WO2002046439A2 - Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr - Google Patents

Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr Download PDF

Info

Publication number
WO2002046439A2
WO2002046439A2 PCT/EP2001/014154 EP0114154W WO0246439A2 WO 2002046439 A2 WO2002046439 A2 WO 2002046439A2 EP 0114154 W EP0114154 W EP 0114154W WO 0246439 A2 WO0246439 A2 WO 0246439A2
Authority
WO
WIPO (PCT)
Prior art keywords
plant
root
nucleic acid
expression
promoter
Prior art date
Application number
PCT/EP2001/014154
Other languages
English (en)
Inventor
Ben Scheres
Renze Heidstra
Original Assignee
Universiteit Utrecht
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Utrecht filed Critical Universiteit Utrecht
Priority to US10/433,731 priority Critical patent/US20040067506A1/en
Priority to AU2002237224A priority patent/AU2002237224A1/en
Priority to CA002430617A priority patent/CA2430617A1/fr
Publication of WO2002046439A2 publication Critical patent/WO2002046439A2/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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 the field of plant molecular biology, more particularly to the root-specific gene expression in plants.
  • a root specific operon comprising a transcriptional regulatory promoter that contributes to root-specific gene expression and its operably linked gene encoding a novel Leucin Rich Repeat (LRR) receptor-like kinase.
  • Said transcriptional regulatory promoter may be used for driving root-specific expression of at least one gene of interest in a transgenic plant and said encoded LRR receptor-like kinase gene may be used to alter the features and/or to confer a selective advantage to transgenic plants.
  • Initiation of transcription is generally understood to be the predominant controlling factor in determining expression of a gene.
  • the transcriptional control elements which may interact with DNA binding proteins, are generally embedded in the sequence 5'-flanking or upstream of the transcribed gene. These DNA sequence elements promote the formation of transcriptional regulatory complexes that either activate or repress the expression of the gene 3' downstream of the promoter.
  • the sequences of the promoter vary in length and base pair composition from gene to gene.
  • many regulatory sequence elements can be embedded that control the promoter activity. These specific elements, called DNA- boxes, contribute to the promoter a defined feature or a defined activation pattern. Many of these boxes are recognition sites to which regulatory transcription factors can bind and are part of a tight control mechanism of the promoter.
  • Numerous plant promoter DNA boxes have been described, such as tissue-specific boxes (Chaubet et al. 1996), pyrimidin boxes (Gubler & Jacobsen 1992), which influence the level of expression or G- boxes (Dolferus et al. 1994) which reduce promoter activity by cold or dehydratation.
  • Constitutive promoters are promoters that drive the expression of genes in a continuous manner, whereas ubiquitous promoters drive the expression of the gene throughout the entire plant.
  • Expression of heterologous DNA sequences in a host is dependent upon the presence of an operably linked promoter that is also present in that host, whereby the choice of promoter will determine when, where or how strong the heterologous DNA sequence will be expressed in the host organism.
  • those skilled in the art desire to limit the expression of the transfected DNA sequence to a certain time-period (e.g. to a particular phase of plant development), to a defined tissue of the host (e.g. leaves), or to a certain expression level.
  • Controlling the expression of the heterologous genes in transgenic plants is considered to provide several advantages to the plant over ubiquitous and constitutive expression. There is a broad need in many industrial applications for transcriptional control elements capable of driving tissue specific gene-expression in plants. It is therefore important that such control elements are continuously provided and that new control elements with their specific transcriptional features are isolated and characterized.
  • root-preferred promoters are not only limited to tissue specific expression, but are also subjected to a tight control mechanism deciding when the promoter should be active. This decision depends on the function and the effects of the gene product that is switched on/off by the related promoter. Some gene products are switched on/off at a certain developmental stage of the plant, at a certain moment of the cell cycle, in stress situations originating from weather factors, upon injury caused by pathogens or upon a change in the environmental resources.
  • a typical example is the ARSK1 gene of Arabidopsis thaliana, that is activated when the roots are exposed to air during growth (dehydration) or by treatment of roots with abcisic acid or salt (Hwang et al. 1995).
  • a constitutive promoter will be needed.
  • the gene product of the transgene is to be isolated and purified from the plant for commercial purposes.
  • Clavata (civ) mutants accumulate massive pools of undifferentiated cells in the central zone of shoot and floral meristem.
  • the CLAVATA genes show a shoot and floral meristem specific expressionpattern. Root meristems of c-V-mutants are unaffected (Clark, Running, & Meyerowitz 1993).
  • the CLAVATA loci ⁇ CLV1, CLV2 and CLV3) appear to promote the transition toward differentiation of cells at the shoot and floral meristems, and/or to restrict the proliferation of cells at the center of these meristems (Clark et al. 1993).
  • CLV1 acts with CLV3 and WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) to maintain the integrity of the central zone which acts as a reservoir of stem cells.
  • WUSCHEL WUSCHEL
  • STM SHOOT MERISTEMLESS
  • the CLAVATA1 gene resides on the Arabidopsis thaliana chomosome 1.
  • Clv1 mRNA is found in the L3 tunica cell layer of the central zone and encodes a protein with leucine- rich repeats (LRR, of a type found in a number of proteins, notably the products of several plant-pathogen resistance genes), a putative serin/threonin kinase domain and a predicted extracellular receptor domain.
  • LRR leucine- rich repeats
  • Expression of the clv1 gene is initiated in the heart stage of the embryo, when cotyledonary primordia are apparent, and is independent of the STM (SHOOT MERISTEMLESS) activity (Long & Barton 1998).
  • GmCLVAI and GmCLVI B Yamamoto, Karakaya, & Knap 2000. It is the aim of the present invention to isolate novel promoters and genes which are useful for altering features of transgenic plants and thus confer selective advantage to transgenic plants.
  • a DNA operon was isolated from Arabidopsis thaliana, which comprises a novel root-specific promoter, driving a gene encoding a Leucine Rich Repeat (LRR) receptor-like kinase.
  • LRR Leucine Rich Repeat
  • the surprisingly specific expression pattern, as well as the very strong expression level, makes the newly found promoter a very attractive and selective tool to drive root-specific expression of any gene of interest.
  • the ability to target meristem cells of the root makes this promoter clearly distinct from the root-specific promoters presently known in the art.
  • Another surprising aspect of the invention is the identification of the new LRR receptor- like kinase gene, which is the first gene identified in roots showing homology with a clavata-gene. This gene is specifically expressed in roots in a specific developmental stage of the root cells (namely in the meristematic phase) and can therefore contribute agronomic interesting features to a transgenic plant when transfected herein.
  • the root-specific promoter is further denominated as ROOT CLAVATA HOMOLOG 1 promoter (RCHI prom) and the LRR receptor-like kinase as RCH1.
  • the invention embodies an isolated DNA sequence with nucleotide sequence as given in SEQ ID NO 1 ( Figure 5) comprising a novel root specific regulatory promoter sequence with nucleotide sequence as given in SEQ ID NO 2 ( Figure 6) or SEQ ID NO 18 ( Figure 13), and an isolated DNA sequence with nucleotide sequence as given in SEQ ID NO 3 ( Figure 8), encoding a novel ROOT CLAVATA HOMOLOG 1 gene, with amino acid sequence given in SEQ ID NO 4 or SEQ ID NO 19 ( Figure 9).
  • the invention embodies a method for modifying cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology comprising the modification of expression in particular cells of the root of a particular transgene operably linked to the RCH1 promoter sequence of the present invention.
  • Provided in the current invention are methods to effect expression of any gene of interest in particular cells of the plant root.
  • the present invention also relates to a method to effect expression of a ROOT CLAVATA HOMOLOG 1 protein or a homologue or a derivative thereof in a plant cell, tissue or organ.
  • the present invention clearly extends to any plant produced by the inventive methods described herein.
  • nucleic acid specifically hybridizing, preferably under stringent conditions, with the nucleotide sequence as defined in (a) or (b),
  • nucleic acid encoding a protein comprising the amino acid sequence as given in SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 19,
  • nucleic acid encoding a protein as defined in SEQ ID NO 4, SEQ ID NO 19 or nucleic acid as defined in any one of (a) to (h), said nucleic acid interrupted by intervening DNA sequences.
  • sequence as depicted in SEQ ID NO 1 is part of a sequence deposited under the
  • the present invention relates to an isolated nucleic acid comprising a novel root-specific promoter sequence, termed RCHIprom, isolated from Arabidopsis thaliana and consisting of the nucleic acid as represented in SEQ ID NO 2 or SEQ ID NO 18, or a functional part of said sequence, which is able to regulate gene expression in a root specific manner as SEQ ID NO 2 or SEQ ID NO 18 itself.
  • RCHIprom novel root-specific promoter sequence
  • said isolated DNA sequence provides a novel type of a root-specific promoter, which differs from other known root-specific promoters in the fact that it is unexpectedly active in only distinct cells of the root, namely the meristem of the main and the lateral root, as well as in the vascular tissue of the main and lateral root and in the lateral root primordia.
  • the novelty of the RCH1 promoter sequence relative to known root-specific promoters is substantiated by its unique expression pattern in plant roots.
  • the analysis of the RCH1 expression patterns was done using a RCH1prom-GUS hybrid operon that was transformed to Arabidopsis thaliana as outlined in Example 2. Analogous experiments with the RCH1prom-GUS hybrid operon were performed to establish the expression pattern of RCH1 in embryo's (Example 8 and Figure 15).
  • the RCH1 promoter is active in the meristem of the main and the lateral roots, as well as in the vascular tissue of the main and the lateral root and in the lateral root primordia.
  • the promoter of the present invention is the first known promoter that regulates a CLAVATA HOMOLOG gene in roots, and is clearly distinct from other described CLAVATA-operons which are all active in the shoot and floral meristem.
  • the present data thus clearly distinguish RCHIprom from other plant root-specific promoters, which are not expressed in the meristem of the main and lateral root, in the vascular tissue of the main and lateral root and in the lateral root primordia.
  • RCHI promoter is strongly active in the endodermis, cortex, epidermis, lateral root cap, in short in the "division zone".
  • the RCH1 promoter is also active but in a lower manner, in the quiescent center and in the vascular tissue.
  • a further characteristic of the RCH1 promoter is its very high expression level. Based on the incubation time of the RCH1 prom-GUS hybrid operon transformed plant with the GUS substrate (1 hour) and the very high output signal in the root of that plant (Example 2, Figure 7D), it is clear for persons skilled in the art that the RCH1 promoter is very strong.
  • a further characteristic of the present invention is that the activity of the RCH1 promoter in the meristem cells stops when these cells start to differentiate and to elongate to become cells of specified root tissue other than root vascular tissue.
  • the promoter is activated again in cells of the pericycle that form lateral root primordia. This expression is extended in the meristem of the lateral root, but again, the cells that differentiate into lateral root tissue other than vascular tissue do not retain RCH1 promoter activity.
  • Functional parts of the promoter of the invention will have at least one of these characteristics.
  • the invention thus relates to an isolated nucleic acid having transcriptional regulatory root specific, root-meristem specific or root-vascular-tissue specific promoter activity comprising at least part of the DNA sequence as given in any of SEQ ID 1 , 2 or 18 and a second transcriptional regulatory sequence.
  • said second trancriptional regulatory sequence is for instance an inducible box or sequence, which is placed within the RCH1 promoter, wherein the function of the RCH1 promoter is not hampered but instead can be regulated, for instance by induction.
  • the RCH1 promoter is not used to confer root-specificity to other promoters, but a second sequence is used to confer an additional regulatory feature to the RCH1 promoter.
  • said second transcriptional regulatory sequence is a promoter sequence not normally exhibiting root-specificity, thereby resulting in a hybrid promoter with root-specificity.
  • another embodiment of the present invention is to confer root-specificity, root-meristem-specificity, and root-vascular-tissue-specificity and/or root abundant gene expression to other promoter sequences.
  • This can be achieved by fusing elements of the here-disclosed RCH1 promoter to the promoter sequence of interest preferably the widely used ubiquitin promoter.
  • Such modifications can be achieved by routine experimentation by those skilled in the art.
  • the invention preferably relates to any of the isolated nucleic acids as described above which is DNA, cDNA, genomic DNA, synthetic DNA or RNA wherein T is replaced by U.
  • the invention also relates to a nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with any of the nucleic acids of the invention.
  • the invention also relates to a nucleic acid molecule of at least 15 nucleotides in length specifically amplifying a nucleic acid of the invention.
  • the invention relates to a vector comprising a nucleic acid of the invention.
  • Said vector may be an expression vector wherein the (said) nucleic acid sequence encoding a novel LRR receptor-like kinase or an immunologically active and/or functional fragment thereof, is operably linked to one or more control sequences allowing the expression in prokaryotic and/or eukaryotic host cells.
  • the invention also relates to a vector comprising at least part of a nucleic acid according to the invention and wherein a transcriptional regulatory root promoter of the invention or functional part(s) thereof, is operably linked to one or more genes of interest.
  • the invention also relates to a host cell containing a nucleic acid molecule of the invention or a vector of the invention wherein said nucleic acid or vector has been introduced by transformation, transfection or infection.
  • Preferred host cells that are part of the invention are bacterial, insect, fungal, plant or animal cells. Therefore, in a preferred embodiment, the invention relates to an isolated nucleic acid as represented in SEQ ID NO 3 encoding a LRR receptor-like kinase characterized by an amino acid sequence as given in SEQ ID NO 4 or 19.
  • said isolated nucleic acid encodes a plant ROOT CLAVATA HOMOLOG 1 (RCH1) of a novel type which is unexpectedly expressed in roots.
  • Said novel plant RCH1 shows significant homology with CLAVATA 1 , a protein involved in the maintenance of floral and shoot meristem cells.
  • RCH1 is 39% identical to CLV1. Additional to 39% of identical amino acids, there is 49% of the amino acids that belong to the same class of amino acids and therefore have the same physical and chemical properties.
  • the classification of RCH1 as a novel type of CLAVATA is documented further in figures 11 and 12.
  • the resistance genes (RPS5, RPS2, RPP8, RPM1 , RPS4, N, RPP5) are described in "Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding super family. Plant J. 1999 Nov;20(3):317-32".
  • the present invention relates to a ROOT CLAVATA HOMOLOG 1 gene that differs from other known CL,4 .TA-like genes, in the fact that it is active in the root meristem, whereas the other known CLAVATA genes and CLAVATA homologs (like ERECTA, BRASSINOLIDE INSENSITIVE and HAESA) are only active in the shoot and floral meristem.
  • the invention also relates to nucleic acids encoding a functional plant LRR receptor-like kinase comprising one or more protein regions, distinguishing said plant LRR receptor-like kinase from those LRR receptor-like kinases known in the art.
  • the invention thus relates to an isolated nucleic acid selected from the group consisting of:
