WO2014127835A1 - Gène de résistance dérivé d'une plante - Google Patents

Gène de résistance dérivé d'une plante Download PDF

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WO2014127835A1
WO2014127835A1 PCT/EP2013/053642 EP2013053642W WO2014127835A1 WO 2014127835 A1 WO2014127835 A1 WO 2014127835A1 EP 2013053642 W EP2013053642 W EP 2013053642W WO 2014127835 A1 WO2014127835 A1 WO 2014127835A1
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plant
sequence
nucleic acid
expression
gene
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PCT/EP2013/053642
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Christian Jung
Sarah JÄGER
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Christian-Albrechts-Universität Zu Kiel
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • 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 beet cyst nematode (BCN) Heterodera schachtii Schmidt Resistance Gene Hsl-2. It further relates to methods and materials employing the gene, and processes for identifying or producing other related genes. It also relates generally to methods for identifying plant protective agents which are capable of inducing the Hsl-2 gene or the activity of its encoded protein. Furthermore, the present invention relates to transgenic plants which become resistant to nematodes, in particular Heterodera schachtii because of the expression of an Hsl-2 transgene.
  • BCN beet cyst nematode
  • Hsl-2 Heterodera schachtii Schmidt Resistance Gene
  • Sugar beet ⁇ Beta vulgaris L. ssp. vulgaris is a biennial dicotyledonous plant that produces during its first growing season a leaf rosette and a large taproot with a sugar content of 15 - 20%.
  • the nutrients are used for the formation of reproductive organs induced by long day conditions after exposure to low temperatures, which is called vernalization (Marlander et al., 2011).
  • vernalization Marlander et al., 2011
  • Important yield reducing diseases and pathogens of the sugar beet crop are the fungi Rhizoctonia solani, Cercospora beticola, Erisyphe betae, and Ramularia beticola, the rhizomania virus (beet nectrotic yellow vein virus) and the beet cyst nematode (BCN) Heterodera schachtii (Biancardi et al., 2010).
  • BCN biotrophic beet cyst nematode
  • the line Pro3 carries a translocation from P. procumbens chromosome 7 that includes the resistance gene Hs2pro-7.
  • the translocation line TR520 has been generated by a cross of the resistant homozygous translocation line A906001 with the sugar beet line 930190, resulting in the Fl population 940081.
  • the translocation line TR520 has been derived from a backcross (BC4) between 940081 and 930190, and it is hemizygous for the translocation.
  • TR520 and TR363 (formerly Pro 4) bear monogenic resistance derived from chromosome 1 of P. procumbens (Jung and Wricke, 1987; Kleine et al., 1995).
  • the wild beet translocation of the resistant line TR520 has been extensively studied for more than 20 years and has been estimated to be -1,500 kb in size.
  • a first nematode resistance gene Hslpro-1 has been positionally cloned (Cai et al., 1997).
  • the existence of a second nematode resistance gene is suggested as the resistant line TR363 shares homologous sequences with TR520 but does not carry the gene Hslpro-1.
  • nematode resistant lines (TR520 and TR363) carrying translocations of the wild beet Patellifolia procumbens were sequenced using next-generation sequencing (NGS) technology and two susceptible (TR659 and TR320), sugar beet translocation lines which share partly homologous sequences from P. procumbens chromosome 1, were the focus of experiments performed in accordance with the present invention.
  • NGS next-generation sequencing
  • TR659 and TR320 sugar beet translocation lines which share partly homologous sequences from P. procumbens chromosome 1
  • a second gene for nematode resistance ⁇ Hsl-2 is located on a region shared by both resistance carrying translocations.
  • Hsl-2 a Beet Cyst Nematode Resistance Gene has been cloned and characterized at the molecular level.
  • the gene was identified by a particular combined positional cloning and candidate gene approach.
  • ORF 803 which shows an overall sequence similarity of 34% on the amino acid level to a mitogen activated protein kinase kinase kinase (MAP3K) that is known to be involved in plant resistance response was identified on a region shared by the resistant translocation lines TR520 and TR363.
  • RT-PCR reverse transcriptase-PCR
  • procumbens reference assemblies clearly showed an affiliation to the wild beet genome.
  • the BpPIPl gene could be identified on scaffold 42352 of the translocation line TR520. This scaffold overlaps with scaffold 20220 of P. procumbens and is integrated into super contig 2-3, linked to the distal side of BAC 74E5 in section E and H. Both sections are not present on translocation TR363 but on both susceptible translocation lines TR320 and TR659. In conclusion, it could not be confirmed that any of these genes is the nematode resistance gene Hsl-2 based on functional analysis of ORF 702 and on sequence analyses of cZR-3, cZR-7 and BpPIPl.
  • translocation lines TR520 and TR363 new gene candidates have been identified and characterized after whole genome shotgun (WGS) sequencing of the translocation lines TR520 and TR363.
  • WGS whole genome shotgun
  • the sequence of the BAC based physical map of the translocation line TR520 was extended by 1,193 kpb.
  • the translocation lines TR520 and TR363 were sequenced by a WGS approach using an Illumina HiSeq2000 sequencer. This resulted in ⁇ 87 Gbp raw sequence data ( ⁇ 110 fold genome coverage / line).
  • a hybrid assembly was performed, combining short reads of the resistant translocation lines TR520 and TR363, 1,032 kbp BAC and YAC sequences of the physical map of the translocation line TR520, and draft reference assemblies of a sugar beet and a wild beet whole genome sequence.
  • 477 kbp of new sequences were identified anchored to the physical map.
  • the total sequence of the physical map adds up to 1 ,509 kbp.
  • 13 new super scaffolds were assembled that integrate scaffolds and contigs of P. procumbens and the translocations TR520 and TR363 that are not anchored to the physical map.
  • the new sequences identified (716 kbp) increase the size of the TR520 translocation to 2,226 kbp, corresponding to ca. 150% of the previously estimated value (1,500 kbp).
  • ORF 803 which shows an overall sequence similarity of 34% on amino acid level to a mitogen activated protein kinase kinase kinase (MAP3K).
  • MAP3K mitogen activated protein kinase kinase kinase
  • the present invention relates to a nucleic acid molecule encoding a polypeptide which is capable of conferring resistance against sedentary nematodes in a plant in which said polypeptide is expressed, said nucleic acid molecule comprising or consisting of a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences encoding at least one transglutaminase domain of Hsl-2 corresponding to amino acid position 75 to 321 or 536 to 601 of Figure 7 (SEQ ID NO: 2); or the transglutaminase domain corresponding to amino acid position 131 to 336 of Figure 8 (SEQ ID: 4), or an amino acid sequence at least 60 % homologous to any one thereof and/or a protein kinase domain of Hsl-2 corresponding to amino acid position 937 to 1071 of Figure 7 (SEQ ID NO: 2) or amino acid position 666 to 919 of Figure 8 (SEQ ID NO: 4) or an amino acid sequence at least 90 % homologous to either domain;
  • nucleotide sequence encoding a protein derived from the protein encoded by a nucleotide sequence of any one of (a) or (b) by way of substitution, deletion and/or addition of one or several amino acids of the amino acid sequence encoded by the nucleotide sequence of (a) or (b), wherein said nucleotide sequence is at least 40%> homologous to the nucleotide sequences of (a) or (b);
  • nucleotide sequence obtainable by screening an appropriate library under stringent conditions with a probe having at least 15 consecutive nucleotides of a nucleotide sequence of any one of Figures 10 and 11 (SEQ ID NOS: 1 and 3) or comprising any one of the sequences set forth in Table 2.
  • nucleic acid molecule encoding a polypeptide which is capable of conferring resistance against a pathogen, such as nematodes, in a plant into which said polypeptide is expressed.
  • Nucleic acid molecules according to the present invention may be provided in recombinant form or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function.
  • the nucleic acid molecules (and their encoded polypeptide products) may also be (i) isolated and/or purified from their natural environment (although not necessarily in pure form per se), or (ii) in substantially pure or homogeneous form.
  • Nucleic acid according to the present invention may include DNA, cDNA, RNA, genomic DNA, preferably the intact gene, and may be wholly or partially synthetic (constructs). Where a DNA sequence is specified, e.g. with reference to a Figure or SEQ ID NO, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Also encompassed is the complement of the various disclosed sequences, which may be used in probing experiments, PCR or in down-regulation of the sequence.
  • a particular aspect of the invention is a nucleic acid molecule having the sequence all or part of the sequence shown in Figure 10 (SEQ ID NO: 1) including (where appropriate) both coding and/or non-coding regions.
  • Figure 10 SEQ ID NO: 1
  • ORF open reading frame
  • the Hsl-2 gene (ORF803) identified in accordance with the present invention is present in a region shared of the two nematode resistant lines TR520 and TR363 and in full length seems to be exclusively expressed in both resistant lines.
  • the deduced amino acid sequence shows a predicted amino acid sequence with high sequence similarity to a MAP3K.
  • MAP3K stands for mitogen activated protein kinase kinase kinase which is member of the mitogen-activated protein kinase (MAPK) cascade.
  • MAPK mitogen activated protein kinase kinase kinase which is member of the mitogen-activated protein kinase (MAPK) cascade.
  • MAPK mitogen-activated protein kinase
  • this gene may have putative functions in signaling cascades in resistance pathways such as PTI.
  • the Rhgl locus from soybean encodes for an LRR-receptor kinase and is discussed to be involved in resistance reactions against the soybean cyst nematode (Heterodera glycines) (Melito et al., 2010; Kandoth et al., 2011)
  • the Pto gene from tomato is an intracellular serine/threonine protein kinase that activates resistance response to Pseudomonas syringae infection in tomato (Oh and Martin, 2011).
  • ORF803 shows homology to genes involved in plant resistance responses and may therefore be relevant in the sugar beet cyst nematode pathosystem.
  • EDRl and CTRl represent two of the best studied A. thaliana MAP3K and are known to be involved as negative regulators in ethylene-mediated signaling and defense response (Frye and Innes, 1998; Frye et al., 2001; Huang et al., 2003; Lin and Grierson, 2010; Lin et al., 2008; Tang et al, 2005).
  • the role of MAP3K in plant defense response is established.
  • the defense response of PTI in plants relies on the recognition of pathogen specific molecules and is accomplished by receptor kinases following a signaling cascade involving additional kinases, e.g.
  • the genomic sequence of ORF803 was screened against transcriptome data, i.e. see DNA sequences derived from R A of inoculated root material of the nematode resistant translocation line TR520 and revealed an mRNA sequence (52364_c0_seq2) encoding an amino acid sequence having a sequence similarity of up to 100% but lacking some of the exons predicted from the genomic sequence of ORF803; see also Figure 6.
  • nucleotide sequence 52364_c0_seq2 represents the actual mRNA resulting from the transcription and processing of the RNA transcribed from the Hsl-2 gene, i.e. see the genomic sequence of ORF803. This conclusion is also supported by the fact that the amino acid sequence deduced from the mRNA in the protein kinase domain has a greater stretch of homology to the corresponding amino acid sequence of MAP3K of A. thaliana compared to the amino acid sequence deduced from the genomic ORF803; see Figure 7.
  • the deviations of the coding sequence deduced from genomic DNA from the nucleotide sequence of the mRNA can also be explained by the fact that the genomic sequence of ORF803 is located at the end of scaffold 26267 of the "TR502 de novo" assembly that contains gaps, i.e. some of 3' sequences of the ORF803, i.e. the Hsl-2 gene is missing and with respect to the coding sequence have thus to be corrected and supplemented with those of the mRNA sequence52364_c0_seq2. For this reason, the genomic sequence is to be reevaluated, for example by re-sequencing of the corresponding genomic DNA from P.
  • Hsl-2 in particular when stated in italics refers to ORF803g, i.e. the genomic DNA sequence illustrated in Figure 10 and depicted in Figure 10 (SEQ ID NO: 1).
  • Hsl-2 also refers to the gene encoding the amino acid sequence of ORF803mRNA (52364_c0_seq2) shown in Figure 8 and depicted in SEQ ID NO: 4.
  • Hsl-2 protein refers to the polypeptide encoded by the Hsl-2 gene, i.e.
  • the protein comprising at least one exon identified in the Hsl-2 gene (ORF803g) and/or encoded by the ORF803mRNA (52364_c0_seq2); see also Figure 11.
