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• • 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
• • 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

Definitions

• the present invention relates to transgenic plants and plant cells comprising a gene encoding an NRCl protein (NB-LRR Required for HR-associated Cell Death 1) integrated in its genome and methods for making such plants and cells. Especially, NRCl protein (NB-LRR Required for HR-associated Cell Death 1) integrated in its genome and methods for making such plants and cells. Especially, NRCl protein (NB-LRR Required for HR-associated Cell Death 1) integrated in its genome and methods for making such plants and cells. Especially
• Solanaceae plants and plant parts with enhanced disease resistance are provided. Also provided are isolated nucleic acid molecules encoding
• NRCl proteins according to the invention, vectors comprising these, as well as isolated NRCl proteins themselves. Further, plant cells and plants comprising one or more mutations in an endogenous NRCl allele are provided, whereby the mutation(s) confer enhanced diseases resistance to the plants and plant cells.
• R genes plant resistance genes
• NB nucleotide binding site
• LRR leucine-rich repeats
• This group consists of genes encoding CC-NB-LRR proteins, containing a coiled-coil domain and genes that encode proteins that have a domain similar to mammalian Toll and interleukin (IL) receptors, the so-called TIR-NB-LRR proteins (Hammond-Kosack and Jones, 1997, supra).
• IL interleukin
• VGS virus-induced gene silencing
• SGTl is involved in multiple defense pathways, such as N-, Rx- and Pto-mediated HR and resistance, and Cf-4- and Cf-9-mediated HR
• SGTl is an interactor of SKPl, which is a component of the SCF E3- ligase complex that is involved in ubiquitination of proteins, a modification which targets them for degradation (Schwechheimer and Schwager, 2004, Plant Cell Reports 23, 353-364).
• MAPKs mitogen activated protein kinases
• NtWIPK wound- induced protein kinase
• NtSIPK salicylic acid- induced protein kinase
• benthamiana inhibits the Cf-9/Avr9- and Cf-4/Avr4-dependent HR (Romeis et al., 2001, EMBO J. 20, 5556-5567) and VIGS of LeACIKl (Avr/Cf-induced kinase 1) in tomato results in decreased C. fulvum resistance (Rowland et al., 2005, Plant Cell 17, 295-310).
• the activation of kinases during defense and the decreased resistance upon 'knock-down' of their encoding genes supports their function in defense activation.
• MAPKKK ⁇ was identified as a positive regulator of both resistance and disease (Del Pozo et al, 2004, EMBO J. 23, 3072-3082).
• Lu et al. (2003, EMBO J. 22, 5690-5699) performed VIGS using 4992 cDNAs from a normalized N benthamiana cDNA library cloned into a PVX vector.
• 79 (1.6%) corresponded to genes required for Pto-mediated HR, whereas silencing of only six of them also impaired Pto-mediated resistance against Pseudomonas syringae.
• VIGS using a cDNA corresponding to HSP90 abolished not only Pto-mediated HR but also Pto-, Rx- and N-mediated resistance, indicating that HSP90 is required in multiple disease resistance pathways.
• the same set of cDNAs was also used for VIGS in ⁇ -transgenic N. benthamiana, after which the plants were inoculated with a GFP-tagged strain of TMV. Resistance against TMV was most significantly suppressed upon silencing using a cDNA fragment derived from a CC- NB-LRR-encoding gene, referred to as NRGl (N requirement gene 1) (Peart et al., 2005, Curr. Biol. 15, 968-973).
• NRGl was shown to be specifically required for N gene function, indicating that CC-NB-LRR proteins do not only act as resistance proteins involved in recognition of avirulence factors, but are also involved in the signaling pathway initiated by the TIR-NB-LRR protein N, which eventually leads to resistance (Peart et al., 2005, supra).
• the tobacco NRGl protein functions downstream of the plant's defense signaling cascade initiated by a resistance protein, it has the drawbacks that it is specifically involved in TV-mediated resistance against tobacco mosaic virus (TMV) and is not a general co factor of disease resistance (Rx- and Pto-mediated resistance against PVX and Pseudomonas syringae were unaffected by NRGl silencing), whereby it may not be suitable for creating broad pathogen resistance in crops such as tomato.
• TMV tobacco mosaic virus
• HR refers to the hypersensitive response, i.e. local plant cell death, seen as either microscopic lesions (as described by Rivas and Thomas, 2005, Ann Rev Phytopath 43: 395-436) and/or macroscopic lesions. Hypersensitive cell death is usually associated with other plant responses, such as production of reactive oxygen species and the activation of defense related genes in cells surrounding the HR lesion.
• Plant pathogens refer to biotic agents which are capable of causing disease on plants, such as plant pathogenic fungi, bacteria, viruses, oomycetes, mycoplasma like organisms, nematodes, white fly and aphids and the like. Generally all strains, races or pathovars of a pathogen species which are capable of causing disease on host tissue are included herein.
• Biotrophic plant pathogens or “biotroph” refers to a pathogen that keeps the host plant cells alive and relies on living cells for growth and tissue colonization.
• Hemibiotrophic plant pathogen or “hemibiotroph” refers to a plant pathogen which keeps the host cells alive during at least part of its life cycle.
• Necrotrophic plant pathogen refers to a plant pathogen which actively kills plant cells upon tissue colonization, by producing toxic enzymes, proteins or metabolites that kill host cells.
• Elicitor independent HR refers to a hypersensitive response which develops without a pathogen or a pathogen elicitor (e.g. a fungal Avr protein) being present.
• a constitutively active NRCl protein one may also distinguish between "constitutive HR”, whereby reference is made to the development of HR lesions in the absence of pathogens or pathogen elicitor proteins, and "induced HR”, whereby reference is made to the development of HR lesions following the presence of an inducing stimulus (e.g. following induction of the promoter which drives expression of the nucleic acid sequence encoding the NRCl protein, or variant thereof).
• “Solanaceae” refers herein to plant genera, species, and varieties thereof, belonging to the family Solanaceae. These include species belonging to the genus Solarium (including Solarium lycopersicum, which used to be known as Lycopersicon esculentum), Nicotiana, Capsicum, Petunia and other genera.
• Disease resistance refers herein to various levels of disease resistance or tolerance of a plant, including moderate resistance and high resistance or complete resistance to one or more pathogens. It can be measured and optionally quantified by comparison of pathogen caused symptoms (such as frequency and/or size of HR lesions, fungal mycelium, etc.) relative to those seen in susceptible control plants when grown under identical disease pressure. Such disease bioassays can be carried out using known methods. Disease resistance can also be indirectly measured as higher yield of resistant plants compared to susceptible plants when grown under disease pressure. "Enhanced disease resistance” refers to any statistically significant increase in disease resistance of a plant or plant tissue compared to a suitable control. Both a qualitative increase (e.g. from susceptible to resistant) and a quantitative increase are encompassed herein.
• a plant having enhanced disease resistance to at least one pathogen is a plant comprising at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% higher levels of resistance to the pathogen than the control plant, using appropriate bioassays and/or field assays for assessing disease resistance.
• “Broad spectrum” disease resistance refers to enhanced resistance against at least two, three, four, or more pathogens of different pathogen species.
• a host plant having enhanced resistance to several biotrophic and/or hemibiotrophic and/or necrotrophic pathogen species would be considered to have broad spectrum resistance.
• “Pathogen caused symptoms” include any symptoms of disease, such as mycelium growth/biomass on/in the host tissue, bacterial growth/biomass, size and/or frequency of necrotic or chlorotic lesions on plant tissue, size and/or frequency of cankers, etc.
• nucleic acid sequence refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention.
• isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
• protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin.
• a “fragment” or “portion” of a protein may thus still be referred to as a "protein”.
• An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
• “Functional”, in relation to NRCl proteins (or variants, such as orthologs or mutants, and fragments), refers to the capability to modify the (quantitative and/or qualitative) development of HR lesions and/or the level of disease resistance by modifying the expression level of the NRCl -encoding gene (e.g. by overexpression or silencing) in a plant.
• the functionality of a putative NRCl protein obtained from plant species X can be tested by various methods. If the protein is functional, silencing of the NRCl gene encoding the protein in plant species X, using e.g.
• VIGS or gene silencing vectors will lead to a reduction or suppression of pathogen- or elicitor induced HR lesions and/or a reduction of pathogen resistance, as shown in the Examples for tomato.
• complementation with a functional NRCl protein will be capable of restoring HR lesions and/or pathogen resistance.
• transient or stable (over)expression in species X of the gene encoding the NRCl protein (optionally together with a posttranscriptional gene silencing inhibitor) will lead to the development of elicitor independent HR lesions and/or enhanced disease resistance, especially against biotrophic and/or hemi-biotrophic pathogens. See also the Examples.
• gene means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
• a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3 'non- translated sequence comprising e.g. transcription termination sites.
• a “chimeric gene” refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature.
• the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region.
• the term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).
• a "3 ' UTR” or “3 ' non-translated sequence” refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises for example a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof).
• a polyadenylation signal such as e.g. AAUAAA or variants thereof.
• the mRNA transcript may be cleaved downstream of the polyadenylation signal and a po Iy(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).
• “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi).
• An active protein in certain embodiments refers to a protein being constitutively active.
• the coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.
• the DNA sequence is preferably present in the form of an antisense DNA or an inverted repeat DNA, comprising a short sequence of the target gene in antisense or in sense and antisense orientation.
• “Ectopic expression” refers to expression in a tissue in which the gene is normally not expressed.
• a “transcription regulatory sequence” is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcription regulatory sequence.
• a transcription regulatory sequence as herein defined will thus comprise all of the sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers.
• promoter elements e.g. attenuators or enhancers.
• regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition.
• promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
• a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
• an “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated.
• a “tissue specific” promoter is only active in specific types of tissues or cells.
• a “promoter active in plants or plant cells” refers to the general capability of the promoter to drive transcription within a plant or plant cell. It does not make any implications about the spatiotemporal activity of the promoter.
• operably linked refers to a linkage of polynucleotide elements in a functional relationship.
• a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.
• Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein".