  • nucleic acid consisting of at least part, preferably at least a functional part, of the DNA sequence as given in SEQ ID NO 1 or 3, or the complement thereof, (b) a nucleic acid encoding a protein as given in SEQ ID NO 4 or 19 or encoding a fragment of said protein, wherein said fragment comprises the sequence as represented in SEQ ID NO 5, and
  • nucleic acid encoding a protein with an amino acid sequence which is at least 60%, preferably at least 65 %, 70 %, 75%, 80%, 85%, more preferably at least 90% or 95% identical to the protein as given in SEQ ID NO 4 or 9, wherein said amino acid sequence comprises the sequence as represented in SEQ ID NO 5, characterised in that said nucleic acid encodes a novel LRR receptor-like kinase protein or an immunologically active and/or functional fragment of such a protein, and further provided that said nucleic acid is not one of the nucleic acids as deposited under the GenBank Accession numbers AB017061 or AQ966419.
  • the present invention also relates to an isolated LRR receptor-like kinase (protein) comprising one of the polypeptides selected from the group consisting of:
  • polypeptide comprising the amino acid sequence as given in SEQ ID NO 5, and, (c) a polypeptide comprising the sequence represented in SEQ ID NO 5 and encoded by a nucleic acid as given in SEQ ID NO 1 or 3, or a homologue or a derivative of said protein, or a fragment or an immunologically active and/or functional fragment thereof, provided that said homologue is not the amino acid sequence as described under the GenBank Accession number BAB10317.1.
  • homologue when referring to homologues of the LRR receptor-like kinase (protein), a polypeptide is meant which is at least 65% identical to the amino acid sequence of RCH1 as represented in SEQ ID NO 4 or 19.
  • the invention relates to a protein consisting of an amino acid sequence as given in SEQ ID NO 4 or SEQ ID NO 19. More specifically, the present invention relates to a root CLAVATA homologue or a functional homologue thereof. A preferred fragment of said protein is represented in SEQ ID NO 5.
  • a further embodiment of the invention comprises homologues, derivatives, immunologically active and/or functional fragments of the LRR receptor-like kinase (protein) according to the invention, and proteins comprising said homologues, derivatives, immunologically active and/or functional fragments of said LRR receptor like kinase (protein).
  • the present invention also relates to an isolated polypeptide comprising the sequence represented in SEQ ID NO 5 encodable by a nucleic acid molecule of the invention as defined above, or a homologue or a derivative thereof, or a fragment or an immunologically active and/or functional fragment thereof.
  • the invention relates to a polypeptide, encodable by a nucleic acid molecule of the invention and which has an amino acid sequence as given in SEQ ID NO 4 or SEQ ID NO 19 or comprising an amino acid sequence as given in SEQ ID NO 5, or a homologue or a derivative thereof, or an immunologically active and/or functional fragment thereof.
  • any of said proteins can be produced in a biological system, e.g. a cell culture.
  • any of said proteins is chemically manufactured e.g. by solid phase peptide synthesis.
  • Said proteins or fragments thereof can be part of a fusion protein as is the case in e.g. a two-hybrid assay which enables e.g. the identification of proteins interacting with the LRR receptor-like kinase according to the present invention. Therefore, according to another embodiment, the invention also relates to a method for producing a polypeptide or a protein of the invention comprising culturing a host cell further specified above under conditions allowing the expression of the polypeptide and recovering the produced polypeptide from the culture.
  • the proteins or fragments thereof obtained by a method of the invention are furthermore useful e.g. to modulate the interaction between a LRR receptor-like kinase according to the invention and a ligand of the receptor and/or other identified interacting protein partners.
  • Chemically synthesized peptides are particularly useful e.g. as a source of antigens for the production of antisera and/or antibodies.
  • the current invention thus furthermore encompasses antisera and/or antibodies specifically recognizing the LRR receptor-like kinase according to the invention or immunologically active parts or epitopes thereof.
  • Said antisera and/or antibodies are useful in many areas related to the invention including: (i) identification in any organism, preferably plants, of other LRR receptor-like kinase and their genes according to the invention; (ii) quantification of synthesis in organisms and/or recombinant organisms of the LRR receptor-like kinase according to the invention; (iii) purification of the LRR receptor-like kinase according to the invention; (iv) immunoprecipitation of the LRR receptor-like kinase according to the invention e.g.
  • the invention also relates to a method for the production of transgenic plants, plant cells or plant tissues comprising the introduction of a nucleic acid molecule of the invention in an expressible format or a vector as described earlier in said plant, plant cell or plant tissue.
  • the invention also relates to a method as described above further comprising regenerating a plant from said plant cell.
  • the invention also relates to a transgenic plant cell comprising a nucleic acid sequence of the invention which is operably linked to regulatory elements allowing transcription and/or expression of said nucleic acid in plant cells or obtainable by one of the methods described above.
  • a transgenic plant cell comprising a nucleic acid sequence of the invention which is operably linked to regulatory elements allowing transcription and/or expression of said nucleic acid in plant cells or obtainable by one of the methods described above.
  • one of the nucleic acids of the invention is stably integrated into the genome of said plant cell.
  • the invention further relates to a transgenic plant or plant tissue comprising said plant cells and to a harvestable part of said plant or tissue. Said harvestable part of said plant or tissue is preferably selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes, tubers and bulbs.
  • the invention further extends to the progeny derived from any of the plants or plant parts described above.
  • the present invention is applicable to any plant, in particular a monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassi
  • the present invention relates to a method for conferring root-specificity and/or root-meristem-specificity and/or root-vascular-tissue- specificity and/or root-endodermis-specificity and/or root-cortex-specificity and/or root- epidermis-specificity and/or lateral-root cap-specificity and/or abundant expression in roots to other promoter sequences comprising the fusion of at least part of the DNA sequence as given in SEQ ID NO 1 , 2 or 18 to a (second) transcriptional regulatory promoter sequence normally not exhibiting root-specificity.
  • the invention further relates to method for root-specific expression of a gene(s) of interest comprising operably linking of said gene(s) of interest to a transcriptional regulatory root-specific promoter comprising at least part of the nucleic acids as given in SEQ ID NO 1 , 2 or 18 as defined above, and possibly in combination/or a (second) transcriptional regulatory promoter sequence not normally exhibiting root-specificity, as defined earlier.
  • the invention further relates to a method for modifying cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology comprising the modification of expression in the meristem of the main and the lateral roots, or in the vascular tissue of the root, or in the lateral root primordia of a gene(s) of interest operably linked to a transcriptional regulatory root promoter as defined earlier.
  • tissue-specific expression of any gene of interest in plants is considered to be of great agronomic importance.
  • This art can be used to exclude expression in eatable parts of the plant, which averts the uptake of ectopic components.
  • the expression of the transgene can be restricted to the relevant tissues of the plant so the total organism does not spend too much energy in superfluous production of the foreign component.
  • This technique can also reduce potential yield loss by limiting the expression of some pernicious, yet useful agronomic genes, to specific tissues only.
  • Said genes of interest, which can be expressed in a root-specific manner under the control of the present invention can also be involved in phytoremediation, enhanced root-growth (like cell cycle genes or cytokinin oxidase) or disease tolerance.
  • the invention described here provides a new transcriptional regulatory element which can be operably linked to a gene effecting and/or modifying root metabolites which can be involved in phytoremediation.
  • Root specific expression of the aluminium-resistance gene that give rise to said metabolites can therefore allow plants to grow on acid soils, like those of south-eastern United States, Central and south America, North Africa and parts of India and China, where the aluminium is set free as an ion that poisons plant roots.
  • the promoter of the present invention also provides a tool to be used in a strategy for environmental remediation.
  • a metal-resistance gene such as the TaPCSI or the CAD1 gene, contributing cadmium tolerance, can be expressed specifically in roots to enable enhanced growth of transgenic plants on soils contaminated with cadmium.
  • the metals can consequently be extracted from the soil and stored in the plant material, which could then be harvested and incinerated.
  • the present invention relates to a method for phytoremidiation or environmental remediation comprising the expression of a gene(s) of interest under the control of a transcriptional regulatory root promoter as defined earlier.
  • cytokinin oxidase can be operably linked to the promoter of the present invention as described in example 6, which will enhance root growth without negatively affecting shoot growth.
  • This enhanced root growth in the plant can be used for bioremidiation or phytoremediation.
  • the said gene of interest that can be operably linked to the promoter of the present invention, can be a root transcription factor, such as Alfin.
  • Alfin binds to promoter fragments of predominantly root-specific and salt inducible genes and its activity has major implications for crop plant yield (Winicov 2000) (Winicov & Bastola 1999)).
  • the present invention provides a method for enhancing plant growth and crop yield.
  • the promoter of the present invention provides a useful tool to be used for the introduction of resistance to soil-borne pathogen attack to plants, a widespread target for genetic improvement of crop plants.
  • Certain disease- inducing microorganisms attack the belowground plant tissue and any genetic modification to contribute resistance to such organisms will require expression of the resistance-gene in the roots.
  • the coding region that is operably linked to the promoter of the present invention preferably encodes a protein which is toxic to root-attacking organisms* and more preferably the protein is an insecticidal endotoxin of Bacillus thu ngiensis.
  • Corn root worm (Diabrotica undecimpunctata howardi Barber) for example is a particularly difficult pest to control or to eradicate. It attacks the plant below the soil line, where insecticides are difficult or impossible to apply effectively. Corn root worm can be eradicated by root- * specific expression of the Cry III gene, which produces a component, specifically toxic for coleoptera. Also root-specific expression of the limonene cyclase gene in combination with GPPP synthetase would be larvicidal.
  • the invention disclosed here provides a method for conferring enhanced resistance to pathogens to a transgenic plant which pathogens attack the belowground plant tissue comprising the expression of a gene(s) of interest under the control of a transcriptional regulatory root-specific promoter as defined earlier.
  • the invention provides a method to enhanced freezing tolerance comprising the expression of a gene(s) of interest under the control of a transcriptional regulatory root-specific promoter as defined earlier.
  • the present invention provides a method to contribute to a transgenic plant stress-tolerance. Further advantages to root-preferred gene expression include the production of useful proteins in an industrial setting. Light-sensitive proteins may be synthesized in root tissue such that said proteins are not exposed to light. Therefore the present invention also relates to a method for the production of light-sensitive proteins comprising the expression of a gene encoding said light-sensitive protein under the control of a transcriptional regulatory root-specific promoter of the invention.
  • RCH1 promoter for applications based on the inhibition of expression of native DNA sequences within the plant's root to achieve a desired phenotype resulting from the silencing of the native gene.
  • inhibition might be accomplished with transformation of the plant to comprise a transcriptional regulatory root-specific promoter of the present invention operably linked to e.g. an antisense nucleotide sequence to the gene of interest, a gene silencing construct or a ribozyme.
  • a transcriptional regulatory root-specific promoter of the present invention operably linked to e.g. an antisense nucleotide sequence to the gene of interest, a gene silencing construct or a ribozyme.
  • plants secure the formation of organs throughout their life span by developing and maintaining a collection of stem cells termed the meristem.
  • the promoter of the present invention offers the opportunity to deliver a certain protein specifically to these cells. For example overexpression of genes that influence the proliferation and/or the differentiation of the root meristem could contribute to a higher turnover
  • the promoter of the present invention drives the Arabidopsis thaliana gene ROOT CLAVATA HOMOLOG 1 (RCH1). Since the c/avat ⁇ -genes are key role players in shoot- meristem maintenance, persons skilled in the art may recognize the possible involvement of the RCH1 operon in root meristem maintenance. Therefor one could assume that the control of this promoter is influenced by signals that promote meristem maintenance. This might be an advantage when aiming to stimulate the root meristem- formation, which eventually could result in more roots, higher uptake of resources and in higher crop yield.
  • the invention relates to methods for stimulating meristem formation and/or maintaining root meristem comprising the expression of a gene that influences the proliferation and/or differentiation of the root mersiteme under the control of a transcriptional regulatory root (-specific) promoter of the invention.
  • the present invention therefore relates to a method for stimulating root meristem formation or for root-meristeme maintenance comprising the expression in particular cells, tissues or organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase, for instance the LRR receptor-like kinase of the invention, wherein optionally said nucleic acid is operably linked to a plant-operable promoter sequence, for instance said plant-operable promoter being the transcriptional regulatory root promoter as defined earlier.
  • the combined gene expression data and homology to CLAVATA 1 indicate that the novel plant LRR receptor-like kinase of the invention, RCH1 (SEQ ID No. 3, 4, 19), is very likely to exert a function that is involved in the development and/or maintenance of the root meristem.
  • RCH1 SEQ ID No. 3, 4, 19
  • preferred genes to be expressed in the methods for stimulating meristem formation and/or for root meristeme maintenance are genes encoding the polypeptide(s) of the invention.
  • the present invention also relates to a method for enhancing root formation and/or root growth comprising the expression in particular cells, tissues or organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase, for instance the LRR receptor-like kinase of the invention, wherein optionally said nucleic acid is operably linked to a plant- operable promoter sequence, for instance said plant-operable promoter being the transcriptional regulatory root promoter as defined earlier.
  • the present invention therefore relates to a method for enhancing overall growth and yield comprising the expression in particular cells, tissues or organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase, for instance the LRR receptor-like kinase of the invention, wherein optionally said nucleic acid is operably linked to a plant- operable promoter sequence, for instance said plant-operable promoter being the transcriptional regulatory root promoter as defined earlier.
  • the present invention also relates to a method for enhancing overall growth and yield comprising the expression of a protein of the invention under the control of a transcriptional regulatory root (or root-specific) promoter of the invention.
  • the present invention further relates to a method to confer pathogen resistance to a transgenic plant comprising the expression in particular cells, tissues or organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase, for instance the LRR receptor-like kinase of the invention and defined above, wherein optionally said nucleic acid is operably linked to a plant-operable promoter sequence, for instance said plant- operable promoter being the transcriptional regulatory root promoter as defined earlier.
