIAQAL
where X can be any one or more amino acids, with X preferably being any 1 to 3 amino acids, with X more preferably being any one amino acid (SEQ ID No.: 16)
In an especially preferred embodiment, the consensus sequence reads:
P
P
S
N
W
G
IR
IVPE
R
KA
F
V
I
ERFGK
YXTTLPSGIHFLIPFVDRIAYVHSLKE
E
A
IPI
PNQT
AITKDNV
S
I
H
I
DGVL
YVKI
V
DP
KL
ASYGV
EN
PI
Y
AV
I
QLAQ
TTMRSELGKITLDKTFEERD
T
LN
EK
IV
EA
IN
V
AA
KD
WGL
Q
C
LR
YEIRDI
M
PPXGVRXAMEMQAEAERKKRAQILESEGERQAHINXADGKKSSVILXSEA
A
MMDQV
NRA
Q
G
E
AEAILA
RAQ
ATA
K
G
LXLV
S
QSLKEXGGXEAASLRVAEQ
Y
IXAFGNIAKEGTTMLLPSXAXNPASMIAQAL
where X can be any one or more amino acids, with X preferably being any 1 to 3 amino acids, with X more preferably being any one amino acid and where the following alternative bases are comprised:
Position 3 might also show V, T or A. Position 36 might also show V or M. Position 96 might also show L, V or M. Position 182 might also show S (SEQ ID No.: 17)
The present invention therefore also relates to nucleic acid sequences which code for the above-shown consensus sequence with the SEQ ID No. 15, preferably SEQ ID No. 16, more preferably SEQ ID No. 17, and to their use in the methods according to the invention for the generation of transgenic plants with an increased pathogen resistance by reducing the content and/or the activity of at least one stomatin STM1. In this context, the consensus sequence shown is preferably characteristic of stomatin STM1 from barley and preferably also for stomatin STM1 from other plants.
Accordingly, another subject matter of the invention relates to transgenic plants, transformed with at least
In one embodiment, the plant according to the invention or the plant used in accordance with the invention is not Arabidopsis thaliana.
Host or starting organisms which are preferred as “transgenic organisms” are mainly plants in accordance with the above definition. In one embodiment, the transgenic organism is a mature plant, seed, shoot and seedling, and parts, propagation material and cultures derived therefrom, for example cell cultures. “Mature plants” means plants at any desired developmental stage beyond the seedling. “Seedling” means a young immature plant in an early developmental stage. Plants which are especially preferred as host organisms are plants to which the method according to the invention of obtaining a pathogen resistance in accordance with abovementioned criteria can be applied. In one embodiment, the plant is a monocotyledonous plant such as, for example, wheat, oats, sorghum and millet, barley, rye, maize, rice, buckwheat, sorghum, triticale, spelt or sugar cane, in particular selected from the species Hordeum vulgare (barley), Triticum aestivum (wheat), Triticum aestivum subsp. spelta (spelt), Triticale, Avena sativa (oats), Secale cereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharum officinarum (sugar cane) and Oryza sativa (rice).
The generation of the transgenic organisms can be accomplished with the above-described methods for the transformation or transfection of organisms.
Another subject matter of the invention relates to the transgenic plants described in accordance with the invention which additionally have an increased Bax inhibitor 1 activity, with plants which have an increased Bax inhibitor 1 activity in mesophyll cells or root cells being preferred, with transgenic plants which belong to the family Poaceae and which have an increased Bax inhibitor 1 activity in mesophyll cells or root cells being especially preferred, with transgenic plants selected among the plant genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea, Saccharum and Oryza being even more preferred, and with the plant species Hordeum vulgare (barley), Triticum aestivum (wheat), Triticum aestivum subsp.spelta (spelt), Triticale, Avena sativa (oats), Secale cereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharum officinarum (sugar cane) and Oryza sativa (rice) being preferred most of all.
Another subject matter of the invention relates to the use of the transgenic organisms according to the invention and of the cells, cell cultures, parts—such as, for example in the case of transgenic plant organisms, roots, leaves and the like—and transgenic propagation material such as seeds or fruits derived therefrom for the preparation of foodstuffs or feedstuffs, pharmaceuticals or fine chemicals.