• a “chimeric protein” or “hybrid protein” is a protein composed of various protein "domains” (or motifs) which is not found as such in nature but which a joined to form a functional protein, which displays the functionality of the joined domains (for example a Coiled Coil domain (CC), a nucleotide binding domain (NB- ARC) and a Leucine Rich Repeat (LRR) region may be combined).
• a chimeric protein may also be a fusion protein of two or more proteins occurring in nature.
• domain as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain.
• target peptide refers to amino acid sequences which target a protein, or protein fragment, to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or to the extracellular space or apoplast (secretion signal peptide).
• a nucleic acid sequence encoding a target peptide may be fused (in frame) to the nucleic acid sequence encoding the amino terminal end (N-terminal end) of the protein or protein fragment, or may be used to replace a native targeting peptide.
• a "nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell.
• the vector backbone may for example be a binary or superbinary vector (see e.g. US 5591616, US 2002138879 and WO95/06722), a co-integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g.
• Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like (see below).
• a “host cell” or a “recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, especially comprising a chimeric gene encoding a desired protein or a nucleic acid sequence which upon transcription yields an antisense RNA or an inverted repeat RNA (or hairpin RNA) for silencing of a target gene/gene family, having been introduced into said cell.
• the host cell is preferably a plant cell or a bacterial cell.
• the host cell may contain the nucleic acid construct as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell.
• host may also refer to the host plant species which a pathogen is able to invade or infect, but this will be clear from the context. Plant species are classified as “host” or “non-host” species in relation to a pathogen. "Non-host” species are completely immune to pathogen infection of all races or strains of a pathogen, even under optimum conditions for disease development.
• the "host” species are also referred to as the "host range" of a pathogen and are immune to certain (but not all) races of a pathogen.
• selectable marker is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker.
• Selectable marker gene products confer for example antibiotic resistance, or more preferably, herbicide resistance or another selectable trait such as a phenotypic trait (e.g. a change in pigmentation) or a nutritional requirements.
• reporter is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, GUS and the like.
• ortholog of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of the tomato nrcl gene may thus be identified in other plant species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and functional analysis.
• homologous and heterologous refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism, especially in the context of transgenic organisms.
• a homologous sequence is thus naturally found in the host species (e.g. a tomato plant transformed with a tomato gene), while a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants).
• a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants).
• the term "homolog” or “homologous” may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs).
• Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60 0 C. Lowering the salt concentration and/or increasing the temperature increases stringency.
• Tm thermal melting point
• Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20 min, or equivalent conditions.
• Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50 0 C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
• Sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimises the number of gaps.
• the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
• Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or Emboss Win version 2.10.0 (using the program "needle").
• percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc.
• the verb "to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
• reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
• the indefinite article “a” or “an” thus usually means “at least one”. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.
• plant includes plant cells, plant tissues or organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, fruit (e.g. harvested tomatoes), flowers, leaves, seeds, roots, root tips and the like.
• the present inventors have used cDNA-AFLP analysis, in combination with VIGS (Virus Induced Gene Silencing), to identify genes involved in Cf-4/Avr4-dQpmdQnt HR and disease resistance.
• VIGS Virus Induced Gene Silencing
• NRCl CC-NB-LRR type resistance protein analog
• NRCl is involved in multiple HR and multiple disease resistance / cell death signaling pathways, such as Cf-9/Avr-9-, LeEix2/Eix-, Pto/AvrPto- and Rx/CP- initiated HR (see Examples). Further tests are being conducted to determine whether NRCl is also involved in other HRs, such as the Mz ' -mediated HR (conferring resistance to nematodes, white fly and aphid- induced HR; see US 6613962 and EP0937155B1).
• Mz ' -mediated HR conferring resistance to nematodes, white fly and aphid- induced HR; see US 6613962 and EP0937155B1.
• NRCl is involved in HR pathways triggered by both extra- and intracellular disease resistance proteins which belong to different classes: extracellular receptor like proteins (RLPs, such as Cf-4, Cf-9 and LeEix2), Ser/Thr protein kinases such as Pto and a CC-NB-LRR protein (Rx), which confer resistance to respectively fungi ⁇ Cladosporium fulvum and Trichoderma viride), a bacterium (Pseudomonas syringae pv tomato) or a virus (PVX).
• RLPs extracellular receptor like proteins
• Ser/Thr protein kinases such as Pto
• Rx CC-NB-LRR protein
• the NRCl protein (and the NRCl gene encoding it) can be used to confer or enhance plant resistance against a variety of pathogens, especially biotrophic and hemi- biotrophic plant pathogens, but also necrotrophic plant pathogens such as Botrytis species.
• expression of NRCl leads to enhanced resistance, especially against pathogens biotrophic and/or hemibiotrophic pathogens, i.e. all pathogens which obtain nutrients from living cells.
• the knock-down (gene silencing) or knock-out e.g.
• NRCl genes can be used to confer or enhance resistance against necrotrophic pathogens, as the pathway leading to necrosis is affected and necrotophic pathogens require this pathway.
• an increase or a decrease in NRCl expression levels may be used to enhance resistance.
• both approaches may be used in one plant, e.g. under control of different promoters.
• NRCl can be expressed under control of a promoter induced by a (hemibiotrophic pathogen, to confer resistance to biotrophic and/or hemibiotrophic leaf pathogens, while at the same time endogenous NRCl gene (or gene family) can be silenced in certain tissues, or upon induction by a necrotroph using a promoter which is inducible by necrotrophic pathogens or wounding.
• NRC1 D481V a constitutively active NRCl protein
• HR elicitor independent cell death
• NRCl protein obtained from tomato shows low sequence identity (less than 25%) to NRGl of tobacco. NRCl also contains a larger number of Leucine Rich Repeats (LRR) than NRGl.
• LRR Leucine Rich Repeats
• the protein structure of NRCl is shown in figure 1 and SEQ ID NO: 2.
• nucleic acid sequences and amino acid sequences of NRCl proteins are provided (including orthologs), as well as methods for isolating or identifying orthologs of NRCl in other plant species, such as other Solanaceae, preferably potato.
• methods for isolating or identifying other NRCl alleles, such as alleles from other tomato species, varieties, lines or accessions are provided herein.
• NRCl proteins comprise the protein depicted in SEQ ID NO: 2 (wild type) and 4 (constitutive mutant), as well as fragments and variants thereof.
• Variants of NRCl include, for example, proteins having at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99%, or more, amino acid sequence identity (over the entire length) to SEQ ID NO: 2 and/or 4. Amino acid sequence identity is determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above.
• NRCl can be obtained from various sources, such as existing sequence databases, from other plant species (especially other species of Solanaceae, such as potato) or other varieties or they can be made by de novo synthesis, mutagenesis and the like.
• SEQ ID NO: 4 a constitutively active NRCl mutant, which is a variant of SEQ ID NO: 2, and was made by targeted mutagenesis using overlap PCR (see Examples).
• the NRCl proteins according to the invention may, thus, be isolated from natural sources, synthesized de novo by chemical synthesis (using e.g. a peptide synthesizer such as supplied by Applied Biosystems) or produced by recombinant host cells by expressing the nucleic acid sequence encoding the NRCl protein, fragment or variant.
• NRCl variants may comprise conservative amino acid substitutions within the categories basic (e. g. Arg, His, Lys), acidic (e. g. Asp,Glu), nonpolar (e. g. Ala, VaI, Trp, Leu, He, Pro, Met, Phe, Trp) or polar (e. g. GIy, Ser, Thr, Tyr, Cys, Asn, GIn).
• basic e. g. Arg, His, Lys
• acidic e. g. Asp,Glu
• nonpolar e. g. Ala, VaI, Trp, Leu, He, Pro, Met, Phe, Trp
• polar e. g. GIy, Ser, Thr, Tyr, Cys, Asn, GIn
• non-conservative amino acid substitutions fall within the scope of the invention.
• any NRCl protein, variant or fragment can be determined using various methods.
• transient or stable overexpression in plant cells can be used to test whether the protein has activity in planta. Functionality is preferably tested in the same plant species from which the protein is obtained.
• transient or stable expression can be used to determine whether an HR develops and/or whether resistance is enhanced, indicating functionality.
• silencing of the endogenous genes or gene family will show whether the NRCl protein is functional.
• VIGS can be used in a variety of Solanaceae, such as potato, tomato and tobacco (see Brigneti et al., 2004, Plant Journal 39: 264; Faivre-Rampant et al.
• silencing vectors comprising sense and/or antisense fragments of an NRCl gene can be used to transform plant cells (see below), followed by an assay to determined whether the capability to develop HR lesions and/or disease resistance is modified.
• NRCl proteins which are constitutively active in plant cells, such as the NRCl protein provided in SEQ ID NO: 4, which comprises a single amino acid substitution in the MHD domain (D481V) (see Figure 1).
• the constitutive activity can be tested by determining whether the protein is capable of eliciting an HR in plant tissue, in the absence of elicitor. For example, Agro infiltration of a 35S:NRC1 construct, as described in the Examples, can be used to infiltrate leaf tissue.
• NRCl proteins can be made, by either random mutagenesis followed by activity testing (as described in Bendahame et al., 2002, p 196) or by site directed mutagenesis of single amino acids in the MHD domain (any one of amino acids VHD or VHDM may be replaced with another amino acid), the NB-ARC domain, e.g. in the RNBS-D domain (amino acids FL YFGTFPRGY), or one of the 13 LRR domains (see Figure 1).
• nucleic acid sequences encoding constitutively active NRCl proteins can be obtained from plants, for example by mutagenizing seeds and screening these for the presence of a spontaneous lesion phenotype (for example microscopic lesions), see e.g. Sharino et al. (2002, The Plant Cell 14: 3149-3162) and further below.
• a spontaneous lesion phenotype for example microscopic lesions
• chimeric NRCl proteins comprise at least a CC domain, a NB-ARC domain and preferably at least 13 LRRs.
• a CC-, NB- ARC- and LRR- domain preferably refers to amino acid motifs comprising at least 30, 40, 50, 60, 70, 80, 90, 95, 98, 99%, or more, amino acid sequence identity to amino acids 1-150, to amino acids 151-508, or to amino acids 509-846 of SEQ ID NO: 2 respectively.