  • Many pathogen resistance genes are LRR receptor-like proteins, which initiate a signal transduction pathway in the root cells, leading to the activation of defense mechanisms against the pathogen.
  • the novel LRR receptor-like kinase might contribute to pathogen resistance to the transgenic plant when transformed herein.
  • Yet another embodiment of the invention relates to methods to perform a functional analysis of the DNA sequence according to the present invention, encoding a novel LRR receptor-like protein.
  • a functional analysis of the DNA sequence according to the present invention encoding a novel LRR receptor-like protein.
  • introduction of the antisense sequence of the DNA of the invention, as well as the overexpression of its sense sequence in a transgenic plant can elucidate a specific feature in this transgenic plant.
  • the loss of function of this gene of the invention by introduction of dominant negative mutations can result in interesting characteristics of the mutant plant.
  • Another aspect of the present invention is the revelation of a new target for herbicides or growth regulators.
  • said gene of the invention is an essential gene for plant cell viability, it can be a target for a herbicide or growth regulator.
  • the invention relates to a method for identifying and obtaining proteins interacting with a (LRR receptor-like kinase) polypeptide of the invention comprising a screening assay wherein said polypeptide is used or expressed.
  • the invention relates to a method for identifying and obtaining proteins interacting with a an LRR receptor-like kinase (protein) comprising a two-hybrid screening system wherein a nucleic acid encoding a polypeptide of the invention as a bait and a cDNA library as prey are expressed.
  • the invention also relates to a method for modulating the interaction between an LRR receptor-like kinase (protein) of the invention and interacting proteins obtainable by a method as described above.
  • the invention further relates to a method for identifying and obtaining compounds interacting with an LRR receptor-like kinase (protein) comprising the steps of: a) providing a two-hybrid screening system wherein a polypeptide or protein of the invention and a protein interacting with said (LRR receptor-like kinase) polypeptide or protein or an interacting protein obtainable by a method of claim as claimed above are expressed, b) interacting said compound with the complex formed by the expressed proteins as defined in a), c) detecting a second complex, wherein the presence of said second complex identifies a compound which specifically binds to one of said polypeptides or said second complex, and d) identifying the compound.
  • a providing a two-hybrid screening system wherein a polypeptide or protein of the invention and a protein interacting with said (LRR receptor-like kinase) polypeptide or protein or an interacting protein obtainable by a method of claim as claimed above are expressed
  • the invention also relates to a method for identifying compounds or mixtures of compounds which specifically bind to a polypeptide of the invention, comprising: a) combining a polypeptide of the invention with said compound or mixtures of compounds under conditions suitable to allow complex formation, and, b) detecting complex formation, wherein the presence of a complex identifies a molecule which specifically binds said polypeptide.
  • the invention further relates to the use of a molecule identified by means of one of the methods described above as a plant growth regulator or herbicide.
  • the invention also relates to a method for the production of a plant growth regulator or herbicide composition comprising the steps of one of the methods described above and formulating the compounds obtained from said steps in a suitable form for the application in agriculture or plant cell or tissue culture.
  • the invention also relates to the use of any of the nucleic acid molecules, vectors, polypeptides or antibodies of described herein for modifying cell fate and/or plant development and/or plant morphology and/or plant biochemistry and/or plant physiology.
  • the invention also relates to a diagnostic composition comprising at least one of the nucleic acid molecules, vectors, polypeptides or antibodies of the invention.
  • pyroglutamic acid formation oxidation, deamidation, dehydration, glycosylation (e.g. pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.), acylation and radiolabels (e.g. 125 l, 131 l, 35 S, 14 C, 32 P, 33 P, 3 H) as well as non-naturally occurring amino acid residues, L-amino acid residues and D-amino acid residues.
  • glycosylation e.g. pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.
  • acylation and radiolabels e.g. 125 l, 131 l, 35 S, 14 C, 32 P, 33 P, 3 H
  • “Homologues” or “Homologs” of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to the said protein with respect to which they are a homolog, without altering one or more of its functional properties, in particular without reducing the activity of the resulting.
  • a homolog of said protein will consist of a bioactive amino acid sequence variant of said protein.
  • amino acids present in the said protein can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break ⁇ -helical structures or ⁇ -sheet structures, and so on.
  • An overview of physical and chemical properties of amino acids is gjven in Table 1.
  • Substitutional variants of a protein of the invention are those in which at least one residue in said protein amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 -10 amino acid residues, and deletions will range from about 1-20 residues.
  • amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra. Table 1: Properties of naturally occurring amino acids.
  • Insertional amino acid sequence variants of a protein of the invention are those in which one or more amino acid residues are introduced into a predetermined site in said protein. Insertions can comprise amino-terminal and/or carboxy-terminal fusions as well as intra- sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 10 residues.
  • amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine) 6 -tag, glutathione S-transferase, protein A, maltose-binding protein, dihydrofolate reductase, Tag «100 epitope (EETARFQPGYRS), c-myc epitope (EQKLISEEDL), FLAG ® -epitope (DYKDDDK), lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C epitope (EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).
  • Deletional variants of a protein of the invention are characterised by the removal of one or more amino acids from the amino acid sequence of said protein.
  • homologues of the peptides or polypeptides of the invention contain a number of amino acid substitutions, deletions and/or additions relative to said peptide or polypepthide such that said homologues are still substantially different from the peptides or polypeptides known in the art.
  • the difference between proteins can be calculated in terms of % identidty according to methods well known in the art.
  • the homologues of the peptides or polypeptides of the present invention comprise the amino acid sequence represented in SEQ ID NO 5, optionally comprising minor amino acid substitutions such as those described in table 1.
  • Other homologues of the polypeptides of the invention have an amino acid sequence which is at least 65 % identical to the sequence represented in SEQ ID NO 4 or 19.
  • Amino acid variants of a protein of the invention may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations.
  • the manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, OH), QuickChange Site Directed mutagenesis kit (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
  • “Derivatives” of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which comprise at least about five contiguous amino acid residues of said polypeptide but which retain the biological activity of said protein and which may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of said polypeptide.
  • a derivative may comprise one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring form of said polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection.
  • the derivates of the proteins of the present invention comprise the amino acid acid sequence as represented in SEQ ID NO 5.
  • immunologically active is meant that a molecule or specific fragments thereof such as epitopes or haptens are recognized by, i.e. bind to antibodies.
  • the immunologically active or fragments of the proteins of the present invention comprise the amino acid acid sequence as represented in SEQ ID NO 5.
  • functional fragment or “functional homologue” is meant a protein which comprises at least about 5,10, 20, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050, 1100, 1110, 1120, 1130, 131 , 1132, 1133 or 1134 contiguous amino acid residues of said polypeptide but which retains the biological activity of said protein.
  • the functional fragments or functional homologues of the polypeptide or proteins of the invention comprise the amino acid sequence as represented in SEQ ID NO 5 but wherein minor amino acid substitutions are allowed (see table 1 ).
  • Antibodies include monoclonal, polyclonal, synthetic or heavy chain camel antibodies as well as fragments of antibodies such as Fab, Fv or scFv fragments.
  • Monoclonal antibodies can be prepared by the techniques as described in e.g. Liddle and Cryer (1991 ) which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized animals.
  • antibodies or fragments thereof to a molecule or fragments thereof can be obtained by using methods as described in e.g.
  • antibodies directed against small peptides such as fragments of a protein of the invention
  • said peptides are generally coupled to a carrier protein before immunization of animals.
  • a carrier protein include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin and Tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • ovalbumin ovalbumin
  • Tetanus toxoid Tetanus toxoid.
  • the carrier protein enhances the immune response of the animal and provides epitopes for T-cell receptor binding sites.
  • the term "antibodies” furthermore includes derivatives thereof such as labelled antibodies.
  • Other uses of antibodies and especially of peptide antibodies include the study of proteolytic processing (Loffler et al. 1994, Woulfe et al. 1994), determination of protein active sites (Lerner 1982), the study of precursor and post-translational processing (Baron and Baltimore 1982, Lerner et al. 1981 , Semler et al. 1982), identification of protein domains involved in protein-protein interactions (Murakami et al. 1992) and the study of exon usage in gene expression (Tamura et al. 1991).
  • Embodied in the current invention are antibodies recognizing a LRR receptor-like kinase polypeptide or protein, or homolog, derivative or fragment thereof as defined supra or specific epitopes of said polypeptide or protein.
  • the terms "gene(s)”, “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “DNA sequence(s)” or “nucleic acid molecule(s)”, when used herein refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric form of any length. Said terms furthermore include double-stranded and single-stranded DNA and RNA.
  • Said terms also include known nucleotide modifications such as methylation, cyclization and 'caps' and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Modifications of nucleotides include the addition of acridine, amine, biotin, cascade blue, cholesterol, Cy3 ® , Cy5 ® , Cy5.5 ® Dabcyl, digoxigenin, dinitrophenyl, Edans, 6-FAM, fluorescein, 3'- glyceryl, HEX, IRD-700, IRD-800, JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S ® , SE, BODIPY ® , Marina Blue ® , Pacific Blue ® , Oregon Green ® , Rhodamine Green ® , Rhodamine Red ® , Rhodol Green
  • Polynucleotide backbone modifications include methylphosphonate, 2'-OMe- methylphosphonate RNA, phosphorothiorate, RNA, 2'-OMeRNA.
  • Base modifications include 2-amino-dA, 2-aminopurine, 3'-(ddA), 3'dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8- oxo-dA, N 6 -Me-dA, abasic site (dSpacer), biotin dT, 2'-OMe-5Me-C, 2'-OMe-propynyl-C, 3'-(5-Me-dC), 3'-(ddC), 5-Br-dC, 5-l-dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, 7-deaza-dG, 8-Br
  • PNAs peptide nucleic acids
  • Said terms also encompass peptide nucleic acids (PNAs), a DNA analogue in which the backbone is a pseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than a sugar.
  • PNAs mimic the behaviour of DNA and bind complementary nucleic acid strands.
  • the neutral backbone of PNA results in stronger binding and greater specificity than normally achieved.
  • the unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.
  • telomere sequence refers to the sequence of a double-stranded DNA molecule that is homologous to a mRNA transcript thereof.
  • anti-sense strand contains an inverted sequence which is complementary to that of the "sense strand”.
  • a "coding sequence” or “open reading frame” or “ORF” is defined as a nucleotide sequence that can be transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences, i.e. when said coding sequence or ORF is present in an expressible format. Said coding sequence of ORF is bounded by a 5' translation start codon and a 3' translation stop codon.
  • a coding sequence or ORF can include, but is not limited to RNA, mRNA, cDNA, recombinant nucleotide sequences, synthetically manufactured nucleotide sequences or genomic DNA. Said coding sequence or ORF can be interrupted by intervening nucleic acid sequences. Table 2. Degeneracy of the genetic code.
  • Genes and coding sequences essentially encoding the same protein but isolated from different sources can consist of substantially divergent nucleic acid sequences.
  • substantially divergent nucleic acid sequences can be designed to effect expression of essentially the same protein.
  • Said nucleic acid sequences are the result of e.g. the existence of different alleles of a given gene, of the degeneracy of the genetic code or of differences in codon usage.
  • amino acids such as methionine and tryptophan are encoded by a single codon whereas other amino acids such as arginine, leucine and serine can each be translated from up to six different codons.
  • Agrobacterium tumefaciens (a bacterium), A. thaliana, M. sativa (two dicotyledonous plants) and Oryza sativa (a monocotyledonous plant). These examples were extracted from (http://www.kazusa.or.jp/codon).
  • the codon GGC (for glycine) is the most frequently used codon in A. tumefaciens (36.2 %->), is the second most frequently used codon in O. sativa but is used at much lower frequencies in A. thaliana and M. sativa (9 %-> and 8.4 %->, respectively).
  • GGC codon is most preferably used in A. tumefaciens and O. sativa. However, in A. thaliana this is the GGA (and GGU) codon whereas in M. sativa this is the GGU (and GGA) codon.
  • Hybridization is the process wherein substantially homologous complementary nucleotide sequences anneal to each other.
  • the hybridization process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • Tools in molecular biology relying on such a process include the polymerase chain reaction (PCR; and all methods based thereon), subtractive hybridization, random primer extension, nuclease S1 mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs, and DNA sequence determination.
  • the hybridization process can also occur with one of the complementary nucleic acids immobilized to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA.
  • the hybridization process can furthermore occur with one of the complementary nucleic acids immobilized to a solid support such as a nitrocellulose or nylon membrane or immobilized by e.g. photolitography to e.g. a silicious glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • Tools in molecular biology relying on such a process include RNA and DNA gel blot analysis, colony hybridization, plaque hybridization, in situ hybridization and microarray hybridization.
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • the stringency of hybridization is influenced by conditions such as temperature, salt concentration and hybridization buffer composition.
  • High stringency conditions for hybridization include high temperature and/or low salt concentration (salts include NaCI and Na 3 -citrate) and/or the inclusion of formamide in the hybridization buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridization buffer and/or exclusion of compounds such as dextran sulfate or polyethylene glycol (promoting molecular crowding) from the hybridization buffer.
  • hybridization conditions are described in e.g. Sambrook et al. (1989) but the skilled craftsman will appreciate that numerous different hybridization conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid sequence. Sufficiently low stringency hybridization conditions are particularly preferred to isolate nucleic acids heterologous to the DNA sequences of the invention defined supra. Elements contributing to said heterology include allelism, degeneration of the genetic code and differences in preferred codon usage as discussed supra.
