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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8291—Hormone-influenced development
  • C12N15/8294—Auxins
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8291—Hormone-influenced development
  • C12N15/8295—Cytokinins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to methods for inhibiting the unwanted action of hormones in plants.
• the invention further relates to plants which are capable of inhibiting the unwanted action of hormones in one or more of their tissues and to methods for obtaining such plants.
• the invention relates to constructs for use in the transformation of plants to make them capable of inhibiting unwanted hormone action.
• a similar process is grafting. Many plants can be grafted (fruit bearing vegetables like cucumber, melon, egg plant, tomato and many trees and ornamental plants) and thereby be propagated vegetatively. For the same reasons as mentioned for rooting, this is undesirable. It is thus also an object of the invention to provide means to avoid unwanted grafting.
• Control of plant-parasitic nematodes is currently based on three principal approaches: crop rotation, use of agrochemicals and host resistance.
• crop rotation use of agrochemicals and host resistance.
• the use of resistant crops or varieties is the most economical and environmentally sound method, but for a number of reasons, this approach is not always feasible: Meloido ⁇ vne species have a very wide host range, while many cyst nematode species can survive prolonged periods without host plant, making it often very difficult to devise safe crop rotations. For many crops there are no good resistant varieties available and resistance is often broken by new pathotypes or races of the nematodes .
• the third object of the invention is therefore to provide plants that are resistant to nematodes and other pathogens.
• Root regeneration in cuttings or other plant parts is induced by the action of auxins.
• auxins are the sole group of plant hormones involved in this induction process .
• a key process in grafting is differentiation of vascular tissue. This differentiation process is regulated by auxins. Application of (very low) concentrations of auxin to scion and rootstock leads to better growing together of the two plant parts.
• the sedentary cyst and root-knot nematodes establish and maintain an intimate relation with their host. After root penetration, a cell in the region of the vascular cylinder is chosen as feeding cell. In response to stimulation by the nematode, caused by salivary secretions, a series of dramatic cellular changes is triggered in and around the initial feeding cell. Depending on the nematode species, this cell either develops into a syncytium (for cyst nematodes) or several cells are stimulated to form giant cells. Syncytia are formed by fusion of cells, giant cells go through repeated nuclear divisions without cell division.
• Both types of feeding structures show similar adaptations to their function: they are multinucleated and hypertrophic, with dense granular cytoplasm, an increased number of mitochondria and an increased amount of rough endoplasmatic reticulum. Auxins are involved in the formation of feeder cells and syncytia.
• nematodes Similar processes as described above for nematodes occur upon infection by certain insects or fungi. Insects like aphids, Diptera, Lepidoptera, Hymenoptera (wasps and saw flies) , which lay eggs in plants, leads to gall formation. Some fungi, like Fusarium, depend on formation of transfer cells for their interaction with the plant. The auxins are also involved in the formation of galls and transfer cells.
• the bacteria Aqrobacterium tumefaciens and Agrobacterium rhizogenes cause the plant diseases crown gall disease and hairy root syndrome. Both of these can be inhibited with the method of the invention.
• the processes of rooting, grafting and infection as well as many other plant processes are regulated by plant hormones.
• the occurring hormone action may be unwanted. It was found according to the invention that such unwanted hormone action can be inhibited by the action of one or more antagonistic substance in the cells subject to such unwanted action.
• the invention relates according to a first aspect thereof to a method for inhibiting the unwanted action of a hormone in plants, comprising the local expression in the plant of one or more hormone antagonists and/or molecules facilitating the action of the hormone antagonist and/or molecules interfering with the biosynthesis or catabolism of the hormone at the site of the hormone action.
• the invention relates to a method of the above kind, wherein for local expression of one or more antagonists the plant is transformed with a gene or the genes that encodes or encode an enzyme or the enzymes that is or are involved in a keystep or the key- steps in the pathway that leads to the synthesis of the hormone antagonist, which gene is under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone.