• Domains may thus be exchanged (domain swapping) between NRCl proteins or between NRCl proteins and other CC-NB-LRR or TIR-NB-LRR proteins, as long as the functionality of the resulting chimeric protein is essentially similar to that of NRCl, or preferably to NRC1 D481V .
• the chimeric protein retains the ability to confer or enhance disease resistance when it is produced by recombinant plant cells, as described below.
• fragments of NRCl proteins and of variants of NRCl proteins comprise fragments of 100, 150, 200, 300, 400, 500, 600, 700, 800, 850, 855 contiguous amino acids or more.
• fragments are functional in plant tissue, i.e. they are capable of conferring or enhancing pathogen resistance when produced in plant cells. Fragments may also be used to make chimeric proteins, as described above.
• nucleic acid sequences encoding any of the above proteins, variants or fragments are provided, such as cDNA, genomic DNA and RNA sequences. Due to the degeneracy of the genetic code various nucleic acid sequences may encode the same amino acid sequence. Any nucleic acid sequence encoding NRCl proteins or variants are herein referred to as "NRCl".
• the nucleic acid sequences provided include naturally occurring, artificial or synthetic nucleic acid sequences. Examples of nucleic acid sequences encoding NRCl proteins are provided for in SEQ ID NO: 1 and 3. It is understood that when sequences are depicted in as DNA sequences while RNA is referred to, the actual base sequence of the RNA molecule is identical with the difference that thymine (T ) is replace by uracil (U).
• variants and fragments of NRCl nucleic acid sequences such as nucleic acid sequences hybridizing to NRCl nucleic acid sequences under stringent hybridization conditions as defined.
• variants of NRCl nucleic acid sequences also include nucleic acid sequences which have a sequence identity to SEQ ID NO: 1 or 3 (over the entire length) of at least 50% or more, preferably at least 55%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.8% or more.
• variants of NRCl encode constitutively active NRCl proteins as described. It is clear that many methods can be used to identify, synthesise or isolate variants or fragments of NRCl nucleic acid sequences, such as nucleic acid hybridization, PCR technology, in silico analysis and nucleic acid synthesis, and the like.
• the nucleic acid sequence, particularly DNA sequence, encoding the NRCl proteins of this invention can be inserted in expression vectors to produce high amounts of NRCl proteins (or e.g. chimeric NRCl proteins), as described below.
• the NRCl DNA sequences can be codon-optimized by adapting the codon usage to that most preferred in plant genes, particularly to genes native to the plant genus or species of interest (Bennetzen & Hall, 1982, J. Biol. Chem. 257, 3026-3031; Itakura et ah, 1977 Science 198, 1056-1063.) using available codon usage tables (e. g. more adapted towards expression in cotton, soybean corn or rice).
• Codon usage tables for various plant species are published for example by Ikemura (1993, In “Plant Molecular Biology Labfax", Croy, ed., Bios Scientific Publishers Ltd.) and Nakamura et al. (2000, Nucl. Acids Res. 28, 292.) and in the major DNA sequence databases (e.g. EMBL at Heidelberg, Germany). Accordingly, synthetic DNA sequences can be constructed so that the same or substantially the same proteins are produced.
• EMBL at Heidelberg, Germany e.g. EMBL at Heidelberg, Germany
• the NRCl nucleic acid sequences can be modified so that the N-terminus of the NRC 1 protein has an optimum translation initiation context, by adding or deleting one or more amino acids at the N-terminal end of the protein.
• the proteins of the invention to be expressed in plants cells start with a Met- Asp or Met- Ala dipeptide for optimal translation initiation.
• An Asp or Ala codon may thus be inserted following the existing Met, or the second codon, VaI, can be replaced by a codon for Asp (GAT or GAC) or Ala (GCT, GCC, GCA or GCG).
• the DNA sequences may also be modified to remove illegitimate splice sites.
• “Fragments” of NRCl nucleic acid sequences include fragments of at least 10, 12, 15, 16, 18, 20, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000 or more consecutive nucleotides of SEQ ID NO: 1 or 3, or of variants of SEQ ID NO: 1 or 3. Short fragments can for example be used as PCR primers or hybridization probes.
• PCR primers and/or probes and kits for detecting the NRCl DNA or RNA sequences are provided.
• Degenerate or specific PCR primer pairs to amplify NRCl DNA from samples can be synthesized based on SEQ ID NO: 1 or 3 (or variants thereof) as known in the art (see Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany).
• any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of SEQ ID NO: 1 or 3 (or the complement strand) may be used as primer or probe.
• DNA fragments of SEQ ID NO: 1 or 3 can be used as hybridization probes.
• An NRCl detection kit may comprise either NRCl specific primers and/or NRCl specific probes, and an associated protocol to use the primers or probe to detect NRCl DNA in a sample. Such a detection kit may, for example, be used to determine, whether a plant has been transformed with an NRCl gene (or part thereof) of the invention. Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein.
• a method for identifying and using orthologs or alleles of the tomato NRCl gene (SEQ ID NO: 1 and 3) is provided.
• the method comprises the steps of: a) obtaining or identifying a nucleic acid sequence comprising at least 70% nucleic acid identity to SEQ ID NO: 1 and/or 3 (or a higher percentage sequence identity, as indicated above), b) optionally modifying the nucleic acid sequence to encode a constitutively active
• NRCl protein and c) using the nucleic acid sequence of a) to generate expression and/or silencing vectors, or using the nucleic acid sequence of b) to generate expression vectors, d) using one or more vectors of c) to transform a plant or plant cell(s), preferably of the plant species from which the nucleic acid was obtained, e) analysing the capability of the transformed plant / plant tissue to develop HR lesions (i.e.
• the HR lesion phenotype which can optionally be quantified) and/or the disease resistance of the transformants in order to determine or verify the gene function in planta and/or to generate transgenic plants having enhanced disease resistance; f) optionally selecting those alleles or orthologs for further use which confer enhanced disease resistance to the transgenic plant but which, upon expression, confer a weak HR phenotype (i.e. cause no or a reduced HR lesion phenotype).
• NRCl alleles or orthologs which upon expression in plants result in fewer and/or smaller HR lesions than seen upon expression of SEQ ID NO: 1 or 3, or upon expression of the wild type NRCl allele obtained from the host species to be transformed, can be identified using this method. Most preferably, NRCl alleles or orthologs are identified which cause no HR lesions, or at least no macroscopically visible HR lesions, upon expression, but which still confer enhanced disease resistance.
• the HR phenotype of different NRCl alleles and/or orthologs can be compared by making expression vectors using the same promoters, transforming a host plant with these, and by comparing the HR lesion phenotype between these transgenic plants.
• the HR lesion phenotype of transformants expressing SEQ ID NO: 1 or 3 is preferably used as reference.
• the wild type allele obtained from the host species to be transformed can be used as reference.
• Those alleles which provide fewer and/or smaller HR lesions compared to SEQ ID NO: 1 or 3, or fewer and/or smaller HR lesions than caused by expression of the wild type allele obtained from the species transformed, can then be selected for further use.
• transgenic plants expressing these can be made as described below.
• Especially alleles from tomato and orthologs from potato may be obtained or identified using e.g. M?C7-specific PCR primers or probes, or bio informatics analysis in silico. Also genetic mapping may be used to map the NRCl locus in the plant (e.g. tomato or potato) genome, whereby sequences may be obtained by linking the genomic map to existing genome sequencing databases (e.g. developed in the tomato sequencing project). Such alleles and/or orthologs may be especially suitable for generating plants with enhanced disease resistance.
• these orthologs are preferably used to generate plants having enhanced resistance to Phytophthora infestans.
• nucleic acid sequences encoding NRCl proteins (including variants or fragments), as described above, are used to make chimeric genes, and vectors comprising these for transfer of the chimeric gene into a host cell and production of the NRCl protein(s) in host cells, such as cells, tissues, organs or organisms derived from transformed cell(s).
• vectors for the production of NRCl protein (or protein fragments or variants) in plant cells are herein referred to as i.e. "expression vectors”.
• Host cells are preferably plant cells and, but microbial hosts (bacteria, e.g. Agrobacterium, yeast, fungi, etc.) are also envisaged.
• Any plant may be a suitable host, such as monocotyledonous plants or dicotyledonous plants, but most preferably the host plant belongs to the family Solanaceae.
• the plant belongs to the genus Solarium (including Ly coper sico ⁇ ), Nicotiana, Capsicum, Petunia and other genera.
• the following host species may suitably be used: Tobacco ⁇ Nicotiana species, e.g. N. benthamiana, N. plumbaginifolia, N. tabacum, etc.), vegetable species, such as tomato (L. esculentum, syn. Solanum lycopersicum) such as e.g. cherry tomato, var. cerasiforme or currant tomato, var.
• pimpinellifolium or tree tomato (S. betaceum, syn. Cyphomandra betaceae), potato (Solanum tuberosum), eggplant (Solanum melongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum) and naranjilla (Solanum quitoense), peppers (Capsicum annuum, Capsicum frutescens, Capsicum baccatum), ornamental species (e.g. Petunia hybrida, Petunia axillaries, P. integrifolia).
• the plant may belong to any other family, such as to the Cucurbitaceae or Gramineae.
• Suitable host plants include for example maize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeum vulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B.
• rapa, etc sunflower (Helianthus annus), saffiower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species, e.g. O. sativa indica cultivar-group or japonica cultivar-group), forage grasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species (Pinus, poplar, fir, plantain, etc), tea, coffea, oil palm, coconut, vegetable species, such as pea, zucchini, beans (e.g.
• Phaseolus species cucumber, artichoke, asparagus, broccoli, garlic, leek, lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearing plants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime, nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g.
• Rose Petunia, Chrysanthemum, Lily, Gerbera species
• herbs mint, parsley, basil, thyme, etc.
• woody trees e.g. species of Populus, Salix, Quercus, Eucalyptus
• fibre species e.g. flax (Linum usitatissimuni) and hemp ⁇ Cannabis sativa
• model organisms such as Arabidopsis thaliana.