  • the current invention embodies the use of the inventive DNA sequences encoding a LRR receptor-like kinase, homolog, derivative and/or immunologically fragment thereof as defined higher in any method of hybridization .
  • the current invention furthermore also relates to DNA sequences specifically hybridizing to said inventive DNA sequences.
  • DNA sequences as defined in the current invention can be interrupted by intervening sequences.
  • intervening sequences is meant any nucleic acid sequence which disrupts a coding sequence comprising said inventive DNA sequence or which disrupts the expressible format of a DNA sequence comprising said inventive DNA sequence. Removal of the intervening sequence restores said coding sequence or said expressible format.
  • intervening sequences include introns, mobilizable DNA sequences such as transposons and DNA tags such as e.g. a T-DNA.
  • mobilizable DNA sequence is meant any DNA sequence that can be mobilized as the result of a recombination event.
  • either the protein may be introduced directly to said cell, such as by microinjection or ballistic means or alternatively, an isolated nucleic acid molecule encoding said protein may be introduced into said cell, tissue or organ in an expressible format.
  • the DNA sequence of the invention comprises a coding sequence or open reading frame (ORF) encoding a LRR receptor-like kinase or a homolog or derivative thereof or an immunologically active fragment thereof as defined supra.
  • the preferred protein of the invention comprises the amino acid sequence of said LRR receptor-like kinase.
  • vector or “vector sequence” is meant a DNA sequence which can be introduced in an organism by transformation and can be stably maintained in said organism.
  • Vector maintenance is possible in e.g. cultures of Escherichia coll, A. tumefaciens, Saccharomyces cerevisiae or Schizosaccharomyces pombe.
  • Other vectors such as phagemids and cosmid vectors can be maintained and multiplied in bacteria and/or viruses.
  • Vector sequences generally comprise a set of unique sites recognized by restriction enzymes, the multiple cloning site (MCS), wherein one or more non-vector sequence(s) can be inserted.
  • MCS multiple cloning site
  • non-vector sequence is accordingly meant a DNA sequence which is integrated in one or more of the sites of the MCS comprised within a vector.
  • “Expression vectors” form a subset of vectors which, by virtue of comprising the appropriate regulatory sequences enabling the creation of an expressible format for the inserted non-vector sequence(s), thus allowing expression of the protein encoded by said non-vector sequence(s).
  • Expression vectors are known in the art enabling protein expression in organisms including bacteria (e.g. E. coif), fungi (e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g. baculoviral expression vectors), animal cells (e.g. COS or CHO cells) and plant cells (e.g. potato virus X-based expression vectors, see e.g. Vance et al. 1998 - WO9844097).
  • bacteria e.g. E. coif
  • fungi e.g. S. cerevisiae, S. pombe, Pichia pastoris
  • insect cells e.g. baculoviral expression vectors
  • the current invention clearly includes any vector or expression vector comprising a non- vector DNA sequence comprising the promoter sequence according to the present invention or a non)vector sequence encoding a LRR receptor-like kinase, homolog, derivative and/or immunologically active fragment thereof as defined supra.
  • chemical protein synthesis can be applied. Synthetic peptides can be manufactured in solution phase or in solid phase. Solid phase peptide synthesis (Merrifield 1963) is, however, the most common way and involves the sequential addition of amino acids to create a linear peptide chain.
  • Solid phase peptide synthesis includes cycles consisting of three steps: (i) immobilization of the carboxy-terminal amino acid of the growing peptide chain to a solid support or resin; (ii) chain assembly, a process consisting of activation, coupling and deprotection of the amino acid to be added to the growing peptide chain; and (iii) cleavage involving removal of the completed peptide chain from the resin and removal of the protecting groups from the amino acid side chains.
  • Common approaches in solid phase peptide synthesis include Fmoc/tBu (9-fluorenylmethyIoxycarbonyl/t-butyl) and Boc (t-butyloxycarbonyl) as the amino-terminal protecting groups of amino acids.
  • Amino acid side chain protecting groups include methyl (Me), formyl (CHO), ethyl (Et), acetyl (Ac), t-butyl (t-Bu), anisyl, benzyl (Bzl), trifluroacetyl (Tfa), N-hydroxysuccinimide (ONSu, OSu), benzoyl (Bz), 4-methylbenzyl (Meb), thioanizyl, thiocresyl, benzyloxymethyl (Bom), 4-nitrophenyl (ONp), benzyloxycarbonyl (Z), 2-nitrobenzoyl (NBz), 2-nitrophenylsulphenyl (Nps), 4-toluenesulphonyl (Tosyl.Tos), pentafluorophenyl (Pfp), diphenylmethyl (Dpm), 2-chIorobenzyloxycarbonyl (Cl-Z), 2,4,5-trichlorophenyl,
  • a number of guidelines is available to produce peptides that are suitable for use in biological systems including (i) limiting the use of difficult amino acids such as cys, met, trp (easily oxidized and/or degraded during peptide synthesis) or arg; (ii) minimize hydrophobic amino acids (can impair peptide solubility); and (iii) prevent an amino-terminal glutamic acid (can cyclize to pyroglutamate).
  • difficult amino acids such as cys, met, trp (easily oxidized and/or degraded during peptide synthesis) or arg
  • minimize hydrophobic amino acids can impair peptide solubility
  • an amino-terminal glutamic acid can cyclize to pyroglutamate
  • “expressible format” is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an intracellular or extracellular signal, such as an environmental stimulus or stress (mitogens, anoxia, hypoxia, temperature, salt, light, dehydration, etc) or a chemical compound such as IPTG (isopropyl- ⁇ -D- thiogalactopyranoside) or such as an antibiotic (tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.
  • an environmental stimulus or stress mitogens, anoxia, hypoxia, temperature, salt, light, dehydration, etc
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • an antibiotic tetracycline, ampicillin, rifampicin, kanamycin
  • hormone e.g.
  • gibberellin gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid, ethylene, abscisic acid etc), hormone analogue (iodoacetic acid (IAA), 2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongst others.
  • expression of a functional protein may also require one or more post- translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others. All such processes are included within the scope of the term "expressible format".
  • expression of a protein in a specific cell, tissue, or organ, preferably of plant origin is effected by introducing and expressing an isolated nucleic acid molecule encoding said protein, such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame, to said cell, tissue or organ, wherein said nucleic acid molecule is placed operably in connection with suitable regulatory sequences including a promoter, preferably a plant-expressible promoter, and a terminator sequence.
  • suitable regulatory sequences including a promoter, preferably a plant-expressible promoter, and a terminator sequence.
  • regulatory sequence refers to control DNA sequences which are necessary to affect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism.
  • control sequences In prokaryotes, control sequences generally include promoters, ribosomal binding sites, and terminators. In eukaryotes generally control sequences include promoters, terminators and enhancers or silencers.
  • control sequence is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components and which determines when, how much and where a specific gene is expressed.
  • Reference herein to a "promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences derived from a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e.
  • promoter also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • nucleic acids in relation to the above defined transcriptional regulatory sequences, refers to a part or parts of the nucleic acid having the activity to specifically drive or promote transcription from sequences which are located downstream of said nucleic acid sequence.
  • Promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected.
  • Such regulatory elements may be placed adjacent to a heterologous promoter sequence to drive expression of a nucleic acid molecule in response to e.g. copper, glucocorticoids, dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin, wounding, ethylene, jasmonate or salicylic acid or to confer expression of a nucleic acid molecule to specific cells, tissues or organs such as meristems, leaves, roots, embryo, flowers, seeds or fruits.
  • the promoter preferably is a plant-expressible promoter sequence. Promoters, however, that also function or solely function in non- plant cells such as bacteria, yeast cells, insect cells and animal cells are not excluded from the invention.
  • plant-expressible is meant that the promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ.
  • plant-operable and “operable in a plant” when used herein, in respect of a promoter sequence shall be taken to be equivalent to a plant-expressible promoter sequence.
  • a "regulatable promoter sequence” is a promoter that is capable of conferring expression on a structural gene in a particular cell, tissue, or organ or group of cells, tissues or organs of a plant, optionally under specific conditions, however does generally not confer expression throughout the plant under all conditions.
  • a regulatable promoter sequence may be a promoter sequence that confers expression on a gene to which it is operably connected in a particular location within the plant or alternatively, throughout the plant under a specific set of conditions, such as following induction of gene expression by a chemical compound or other elicitor.
  • the regulatable promoter used in the performance of the present invention confers expression in a specific location within the plant, either constitutively or following induction, however not in the whole plant under any circumstances.
  • promoters include cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, cell cycle specific gene promoter sequences, inducible promoter sequences and constitutive promoter sequences that have been modified to confer expression in a particular part of the plant at any one time, such as by integration of said constitutive promoter within a transposable genetic element (Ac, Ds, Spm, En, or other transposon).
  • a "constitutive promoter” is a promoter that is transcriptionaly active in an organism, preferably a plant, during most, but not necessarily all phases of its growth and development.
  • a "ubiquitous promoter” is a promoter that is transcriptionally active throughout most, but not necessarily all parts of an organism, preferably a plant.
  • weak promoter a promoter that drives expression of a coding sequence at a low level.
  • low level is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts.
  • strong promoter drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1 ,000 transcripts.
  • tissue-specific shall be taken to indicate that expression is predominantly in a particular cell or cell-type, preferably of plant origin, albeit not necessarily exclusively in said cell or cell-type.
  • tissue-specific shall be taken to indicate that expression is predominantly in a particular tissue or tissue-type, preferably of plant origin, albeit not necessarily exclusively in said tissue or tissue-type.
  • organ-specific shall be taken to indicate that expression is predominantly in a particular organ, preferably of plant origin, albeit not necessarily exclusively in said organ.
  • Root specific means that the promoter is expressed in the root only and not in other tissues of the plant.
  • root-preferred it is intended that expression of the heterologous nucleotide sequence is most abundant root, but could also have low expression levels elsewhere in the plant. While some level of expression of the heterologous nucleotide sequence occurs in other plant tissue types, expression occurs most abundantly in the root including primary, lateral and adventitious roots.
  • root structure any part of the root structure, including, but not limited to, the root cap, apical meristem, protoderm, ground meristem, procambium, endodermis, cortex, vascular cortex, epidermis, and the like.
  • cell cycle specific shall be taken to indicate that expression is predominantly cyclic and occurring in one or more, not necessarily consecutive phases of the cell cycle albeit not necessarily exclusively in cycling cells, preferably of plant origin.
  • the term ubiquitous shall be taken to indicate that expression is throughout the entire oranism.
  • an "inducible promoter” is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical, environmental, or physical stimulus.
  • a “constitutive promoter” is a promoter that is transcriptionally active throughout most, but not necessarily all parts of an organism, preferably a plant, during most, but not necessarily all phases of its growth and development.
  • Those skilled in the art will readily be capable of selecting appropriate promoter sequences for use in regulating appropriate expression of the LRR receptor-like kinase as described supra from publicly-available or readily-available sources, without undue experimentation.
  • Placing a nucleic acid molecule under the regulatory control of a promoter sequence, or in operable connection with a promoter sequence means positioning said nucleic acid molecule such that expression is controlled by the promoter sequence.
  • a promoter is usually, but not necessarily, positioned upstream, or at the 5'-end, and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting (i.e., the gene from which the promoter is derived).
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting (i.e., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.
  • “Expression” means the production of a protein or nucleotide sequence in the cell itself or in a cell-free system. It includes transcription into an RNA product, post-transcriptional modification and/or translation to a protein product or polypeptide from a DNA encoding that product, as well as possible post-translational modifications.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double- stranded nucleic acid is preferably used.
  • promoters suitable for use in gene constructs of the present invention include those listed in Table 3, amongst others.
  • the promoters listed in Table 5 are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention.
  • constitutive promoters or promoters that induce expression throughout the entire plant it is preferred . that such sequences are modified by the addition of nucleotide sequences derived from one or more of the tissue-specific promoters listed in Table 8, or alternatively, nucleotide sequences derived from one or more of the above- mentioned tissue-specific inducible promoters, to confer tissue-specificity thereon.
  • the CaMV 35S promoter may be modified by the addition of maize Adh1 promoter sequence, to confer anaerobically-regulated root-specific expression thereon, as described previously (Ellis ef al., 1987).
  • Another example describes conferring root specific or root abundant gene expression by fusing the CaMV35S promoter to elements of the maize glycine-rich protein GRP3 gene (Feix and Wulff 2000 - WO0015662). Such modifications can be achieved by routine experimentation by those skilled in the art.
  • the term "terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription.
  • Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
  • terminators particularly suitable for use in the gene constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-IA gene terminator, and the rbcs-3A gene terminator sequences, amongst others.
  • NOS nopaline synthase
  • OCS Agrobacterium tumefaciens octopine synthase
  • CaMV Cauliflower mosaic virus
  • t3'Bt2 Oryza sativa ADP-glucose pyrophosphorylase terminator sequence
  • Ectopic expression or “ectopic overexpression” of a gene or a protein are conferring to expression patterns and/or expression levels of said gene or protein normally not occurring under natural conditions.
  • Ectopic expression can be achieved in a number of ways including operably linking of a coding sequence encoding said protein to an isolated homologous or heterologous promoter in order to create a chimeric gene and/or operably linking said coding sequence to its own isolated promoter (i.e.
  • ectopic co-expression is meant the ectopic expression or ectopic overexpression of two or more genes or proteins. The same or, more preferably, different promoters are used to confer expression of said genes or proteins.
  • the promoter sequence used in the context of the present invention is operably linked to a coding sequence or open reading frame (ORF) encoding a LRR receptor-like kinase or a homolog, derivative and/or an immunologically active fragment thereof as defined supra.
  • ORF open reading frame
  • Dominant negative version or variant refers to a mutant protein, which interferes with the activity of the corresponding wild-type protein.