  • ORF803g the protein comprising at least one exon identified in the Hsl-2 gene
  • ORF803mRNA 52364_c0_seq2
  • Figure 11 the protein comprising at least one exon identified in the Hsl-2 gene (ORF803g) and/or encoded by the ORF803mRNA (52364_c0_seq2); see also Figure 11.
  • nucleotide and the amino acid sequence identified in accordance with the present invention and represented in the appended Figures as well as sequence listing relate to the preferred embodiments of the present invention, due to genomic variance, silent mutations, conservative amino acid substitutions, deletions, additions and the like which remain the biological activity of the Hsl- 2 polypeptide, i.e. capability of conferring resistance to nematodes una
  • the Hsl-2 sequences which may for instance be mutants or other derivatives, or naturally occurring Hsl-2 homologues such as allelic variants, paralogues (from the same species, but at a different location e. g. pseudoalleles at linked loci), or orthologues (related genes from different species).
  • the variant encodes a product which is homologous (similar) to Hsl-2, which may be isolated or produced on the basis of that sequence, and is capable of conferring pathogen resistance against one or more pathogens.
  • the nucleic acid molecule of the present invention is derived from a wild species of the section Procumbens of the genus Beta.
  • the nucleic acid molecule of the present invention preferably confers resistance against nematodes in a plant selected from the group of families consisting of Solanaceae, Amaranthaceae, and Brassicaceae. Most preferably, the nucleic acid molecule of the present invention confers resistance against sedentary nematodes in plants of species Beta vulgaris and/or Brassica spec.
  • Resistance gene activity can be tested by conventional methods known in the art, as appropriate to the nature of the resistance being investigated.
  • Example methods can be found in the following publications: bacterial (Grant, (1995) Science 269, 843-846); fungal (Dixon, (1996) Cell 84, 451-459; Jones, (1994) Science 266, 789- 793; Thomas, (1997) The Plant Cell 9, 2209-2224; nematode and viral (Whitham, (1994) Cell 78, 1101-1115).
  • activity is tested by complementation of the trait in a plant; see Example 5. This can be achieved by using the isolated gene or for example by coupling the putative active variant to a promoter and terminator for expression in plants and transforming it into a susceptible plant that lacks a given resistance trait.
  • the activity of the Hsl-2 variant is then confirmed by challenge with the appropriate pathogen.
  • a transient expression assay can be used to test for activation of the Hsl-2 variant analogous to the assay used by Mindrinos, (1994) Cell 78, 1089-1099. Briefly, the putative active Hsl-2 variant is co-expressed from a plasmid with a pathogen-derived gene which is an elicitor of the resistance specified by the putative Hsl-2 homologue and a reporter gene (e.g. GUS). If the variant is activated by the continuous expression of the pathogen derived gene, then an HR would result and the reporter gene activity would be abolished. If no activity was initiated, then the reporter gene would be detectable.
  • resistance gene activity is tested as illustrated in the Examples, e.g. in a complementation study using sugar beet hairy roots beet hairy roots and transgenic Arabidopsis thaliana exposed to beet cyst nematode (BCN) Heterodera schachtii Schmidt.
  • Plant parasitic nematodes comprise about 15% of the phylum Nematoda and are responsible for global agricultural losses of $ 157 billion annually (Abad et al., 2008). All agriculturally important crops, such as soybean, rice, maize, wheat, potato, tomato, and beet, are hosts for one or more species of nematodes.
  • the nucleic acid molecule of the present invention is capable of conferring resistance in a plant is selected from the group of genera consisting of Beta, Brassica, and Solarium.
  • the largest and economically most significant groups of plant parasitic nematodes are comprised in the order Tylenchida as the sedentary endoparasitic nematodes of the genera Meloidogyne, Heterodera and Globodera as well as the migratory endoparasitic nematodes of the genus Pratylenchus (Abad and Williamson, 2010; Fuller et al., 2008).
  • the nucleic acid molecule of the present invention preferably confers resistance against sedentary nematodes selected from the group of genera consisting of Meloidogyne, Heterodera, and Globodera.
  • the nucleic acid molecule of the present invention confers resistance against Heterodera schachtii.
  • Similarity or homology between the variant and Hsl-2 may be as defined and determined by the TBLASTN program, of Altschul, (1990) J. Mol. Biol. 215, 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 10, January 1999, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711 ), which has been used to calculate sequence homologies in the present application. DNASTAR software using the CLUSTAL method with PAM250 residue weight table (gap penalty 10, gap length 10) may also be used. Homology (or similarity, or identity) may be at the nucleotide sequence and/or the expressed amino acid sequence level.
  • the nucleic acid and/or amino acid sequence shares homology with the coding sequence or the amino acid sequence encoded by the nucleotide sequence of Figure 10 or 11 (SEQ ID NO: 1 or 3) or other sequences set out herein, preferably at least about 40%>, 50%>, or 60%>, or 70%>, or 80%> homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
  • Homology may be over the full- length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about e. g. 20, 100, 200, 300, 500, 600 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.
  • Naturally occurring Hsl-2 variants may be isolated, in the light of the present disclosure, without burden from any suitable plant.
  • Naturally occurring Hsl-2 variants may be isolated, from e.g. genomic or cDNA.
  • the putative resistance genes can be obtained using materials ⁇ e.g. primers or probes) based on regions peculiar to Hsl-2, for instance designated in Figures 7 and 8.
  • the Hsl-2 gene identified according to the present invention in P. procumbens is expected to define a novel class of plant resistance genes. Corresponding genes encoding proteins displaying similar properties should therefore be present in other plants as well.
  • Nucleic acid molecules of the invention can be obtained, e.g., by hybridization of the above- described nucleic acid molecules with a (sample of) nucleic acid molecule(s) of any source.
  • Nucleic acid molecules hybridizing with the above-described nucleic acid molecules can in general be derived from any plant possessing such molecules, preferably form dicotyledonous plants, in particular from any plant of interest in agriculture, horticulture or wood culture, such as crop plants, namely those of the family Solanaceae, such as potato and tomato, Amaranthaceae, and Brassicaceae such as section Procumbens of the genus Beta but also from plants such as manioc, leguminous plants, oil producing plants, such as oilseed rape, linenseed, etc., plants using polypeptide as storage substances, such as soybean, plants using sucrose as storage substance, such as sugar beet or sugar cane, trees, ornamental plants as well as plants that can be used for the production of biomass, re
  • nucleotide sequence information provided herein may be used in a data base ⁇ e.g. of ESTs, or STSs, or other genomic sequence information) search to find homologous sequences, expression products of which can be tested for pathogen resistance activity e.g. using methods based on the transient assays of the present invention, or conventional phenotype assays in transgenic plants.
  • the transgenic plant, plant cell, plant tissue, harvestable part of the plant and the like described above comprise further genes of foreign origin, i.e. recombinant nucleic acid molecules encoding polypeptides that induce pathogen resistance, for example resistance against bacteria, fungi, viruses or nematodes.
  • the plant and plant cell of the present invention or any derivative thereof comprise a recombinant nucleic acid molecule which translated region is identical or at least 60 % homologous to the sequence of the Hsl pr ° ⁇ ! gene from B. procumbens described in Cai et al, 1997 and in US patent US 6,294,712 Bl, the disclosure content of which is incorporated herein by reference.
  • nucleic acid molecule of the present invention may be transformed concomitantly either in the same, for example in form of a binary vector for expression in plants such as used in the experiments described in US 6,294,712 Bl or with another suitable vector into the desired plant or subsequently.
  • a transgenic plant carrying the Hsl pr ° ⁇ ! gene prepared in accordance with the methods described in US patent US 6,294,712 Bl may be used and transformed with a vector of the present invention carrying the Hsl-2 gene or corresponding nucleic acid molecule disclosed herein.
  • nucleic acid molecules i.e. genes and open reading frames that are found to be present on chromosome 9 in the genome of the resistance carrying translocation lines TR520 and TR363 may be introduced into the plant of the present invention, which may contribute to the efficacy of pathogen and in particular nematode resistance of the plants.
  • probes based on the sequence may be used e.g. in southern blotting. For instance DNA may be extracted from cells taken from plants displaying the appropriate resistance trait and digested with different restriction enzymes. Restriction fragments may then be separated (e.g. by electrophoresis on an agarose gel) before denaturation and transferred to a nitrocellulose filter. Labeled probe may be hybridized to the DNA fragments on the filter and binding determined.
  • Preliminary experiments may be performed by hybridizing under low stringency conditions.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridizations identified as positive which can be investigated further.
  • hybridizations may be performed using a hybridization solution comprising: 5X SSC (wherein SSC - 0.15M sodium chloride; 0.15 M sodium citrate; pH 7), 5x Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05%) sodium pyrophosphate and up to 50%> formamide.
  • Hybridization is carried out at 37- 42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1 % SOS; (2) 15 minutes at room temperature in 2X SSC and 0.1 % SOS; (3) 30 minutes- 1 hour at 37°C in 1 X SSC and 1% SOS; (4) 2 hours at 42-65°C in IX SSC and 1% SOS, changing the solution every 30 minutes.
  • Tm 81.5°C + 16.6Log [Na+] + 0.41 (% G+Q-0.63 (% 1 formamide)-600/#bp in duplex.
  • the Tm of a DNA duplex decreases by 1-1. 5°C with every 1 % decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention. It is well known in the art to increase stringency of hybridization gradually until only a few positive clones remain. Other suitable conditions include, e. g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in O. lx SSC, 0.1 % SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2,6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1 X SSC, 0.1 % SDS.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labeled.
  • Other methods not employing labeling of probe include amplification using PCR (including, where appropriate, RACE PCR), RNase protection and allele specific oligonucleotide probing.
  • the identification of successful hybridization is followed by isolation of the nucleic acid which has hybridized, which may involve one or more steps of PCR or amplification by cloning in a vector that replicates in a suitable host. In each case, if need be clones (e.g.
  • lambda, cosmid, plasmid, BACs, biBACS) or fragments identified in the search can be extended or supplemented. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mR A preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence (see, e.g., "Principles of Genome Analysis" by S B Primrose (1995) Pub. Blackwell Science Ltd, Oxford, UK).
  • the original DNA source e.g. a clone library, mR A preparation etc.
  • nucleic acid molecules or corresponding genes can then be tested for functionality, for example, as described in the examples.
  • One scheme for isolating Hsl-2 homologues is as follows:
  • Resistance gene coding activity can then be tested as described above or in the examples.
  • a nucleic acid encoding an Hsl-2 derivative may be produced comprising the step of modifying a nucleic acid molecule encoding Hsl-2.
  • the derivative may include changes to the nucleic acid molecule which make no difference to the encoded amino acid sequence (i.e. degenerative equivalent).
  • Changes to a sequence, to produce a mutant or derivative may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.
  • a variant nucleic acid may encode an amino acid sequence including additional amino acids at the C-terminus and/or N-terminus.
  • parts or fragments (however produced) corresponding to portions of the sequences provided, and which encode polypeptides having biological activity, for instance pathogen resistance and/or the ability to raise or bind Hsl-2- binding antibodies.
  • changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage; other sites which are required for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc.
  • Leader or other targeting sequences may be added to the expressed protein to determine its location following expression.
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.
  • homologues having non-conservative substitutions.
  • substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure.
  • regions which are critical in determining the peptides conformation or activity such changes may alter the properties of the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e. g. altered stability or specificity, in particular broader specificity.
  • Mutants having these properties can then be selected as described above.
  • Other methods may include mixing or incorporating sequences from related resistance genes into the Hsl-2 sequence.
  • restriction enzyme fragments of Hsl-2 could be ligated together with fragments of an Hsl-2 homologue or even of an unrelated gene to generate recombinant versions of Hsl-2.
  • An alternative strategy for modifying Hsl-2 would employ PCR as described above (Ho et al, 1989 Gene 77, 51- 59) or DNA shuffling (Crameri et al clove 1998 Nature 391).
  • the methods of the invention, described above may include hybridization of one or more ⁇ e.g.
  • oligonucleotides, probes or primers form a further part of the present invention.
  • An oligonucleotide for use in probing or PCR may be about 30 or fewer nucleotides in length ⁇ e.g. 18, 23, 24 or 25).
  • Generally specific primers are upwards of 14 or 15 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred.
  • the present invention also relates to an isolated nucleic acid molecule of at least 15, preferably 16 or 17 and more preferably 18, 19, 20, 21 or 22 nucleotides in length that specifically hybridizes under stringent conditions to the nucleotide sequence set forth in Figure 10 and 11 (SEQ ID NO: 1 or 3) or with a complementary sequence thereof.