• a crop plant i.e. plant species which is cultivated and bred by humans.
• a crop plant may be cultivated for food purposes (e.g. field crops), or for ornamental purposes (e.g. production of flowers for cutting, grasses for lawns, etc.).
• a crop plant as defined herein also includes plants from which non-food products are harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork and the like.
• chimeric genes and vectors for, preferably stable, introduction of NRCl protein encoding nucleic acid sequences into the genome of host cells is generally known in the art.
• the nucleic acid sequence encoding a NRCl protein (or variant or fragment) is operably linked to a promoter sequence, suitable for expression in the host cells, using standard molecular biology techniques.
• the promoter sequence may already be present in a vector so that the NRCl nucleic sequence is simply inserted into the vector downstream of the promoter sequence.
• the vector is then used to transform the host cells and the chimeric gene is inserted in the nuclear genome or into the plastid, mitochondrial or chloroplast genome and expressed there using a suitable promoter (e.
• a chimeric gene comprises a suitable promoter for expression in plant cells or microbial cells (e.g. bacteria), operably linked thereto a nucleic acid sequence encoding a NRCl protein according to the invention, optionally followed by a 3 'nontranslated nucleic acid sequence.
• the NRCl nucleic acid sequence preferably the NRCl chimeric gene, encoding an functional NRCl protein (or in certain embodiments a constitutively active NRCl protein), can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that has an altered phenotype due to the presence of the NRCl protein in certain cells at a certain time.
• a T-DNA vector comprising a nucleic acid sequence encoding a NRCl protein, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO84/02913 and published European Patent application EP 0 242 246 and in Gould et al. (1991, Plant Physiol. 95,426-434).
• the construction of a T-DNA vector for Agrobacterium mediated plant transformation is well known in the art.
• the T-DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP 0 116 718.
• Preferred T-DNA vectors each contain a promoter operably linked to NRCl encoding nucleic acid sequence (e.g. encoding SEQ ID NO: 2 or SEQ ID NO: 4) between T- DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al. (1984, EMBO J 3,835-845).
• vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247), pollen mediated transformation (as described, for example in EP 0 270 356 and WO85/01856), protoplast transformation as, for example, described in US 4,684, 611, plant RNA virus- mediated transformation (as described, for example in EP 0 067 553 and US 4,407, 956), liposome-mediated transformation (as described, for example in US 4,536, 475), and other methods.
• direct gene transfer as described, for example in EP 0 223 247)
• pollen mediated transformation as described, for example in EP 0 270 356 and WO85/01856
• protoplast transformation as, for example, described in US 4,684, 611
• plant RNA virus- mediated transformation as described, for example in EP 0 067 553 and US 4,407, 95
• liposome-mediated transformation as described, for example in US 4,536, 475
• transformation of the nuclear genome also transformation of the plastid genome, preferably chloroplast genome, is included in the invention.
• plastid genome transformation is that the risk of spread of the transgene(s) can be reduced. Plastid genome transformation can be carried out as known in the art, see e.g. Sidorov VA et al. 1999, Plant J.19: 209-216 or Lutz KA et al. 2004, Plant J. 37(6):906- 13.
• the resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants containing the transgene.
• Single copy transformants can be selected, using e.g. Southern Blot analysis or PCR based methods or the Invader® Technology assay (Third Wave Technologies, Inc.). Transformed cells and plants can easily be distinguished from non-transformed ones by the presence of the chimeric gene.
• the sequences of the plant DNA flanking the insertion site of the transgene can also be sequenced, whereby an "Event specific" detection method can be developed, for routine use. See for example WO0141558, which describes elite event detection kits (such as PCR detection kits) based for example on the integrated sequence and the flanking (genomic) sequence.
• the NRCl nucleic acid sequence is inserted in a plant cell genome so that the inserted coding sequence is downstream (i.e. 3') of, and under the control of, a promoter which can direct the expression in the plant cell. This is preferably accomplished by inserting the chimeric gene in the plant cell genome, particularly in the nuclear or plastid (e. g. chloroplast) genome.
• the constitutive production of the NRCl protein may leads to the induction of cell death (e.g. microscopic lesions and/or macroscopic lesions) and/or may lower yield (see e.g. Rizhsky and Mittler, Plant MoI Biol, 2001 46: 313-23), it is in one embodiment preferred to use a promoter whose activity is inducible.
• inducible promoters are wound-inducible promoters, such as the MPI promoter described by Cordera et al. (1994, The Plant Journal 6, 141), which is induced by wounding (such as caused by insect or physical wounding), or the COMPTII promoter (WO0056897) or the PRl promoter described in US6031151.
• the promoter may be inducible by a chemical, such as dexamethasone as described by Aoyama and Chua (1997, Plant Journal 11: 605-612) and in US6063985 or by tetracycline (TOPFREE or TOP 10 promoter, see Gatz, 1997, Annu Rev Plant Physiol Plant MoI Biol. 48: 89-108 and Love et al. 2000, Plant J. 21: 579-88).
• Other inducible promoters are for example inducible by a change in temperature, such as the heat shock promoter described in US 5,447, 858, by anaerobic conditions (e.g. the maize ADHlS promoter), by light (US6455760), by pathogens (e.g.
• a pathogen inducible promoter is used, as thereby the NRCl protein (or variant or fragment) will only be produced following pathogen attack of the plant tissue.
• promoters of genes which are upregulated quickly after pathogen attack are desired.
• Pathogen inducible promoters include, for example, the hsr203J, str246C and sgd24 promoters from tobacco, EAS4 promoter described by Yin et al. (1997, Plant Physiology 115(2):437-51), the tapl or tap2 promoter (Mohan et al., 1993, Plant MoI Biol. 1993 22 :475-90), the gstl promoter or variants thereof (Martini et al.
• the promoter is inducible by a number of pathogens, i.e. it is inducible by a broad range of pathogens of the host plant.
• a different promoter may be most suitable.
• the promoter when tomato is used as a host, the promoter is preferably induced upon at least one, but preferably more than one tomato pathogen.
• a promoter which is inducible by one or more fungal plant pathogens and/or bacterial plant pathogens is preferred.
• Pathogens of tomato include, for example, the following fungal and bacterial species and viruses (non-limiting): Botrytis cinerea (fungus / necrotroph); Colletotrichum coccodes (fungus / necrotroph); Alternaria alternata (fungus); Alternaria solani (fungus / necrotroph); Stemphylium solani; Phytophthora infestans (oomycte / hemibiotroph); Septoria tycopersici; Cladosporium fulvum, (fungus / hemibiotroph); Phytophthora parasitica; Oidium lycopersicum (biotroph); Fusarium oxysporum; Sclerotium rolfsii; Pythium; Rhizoctonia (fungus / necrotroph); Corynebacterium michiganense (bacterium); Pseudomonas syringae pv tomato or pv syringa
• Pathogens of potato include, for example, various fungi, bacteria, nematodes and viruses, such as: Phytophthora infestans (oomycte / hemibiotroph), nematodes (biotrophic); Erwinia carotovora (bacterium); Colletotrichum coccodes (fungus); Rhizoctonia solani (fungus / necrotroph); Verticillium dahliae (fungus); Streptomyces scabies; Alternaria solani (fungus / necrotroph) ; Pythium; Spongospora subterranean; PVX and PVY; Potato Leafroll Virus (PLRV); etc.
• Phytophthora infestans oomycte / hemibiotroph
• nematodes biotrophic
• Erwinia carotovora bacterium
• Colletotrichum coccodes fungus
• Rhizoctonia solani fungus /
• the promoter is preferably inducible by one or more of the above pathogens, most preferably at least by one or more of the above biotrophic and/or hemibiotrophic pathogens.
• a host plant may comprise various NRCl transgenes, each under control of a different pathogen inducible promoter, to ensure that NRCl protein is produced following attack by a variety of pathogens.
• one promoter may be inducible by Phytophthora and one by Cladosporium.
• the word "inducible” does not necessarily require that the promoter is completely inactive in the absence of the inducer stimulus. A low level non-specific activity may be present, as long as this does not result in severe yield or quality penalty of the plants.
• Inducible thus, preferably refers to an increase in activity of the promoter, resulting in an increase in transcription of the downstream NRCl coding region following contact with the inducer.
• the most preferred combination herein is the use of a pathogen inducible promoter, operably linked to an NRCl nucleic acid sequence which encodes a constitutively active NRCl protein, as described above.
• a pathogen inducible promoter operably linked to an NRCl nucleic acid sequence which encodes a constitutively active NRCl protein, as described above.
• the constitutively active NRCl upon pathogen attack the constitutively active NRCl will be expressed resulting in a local HR (restricted to the site of pathogen attack) preventing further growth of any (hemi)-biotrophic pathogen.
• constitutive promoters may be used, such as the strong constitutive 35S promoters or enhanced 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al, 1981, Nucleic Acids Research 9, 2871-2887), CabbB-S (Franck et al, 1980, Cell 21, 285-294) and CabbB-JI (Hull and Howell, 1987, Virology 86,482-493); the 35S promoter described by Odell et al (1985, Nature 313, 810-812) or in US5164316, promoters from the ubiquitin family (e.g.
• rice actin promoters such as the promoter described by Zhang et ⁇ /.(1991, The Plant Cell 3, 1155-1165) and the promoter described in US 5,641,876 or the rice actin 2 promoter as described in WO070067; promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al 1998, Plant MoI. Biol.
• the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g., pAdhlS (GenBank accession numbers X04049, X00581), and the TRl' promoter and the TR2' promoter (the "TRl' promoter” and "TR2' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al, 1984, EMBO J 3, 2723-2730), the Figwort Mosaic Virus promoter described in US6051753 and in EP426641, histone gene promoters, such as the Ph4a748 promoter from Arabidopsis (PMB 8: 179-191), or others.
• the AA6 promoters as described in PCT/NL2005/050083 (filed 16 December 2005
• tissue preferred / tissue specific including developmentally regulated promoters
• leaf preferred epidermis preferred
• root preferred for example leaf preferred
• flower tissue e.g. tapetum or anther preferred, seed preferred, pod preferred, etc.