  • Downregulation of expression means lowering levels of gene expression and/or levels of active gene product and/or levels of gene product activity. Decreases in expression may be accomplished by e.g. the addition of coding sequences or parts thereof in a sense orientation (if resulting in co-suppression) or in an antisense orientation relative to a promoter sequence and furthermore by e.g. insertion mutagenesis (e.g. T-DNA insertion or transposon insertion) or by gene silencing strategies as described by e.g. Angell and Baulcombe (1998 - WO9836083), Lowe et al. (1989 - WO9853083), Lederer et al.
  • insertion mutagenesis e.g. T-DNA insertion or transposon insertion
  • Modulating, including lowering, the level of active gene products or of gene product activity can be achieved by administering or exposing cells, tissues, organs or organisms to said gene product, a homolog, analogue, derivative and/or immunologically active fragment thereof.
  • Immunomodulation is another example of a technique capable of downregulation levels of active gene product and/or of gene product activity and comprises administration of or exposing to or expressing antibodies to said gene product to or in cells, tissues, organs or organisms wherein levels of said gene product and/or gene product activity are to be modulated.
  • Such antibodies comprise "plantibodies", single chain antibodies, IgG antibodies and heavy chain camel antibodies as well as fragments thereof.
  • Modulating, including lowering, the level of active gene products or of gene product activity can futhermore be achieved by administering or exposing cells, tissues, organs or organisms to an inhibitor or activator of said gene product or the activity thereof.
  • inhibitors or activators include proteins (comprising e.g. proteinases and kinases) and chemical compounds identified according to the current invention as described supra.
  • agonist refers to a substance that can be either a protagonist or an antagonist, i.e. can have either positive or negative effects, can be an enhancer or an inhibitor or a modulator as well.
  • the downregulation of the expression of a LRR receptor-like kinase gene as defined higher.
  • the invention further comprises downregulation of levels of a LRR receptor-like kinase or of a LRR receptor-like kinase activity whereby said LRR receptor-like kinase has been defined supra.
  • cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology is meant that one or more developmental and/or morphological and/or biochemical and/or physiological characteristics of a plant is altered by the performance of one or more steps pertaining to the invention described herein.
  • Cell fate refers to the cell-type or cellular characteristics of a particular cell that are produced during plant development or a cellular process therefor, in particular during the cell cycle or as a consequence of a cell cycle process.
  • Plant morphology or the term “plant morphological characteristic” or similar term will, when used herein, be understood by those skilled in the art to refer to the external appearance of a plant, including any one or more structural features or combination of structural features thereof.
  • Such structural features include the shape, size, number, position, colour, texture, arrangement, and pattemation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, stem, leaf, shoot, petiole, trichome, flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma, aerenchyma, sieve element, phloem or vascular tissue, amongst others.
  • Plant physiology or the term “plant physiological characteristic” or similar term will, when used herein, be understood to refer to the functional processes of a plant, including developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fiber production, secondary growth or wood production, amongst others; responses of a plant to externally-applied factors such as metals, chemicals, hormones, growth factors, environment and environmental stress factors (eg. anoxia, hypoxia, high temperature, low temperature, dehydration, light, daylength, flooding, salt, heavy metals, amongst others), including adaptive responses of plants to said externally-applied factors.
  • developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fiber production, secondary growth or wood production, amongst others
  • Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T- DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et al.(1985), Dodds et al.
  • a whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • existing meristematic tissue e.g., apical meristem, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
  • the plant is produced according to the inventive method is transfected or transformed with a genetic sequence, or amenable to the introduction of a protein, by any art-recognized means, such as microprojectile bombardment, microinjection, transformation (including the 'flower dip' transformation method; Bechtold and Pelletier 1998, Trieu et al. 2000), protoplast fusion, or electroporation, amongst others.
  • any art-recognized means such as microprojectile bombardment, microinjection, transformation (including the 'flower dip' transformation method; Bechtold and Pelletier 1998, Trieu et al. 2000), protoplast fusion, or electroporation, amongst others.
  • said plant is produced by .4gr ⁇ acter/u/7.-mediated transformation.
  • the “seedling” is the juvenile plant that arises from the mature embryo after seed germination.
  • “differentiation of a cell” it is understood that the cell develops unique features to be engaged for a specific function. Usually differentiation is irreversible. transformation or agrolistic transformation of plants, yeast, moulds or filamentous fungi is based on the transfer of part of the transformation vector sequences, called the T-DNA, to the nucleus and on integration of said T-DNA in the genome of said eukaryote.
  • Agrobacterium is meant a member of the Agrobacteriaceae, more preferably Agrobacterium or Rhizobacterium and most preferably Agrobacterium tumefaciens.
  • T-DNA or transferred DNA, is meant that part of the transformation vector flanked by T-DNA borders which is, after activation of the Agrobacterium vir genes, nicked at the T-DNA borders and is transferred as a single stranded DNA to the nucleus of an eukaryotic cell.
  • T-DNA borders “T-DNA border region”, or “border region” are meant either right T-DNA border (RB) or left T-DNA border (LB).
  • T-DNA transformation vector or "T-DNA vector” is meant any vector encompassing a T-DNA sequence flanked by a right and left T-DNA border consisting of at least the right and left border core sequences, respectively, and used for transformation of any eukaryotic cell.
  • T-DNA vector backbone sequence or "T-DNA vector backbone sequences” is meant all DNA of a T-DNA containing vector that lies outside of the T-DNA borders and, more specifically, outside the nicking sites of the border core imperfect repeats.
  • the current invention includes optimized T-DNA vectors such that vector backbone integration in the genome of a eukaryotic cell is minimized or absent.
  • optimal T- DNA vector is meant a T-DNA vector designed either to decrease or abolish transfer of vector backbone sequences to the genome of a eukaryotic cell.
  • Such T-DNA vectors are known to the one familiar with the art and include those described by Hanson et al.
  • the current invention clearly considers the inclusion of a DNA sequence comprising the promoter sequence of the present invention encoding a LRR receptor-like kinase, homolog, derivative or immunologically active fragment thereof as defined supra, in any
  • T-DNA vector comprising binary transformation vectors, super-binary transformation vectors, co-integrate transformation vectors, Ri-derived transformation vectors as well as in T-DNA carrying vectors used in agrolistic transformation.
  • binary transformation vector is meant a T-DNA transformation vector comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in the eukaryotic cell to be transformed; and a vector backbone region comprising at least origins of replication active in E. coli and
  • Agrobacterium and markers for selection in E. coli and Agrobacterium are markers for selection in E. coli and Agrobacterium.
  • the T-DNA borders of a binary transformation vector can be derived from octopine-type or nopaline-type Ti plasmids or from both.
  • the T-DNA of a binary vector is only transferred to a eukaryotic cell in conjunction with a helper plasmid.
  • helper plasmid Also known in the art are multiple binary vector Agrobacterium strains for efficient co-transformation of plants
  • helper plasmid is meant a plasmid that is stably maintained in Agrobacterium and is at least carrying the set of vir genes necessary for enabling transfer of the T-DNA.
  • Said set of vir genes can be derived from either octopine-type or nopaline-type Ti plasmids or from both.
  • “super-binary transformation vector” is meant a binary transformation vector additionally carrying in the vector backbone region a vir region of the Ti plasmid pTiBo542 of the super-virulent A. tumefaciens strain A281 (Hiei et al. 1994 -
  • co-integrate transformation vector is meant a T-DNA vector at least comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in plants; and a vector backbone region comprising at least origins of replication active in Escherichia coli and Agrobacterium, and markers for selection in E. coli and Agrobacterium, and a set of vir genes necessary for enabling transfer of the T-DNA.
  • the T-DNA borders and said set of vir genes of a said T-DNA vector can be derived from either octopine-type or nopaline-type Ti plasmids or from both.
  • Ra-derived plant transformation vector is meant a binary transformation vector in which the T-DNA borders are derived from a Ti plasmid and said binary transformation vector being used in conjunction with a 'helper' Ri-plasmid carrying the necessary set of vir genes.
  • selectable marker gene or “selectable marker” or “marker for selection” includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp 1 ), tetracycline resistance gene (Tc r ), bacterial kanamycin resistance gene (Kan r ), phosphinothricin resistance gene, neomycin phosphotransferase gene (npfll), hygromycin resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff ef al, 1997), and luciferase gene, amongst others.
  • Amicillin resistance Amp 1
  • Tc r tetracycline resistance gene
  • Kan r bacterial kanamycin resistance gene
  • phosphinothricin resistance gene phosphinothricin resistance gene
  • neomycin phosphotransferase gene npfll
  • hygromycin resistance gene ⁇ -glucuronidas
  • agrolistic transformation or “agrolistic transfer” is meant here a transformation method combining features of Agrobacterium-med ⁇ ated transformation and of biolistic DNA delivery.
  • a T-DNA containing target plasmid is co-delivered with DNA/RNA enabling in planta production of VirDI and VirD2 with or without VirE2 (Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton 1997 - WO9712046).
  • the present invention further describes an approach to remove from transformed cells a stably integrated foreign DNA sequence by recombination involving a recombinase and recombination sites.
  • foreign DNA any DNA sequence that is introduced in the host's genome by recombinant techniques.
  • Said foreign DNA includes e.g. a T-DNA sequence or a part thereof such as the T-DNA sequence comprising the selectable marker in an expressible format.
  • Foreign DNA furthermore include intervening DNA sequences as defined supra.
  • recombination event is meant either a site-specific recombination event or a recombination event effected by transposon 'jumping'.
  • recombinase is meant either a site-specific recombinase or a transposase.
  • site is meant either site-specific recombination sites or transposon border sequences.
  • site specific recombination event is meant an event catalyzed by a system generally consisting of three elements: a pair of DNA sequences (the site-specific recombination sequences or sites) and a specific enzyme (the site-specific recombinase). The site-specific recombinase catalyzes a recombination reaction only between two site-specific recombination sequences depending on the orientation of the site-specific recombination sequences.
  • Sequences intervening between two site-specific recombination sites will be inverted in the presence of the site-specific recombinase when the site-specific recombination sequences are oriented in opposite directions relative to one another (i.e. inverted repeats). If the site-specific recombination sequences are oriented in the same direction relative to one another (i.e. direct repeats), then any intervening sequences will be deleted upon interaction with the site-specific recombinase.
  • site-specific recombination sequences are present as direct repeats at both ends of a foreign DNA sequence integrated into a eukaryotic genome, such integration of said sequences can subsequently be reversed by interaction of the site-specific recombination sequences with the corresponding site specific recombinase.
  • site specific recombinase systems can be used including but not limited to the Cre/lox system of bacteriophage P1 , the FLP/FRT system of yeast, the Gin recombinase of phage Mu, the Pin recombinase of E.
  • Recombinases generally are integrases, resolvases or flippases. Also dual-specific recombinases can be used in conjunction with direct or indirect repeats of two different site-specific recombination sites corresponding to the dual-specific recombinase (Baszczynski et al. 1999 - WO9925840).
  • the preferred site-specific recombinase systems are the bacteriophage P1 Cre/lox, the yeast FLP/FRT and the Z. rouxii R/RS systems.
  • a recombinase (Cre, FLP or R) interact specifically with its respective site-specific recombination sequence (lox, FRT, or RS respectively) to invert or excise the intervening sequences.
  • the site-specific recombination sequences for each of these two systems are relatively short (34 bp for lox and 47 bp for FRT).
  • Site-specific recombination systems have many applications in plant molecular biology including methods for control of homologous recombination (e.g. Hodges et al. 1996 - US5527695), for targeted insertion, gene stacking, etc. (Baszczynski et al. 1999 - WO9925821) and for resolution of complex T-DNA integration patterns or for excision of a selectable marker (Ow et al. 1999 - WO9923202).
  • the site-specific recombination sequences must be linked to the ends of the DNA to be excised or to be inverted, the gene encoding the site specific recombinase may be located elsewhere.
  • the recombinase gene could already be present in the eukaryote's DNA or could be supplied by a later introduced DNA fragment either introduced directly into cells, through crossing or through cross-pollination.
  • a substantially purified recombinase protein could be introduced directly into the eukaryotic cell, e.g. by micro-injection or particle bombardment.
  • the site-specific recombinase coding region will be operably linked to regulatory sequences enabling expression of the site-specific recombinase in the eukaryotic cell.
  • transposase-mediated recombination a recombination event catalyzed by a system consisting of three elements: a pair of DNA sequences (the transposon border sequences) and a specific enzyme (the transposase).
  • the transposase catalyzes a recombination reaction only between two transposon border sequences which are arranged as inverted repeats.
  • a number of different transposon/transposase systems can be used including but not limited to the Ds/Ac system, the Spm system and the Mu system.
  • Ds/Ac and the Spm system originate from corn but it has been shown that at least the Ds/Ac and the Spm system also function in other plants (Fedoroff et al. 1993, Schlappi et al. 1993, Van Sluys et al. 1987).
  • Preferred are the Ds- and the Spm-type transposons which are delineated by 11 bp- and 13 bp- border sequences, respectively.
  • transposon border sequences must be linked to the ends of the DNA to be excised, the gene encoding the transposase may be located elsewhere.
  • the recombinase gene could already be present in the eukaryote's DNA or could be supplied by a later introduced DNA fragment either introduced directly into cells, through crossing or through cross-pollination.
  • a substantially purified transposase protein could be introduced directly into cells, e.g. by microinjection or by particle bombardment.
  • transposon border sequences are included in a foreign DNA sequence such that they lie outside said DNA sequence and transform said DNA into a transposon-like entity that can move by the action of a transposase.
  • the genetic element is preferably induced to mobilize, such as, for example, by the expression of a recombinase protein in the cell which contacts the integration site of the genetic element and facilitates a recombination event therein, excising the genetic element completely, or alternatively, leaving a "footprint", generally of about 20 nucleotides in length or greater, at the original integration site.
  • Those hosts and host parts that have been produced according to the inventive method can be identified by standard nucleic acid hybridization and/or amplification techniques to detect the presence of the mobilizable genetic element or a gene construct comprising the same.