• Auxin action in the above mentioned processes of rooting, grafting and infection can be antagonized by cytokinins and ethylene . This means that an increase in the cytokinin or ethylene level inhibits rooting of cuttings, growing together of plant parts and infection by nematodes or other pathogens .
• the antagonist hormone cytokinin can be expressed in the relevant tissue of a plant by transformation of the plant with the isopentenyltransferase gene (ipt-qene, a gene encoding the biosynthesis of the cytokinin isopentenyladenine which, in plant tissue, is converted into the highly active cytokinin zeatin) .
• ipt-qene a gene encoding the biosynthesis of the cytokinin isopentenyladenine which, in plant tissue, is converted into the highly active cytokinin zeatin
• an increase in the ethylene level can be obtained via the ACC-synthase and ACC-oxidase gene.
• the ACC-synthase gene catalyzes the conversion of S- adenosyl-L-methionine to 1-aminocyclopropane-l-carboxylic acid, ACC, which is the rate limiting step in the ethylene synthesis in the plant tissue.
• the ACC-oxidase gene converts ACC into ethylene. Since ethylene antagonizes auxin activity, expression of the ACC- synthase and ACC-oxidase gene at the proper time and place in the plant tissue will also inhibit rooting of cuttings, growing together of plant parts and infection of nematodes.
• the present inventors demonstrated the antagonistic effect on auxin- induced rooting by showing that rooting experiments with (low concentrations of) ethylene gas were not successful. Since the ACC-oxidase is constitutively present in most tissues the ACC- synthase gene is preferred.
• genes involved in the sensitivity of the plant tissue for cytokinins or ethylene can be used to inhibit auxin action.
• Such genes encode what are called herein "molecules facilitating the action of the hormone antagonist" at the site of the hormone action.
• molecules are for example hormone receptors.
• examples of such genes are the gene encoding the ethylene receptor ETR-1 (Chang et al . , Science 262:539 (1993) and Ecker, Science 268:667-675 (1995)) or the gene encoding the putative cytokinin receptor CKI1 (Kakimoto, Science 274:982-985 (1996)).
• ETR-1 ethylene receptor ETR-1
• Ecker Science 268:667-675
• genes are also to be used in combination with an appropriate gene promoter (e.g. wounding, auxin, nematode or other pathogen related) .
• the action of hormones in the plant can also be inhibited by stimulating the catabolism of the hormone, thus leading to a lower concentration thereof.
• Catabolism involves enzymes, which can be expressed to stimulate the catabolism.
• genes are also under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone.
• auxins such a gene encodes for example IAA-oxidase, which can be isolated from any plant material.
• the unwanted hormone action can thus be inhibited by inhibiting the genes that encode enzymes that are involved in the biosynthesis of the hormone.
• enzymes involved in the catabolism of the hormone can be provided, induced or increased.
• Inhibition of biosynthesis genes can be accomplished by the introduction into the plant of genes that encode molecules inhibiting the biosynthesis. Such genes are again under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone to avoid a systemic effect of the inhibition.
• Examples of enzymes to be inhibited in the auxin biosynthesis are tryptophan decarboxylase and IBA- synthase (which can be isolated from any plant material) .
• Inhibition can inter alia be accomplished by co- suppression or antisense expression of the gene.
• Several interactions between plants and pathogens involve the formation of specialized cells that produce nutrition for the pathogen. This process is well- known for certain plant parasitic nematodes, like root- knot and cyst nematodes.
• Plant-pathogenic bacteria and fungi and some insects are also capable of inducing changes in the plant at cellular or tissue level, giving raise to specialized feeding structures. These feeding cells or transfer cells have in common that they are metabolically highly active and that plant defense reactions, like the production of phytoalexines (toxic secondary metabolites) is suppressed. In all cases the development and reproduction of the plant pathogens or plant-feeding insects is dependent on the proper functioning of the transfer cells. Differentiation of cell function and morphology and up-regulation of cell metabolism involves activity of plant hormones of the auxin-group. An inhibition of the auxin-related changes in cell metabolism, functioning and morphology disrupts the process of feeder cell formation and/or functioning and, through this, nutrition of the pathogen. Depending on the type of pathogen-plant relation, this will either inhibit pathogen establishment in the plant and/or pathogen propagation. Therefore, an inhibition of pathogen-induced auxin activity will prevent both pathogen damage to the plant and the build-up of pathogen populations .