In one embodiment, the invention furthermore relates to a method for the recombinant production of pharmaceuticals or fine chemicals in host organisms, where a host organism or a part thereof is transformed with one of the above-described nucleic acid molecule expression cassettes and this expression cassette comprises one or more structural genes which code for the desired fine chemical or catalyse the biosynthesis of the desired fine chemical, where the transformed host organism is grown and where the desired fine chemical is isolated from the growth medium. This method can be applied widely to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavorings, aroma substances and colorants. Especially preferred is the production of tocopherols and tocotrienols and carotenoids. The growing of the transformed host organisms and the isolation from the host organisms or the growth medium are accomplished by methods known to the skilled worker. The production of pharmaceuticals such as, for example, antibodies or vaccines, is described in Hood E E, Jilka J M (1999). Curr Opin Biotechnol. 10(4):382-6; Ma J K, Vine N D (1999). Curr Top Microbiol Immunol. 236:275-92.
In accordance with the invention, the expression of a structural gene can, of course, also take place, or be influenced, independently of carrying out the method according to the invention or using the subject matters according to the invention.
Sequences
General Methods:
The chemical synthesis of oligonucleotides can take place for example in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, page 896-897). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of E. coli cells, culturing of bacteria, replication of phages and sequence analysis of recombinant DNA are carried out as described in Sambrook et al. (1989) Cold Spring Harbour Laboratory Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules takes place using a laser fluorescence DNA sequencer from the company MWG-Licor by the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467).
The barley variety Golden Promise is from Patrick Schweizer, Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben. The variety Pallas and the backcrossed line BClngrid-mlo5 was provided by Lisa Munk, Department of Plant Pathology, Royal Veterinary and Agricultural University, Copenhagen, Denmark. Its preparation is described (Kølster P et al. (1986) Crop Sci 26: 903-907).
Unless otherwise described, the seed which has been pregerminated for 12 to 36 hours in the dark on moist filter paper is placed in batches of 5 grains along the edge of a square pot (8×8 cm) in Fruhstorfer soil type P, covered with soil and watered regularly with tapwater. All plants are grown in controlled-environment cabinets or chambers at from 16 to 18° C. for 5 to 8 days, at a relative atmospheric humidity of from 50 to 60% and in a 16/8-hour photo period with 3000 and 5000 lux, respectively (50 and 60 μmols−1m−2 photon flux density, respectively) and employed in the experiments in the seedling stage. In the case of experiments where primary leaves are treated, the latter are fully developed.
Before the plants are subjected to the transient transfection experiments, they are grown in controlled-environment cabinets or chambers at a daytime temperature of 24° C., night-time temperature of 20° C., relative atmospheric humidity of 50 to 60% and a 16/8-hour photo period with 30 000 lux.
Powdery mildew of barley Blumeria graminis (DC) Speer f.sp. hordei Em. Marchal der Rasse A6 (Wiberg A (1974) Hereditas 77: 89-148) (BghA6) is used to inoculate barley plants. The mildew was provided by the Institut für Biometrie, JLU Gieβen. The inoculum is maintained in controlled-environment cabinets under conditions which are identical to those which have been described above for the plants by transferring the conidia from infected plant material to 7-day old barley plants cv. Golden Promise which have been raised at regular intervals, at a density of 100 conidia/mm2.
The inoculation with BghA6 is carried out using 7-day-old seedlings by shaking the conidia of infected plants in an inoculation tower at a density of approximately 100 conidia/mm2 (unless otherwise stated).
Total RNA is extracted from 8 to 10 primary leaf segments (5 cm in length) by means of “RNA extraction buffer” (AGS, Heidelberg, Germany).
To this end, central primary leaf segments 5 cm in length are harvested and homogenized in liquid nitrogen using a pestle and mortar. The homogenate is stored at −70° C. until the RNA is extracted.
Total RNA is extracted from the frozen leaf material with the aid of an RNA extraction kit (AGS, Heidelberg). To this end, 200 mg of the frozen leaf material is covered with 1.7 ml of RNA extraction buffer (AGS) in a microcentrifuge tube (2 ml) and immediately subjected to thorough mixing. After the addition of 200 μl of chloroform, the mixture is again mixed thoroughly and shaken for 45 minutes at room temperature on an orbital shaker at 200 rpm. Thereafter, the mixture is centrifuged for 15 minutes at 20 000 g and 4° C. in order to separate the phases, the aqueous top phase is transferred into a fresh microcentrifuge tube, and the bottom phase is discarded. The aqueous phase is again purified with 900 μl of chloroform by homogenizing 3 times for 10 seconds and recentrifuging (see above) and removing the top phase. To precipitate the RNA, 850 μl of 2-propanol are then added, the mixture is homogenized and placed on ice for 30 to 60 minutes. Thereafter, the mixture is centrifruged for 20 minutes (see above), the supernatant is carefully decanted off, 2 ml of 70% strength ethanol (−20° C.) are added, using a pipette, and the batch is mixed and again centrifuged for 10 minutes. The supernatant is then again decanted off and the pellet is carefully freed from residual fluid, using a pipette, and then dried in a stream of pure air on a sterile workbench. Thereafter, the RNA is dissolved in 50 μl of DEPC water on ice, and the batch is mixed and centrifuged for 5 minutes (see above). 40 μl of the supernatant are transferred into a fresh microcentrifuge tube as RNA solution and stored at −70° C.