  • the oligonucleotide and primer or probe of the present invention does not hybridize to the conserved sequences and motifs shown in Figures 7 and 8, as well as Figure 10.
  • the nucleic acid molecule described above is in the form of a recombinant and preferably replicable vector.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally ⁇ e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic ⁇ e.g. higher plant, mammalian, yeast or fungal cells).
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as -a microbial, e.g. bacterial, or plant cell.
  • a host cell such as -a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bifunctional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this-may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3-direction on the sense strand of double- stranded DNA).
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the coding region of the Hsl-2 gene, or a variant ⁇ e.g. mutant, derivative or allele) thereof.
  • a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the coding region of the Hsl-2 gene, or a variant ⁇ e.g. mutant, derivative or allele
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • One embodiment of the present invention provides a gene construct, preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Same inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic.
  • an inducible (or “switchable”) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero). Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype.
  • Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, /ac, trp or tac promoter in E.
  • coli and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL 1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • suitable expression vectors are known in the art such as OkayamaBerg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNA 1, pcDNA3 (Invitrogen), pSPORTl (GIBCO BRL).
  • Plant vectors Particularly of interest in the present context are plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984) and Guerineau and Mullineaux (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148).
  • Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 358 (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a and 1990b); the cauliflower mosiac promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, e.g. inner phloem, flower primordia, branching points in root and shoot (Medford, 1992) and the A. thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al., 1992).
  • Other promoters include the rice actin promoter.
  • the promoter may include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression.
  • the vectors of the present invention may include the Hsl-2 gene or a variant thereof, in addition to various sequences required to give them replicative, integrative and/or expression functionality. Such vectors can be used, for instance, to make plants into which they are introduced resistant to H. schachtii or other nematodes.
  • the present invention also provides methods comprising introduction of the Hsl-2 constructs discussed above (such as vectors) into a host cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus, an effective exogenous inducer.
  • the vectors described above may be introduced into hosts by any appropriate method e.g. conjugation, mobilization, transformation, transfection, transduction or electroporation, as described in further detail below.
  • a host cell containing nucleic acid or a vector according to the present invention especially a plant or a microbial cell.
  • the host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the molecules are placed under the control of regulatory elements which ensure the expression in plant cells. These regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the plant species to be transformed.
  • such regulatory elements comprise a promoter active in plant cells.
  • constitutive promoters such as the 35S promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689).
  • tissue specific promoters see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251).
  • tissue specific promoters see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251).
  • promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley etc. Inducible promoters may be used in order to be able to exactly control expression.
  • inducible promoters examples are the promoters of genes encoding heat shock proteins. Also microspore-specific regulatory elements and their uses have been described (W096/16182). Furthermore, the chemically inducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991), 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366).
  • the regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells. Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A-tail to the transcript which may improve its stability; for literature see also supra.
  • nucleic acid molecule according to the invention is expressed in sense orientation it is in principle possible to modify the coding sequence in such a way that the protein is located in any desired compartment of the plant cell.
  • these include the endoplasmatic reticulum, the vacuole, the mitochondria, the plastids, the apoplast, the cytoplasm etc. Methods how to carry out this modifications and signal sequences ensuring localization in a desired compartment are well known to the person skilled in the art.
  • Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment and other methods known in the art.
  • the vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border”-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome.
  • Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res.13 (1985), 4777; Bevan, Nucleic. Acid Res. l2(1984), 8711; Koncz, Proc. Natl. Acad. Sei. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol.
  • Agrobacterium tumefaciens Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.
  • the resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known by a skilled person. This can be found, for example, in Hood, Molecular Breeding 3 (1997), 291-306; Coleman, Proc. Natl. Acad. Sei. USA 94 (1997), 7094-7097; Shilito, Biotechnology 7 (1989), 581-587.
  • the plants which can be modified according to the invention and which either show overexpression of a protein according to the invention or a reduction of the synthesis of such a protein can be derived from any desired plant species.
  • They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil producing plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, sugar beet, sugar cane, leguminous plants (e.g. beans, peas etc.), wood producing plants, preferably trees, etc.
  • crop plants e.g. maize, rice, barley, wheat, rye, oats etc.
  • potatoes oil producing plants
  • oil producing plants e.g. oilseed rape, sunflower, pea nut, soy bean, etc.
  • cotton e.g. oilseed rape, sunflower, pea nut, soy bean, etc.
  • sugar beet e.g. beans, peas etc.
  • a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practizing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
  • selectable genetic markers may be used consisting of chimeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.
  • a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a vector comprising a nucleic acid of the present invention (e.g. Hsl-2 or Hsl-2 variant) into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome.
  • a nucleic acid of the present invention e.g. Hsl-2 or Hsl-2 variant
  • the invention further encompasses a host cell transformed with a nucleic acid molecule or a vector according to the present invention, especially a plant or a microbial cell.
  • a host cell transformed with a nucleic acid molecule or a vector according to the present invention, especially a plant or a microbial cell.
  • the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.
  • heterologous is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
  • a heterologous gene may be additional to a corresponding endogenous gene.
  • Nucleic acid heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species.
  • the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant.
  • a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol. I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
  • Plants which include a plant cell according to the invention are also provided, along with any part or propagate thereof, seed, selfed or hybrid progeny and descendants.
  • a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
  • the transgenic plant of the invention upon the presence of the Hsl-2 gene of the invention attained resistance or improved resistance against a pathogen the corresponding wild-type plant was susceptible to.
  • the term "resistance” covers the range of protection from a delay to complete inhibition of disease development.
  • Examples for pathogens of importance comprise nematodes; see also supra.
  • the transgenic plant of the invention attains resistance to H. schachtii.
  • the present invention embraces all of the following: a clone of such a plant, seed, selfted or hybrid progeny and descendants (e.g. Fl and F2 descendants) and any part of any of these, such as tissue, cuttings, harvestable parts such as fruits and seed.
  • a clone of such a plant seed, selfted or hybrid progeny and descendants (e.g. Fl and F2 descendants) and any part of any of these, such as tissue, cuttings, harvestable parts such as fruits and seed.
  • the invention also provides a plant propagate from such a plant that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • the sequences disclosed herein may be used to facilitate selection of plants into which it is desired to introduce the resistance trait using conventional plant breeding methods. Progeny from crosses which carry the gene may be readily identified by screening on the basis of the Hsl-2 sequence, particularly the Hsl-2 signature sequence.
  • the present invention also encompasses the expression product of any of the Hsl-2 or variant nucleic acid sequences disclosed above, and methods of making the expression product by expression from encoding nucleic acid molecules therefore under suitable conditions, which may be in suitable host cells in vitro, or chemically synthesized, in particular if antigens for raising antibodies are desired.
  • the present invention also relates to antibodies and equivalent binding molecules specifically recognizing the Hsl-2 protein of the present invention.
  • Antibodies of the present invention may be monoclonal or polyclonal antibodies. Antibodies may be raised to a purified Hsl-2 /variant polypeptide or peptide by any method known in the art (for an overview see e.g. Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988). Such antibodies, or fragments or derivatives thereof, can be used to bind Hsl-2 or in the identification and/or isolation of proteins homologous to Hsl-2 (i.e. which share epitopes therewith), which in turn can provide the basis of an alternative method to those described above to isolate their encoding genes.
  • the antibodies of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab) 2 , as well as in single chains; see e.g. WO88/09344.
  • the antibody of the present invention is preferably raised against and recognizes, respectively, a unique epitope of the Hsl-2 protein.
  • a fragment of Hsl-2 protein is used as an antigen which lacks the conserved transglutaminase and/or protein kinase domain.
  • an antigen is preferably used which amino acid sequence does not have more than 8, preferably no more than 7, more preferably no more than 6, still more preferably no more than 5, and still more preferably no more than 4 consecutive amino acids in common with any of the reference proteins shown in Figure 7 and 8.
  • the antibody of the present invention is specific for the Hsl-2 protein in that the antibody does not recognize MAP3K, CTR1 and EDR1 protein, in particular those with the amino acid sequences shown in Figure 7 and 8.
  • Means and methods for producing Hsl-2 protein specific antibodies are well known to the person skilled in the art and described for example in the references cited above.
  • aptamers that bind to the Hsl-2 polypeptide of the invention may be employed.
  • the preparation of aptamers is known to the person skilled in the art; see, e.g., Thomas, and Dinshaw (2000) Adaptive recognition by nucleic aptamers. Science 287:820-825.
  • the invention further provides a method of influencing or affecting a resistance trait in a plant, whereby the method includes the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above (e.g. Hsl-2 or Hsl-2 variant, in each case plus an optional elicitor) within cells of the plant.
  • a heterologous nucleic acid sequence as discussed above (e.g. Hsl-2 or Hsl-2 variant, in each case plus an optional elicitor) within cells of the plant.
  • antisense technology which is reviewed in Bourque, (1995), Plant Science 105,125-149 and Flavell, (1994) PNAS USA 91, 3490-3496.
  • An alternative to antisense is to use a copy of all or part of the target gene inserted in sense, that is the same orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; see, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al, (1990) The Plant Cell 2, 279-289; Zhang et al, (1992) The Plant Cell 4, 1575-1588, and US-A-5,231,020.
  • the invention also relates to a transgenic plant cell - and to transgenic plants comprising such plants cells - which contain, preferably stably integrated into the genome, a nucleic acid molecule according to the invention or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of an Hsl-2 protein.
  • the reduction is achieved by an antisense, sense, ribozyme, R Ai, co-suppression, dominant mutant effect, or knock out mutant in the Hsl-2 gene.
  • the invention provides a method which includes expressing of nucleic acid sequences shown in Figure 10 and 11 (SEQ ID NO: 1 or 3) or a variant thereof within the cells of a plant (thereby producing the encoded polypeptide), following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.
  • a method may be used to introduce nematode resistance into the plant whereby an Hsl-2- mediated resistance is triggered by contact with an appropriate elicitor or other initiator or inducer.
  • the elicitor or other trigger may be encoded directly by the invading nematode such as H. schachtii or certain other nematodes.
  • Hsl-2 variants of the present invention.
  • modification of the Hsl-2 (variant) sequence may allow triggering by a non-natural elicitor, if this is preferred.
  • the methods for establishing gene for gene compatibility between elicitor and resistance gene, are characterized in that they include the steps of: (a) causing or permitting the co-expression in cell of Hsl-2 or an Hsl-2 derivative with the elicitor, (b) observing said cell for an response such as hypersensitivity (HR) and cell death or eliciting factors affecting the nematode, (c) correlating the result of the observation made in (b) with the specificity of the Hsl-2 or the Hsl-2 derivative for the elicitor.
  • HR hypersensitivity
  • the present invention also relates to such transgenic plants which are more sensitive to nematode infection compared to a corresponding wild type plant.
  • the present invention relates to harvestable parts and propagation material of such plants; see also supra.
  • an Hsl-2 gene has been isolated which upon transformation into a susceptible plant confers resistance to nematodes, in particular H schachtii. Since the genomic clone the corresponding DNA sequence of which is depicted in Figure 10 (SEQ ID NO: l) is able to give rise to this effect, it is apparent that the regulatory sequences of the Hsl- 2 gene necessary and sufficient to mediate the expression of the Hsl-2 polypeptide upon pathogen infection are contained in the isolated DNA sequence.
  • the present invention also relates to a regulatory sequence of a promoter naturally regulating the expression of a nucleic acid molecule of the invention described above or of a nucleic acid molecule homologous to a nucleic acid molecule of the invention, said regulatory sequence being capable of conferring or modulating the expression of a heterologous DNA sequence upon pathogen infection, preferably comprising the DNA sequence of Figure 10 (SEQ ID NO. 1), preferably from nucleotides from nucleotides 1 to 7845 or from nucleotides 1 to 3920 or a homologous DNA sequence and/or at least one regulatory element depicted in Table 5.
  • SEQ ID NO. 1 the DNA sequence of Figure 10
  • regulatory sequence refers to sequences which influence the specificity and/or level of expression, for example in the sense that they confer cell and/or tissue specificity. Such regions can be located upstream of the transcription initiation site, but can also be located downstream of it, e.g., in transcribed but not translated leader sequences, or in introns.