• nematode infection the use of a gene construct consisting of a cytokinin or ethylene synthesis-related gene and a nematode-inducible promotor can block the development of feeding sites by counteracting the auxin effect necessary for differentiation of the cells involved. This strategy inhibits feeding site formation, and thereby disrupts the life cycle of the nematodes, that are wholly dependent on a functioning feeding site. This way the resistance becomes independent of the infecting nematode. It is thus considered that the type of resistance obtained through the present invention will not lead to resistance problems when new pathogens arise.
• cytokinins and ethylene are not toxic, neither to the plant nor to mammals consuming the plant, the problem of very strict control of the introduced gene is not as great as in using cytotoxin-producing genes to destroy feeding cells. Furthermore, the level of gene- expression necessary to obtain the effect envisioned is not very high, since cytokinins and ethylene, being plant hormones, are active at low concentrations. In this way, problems with too low expression of transferred genes are avoided. Therefore this approach will yield nematode- resistant plants, in which the resistance is only expressed in the few cells under direct influence of the nematodes, using non-toxic compounds naturally occurring in plants.
• auxins as the hormone displaying the unwanted action and cytokinins or ethylene as the antagonist
• other combinations fall within the invention.
• the antagonist is a cytokinin and the hormone is ethylene, or wherein the antagonist is ethylene and the hormone is a cytokinin, or wherein the antagonist is an auxin and the hormone is ethylene, a cytokinin or a gibberellin, or wherein the antagonist is a gibberellin and the hormone is abscisic acid (ABA) , or wherein the antagonist is abscisic acid and the hormone is an auxin, a cytokinin, ethylene or a gibberellin.
• ABA abscisic acid
• the invention further relates to a method for introducing a mechanism of inhibiting unwanted hormone action in plants, comprising the steps of: a) transforming a plant cell with a DNA construct, comprising a gene or the genes encoding an enzyme or the enzymes that is or are involved in a keystep or the keysteps in the pathway that leads to the synthesis of a hormone antagonist, which gene is under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone; and b) regenerating a transgenic plant out of the transformed plant cell.
• the method can comprise the steps of : a) transforming a plant cell with a DNA construct, comprising a gene or the genes encoding or leading to molecules facilitating the action of a hormone antagonist at the site of the hormone action, which gene is under the control of a promoter, which is specifically induced by the unwanted action of the plant hormone; and b) regenerating a transgenic plant out of the transformed plant cell.
• a method comprising the steps of : a) transforming a plant cell with a DNA construct, comprising one or more genes encoding molecules that inhibit genes involved in the biosynthesis of the hormone and/or comprising one or more genes encoding molecules involved in the catabolism of the hormone, which gene is under the control of a promoter, which is specifically induced by the unwanted action of the plant hormone; and b) regenerating a transgenic plant out of the transformed plant cell.
• the genes involved in the biosynthesis of the hormone are for example encoding tryptophan decarboxylase or IBA-synthase, both of which are involved in the biosynthesis of auxins.
• the expression of these genes is inhibited by inhibitors encoded by gene(s) which is (are) under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone .
• An example of a gene involved in the catabolism of the hormone is the gene for IAA-oxidase, which is involved in the catabolism of auxins, and is under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone. All three methods result in plants which are capable to inhibit unwanted hormone action and can therefore also be considered a method for obtaining the plants .
• the invention further provides DNA constructs suitable for the transformation of plant cells to introduce a mechanism of inhibiting unwanted hormone action, comprising a gene or the genes encoding an enzyme or the enzymes that is or are involved in a keystep or the keysteps in the pathway that leads to the synthesis of a hormone antagonist, which gene is under the control of a promoter, which is specifically induced by the cause of the unwanted action of the plant hormone.