  • the term "footprint” shall be taken to refer to any derivative of a mobilizable genetic element or gene construct comprising the same as described herein which is produced by excision, deletion or other removal of the mobilizable genetic element from the genome of a cell transformed previously with said gene construct.
  • a footprint generally comprises at least a single copy of the recombination loci or transposon used to promote excision.
  • a footprint may comprise additional sequences derived from the gene construct, for example nucleotide sequences derived from the left border sequence, right border sequence, origin of replication, recombinase-encoding or transposase-encoding sequence if used, or other vector-derived nucleotide sequences. Accordingly, a footprint is identifiable according to the nucleotide sequence of the recombination locus or transposon of the gene construct used, such as, for example, a sequence of nucleotides corresponding or complementary to a lox site, frt site or RS site.
  • cell cycle means the cyclic biochemical and structural events associated with growth and with division of cells, and in particular with the regulation of the replication of DNA and mitosis.
  • Cell cycle includes phases called: GO, Gap1 (G1), DNA synthesis (S), Gap2 (G2), and mitosis (M). Normally these four phases occur sequentially, however, the cell cycle also includes modified cycles wherein one or more phases are absent resulting in modified cell cycle such as endomitosis, acytokinesis, polyploidy, polyteny, and endoreduplication.
  • cell cycle interacting protein means a protein which exerts control on or regulates or is required for the cell cycle or part thereof of a cell, tissue, organ or whole organism and/or DNA replication. It may also be capable of binding to, regulating or being regulated by cyclin dependent kinases or their subunits.
  • the term also includes peptides, polypeptides, fragments, variant, homologs, alleles or precursors (eg preproproteins or preproteins) thereof.
  • cell cycle control genes refers to any gene or mutant thereof which exerts control on or are required for: chromosomal DNA synthesis and for mitosis (preprophase band, nuclear envelope, spindle formation, chromosome condensation, chromosome segregation, formation of new nuclei, formation of phragmoplast, duplication of microtubule-organizing center, etc) meiosis, cytokinesis, cell growth, endoreduplication, cell cycle control genes are also all genes exerting control on the above: homologs of CDKs, cyclins, E2Fs, Rb, CKI, Cks, and also any genes which interfere with the above, cyclin D, cdc25, Wee1 , Nim1 , MAP kinases, etc.
  • cell cycle control genes are all genes involved in the control of entry and progression through S phase. They include, not exclusively, genes expressing "cell cycle control proteins” such as cyclin dependent kinases (CDK), cyclin dependent kinase inhibitors (CKI), D, E and A cyclins, E2F and DP transcription factors, pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Ore proteins, cdc45, components of SCF ubiquitin ligase, PCNA, DNA-polymerase.
  • CDK cyclin dependent kinases
  • CKI cyclin dependent kinase inhibitors
  • D cyclin dependent kinase inhibitors
  • E2F and DP transcription factors DP transcription factors
  • pocket proteins CDC7/DBF4 kinase
  • CDC6, MCM2-7 or MCM2-7
  • Ore proteins cdc45
  • components of SCF ubiquitin ligase PCNA, DNA-
  • cell cycle control protein include cyclins A, B, C, D and E including CYCA1 ;1 , CYCA2;1 , CYCA3;1 , CYCB1 ;1 , CYCB1 ;2, CYC B2;2, CYCD1 ;1 , CYCD2;1 , CYCD3;1 , and CYCD4;1 (Evans et al. 1983;Francis et al. 1998;Labbe et al. 1989;Murray & Kirschner 1989;Renaudin et al. 1996;Soni et al. 1995;Sorrell et al.
  • CKI cyclin dependent kinase inhibitor proteins
  • ICK1 Wang, Fowke, & Crosby 1997), FL39, FL66, FL67 (PCT/EP98/05895), Sid , Far1 , Rum1 , p21 , p27, p57, p16, p15, p18, p19 (Elledge 1996;Pines 1995), p14 and p14ARF; p13sud or CKSIAt (De Veylder et al.
  • Cdc2MsB Hirt et al. 1993
  • CdcMs kinase Bogre et al. 1997) cdc2 T14Y15 phosphatases such as Cdc25 protein phosphatase or p80cdc25 (Bell et al. 1993;EIIedge 1996;Kumagai & Dunphy 1991 ;Russell et al.
  • cell cycle control proteins that are involved in the formation of a pre-replicative complex at one or more origins of replication, such as, but not limited to, ORC, CDC6, CDC14, RPA and MCM proteins or in the regulation of formation of this pre-replicative complex, such as, but not limited to, the CDC7, DBF4 and MBF proteins.
  • cell cycle control protein shall further be taken to include any one or more of those proteins that are involved in the turnover of any other cell cycle control protein, or in regulating the half-life of said other cell cycle control protein.
  • protein turnover is to include all biochemical modifications of a protein leading to the physical or functional removal of said protein.
  • Such modifications are phosphorylation, ubiquitination and proteolysis.
  • Particularly preferred proteins which are involved in the proteolysis of one or more of any other of the above-mentioned cell cycle control proteins include the yeast- derived and animal-derived proteins, Skp1 , Skp2, Rub1 , Cdc20, cullins, CDC23, CDC27, CDC16, and plant-derived homologues thereof (Cohen-Fix & Koshland 1997;Hochstrasser 1998;Krek 1998;Lisztwan et al. 1998) and Plesse et al in (Francis et al. 1998)).
  • cell cycle control genes shall further be taken to include any one or more of those gene that are involved in the transcriptional regulation of cell cycle control gene expression such as transcription factors and upstream signal proteins. Additional cell cycle control genes are not excluded.
  • the term “cell cycle control genes” shall further be taken to include any cell cycle control gene or mutant thereof, which is affected by environmental signals such as for instance stress, nutrients, pathogens, or by intrinsic signals such as the animal mitogens or the plant hormones (auxins, cytokinins, ethylene, gibberellic acid, abscisic acid and brassinosteroids).
  • cell cycle progression refers to the process of passing through the different cell cycle phases.
  • cell cycle progression rate accordingly refers to the speed at which said cell cycle phases are run through or the time spans required to complete said cell cycle phases.
  • pathogen is meant those organisms that have a negative effect on the physiological state of the plant or a part thereof. Some pathogens are for instance viruses, bacteria, fungi, and parasitic plants, with plant “pests” is meant the group of nematodes as well as insects, which are able to attack and to damage the plant.
  • Plant cell comprises any cell derived from any plant and existing in culture as a single cell, a group of cells or a callus.
  • a plant cell may also be any cell in a developing or mature plant in culture or growing in nature.
  • Plants comprises all plants, including monocotyledonous and dicotyledonous plants.
  • yeast two-hybrid assay is meant an assay that is based on the observation that many eukaryotic transcription factors comprise two domains, a DNA-binding domain (DB) and an activation domain (AD) which, when physically separated (i.e. disruption of the covalent linkage) do not effectuate target gene expression.
  • DB DNA-binding domain
  • AD activation domain
  • Two proteins able to interact physically with one of said proteins fused to DB and the other of said proteins fused to AD will re-unite the DB and AD domains of the transcription factor resulting in target gene expression.
  • the target gene in the yeast two-hybrid assay is usually a reporter gene such as the ⁇ -galactosidase gene. Interaction between protein partners in the yeast two-hybrid assay can thus be quantified by measuring the activity of the reporter gene product (Bartel and Fields 1997).
  • a mammalian two-hybrid system can be used which includes e.g. a chimeric green fluorescent protein encoding reporter gene (Shioda et al. 2000).
  • Yet another alternative consists of a bacterial two- hybrid system using e.g. HIS as reporter gene (Joung et al. 2000).
  • sequence will be a maximum of about 50 amino acids in lenght, preferably a maximum of about 60 amino acids. It is usually desirable to select sequences of at least about 10, 12 or 15 amino acids or nucleotides, up to a maximum of about 20 or 25 amino acids or nucleotides.
  • folding simulations and computer redesign of structural motifs of the protein of the invention can be performed using appropriate computer programs (Olszewski et al.1996, Hoffman et al.1995). Computer modeling of protein folding can be used for the conformational and energetic analysis of detailed peptide and protein models (Monge et al. 1995, Renouf et al. 1995).
  • the appropriate programs can be used for the identification of interactive sites of the LRR receptor-like kinase of the present invention by computer assistant searches for complementary peptide sequences (Fassina and Melli 1994).
  • Further appropriate computer systems for the design of protein and peptides are described in the prior art, for example in Berry and Brenner (1994), Wodak (1987), Pabo and Suchanek (1986).
  • the results obtained form the above-described computer analysis can be used for, e.g. the preparation of peptidomimetics of the protein of the invention or fragments thereof.
  • Such pseudopeptide analogues of the natural amino acid sequence of the protein may very efficiently mimic the parent protein (Benkirane et al. 1996).
  • incorporation of easily available achiral ⁇ -amino acid residues into a protein of the invention or a fragment thereof results in the substitution of amino bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic (Banerjee et al. 1996).
  • Superactive peptidomimetic analogues of small peptide hormones in other systems are described in the prior art (Zhang et al. 1996).
  • Appropriate peptidomimetics of the protein of the present invention can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive amine alkylation and testing the resulting compounds, e.g., for their binding, kinase inhibitory and/or immunlogical properties. Methods for the generation and use of peptidomimetic combinatioral libraries are described in the prior art, for example in Ostresh et al. (1996) and Dorner et al. (1996).
  • a three-dimensional and/or crystallographic structure of the protein of the invention can be used for the design of peptidomimetic inhibitors of the biological activity of the protein of the invention (Rose et al. 1996, Ruterber et al. 1996).
  • To align sequences we used the Clustal V method described in Higgins and Sharp (Higgins DG & Sharp PM, Comput Appl Biosci. 1989 Apr;5(2)151 -3).
  • the Clustal Method groups sequences into clusters by examining sequence distances between all pairs. Clusters are aligned as pairs then collectively as sequence groups to produce the overall alignment. After the multiple alignment is completed, a neighbor-joining method is employed to reconstruct phylogeny for the putative alignment. 1. Residue weight table
  • the Clustal method uses weight tables to construct multiple alignments. Residue weight tables are used in scoring protein and nucleotide alignments so that slightly mismatched residues, such as lie vs. Val amino acids or G vs. R nucleotides, are not scored the same as total mismatches. If your sequences are protein, or a mixture of protein and DNA, choose between
  • iPAM250 is recommended for the Clustal method and is ideal for longer sequences or highly diverged sequences.
  • Pairwise alignment parameters may include K-tuple, Gap Penalty, Window and Diagonals Saved:
  • K-tuple expressed as the unit's length in nucleotides. Any stretch of K or more adjacent nucleotides that form an exact match in both sequences is a K-tuple. A lower K-tuple will find smaller matching regions; a higher value will find fewer, but better, matching regions of similarity.
  • Gap Penalty the amount deducted from the score for each gap in the alignment. Gaps of different sizes carry the same penalty.
  • a phenogram averages the distances between ancestors in the tree. Dotted lines indicate a negative branch length, which may be a byproduct of averaging. In the cladogram, branch distances correspond to sequence divergence.
  • the compounds to be obtained or identified in the methods of the invention can be compounds that are able to bind to any of the nucleic acids, peptides or proteins of the invention.
  • Other interesting compounds to be identified are compounds that modulate the expression of the genes or the proteins of the invention in such a way that either the expression of said gene or protein is enhanced or decreased by the action of said compound. Alternatively the compound can exert his action by directly or indirectly enhancing or decreasing the activity of any of the proteins of the invention.
  • Said compound or plurality of compounds may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compound(s) may be known in the art but hitherto not known to be capable of suppressing or activating cell cycle interacting proteins.
  • the reaction mixture may be a cell free extract of may comprise a cell or tissue culture. Suitable set ups for the method of the invention are known to the person skilled in the art and are, for example, generally described in Alberts et al., Molecular Biology of the Cell, third edition (1994), in particular Chapter 17.
  • the plurality of compounds may be, e.g., added to the reaction mixture, culture medium or injected into the cell.
  • a sample containing a compound or a plurality of compounds is identified in the method of the invention, then it is either possible to isolate the compound form the original sample identified as containing the compound capable of acting as an agonist, or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample.
  • the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s).
  • said sample comprises substances or similar chemical and/or physical properties, and most preferably said substances are identical.
  • the compound identified according to the above described method or its derivative is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
  • Figure 1 Models for stem-cell maintenance in the root and shoot meristem.
  • the quiescent center functions as an organizing center. Those cells in immediate contact with the QC are stem cells (dark shading), which are initials for all cell files: endodermis (end), cortex (cort), epidermis (epi), lateral root cap (Ire) and columella (col),
  • WUS is functionally equivalent to the QC in the root meristem in maintaining the presence of stem cells (dark shading). Long- range signaling for stem cell maintenance is also postulated for ZLL, which is expressed in the vascular cells.
  • CLV3 is expressed in the presumptive stem cells that the initials for the L1 (epidermis), L2 (subepidermal tissues, i.e. mesophyll cells) and L3 (subepidermal tissues, i.e. vascular cells) tissue layers (Schnittger, Schellmann, & Hulskamp 1999).
  • CLV1 is found in the L3 tunica layer of the central zone.
  • Figure 2 List of the degenerative primers used for RT-PCR of root-specific members of the LRR receptor kinase family.
  • PCR fragments obtained upon amplification of R and C cDNAs with degenerate primers are separated on agarose gel. DNA fragments of different size can be detected in the R 22 compared to C 22 PCRs.
  • M represents the 100 bp marker (Boehringer).
  • C and R indicated the origin of the cDNA sample used in the PCR and the numbers below indicate the primer combination used, e.g. C 23 represents hypocotyl/cotyledon cDNA amplified with primers LRR2 and KIN3.
  • RCH1 F and RCH1 R primers As a control for cDNA quality and quantity as well as relative expression level, ubiquitin (Ubi) mRNA was amplified for 20 cycles under the same conditions using the UBIF and UBIR primers.
  • Figure 5 The nucleotide sequence of the RCH1 operon.
  • This figure shows het nucleotide sequence of SEQ ID NO 1 that contains the genomic RCH1 clone.
  • FIG. 1 The nucleotide sequence of the RCH1 promoter region.
  • This fragment of the genomic RCH1 clone is about 3498 base pairs and is identified as SEQ ID NO 2.
  • Two putative ATG start codons of the RCH1 protein on position 3479- 3481 and on position 3491-3493 could be identified and are represented in bold.
  • the underlined sequence corresponds to SEQ ID NO 18.
  • GUS Glucuronidase
  • Figure 8 The nucleotide sequence of the RCH1 gene.
  • This fragment of the genomic RCH1 clone is about 3960 base pairs long and is identified as SEQ ID NO 3.
  • Two putative ATG start codons of the RCH1 protein on position 1 -3 and on position 13-15 could be identified and are represented in bold. Also the TAA stopcodon on position 3958-3960 is represented in bold.
  • Figure 9 The amino acid sequence of the open reading frame encoding RCH1 protein.
  • FIG. 10 Amino acid alignment of the RCH1 protein the Arabidopsis thaliana CLV1 receptor kinase like protein.
  • RCH1 promoter is strongly active in endodermis, cortex, epidermis, lateral root cap, in short: in the "division zone".
  • the RCH1 promoter is also active but in a low manner, in the quiescent center and in the vascular tissue.
  • Example 1 Isolation of the RCH1 operon from root tissue of Arabidopsis thaliana .
  • Plant organ development depends on the presence of distally positioned groups of continuously dividing cells, the shoot and root apical meristems.