  • promoter within the meaning of the present invention refers to nucleotide sequences necessary for transcription initiation, i.e. RNA polymerase binding and successful start of processive transcript formation, and may also include, for example, the TATA box.
  • nucleic acid molecule homologous to a nucleic acid molecule of the invention includes promoter regions and regulatory sequences of other Hsl-2 genes, such as genes from other species, for example, tomato which are homologous to P. procumbens Hsl-2 genes and which display substantially the same expression pattern.
  • promoters are characterized by their capability of conferring preferably exclusively expression of a heterologous DNA sequence in a plant upon pathogen infection.
  • the term "capable of conferring or modulating the expression of a heterologous DNA sequence upon pathogen infection” as used herein means that said promoter is capable of controlling the expression of a heterologous DNA sequence in plants at infection sites, analogous or closely related to the controlled expression of pathogen related genes which are involved in the natural resistance in most incompatible host/pathogen interactions, such as the hypersensitive cell death or otherwise pathogen specific response at infection sites of a part of a plant.
  • the regulatory sequence of the invention is characterized by its capability of mediating localized transcriptional activation selectively in response to pathogen attack or in response to stimuli that mimic pathogen attack such as elicitors prepared from, e.g., pathogens such as nematodes or derivatives thereof.
  • the transcriptional activation by the regulatory sequence of the invention may also occur in cells surrounding the actual infection site due to cell-cell interactions.
  • the regulatory sequence of the invention and chimeric promoters comprising such sequences may advantageously not or only to a small extent be inducible upon other stimuli such as abiotic stress.
  • the induction from the chimeric promoter upon pathogen attack or elicitor treatment is at least about 10- fold higher, preferably 20-fold higher and particularly 30-fold higher than its activation, if any, by abiotic stress.
  • the expression specificity conferred by the regulatory sequences of the invention may not be limited to local gene expression due to pathogens, for example, they may be combined with further regulatory sequences that provide for tissue specific gene expression.
  • the particular expression pattern may also depend and the plant/vector system employed.
  • expression of heterologous DNA sequences driven by the regulatory sequences of the invention predominantly occurs upon pathogen infection or treatment with a corresponding elicitor unless certain elements of the invention were taken and designed by the person skilled in the art to control the expression of a heterologous DNA sequence in certain cell types.
  • regulatory sequences from other species can be used that are functionally homologous to the regulatory sequences of the promoter of the above defined Hsl-2 specific nucleic acid molecules, or promoters of genes that display an identical or similar pattern of expression.
  • the particular expression pattern may also depend on the plant/vector system employed.
  • expression of heterologous DNA sequences driven by the regulatory sequences of the invention predominantly occurs in any cell infected by a particular pathogen unless certain elements of the regulatory sequences of the invention, were taken and designed by the person skilled in the art to control the expression of a heterologous DNA sequence in a particular tissue or otherwise controlled manner.
  • novel regulatory sequences of Hsl-2 genes can be isolated and have been exemplified for the regulatory sequence of the Hsl-2 gene of P. procumbens.
  • genomic DNA can be digested with appropriate restriction enzymes, denatured and allowed to anneal to a reverse primer derived from the cDNA sequence of the invention.
  • a blunt-ended adaptor can be ligated and PCR can be performed using a nested reverse primer derived from the mentioned cDNA, and a forward primer derived from the adaptor sequence.
  • a physical map of the genomic sequences upstream the coding region can be constructed by mean of genomic southern analysis.
  • genomic DNA can be digested with selected restriction enzymes, genomic fragments containing a piece of the upstream sequences and the coding sequence can be gel purified and self-ligated in a large volume to favor the formation of circular molecules, that can subsequently be amplified by PCR with forward and reverse primers, derived from the coding sequence of the gene.
  • the transcription start site can be determined by standard procedures well known to everyone skilled in the art, such as 5'-RACE, primer extension or SI mapping. To define czs-regulatory elements upstream of the transcription start site ⁇ i.e.
  • the respective region is fused to marker genes such as genes encoding GUS or GFP, and 5' deletion derivatives of these construct are generated. They are transformed into suitable plant material, and the expression of the marker gene depending on the remaining upstream sequence (putative promoter) is determined.
  • DNA sequences comprising the nucleotide sequence as depicted in Figure 10 (SEQ ID NO. 1) from nucleotides 1 to 20779 or (a) part(s) thereof, preferably from nucleotides 1 to 7845 or from nucleotides 1 to 3920 or a homologous DNA sequence; DNA sequences comprising at least 14 consecutive nucleotides of the nucleotide sequence as depicted in Figure 10 (SEQ ID NO: 1) from nucleotides 1 to 20779;
  • DNA sequences comprising at least one transcriptional initiation sequence and at least one motif for the function selected from the group consisting of root nodule expression, elicitor responsiveness, pathogen-induced gene expression, defense-related gene expression, guard cell expression, binding site of WR Y transcription factors in pathogenesis related genes, binding site of WRKY transcription factors in NPR1 gene expression or any combination thereof as set forth in Table 5, preferably at a copy number, distance and composition substantially identical or equivalent to those found in the Hsl-2 gene;
  • DNA sequences of a gene of fragment thereof obtainable by screening an appropriate genomic DNA library with a probe having a nucleotide sequence as defined for the nucleic acid molecule of the present invention supra and in claim 1;
  • DNA sequences comprising nucleotide sequences which are conserved in (a), (b) and
  • the DNA sequence comprising the regulatory sequence of the present invention may fulfill one or more of the above-mentioned features, for example comprising the motifs referred to in section (c) and hybridizing to the promoter region depicted in Figure 10.
  • the motifs referred to in section (c) and shown in Table 5 may be present at nucleotide positions and at a copy number, respectively, shown for the Hsl- 2 promoter depicted in Figure 10 and identified in Table 5, or at any other position, distance to each other and copy number as long as the corresponding DNA sequence displays a substantially identical or similar pattern of expression as the Hsl-2 promoter and the expression of the Hsl-2 gene in P. procumbens or the two resistant translocation lines.
  • the DNA sequence of the present invention comprises a promoter fragment with a nucleotide sequence of position 1 to 3920 or of a nucleotide sequence homologous thereto, or a fragment thereof containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 of any one of the sequence motifs indicated in Figure 10 and referred to in Table 5. Verification of the DNA sequences comprising the regulatory sequence of the present invention can be done by methods well known to the person skilled in the art and illustrated in the appended Examples.
  • the promoter of the present invention comprising the entire 5' non translated nucleotide sequence up to the ATG at position 7845 or any derivative, deletion mutant or homologous nucleotide sequence thereof can be fused to a reporter gene such as GUS or fluorescent protein, preferably such that the ATG and probably some more of the coding region of exon 1 are fused in frame to the coding sequence of the reporter gene since it is known that for example the enzymatic activity of GUS is not affected by additional foreign amino acids at its N-terminus.
  • a reporter gene such as GUS or fluorescent protein
  • homologous regulatory sequences differ at one or more positions from the regulatory sequence of (a) or (b) but still have the same specificity, namely they comprise the same or similar sequence motifs, preferably 6 to 10 nucleotides in length, responsible for the above described expression pattern.
  • such regulatory sequences hybridize to one of the above-mentioned regulatory sequences, most preferably under stringent conditions.
  • regulatory sequences which share at least 85%, more preferably 90- 95%, and most preferably 96-99%) sequence identity with one of the above-mentioned regulatory sequences and have the same or substantially the same specificity.
  • Such regulatory sequences also comprise those which are altered, for example by one or more nucleotide deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison to the above- described nucleotide sequence. Methods for introducing such modifications in the nucleotide sequence of the regulatory sequences of the invention are well known to the person skilled in the art.
  • regulatory elements may be added to the regulatory sequences of the invention.
  • transcriptional enhancers and/or sequences which allow for induced expression of the regulatory sequences of the invention may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gatz, supra.
  • the regulatory sequence of the invention may be derived from the Hsl-2 genes of P.
  • nucleotide sequence of the invention can be compared as appropriate computer programs known in the art such as BLAST, which stands for Basic Local Alignment Search Tool (Altschul, 1997; Altschul, J. Mol. Evol. 36 (1993), 290-390; Altschul, J. Mol. Biol. 215 (1990); 403-410), can be used to search for local sequence alignments.
  • BLAST produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologues. With such means it is possible to identify conserved nucleotide sequences that may play a role in pathogen specific expression.
  • said regulatory sequence is part of a recombinant DNA molecule.
  • the regulatory sequence in the recombinant DNA molecule is operatively linked to a heterologous DNA sequence.
  • heterologous with respect to the DNA sequence being operatively linked to the regulatory sequence of the invention means that said DNA sequence is not naturally linked to the regulatory sequence of the invention.
  • Expression of said heterologous DNA sequence comprises transcription of the DNA sequence, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably plant cells, are well known to those skilled in the art.
  • poly-A signals usually comprise poly-A signals ensuring termination of transcription and stabilization of the transcript, see also supra.
  • Additional regulatory elements may include transcriptional as well as translational enhancers; see supra.
  • the heterologous DNA sequence of the above-described recombinant DNA molecules encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme.
  • the recombinant DNA molecule of the invention can be used alone or as part of a vector to express heterologous DNA sequences, which, e.g., encode proteins for, e.g., seed storage proteins, toxins, antibodies ("plantibodies”) or diagnostics of Hsl-2 related gene expression.
  • the recombinant DNA molecule or vector containing the DNA sequence encoding a protein of interest is introduced into the cells which in turn produce the protein of interest.
  • resistance genes represent an effective resource for nematode control.
  • they are often not available for a particular host, and attempts to transfer cloned resistance genes to new hosts showed limited success, as the unsuccessful transfer of the tomato gene Hero to potato shows (Sobczak et al, 2005). Consequently, other strategies had been investigated, as the expression of transgenic proteins that are harmful to the nematodes.
  • transgenic proteinase inhibitors Plant proteins with toxic effects exist, which reduce nematode growth and fertility.
  • cysteine Pis defined as cystatins
  • cystatins had been analyzed in tomato hairy roots and A. thaliana resulting in the reduced development of the females.
  • Analysis of sugar beet hairy roots expressing sweet potato serine PI sporamin showed inhibited growth and development of H. schachtii females (Cai et al., 2003).
  • cytolytic ⁇ -endotoxin (Cry-toxin) proteins from Bacillus thuringiensis had been found to exhibit toxicity against nematodes, first demonstrated for the free-living nematode Caenorhabditis elegans (Borgonie et al, 1996). Results from successful expression experiments in tomato roots suggested this approach to be rather promising with regard to plant-parasitic nematode control (Li et al., 2008b).
  • RNA interference can trigger the silencing of target genes by mRNA degradation.
  • Fire and Mello et al. (1998) first described the promising technique in C. elegans and had been awarded the Nobel Prize in physiology or medicine in 2006. To date, more than twenty studies of successful RNAi in root knot and cyst nematodes have been published (Jones et al., 2011).
  • Vanholme et al. (2007) identified a pectate lyase secreted by this nematode in the migratory stage.
  • Post-transcriptional gene silencing against the gene by soaking the nematodes for 24 h in double stranded RNA solution resulted in a reduction of the infectiveness.
  • incognita parasitism genes HSP90, isocitrate lyase, and three proteinase genes, resulted in a reduction in egg number per root gram 45 days after nematode infection of 28 - 37%, 67 - 76%, and 30 - 40%>, respectively (Grossi-de-Sa et al., 2012).
  • the regulatory sequences of the invention can be operatively linked to sequences encoding Barstar and barnase, respectively, for use in the production of HR response in plants upon infection by the nematode.
  • the protein put under the control of the promoter of the present invention can be a scorable marker, e.g., luciferase, green fluorescent protein or ⁇ -galactosidase.
  • a scorable marker e.g., luciferase, green fluorescent protein or ⁇ -galactosidase.
  • This embodiment is particularly useful for simple and rapid screening methods for compounds and substances described herein below capable of modulating HS1-2 gene expression.
  • a transgenic plant can be cultured in the presence and absence of a candidate compound in order to determine whether the compound affects the expression of genes which are under the control of regulatory sequences of the invention, which can be measured, e.g., by monitoring the expression of the above-mentioned marker.
  • other marker genes may be employed as well, encoding, for example, a selectable marker which provides for the direct selection of compounds which induce or inhibit the expression of said marker.
  • the regulatory sequences of the invention may also be used in methods of antisense approaches.