The RNA concentration is determined photometrically. To this end, the RNA solution is diluted 1:99 (v/v) with distilled water and the absorbance (Photometer DU 7400, Beckman) is measured at 260 nm (E260 nm=1 at 40 μg RNA/ml). In accordance with the calculated RNA contents, the concentrations of the RNA solutions are subsequently standardized with DEPC water to 1 μg/μl and verified in an agarose gel.
To verify the RNA concentrations in a horizontal agarose gel (1% agarose in 1×MOPS buffer with 0.2 μg/ml ethidium bromide), 1 μl of RNA solution is treated with 1 μl of 10×MOPS, 1 μl of color marker and 7 μl of DEPC water, separated according to size at a voltage of 120 V in the gel in 1×MOPS running buffer in the course of 1.5 hours and photographed under UV light. Any differences in concentration of the RNA extracts are standardized with DEPC water, and the standardization is again verified in the gel.
A contig for stomatin STM1 was constructed from the publicly available EST sequences
Cycler Program:
Mix:
The end-to-end PCR yielded a product of 1086 bp. The PCR product obtained was isolated via a 1% strength agarose gel, extracted from the gel, cloned into pCR4-Topo (Invitrogen Life Technologies) by means of T-overhand ligation and sequenced.
Biological Material
Barley near-isogenic lines (NILs) of the cultivars cv Ingrid (Mlo) and Ingrid BC7 mlo5 or barley cv Golden Promise were grown in controlled-environment chambers in pots filled with potting compost (provenance: IPK Gatersleben) (16 hours light from metal halogen lamps; 8 hours darkness, relative atmospheric humidity of 70%, constant temperature of 18° C.). Blumeria graminis DC Speer f.sp. hordei (Bgh) (isolate 4.8 comprising AvrMla-9 was grown at 22° C. and 16 hours light by weekly transfer to fresh barley leaves of the cultivar cv. Golden Promise. Blumeria graminis DC Speer f.sp. tritici Em Marchal (Bgt) of the Swiss isolate FAL (Reckenholz) was propagated at 22° C. and 16 hours light by weekly transfer to fresh leaves of wheat of the cultivar cv. Kanzler.
Plasmid Vectors
The vector pIPKTA38 was used as entry vector for the Gateway™ cloning system (Invitrogen). The vector is a pENTR1a derivative where the ccdB gene had been removed and a novel multiple cloning site had been inserted. The destination vector used was pIPKTA30N, which is based on a pUC18 background and which comprises a constitutive promoter, terminator and two Gateway cassettes comprising attR sites, ccdB gene and a chloramphenicol resistance gene. The two cassettes are arranged in opposite directions and separated from one another by a spacer from the wheat RGA2 gene (accession number AF326781). This vector system permits a one-step transfer of two copies of a PCR fragment via entry vector into the dsRNAi vector by means of Gateway LR clonase reaction (Invitrogen).
PCR and Primer Design
EST sequences of the target gene were amplified via PCR. Purified DNA from the selected cDNA clones was used as template for the PCR reaction. The primers were derived with the aid of the software package “Primer3” in the batch-file mode using the 5′-EST sequences. The EST sequences were typically amplified with a universal forward primer and a reverse EST-specific primer. The amplificates were in the range of from 400-700 bp. The primers were 20-22 bp in length and had a Tm of approx. 65° C. The PCR reactions were carried out in 96-well microtiter plates using a DNA polymerase which produces blunt ends (ThermalAce; Invitrogen). The PCR products were purified with the aid of the MinElute UF Kit (Qiagen, Hilden, Germany) and eluted with 25 μl of water.
Ligation into the Entry Vector
The PCR fragments were cloned into the Swa I cleavage site of this vector pIPKTA38. The ligation was carried out at 25° C. in the presence of the N U T4 DNA ligase (MBI Fermentas) and 5 U of Swa I per reaction. To optimize the reaction conditions for Swa I, the buffer was supplemented with NaCl to a final concentration of 0.05 M. After 1 h, the reaction was terminated by heating for 15 minutes at 65° C. Thereafter, an additional 5 U of Swa I were added in order to suppress a religation of the plasmid. The Swa I buffer was supplemented with additional NaCl to a final concentration of 0.1 M. The reaction mixtures were incubated for a further hour at 25° C.