• the NRCl gene is expressed only in cells of the specific tissue(s) or organ(s) and/or only during a certain developmental stage.
• the NRCl gene(s) can be selectively expressed in the leaves of a plant by placing the coding sequence under the control of a light-inducible promoter such as the promoter of the ribulose-1, 5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254, 799 or Arabidopsis as disclosed in US5034322.
• a light-inducible promoter such as the promoter of the ribulose-1, 5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254, 799 or Arabidopsis as disclosed in US5034322.
• the promoter of the endogenous NRCl gene is used.
• the promoter of the tomato NRCl gene may be isolated and operably linked to the coding region encoding NRCl protein of SEQ ID NO: 2 or 4.
• the NRCl promoter (the upstream transcription regulatory region of SEQ ID NO: 1 and 3) can be isolated from tomato plants using known methods, such as TAIL-PCR (Liu et al 1995, Genomics 25(3):674-81; Liu et al 2005, Methods MoI Biol. 286:341-8), Linker-PCR, or Inverse PCR (IPCR).
• the NRCl coding sequence is preferably inserted into the plant genome so that the coding sequence is upstream (i.e. 5') of suitable 3' end nontranslated region ("3 'end" or 3'UTR).
• suitable 3'ends include those of the CaMV 35S gene ("3' 35S"), the nopaline synthase gene ("3' nos”) (Depicker et al., 1982 J. Molec. Appl.
• 3'ocs the octopine synthase gene
• 3' gene 7 the T-DNA gene 7
• SEQ ID NO: 3 the 3'UTR of the tomato NRCl gene is used, as shown in SEQ ID NO: 3, from nucleotide 2748 to nucleotide 3168, and as shown in SEQ ID NO: 5.
• the NRCl 3'UTR is also an embodiment in itself herein, as it may also be used as 3'UTR in combination with other coding regions.
• any variant or fragment of SEQ ID NO: 5 is provided.
• a variant of SEQ ID NO: 5 includes nucleic acid sequences comprising at least 40, 50, 60, 70, 80, 90, 95, 98, 99% or more nucleic acid sequence identity to SEQ ID NO: 5 (as determined using the Needleman and Wunsch algorithm and the GAP penalties as defined above).
• Fragments include any nucleotide sequences comprising at least 30, 50, 100, 150, 200, 300, 400 or more consecutive nucleotides of SEQ ID NO: 5, or of a variant of SEQ ID NO: 5.
• T-DNA vector into Agrobacterium can be carried out using known methods, such as electroporation or triparental mating.
• a NRC 1 encoding nucleic acid sequence can optionally be inserted in the plant genome as a hybrid gene sequence whereby the NRCl sequence is linked in- frame to a (US 5,254, 799; Vaeck et al., 1987, Nature 328, 33-37) gene encoding a selectable or scorable marker, such as for example the neo (or nptll) gene (EP 0 242 236) encoding kanamycin resistance, so that the plant expresses a fusion protein which is easily detectable.
• a selectable or scorable marker such as for example the neo (or nptll) gene (EP 0 242 236) encoding kanamycin resistance
• NRCl nucleic acid sequence encoding a NRCl protein (or variant or fragment)
• a NRCl nucleic acid sequence can also be used to transform microorganisms, such as bacteria (e.g. Escherichia coli, Pseudomonas, Agrobacterium, Bacillus, etc.), fungi, or algae or insects, or to make recombinant viruses. Transformation of bacteria, with all or part of a NRCl nucleic acid sequence of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Maillon et al. (1989, FEMS Microbiol. Letters 60, 205-210.) and WO 90/06999.
• the codon usage of the nucleic acid sequence may be optimized accordingly (as described for plants above). Intron sequences should be removed and other adaptations for optimal expression may be made as known.
• the DNA sequence of the NRCl nucleic acid sequence can be further changed in a translationally neutral manner, to modify possibly inhibiting DNA sequences present in the gene part and/or by introducing changes to the codon usage, e. g., adapting the codon usage to that most preferred by plants, preferably the specific relevant plant genus, as described above.
• the NRCl proteins are targeted to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or are secreted from the cell, potentially optimizing protein stability and/or expression.
• the protein may be targeted to vacuoles.
• the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the NRCl protein coding region of the invention.
• Particularly preferred peptides to be included in the proteins of this invention are the transit peptides for chloroplast or other plastid targeting, especially duplicated transit peptide regions from plant genes whose gene product is targeted to the plastids, the optimized transit peptide of Capellades et al. (US 5,635, 618), the transit peptide of ferredoxin-NADP+oxidoreductase from spinach (Oelmuller et al., 1993, MoI. Gen. Genet. 237,261-272), the transit peptide described in Wong et al. (1992, Plant Molec. Biol. 20, 81-93) and the targeting peptides in published PCT patent application WO 00/26371.
• peptides signalling secretion of a protein linked to such peptide outside the cell such as the secretion signal of the potato proteinase inhibitor II (Keil et al., 1986, Nucl. Acids Res. 14,5641- 5650), the secretion signal of the alpha- amylase 3 gene of rice (Sutliff et al., 1991, Plant Molec. Biol. 16,579-591) and the secretion signal of tobacco PRl protein (Cornelissen et al., 1986, EMBO J. 5,37-40).
• Particularly useful signal peptides in accordance with the invention include the chloroplast transit peptide (e.g.
• an appropriate secretion signal peptide may be fused to the amino terminal end (N- terminal end) of the NRCl protein.
• Putative signal peptides can be detected using computer based analysis, using programs such as the program Signal Peptide search (SignalP Vl.1 or 2.0)(Von Heijne, Gunnar, 1986 and Nielsen et al, 1996).
• NRCl encoding nucleic acid sequences are co-expressed in a single host, optionally under control of different promoters.
• a co-expressing host plant is easily obtained by transforming a plant already expressing NRCl protein of this invention, or by crossing plants transformed with different NRCl proteins of this invention.
• several NRCl protein encoding nucleic acid sequences can be present on a single transformation vector or be co-transformed at the same time using separate vectors and selecting transformants comprising both chimeric genes.
• one or more NRCl encoding genes may be expressed in a single plant together with other chimeric genes, for example encoding other proteins which enhance disease resistance or which are involved in the disease resistance signalling pathway, or others.
• each parent plant can express a single protein. Upon crossing the parent plants to produce hybrids, both proteins are combined in the hybrid plant.
• the transgenic plants of the invention are also transformed with a DNA encoding a protein conferring resistance to herbicide, such as a broad-spectrum herbicide, for example herbicides based on glufosinate ammonium as active ingredient (e.g. Liberty® or BASTA; resistance is conferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) or glyphosate (e.g. RoundUp®; resistance is conferred by EPSPS genes, see e.g. EPO 508 909 and EP 0 507 698).
• herbicide resistance genes or other genes conferring a desired phenotype
• selectable marker further has the advantage that the introduction of antibiotic resistance genes can be avoided.
• selectable marker genes may be used, such as antibiotic resistance genes. As it is generally not accepted to retain antibiotic resistance genes in the transformed host plants, these genes can be removed again following selection of the transformants.
• Another site specific recombination systems is the FLP/FRT system described in EP686191 and US5527695. Site specific recombination systems such as CRE/LOX and FLP/FRT may also be used for gene stacking purposes. Further, one-component excision systems have been described, see e.g. WO9737012 or WO9500555).
• a transgenic plant with enhanced disease resistance can be generated by transforming a plant host cell with a nucleic acid sequence encoding at least one NRCl protein under the control of a suitable promoter, as described above, and regenerating a transgenic plant from said cell.
• Preferred promoters are promoters which are inducible by external biotic and/or abiotic stimuli. Especially promoters which are pathogen inducible are preferred, as described above.
• Preferred promoter - NRCl combinations are: a) a pathogen inducible promoter - nucleic acid sequence encoding a constitutively active NRCl protein; b) a pathogen inducible promoter - nucleic acid sequence encoding a wild type NRCl protein; c) the promoter of a plant NRCl gene (preferably of the same species which is to be transformed) - nucleic acid sequence encoding a constitutively active NRCl protein; d) the promoter of a plant NRCl gene (preferably of the same species which is to be transformed) - nucleic acid sequence encoding a wild type NRCl protein; e) a biotic stress inducible promoter (e.g.
• insect wounding inducible, pathogen inducible, etc. - nucleic acid sequence encoding a constitutively active NRCl protein
• a biotic stress inducible promoter e.g. insect inducible, pathogen inducible, etc.
• a constitutive promoter e.g. 35S promoter
• a constitutive promoter e.g. 35S promoter
• a constitutive promoter e.g. 35S promoter
• the transgenic plant may show either constitutive HR lesions or inducible HR lesions, and enhanced disease resistance to one or more pathogens.
• no HR lesions or "weak" HR lesions such as smaller lesions, e.g. micro-lesions, and/or a low lesion frequency
• NRCl alleles or orthologs which, upon expression in host plants under control of the identical promoters, result in fewer and/or smaller HR lesions than SEQ ID NO: 1 or 3, or than the expression of the wild type NRCl allele obtained from the same host species which is transformed, are particularly preferred herein, especially in approaches g) and h) above.
• NRCl alleles conferring a "weak HR phenotype" in a given host.
• NRCl alleles or orthologs can be identified and/or isolated as described herein above.
• the HR phenotype of different NRCl alleles and/or orthologs can be compared by making expression vectors using these (preferably all nucleic acids which are to be compared are operably linked to the identical promoters, e.g. 35S), transforming plants or plant tissue with these, and by comparing the HR lesion phenotype between these plants.
• the HR lesion phenotype of transformants expressing SEQ ID NO: 1 or 3 is preferably used as reference and any allele resulting in fewer and/or smaller HR lesions upon expression under control of the same promoter is an allele conferring a weak HR phenotype.
• the HR lesion phenotype can be compared and optionally quantified using various methods, such as microscopy (optionally staining dead cells), visual scoring, counting lesions to calculate the number per cm 2 , measuring the diameter of HR lesions, etc.
• the transgenic plants of the invention comprise enhanced disease resistance against one or more pathogens, especially biotrophic and/or hemibiotrophic pathogens of the transgenic plant species.
• transgenic tomato or potato plants comprise enhanced resistance to at least one, or more, of the fungal, bacterial, nematode species and/or viral pathogens listed above, most preferably at least against one or several biotrophic and/or hemibiotrophic species.
• Disease resistance or “increased/enhanced disease resistance” is used herein to refer to an enhanced ability of transformants (compared to wild type or control transformants) to withstand the attack of one or more plant pathogens, or in other words, it refers to a significant reduction in disease symptoms in transformants compared to non-transformed (or empty-vector transformed) controls.
• Disease resistance or enhanced disease resistance may be determined using a variety of methods. Often disease symptoms are scored visually (either in bioassays or in the field) by assessing the disease symptoms at one or more time points after inoculation or contact with a pathogen. Alternative methods include methods whereby the pathogen is detected and optionally quantified.
• a transgenic plant may thus show enhanced disease resistance if the amount of pathogen detected in/on the tissue is significantly less compared to controls, or if the pathogen spread is significantly slower than in controls.
• a significant increase in average yield of transformants e.g. at least 1%, 2%, 5%, 10% or more
• equivalent disease pressure preferably in the field
• a plurality of transformed plants expressing NRCl protein show enhanced disease resistance if they show a significant reduction of disease symptoms, compared to the untransformed or empty- vector transformed controls.
• statistical analysis is required to determine whether significant difference exist.
• one or more disease symptoms are on average at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, or even 100% lower in NRCl transformants than in the control plants.
• the disease assay is different for every host- pathogen combination, no specific protocol can be provided, but the skilled person knows how to determine whether transformants show significantly enhanced disease resistance to one or more pathogens. Bioassays as known in the art for each plant- pathogen combination can be used to compare resistance of transgenic plants to suitable controls.
• the NRCl protein may in some embodiments result in HR lesions in the absence of pathogen (for example if the NRCl gene is under the control of a constitutive promoter): it may in certain embodiments be important to differentiate between symptoms caused by NRCl expression and symptoms caused by pathogen infection and spread. It may, therefore, be preferred to use methods which detect the pathogen itself (rather than necrosis on the plant tissue) and to compare the amount of pathogen present or the speed of pathogen spread. For example, bioassays may be used wherein the pathogen can be detected by staining. In the examples a transgenic Cfulvum race is used which expresses GUS. Fungal mycelium can, therefore, be visualized using X- glue staining of the inoculated plant tissue.
• transgenic plants which express several NRCl proteins, preferably under the control of different promoters, such as different pathogen inducible promoters.
• the disease resistance phenotype can be fine-tuned by expressing a suitable amount of NRCl protein at a suitable time and location. Such fine-tuning may be done by determining the most appropriate promoter for a particular host-pathogen combination and also by selecting transgenic "events" which show the desired expression level. A too low level of NRCl protein or too slow induction of NRCl protein production following pathogen attack may be insufficient to enhance disease resistance levels. On the other hand, a too high protein level or expression at times and locations devoid of pathogen attack, may result in agronomically undesired phenotypes, such as lesions in leaves or fruit in the absence of pathogens and yield penalties.
• Optimal NRCl alleles may be isolated or identified as described, e.g. alleles providing high resistance levels and only a weak HR phenotype.
• Transformants expressing desired levels of the NRCl protein are selected by e.g. analysing copy number (Southern blot analysis), mRNA transcript levels (e.g. RT-PCR using NRCl primer pairs or flanking primers) or by analysing the presence and level of NRCl protein in various tissues (e.g. SDS-PAGE; ELISA assays, etc).
• single copy transformants are preferably selected and the sequences flanking the site of insertion of the chimeric gene is analysed, preferably sequenced to characterize the "event”.
• High or moderate NRCl expressing transgenic events are selected for further crossing / backcrossing / selfing until a high performing elite event with a stable NRCl transgene is obtained.
• Transformants expressing one or more NRCl genes according to the invention may also comprise other transgenes, such as other genes conferring disease resistance or conferring tolerance to other biotic and/or abiotic stresses.
• other transgenes may either be introgressed into the NRCl transformants, or the NRCl transformants may be transformed subsequently with one or more other genes, or alternatively several chimeric genes may be used to transform a plant line or variety. For example, several chimeric genes may be present on a single vector, or may be present on different vectors which are co-transformed.
• NRCl genes are combined with one or more NRCl genes according to the invention: known disease resistance genes, especially genes conferring enhanced resistance to necrotophic pathogens, virus resistance genes, insect resistance genes, abiotic stress resistance genes (e.g. drought tolerance, salt tolerance, heat- or cold tolerance, etc.), herbicide resistance genes, and the like.
• the stacked transformants may thus have an even broader biotic and/or abiotic stress tolerance, to pathogen resistance, insect resistance, nematode resistance, salinity, cold stress, heat stress, water stress, etc.
• NRCl silencing approaches may be combined with NRCl expression approaches in a single plant. For example, NRCl overexpression in roots or tubers may confer or enhance root or tuber resistance to soil pathogens. At the same time downregulation of NRCl in aerial parts may confer or enhance resistance to necro trophic pathogens (or vice versa).
• NRCl gene it is also possible to introduce or introgress the NRCl gene into a plant breeding line which already has a certain level of disease resistance.
• Whole plants, seeds, cells, tissues and progeny (such as Fl hybrids, F2 seeds/plants, etc.) of any of the transformed plants described above are encompassed herein and can be identified by the presence of the transgene in the DNA, for example by PCR analysis using total genomic DNA as template and using NRCl specific PCR primer pairs. Also “event specific" PCR diagnostic methods can be developed, where the PCR primers are based on the plant DNA flanking the inserted chimeric gene, see US6563026. Similarly, event specific AFLP fingerprints or RFLP fingerprints may be developed which identify the transgenic plant or any plant, seed, tissue or cells derived there from.
• the transgenic plants according to the invention preferably do not show non-desired phenotypes, such as yield reduction, enhanced susceptibility to diseases (especially to necrotrophs) or undesired architectural changes (dwarfing, deformations) etc. and that, if such phenotypes are seen in the primary transformants, these can be removed by normal breeding and selection methods (crossing / backcrossing / selfing, etc.). Any of the transgenic plants described herein may be homozygous or hemizygous for the transgene.
• NRCl gene silencing approaches and gene silencing vectors It is a further embodiment of the invention to provide plants with enhanced disease resistance, especially against necrotrophic pathogens, whereby the plant is transformed with an NRCl gene silencing vector. Without limiting the scope of the invention, it is thought that silencing of endogenous NRCl genes or gene families results in the inability of the transgenic plant to trigger and/or mount an HR response. As necrotrophic pathogens require cell death for their growth and development, such plants may comprise enhanced resistance to one or more necrotrophic pathogens.
• Gene silencing refers to the down-regulation or complete inhibition of gene expression of one or more target genes (e.g. endogenous NRCl genes).
• target genes e.g. endogenous NRCl genes.
• inhibitory RNA to reduce or abolish gene expression is well established in the art and is the subject of several reviews (e.g Baulcombe 1996, Stam et al. 1997, Depicker and Van Montagu, 1997).
• technologies available to achieve gene silencing in plants such as chimeric genes which produce antisense RNA of all or part of the target gene (see e.g. EP 0140308 Bl, EP 0240208 Bl and EP 0223399 Bl), or which produce sense RNA (also referred to as co-suppression), see EP 0465572 Bl.
• a vector according to the invention may, therefore, comprise a transcription regulatory region which is active in plant cells operably linked to a sense and/or antisense DNA fragment of a NRCl gene according to the invention.
• stretches of the target gene sequence such as 17, 18, 19, 20, 21, 22 or 23 nucleotides of coding or non-coding sequence are sufficient. Longer sequences can also be used, such as 50, 100, 200 or 250 nucleotides or more.
• the short sense and antisense fragments are separated by a spacer sequence, such as an intron, which forms a loop (or hairpin) upon dsRNA formation.
• Any short stretch of SEQ ID NO: 1 or 3, or variants thereof, may be used to make a NRCl gene silencing vector and a transgenic plant in which one or more NRCl genes are silenced in all or some tissues or organs (depending on the promoters used).
• a convenient way of generating hairpin constructs is to use generic vectors such as pHANNIBAL and pHELLSGATE, vectors based on the Gateway® technology (see Wesley et al. 2004, Methods MoI Biol. 265:117-30; Wesley et al. 2003, Methods MoI Biol. 236:273-86 and Helliwell & Waterhouse 2003, Methods 30(4):289-95.), all incorporated herein by reference.
• NRCl family members in a host plant or plant parts can be silenced.
• transgenic plants comprising a transcription regulatory element operably linked to a sense and/or antisense DNA fragment of a NRCl gene and exhibiting enhanced resistance to one or more pathogens, especially necro trophic pathogens.
• plants having enhanced resistance to one or more biotrophic and/or hemi- biotrophic pathogens and to one or more necrotrophic pathogens are provided.
• Such plants can be generated by choosing appropriate promoter - NRCl gene combinations.
• a functional NRCl protein may be produced in a certain tissue at a certain time (e.g. upon induction or in aerial plant parts), providing resistance to biotrophic and/or hemibiotrophic pathogens, while the endogenous NRCl gene(s) are silenced in a different tissue and/or at a different time (e.g. in seedlings, in roots or tubers, etc.), thereby providing resistance to one or more necrotrophic pathogens.
• a single plant may, therefore, comprise both a chimeric NRCl expressing transgene and an NRCl silencing gene.
• Mutant alleles and plants according to the invention It is also an embodiment of the invention to use non-transgenic methods, e.g. mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al, 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference) and selection to generate plant lines which produce higher levels of one or more NRCl proteins according to the invention and/or which produce a constitutively active NRCl protein as described.
• TILLING Targeting Induced Local Lesions IN Genomics; McCallum et al, 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference
• NRC 1 protein levels in the mutant or parts of the mutant are at least about 2, 5, 10, 15%, or more, increased in the mutant compared to non-mutant plants.
• TILLING uses traditional chemical mutagenesis (e.g. EMS mutagenesis) followed by high-throughput screening for mutations (e.g. using CeI 1 cleavage of mutant-wild type DNA heteroduplexes and detection using a sequencing gel system), see e.g.
• non-transgenic plants, seeds and tissues comprising an enhanced NRCl gene expression in one or more tissues and comprising one or more of the NRCl pheno types according to the invention (enhanced disease resistance and/or HR lesions) and methods for generating and identifying such plants is encompassed herein.
• the method comprises in one embodiment the steps of mutagenizing plant seeds (e.g. EMS mutagenesis), pooling of plant individuals or DNA, PCR amplification of a region of interest, heteroduplex formation and high-throughput detection, identification of the mutant plant, sequencing of the mutant PCR product. It is understood that other mutagenesis and selection methods may equally be used to generate such mutant plants. Seeds may for example be radiated or chemically treated and the plants screened for a modified phenotype, such as enhanced disease resistance and/or HR lesions.
• the plant materials are natural populations of the species or related species that comprise polymorphisms or variations in DNA sequence at the NRCl orthologous coding and/or regulatory sequence.
• Mutations at the NRCl gene target can be screened for using a ECOTILLING approach (Henikoff et al 2004, supra).
• ECOTILLING approach Henikoff et al 2004, supra.
• natural polymorphisms in breeding lines or related species are screened for by the above described TILLING methodology, in which individual or pools of plants are used for PCR amplification of the NRCl target, heteroduplex formation and high- throughput analysis. This can be followed up by selecting of individual plants having the required mutation that can be used subsequently in a breeding program to incorporate the desired ⁇ /7?C7-orthologous allele to develop the cultivar with desired trait.
• Mutant plants can be distinguished from non-mutants by molecular methods, such as the mutation(s) present in the DNA, NRCl protein levels, NR Cl RNA levels etc, and by the modified phenotypic characteristics.
• the non-transgenic mutants may be homozygous or heterozygous for the mutation.
• SEQ ID NO 1 coding region of the tomato NR Cl gene
• SEQ ID NO 2 amino acid sequence of the tomato NRCl protein
• SEQ ID NO 3 full length cDNA of the tomato NRCl gene (including 5' and 3' UTR)
• SEQ ID NO 4 amino acid sequence of the tomato NRC1 D481V protein
• SEQ ID NO 5 3'UTR of the tomato NRCl gene
• the first 150 amino acid residues represent the coiled-coil (CC) domain and residues that are predicted to form the CC structure are underlined.
• Residues 151 to 508 comprise the nucleotide-binding (NB-ARC) domain, with the following motifs (underlined and labeled): KinaselA (P loop), RNBS-A, Kinase 2, RNBS-B, RNBS-C, GLPL, RNBS-D and MHD.
• Residues 509 to 846 comprise the 13 imperfect leucine- rich repeats (LRRs); the conserved hydrophobic and proline residues are shown in bold. Below the protein sequence the LRR consensus motif is indicated: T indicates a conserved aliphatic residue, 'c' indicates a conserved charged residue and 'P' indicates a conserved proline residue.
• FIG. 2A - NR Cl Is Required for Full Cf-4-Mediated HR of Tomato to Cladosporium fulvum CfD tomato and Q ⁇ -containing tomato plants were inoculated with the indicated TRV constructs and plants were analyzed three weeks after the onset of VIGS.
• Leaflets of TRV-infected Q ⁇ -containing tomato plants were injected with Avr4 protein and examined for the development of an HR.
• the number of sites mounting an HR on TRV:00-infected plants was set to 100%.
• Each error bar represents the standard error from four independent experiments.
• Figure 3 Inoculation of N. benthamiana with TRV:NRC1 affects Cf/ Ayr-, LeEix2/tvEix-. Pto/AyrPto-and Rx/CP-Induced HR N. benthamiana was inoculated with TRV:00 (empty vector), TRV:NRC1 and TRV:SGT1. Three weeks later leaves were infiltrated with Agrobacteria expressing HR-inducing proteins and pictures were taken at 4 days post infiltration. First, second and third column: leaves of N.
• N. benthamiana expressing the Cf-4 resistance gene was agroinfiltrated with the indicated genes.
• Second column agroinfiltration with the gene encoding a constitutively active form of LeMEK2 (MEK2DD).
• NRC1 K191R (K191R): inactive P-loop mutant of NRCl; NRC1 D481V (D481V): constitutively active NRCl (mutated in the MHD motif); NRC 1 K191R/D481V (K191R/D481V): double mutant of NRCl. Pictures were taken three days post agroinfiltration. (C) Agroinfiltration of Avr4 and the gene encoding constitutively active NRC1 D481V (D481V). Pictures were taken three days post agroinfiltration. Figure 5 - Model for NRCl Mediated Cell Death Signaling
• Cf-4/Avr4 mediated cell death signals in an EDSl-, NRCl-, MEK2-, and SGTl/RARldependent manner.
• benthamiana Pto (line 38-12 (Rommens et al., 1995, Plant Cell 7, 1537-1544)) (Pedley and Martin, 2003, Annu. Rev. Phytopathol. 41, 215-243) and Rx (N. benthamiana: Rx (line Rx- 18) (Bendahmane et al., 1999, Plant Cell 11, 781-791), respectively.
• Agroinfiltration was performed in N. benthamiana expressing the resistance gene Cf-4 (N. benthamiana: Cf-4).
• Avr4-HIS-FLAG-tagged protein was treated with enterokinase EK-max according to the manufacturer's recommendations (Invitrogen, Breda, NL) and 5 ⁇ M Avr4 protein in water, supplemented with 0.2% tween (v/v) was used for injections.
• enterokinase EK-max enterokinase EK-max
• 5 ⁇ M Avr4 protein in water, supplemented with 0.2% tween (v/v) was used for injections.
• Three to five days post agroinfiltration or protein injection the leaves were examined for the development of an HR, or assayed for ⁇ -glucuronidase (GUS) activity.
• GUS ⁇ -glucuronidase
• TRV:222-UTR part of the 3'-UTR of NRCl was amplified using primers 222-3 'UTR- F (5 '-GTGGATCCGCAGGTTCAACCAGCCTGGT-S'; Bam ⁇ l site underlined) and 222-3 'UTR-R (5 '-GTGGTACCCAAGTGACTTGTTCTGCTGT-S'; AspllS site underlined) and to construct TRV:222-LRR, part of the NRCl region coding the LRRs was amplified using primers
• 222-LRR-F (5 '-GTGGATCCGTTAAGAGGCTGCAATTTCT-S'; Bam ⁇ l site underlined) and 222-LRR-R (5 '-GTGGTACCGATCTTCTCAAGTTTATCAC-S '; AspllS site underlined).
• TRViPrf construction has been described (Ekengren et al., 2003, Plant J. 36, 905-917). All plasmids were transformed to A.
• tumefaciens strain GV3101 by electroporation (Takken et al., 2000, Plant J. 24, 275- 283).
• VIGS VIGS in tomato
• cotyledons of ten- to twelve-day-old tomato seedlings were agroinfiltrated with a mixture of pTRV-RNAl and the pTRV-RNA2- derived constructs (combined in a 1:1 ratio) (Liu et al., 2002, supra).
• For each TRV construct either four Q ⁇ -containing tomato plants (CfO plants transformed with Hcr9- 4D ⁇ Cf-4)) (Thomas et al., 1997, Plant Cell 9, 2209-2224), resistant to C.
• CfO plants transformed with Hcr9-9C (Cf-9) CfO plants transformed with Hcr9-9C (Cf-9)
• CfO plants transformed with Hcr9-9C (Cf-9) CfO plants transformed with Hcr9-9C (Cf-9)
• As control CfO tomato plants (MM-ClD) fully susceptible to C fulvum, either TRV:00- or TRV:NRC1- inoculated were used.
• TRV:00- or TRV:NRC1- inoculated were used for disease assays.
• three weeks post TRV inoculation CfO and Q ⁇ -containing plants were inoculated with C. fulvum (De Wit, 1977, Neth. J. Plant Path. 83, 109-122).
• a C fulvum race 5- pGPDv.GUS was used (expressing Avr4 and the ⁇ -glucuronidase gene under control of the constitutive GPD promoter). Colonization of the leaflets was assessed two weeks later by X-gluc staining.
• leaflets of the second, third or fourth compound leaf of the plants were used for RT-PCR analysis to test for 'knock down' of the gene of interest (see below).
• HR assays leaflets of the third compound leaf of TRV- infected Cf-4- or C/ ⁇ P-containing plants were injected with Avr4 or Avr9, respectively. Both elicitors were injected into leaflets with a micro-syringe (Ito Corporation, Fuji, Japan).
• Avr4 was injected at a concentration of 10 ⁇ M. at ten sites per leaflet and four leaflets per plant.
• For Avr9 eight times diluted apoplastic fluid containing about lO ⁇ M of Avr9, isolated from a compatible interaction between race 5 of C. fulvum and CfO plants, was injected at eight sites per leaflet and four leaflets per plant.
• Resistance against Pseudomonas syringae pv. tomato was assayed in tomato RG-PtoR (Pto/Pto, Prf/Prf), inoculated with TRV:00, TRV:Prf or TRV:NRC1. The inoculation procedure and the determination of bacterial colonization of the leaves were performed as described previously (Ekengren et al., 2003, supra).
• NRCl cDNA was PCR-amplified using primers 222-Start-F (5 '-GGGATCCATGGTTGATGTAGGGGTTGA-S ') and 222-Stop-R (5 '-GTCACTGCAGACCTTTCTAAGAAGCTGTCTG-S '). thereby introducing Ncol and Pstl restriction sites, respectively (restriction sites underlined).
• the PCR fragment was ⁇ /coI/i-t ⁇ l-digested and inserted into Ncol/Pstl- digested pRH80 (Van der Hoorn et al., 2000, MoI. Plant-Microbe Interact. 13, 439- 446).
• the construct was Xbal/Kpnl-digQStQd and the resulting fragment containing the 35S promoter, the NRCl open reading frame and the NOS terminator (tNOS), was cloned into the pMOG800 binary vector (Honee et al., 1998, Plant Physiol. 117, 809-820) to create plasmid NRCl(wt).
• the D481V mutation was introduced by overlap extension PCR (Higuchi et al., 1988, Nucleic Acids Res. 16, 7351-7367) using the NRClwt plasmid as a template and flanking primers 222-Start-F and 222-Stop-R and mismatch primers
• 222MHD-F (5 '-CAAAACTTGTCGTGTTCATGTCATGTTGTATGAG-S ') and 222MHD-R (5 '-CC AGC AAAACTC AT AC AAC ATGAC ATGAAC ACGAC-3 ') (mutation underlined).
• the fragment was TVcoI/ ⁇ tl-digested, inserted into pRH80 and the 35S-NRC1 D481V - tNOS fragment was excised and subsequently inserted into pMOG800 as described above.
• the P-loop mutant NRC1 KI9IR and the inactive double mutant NRC1 K191R/D481V WQVQ created.
• the Kl 9 IR mutation was introduced using mismatch primers
• 222Ploop-F (5 '-GGAATGCCTGGTCTTGGCAGAACTACACTAGC-S ') and 222Ploop-R (5'- GCT AGTGT AGTTCTGCC AAGACC AGGC ATTCC-3') (mutation underlined) with respectively plasmid NRCl (wt) and NRC1 D481V as a template. All constructs were sequence- verified and transformed to A. tumefaciens strain GV3101.
• Genomic DNA from N. benthamiana was isolated using the QIA-Gen DNA extraction protocol (Qiagen, Venlo, NL), whereas for tomato the standard protocol described by (Sambrook and Russell, 2001, Molecular cloning: A Laboratory Manual, 3rd ed. (Cold Spring Harbor, NY, U.S.A.: Cold Spring Harbor Laboratory Press) was used.
• the DNA was digested with BamHl, Hindlll, EcoRl, EcoRN or Xbal.
• the N benthamiana gel- blot was hybridized with the 32 P-labeled (Prime-a-gene Labeling System, Promega, Madison, WI) 252 bp fragment present in the TRV:NRC1 vector and the DNA gel-blot of tomato was hybridized with a 32 P-labeled probe of 1293 bases corresponding to nucleotides 1876 to 3168 of the full length NRCl cDNA. Sites for the restriction enzymes used are not present in the probes. Low stringency refers to washing at 55°C in 2x SSC and 0.5% SDS. High stringency conditions consist of washing at 65°C in 0.5x SSC and 0.5% SDS.
• the primers that were used do not cover the fragment which is inserted in TRV:NRC1.
• Actin primers ActinFnrl82: 5 '-TATGGAAACATTGTGCTCAGTGG-S ' and ActinRnrl 83 : 5 '-CCAGATTCGTCATACTCTGCC-3 ') were used to check for the presence of equal amounts of cDNA in the PCR reactions.
• Example 2 Results 2.1 Tomato NRCl; a CC-NB-LRR Protein cDNA-AFLP analysis was performed, followed by VIGS of the identified fragments of tomato in N benthamiana:Cf-4. 20 cDNA fragments were identified of which VIGS affects the Cf-4/Avr4-mduced HR.
• NRCl the full length cDNA was isolated, as depicted in SEQ ID NO: 3. The open reading frame is shown in SEQ ID NO: 1, which encodes the NRCl protein depicted in SEQ ID NO: 2.
• NRCl The predicted primary structure of the NRCl protein (SEQ ID NO: 2) typically resembles that of CC-NB-LRR resistance proteins ( Figure 1).
• NRCl has an amino- terminal coiled-coil (CC) domain, an NB-ARC (Nucleotide Binding adapter shared by Apaf-1, R proteins and CED4) domain (Van der Biezen and Jones, 1998, Curr. Biol. 8, R226-R227; Aravind et al, 1999, Trends Biochem. Sci. 24, 47-53) and 13 imperfect leucine-rich repeats (LRRs).
• CC amino- terminal coiled-coil
• NB-ARC Nucleotide Binding adapter shared by Apaf-1, R proteins and CED4 domain
• LRRs 13 imperfect leucine-rich repeats
• the 252 bp cDNA-AFLP fragment present in the TRV:NRC1 vector used for VIGS codes for amino acids 599-681, which are located in LRRs four to seven.
• NRCl -Silenced Tomato Is Affected in Cf-4-Mediated HR and Disease Resistance To investigate the function of NRCl in HR-signaling and resistance to C. fulvum, the inventors performed VIGS in tomato, since this plant is the only host for this fungus. Ten-day-old tomato seedlings were agroinfiltrated with TRV:NRC1 and three weeks post infiltration RNA was isolated from potentially silenced leaflets and analyzed by RT-PCR. The NRCl transcript levels varied in different TRV:NRC1 -infected plants, but in most cases they were lower than in the TRV:00-infected plants, indicating that 'knock-down' of NRCl expression had occurred (data not shown).
• TRV:222-LRR and TRV:222- UTR-infected plants this percentage was similar (56% and 48%, respectively) ( Figure 2), confirming the function of NRCl in Cf-4/Avr4-mducQd HR, also in tomato. Similar results were obtained upon VIGS of Nrcl in C ⁇ P-containing tomato and subsequent injections of apoplastic fluid containing Avr9 (not shown).
• VIGS of Cf-9 in Cf-9- containing tomato we used the TRV:Cf-4 construct, since the 404 bp Cf-4 fragment codes for the highly conserved LRRs 15 to 21, enabling silencing of both Cf-4 as well as the homologous Cf-9 resistance gene (Van der Hoorn et al., 2001, supra).
• TRV:Cf-4-infected Cf-4 plants patches of blue staining indicate compromised Q ⁇ -mediated resistance (not shown).
• TRV:NRC1 -infected plants small patches of blue staining indicate loss of full resistance against the fungus.
• Microscopical analysis revealed intercellular growth of fungal hyphae in TRV:Cf-4- and TRV:NRC1 -infected plants, but not in the TRV:00- infected control plants. All CfO plants displayed extensive colonization by C. fulvum, indicating that neither the TRV infection itself, nor VIGS using TRV:NRC1 affects the susceptibility of these plants to the fungus.
• AvrPto from the bacterial pathogen Pseudomonas syringae pv tomato and the gene encoding the coat protein (CP) of potato virus X (PVX) were agroinfiltrated in TRV-infected N. benthamiana expressing the resistance gene Pto (Pedley and Martin, 2003, Annu. Rev. Phytopathol. 41, 215-243) and Rx (Bendahmane et al, 1999, Plant Cell 11, 781-791), respectively.
• plants infected with TRV:00 showed an HR, while the HR was abolished in TRV: SGTl -infected plants.
• TRV:NRC1 -infection resulted in a severely suppressed Pto/ AvrPto- as well as Rx/CP- induced HR, indicating that in N. benthamiana an NRCl protein is required for HR signaling activated by several R/Avr gene-for-gene combinations (Figure 3).
• Figure 3 To exclude the possibility that the compromised HR in TRV:NRC1 -infected N. benthamiana results from a decreased transformation efficiency by Agrobacterium, the inventors infiltrated TRV:00- and TRV:NRC1 -infected N. benthamiana: Cf-4 with Agrobacterium expressing the ⁇ -glucuronidase (GUS) gene (Van der Hoorn et al., 2000, supra).
• GUS ⁇ -glucuronidase
• TRV:00- and TRV:NRC1 -infected plants Three days post infiltration a similar intensity of the blue staining in TRV:00- and TRV:NRC1 -infected plants revealed that the transformation efficiency of the plants by Agrobacterium is not affected (data not shown). In addition, the TRV:NRC1 -infected plants also showed a reduced HR upon injection with Avr4 protein, while in TRV:00-infected plants a clear HR developed within 2 days.
• NRCl is required not only for Cf-4/Avr4-mduced HR, but also for HR induced by several additional R/Avr combinations, NRCl appears to be involved in a common HR signaling pathway.
• a typical host response that precedes the initiation of the HR includes activation of MAPK cascades (Romeis et al, 2001, EMBO J. 20, 5556-5567; Del Pozo et al., 2004, EMBO J. 23, 3072-3082; Pedley and Martin, 2005, Plant Biol. 8, 541-547).
• NRC1 D481V Epistasis experiments employing NRC1 D481V were performed to further investigate which genes are required for HR signaling by this protein, and thereby determine its putative position in an HR pathway.
• VIGS genes known to be generally involved in HR signaling, such as SGTl and RARl (Required for Mlal2 resistance) (Shirasu and Schulze-Lefert, 2003, Trends Plant Sci. 8, 252-258)
• N. benthamiana:Cf-4 was silenced for NDRl (non race-specific disease resistance) (Century et al, 1995, Proc. Natl. Acad. Sci.
• NRCl D481v -induced HR was not compromised in TRV:00- and TRV:NDR1 -infected plants, and also not in TRV:Cf-4-infected plants.
• NRC 1 D48i v stin induces an HR J n TRV:EDS1 -infected plants, indicating that NRCl is functional downstream of EDSl ( Figure 4C; dark circles).
• the NRCl D481v -induced HR is compromised in plants silenced for MEK2, showing that NRCl requires the MAP kinase cascade for its signaling and can be positioned upstream of these kinases.
• VIGS of RARl and SGTl also compromises D481V-induced HR, similar to the HR induced by Avr4 ( Figure 4C; light circles).
• NRCl is required for HR signaling initiated by Cf-4 and can be positioned upstream of the MAPK cascade and downstream of EDSl.
• NRCl In order to determine whether NRCl is required for Mz ' -mediated resistance against nematodes, white fly and aphids, a constitutively active form of Mi (see US 6613962 and EP0937155B1) is agroinfiltrated into NRCl silenced plants. A decreased HR in NRCl silenced plants indicates that NRCl is also required for Mi-mediated HR and that (over)expression of NRCl can be used to generate transgenic plants having enhanced resistance against nematodes, white fly and aphids.

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