  • the antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and optionally up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to a portion of a specific mRNA sequence and/or DNA sequence of the gene of interest. Standard methods relating to antisense technology have been described; see, e.g., Klann, Plant Physiol. 112 (1996), 1321-1330 and supra.
  • the antisense RNA binds to its target sequence within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein encoded by the mRNA.
  • the invention relates to nucleic acid molecules of at least 15 nucleotides in length hybridizing specifically with a regulatory sequence as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences having no or substantially different regulatory properties. Such nucleic acid molecules may be used as probes and/or for the control of gene expression.
  • nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of regulatory sequences according to the invention. Another application is the use as a hybridization probe to identify regulatory sequences hybridizing to the regulatory sequences of the invention by homology screening of genomic DNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a regulatory sequence as described above may also be used for repression of expression of a gene comprising such regulatory sequences, for example due to an antisense, RNAi, cosupression or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-81 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)- mRNA of a gene comprising a regulatory sequence of the invention.
  • nucleic acid probe with an appropriate marker for specific applications, such as for the detection of the presence of a nucleic acid molecule of the invention in a sample derived from an organism.
  • the above described nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
  • nucleic acid molecules may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches; see supra.
  • the present invention also relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a regulatory sequence or corresponding recombinant DNA molecule of the invention.
  • said vector is an expression vector and/or a vector further comprising a selection marker for plants.
  • selector markers see supra.
  • Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley lnterscience, N.Y. (1989).
  • the recombinant DNA molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the present invention furthermore relates to host cells transformed with a regulatory sequence, a DNA molecule or vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the regulatory sequence, vector or recombinant DNA molecule of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred cells are plant cells.
  • the present invention provides a method for the production of transgenic plants, plant cells or plant tissue comprising the introduction of a nucleic acid molecule, recombinant DNA molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • a nucleic acid molecule, recombinant DNA molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • further regulatory sequences such as poly A-tail may be fused, preferably 3' to the heterologous DNA sequence, see also supra.
  • Further possibilities might be to add transcriptional or translational enhancers that increase gene expression, or sequences that increase mRNA stability.
  • the present invention relates also to host cells, in particular transgenic plant cells which contain, preferably stably integrated into the genome, a regulatory sequence, a recombinant DNA molecule or vector according to the invention. Furthermore, the present invention also relates to transgenic plants and plant tissue, seed or plant part comprising the above described transgenic plant cells and regulatory sequence, recombinant DNA molecule or vector according to the invention, respectively.
  • the transgenic plant of the present invention is preferably selected from the group of genera consisting of Beta and Brassica, most preferably the plant is Beta vulgaris.
  • Means and methods for the transformation of plants are known to the person skilled in the art and are also described in US patent 6,294,712 Bl; see in particular Examples 7 and 9 for Brassica; Example 12 for sugar-beet; Example 14 for Brassica napus and Example 15 for potato, the disclosure content of which is incorporated herein by reference.
  • the present invention relates to a method for the identification of a plant protective agent comprising the steps of:
  • read out system in context with the present invention means a DNA sequence which upon transcription and/or expression in a cell, tissue or organism provides for a scorable and/or selectable phenotype.
  • read out systems are well known to those skilled in the art and comprise, for example, recombinant DNA molecules and marker genes as described above.
  • plurality of compounds in a method of the invention is to be understood as a plurality of substances which may or may not be identical. Said compound or plurality of compounds may be inorganic or organic, naturally occurring or man made compounds and may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compound(s) may be known in the art but hitherto not known to be capable of suppressing or activating and/or enhancing the transcription of an Hsl-2 gene.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into the plant, plant cells or tissue or sprayed onto the plant or supplied in the soil.
  • 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 from the original sample identified as containing the compound capable of suppressing or activating and/or enhancing the transcription of a Hsl-2 gene, 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 of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the compound identified according to the above described method is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
  • the compounds which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Gell 79 (1994), 193-198 and references cited supra).
  • expression libraries e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like
  • genes encoding a putative regulator of an Hsl-2 gene may be identified using, for example, insertion mutagenesis using, for example, gene targeting vectors known in the art (see, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritze and Walden, Gene activation by T-DNA tagging. In Methods in Molecular Biology 44 (Gartland, K.M.A. and Davey, M.R., eds) Totowa: Human Press (1995), 281-294) or transposon tagging (Ghandlee, Physiologia Plantarum 78 (1990), 105-115). Said compounds can also be functional derivatives or analogues of known inhibitors or activators.
  • Determining whether a compound is capable of suppressing or activating and/or enhancing the transcription of an Hsl-2 gene can be done, for example, in plants by monitoring the reporter gene. It can further be done by monitoring the phenotypic characteristics of the transgenic plant of the invention contacted with the compounds and compare it to that of wild-type plants. In an additional embodiment, said characteristics may be compared to that of a transgenic plant contacted with a compound which is either known to be capable or incapable of suppressing or activating and/or enhancing Hsl-2 gene expression or the activity of the protein.
  • the compounds identified according to the method of the invention are expected to be very beneficial since promoters that have been known so far are only of limited use due to the non- or not tightly regulated pathogen specificity of their regulatory sequences.
  • the inhibitor or activator identified by the above-described method may prove useful as a herbicide, pesticide and/or as a plant growth regulator.
  • the invention relates to a compound obtained or identified according to the method of the invention.
  • useful compounds can be, for example, transacting factors which bind to the regulatory sequence of the invention. Identification of transacting factors can be carried out using standard methods in the art (see, e.g., Sambrook, supra, and Ausubel, supra). To determine whether a protein binds to the regulatory sequences of the invention, standard DNA footprinting and/or native gelshift analyses can be carried out.
  • the regulatory sequence can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
  • modulation of its binding to the regulatory sequences of the invention can be pursued, beginning with, for example, screening for inhibitors against the binding of the transacting factor to the regulatory sequences of the present invention.
  • Activation or repression of Hsl-2 genes could then be achieved in plants by applying of the transacting factor (or its inhibitor) or the gene encoding it, e.g. in a vector for transgenic plants.
  • the active form of the transacting factor is a dimer
  • dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
  • further components in the pathway leading to activation ⁇ e.g. signal transduction) or repression of a gene under the control of the regulatory sequences of the present invention can then be identified. Modulation of the activities of these components can then be pursued, in order to develop additional drugs and methods for modulating the expression of a gene under the control of the regulatory sequences of the present invention.
  • the compound identified according to the above described method or its analog or derivative is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
  • it can be combined with an agriculturally acceptable carrier known in the art.
  • the plant protection composition can be prepared by employing the above-described method of the invention and synthesizing the compound identified as inhibitor or activator in an amount sufficient for use in agriculture.
  • the present invention also relates to a method for the preparation of an agricultural plant protection composition comprising the above described steps of the method of the invention and synthesizing the compound so identified or an analog or derivative thereof.
  • the compound identified by the above- described method may be preferentially formulated by conventional means commonly used for the application of, for example, herbicides and pesticides or agents capable of inducing systemic acquired resistance (SAR).
  • SAR systemic acquired resistance
  • certain additives known to those skilled in the art comprising stabilizers or substances which facilitate the uptake by the plant cell, plant tissue or plant may be used, for example, carborundum, or 0.01 % SDS (sodium dodecylsulfate) solution.
  • the present invention relates to a method for identifying and obtaining an avirulence or a virulence factor of a pathogen comprising the steps of: (a) screening the Hsl-2 protein of the present invention or a fragment thereof against a peptide or protein expression library derived from a pathogen in a readout system under suitable conditions which permit interaction of the protein and peptide in said readout system;
  • the described nucleic acid molecules may also be used for several other applications, for example, for the identification of nucleic acid molecules which encode proteins which interact with the Hsl-2 proteins described above. This can be achieved by assays well known in the art, for example, as described in Sco field (Science 27 4 (1996), 2063-2065) by use of the so-called yeast "two-hybrid system". In this system the protein encoded by the nucleic acid molecules according to the invention or a smaller part thereof is linked to the DNA-binding domain of the GAL4 transcription factor.
  • a yeast strain expressing this fusion protein and comprising a lacZ reporter gene driven by an appropriate promoter, which is recognized by the GAL4 transcription factor, is transformed with a library of cDNAs which will express plant proteins or peptides thereof fused to an activation domain.
  • yeast two-hybrid system originally has been described by Fields and Song (Nature 340 (1989), 245-246; see also for review Vidal, M, in Bartei, P.L. and Fields, S. (eds.), The yeast two-hybrid system. Oxford University Press, New York, NY, (1997), 109-14 7).
  • a modified version of the yeast two-hybrid system has been described by (Gyuris, Cell 75 (1993), 223- 232; Zervos, Cell 72 (1993), 223-232).
  • a domain of the polypeptide is used as bait for binding compounds. Positives are then selected by their ability to grow on plates lacking leucine, and then further tested for their ability to turn blue on plates with X-gal, as previously described in great detail; see also international application WO 95/31544.
  • a modified version is the "reverse yeast two hybrid system" which allows selection for interaction of defective alleles using a negative selection strategy as, for example, described in (Vidal, Proc. Natl Acad Sei. USA 93 (1996), 10321- 10326; Vidal, Proc. Natl Acad Sei. USA 93 (1996), 10315-10320). This system uses the counter selectable reporter gene URA3.
  • Yeast cells expressing Ura3p convert the compound 5-flouroorotic acid (FOA) into the toxic derivative 5-flourouracil.
  • a two-hybrid interaction which leads to activation of the URA3 reporter gene can, thus, be counter-selected in the presence of FOA and loss of function mutants can be specifically selected out of a large pool of wild type alleles.
  • the complex is able to direct expression of the reporter gene.
  • the nucleic acid molecules according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with Hsl-2 proteins. This method can also be employed for identifying inhibitors and activators as described above.
  • yeast three-hybrid system As described (SenGupta, Proc. Natl. Acad. Sci. USA 93 (1996), 8496-8501).
  • the yeast three-hybrid selection system was developed for isolating the genes of the proteins that interact with RNA, and to study RNA-protein interactions.
  • This system based on the yeast two-hybrid system, consists of a DNA-binding domain fused to a known RNA-binding protein, an activation domain fused to a prospective RNA-binding protein, and a hybrid RNA. Transcription of reporter genes only occurs when both hybrid proteins interact with the hybrid RNA.
  • the invention also relates to compositions comprising at least one of the aforementioned nucleic acid molecules and/or comprising a nucleic acid molecule which is complementary for such a nucleic acid molecule, a vector of the invention, a Hsl-2 protein of the invention or an immunologically or biologically active fragment thereof or an antibody or aptamer specifically recognizing such a protein or fragment, a regulatory sequence or recombinant DNA, or a corresponding vector of the invention, a compound designed orientated according to the protein of the invention and/or identified according to the method described above and/or an antibody specifically recognizing such a compound or a regulatory sequence of the invention, and optionally suitable means for detection or suitable means for plant cell and tissue culture.
  • Diagnostic compositions may be used for methods for detecting expression of Hsl-2 gene by detecting the presence of corresponding mR A which comprises isolation of mR A from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid probe as described above under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the gene by the cell.
  • Further methods of detecting the presence of a protein according to the present invention comprise immunotechniques well known in the art, for example enzyme linked immunosorbent assay and histochemical assays such as in situ hybridization and detection of the subject protein by an appropriate antibody.
  • the present invention relates to a kit comprising at least one of the aforementioned nucleic acid molecules, vectors, proteins, compounds, antibodies, or aptamers of the invention.
  • the kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic plant cells, plant tissue or plants.
  • the kit may include buffers and substrates for reporter genes that may be present in the recombinant gene or vector of the invention.
  • the kit of the invention may advantageously be used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications referred to herein, e.g., in the diagnostic field or as research tool.
  • kits of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
  • the kit or its ingredients according to the invention can be used in plant cell and plant tissue cultures, for example, for any of the above described methods for detecting inhibitors and activators of Hsl-2 genes.
  • the kit of the invention and its ingredients are expected to be very useful in breeding new varieties of, for example, plants which display improved properties such as nutrient value or disease resistance.
  • regulatory sequences, recombinant DNA molecules, vectors and compounds of the present invention can be employed to produce transgenic plants with a desired trait; see for review TIPTEC Plant Product & Crop Biotechnology 13 (1995), 312-397.
  • nucleic acid molecules according to the invention are useful for the alteration or modification of plant/pathogen interaction.
  • pathogen includes, for example, bacteria, viruses and fungi as well as protozoa.
  • said pathogen is a nematode, in particular H. schachtii.
  • the present invention relates to the use of a nucleic acid molecule, vector, host cell, protein, a regulatory sequence, a recombinant DNA molecule, a vector, a compound, an aptamer and/or the antibody of the invention for use in a screening method for the identification of virulence and avirulence genes of pathogens, for screening plant protective compounds, for inducing pathogen resistance in plants, as a marker in marker-assisted plant breeding.
  • the regulatory sequence or a recombinant DNA molecule of the present invention is preferably used for the expression of a heterologous DNA sequence.
  • FIG. 1 Flowchart of the whole genome shotgun (WGS) sequencing approach of two nematode resistant translocation lines TR520and TR363.
  • WGS whole genome shotgun
  • the following hybrid assembly strategy was used to identify translocation specific sequences and is shown in the lower part of the flowchart. Two different assembly strategies were used and two assemblies for each line were generated. Sequence analysis was performed by using the four translocation genome assemblies and four reference assemblies of, P. procumbens, B. vulgaris RefBeet-0.4 and RefBeet-0.9, and the physically mapped sequences (physical map data: BACs and YACs).
  • Figure 2 A sequence-based physical map of the P. procumbens translocation of line
  • TR520 The map was aligned with molecular markers (CAU numbers) derived from bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) ends, and scaffold sequences obtained by WGS sequencing. A minimal tiling path of YACs and BACs, and the scaffolds from WGS sequencing are integrated into this map.
  • CAU numbers molecular markers
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • 'Sections' denote translocation specific sequences present or absent on different translocation lines identified by marker analysis.
  • the term 'super scaffold' (SS) is used for assembled contigs and scaffolds of different genome assemblies of P. procumbens and of the translocation lines TR520 and TR363 of the WGS data.
  • 'Super contigs' are the largest types of sequence assemblies on the physical map and incorporate BACs and super scaffolds of the WGS sequencing whereas the contigs of the physical map presented by Capistrano (2009) are named 'BAC contigs' in the following. Shaded areas highlight the different sequence resources mapped to the translocation: YACs, BACs and WGS scaffolds. Regions in common between both resistant translocations and absent from the susceptible translocation are highlighted in yellow.
  • Figure 3 Pairwise comparison of the predicted ORF of the mRNA sequence
  • EDR1 ENHANCED DISEASE RESISTANCE 1 protein
  • Ath Arabidopsis thaliana
  • ABR45974.1 CONSTITUTIVE TRIPLE RESPONSEl
  • CTR1 CONSTITUTIVE TRIPLE RESPONSEl
  • XP 002279319.2 of Vitis vinifera
  • MAP3K mitogen-activated protein kinase kinase kinase
  • Figure 4 (A) Graphical overview of the predicted exon-intron structure of ORF 803 using
  • EDR1 ENHANCED DISEASE RESISTANCE 1 gene
  • ABR45974.1 A. thaliana
  • CTR1 CONSTITUTIVE TRIPLE RESPONSEl
  • Vv V. vinifera
  • MAP3K mitogen-activated protein kinase kinase kinase
  • ORF803 m RNA Amino acid sequence alignment of the predicted protein the of mRNA sequence 52364_co_seq2, denoted as ORF803 m RNA, that shows high sequence similarity to ORF803, with the amino acid sequences of the ENHANCED DISEASE RESISTANCE 1 gene (EDR1) of A. thaliana (Ath) (ABR45974.1), the CONSTITUTIVE TRIPLE RESPONSEl (CTR1) like gene (XP 002279319.2) of V. vinifera. Vv) and a mitogen-activated protein kinase kinase kinase (MAP3K) from A.
  • EDR1 ENHANCED DISEASE RESISTANCE 1 gene
  • ABR45974.1 ABR45974.1
  • CTR1 CONSTITUTIVE TRIPLE RESPONSEl
  • MAP3K mitogen-activated protein kinase kinase kinase
  • Figure 9 Amino acid sequence alignment of the predicted protein the of mRNA sequence
  • FIG. 10 Genomic sequence of the ORF803 of Scaffold26267.
  • the genomic sequence including a part of the putative promoter region upstream of the ATG (5' prime region) of Scaffold26267 is shown. Regulatory elements within the putative promoter region are highlighted in light grey.
  • the transcription start site ATG is highlighted in in light grey, underlined and bold. Exons within the genomic sequence are hightlighted in light grey and underlined.
  • Figure 11 Nucleic acid sequence of the mRNA sequence 52364_co_seq2.
  • the transcription start site ATG is highlighted in bold and an underlining.
  • the predicted amino acid sequences of the CDR are denoted under the genetic code, marking the transcription stop codon by an asterisk.
  • translocation lines carrying translocations from chromosome 1 of P. procumbens were used for WGS sequencing.
  • the translocation of the line TR520 was estimated to be ⁇ 1.5 Mbp in size.
  • the line A906001 (seed code 940043) was sequenced which carries essentially the same translocation as the line TR520 in a homozygous form.
  • the line TR363 (seed code 9300363) is hemizygous for the P. procumbens translocation and estimated to have a translocation of different size (Kleine et al., 1998). Physical mapping and marker analysis by Schulte et al. (2006) and Capistrano (2009) suggested that the two lines share ⁇ 700 kbp of the P. procumbens translocation. Both carry the nematode resistance gene Hsl-2 and both are resistant to the BCN H. schachtii.
  • TR659 and TR320 Two susceptible translocation lines, TR659 and TR320, were identified after a marker based screening of 578 Ml offspring of 400 gy gamma irradiated seeds of the line 950631, which is essentially the same as the resistant translocation line TR520. Both lines had lost most of the translocation and show a susceptible phenotype .
  • Table 2 Primer sequences, amplicon sizes, and primer locations.
  • the assembly size of the B. vulgaris RefBeet-0.4 is 967 Mbp, includes 35,475 sequences, and has a N50 size of 358 kbp.
  • the assembly size is 590 Mbp and includes 82,305 sequences.
  • the N50 size is 759 kbp.
  • the assembly size of P. procumbens adds up to 641 Mbp, with 94,485 sequences, and a N50 size of 38 kbp.
  • the two nematode resistant translocation lines TR520 and TR363 were used for sequencing on the Illumina HiSeq2000 platform and subsequently, two hybrid assembly strategies were applied.
  • the paired-end sequencing of the translocation lines TR520 and TR363 using the Illumina HiSeq 2000 platform resulted in 88.6 Gbp and 85.5 Gbp high quality 100 bp-reads, respectively. This represents 116.6 (TR520) and 112.5-fold (TR363) coverage of the sugar beet translocation genome (758 Mbp + ⁇ 2 Mbp translocation).
  • Sequence reads generated in WGS sequencing are assembled into contiguous sequences and are denoted as contigs.
  • Contigs are organized into scaffolds on the basis of linking information provided by read pairs. Therefore, scaffolds provide the contig order and orientation as well as the size of gaps between the contigs (Green, 2001).
  • the term 'super scaffold' is used for assembled contigs and scaffolds of different genome assemblies of P. procumbens and of the translocation lines TR520 and TR363 of the WGS data.
  • 'Super contigs' are the largest types of sequence assemblies on the physical map and incorporate BACs and super scaffolds of the WGS sequencing whereas the contigs of the physical map presented by Capistrano (2009) are named 'BAC contigs' in the following.
  • the aim of the project was to select sequence reads from the P. procumbens translocation.
  • Two different assembly strategies were designed to identify translocation-specific read pairs.
  • One strategy is based on mapping the total number of reads to the draft genome sequence of B. vulgaris (RefBeet-0.4) using the Burrows- Wheeler- Aligner (BWA), allowing two mismatches (Burrows and Wheeler, 1994). It was expected that most of the sugar beet reads to map to the draft reference assembly. The actual result was that 68.7% (TR520) and 70.6% (TR363) reads could be mapped to the sugar beet reference genome. Thus, the fractions of unmapped reads were 546 million (TR520) and 506 million (TR363).
  • Figure 1 shows the workflow of the WGS sequencing approach.
  • Genomic leaf DNA of the lines TR520 and TR363 was isolated using the CTAB protocol according to Rogers and Bendich (1985).
  • the requirements for sequencing on the Illumina HiSeq 2000 platform for DNA were OD260/280 >1.8 and a concentration of > 60 ng/ ⁇ (measured on a Nanodrop spectrophotometer) in a total volume of 20 ⁇ .
  • Genomic leaf DNA of the translocation lines TR520 and TR363 as shown in Table 1 was used for sequencing-library preparation using the Illumina TruSeq DNA sample preparation kit. Each library was sequenced (paired-end, 2x100 bp) on two lanes of the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA, USA) at the sequencing unit of the Institute of Clinical Molecular Biology (ICMB), Kiel, Germany.
  • Plants were grown in the greenhouse for two weeks, transferred to quartz sand, and three days later inoculated with 500 H. schachtii J2-larvae. Roots were sampled from inoculated plants one and three days after inoculation, and were subsequently frozen in liquid nitrogen. RNA Isolation and cDNA Synthesis
  • RNA from A. thaliana plants was generated using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany).
  • the cDNA synthesis was performed using the Superscript II First-Strand Synthesis System (Invitrogen, Düsseldorf) according to the manufacturer's protocol. 1 ⁇ g total RNA was used as template for cDNA synthesis.
  • Root material of inoculated TR520 (940043) plants was harvested one and three days post inoculation (dpi), total RNA was isolated and separated on agarose gels.
  • a RNA sequencing library was constructed using the Illumina TruSeq RNA sample preparation kit and subsequently sequenced (2x100 bp) on the Illumina HiSeq 2000 system at the sequencing unit of the Institute of Clinical Molecular Biology (ICMB). Bioinformatical Analysis
  • One of the strategies to separate the translocation specific read pairs is based on mapping the total number of reads against the draft genome assembly of B. vulgaris (RefBeet-0.4) using the Burrows- Wheeler- Aligner (BWA) (0) (Burrows and Wheeler, 1994).
  • the unmapped reads were extracted and assembled by SOAPdenovo (Li et al., 2008; Li et al, 2009b).
  • the output datasets are denoted in the following as 'TR520 unmapped de novo' and 'TR363 unmapped de novo '.
  • all short reads were directly assembled de novo by SOAPdenovo (Li et al., 2008; Li et al., 2009b).
  • the output datasets were denoted as 'TR520 de novo' and 'TR363 de novo'.
  • the reference assemblies of RefBeet-0.4, RefBeet-0.9, P. procumbens, and the new generated assemblies of the translocation lines TR520 and TR363 were used to create a BLAST databases of each via the CLC bio Genomics Workbench. Additionally a BLAST database was generated from the BAC-, BAC-end, and YAC-end sequences. In order to identify new translocation specific sequences, the datasets 'TR520 de novo' and 'TR363 unmapped de novo' were mapped to both the P. procumbens reference assembly and to the physical map data using local nucleotide BLAST (blastn) of the CLC bio Genomics Workbench.
  • the resulting datasets were separated by (1) bit score >1400, (2) e-value smallest to largest, and (3) identity threshold largest to smallest.
  • Promoter-specific regulatory elements as shown in Figure 10 were determined by using the signal scan search of the program PLACE (Plant Cis Acting Regulatory Elements, 2012) with default settings (Higo et al., 1999).
  • the multi FASTA file of the mRNA assembly was transformed to a BLAST database. All sequences anchored and not anchored to the physical map were blasted (blastn) against this database. The resulting output was filtered by score > 100. Significant aligned sequences of the mRNA dataset were extracted and analyzed by tblastx using the non-redundant protein database and default settings on the NCBI platform.
  • the amplification of the fragment is performed by PCR using the following protocol: 3 min at 94°C, 25-35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 64°C, and elongation for 1 min at 72°C, and a final elongation step for 10 min at 72°C.
  • the PCR is performed with cDNA of inoculated roots of the resistant genotype A906001 (seed code: 940043).
  • the corresponding insert is sub-cloned into the binary vector pAM194 using the integrated Xhol restriction sites. After restriction and cloning, the sequence is under the control of the constitutive 35 S promoter, a 35 S terminator, and the reporter gene GUS (KWS Saat AG, Einbeck, Germany) (Kifle et al., 1999).
  • PCR amplification is performed using one sequence specific primer and one vector specific primer, following sequencing of the amplified products.