The resulting recombinant pIPKTA38-EST clones were employed for the transformation of chemically competent E. coli DH10B cells in 96-well PCR microtiter plates (5 μl of ligation mixture per 20 μl of competent cells) and plated onto LB agar plates with kanamycin. One colony of each cloning reaction was picked and taken up in 1.2 ml of LB+kanamycin liquid culture and distributed in 96-deep-well plates. The plates were covered with an air-permeable film and incubated for 18 hours at 37° C. on a shaker. Thereupon, the deep-well plates were centrifuged for 10 minutes at 750 g, and the pellets were used for isolating the plasmid by means of the NucleoSpin Robot-96 plasmid kit (Macherey-Nagel). The presence of the pIPKTA38 plasmid was verified via restriction digest with EcoRI. The positive pIPKTA38 clones were employed as donor vector in the LR reaction.
LR Reaction and Preparation of RNAi Constructs
EST fragments in pIPKTA38 were cloned as inverted repeats into the RNAi destination vector pIPKTA30N via a single LR recombination reaction. The reaction volume was reduced to 6 μl and comprised 1 μl of the pIPKTA38 donor clone, 1 μl pIPKTA30N destination vector (150 ng/μl), 0.8 μL clonase enzyme mix and 3.2 μl of H2O. The reaction was incubated overnight at room temperature, and 5 μl of it were transformed into 20 μl of chemically competent E. coli cells in 96-well PCR plates. Two 96-deep-well plates with LB+ampicillin were half-filled with half the volume of the transformation mix, sealed with an air-permeable film and incubated for 24 hours at 37° C. on a plate shaker. Thereafter, the deep-well plates were centrifuged for 10 minutes at 750 g, and the pellets of two duplicates of each clone were combined and subjected to the plasmid preparation. The NucleoSpin Robot-96 plasmid kit (Macherey-Nagel) was used for this purpose. The DNA quantity was on average 20-30 μg of DNA per clone.
Particle Bombardment and Inoculation with Fungal Spores
Segments of primary leaves of 7-day-old barley seedlings were placed on 0.5% w/v Phytoagar (Ducheva) in water comprising 20 ppm of benzimidazole and bombarded with gold particles (diameter 1 μm) in a PDS-1000/He system (Bio-Rad, Munich, Germany) using the Hepta adaptor with a helium pressure of 900 psi. Seven leaf segments were employed per bombardment. The particle coating with 0.5 M Ca(NO3)2 was carried out as described by Schweizer et al., 1999, except that the stock solution comprised 25 mg ml−1 gold. After the coating, all of the supernatant was removed, and the particles were resuspended in 30 μl of pure ethanol. 2.18 mg of gold microcarrier were employed per bombardment. Four hours after the bombardment, the leaf segments were placed on 1% w/v Phytoagar (Ducheva) in water comprising 20 ppm of benzimidazole in 20×20 cm plates and weighted down at both ends.
The leaf segments were inoculated with spores of Bgt and Bgh 48 hours or 96 hours after the particle bombardment. The plasmid pUbiGUS, which comprises the β-glucuronidase (GUS) gene under the control of the maize ubiquitin promoter, was employed as reporter construct for transformed epidermal cells. 40 hours after the inoculation, the leaf segments were stained on GUS activity and destained for 5 minutes with 7.5% w/v trichloroacetic acid and 50% methanol. The GUS staining solution has been described in Schweizer et al. 1999.
To evaluate the interaction of phenotypes, GUS-stained cells were counted under an optical microscope, and the number of haustoria in these transformed cells was determined, whereby the haustorial index is derived. As an alternative, the number of GUS-stained cells which comprised at least one haustorium was determined, and the susceptibility index was calculated thereby.
see
| Number | Date | Country | Kind |
|---|---|---|---|
| 06100304 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/050062 | 1/4/2007 | WO | 00 | 7/8/2008 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2007/080143 | 7/19/2007 | WO | A |
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| 6506559 | Driver et al. | Jan 2003 | B1 |
| 20050132439 | Kogel et al. | Jun 2005 | A1 |
| Number | Date | Country |
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| WO-0015817 | Mar 2000 | WO |
| WO-03020939 | Mar 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090222944 A1 | Sep 2009 | US |