  • Laser ablation studies suggest that a balance between signals for proper differentiation and short range signals from the mitotically inactive quiescent centre (QC), required to keep cells less differentiated, is important to maintain the root meristem (van den Berg et al. 1995;van den Berg et al. 1997).
  • QC mitotically inactive quiescent centre
  • CLV WUSCHEL and CLAVATA
  • the CLV1 gene encodes a leucine rich repeat (LRR) receptor kinase (Clark, Williams, & Meyerowitz 1997), a member of a large gene family (Lease, Ingham, & Walker 1998).
  • Other members are ERECTA (ER) (Torii et al. 1996), BRASSINOLIDE INSENSITVE (BRl) (Li et al. 1997) and HAESA (HAE) (Jinn et al. 2000), genes which have a function in plant development
  • ERECTA ER
  • BRl BRASSINOLIDE INSENSITVE
  • HAE HAESA
  • PCR products were generated from root tip and hypocotyl/cotyledon cDNA pools using degenerate primers, resulting in the isolation of the root specific ROOT CLAVATA HOMOLOG 1 (RCH1).
  • the promoter region of this gene was translationally fused to ⁇ -glucoronidase-green fluorescent protein (GUS/GFP) conferring root specific expression of these reporter genes (see Example 2). Designing degenerate primers
  • LRR receptor kinase gene family To RT-PCR root specific members of the LRR receptor kinase gene family we compared the sequences of the CLV1, ER and HAE genes and their encoded proteins.
  • the degenerate LRR primers were designed against the NxLxGxlP encoding region of the xLxxNxLxGxlPxxLxxLxxL consensus for the LRR receptor-like kinases.
  • Degenerate kinase primers were disigned for different homologous regions of the kinase domain. The primers are listed in Figure 2.
  • RNA Isolation For each RNA isolation the roottips and the hypocotyl/cotyledon part including the shoot apical meristem of Arabidopsis thaliana (ColO) plants, 4 days after germination, were collected.
  • RT Reverse transcription
  • R root tip
  • C hypocotyl/cotyledon
  • the amplified R and C cDNAs were separated and compared with agarose gel electrophoresis ( Figure 3).
  • the root specific fragments e.g. fragments present in R 22 and not in C 22 , were isolated from gel, cloned into the pGEM-T vector (Promega) and transformed into E. coli (strain DH5 ⁇ ), using standard cloning procedures.
  • RCH1 in root tip (R), hypocotyl/cotyledon (C) and flowers (F) were determined using RT-PCR.
  • Primers specific for the RCH1 sequence were designed (RCH1F and RCH1R, Figure 2).
  • the PCR samples were analyzed with agarose gel electrophoresis.
  • Ubiquitin (Ubi) mRNA was used as an internal control (Horvath et al. 1993).
  • Polyubiquitin genes consist of multiple units (Callis et al. 1995) and the Ubi primers hybridize with the ends of each single unit.
  • Example 2 Promoter isolation and promoter fusion construct.
  • This deposit was given the accession number LMBP 5582CB by the international depositary authority.
  • a restriction map was constructed of the genomic clone and a 7 kb Xhol fragment, containing 3.5 kb promoter and part of the RCH1 coding sequence, was subcloned into the expression vector pBS (Stratagene, La Jolla, CA).
  • the 3.5 kb ' promoter region including the first amino acids of the putative RCH1 ORF is set forth in Figure 5 and is identified as SEQ ID NO 2.
  • This promoter region was amplified using the primers M13F and PRCH1 R and Pfu Taq polymerase (Stratagene, La Jolla, CA) in the manufacturer specified conditions.
  • the promoter region was subsequently cloned into the binary pCAMBIA3383Xb vector (Roberts et al. 2000) creating a translational fusion with the GUS/GFP reporter gene.
  • GUS histochemical assay Arabidopsis thaliana plants were transformed with this construct according to the vacuum transformation method (Bechtold & Pelletier 1998). Transformants were selected for phosphinothricin-tripeptide (PIT, Duchefa, NL) resistance. Histochemical localization of ⁇ -Glucuronidase activity (GUS) was performed using the substrate 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc, Biosynth AG, Staad, Switzerland). X-gluc was dissolved in n-dimethyl-formamide and diluted to 0.5 mg/ml in 50 mM NaP04, pH 7.2 with 0.1% Triton X-100.
  • Oxidative dimerization of the produced indoxyl derivative was enhanced by adding the oxidation catalyst K + ferricyanide/ferrocyanide to a final concentration of 0.5 mM (0.5 mM K 4 Fe(CN) 6 .H 2 O/0.5 mM K 3 Fe(CN) 6 ).
  • the GUS reaction was incubated at 37°C over several time periods, and pictures were taken from the cloured plant-tissues ( Figures 7 and 15).
  • Figure 7 the very specific expression pattern of the RCH1 promoter is visualized by the GUS reporter gene activity.
  • Persons skilled in the art will also agree to the fact that the expression level of the RCH 1 promoter is very high, since after one hour the saturation level of staining is reached in the meristem of the main root from a 1 week old seedling ( Figure 7 D).
  • RCH1 ::GFP expression in roots is shown in Figure 16.
  • Example 3 Alignment.
  • Sequence alignment of one complete register sequence against a target sequence as in figure 10 was done using the program GAP of the GCG package.
  • the algorithm of Needleman and Wunsch is applied here to find the alignment of two complete sequences (Needleman and Wunsch, JMB 48(3): 443-453, 1970).
  • the used parameters during the alignment were Gap Weight: 8, Average Match: 2.912, Length Weight: 2, Average Mismatch: -2.003, Quality: 1388, Length: 1149, Ratio: 1.416, and Gaps: 25.
  • the Clustal method uses weight tables to construct multiple alignments. Residue weight tables are used in scoring protein and nucleotide alignments so that slightly mismatched residues, such as lie vs. Val amino acids or G vs. R nucleotides, are not scored the same as total mismatches.
  • the PAM250 weight table was used for the alignment and pylogenetic trees in figure 11 and 12 .
  • Other method parameters for pairwise alignment were K-tuple: 1 , Gap Penalty: 3, Window: 5 and Diagonals: 5.
  • Other method parameters for multiple alignment include Gap Penalty: 10 and Gap Length Penalty: 10. To view phylogenetic relationships compatible with multiple sequence alignments a Phylogenetic Tree was calculated.
  • the length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
  • the phylogenetic tree gives a first estimate of the relationship between homologous sequences. A cladogram presentation of the phylogenetic tree is shown in figures 11 and 12.
  • Example 5 Root-specific reporter gene expression in Rice mediated by the Arabidopsis thaliana RCH 1 promoter.
  • CLAVATA homologs are found in many other plant species from both the groups of monocots and dicots. Therefore one can assume that also the promoter of RCH1 is functional in those other plant species, and possibly exerts the same specific expression- pattern.
  • the aforementioned promoter-reporter gene construct wherein the GUS/GFP chimeric gene is operably linked to the RCH1 promoter, will be transformed to rice using the standard transformation procedures well known to the persons skilled in the art and outlined in the following paragraph. After several time periods ranging from 1 day to 1 or more weeks, the seedling will be checked for the expression of the GUS reporter gene. This will be done by growing the seedlings in organogenesis medium, and staining them with glucuronidase for several time periods ranging from 1 hour to 5 hours.
  • the Promoter-GUS hybrid gene outlined in Example 2 can be transformed to Agrobacterium tumefaciens strain LBA4404 or C58 by means of electroporation and subsequently transformed bacterial cells can be selected on a solid agar medium containing the appropriate antibiotics.
  • Example 6 Expression of a cytokinine oxidase cDNA under the root-specific RCH1 promoter in transgenic Tobacco and Rice leads to increased root production.
  • the AtCKXI gene (located in the database with the accession number: AC002510, Arabidosis thaliana chromosome II section 225 of 255 of the complete sequence, sequence from clone T32G6) can be cloned under the control of the RCH1 promoter of Arabidopsis, in a binary vector.
  • the ZmCKXI from maize (accession number AF044603, (Morris et al. 1999) can be operably linked to the RCH1 promoter a binary vector.
  • These genes can be transfected into Tobacco or Rice plants, using agrobacterium-mediated gene transfer.
  • the transgenic plants expressing the ATCKX1 or the ZmCKXI specifically in roots are expected to show increased root production without negatively affecting shoot development and without inducing premature leaf senescence.
  • the length of the RCH1 promoter region (SEQ ID NO 2), which confers to root specific expression patterns, can be reduced to a "minimal promoter" without altering the root specific expression patterns or other features of the promoter such as constitutive and strong activity.
  • the length of the promoter region was reduced to a minimal promoter sequence ranging from the -1 base pair just before the start codon at position 3479-3481 of SEQ ID NO. 2 to -842 base pairs before said start codon.
  • Said minimal promoter is represented in SEQ ID NO. 18. This minimal promoter was PCR amplified and translationally fused to the GUS reporter gene.
  • Example 2 With a GUS histochemical assay (example 2) the same expression pattern as the large promoter region was demonstrated (results not shown). The difference is that the sequence before the Hindi 11 site at the beginning of the minimal promoter is not necessary for the root specific expression pattern.
  • Siliques of Arabidopsis plants transformed with RCH1prom::GUS were opened at different stages of development and the embryo's were surgically taken out of the ovules and transferred to X-gluc staining solution. Embryo's were stained for 0.5h - 4h before pictures were taken using light microscopy, Fig 15 A and B are at 25X and 40X magnification respectively, stained for 0.5 h. Longer staining did not reveal expression outside the root promeristem region.
  • RCH1 is active very early: RCH1 expression was first observed in torpedo stage embryo's in the root promeristem and was maintained in this region during the later stages of development. This figure shows that as soon as a meristem is formed RCH1 is expressed and provides further evidence that RCH1 expression is correlated with the appearance and presence of a root meristem from the embryonic stage onwards.
  • AB028621 (see figure 12, phylogenetic tree) is determined to be RCH1.2 protein and is also used by the inventors for further experiments. Double knock outs of RCH1 and RCH1.2 were made and also other closely related sequences (AB011476.pro, T05050.pro and AC015446.pro) are used in knock out experiments or in antisense expression.
  • RCH1 in normal cells correlates with the zone of rapidly dividing cells and is involved in the maintenance of meristem cells and dividing cells. Improved activity of RCH1 in a host cell results in the maintenance of cell division in that host cell and thus works against the differentiation of that cell. On the contrary diminished RCH1 activity in a host cell results in a diminished cell division in that host cell and thus in the loss of the amount of dividing cells in the meristem.
  • the coding region of the RCH1 gene is fused to a strong promoters that is inducible or constitutive and/or ubiquitous or tissue specific.
  • the RCH1 coding region is cloned under control of other meristemic promoters such as the promoters of
  • the RCH1 gene and the RKN gene are operationally linked to the root-specific promoter PYK10, a constitutive ubiquitin or GOS 2 promoter and Cdc2a promoter and expressed in Arabidopsis or in rice.
  • the coding region of the RCH1 gene cloned in a sense and antisense orientation in order to form a hairpin that induces gene-silencing in the host cell.
  • Downregulation or upregulation of the RCH1 in combination with downregulation or upregulation of other genes e.g.: With WUSHEL: In the shoot meristem a balance between the WUSCHEL and CLAVATA (CLV) genes has been implicated to play an essential role in regulation cell division and differentiation (Clark et al. 1993;Fletcher et al. 1999;Schoof et al. 2000). The whole meristem maintenance is controlled by these genes and they seem to have an opposite function keeping the balance between cell division and differentiation. Therefore, WUSCHEL or a root WUSCHEL homologue is a preferred partner to be co-expressed with RCH1.
  • CLV CLAVATA
  • RCH1 is upregulated in combination with the downregulation of WUSCHEL in a root specific manner.
  • the obtained effect of this co- expression is a better control on the meristem maintenance.
  • RCH1 family members Possibly the function of RCH1 is redundant and other closely related genes can take over its function. Therefore the RCH1 gene ectopically expressed in combination with closely related genes in order to downregulate or upregulate the activity of the encoded protein.
  • the RCH1 homologues used for co-expression are for example AB028621.pro, AB011476.pro, T05050.pro and AC015446 (see figure 12 and table 4). The obtained effect is a better effect as described for the ectopic expression of RCH1 alone.
  • Clavata homologues are for example AB028621.pro, AB011476.pro, T05050.pro and AC015446 (see figure 12 and table 4). The obtained effect is a better effect as described for the ectopic expression of R
  • Clavata 1 is a receptor that is involved in a signal pathway probably through the binding of a ligand (e.g. Clavata 3).
  • a particular useful application of the present invention is the co-expression of RCH1 with the root Clavata 3 homologue RCH3. The obtained effect is a better control on meristem maintenance.
  • Example 10 Domain swapping
  • the N-terminal LRR domain of RCH1 functions as an extracellular perception domain and a C-terminal kinase domain functions in an intracellular signal transduction pathway.
  • the domains of RCH1 are swapped with the domains of another protein.
  • the LRR domain of RCH1 is replaced by the LRR domain of BRI.
  • the effect obtained is the activation of a different pathway, which is normally not activated by RCH1 , because the kinase domain of RCH1 now responds to another external signal that normally is not recognized by RCH1.
  • Clark S.E. Running M.P., & Meyerowitz E.M. (1993) CLAVATA1 , a regulator of meristem and flower development in Arabidopsis. Development 119, 397-418. Clark S.E., Williams R.W., & Meyerowitz E.M. (1997) The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89, 575-585.
  • the M015 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.
  • CDKs cyclin-dependent kinases
  • Haseloff J. et al. (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly.
  • the cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.
  • SbPRPI soybean cell wall protein gene, in roots of transgenic tobacco and cowpea. Plant Mol.Biol. 21 , 109-1 19.
  • Torii K.U. et al. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats. Plant Cell 8, 735-746. Trieu AT. et al. (2000) Technical Advance: Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 22, 531-541. Uribe X. et al. (1998) Maize alpha-tubulin genes are expressed according to specific patterns of cell differentiation. Plant Mol.Biol. 37, 1069-1078. van den Berg C. et al. (1995) Cell fate in the Arabidopsis root meristem determined by directional signalling .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne le domaine de la biologie moléculaire végétale et plus particulièrement l'expression de gènes spécifiques des racines. L'invention concerne notamment des acides nucléiques pour un nouveau promoteur et un nouvel acide nucléique régulateurs transcriptionnels spécifiques des racines et des séquences de protéines codant pour une nouvelle protéine kinase de type récepteur du domaine LRR (à répétitions riches en leucines), spécifiée comme RCH1 (root clavata 1 homolog). L'invention concerne également des compositions contenant des acides nucléiques, des polypeptides, des anticorps et des vecteurs. L'invention concerne en outre des méthodes permettant de modifier le sort des cellules et/ou le développement de la plante et/ou la morphologie de la plante et/ou la biochimie de la plante et/ou la physiologie de la plante en modifiant l'expression dans des cellules, tissus ou organes particuliers, d'une plante de la nouvelle kinase de type récepteur du domaine LRR ou en exprimant un gène d'intérêt sous le contrôle du nouveau promoteur régulateur transcriptionnel spécifique des racines. L'invention concerne enfin des composés interagissant avec les nouveaux polypeptides destinés à être utilisés comme herbicides ou régulateurs de la croissance.
PCT/EP2001/014154 2000-12-04 2001-12-04 Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr WO2002046439A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/433,731 US20040067506A1 (en) 2000-12-04 2001-12-04 Novel root specific promoter driving the expression of a novel lrr receptor-like kinase
AU2002237224A AU2002237224A1 (en) 2000-12-04 2001-12-04 A novel root specific promoter driving the expression of a novel LRR receptor-like kinase
CA002430617A CA2430617A1 (fr) 2000-12-04 2001-12-04 Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP00870288.8 2000-12-04
EP00870288 2000-12-04
US25620400P 2000-12-15 2000-12-15
US60/256,204 2000-12-15