  • the construct is transformed into competent cells of Agrobacterium rhizogenes strain Ril5834 and A. tumefaciens strain GV2260 by electroporation using the Gene Pulser II (Bio-rad, Kunststoff, Germany) with the following conditions: 200 R, 2.5 kV, 25 ⁇ .
  • Leaf stalks of the susceptible sugar beet line 930176 and the resistant translocation line 930363 are used for sugar beet hairy root transformation.
  • Leaf stalks are sterilized by submergence in 5% (w/v) CaCl 2 0 2 for 10 min, followed by treatment with 70% (v/v) ethanol for 5 min. After two washing steps with sterile water, the sterilized leaf stalks are cut into 2 cm pieces and incubated for 5 min with A. rhizogenes solution. The soaked explants are dried on sterilized Whatman filter paper before cultivation on solid 1 ⁇ 2 B5 Gamborg agar medium.
  • Transgenic A. thaliana plants are generated by using the floral dip transformation protocol (Clough and Bent, 1998). Seeds of the A. thaliana C24 ecotype are grown for three to four weeks (24°C, 16/8 h light/dark) until flowering. Developing floral tissues of the TO generation are dipped into the transformed A. rhizogenes inoculation medium for 2 min. Tl seeds are harvested and stored at 4 °C. The seeds are surface sterilized by washing in 70% ethanol for 1 min and incubated in sterilization solution containing 2.625% NaCl and 0.05% Tween for further 5 min, followed by three washing steps with sterile H 2 0 each for 30 sec. Sterile seeds are plated on 0.2 KNOP agar plates containing 50 ⁇ g/ml kanamycin. Selected vital Tl plants are transferred and isolated for growing in single pots, selfed, and T2 seeds are harvested.
  • the nematode resistance test is performed in vitro according to the protocol described by Sijmons (1991). Sugar beet hairy roots of 1 cm length are transferred to Petri dishes containing 0.2x KNOP medium supplemented with Daishin agar and T2 seeds as well as unmodified seeds of ecotype C24 as controls are sown in 6-well plates. After 14 to 16 days of growing in the climate chamber (22 °C, 16/8h light/dark photoperiod) each A. thaliana plant is inoculated with 100 sterilized H. schachtii SchachO J2-larvae.
  • HgCl 2 + which includes antibiotics (Streptomycin sulphate (0.2 mg/ml), Cefotaxime (0.25 mg/ml) and Miconazole (0.025 mg/ml))
  • the hairy roots are inoculated after 10-14 days of growth (22 °C; dark) with 200 sterilized H. schachtii SchachO J2 larvae.
  • Nematodes on the inoculated plants and clones are evaluated by counting the cysts under the stereomicroscope 28 days after inoculation.
  • the nematode pathotype H. schachtii SchachO is propagated on in vitro stock cultures of mustard (Sinapis alba cv. Albatros) roots grown on 0.2x KNOP medium, supplemented with 2% (w/v) sucrose and 0.8% (w/v) Daishin agar under sterile conditions, and on B. vulgaris (seed code: 930176) in the greenhouse under non-sterile conditions.
  • Fully developed cysts are harvested from the roots with 50 ⁇ gauze. Soaking the cysts in 3 mM ZnCl 2 for eight to ten days stimulated hatching of juveniles. The larvae are harvested with 10 ⁇ gauze, surface-sterilized with 0.05%> HgCl 2 solution for 30 seconds, washed four times in sterile water, resuspended in 0.2%> (w/v) Gelrite (Duchefa, Harlem, Netherlands), and used directly for inoculation experiments.
  • Another sterilization step is performed, for non- sterile J2 larvae, including incubation for 10 min in a solution containing streptomycin sulfate (0.2 mg/ml), cefotaxime (0.25 mg/ml), and miconazole (0.025 mg/ml).
  • ORF 803 is evaluated by qualitative reverse transcriptase-PCR (RT-PCR) on cDNA of the hairy root clones and A. thaliana plants.
  • RT-PCR amplification 0.5 ⁇ of the first-strand reaction is used with the following standard PCR-program: 3 min at 94°C, followed by 32 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 58°C and elongation for 1 min at 72°C, and finally 10 min elongation at 72°C.
  • Primers are specifically designed for exon regions, see Table 3. Table 3: Primers used for expression analysis of ORF 803 in resistant and susceptible plants, in sugar beet hairy root clones and in transgenic A. thaliana plants.
  • the histochemical GUS test is performed with in vitro cultured sugar beet hairy roots and A. thaliana leaves.
  • the plant material is incubated overnight at 37 °C in 10 ml X-Gluc solution containing 50 mM Na 3 P0 4 buffer (pH 7.0), 0.2 mg/ ml X-Gluc (5-bromo-4-chloro-3- indolyl ⁇ -D-glucuronide), and 20 ⁇ Triton.
  • the solution is removed, and the plant material is washed several times with 70% ethanol. Blue staining is evaluated using a stereomicroscope (Stemi SV 11, Zeiss, Germany).
  • the physical map of TR520 is composed of three BAC contigs (Capistrano, 2009).
  • One aim was to select translocation-specific sequences anchored to BACs or YACs to extend the physical map. Scaffold walking was performed in order to identify new sequences linked to the physical map in the following way: BLAST output datasets were screened for significant alignments of the translocation lines TR520 and TR363 to BACs or YACs and significant alignments to P. procumbens.
  • the identified sequences, scaffolds or contigs were extended by BLAST analyses against the datasets 'TR520 unmapped de novo', 'TR520 de novo', 'TR363 unmapped de novo', 'TR363 de novo', and P.
  • procumbens in order to identify new scaffolds that overlap on the proximal (centromeric) or distal (telomeric) end. In this way super scaffolds could be generated.
  • additional sequence information anchored to BAC contig 1 , BAC contig 2, and BAC contig 3 could be obtained. Consequently, super contigs were generated by incorporation of super scaffolds to the BAC contigs.
  • ORF prediction and the exon-intron structure was determined using the ORF prediction web-based tools FGENESH (Softberry FGENESH, 2007) or GEN SCAN (GENSCAN, 2009). Sequence similarities to known proteins from other organisms were identified using the tool protein BLAST with the non-redundant protein database on the NCBI platform.
  • ORF prediction of the non-anchored sequences resulted in 134 ORFs.
  • Significant similarities to known genes from other organisms were identified by BLAST (blastp) using the non-redundant protein database on the NCBI platform.
  • a number of 44 ORFs out of the predicted 134 ORFs showed no significant similarity to known proteins from other organisms in the non-redundant protein database of NCBI.
  • Sequence similarity to unclassified hypothetical proteins was identified in 25 predicted ORFs, and a number of 20 ORFs showed significant similarity to retroelements or transposons. For 45 predicted ORFs, significant similarities to proteins from other organisms were determined.
  • ORF 801 - ORF 811 were selected for further analysis in accordance to their position on the physical map and with sequence similarity to known genes involved in plant stress-response.
  • ORFs were selected as resistance gene candidates for Hsl-2 due to their homology to genes known to be involved in plant response to nematodes or plant stress response.
  • the searching criteria were as follows: plants have evolved two systems to defend themselves against pathogens: the pathogen-associated molecular patterns (PAMP) triggered immunity (PTI) and the effector-triggered immunity (ETI).
  • PAMP pathogen-associated molecular patterns
  • PTI pathogen-associated molecular patterns
  • ETI effector-triggered immunity
  • Pattern recognition receptors PRRs
  • RLKs receptor-like kinases
  • RLPs receptor-like proteins
  • resistance genes encode proteins that mostly consist of a nucleotide-binding site (NBS) and an LRR motif for effector detection (Gohre and Robatzek, 2008).
  • NBS nucleotide-binding site
  • LRR motif for effector detection Gohre and Robatzek, 2008.
  • Many of the cloned nematode resistance genes belong to the group of NBS -LRR genes, such as Mi-1.2, Gpa2, Grol-4, or Ma (Claverie et al., 2011; Milligan et al., 1998; Paal et al., 2004; van der Vossen et al., 2000; Vos et al., 1998).
  • SAR systemic acquired resistance
  • PR pathogenesis-related proteins
  • ISR induced systemic resistance
  • the ORFs mentioned were from newly assembled super scaffolds both anchored and non- anchored to the physical map (super contig 1 and super contig 2-3, and super scaffold 1 - super scaffold 13). Their numbers start with an '8'.
  • the exon/intron structure was determined by transferring selected non-anchored sequences of the TR520 and TR363 assemblies to the web-based tools FGENESH (Softberry FGENESH, 2007) or GENSCAN (GENSCAN, 2009).
  • ORFs were selected due to their sequence similarity to known proteins involved in plant stress response. Primers were designed based on exon regions of the ORFs. Selected ORFs were mapped to the physical map by PCR experiments using genomic DNA of B. vulgaris, P. procumbens, and the translocation lines TR520, TR363, TR659, and TR320. The transcriptional activities were analyzed by RT-PCR amplification using cDNA of inoculated root material of the translocation line.
  • RNA of inoculated root material of the nematode resistant translocation line TR520 was isolated and RNA-Seq was performed. During this process, mRNA is reversely transcribed to cDNA which subsequently is sequenced. The de novo assembly of the short reads was performed with the software Trinity (Grabherr et al., 2011) and consists of 213,385 sequences, of which the longest is 17,309 bp in size. In the following it is referred to these data as the transcriptome data.
  • transcriptome data were blasted (BLASTn) against the previously identified genomic sequence of each candidate ORF, and the hit sequences of the transcriptome data with the highest score were separated.
  • the selected sequences were analyzed for homology to annotated proteins by tblastx using the non-redundant protein database at the NCBI platform.
  • Primers were designed from the predicted exons of the ORFs, see Figure 9. These primer sequences were aligned to the assembly of the transcriptome data. As a result significant hits were determined for all primer sequences, which verify that all primers used for RT-PCR, see Figure 6 are located on exon sequences.
  • ORF 803mRNA an alignment of ORF 803 mRNA to the proteins of a MAP3K of A. thaliana and to the MAP3K proteins CTR1 and EDR1, which were previously used for alignments with ORF803 is shown. Pairwise comparisons between the sequences showed the overall sequence similarity, which was calculated using the CLC bio Genomics Workbench.
  • ORF803mRNA shows a sequence similarity of 48.3% to MAP3K, 54.5% to CTR1 and 34.7% to EDR1.
  • the overall sequence similarity of ORF803mRNA to MAP3K, CTR1 and EDR1 is higher than determined for the predicted protein sequence of ORF803.
  • ORFs that are present on the resistant and absent from the susceptible translocation lines were of major interest for further studies. None of the selected ORFs was present only on the resistant translocation lines TR520 and TR363, and absent from the susceptible ones. To find out whether ORFs are differentially expressed, differences between gDNA and cDNA were determined for six ORFs by RT-PCR. ORF 803 was found to be present on the translocation lines TR520, TR363, and TR659 (primer combination H42H43). Interestingly, this sequence was the only that was differentially expressed between the resistant (TR520 and TR363) and the susceptible genotypes (TR659 and TR320). Therefore, this ORF was further investigated.
  • Figure 4A gives an overview of the predicted exon-intron structure of ORF803, the promoter region and the primer combinations designed. A number of 18 exons were predicted by the software FGENESH (Softberry FGENESH, 2007) using the dicotyledonous dataset, see Figure 4B.
  • RT-PCR was additionally performed with the primer combinations H42H132, located on the predicted exon 1 and exon 4, and H133H134 present on predicted exon 10, using cDNA of inoculated root material, see Table 2 of B. vulgaris (930176), P. procumbensl (35535), and the translocation lines TR520, TR363, TR659, and TR320, see Figure 4 A and B. Amplicons were observed for P. procumbens and the lines TR520 and TR363, using the primer combinations H42H43, which amplified on exon 1, and H42H132, which amplified from exon 1 to exon 4, see Figure 4 D and E.
  • amplicons were determined for P. procumbens, the resistant translocation lines TR520 and TR363, and the susceptible translocation line TR659, see Figure 4 C to E.
  • RT-PCR amplicons of the conserved transglutaminase domain were found only in the resistant translocation lines TR520 and TR363 (primer combinations H42H43 and H42H132).
  • the primer combination H133H134 located on exon 10 amplified cDNA from both resistant lines and the susceptible translocation line TR659, see Figure 4 C to E. These might indicate differential splicing of the gene.
  • Alternative splicing increases the protein diversity by differential splicing of precursor (pre) mRNA to various mRNA isoforms for example by post-transcriptional regulation such as exon skipping or intron retention (Syed et ah, 2012).
  • ORF803 The predicted protein sequence of ORF803 shows homology to the following proteins: CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) (XP 02279319.2) from V. vinifera, ENHANCED DISEASE RESISTANCE 1 (EDR1) (ABR45974.1) from A. thaliana, and a mitogen activated protein kinase kinase kinase (MAP3K) (CAB8658.1) from A. thaliana, see Table 4. All proteins are known to be involved in the regulation of ethylene signaling and stress response (Tang et ah, 2005; Huang et ah, 2003; Lin et al., 2010).
  • the predicted protein sequence of ORF803 was aligned with the protein sequences of EDR1 , CTR1, and a MAP3K of A. thaliana because these genes showed the highest identity to ORF 803 and were involved in biotic stress response, see Figure 7.
  • the transglutaminase domain in the N-terminal region of the protein of ORF 803 was similar to EDR1, CTR1 , and MAP3K, shown as underlined in the alignment in Figure 7.
  • the kinase domain in the C-terminal region is highly conserved between MAP3K, CTR1, EDR1, and ORF 803, shown as bold and underlined in Figure 7.
  • Table 4 Analysis of the predicted amino acid sequence of ORF803 using the protein BLAST database for homology search to known proteins from other organisms and conserved domains on the NCBI platform.
  • ORF 803 The predicted protein sequence of ORF 803 showed an overall sequence similarity of 35.48%) to CTRl, 24.18% to EDRl, and 34.9% to MAP3K, see Figure 5.
  • ORF803 indeed seemed to be the sequence for the Hsl-2 gene due to its sequence similarity to MAP3K and its expression in both resistant translocation lines (TR520, TR363).
  • ORF803 is alternatively spliced.
  • Alternative splicing is a mechanism to increase the protein diversity by generation of various protein iso forms originating from a single gene (Stamm et al., 2005).
  • A. thaliana 42% of all intron- containing genes are supposed to be alternatively spliced (Filichkin et al., 2010).
  • An amount of approximately 60% - 70% of alternative splicing takes place in translated regions of the mRNA and might have effects on protein stability, signaling activities, and binding properties (Stamm et al., 2005).
  • Alternative splicing events can be classified to four subgroups: during 'exon skipping' exons are spliced out of the precursor (pre-) mRNA; 'selection of alternative 5' or 3' splice sites' is the result of additional splice sites of one end of an exon, and 'intron retention' means that introns are incorporated into the mRNA (Keren et al., 2010; Kim et al., 2008).
  • Ner-Gaon et al. (2004) examined A. thaliana EST data from public databases such as TIGR (The Institute for Genomic Research TIGR, 2012) in order to determine different types of alternative splicing. They found intron retention as the major alternative splicing events in A. thaliana (30%>), which was unexpected, since in human, only 2 - 5% of alternative splicing events are the result of intron retention. Interestingly, transcripts involved in stress and stimuli response were overrepresented among alternatively spliced transcripts, whereas transcripts related to metabolism and cell maintenance were underrepresented (Ner-Gaon et al., 2004). Iida et al. (2004) reported about biotic stresses affecting alternative splicing profiles in A. thaliana.
  • the disease resistance gene RESISTANCE TO PSEUDOMONAS SYRINGAE4 produces multiple transcripts via alternative splicing.
  • the alternative transcript was up-regulated during resistance response in A. thaliana Columbia (Col-0) (Zhang and Gassmann, 2007).
  • Wdreb2 produces three alternatively spliced forms. They are differentially expressed and activated by several abiotic stresses (Egawa et al., 2006).
  • ORF803 in contrast to amplification with gDNA, exon 1 and exon 4 could not be amplified using the primer combination H42H43 and H42H132, respectively, by RT-PCR in the susceptible line TR659; see Figure 4.
  • transcripts of ORF803 that include exon 1 to exon 4, where a major part of the transglutaminase domain is located, are transcribed during stress response in contrast to transcripts with the alternative spliced sites. Since the susceptible translocation lines TR659 and TR320 were produced by gamma irradiation and the generation of alternative spliced mRNAs is regulated by proteins which bind to czs-acting sites on the pre-mRNA, a mutation in a specific binding site might also be a possible scenario (David and Manley, 2008).
  • ORF803 is located on scaffold 26267 of the 'TR520 de novo' assembly, which is incorporated into super contig 1.
  • the continuous sequence upstream of the ATG until the next gap (3,922 bp) on scaffold 26267 was selected for further analyses of regulatory elements using the PLACE database (Higo et al, 1999; Plant Cis Acting Regulatory Elements, 2012). Regulatory elements, which could indicate root specific expression or are related to disease and defense response were selected and are shown in Table 5. Five different elements were identified that could indicate a root specific expression of the gene. Additionally, six different motifs for defense related gene expression were determined. Among them, elicitor responsiveness motifs and binding sites for WR Y transcription factors were identified, which can act as positive or negative regulators in defense responses (Eulgem and Somssich, 2007; Koschmann et al., 2012).
  • Table 5 Overview of selected root and disease specific regulatory elements present in the promoter region (3,922 bp) of ORF803 that were identified using the web-based program PLACE (Higo et al., 1999; Plant Cis Acting Regulatory Elements, 2012). Name of regulatory element, sequence, copy number on + and - strand and functions are given.
  • ORF803 constitutive expression of ORF803 in sugar beet hairy roots and A. thaliana, as R-genes are often expressed constitutively in plants (Gu et al., 2005).
  • the coding sequence of ORF803 was under the control of the constitutive 35S promoter, a 35S terminator and the reporter gene GUS in the binary vector pAM194 (Kifle et al., 1999; KWS Saat AG, Einbeck, Germany).
  • the A. rhizogenes strain Ril5834 was used to transform different sugar beet accessions.
  • the sugar beet accession 930176 was chosen for hairy root transformation with ORF 803 because of its susceptible phenotype. To generate hairy root clones that serve as controls, the susceptible line 930176 and the resistant line 930363 is inoculated with untransformed A. tumefaciens to induce hairy root growth.
  • Sugar beet hairy root clones are grown on 1 ⁇ 2 B5 medium containing 150 ⁇ g/ml cefotaxime for 21 days. They are numbered in the following way e.g. 1, 2, 3, 4. Then they are further cultivated by cutting into 2-4 cm pieces. Those sub-clones are numbered in the following way e.g. 1 1, 2 1, 2 2, 2 3, 3 1.
  • the in vitro nematode resistance test is divided into two experiments. A total of 24 T2 families are selected for nematode inoculation experiments. In experiment 1, a total number of 15 T2 families is tested. In experiment 2, two T2 families of experiment 1 are also included. Thus, the number of tested T2 families sums up to 11. In total, 36 seeds of each T2 family are sown on 0.2x K OP media in six 6-well plates and inoculated with 100 sterilized J2 larvae per plant. Besides that, untransformed plants of the A. thaliana ecotype C24 are grown as controls. During the experiments, leaf material of all T2 plants is harvested for GUS staining. The T2 families segregated into transgenic (GUS positive) and non-transgenic plants. A ⁇ 2 -test is performed with phenotypic data to test deviation from 3 : 1 segregation as expected for a single copy introgression of the transgene.
  • Hsl-2 gene (SEQ ID NO: 1) is isolated from appropriate genomic library, such as BAC, YAC, and ⁇ -library using a PCR fragment from the 5'-region of the Hsl-2 gene as a probe or by PCR amplification with PCR primers containing appropriate restriction sites.
  • the promoter fragment is fused with the GUS gene and introduced into the root of sugar-beets and potatoes by means of the vector pBIN19 and Agrobacterium tumefaciens/Agrobacterium rhizogenes co-transformation.
  • the GUS activity was only detectable in the syncytium.
  • a corresponding GUS activity is not discernible. This shows that the promoter used has a "pathogen-responsive" element or elements, which is or are activated more strongly after nematode attack.
  • the Hsl-2 promoter enables the tissue- specific expression of any gene in host plants, such as for example the sugar-beet. From these experiments it can be concluded that the promoter according to the present invention binds transcription factors which derive from nematodes or which are formed as a result of the infection. Thus, the experiments demonstrate the root-specificity and nematode inducibility of the Hsl-2 promoter. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, sequences or other disclosures) in the Background of the Invention, Description, Examples, and Sequence Listing is hereby incorporated herein by reference.
  • Benfey PN, Ren L, Chua NH., 1990b Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 1990 Jun;9(6): 1677-84.
  • Capistrano G.G.G., 2009: A candidate sequence for the nematode resistance gene Hsl-2 in sugar beet. PhD-thesis, Plant Breeding Institute, Kiel - Germany.
  • Floral dip a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana Plant Journal 16, 735-743.
  • Kandoth, P.K., N. Ithal, J. Recknor, T. Maier, D. Nettleton, T.J. Baum, and M.G. Mitchum, 2011 The soybean Rhgl locus for resistance to the soybean cyst nematode Heterodera glycines regulates the expression of a large number of stress- and defense-related genes in degenerating feeding cells. Plant Physiology 155, 1960-1975.
  • Knecht, K., 2010 Molecular mechanisms of the Hsl pro- 1 -mediated nematode (Heterodera schachtii) resistance and its potential for genetic engineering of plant disease resistance. Thesis; Institut fur Phytopathology, Christian- Albrechts Universitat, Kiel, Germany.
  • the root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes. Plant Cell 10, 1307-1319.
  • NCBI National Center for Biotechnology Information
  • Plesch, G., T. Ehrhardt, and B. Mueller-Roeber, 2001 Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression.
  • TMHMM Server 2012: http://www.cbs.dtu.dk/services/TMHMM/.
  • Vasil et al. Cell Culture and Somatic Cell Genetics of Plants, Vol. 1, II and 111, Laboratory Procedures and Their Applications, Academic Press, 1984 Vasil, et al. (1992) Bio/Technology 10,667-674
  • VfLb29 The Promoter of the Vicia faba L. Leghemoglobin Gene VfLb29 Is Specifically Activated in the Infected Cells of Root Nodules and in the Arbuscule-Containing Cells of Mycorrhizal Roots from Different Legume and Nonlegume Plants. Molecular Plant-Microbe Interactions 17, 62-69.

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Abstract

La présente invention concerne des molécules d'acide nucléique codant pour des polypeptides qui peuvent conférer une résistance contre des pathogènes végétaux tels que des nématodes (par ex. Heterodera schachtii Schmidt et isolats apparentés) et être déclenchés par ceux-ci. En particulier, l'invention concerne des molécules d'acide nucléique liées au gène de résistance au nématode des racines de betterave (BCN) Hs1-2 et certains de ses modes de réalisation comprennent des amorces spécifiques, des vecteurs, des cellules hôtraittes, des polypeptides, des anticorps, des aptamères, des plantes transgéniques, des procédés de production et d'utilisation associés, et des procédés permettant d'influer sur la caractéristique de résistance dans une plante. En outre, l'invention concerne des plantes transgéniques résistantes aux nématodes, en particulier Heterodera schachtii à cause de l'expression d'un transgène Hs1-2. En outre, l'invention concerne des procédés de criblage permettant d'identifier et d'obtenir des composés phytoprotecteurs.
PCT/EP2013/053642 2013-02-22 2013-02-22 Gène de résistance dérivé d'une plante WO2014127835A1 (fr)

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EP3567111A1 (fr) * 2018-05-09 2019-11-13 KWS SAAT SE & Co. KGaA Gène de résistance à un pathogène du genre heterodera
CN112322633A (zh) * 2020-11-12 2021-02-05 华南农业大学 一种水稻根结线虫抗性基因OsBetvI及其应用
WO2021093943A1 (fr) * 2019-11-12 2021-05-20 KWS SAAT SE & Co. KGaA Gène de résistance à un pathogène du genre heterodera
CN113481215A (zh) * 2021-07-06 2021-10-08 清华大学深圳国际研究生院 一种新型四环素抗性基因tetX及其应用
CN113493762A (zh) * 2020-04-08 2021-10-12 中国科学院分子植物科学卓越创新中心 增强植物免疫效应的方法及其用途
EP3913059A1 (fr) 2020-05-19 2021-11-24 Christian-Albrechts-Universität zu Kiel Résistance aux nématodes dans les plantes

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