• Gene-promoters that can be used to regulate expression of the ipt-qene or ACC-synthase gene can be isolated as follows : 1) Inhibition of grafting and rooting. Using cDNA-AFLP or other standard differential screening methods wounding-related genes that are expressed in stem tissue between approximately 0 to 72 hours after cutting can be isolated. The promoter of these genes can be used to regulate expression of the ipt-gene or ACC-synthase gene. Isolation of these promoters can be performed with standard techniques . The DNA sequence of the gene of interest can be used to screen e.g. a genomic-library (but also other methods can be used) from which the promoter sequence can be isolated (the genomic-library is made of the same plant tissue as was used to isolate the gene) .
• the promoter fused to the ipt-gene, ACC- synthase gene or other suitable gene coding for or leading to an antagonist can be used to transform plant tissues with standard techniques: e.g. via Agrobacterium tumefaciens (or other types of rhizobium bacteria) or by using the "particle gun" technique.
• Agrobacterium tumefaciens or other types of rhizobium bacteria
• the "particle gun” technique for other plant species specific (homologous) promoters can be isolated or the promoter will be used heterologously in the other plant tissues.
• nematode-related genes that are expressed in root tissue between approximately 0 to 72 hours after infection by nematodes can be isolated. These promoters can be used to regulate the expression of the ipt-qene or ACC-synthase gene. Isolation of the nematode-induced promoter and synthesis of the gene-construct (ipt-gene or ACC-synthase gene under control of the nematode related promoter) are as described above for regulation of the ipt-qene or ACC- synthase gene by a wounding-related promoter. Preferred embodiments of promoters are listed in Tables 1 and 2 hereinbelow.
• Inhibition of nematode infection with the ipt-qene or ACC-synthase gene can be achieved with the following available promoters that are described in literature
• inhibition as used in this application is intended to encompass both cancellation, decrease and prevention of the unwanted hormone action.
• inhibition can be obtained by antagonizing the plant hormone and/or by facilitating the action of an antagonist and/or by inhibiting biosynthesis of the hormone and/or by stimulating the catabolism of the hormone. All these means of inhibition can be achieved by transforming a plant cell with one or more genes that code for or lead to molecules performing the said functions. All these genes are under the control of a promoter that is specifically induced by the cause of the unwanted hormone action so as to avoid a systemic effect of the inhibition.
• the invention further relates to the transgenic plants prepared according to the claimed methods and to progeny of those plants.
• the invention also provides seeds and somatic embryos of such plants and progeny as well as haploid plants derived from either of those.
• the invention relates to vegetatively propagatable plant parts of those, such as cuttings, grafts or protoplasts for tissue culture regeneration.
• the cuttings and grafts can only be used for pathogen resistant plants, not for plants that can resist rooting and grafting.
• the method of the present invention in its various embodiments has several new and desired characteristics .
• the plant hormone cytokinin is a non-toxic compound, naturally occurring in all higher plants. This excludes toxicity risks both for the plant itself and for consumers.
• the use of a specific pathogen- induced promoter restricts the actual control to the site of the plant influenced by the pathogen only.
• the plant hormone cytokinin is active in very low dosages, which makes the system sensitive and not dependent on strong promoter activity.
• the problem of non-target site expression of the promoter is much smaller than when using toxicity genes coupled to pathogen-related promoters, since cytokinin activity will neither kill the plant nor result in toxicity problems in consumption.
• the gene construct does not give the transformed plant any strong competitive advantage over other plants, in situations without strong disease pressure. This means that the ecological risk of unwanted gene-transfer is minimal.
• the invention uses disruption of plant reactions to the pathogen that are vital to pathogen development. Therefore the risks of the pathogen developing resistance to the control strategy is excluded.
• the present invention will be further elucidated in the following examples, which are given for illustration purposes only.
• the ipt-qene and ACC-synthase gene are used for engineering transgenic plants which: (A) can not grow together, (B) can not be rooted and (C) are resistant against nematodes.
• the mechanism works under defined circumstances. First, is important that the ipt-qene and ACC-synthase gene are expressed at a specific location in the plant at a specific time.
• the expression should start shortly after making of the cutting and last for a few hours up to some days.
• Engineering of plants that are resistant against nematodes needs expression of the ipt-qene or ACC- synthase gene in the root cells that are infected by the nematodes. Again the expression should start shortly after infection and last for a few hours up to some days.
• the present invention can in principle be used for all plants that are subject to undesired vegetative propagation, or susceptible to infections by pathogenic organisms that depend on infection- induced specialized plant cells for their survival, such as nematodes, fungi and insects.
• pathogenic organisms such as nematodes, fungi and insects.
• tobacco is given as a model system, but the teachings in this application can equally well be performed on any plant.
• Agrobacterium The same gene promoter was used in combination with the ipt-qene. This promoter is switched on in root tissue by auxin. It was found by the present inventors that this promoter is switched off by cytokinins. Thus, the ipt-qene with the rolB promoter is switched on by the accumulation of auxin involved in rooting, grafting or nematode infection. This results in the synthesis of a cytokinin which antagonizes the auxin action in the cells in which auxin had accumulated. The cytokinin not only disrupts the auxin-induced processes but after some time it also stops the expression of the ipt-qene . Thereby preventing that too high levels of cytokinins are produced (causing pleiotropic effects) .
• the rolB promoter is as described by Slightom et al., J. Biol. Chem. 261, 108-121 (1986).
• the promoter is auxin sensitive.
• a promoter fragment was generated by means of PCR.
• a Xbal restriction site was introduced at the 5' end A Ncol site at the 3' end.
• the Ncol site contains the start codon of the rolB gene.
• a start codon fusion is obtained by ligating the rolB promoter to the Ncol site of the ipt-qene.
• a NOS terminator was used.
• the complete DNA construct consisting of the following elements:
• DNA constructs with the nematode-inducible promoter TobRB7 can be obtained.
• the TobRB7 promoter is described by Opperman et al . , Science Wash 263, 221-223 (1994).
• the ACC synthase gene is as described by Trebitsh T. et al . , Plant Phys . 113 (3) : 987-995 (1997).
• Tobacco plant cells are transformed with the constructs as described in Example 1 by means of Agrobacterium tumefaciens . (R.B. Horsch et al., Plant Molec. Biology Manual AS 1-9 (1988)).
• DNA construct 1 is used as a control for transformation.
• the GUS gene is a reporter gene that leads to bluish coloration upon expression.
• Figures 1 to 4 show the clear blue to green coloration after nematode infection (as described in Example 3) . Directly after infection the infected cell was colored blue ( Figure 1) and at later stages the whole root-knot was colored blue ( Figure 4) . Because the promoter is activated by auxin, this example shows that auxins are induced upon nematode infection. EXAMPLE 3
• transgenic shoots according to Example 1.2 were cut off and dipped into rooting powder containing 0.2 or 0.5% IBA. It was found that in shoots cut from transgenic plants of the invention adventitious rooting was inhibited.
• Nematode infection In order to challenge the transgenic plants with nematodes, the plants and controls were individually planted in a 1 liter tub. Four holes were made around the stem of the plant. To each hole 0.25 ml of a nematode solution was added amounting to a total nematode load of 4000 per liter potting soil. After 4 weeks de plants were checked for nematode infection. It was found that the plants on the roots of the transgenic plants were strongly reduced compared to wild type tobacco plants.
• the present invention has in general various advantages. The advantage of inhibition of vegetative propagation is for example that cultivated plants (that may be genetically modified) which are protected by "plant breeders rights" can not be vegetatively propagated. This is of great commercial value for plant breeding companies that want to prevent illegal propagation.
• nematode resistance The advantages for nematode resistance are obvious: a decrease in nematode infection leads to an increase in plant productivity, no nematocides have to be used.

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