Publications (1)

Publication Number Publication Date
WO2002046439A2 true WO2002046439A2 (fr) 2002-06-13

Family

ID=26074314

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/014154 WO2002046439A2 (fr) 2000-12-04 2001-12-04 Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr

Country Status (3)

Country Link
AU (1) AU2002237224A1 (fr)
CA (1) CA2430617A1 (fr)
WO (1) WO2002046439A2 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1382682A2 (fr) * 2002-07-17 2004-01-21 Expressive Research B.V. Modulation des voies de développement des plantes
WO2004038027A1 (fr) * 2002-10-24 2004-05-06 Cropdesign N.V. Bioremediation
WO2005035770A1 (fr) * 2003-10-09 2005-04-21 Pioneer Hi-Bred International, Inc. Promoteur du mais a preference radicale denomme crwaq81
WO2014024079A2 (fr) * 2012-08-09 2014-02-13 Basf Plant Science Company Gmbh Plantes exprimant rlk1 résistant aux pathogènes fongiques
WO2014033672A1 (fr) 2012-08-30 2014-03-06 Institut National De La Recherche Agronomique UTILISATION D'UN RÉCEPTEUR KINASE À MOTIFS LysM POUR AMÉLIORER LA RÉPONSE DES PLANTES AUX LIPOCHITOOLIGOSACCHARIDES.
WO2014076659A1 (fr) * 2012-11-15 2014-05-22 Basf Plant Science Company Gmbh Procédé de production de plantes dotées d'une résistance augmentée aux agents pathogènes
WO2020035486A1 (fr) 2018-08-13 2020-02-20 Aarhus Universitet Plantes génétiquement modifiées exprimant des récepteurs hétérologues qui reconnaissent les lipo-chitooligosaccharides
WO2020035488A1 (fr) 2018-08-13 2020-02-20 Aarhus Universitet Récepteurs lysm génétiquement modifiés ayant une spécificité et une affinité d'agoniste modifiées
WO2020115181A1 (fr) 2018-12-06 2020-06-11 Wageningen Universiteit Procédés de modification génétique d'un gène nin de plante la rendant sensible à la cytokinine
WO2020187995A1 (fr) 2019-03-21 2020-09-24 University Of Essex Enterprises Limited Procédés d'amélioration de la biomasse dans une plante par stimulation de la régénération de rubp et le transport d'électrons
US10833198B2 (en) 2019-03-14 2020-11-10 International Business Machines Corporation Confined source drain epitaxy to reduce shorts in CMOS integrated circuits
WO2021233904A1 (fr) 2020-05-19 2021-11-25 Aarhus Universitet Motifs de récepteur lysm
WO2022251428A2 (fr) 2021-05-26 2022-12-01 The Board Of Trustees Of The University Of Illinois Plantes en c4 à efficacité photosynthétique accrue
WO2023201230A1 (fr) 2022-04-11 2023-10-19 The Regents Of The University Of California Procédés de criblage de gain de plante de mutations de fonction et compositions associées

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7838728B2 (en) 2002-07-17 2010-11-23 Expressive Research B.V. Modulating developmental pathways in plants
WO2004007712A3 (fr) * 2002-07-17 2004-03-18 Expressive Res Bv Modulation des processus de developpement des plantes
EP1382682A3 (fr) * 2002-07-17 2004-06-30 Expressive Research B.V. Modulation des voies de développement des plantes
EP1382682A2 (fr) * 2002-07-17 2004-01-21 Expressive Research B.V. Modulation des voies de développement des plantes
WO2004038027A1 (fr) * 2002-10-24 2004-05-06 Cropdesign N.V. Bioremediation
WO2005035770A1 (fr) * 2003-10-09 2005-04-21 Pioneer Hi-Bred International, Inc. Promoteur du mais a preference radicale denomme crwaq81
US7411112B2 (en) 2003-10-09 2008-08-12 Pioneer Hi-Bred International, Inc. Maize promoter named CRWAQ81
US11142774B2 (en) 2012-08-09 2021-10-12 Basf Plant Science Company Gmbh Fungal resistant plants expressing RLK1
WO2014024079A2 (fr) * 2012-08-09 2014-02-13 Basf Plant Science Company Gmbh Plantes exprimant rlk1 résistant aux pathogènes fongiques
WO2014024079A3 (fr) * 2012-08-09 2014-04-03 Basf Plant Science Company Gmbh Plantes exprimant rlk1 résistant aux pathogènes fongiques
US10329580B2 (en) 2012-08-09 2019-06-25 Basf Plant Science Company Gmbh Fungal resistant plants expressing RLK1
US11708583B2 (en) 2012-08-09 2023-07-25 Basf Plant Science Company Gmbh Fungal resistant plants expressing RLK1
WO2014033672A1 (fr) 2012-08-30 2014-03-06 Institut National De La Recherche Agronomique UTILISATION D'UN RÉCEPTEUR KINASE À MOTIFS LysM POUR AMÉLIORER LA RÉPONSE DES PLANTES AUX LIPOCHITOOLIGOSACCHARIDES.
WO2014076659A1 (fr) * 2012-11-15 2014-05-22 Basf Plant Science Company Gmbh Procédé de production de plantes dotées d'une résistance augmentée aux agents pathogènes
WO2020035488A1 (fr) 2018-08-13 2020-02-20 Aarhus Universitet Récepteurs lysm génétiquement modifiés ayant une spécificité et une affinité d'agoniste modifiées
WO2020035486A1 (fr) 2018-08-13 2020-02-20 Aarhus Universitet Plantes génétiquement modifiées exprimant des récepteurs hétérologues qui reconnaissent les lipo-chitooligosaccharides
WO2020115181A1 (fr) 2018-12-06 2020-06-11 Wageningen Universiteit Procédés de modification génétique d'un gène nin de plante la rendant sensible à la cytokinine
US10833198B2 (en) 2019-03-14 2020-11-10 International Business Machines Corporation Confined source drain epitaxy to reduce shorts in CMOS integrated circuits
WO2020187995A1 (fr) 2019-03-21 2020-09-24 University Of Essex Enterprises Limited Procédés d'amélioration de la biomasse dans une plante par stimulation de la régénération de rubp et le transport d'électrons
WO2021233904A1 (fr) 2020-05-19 2021-11-25 Aarhus Universitet Motifs de récepteur lysm
WO2022251428A2 (fr) 2021-05-26 2022-12-01 The Board Of Trustees Of The University Of Illinois Plantes en c4 à efficacité photosynthétique accrue
WO2023201230A1 (fr) 2022-04-11 2023-10-19 The Regents Of The University Of California Procédés de criblage de gain de plante de mutations de fonction et compositions associées

Also Published As

Publication number Publication date
CA2430617A1 (fr) 2002-06-13
AU2002237224A1 (en) 2002-06-18

Similar Documents

Publication Publication Date Title
US20040067506A1 (en) Novel root specific promoter driving the expression of a novel lrr receptor-like kinase
US7427697B2 (en) Sugar beet genes involved in stress tolerance
US7332316B2 (en) Plant cytokinin oxidase
AU2002237249A1 (en) Sugar beet genes involved in stress tolerance
US7619146B2 (en) Method for modifying plant morphology, biochemistry and physiology
WO2002046439A2 (fr) Nouveaux promoteurs specifiques des racines activant l'expression d'une nouvelle kinase de type recepteur du domaine lrr
US20100095398A1 (en) Protection against environmental toxicity through manipulation of the processing of messenger RNA precursors
EP1458874A2 (fr) Procede de modification de la morphologie, biochimie et physiologie des plantes comprenant l'expression d'une cytokinine oxydase des plantes
EP1334121B1 (fr) Gene regulant le developpement des vegetaux et ses utilisations
EP1301532B1 (fr) Inhibiteurs de kinase a dependance de cycline dans des plantes
US20050050591A1 (en) Novel plant cyclin
WO2002053589A2 (fr) Molecules d'acides nucleiques codant des interacteurs dim et utilisations
AU2001265889A1 (en) Protection against environmental toxicity through manipulation of the processing of messenger rna precursors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2002237224

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2430617

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 10433731

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP