IL193760A - Transgenic plants having enhanced disease resistance - Google Patents

Description

193760/2 m?n¾7 irax\n nn^s ^sn o^!uonu nm genie plants comprising enhanced disease resistance FIELD OF THE INVENTION The present invention relates to transgenic plants and plant cells comprising a gene encoding an NRC1 protein (NB-LRR Required for HR-associated Cell Death 1 ), the NRC1 protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70% amino acid identity to the amino acid sequence of SEQ ID NO: 2 over its entire length, operably linked to a promoter active in plant cells, integrated in its genome and methods for making such plants and cells. Especially Solanaceae plants and plant parts (seeds, fruit, leaves, etc.) with enhanced disease resistance are provided. Also provided are isolated nucleic acid molecules encoding NRC1 proteins according to the invention, vectors comprising these, as well as isolated NRC1 proteins themselves. Further, plant cells and plants comprising one or more mutations in an endogenous NRC1 allele are provided, whereby the mutation(s) confer enhanced diseases resistance to the plants and plant cells.

BACKGROUND OF THE INVENTION Active defense of plants, triggered upon recognition of an avirulence factor of a pathogen mediated by a resistance gene, follows the gene-for-gene model (Dangl and Jones, 2001 , Nature 41 1 , 826-833). To date, several plant resistance genes (R genes) have been cloned and based on the structure of the proteins they encode, the genes are divided into several groups (Hammond-Kosack and Jones, 1997, Annu. Rev. Plant Physiol. Plant Molec. Biol. 48, 575-607). Most R genes encode cytoplasmic NB-LRR proteins, containing a nucleotide binding site (NB) and leucine-rich repeats (LRR). 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).

Using such specific resistance genes in breeding programs for durable resistance is problematic since pathogens easily circumvent recognition by mutations in their avirulence factors, thereby preventing induction of active defense (Westerink el al., 2004, Mol. Microbiol. 54, 533-545). Similarity among resistance proteins (R proteins) suggests the existence of common resistance pathways (Shirasu and Schulze-Lefert, 2000, Plant Mol. Biol. 44, 371 -385). Therefore, identification of additional genes required for resistance not only provides information on how such signaling pathways function but might also enable us to identify genes that play a more general role in resistance. For example, by vims-induced gene silencing (VIGS) in Nicotiana benthamiana it was shown that SGT I is involved in multiple defense pathways, such as N-, Rx- and Pto-mcdiatcd HR and resistance, and Cf-4- and Cf-9-medialed HR (Peart et al., 2002, Proc. Nail. Acad. Sci. USA 99, 10865- 10869; Zhang et al., 2004, Plant J. 40, 213-224). SGT I is an intcraclor of SK.P l , which is a component of the SCF E3-ligase complex that is involved in ubiquitinalion of proteins, a modification which targets them for degradation (Schwcchhcimer and Schwager, 2004, Plant Cell Reports 23, 353-364). It is hypothesized that silencing an essential gene of this protein degradation system hampers the ubiquitinalion process, thereby inhibiting the degradation of negative regulators, which is required for defense activation (Azevedo et al., 2002, Science 295, 2073-2076).

In several resistance pathways MAPKs (mitogen activated prolcin kinases) arc activated (Zhang and Klessig, 2001 , Trends Plant Sci. 6, 520-527; Pedley and Martin, 2005, Curr. Opin. Plant Bioi. 8, 5 1 -547). In C/- -containing tobacco plants and cell cultures challenged with Avr9, NtWIPK (wound-induced protein kinase) and NlSIPK (salicylic acid-induced protein kinase) are activated (Romcis et al., 1999, Plant Cell 1 1 , 273-287). VIGS of a NlCDP (calcium-dependent protein kinase) in N. benthamiana inhibits the Cf-9/Avr9- and C -4//l v/'4-dcpendent HR (Romeis et al., 2001 , EMBO J. 20, 5556-5567) and VIGS of LeACI I (Avr/Cf-t'mluccd kinase 1 ) in tomato results in decreased C. fulv m resistance (Rowland et al., 2005, Plant Cell 17, 295-3 10). The activation of kinases during defense and the decreased resistance upon 'knock-down' of their encoding genes supports their function in defense activation.

Following a biased approach, 21 genes known to be involved in defense-related signaling were used for VIGS in tomato and it was found that nine of them are involved in P/omediated resistance. Among these arc two genes encoding MAP s (LeMEKl and LeMEK2) and two genes encoding MAPKs (LeNTF6 and LeWIPK) (Ekcngrcn et al. , 2003, Plant J. 36, 905-917). In another study, over 2400 cD As from a normalized library of N. benthamiana cDNA were cloned in a Potato Virus X-based vector and used for VIGS in N. benthamiana. About 3% of the cDNAs affected P/o-dcpcndcnt HR upon silencing. Among these a MAP KKa was identified as a positive regulator of both resistance and disease (Del Pozo et al., 2004, EM BO 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. 01* the cDNAs, 79 ( t .6%) corresponded to genes required for io-medialed MR, whereas silencing of only six of ihem also impaired /¾>-medialcd resistance against Pseudomonas syringae. VIGS using a cDNA corresponding to HSP90 abolished not only ¾>- mediated HR but also Pto-, Rx- and /V-mcdialcd resistance, indicating that HSP90 is required in multiple disease resistance pathways. The same set of cDNAs was also used for VIGS in N-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-cncoding gene, referred to as NRG I (TV requirement gene 1 ) (Peart et al., 2005, Curr. Biol. 15, 968-973). NRG J 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 avirulcnce factors, but are also involved in the signaling pathway initialed by the TI R-NB-LRR protein N, which eventually leads to resistance (Peart et al., 2005, supra). Thus, although the tobacco NRG I protein functions downstream of the plant's defense signaling cascade initiated by a resistance prolein, it has the drawbacks that it is specifically involved in N-mediated resistance against tobacco mosaic virus (TMV) and is not a general cofactor of disease resistance (Rx-and Λο-mediatcd resistance against PVX and Pseudomonas syringae were unaffected by NRG I silencing), whereby it may not be suitable for creating broad pathogen resistance in crops such as tomato.

Despite the increasing information about disease resistance pathways, there is still a need in identifying genes and proteins which can be used to create plants with durable, broad range disease resistance. It is an object of the invention to provide such nucleic acids, proteins and methods for creating plants, especially plants belonging to the family Solanaceae, with enhanced disease resistance.

GENERAL DEFINITONS "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, oomycetcs, mycoplasma like organisms, nematodes, white fly and aphids and the like. Generally all strains, races or palhovars of a pathogen species which are capable of causing disease on host tissue are included herein, "Biolrophic plant pathogens" or "biolroph" refers to a pathogen that keeps the host plant cells alive and relics on living cells for growth and tissue colonization.

"Hcmibiotrophic plant pathogen" or "hemibiotroph" refers to a plant pathogen which keeps the host ceils alive during at least part of its life cycle.

"Nccrotrophic 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 A vr protein) being present.

When referring to plants expressing an NRC l protein according to the invention (e.g. a constitulively active NRC l 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 o f the nucleic acid sequence encoding the NRC l 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 Solatium (including Solatium tycopersicum, which used to be known as Lycopersicon esculentu ), 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 H R 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 arc encompassed herein. Also encompassed is both a reduction of disease incidence (percentage of plants becoming infected) and/or of disease severity. Preferably, a plant having enhanced disease resistance to at least one pathogen is a plant comprising at least 1 %, 2%, 5%, 10%, 1 5%, 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. For example, a host plant having enhanced resistance to several biotrophic and/or hcmibiolrophic and/or nccrolrophic pathogen species would be considered lo have broad spectrum resistance. "Pathogen caused symptoms" include any symptoms of disease, such as mycelium growth/bio mass 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. The term "nucleic acid sequence" (or nucleic acid molecule) 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. An "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 plast id genome.

The terms "protein" or "polypeptide" arc used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a speci fic mode of action, size, 3 dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred lo 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 NRC l 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 NRC l -cncoding gene (e.g. by ovcrcxprcssion or silencing) in a plant. For example, the functionality of a putative NRC I protein obtained from plant species X can be tested by various methods. If the protein is functional, silencing of the NRCI 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 clicitor induced HR lesions and/or a reduction of pathogen resistance, as shown in the Examples for tomato. Also, complementation with a functional NRC I protein will be capable of restoring HR lesions and/or pathogen resistance. Alternatively, transient or stable (over)cxprcssion in species X of the gene encoding the NRC I protein (optionally together with a posllranscriptional gene silencing inhibitor) will lead to the development of clicitor independent HR lesions and/or enhanced disease resistance, especially against biotrophic and/or hemi-biotrophic pathogens. See also the Examples.

The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an inRNA) 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" (or recombinant 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. For example 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 lo 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" (also often referred to as 3 ' untranslated region, or 3 'cnd) 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 cukaryotic mRNAs) a polyadcnylalion signal (such as e.g. AAUAAA or variants thereof)- After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadcnylation signal and a poly(A) tail may be added, which is involved in Ihc transport of the mRNA to the cytoplasm (where translation lakes place).

"Expression of a gene" refers to the process wherein a DNA region, which is opcrably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically act ive, 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 posltranscriptional gene silencing or RNAi). An active protein in certain embodiments refers to a protein being constilutivcly active. The coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment. In gene silencing approaches, 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. Although mostly the upstream (5') transcription regulatory sequences of a coding sequence arc referred to, regulatory sequences found downstream (3') of a coding sequence arc also encompassed by this definition.

As used herein, the term "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 (he transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependcnl 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 developmental ly 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 spaliolemporal activity of the promolcr.

As used herein, the term "opcrably 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. For instance, a promoter, or rather a transcription regulatory sequence, is opcrably 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 ( B-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. The term "domain" as used herein means any part(s) or domain(s) of the protein with a specific struclure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain. Specific domains can also be used to identify other NRC 1 proteins, such as NRC1 orthologs from other plant species.

The terms "target peptide" refers to amino acid sequences which target a protein, or protein fragment, lo intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or lo the extracellular space or apoplasl (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 lo deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (sec e.g. US 5591616, US 2002138879 and WO95/06722), a co-integrale 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. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream o f the transcript ion regulatory sequence. 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 repeal RNA (or hairpin RNA) for silencing of a target gene/gene lamily, 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 cxtra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell. Throughout the text the Icrm "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 arc 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.

The term "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 phcnolypic trait (e.g. a change in pigmentation) or a nutritional requirements. The term "reporter" is mainly used to refer to visible markers, such as green fluorescent protein (GFP), cGFP, lucifcrase, GUS and the like.

The term "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 special ion).

Orlhologs of the tomato nrc] 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.

The terms "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). Depending on the context, the term "homolog" or "homologous" may alternatively refer to sequences which arc descendent from a common ancestral sequence (e.g. they may be orlhologs).

"Stringent hybridisation conditions" can be used to identi fy 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 Tm 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 salf concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. l OOnt) arc 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 (Southern blots using a probe of e.g. l OOnt) arc for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°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 Necdlcman and Wunsch global alignment π algorithm to align two sequences over their entire length, maximizing the number of matches and minimises the number of gaps. Generally, the GAP default parameters arc used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Hcnikoff & Henikoff, 1992, PNAS 89, 91 5-919). 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 Accclrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2. 10.0 (using the program "needle"). Alternatively percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word arc included, but items not specifically mentioned are not excluded, in addition, reference to an clement by the indefinite article "a" or "an" docs 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) arc referred to.

As used herein, the term "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 arc 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.

DETAILED DESCRIPTION OF THE INVENTION The present inventors have used cDNA-AFLP analysis, in combination with VIGS (Virus Induced Gene Silencing), to identify genes involved in Q-^/zlt -dcpcndcnt HR and disease resistance. Among the genes of which VIGS resulted in a suppression of the Ii -induccd HR, one tomato gene was identified (referred herein to as NRCI), encoding a CC-NB-LRR type resistance protein analog (herein referred to as NRCI for NB-LRR protein Required for HR-associated Cell death I ). Silencing of NRCI in tomato compromised not only the development of an Avr4 induced HR, but also resistance to the tomato pathogen Cladosporium fulviim. This indicated that the tomato Cf-4 resistance protein (an extracellular receptor like protein) requires a cytoplasmic NB-LRR protein to be functional.

Furthermore, it was surprisingly found that NRCl is involved in multiple HR and multiple disease resistance / cell death signaling pathways, such as Cf-9/A vr-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 Aii'-mediated HR (conferring resistance to nematodes, white fly and aphid-induced HR; sec US 6613962 and EP0937155B 1 ). Thus, NRC l 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 fulv rn and T choderma viride), a bacterium {Pseudomonas syringae pv tomato) or a virus (PVX).

The NRC l protein (and the NRCl gene encoding il) 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 Botiytis species. Especially, expression of NRCl (or variants or fragments thereof, as defined elsewhere) leads to enhanced resistance, especially against pathogens biotrophic and/or hemibiotrophic pathogens, i.e. all pathogens which obtain nutrients from living celts. Without limiting the scope of the invention, it is thought that the knock-down (gene silencing) or knock-out (e.g. by TILLING) of endogenous NRCl genes can be used to confer or enhance resistance against necrotrophic pathogens, as the pathway leading to necrosis is affected and nccrotophic pathogens require this pathway. Thus, depending on the pathogen(s) against which resistance is to be enhanced, either an increase or a decrease in NRCl expression levels may be used to enhance resistance. Optionally both approaches may be used in one plant, e.g. under control of different promoters. For example, NRCl can be expressed under control of a promoter induced by a (hemi)-biotrophic 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 nccrotroph using a promoter which is inducible by necrotrophic pathogens or wounding.

It was further found that, when a constitutively active NRC l protein (NRC I 1 K 1 V) was produced transiently in tomato, the plant tissue showed clicitor independent cell death (HR), showing that expression of a functional NRC l protein can be used to confer or enhance disease resistance in plants.

Proteins and nucleic acid sequences according to the invention The NRC l protein obtained from tomato shows low sequence identity (less than 25%) to NRG l of tobacco. NRC l also contains a larger number of Leucine Rich Repeals (LRR) than NRG l . The protein structure of NRC l is shown in figure 1 and SEQ ID NO: 2.

In one embodiment of the invention nucleic acid sequences and amino acid sequences of NRCl proteins arc provided (including orthologs), as well as methods for isolating or identifying orthologs of NRC l in other plant species, such as other Solanaceae, preferably potato. Equally, methods for isolating or identifying other NRCl alleles, such as alleles from other tomato species, varieties, lines or accessions are provided herein.

In one embodiment NRC l proteins are provided. "NRC l proteins" comprise the protein depicted in SEQ I D NO: 2 (wild type) and 4 (constitutive mutant), as well as fragments and variants thereof. Variants of NRC l 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 Necdleman and Wunsch algorithm and GAP default parameters as defined above. Variants of 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. For example, SEQ ID NO: 4, a constitutively active NRC l mutant, which is a variant of SEQ I D NO: 2, and was made by targeted mutagenesis using overlap PCR (sec Examples), The NRC l 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 NRC 1 protein, fragment or variant.

NRC l 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, Val, Trp, Leu, He, Pro, Mel, Phc, Trp) or polar (c. g. Gly, Ser, Thr, Tyr, Cys, Asn, Gin). In addition non-conservative amino acid substitutions Tall within the scope of the invention.

The functionality of any NRC1 protein, variant or fragment, can be determined using various methods. For example, transient or stable ovcrcxpression in plant cells can be used to test whether the protein has activity in plantci. Functionality is preferably tested in the same plant species from which the protein is obtained. Thus, for example transient or stable expression can be used to determine whether an HR develops and/or whether resistance is enhanced, indicating functionality. Alternatively, silencing of the endogenous genes or gene family will show whether the NRC l protein is functional. For example, V1GS can be used in a variety of Solanaceae, such as potato, tomato and tobacco (see Brigncti et al., 2004, Plant Journal 39: 264; Faivre-Rampant el al. Plant Physiology 134: 1308- 1316; Baulcombe 1999, Curr. Opinion. Plant Biol. 2: 109-1 13; Lu et al. 2003, EMBO J. 22:5690-5699), in model organisms such as Arabtdopsis (Turnagc et al. 2002, Plant J. 30: 107- 1 14), in monocots such as barley (Holzbcrg et al. 2002, Plant J. 30: 315-327). Altcmativeiy, silencing vectors comprising sense and/or antisensc 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.

In a preferred embodiment variants of NRCl include NRC l proteins which arc constitutively active in plant cells, such as the NRC l protein provided in SEQ ID NO: 4, which comprises a single amino acid substitution in the MHD domain (D481 V) (see Figure I ). The constitutive activity can be tested by determining whether the protein is capable of eliciting an HR in plant tissue, in the absence of clicitor. For example, Agroinl ltralion of a 35S.NRC ! construct, as described in the Examples, can be used to infiltrate leaf tissue. Other constitutivcly active NRC I proteins can be made, by either random mutagenesis followed by activity testing (as described in Bendahame et a!., 2002, p ! 96) 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 FLYFGTFPRGY), or one of the 13 LRR domains (sec Figure 1 ). Alternatively, nucleic acid sequences encoding constitutivcly active NRC I proteins can be obtained from plants, for example by mutagenizing seeds and screening these for the presence of a spontaneous lesion phcnotype (for example microscopic lesions), sec e.g. Sharino et al. (2002, The Plant Cell 14: 3149-3 162) and further below.

In one embodiment also chimeric NRC I proteins are provided. Such 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 15 1 -508, or to amino acids 509-846 of SEQ ID NO: 2 respectively. Domains may thus be exchanged (domain swapping) between NRCI proteins or between NRC I 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 NRC I , or preferably to NRC 1 D481 V. Most preferably, 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 NRCI proteins and of variants of NRC I proteins, as described above, comprise fragments of 100, 150, 200, 300, 400, 500, 600, 700, 800, 850, 855 contiguous amino acids or more. Preferably, such fragments are functional in plant tissue, i.e. they arc capable of conferring or enhancing pathogen resistance when produced in plant cells, Fragments may also be used to make chimeric proteins, as described above.

In another embodiment isolated nucleic acid sequences encoding any o f the above proteins, variants or fragments are provided, such as cD A, genomic D A 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 NRC l proteins or variants arc herein referred to as "NRCl". The nucleic acid sequences provided include naturally occurring, arti ficial or synthetic nucleic acid sequences. Examples of nucleic acid sequences encoding NRC l 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 di fference that thymine (T ) is replace by uracil (U).

Also included arc 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. In a preferred embodiment, 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 silica 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 NRC l proteins (or e.g. chimeric NRC l proteins), as described below. For optimal expression in a host 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 (Bennclzen & Hall, 1982, J. Biol. Chetn. 257, 3026-3031 ; Ilakura et al., 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 arc published for example by Ikcmura ( 1993, In "Plant Molecular Biology Lab ax", 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 arc produced. Several techniques for modifying the codon usage to that preferred by the host cells can be found in patent and scientific literature. The exact method of codon usage modification is not critical for this invention.

Small modifications to a DNA sequence such as described above can be routinely made, i.e., by PCR-mediated mutagenesis (Ho et al., 1989, Gene 77, 51 -59., White ct at., 1989, Trends in Genet. 5, 185- 189). More profound modifications to a DNA sequence can be routinely done by de novo DNA synthesis of a desired coding region using available techniques.

Also, the NRCJ nucleic acid sequences can be modified so that the N-tcrminus of the NRC I protein has an optimum translation initiation context, by adding or deleting one or more amino acids at the N-tcrminal end of the protein. Often it is preferred that the proteins of the invention to be expressed in plants cells start with a Mct-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, Val, 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 NRCI nucleic acid sequences include fragments of at least 10, 12, 15, 16, 1 8, 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 I D NO: 1 or 3. Short fragments can for example be used as PGR primers or hybridization probes.

In another embodiment of the invention PCR primers and/or probes and kits for detecting the NRCJ DNA or RNA sequences are provided. Degenerate or specific PCR primer pairs to amplify NRCI DNA from samples can be synthesized based on SEQ I D NO: 1 or 3 (or variants thereof) as known in the art (sec Dieffenbach and Dvekslcr ( 1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPhcrson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany). For example, any stretch of 9, 10, I I , 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of SEQ ID NO: I or 3 (or the complement strand) may be used as primer or probe. Likewise, DNA fragments of SEQ ID NO: 1 or 3 (or variants thereof) can be used as hybridization probes. An NRCl detection kit may comprise cither NRCl speci fic primers and/or NRCl specific probes, and an associated protocol to use the primers or probe to detect NRC! DNA in a sample. Such a detection kit may, for example, be used to determine, whether a plant has been transformed with an NRC! gene (or part thereof) of the invent ion. 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.

In yet another embodiment 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 NRC l 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 ccll(s), preferably of the plant species from which the nucleic acid was obtained, c) analysing the capability of the transformed plant / plant tissue to develop HR lesionvs (i.e. the HR lesion phenotypc, 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; 0 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 phenotypc).

Thus, NRCl alleles or orthologs, which upon expression in plants result in fewer and/or smaller HR lesions than seen upon expression of SEQ I D 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 arc identified which cause no HR lesions, or at least no macroscopically visible HR lesions, upon expression, but which still confer enhanced disease resistance.

The H R phenotypc of different NRCI 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. When different alleles arc compared in transgenic Solanaceae plants (e.g. under control of a constitutive or inducible promoter), the HR lesion phenotype of transformanls expressing SEQ ID JO: 1 or 3 is preferably used as reference. Alternatively, 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. For example 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?C/-spccific PCR primers or probes, or bioinformatics analysis in silico. Also genetic mapping may be used to map the NRCJ 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.

When potato orthologs of NRCJ arc identified in the above method, these orthologs (or variants of these orthologs) are preferably used to generate plants having enhanced resistance to Phytop thor infestans.

Chimeric nencs. expression vectors and recombinant oruanisms according to the invention In one embodiment of the invention nucleic acid sequences encoding NRCI 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 NRCJ prolein(s) in host cells, such as cells, tissues, organs or organisms derived from transformed ccll(s). Vectors for the production of N C 1 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. For example, the plant belongs to the genus Solamtm (including Lycopersicon), Nicotiana, Capsicum, Petunia and other genera. The following host species may suitably be used: Tobacco {Nicotiana species, e.g. N. benthamiana, N. pl mbaginifolia, N. tabacum, etc.), vegetable species, such as tomato (L. esculentum, syn. Solarium lycopersicuin) such as e.g. cherry tomato, var. cerasifonne or currant tomato, var. pimpineUifolium) or tree tomato (S. betaceum, syn. Cyphomandra betaceae), potato (Solanuin tuberosum), eggplant (Solanum melongena), pepino (Solanuin inuricatam), cocona (Solanuin sessiliflorum) and naranjilla (Solanum quitoense), peppers (Capsicum annuum, Capsicum frutescens, Capsicum baccatum), ornamental species (e.g. Petunia hybrida, Petunia axillaries, P. integrifolia).

Alternatively, the plant may belong to any other family, such as to the Cucurbitaceae or Gratnineae. Suitable host plants include for example maize/corn (Zea species), wheat (Triticum species), barley (e.g. Nordeuin vulgare), oat (e.g. A vena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G. hirsutum, G. barhadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc), sunflower (IJeliant us annus), safflowcr, yam, cassava, al falfa (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. Phaseohts species), cucumber, artichoke, asparagus, broccoli, garlic, leck, 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, Gcrbcra species), herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species of Populus, Salix, Quercus, Eucalyptus), fibre species e.g. fiax (Liinim usitatissimum) and hemp {Cannabis aativa), or model organisms, such as Arabidopsis t aliana.

Preferred hosts are "crop plants", 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 (lowers for cutting, grasses for lawns, etc.). A crop plant as defined herein also includes plants from which non-food products arc harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork and the like.

The construction of chimeric genes and vectors for, preferably stable, introduction of NRC I protein encoding nucleic acid sequences into the genome o f host cells is generally known in the art. To generate a chimeric gene the nucleic acid sequence encoding a NRC I 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 NRCI 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 (c. g., Mc Bride et al., 1995 Bio/Technology 13, 362; US 5,693, 507). In one embodiment 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 NRC I protein according to the invention, optionally followed by a 3'nontranslated nucleic acid sequence.

The NRCI nucleic acid sequence, preferably the NRCI chimeric gene, encoding an functional NRC I protein (or in certain embodiments a const ilutivciy active NRCI protein), can be stably inserted in a conventional manner into Ihc nuclear genome of a single plant cell, and the so -trans formed 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 NRC I protein in certain cells at a certain time. In this regard, a T-DNA vector, comprising a nucleic acid sequence encoding a NRC I 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 1 16 71 8, 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-intcgratc vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP 0 1 16 7 1 8.

Preicrred T-DNA vectors each contain a promoter opcrably linked lo NRC I 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 lo the left of the right border sequence. Border sequences are described in Gielcn et al. ( 1984, EMBO J 3,835-845). Of course, other types of 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 WO85/01 856), protoplast transformation as, for example, described in US 4,684, 61 1 , plant RNA virus- mediated transformation (as described, for example in EP 0 067 553 and US 4,407, 956), liposomc-mediated transformation (as described, for example in US 4,536, 475), and other methods. For tomato or tobacco transformation see also An G. et al., 1986, Plant Physiol. 81 : 301 -305; Horsch R.B. et al. , 1988, In: Plant Molecular Biology Manual A5, Dordrecht, Netherlands, Kiuwer Academic Publishers, pp 1 -9; Koomneef M. et al., 1 86, Jn: Nevins D.J. and R.A. Jones, eds. Tomato Biotechnology, New York, NY, USA, Alan R. Liss, Inc. pp 169- 178). For potato transformation see e.g. Sherman and Bevan ( 1 88, Plant Cell Rep. 7: 13- 16).

Likewise, selection and regeneration of transformed plants from transformed cells is well known in the art. Obviously, for different species and even for different varieties or cullivars of a single species, protocols are specifically adapted for regenerating transformants at high frequency.

Besides transformation of the nuclear genome, also transformation of the plastid genome, preferably chloroplast genome, is included in the invention. One advantage plastid genome transformation is that the risk of spread of the transgcnc(s) can be reduced. Plastid genome transformation can be carried out as known in the art, sec e.g. Sidorov VA et al, 1999, Plant J. 19: 209-216 or Lulz 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 WOO 141558, which describes elite event detection kits (such as PCR detection kits) based for example on the integrated sequence and the flanking (genomic) sequence.

The NRCI 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.

As the constitutive production of the NRC I 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 Mol Biol, 2001 46: 313-23), it is in one embodiment preferred to use a promoter whose activity is inducible. Examples o f inducible promoters arc wound-induciblc promoters, such as the MPl promoter described by Cordcra et al. ( 1994, The Plant Journal 6, 1 1 ), which is induced by wounding (such as caused by insect or physical wounding), or the COMPT1I promoter (WO0056897) or the PR1 promoter described in US603 1 15 I . Alternatively the promoter may be inducible by a chemical, such as dexamelhasonc as described by Aoyama and Chua ( 1997, Plant Journal 1 1 : 605-612) and in US6063985 or by tetracycline (TOPFREE or TOP 10 promoter, sec Gatz, 1997, Annu Rev Plant Physiol Plant Mol Biol. 48: 89- 108 and Love ct al. 2000, Planl 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 ADH I S promoter), by light (US6455760), by pathogens (e.g. the gstl promoter of EP759085 or the vstl promoter of EP309862) or by senescence (SAG 12 and SAG 13, see US5689042). Obviously, there arc a range of other promoters available.

In one embodiment preferably, a pathogen inducible promoter is used, as thereby the NRC 1 protein (or variant or fragment) will only be produced following pathogen attack of the plant tissue. Especially, promoters of genes which arc uprcgulalcd 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 ct al. ( 1 97, Planl Physiology 1 15(2):437-5 1), the tap ! or tap2 promoter (Mohan et al., 1993, Plant Mot Biol. 1993 22 :475-90), the gst l promoter or variants thereof (Martini et al. 1993, Mol. Gen. Gen. 236, 179- 186; Hennin C., 1997, Afstudeerwerk, Faculteil Landbouwkundige en Tocgepaste Biologische Wetcnschappen, University of Gent, Belgium), the WRKY promoters (Eulgem ct al., EMBO J., 1999, 18(17):4689-99 and chimeric promoters described in WO0029592). Promoters inducible by a particular plant pathogen may also be identified using known methods, such as cDNA-AFLP®.

Preferably, the promoter is inducible by a number of pathogens, i.e. it is inducible by a broad range of pathogens of the host plant. For each particular host plant species, a different promoter may be most suitable. For example, when tomato is used as a host, the promoter is preferably induced upon al least one, but preferably more than one tomato pathogen. Especially, a promoter which is inducible by one or more fungal plant pathogens and/or bacterial planl pathogens (especially by one or more biotrophic and/or hemi-biotrophic plant pathogens) is preferred.

Detailed descriptions of plant pathogens, the disease symptoms caused by them and their li e cycles can be found for each plant species. For example, tomato pathogens are described in "Compendium of Tomato Diseases" , Editors Jones, Jones, Stall and Ziller, ISBN 0-89054- 120-5, APS Press (http:/www.shopapsprcss/org). Potato pathogens are described in "Compendium of Potato Disease", 2nd edition, Editors Stevenson, Franc and Wcingartner, APS Press, ISBN 0-89054-275-9.

Pathogens of tomato include, for example, the following fungal and bacterial species and viruses (non-limiting): Botiytis cinerea (fungus / nccrotroph); Colletotrichum coccodes (fungus / nccrotroph); Alternaria alternata (fungus); Alternaria solani (fungus / necrolroph); Stemphylium solani; Phytophthora infestans (oomyctc / hemibiotroph); Septoria lycopersici; Ciadosporium fulvum, (fungus / hemibiotroph); Phytophthora parasitica; Oidium lycopersicum (biotroph); Fusarium oxysporum; Sclerotium rolfsii; Pythium; Rhizoctonia (fungus / nccrotroph); Corymbacterium michiganense (bacterium); Pseudomonas syringae pv tomato or pv svringae (bacterium / biotroph); Pseudomonas soianacearum; Pseudomonas corrugate; Clavibacter Xanthomonas campestris (bacterium / biotroph); VerticUlium (fungus), tomato spotted wilt virus (TSWV); Tobacco or tomato mosaic viruses (TobMV, TomMV).

Pathogens of potato include, for example, various fungi, bacteria, nematodes and viruses, such as: Phytophthora infestans (oomyctc / hemibiotroph), nematodes (biotrophic); Erwinia carotovora (bacterium); Colletotrichum coccodes (fungus); Rhizoctonia solani (fungus / nccrotroph); VerticUlium dahliae (fungus); Strepto yces scabies; Alternaria solani (fungus / necrotroph) ; Pythium; Spongospora subterranean; PVX and PVY; Potato Lcafroll Virus (PLRV); etc.

See also http://www.apsnet.org/online/common/toc.asp for plant diseases of various plant species. Thus, in one embodiment 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.

Alternatively, a host plant may comprise various NRCJ transgencs, each under control of a different pathogen inducible promoter, to ensure that NRC I protein is produced following attack by a variety of pathogens. For example, for transformation of tomato, one promoter may be inducible by Phytophthora and one by Ciadosporium.

The word "inducible" docs 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 NRCI coding region following contact with the inducer.

The most preferred combination herein is the use of a pathogen inducible promoter, opcrably linked to an NRCI nucleic acid sequence which encodes a conslilulivcly active NRC I protein, as described above. In this case upon pathogen attack the constitutivcly active NRC I will be expressed resulting in a local HR (restricted to the site of pathogen attack) preventing further growth of any (hcmi)-biotrophic pathogen.

In another embodiment 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 18 1 (Gardner et al., 1981 , Nucleic Acids Research 9, 2871 -2887), CabbB-S (Franck et al., 1980, Cell 21 , 285-294) and CabbB-J I (Hull and Howell, 1 87, Virology 86,482-493); the 35S promoter described by Odcll et al. (1985, Nature 3 13, 810-812) or in US5 I 643 16, promoters from the ubiquitin family (e.g. the maize ubiquitin promoter of Christenscn et al., 1992, Plant Mol. Biol. 18,675-689, EP 0 342 926, see also Cornejo et al. 1993, Plant Mol.Biol. 23, 567-581 ), the gos2 promoter (de Paler et al., 1992 Plant J. 2, 834-844), the emu promoter (Last et al., 1990, Theor. Appl. Genet. 81 ,581 -588), Arabidopsis aclin promoters such as the promoter described by An et al. ( 1996, Plant J. 10, 107.), rice actin promoters such as the promoter described by Zhang et ø/.(.! 991 , The Plant Cell 3, 1 155- 1 165) and the promoter described in US 5,64 1 ,876 or the rice actin 2 promoter as described in WO070067; promoters of the Cassava vein mosaic virus (WO 97/48819, Vcrdaguer et al. 1998, Plant Mol. Biol. 37, 1055- 1067), the pPLEX scries of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g., pAd l S (GenBank accession numbers X04049, X00581 ), and the TRT promoter and the TR2' promoter (the "TR I ' promoter" and "TR2' promoter", respectively) which drive the expression of the and 2' genes, respectively, of the T-DNA (Vclten et al., 1984, EMBO .1 3, 2723-2730), the Figwort Mosaic Vims promoter described in US6051753 and in EP426641 , histonc gene promoters, such as the Ph4a748 promoter from Arabidopsis (PMB 8: 179-191 ), or others. In a preferred embodiment the AA6 promoters, as described in PCT/NL2005/050083 (filed 16 December 2005) are used.

Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (tissue preferred / tissue specific, including developmental ly regulated promoters), for example leaf preferred, epidermis preferred, root preferred, flower tissue e.g. tapctum or anther preferred, seed preferred, pod preferred, etc.), whereby the NRCl gene is expressed only in ceils of the specific tissuc(s) or organ(s) and/or only during a certain developmental stage. For example, 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-induciblc promoter such as the promoter of the ribulosc-1 , 5-bisphosphatc 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.

In one embodiment the promoter of the endogenous NRCl gene is used. For example, the promoter of the tomato NRCl gene may be isolated and opcrably 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: I and 3) can be isolated from tomato plants using known methods, such as TA1L-PCR (Liu et al. 1 95, Genomics 25(3):674-8 l ; Liu et al. 2005, Methods Mol Biol. 286:341 -8), Linkcr-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 nonlranslatcd region ("3'end" or 3'UTR). Suitable 3'ends include those of the CaMV 35S gene ("3' 35S"), the nopaline synthase gene ("3 ' nos") (Dcpickcr et al., 1982 J. Molcc. Appi. Genetics 1 , 561 -573.), the octopinc synthase gene ("3'ocs") (Giclen et al, 1 84, EM BO J 3, 835-845) and the T-DNA gene 7 ("3' gene 7") (Velten and Schell, 1985, Nucleic Acids Research 13, 6981 -6998), which act as 3†-unlranslatcd DNA sequences in transformed plant cells, and others. In one embodiment the 3'UTR of the tomato NRCl gene is used, as shown in SEQ ID NO: 3, from nucleotide 2748 to nucleotide 3 168, and as shown in SEQ I D 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. Equally, any variant or fragment of SEQ ID NO: 5 is provided. A variant of SEQ I D 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.

Introduction of the T-DNA vector into Agrobacterium can be carried out using known methods, such as electroporation or triparcntal mating.

A NRC l 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; Vacck et al.t 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.

All or part of a NRCl nucleic acid sequence, encoding a NRC l protein (or variant or fragment), 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. ( 1 89, FEMS Microbiol. Letters 60, 205-210.) and WO 90/06999. For expression in prokaryotic host cell, the codon usage of the nucleic acid sequence may be optimized accordingly (as described for plants above), lntron sequences should be removed and other adaptations for optimal expression may be made as known.

The DNA sequence of the NRCI 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.

In accordance with one embodiment of this invention, the NRC I proteins (or chimeric 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. Similarly, the protein may be targeted to vacuoles. For this purpose, in one embodiment of this invention, the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the NRCI 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 Ihe plastids, the optimized transit peptide of Capcliades et al. (US 5,635, 618), the transit peptide of ferredoxin-NADP- oxidoreduelase from spinach (Oe!muller et al., 1993, Mol. Gen. Genet. 237,261-272), the transit peptide described in Wong et al. ( 1992, Plant Molcc. Biol. 20, 81 -93) and the targeting peptides in published PCX patent application WO 00/26371. Also preferred are peptides signalling secretion of a protein linked to such peptide outside the cell, such as the secretion signal of the potato proteinase inhibitor II ( eil et ai, 1986, Nucl. Acids Res. 14,5641 -5650), the secretion signal of the alpha- amylase 3 gene of rice (Sutliff et al. , 1991 , Plant olec. Biol. 16,579-591 ) and the secretion signal of tobacco PR1 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. Van Den Brocck et al., 1985, Nature 3 13, 358), or the optimized chloroplast transit peptide of US 5, 10, 471 and US 5,635, 618 causing transport of the protein to the chloroplasts, a secretory signal peptide or a peptide targeting the protein to other plastids, mitochondria, the ER, or another organelle. Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or to the cell wall are found in naturally targeted or secreted proteins, preferably those described by Klosgcn et al. (1989, Mol. Gen. Genet. 217, 1 55- 161 ), KJosgen and Weil ( 1993 , Mol. Gen.

Genet. 225, 297-304), Neuhaus & Rogers ( 1 98, Plant Mol. Biot. 38, 127-144), Bih el al. ( 1999, J. Biol. Chcm. 274, 22884-22894), Morris ct al. ( 1999, Biochem. Biophys. Res. Commun. 255, 328-333), Hesse et al. ( 1989, EM BO J. 8, 2453-2461 ), Tavladoraki cl al. ( 1998, FEBS Lett. 426,62-66.), Tcrashima el al, (1999, Appl. Microbiol. Biotcchnol. 52,516-523), Park et al, ( 1997, J.Biol. Chcm. 272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad. Sci USA 92,9245-9249).

To allow secrelion of Ihe NRC I proteins to the outside of the transformed host cell, an appropriate secretion signal peptide may be fused to the amino terminal end (N-terminal end) of the NRC I protein. Putative signal peptides can be detected using computer based analysis, using programs such as the program Signal Peptide search (SignalP V I .1 or 2.0)(Von Hcijne, Gunnar, 1986 and Nielsen et al., 1996).

In one embodiment, several NRC I 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 NRC I protein of this invention, or by crossing plants transformed with different NRC I proteins of this invention. Alternatively, several NRC I protein encoding nucleic acid sequences can be present on a single transformation vector or be co -tran formed at the same time using separate vectors and selecting transformanls comprising both chimeric genes, Similarly, one or more NRCI 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.

It is understood that the different proteins can be expressed in the same plant, or each can be expressed in a single plant and then combined in the same plant by crossing the single plants with one another. For example, in hybrid seed production, each parent plant can express a single protein. Upon crossing the parent plants to produce hybrids, both proteins arc combined in the hybrid plant.

Preferably, for selection purposes but also for weed control options, 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 glufosinatc 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, sec e.g. EPO 508 909 and EP 0 507 698). Using herbicide resistance genes (or other genes conferring a desired phenolypc) as selectable marker further has the advantage that the introduction of antibiotic resistance genes can be avoided.

Allcrnalively, other 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 transfonnanls. Different technologies exist for removal of transgencs. One method to achieve removal is by flanking the chimeric gene with lox sites and, following selection, crossing the transformed plant with a CRE rccombinase-cxpressing plant (see e.g. EP506763B 1 ). Site specific recombination results in excision o f the marker gene. 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 slacking purposes. Further, one-component excision systems have been described, see e.g. WO9737012 or WO9500555).

Transformed plan! cclls/plants sceds and uses of the nucleic acid sequence and proteins according to the invention In the following part the use of the NRC1 sequences according to the invention to generate transgenic plant cells, plants, plant seeds, etc. and any derivatives/progeny thereof, with an enhanced diseases resistance phenotype is described.

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 NRC 1 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 arc pathogen inducible are preferred, as described above. Preferred promoter - NRCl combinations arc: a) a pathogen inducible promoter - nucleic acid sequence encoding a constitutivcly active NRC t protein; b) a pathogen inducible promoter - nucleic acid sequence encoding a wild type NRC I protein; c) the promoter of a plant NRCI gene (preferably of the same species which is to be transformed) - nucleic acid sequence encoding a constitutivcly active NRC I protein; d) the promoter of a plant NRCI gene (preferably of the same species which is to be transformed) - nucleic acid sequence encoding a wild type NRC I protein; e) a biotic stress inducible promoter (e.g. insect wounding inducible, pathogen inducible, etc.) - nucleic acid sequence encoding a constitutivcly active NRCI protein; f) a biotic stress inducible promoter (e.g. insect inducible, pathogen inducible, etc.) - nucleic acid sequence encoding a wild type MRC 1 protein; g) A constitutive promoter (e.g. 35S promoter) -nucleic acid sequence encoding a wild type NRC I protein; h) A constitutive promoter (e.g. 35S promoter) - nucleic acid sequence encoding an amino-acid sequence comprising at least 70% amino acid sequence identity to SEQ I D NO:2 over the entire length. i) A pathogen- inducible promoter - nucleic acid sequence encoding an amino-acid sequence comprising at least 70% amino acid sequence identity to SEQ JD NO:2 over the entire length. j) The promoter of a plant NRCI gene - nucleic acid sequence encoding an amino- acid sequence comprising at lest 70% amino acid sequence identity to SEQ ID NO:2 over the entire length.

In one embodiment the transgenic plant may show either constitutive HR lesions or inducible HR lesions, and enhanced disease resistance to one or more pathogens. However, it is also envisaged herein that no HR lesions or "weak" HR lesions (such as smaller lesions, e.g. micro-lesions, and/or a low lesion frequency) develop, while the plant still shows enhanced disease resistance. NRCI 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: I or 3, or than the expression of the wild type NRCl allele obtained from the same host species which is transformed, arc particularly preferred herein, especially in approaches g) and h) above. Such altclcs/orthologs can be referred to as NRCl alleles conferring a "weak HR phcnolypc" in a given host. Such 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 arc to be compared are operably linked lo the identical promoters, e.g. 35S), transforming plants or plant tissue with these, and by comparing the HR lesion phenotype between these plants. For Solanaceae transformants, 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 11 R 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 cm2, measuring the diameter of HR lesions, etc.

Preferably, the transgenic plants of the invention comprise enhanced disease resistance against one or more pathogens, especially biolrophic and/or hcniibiotrophic pathogens of the transgenic plant species. Thus, for example 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 lo wild type or control transformants) lo withstand the attack of one or more planl pathogens, or in other words, it refers lo 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 arc scored visually (cither 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. Ultimately, a signi ficant increase in average yield of transformants (e.g. at least 1 %, 2%, 5%, 10% or more) compared to controls, when grown under equivalent disease pressure (preferably in the field) provides an indirect measurement of enhanced disease resistance.

Thus, a plurality of transformed plants expressing NRC 1 protein (or a constitutively active NRC 1 protein) show enhanced disease resistance if they show a significant reduction of disease symptoms, compared to the untransformed or empty- vector transformed controls. Obviously, statistical analysis is required to determine whether significant difference exist. Preferably, one or more disease symptoms are on average at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, or even 100% lower in NRC1 Iransformants than in the control plants. As 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.

As the NRC 1 protein may in some embodiments result in HR lesions in the absence of pathogen (for example if the NRC1 gene is under the control of a constitutive promoter): it may in certain embodiments be important lo differentiate between symptoms caused by NRCI 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 C. f lvum race is used which expresses GUS. Fungal mycelium can, therefore, be visualized using X-glue staining of the inoculated plant tissue. A significant reduction of fungal mycelium in the transgenic plants compared to the controls indicates an enhanced resistance to the fungus.

It is also an embodiment to generate transgenic plants which express several NRC1 proteins, preferably under the control of different promoters, such as di fferent pathogen inducible promoters.

The disease resistance phcnotype can be fine-tuned by expressing a suitable amount of NRC 1 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 NRC I protein or too slow induction of NRC 1 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 agronotnically undcsired phenotypes, such as lesions in leaves or fruit in the absence o f pathogens and yield penalties. However, the skilled person can easily generate plants having enhanced disease resistance, but which at the same time are agronomical acceptable. Optimal NRC I alleles may be isolated or identi fied as described, e.g. alleles providing high resistance levels and only a weak HR phcnotype.

Transformants expressing desired levels of the NRC I protein arc selected by e.g. analysing copy number (Southern blot analysis), mRNA transcript levels (e.g. RT-PCR using NRCI primer pairs or Hanking primers) or by analysing the presence and level of NRC I protein in various tissues (e.g. SDS-PAGE; ELISA assays, etc). For regulatory reasons, preferably single copy transformants arc 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 NRCI expressing transgenic events are selected for further crossing / backcrossing / selling until a high performing elite event with a stable NRCI transgene is obtained.

Transformants expressing one or more NRCI 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. To obtain such plants with "stacked" transgenes, other transgenes may cither be introgressed into the NRCI transformants, or the NRCI 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.

In one embodiment the following genes are combined with one or more NRCI 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 transformanls 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. Also, NRCI silencing approaches may be combined with NRC I expression approaches in a single plant. For example, NRCI ovcrexprcssion in roots or tubers may confer or enhance root or tuber resistance to soil pathogens. At the same time downrcgulation of NRCI in aerial parts may confer or enhance resistance lo necrotrophic pathogens (or vice versa).

It is also possible to introduce or mtrogrcss the NRCI gene into a plant breeding line which already has a certain level of disease resistance. For durability o f disease resistance in the field, it may be desirable lo stack several disease resistance mechanisms in a plant, preferably whereby Ihc resistance sources have different underlying molecular mechanisms.

Whole plants, seeds, cells, tissues and progeny (such as Fl hybrids, F2 seeds/plants, etc.) of any of the transformed plants described above arc 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 lemplalc and using NRCI specific PCR primer pairs. Also "event specific" PCR diagnostic methods can be developed, where the PCR primers arc based on the plant DNA Hanking the inserted chimeric gene, sec US6563026. Similarly, event speci fic AFLP fingerprints or RFLP fingerprints may be developed which identify the transgenic plant or any plant, seed, tissue or cells derived there from.

It is understood that the transgenic plants according to the invention preferably do not show non-desired phenotypes, such as yield reduction, enhanced susceptibility to diseases (especially to nccrolrophs) or undesjred architectural changes (dwarfing, deformations) etc. and that, if such phenotypes arc seen in the primary transfonnants, these can be removed by normal breeding and selection methods (crossing / backcrossing / selfing, etc.). Any o f the transgenic plants described herein may be homozygous or hemizygous for the transgenc.

NRCI gene silencing approaches and gene silencing vectors It is a further embodiment of the invention to provide plants with enhanced disease resistance, especially against necrolrophic pathogens, whereby the plant is transformed with an NRCI gene silencing vector. Without limiting the scope of the invention, it is thought that silencing o f endogenous NRCI 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 o f gene expression of one or more target genes (e.g. endogenous NRCI genes). The use of 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, Depickcr and Van Montagu, 1997). There are a number of technologies available to achieve gene silencing in plants, such as chimeric genes which produce anliscnse RNA of all or part of the target gene (see e.g. EP 0140308 B l , EP 0240208 B l and EP 0223399 B l ), or which produce sense RNA (also referred to as co-suppression), see EP 0465572 B I .

The most successful approach so far has however been the production of both sense and antisensc RNA of the target gene ("inverted repeats"), which forms double stranded RNA (dsRNA) in the cell and silences the target gene. Methods and vectors for dsRNA production and gene silencing have been described in EP 106831 1 , EP 983370 Al , EP 1042462 A l , EP 1071762 A l and EP 1080208 A l . 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. Generally short (sense and antisense) stretches of the target gene sequence, such as 17, J 8, 1 , 20, 21 , 22 or 23 nucleotides of coding or non-coding sequence arc sufficient. Longer sequences can also be used, such as 50, 100, 200 or 250 nucleotides or more. Preferably, the short sense and antisense fragments arc separated by a spacer sequence, such as an intron, which forms a loop (or hairpin) upon dsRNA fonnat ion. 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 arc 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 (sec Wesley et at. 2004, Methods ol Biol. 265: 1 17-30; Wesley et al. 2003, Methods Mot Biol. 236:273-86 and Helliwell & Watcrhouse 2003, Methods 30(4):289-95.), all incorporated herein by reference.

By choosing conserved nucleic acid parts of the NRCl gene, NRCl family members in a host plant or plant parts can be silenced. Encompassed herein arc also transgenic plants comprising a transcription regulatory clement operably linked to a sense and/or antisense DNA fragment of a NRCl gene and exhibiting enhanced resistance to one or more pathogens, especially nccrol ophic pathogens.

Also, plants having enhanced resistance to one or more biotrophic and/or hemi-biolrophic pathogens and to one or more necrotrophic pathogens arc provided. Such plants can be generated by choosing appropriate promoter - NRCl gene combinations. For example 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 gcne(s) are silenced in a different tissue and/or al 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 NRC l 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 ai, 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 NRC I proteins according to the invention and/or which produce a constitutivcly active NRC I protein as described. Without limiting the scope of the invention, it is believed that such plants could comprise point/deletion mutations in the promoter that are binding sites for repressor proteins that would make the host NRCI gene constitutive or higher in expression. Constitutivcly active NRCI mutants will comprise mutations in the coding region, such as the MHD region. Preferably TNRC l protein levels in the mutant or parts o the mutant are at least about 2, 5, 10, 15%, or more, increased in the mutant compared to non-mutant plants. TI LLING uses traditional chemical mutagenesis (e.g. EMS mutagenesis) followed by high-throughput screening for mutations (e.g. using Cel 1 cleavage of mutant-wild type DNA heteroduplexes and detection using a sequencing gel system), see e.g. Hcnikoff et al. Plant Physiology Preview May 21 , 2004. Thus, non-transgenic plants, seeds and tissues comprising an enhanced NRCI gene expression in one or more tissues and comprising one or more of the NRC I phenotypes according to the invention (enhanced disease resistance and/or HR lesions) and methods for generating and identi ying 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, hetcroduplex 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.

In another embodiment of the invention, the plant materials are natural populations of the species or related species that comprise polymorphisms or variations in DNA sequence at the NRCI orthologous coding and/or regulatory sequence. Mutations at the NRCI gene target can be screened for using a ECOTILLING approach (Hcnikoff ct al 2004, supra). In this method natural polymorphisms in breeding lines or related species arc screened for by the above described TILLING methodology, in which individual or pools of plants are used for I'CR amplification of the NRCI target, heterodupicx 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 /W?C/-orthologous allele to develop the cultivar with desired trait.

Mutant plants can be distinguished from non-mutants by molecular methods, such as the mulation(s) present in the DNA, NRC I protein levels, NRCI RNA levels etc, and by the modified phenolypic characteristics.

The non-transgenic mutants may be homozygous or heterozygous for the mutation.

Sequences referred to SEQ ID NO 1 : coding region of the tomato NRCI gene SEQ ID NO 2: amino acid sequence of the tomato NRC I protein SEQ ID NO 3: full length cDNA of the tomato NRCI gene (including 5' and 3 ' UTR) SEQ ID NO 4: amino acid sequence of the tomato NRC 1 D<, ,V protein SEQ ID NO 5: 3'UTR of the tomato NRCI gene Figure legends Figure 1 - Predicted sequence of the NRC I protein The first 150 amino acid residues represent the coiled-coil (CC) domain and residues that are predicted to form the CC structure arc underlined. Residues 151 to 508 comprise the nucleotide-binding (NB-ARC) domain, with the following motifs (underlined and labeled): Kinase 1 A (P loop), RNBS-A, Kinase 2, RNBS-B, RNBS-C, GLPL, RNBS-D and MHD. Residues 509 lo 846 comprise the 13 imperfect leucine-rich repeals (LRRs); the conserved hydrophobic and proline residues arc 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.

Fiaurc 2A - NRC! Is Required for Full GY- -Mcdiatcd HR of Tomato to Cladospori m fitlvinn Cfi) lomalo and Q"- Figure 2B - NRCI Is Required for Full cy- -Mediated Resistance of Tomato to CUulosporium fulvum Non-TRV infected and TRV-infectcd Cf-4 or Cf-0 plants were inoculated with C. /i//vww-pGPD::GUS and two weeks post inoculalion colonization of the Icallets was studied with an X-giuc assay.

Figure 3 - Inoculation of N. benthamiana with TRV:NRC1 affects Cf/Ayr-, LcEix2/tvEix-. Pto/AyrPto-and Rx/CP-induccd HR N. benthamiana was inoculated with TRV:00 (empty vector), TRV:NRC l and TRV:SGT1 . Three weeks later leaves were infiltrated with Agrobacleria expressing HR-inducing proteins and pictures were taken at 4 days post infiltration. First, second and third column: leaves of N. benthainiana expressing the Cf-4 resistance gene agroinfillratcd with Avr4 or a mix of Cf-9 and Avr9, or a mix of LeEix2 and tvEix (combined in a l ; l ratio), respectively. Fourth column: leaves o f" transgenic* N. benthainiana expressing the Pto resistance gene agroinl'iltratcd with A vrPto. Fifth column: leaves of transgenic N. benthamiana expressing the Rx resistance gene agroin filtrated with the gene expressing the coat protein of PVX (CP). The dark circles indicate an HR, light circles indicate a compromised HR.

Figure 4 - Constitutivcly Active NRC I Induces an Elicilor-lndepcndcnt HR and Allows to Position NRCI in a Cell Death Signaling Pathway N. benthamiana expressing the Cf-4 resistance gene was agroinfillratcd with the indicated genes. For panels A and C, three weeks prior to agroinfiltration the plants were inoculated with the indicated TRV constructs. Dark circles indicate an HR, light circles indicate a compromised HR.

(A) Agroinfiltration of genes encoding constitutivcly active MAP J and MAPK. kinases. First column: agroinfiltration with the gene encoding the constitutivcly active kinase domain of LeMAPK Ka ( A K. X- D). Second column: agroinfiltration with the gene encoding a constitutivcly active form of LeME 2 (MEK2DD). Two days post infiltration of MAPKKK-KD or MEK2DD expression was induced by spraying the leaves with estradiol. Pictures were taken four days post agroinflltration.

(B) Agroinflltration of wild-type NRCl (wt) and mutated forms of the gene, under control of the 35S-promoter, either mixed in a 1 : 1 ratio with Agrobaclerium directing expression of the gene encoding silencing suppressor pi 9 (left panel), or alone (right panel). NRCIKI9,R ( 191 R): inactive P-loop mutant of NRCI; NRCID4 IV (D481 V): constitutively active NRCl (mutated in the MHD motif); NRC1K,9,R/D48, y (K191 R/D481 V): double mutant of NRCL Pictures were taken three days post agroinflltration.

(C) Agroinflltration of Avr4 and the gene encoding constitutively active NRC1 48 I V (D481 V). Pictures were taken three days post agroinflltration.

Figure 5 - Model for NRC l Mediated Cell Death Signaling Model based on epistasis experiments combining cell death assays and VIGS in N. benthamiana. Cf-4/Avr4 mediated cell death signals in an EDS1 -, NRC 1-, ME 2-, and SGTl/RARl dependent manner.

It is important to note that those portions of the specification which do not fall within the scope of the claims do not belong to the invention.

The following non-limiting Examples illustrate the different embodiments of the invention. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook el al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel el al, (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

EXAMPLES 1. MATERIAL AND METHODS 1.1 VIGS in N. benthamiana, Agroinflltration, HR and Disease Assays Four-week-old N. benthamiana plants were agroinfiltrated with a 1 : 1 mixture of pTVOO- derived constructs (binary TRV RNA2 vector) and pBintra6 (binary TRV RNA 1 vector) (Ratcliff et al., 2001 Plant J. 25, 237-245), or a 1 : 1 mixture of pTRV- RNA2-dcrivcd constructs and pTRV-RNA l (Liu et a!., 2002, Plant J. 31 , 777-786; Liu et al., 2002, Plant J. 30, 415-429). The following TRV constructs were used: TRV:NRC I , TRV:Cf-4 and TRV:SGT 1 (Peart ct al., 2002, Proc. Natl. Acad. Sci. USA 99, 10865- 10869), all in the TRV vector described by RatclilT et al. (2001 , supra) and TRV:EDS 1 , TRV:MEK2, TRV:RAR 1 and TRV:NDR I (Ekcngrcn et al., 2003, Plant J. 36, 905-917), all in the TRV vector described by Liu et al. (2002, Plant J. 30, 15-429). For each TRV conslrucl, in each experiment four plants were used. A vrPto and CP were agroinfillralcd in TRV-infcctcd N. benthamiana expressing the resistance gene Pto (N. benthamiana: Pto (line 38- 12 (Rommcns ct al., 1995, Plant Cell 7, 1537- 1544)) (Pedley and Martin, 2003, Annu. Rev. Phylopathol. 41 , 215-243) and Rx {N. benthamiana: Rx (line Rx- 18) (Bendahmanc et al„ 1999, Plant Cell I I , 781 -791), respectively. In all other cases Agroinfiltration was performed in N. benthamiana expressing the resistance gene Cf-4 (N. benthamiana: Cf~4). Three weeks post TRV inoculation the ihird, fourth and fifth leaf above the inoculated leaves were challenged with Agrobacterium t mefaciens that directs expression of AvrPto (OD<;oo=0.06) (Tang et al., 1996, Science 274, 2060-2063), CP (OD6o< 0. 12) (Bendahmane ct al., 1999, supra), A vr4 (OD60o=0.03), Cf-9 and Avr9 (mixed in a 1 : 1 ratio, OD60o=0.2) (Van der Hoorn ct al., 2000 Mol. PI ant- Microbe Interact. 13, 439-446), LeEixl and tvEix (mixed in a 1 : 1 ratio, OD^l ) (Ron and Avni, 2004, Plant Cell 16, 1604- 1615), the β-glucuronidase (GUS) gene (OD6Uo=2) (Van dcr Hoorn et al., 2000, Mol. Plant-Microbe Interact. 13, 439-446), NRC1 and pi 9 (mixed in a 1 : 1 ratio, OD60o=l) (Voinnet et al., 2003, Plant J. 33, 949-956), the constitutivcly active NRCID4S, V or the inactive mclKi9iiW4iii v double rnutanl iODft00=2), MAPKKK KD , LeMAPKKK KI (Del Pozo ct al., 2004, EMBO J. 23, 3072-3082) (both at OD600=0.12) or LeMEK2DD and LeMEK2 (Del Pozo et al., 2004, supra) (both at OD60o=0.25). Two days post infiltration of LeMAPKKKo.KD, LeMAPKKKv!™ LeMEK2DD or LeMEK2 the leaves were sprayed with a 7.5 μΜ solution of 17-P-estradiol in water, containing silwet (4 μΙ/100 ml) (Del Pozo ct al., 2004, supra). For protein injections, Avr4-HlS-FLAG-tagged protein was treated with enterokinase E -max according to the manufacturer's recommendations (Invilrogen, Breda, NL) and 5 μΜ Avr4 protein in water, supplemented with 0.2% tween (v/v) was used for injections. Three to live days post agroinfiltration or protein injection the leaves were examined for the development of an HR, or assayed for β-glucuronidase (GUS) activity. 1 ,2 VIGS in tomato. HR and Disease Assays For V IGS in tomato the pTRV-R A I and pTRV-RNA2 vectors described by Liu cl a!. (2002, Plant J. 30, 415-429) were used. The Cf-4 and NRCI fragments were excised from pTVOO by digestion with Bam\-\ \!Aspl \ % and inserted into BamH \/Asp7 \ 8-digcslcd pTRV-R A2 (pYL 156) (Liu el al., 2002, Plant J. 31 , 777-786). To construct TRV:222-UTR, pari of the 3'-UTR of NRCI was amplified using primers 222-3 'UTR-F (5 '-GTGGATCCGC AGGTTCA ACCAGCCTGGT-3 ' ; BamH\ site underlined) and 222-3 ' UTR-R (5'-GTGΌTACCCAAGTGACTTGTTCΊ·GCTGT-3,; Aspm site underlined) and to construct TRV:222-LRR, part of the NRCI region coding the LRRs was amplified using primers 222-LRR-F (5'-GTGGATCCCi ITAAGAGGCTGCAATTTCT-3'; B mYW site underlined) and 222-LRR-R (5'-GTGGTACCGATCTTCTCAAGTTTATCAC-3 '; Aspl\ site underlined). The PCR fragments were Z?«mH l/vi.5/j718-digcsted and inserted into BamW MAspl 18 -digested pTRV-R A2. TRV:Prf construction has been described (Ekcngren ct al., 2003, Plant J. 36, 905-917). All plasmids were transformed to A. tumefaciens strain GV3 I 0J by clcctroporation (Takken el al., 2000, Plant J. 24, 275-283). To establish VIGS in tomato, cotyledons of len- lo twelvc-day-old tomato seedlings were agroinfiltratcd with a mixture of pTRV-R A I and the pTRV-RMA2-derived constructs (combined in a 1 :1 ratio) (Liu et al., 2002, supra). For each TRV construct either four C - The fragment was NcoMPsti -digested, inserted into pRH80 and the 35S-NRCID''S'V-tNOS fragment was excised and subsequently inserted into pMOG800 as described above. In a similar way the P-loop mutant NRC!KI9IR, and the inactive double mutant NRC}K, !R D''S'V were created. Here, the K 1 1 R mutation was introduced using mismatch primers 222Ploop-F (5'-GGAATGCCTGGTCTTGGCAGAACTACACTAGC-3 ') and 222Ploop-R (5 '- GCTAG I GTAGTTCTGCCAAGACCAGGCATTCC-3') (mutation underlined) with respectively p!asmid NRCl(wt) and NRCID4Si y s a lemplatc. All constructs were sequence- verified and transformed io A. t mefaciens strain GV3101. 1.4 DNA Gel-Blot Analysis Genomic DNA from N, benthainiana 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 cd. (Cold Spring Harbor, NY, U.S.A.: Cold Spring Harbor Laboratory Press) was used. The DNA was digested with Bam\ \ \, 1tind\ \ \, EcoR\, EcoRV or Xl)a\. The N. benthainiana gel-blot was hybridized with the 32P-labclcd (Prime-a-genc Labeling System, Promcga, Madison, Wl) 252 bp fragment present in the TRVrNRC i vector and the DNA gel-blot of tomato was hybridized with a 12P-labelcd probe of 1293 bases corresponding to nucleotides 1876 to 3168 of the full length NRC 1 cDNA. Sites for the restriction enzymes used arc 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. 1 .5 RT-PCRs to Show Silcncinu of NRC1 in Tomato Four leaf discs (approximately 100 mg of tissue in total) were collected from the second, third or fourth compound leaf of TRV-infectcd plants. Total RNA was extracted using the QIA-Gen RNAeasy extraction protocol (Qiagen, Venlo, NL) and treated with RNase-Frcc DNasc (Bio-Rad, Vccncndaal, NL). First strand cDNA was synthesized from 1 μ of total RNA using the Bio-Rad cDNA synthesis kit {Bio-Rad, Vcencndaal, NL) and RT-PCR was performed using the following cycles: 95°C for 1 5 sec, 60°C for 45 sec and 72°C for 60 sec. The primers that were used (222F: 5'- TGAGGTATATTGCTTTCTCATCTGAC-3' and 222R: 5 '-AGCTATTTTCCCACGGATGCCCAG-3 ') do not cover the fragment which is inserted in TRV:NRC 1 . Actin primers (ActinFnrl 82: 5'-TATGGAAACATTGTGCTCAGTGG-3 ' and ActinRnrl 3: 5'-CCAGATTCGTCATACTCTGCC-3') were used to check for the presence of equal amounts ol' cDNA in the PCR reactions.

Example 2 - Results 2. 1 Tomato NRC I : 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-m' The predicted primary structure of the NRC I protein (SEQ ID NO: 2) typically resembles that of CC-NB-LRR resistance proteins (Figure 1 ). NRCI has an amino-terminal coilcd-coil (CC) domain, an NB-ARC (Nucleotide Binding adapter shared by Apaf- 1 , R proteins and CED4) domain (Van der Biczen 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). As indicated in Figure 1 , comparison with homologous NB-ARC domains revealed the presence of a inase l A or P-loop motif, four RNBS (Rcsislancc Nucleotide Binding Site) motifs and a GLPL and MHD motif (Meyers et al., 1999, Plant J. 20, 317-332; Meyers et al., 2003, Plant Cell 15, 809-834).

The 252 bp cDNA-AFLP fragment present in the TRV:NRC t vector used for VIGS codes for amino acids 599-681 , which are located in LRRs four to seven.

Low stringency DNA gel-blot analysis of genomic DNA of tomato digested with the BamHI-, HindHI-, EcoRI-, EcoRV- and Xbal, was hybridized with a 1293 bp NRC I cDNA fragment (nucleotides 1876 to 3 168 of SEQ ID NO: 3) covering the NRCI sequence present in the TRV:NRC I -, TRV:NRC I -LRR- and TRV:NRC 1 -UTR constructs (sec below) as a probe. This Southern blot revealed only one prominent band afler a high stringency wash, which indicates that NRCI is a single-copy gene in tomato.

A gel blot of BamH l-, Hindi! 1-, EcoRI-, EcoRV- and Xbal-digcstcd genomic DNA of JV. bent arniana was probed with the tomato NRCI cDNA-AFLP fragment present in the TRV vector and two-three hybridizing bands were found (results not shown) (0.5 x SSC, 0.5% SDS, 65°C). This suggests that there are at least two to three NRCI orthologs present in the genome of N. be tharniana that can be silenced upon inoculation with TRV:NRC 1. 2.2 NRC I -Silenced Tomato Is Affected in Cf-4-Mediated HR and Disease Resistance To investigate the function of NRC I in HR-signaling and resistance to C. f lv tn, the inventors performed VIGS in tomato, since this plant is the only host for this fungus. Ten-day-old tomato seedlings were agroin 11 lira ted with TRV:NRC 1 and three weeks post infiltration RNA was isolated from potentially silenced leaflets and analyzed by RT-PCR. The NRC I transcript levels varied in different TRV:NRC I -infected plants, but in most cases they were lower than in the TRV:00-infected plants, indicating that 'knock-down' of NRCI expression had occurred (data not shown).

To exclude the possibility that the phenotype that we observe in tomato is caused by silencing of additional NB-LRR proteins, we also performed VIGS in tomato using a 360 bp fragment of NRCI targeted to LRRs eight to twelve (TRV:NRC 1 -LRR), and a fragment consisting of 297 bp of the 3'-untranslated region (UTR) of NRCI (TRV:NRC1 -U TR). With these constructs we tested whether NRC I is required for Cf-4-mcdiatcd HR in tomato by Avr4 protein injections in TRV:222-LRR and TRV:222-UTR-infectcd, Cf-4-conlaining tomato plants. Silencing of NRCI (using each of the three constructs) results in a mild phenotype as the tomato plants appeared somewhat smaller than the TRV:00- or TRV:Cf-4-infectcd plants (data not shown). As controls Avr4 protein was injected in TRV:00- and TRV:Cf-4-infectcd plants. In TRV:Cf-4-infectcd plants the percentage of responding Avr4-injectcd sites was 52% (Figure 2), indicating a decreased HR due to silencing of Cf-4. In TRV:222-LRR and TRV:222-UTR-infected plants this percentage was similar (56% and 48%, respectively) (Figure 2), confirming the function of NRC I in Cf-4/Avr4-induccd HR, also in tomato. Similar results were obtained upon VIGS of Nrcl in C/-9-containing tomato and subsequent injections of apoplastic fluid containing Avr9 (not shown). For 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 el al., 2001 , supra).

Further, it was invest igated whether NRC I is also required for full resistance of tomato to C. fulvum. CfO and C/-4-plants were inoculated with T V:00, TRV:Cf-4 and TRV:NRC 1 and after three weeks silenced plants were inoculated with a strain of C. fulvum expressing Avr4 and the β-glucuronidasc (GUS) gene, thereby allowing visualization of fungal growth. Two weeks post C. fulvum inoculation leaves were stained with X-gluc. In leaflets of Cf-4 plants infected with TRV:00 no growth of C. fulvum was detected, whereas in TRV:Cf-4-infectcd Cf-4 plants patches of blue staining indicate compromised C/-4-mediatcd resistance (not shown). Also in TRV:NRC 1 -infected plants small patches of blue staining indicate toss of ful l resistance against the fungus. Microscopical analysis revealed intercellular growth of fungal hyphae in TRV:Cf-4- and TRV:NRC 1 -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 ilsclf, nor VIGS using TRV:NRC 1 affects the susceptibility of these plants to the fungus. 2,3 VIGS of NRC I Affects the HR Induced by Different Matching R Gene/Ayr Gene Combinations In addition to a decreased Cf-4/Avr4-induced HR upon VIGS using NRC I , it was found that also the HR induced by the Infl clicilor of the oomycete pathogen Phytophthora infestans is decreased upon VIGS using NRCI in N. henthamiana. To further investigate the specificity of NRC I in defense signaling, the inventors tested its requirement for the HR induced by additional R/Avr combinations. As controls TRV:00 (empty vector) and TRV:SGTI were included, since SGT 1 is known to be required for the HR induced by several R Avr combinations (Peart el al., 2002, Proc. Natl. Acad. Sci. USA 99, 10865- 10869).

Agroinfiltration of a mix o f Cf-9 and Avr9 (Van der Hoorn ct al., 2000, supra), or a mix ot LeEix2 and tvEix (Ron and Avni, 2004, Plant Cell 16, 1604- 161 ) in TRV:NRC1 -infectcd N. henthamiana resulted in a decreased HR, whereas in the TRVrOO-infcctcd plants the HR developed normally. In TRV:SGT I -infected plants the HR was completely abolished, confirming the observations of Peart et al. (2002, supra) (Figure 3). Also AvrPto from the bacterial pathogen Pseudomonas syringae pv tomato and the gene encoding the coat protein (CP) of potato virus X (PVX) were agro in filtrated in TRV-infccted N. benthamiana expressing the resistance gene Pto (Pedlcy and Martin, 2003, Annu. Rev. Phytopalhol. 41 , 21 5-243) and Rx (Bcndahmanc cl al., 1 99, Plant Cell 1 1 , 781 -7 1 ), respectively. In both cases plants infected with TRV:00 showed an HR, while the HR was abolished in TRV:SGT1 -infected plants. TRV:NRC I -infection resulted in a severely suppressed Pto/AvrPto- as well as Rx/CP-' duccd HR, indicating that in N. benthamiana an NRC1 protein is required for HR signaling activated by several R/A vr gene-for-gene combinations (Figure 3).

To exclude the possibility that the compromised HR in TRV:NRC 1 -infected N. benthamiana results from a decreased transformation efficiency by Agrobacterium, the inventors infiltrated TRV:00- and TRV:NRC I -infected N. benthamiana :Cf-4 with Agrobacterium expressing the β-glucuronidase (GUS) gene (Van dcr Hoorn el al., 2000, supra). Three days post infiltration a similar intensity of the blue staining in TRV:00- and TRV:NRC 1 -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. 2.4 NRC 1 Acts Downstream of EDS 1 and Upstream of the MAPK Cascade in a Cell Death Signaling Pathway Since NRC 1 is required not only for C/- To investigate the requirement of NRC I for the HR initiated by MAPKs, cpislasis experiments in N, benthamiana were performed. Plants were inoculated with TRV:00, TRV:SGT 1 and TRV:NRC 1 and subsequently agroin filtrated with genes encoding the kinase domain o LeMAPKKKa (L eMA PKKKaK0) or constitulivcly active LcMEK2 (LeMEK2DD) (Yang cl al., 2001 , Proc. Natl. Acad. Sci. USA 98, 741 -746; Del Pozo cl al., 2004, EMBO J. 23, 3072-3082.). Two days post agroinfillration expression of the genes was induced by spraying the infiltrated leaves with estradiol. Transient expression of each of the genes results in an HR in TRV:00-infccted plants, whereas in TRV:SGT I -infected plants the HR is decreased (Figure 4A). In TRV:NRC l -infccled plants the HR caused by both conslitutively active kinases is not affected (Figure 4Λ). Agroinfiltration of the corresponding negative controls, LeMA PK KKaKD ' and wild-type LeMEK2 did not result in an HR in any of the TRV-infccted plants (data not shown). These results indicate that SGT l is functional downstream of these MAPKs, whereas the MAPKs act cither downstream or independent of NRC 1 . 2.5 Transient overexprcssion oiNRCi and construction of a constitutivcly active NRC l protein To further investigate which genes arc required for HR signaling by the CC-NB-LRR protein the effect of overexpression of NRCl was investigated. Therefore, the coding sequence (SEQ ID NO: 3 ) of the cD A was fused to the constitutive 35S promoter and inserted into a binary vector, Agroinfiltration of this construct in N. benth miana did not result in an HR, whereas expression of a mix of NRC l and the p 1 silencing inhibitor (Voinnet et al„ 2003, Plant J. 33, 949-956) did provoke an elicitor-independent HR (Figure 4B). Agroinfiltration o f a construct encoding a P-loop mutant of NRC l (K I 91 R) disrupting the P-toop motif, thereby affecting ATP hydrolysis (Tameling et al., 2002, Plant Cell 14, 2929-2939), either with or without p i 9, did not result in an HR (Figure 4B).

The above described data indicated that post transcriptional gene silencing (PTGS) of the NRCl gene may, therefore, prevent the development of an HR in NRCl overcxprcssing tissue. Also, the disruption of the P-loop motif results in a nonfunctional NRCl protein.

Since mutations in the MHD motif of the NB-LRR resistance proteins Rx (D460V) (Bendahmane et al., 2002; Tameling et al., 2002) and 1-2 (D495V) (Bendahmanc et al., 2002, Plant J. 32, 195-204; Tameling et al., 2002, Plant Cell 14, 2929-2939; Van Bcnlem et al., 2005, Plant J. 43, 284-298) result in constitutive activity, the inventors generated a similar mutant of NRCl (NRC 1 W81V). Indeed, agroinfiltration o f N RC ] P4s iv rcsuUcd in an elicitor-indcpendenl HR in leaves of N. benthamiana within three days post infiltration and again no HR was observed upon agroinfiltration of the double mutant NRC 1 KISM R EMS I V (Figure 4B). Furthermore, no HR was induced upon expression of NRC I D4 I V in SGT l -silenced plants (see below). These results indicate that the response induced upon agroinfiltration of NRC 1 D<""V is specifically due to constitutive activity of the NRC 1 protein and that NRC 1 functions in a signal transduction cascade leading to H . 2.6 Epistatis experiments using a constttutively active NCR! protein Epistasis experiments employing NRC1 ws> v 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. In addition to VIGS of genes known to be generally involved in HR signaling, such as SGTl and RARI (Required for Mlal2 resistance) (Shirasu and Schulzc-Lcfert, 2003, Trends Plant Sci. 8, 252-258), N. benthamiana:Cf-4 was silenced for NDR! (non race-specific disease resistance) (Century et al., 1995, Proc. Natl. Acad. Sci. USA 92, 6597-6601), EDS l (enhanced disease susceptibility) (Aarts et al., 1998, Proc. Natl. Acad. Sci. USA 95, 10306- 103 1 1) and MEK2 (a MAP K) (Ekengren ct al., 2003, Plant J. 36, 905-917), and subsequently agroinfiltrated with NRC 1 MSI V or Avr4. Furthermore, VIGS using TRV:00, TRV:Cf-4 and TRV:NRC1 was included as controls. A compromised NRC 1 D4K I V- or Avr4-induced HR indicates 'knock-down' of a gene required for respectively NRC 1- or Cf-4/Avr4- induced HR signaling.

As expected, HR induced upon agroinfiltration of Avr4 was compromised in TRV;Cf-4- and TRV:NRC I -infected plants. Cf-4-mediated signaling also requires EDS l , as plants silenced for this gene displayed a less severe Avr4-induced HR. In addition, the inventors found a reduced HR upon agroinfiltration of Avr4 in plants silenced for EK2, RAR I and SGTl (Figure 4C; light circles). The Avr4-induccd HR is not compromised in TRV:00- and TRV:NDR 1 -infected plants (Figure 4C; dark circles), indicating that NDRI is not required for Cf-4-mcdialcd signaling. Similarly, NRC l D481v-induccd HR was not compromised in TRV:00- and TRV:NDR1 -infected plants, and also not in TRV:Cf-4-infeclcd plants. Interestingly, in contrast to Avr4, N RC ] D4B ] v sli|| induces an H R in TRV: EDS I -infected plants, indicating that NRC I is functional downstream of EDS l (Figure 4C; dark circles). The NRC l l !81v-induced HR is compromised in plants silenced for MEK2, showing that NRC l requires the MAP kinase cascade for its signaling and can be positioned upstream of these kinases. VIOS of RAR l and SGTl also compromises D48 I V-induccd HR, similar to the HR induced by Avr4 (Figure 4C; light circles). Thus, NRC l is required for HR signaling initiated by Cf-4 and can be positioned upstream of the MAPK cascade and downstream of EDS I .

Sec Figure 5 for a model of NRC l mediated cell signaling.

Example 3 - NRC l requirement for M/-mediated resistance In order to determine whether NRC l is required for Λί/'-mcdiated resistance against nematodes, white fly and aphids, a constitutivcly active form of Mi (sec US 6613962 and EP0937 I 55B 1 ) 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)cxprcssion of NRCl can be used to generate transgenic plants having enhanced resistance against nematodes, white fly and aphids.

SEQUENCE LISTING <110> Kevaene NV Wageningen University <120> Disease Resistant Plants <130> 44862 <160> 5 <170> Patentln version 3 . 3 <210> 1 <211> 2664 <212> DNA <213> unknown <220> <223> Tomato nrcl open reading frame <400> 1 atggttgatg taggggttga atttctgtta gagaacttga agcaattggt actggacaat 60 gtggagttaa tcggaggagc taaagatgaa atcgagaatc tgcgtgatga tttgagtgaa 120 ttcaatgcct ttctcaagca agctgcaatg gtccgcagcg aaaacccagt tctcaaagaa 180 ctagtgagga gtatcagaaa agtggtgaat cgtgctgaag atgctgttga taaatttgta 240 attgaagcta aagttcataa agacaaaggg tttaaagggg ttttcgataa acctggacat 300 tatagaagag tgagggatgc agctgtggag attaaaggta tcagagataa aatgagagaa 360 attcggcaaa ataaggcaca tggccttcag gctctacttc aagatcatga tgattcaatc 420 agcagaggtg gagaagagag acagcctcct gtggttgagg aagatgatgt ggtgggcttt 480 gacgatgagg cgcagacggt aatcgaccgt cttcttgaag gatcaggtga tttagaggtt 540 attccagtag ttggaatgcc tggtcttggc aaaactacac tagccactaa gatcttcaag 600 catccgaaga ttgagtacga gttctttact agactttggc tttacgtttc ccaatcatac 660 aagacaagag aattatatct taacatcatc agtaaattca ccggaaacac caaacattgc 720 cgtgatatgt ctgaaaagga tttagctctt aaggtacaag agattttgga agaaggagga 780 aaatacttga ttgtcttgga tgatgtctgg tcgacagatg cttgggatcg tatcaagatt 840 gctttcccga aaaatgacaa gggcaataga gtattgttga ctactcgaga ccaccgtgtt 900 gcaagatatt gcaataggag tccacatgat ttaaaatttc tgactgatga agagagttgg 960 attttactgg agaaaagagc ttttcacaaa gctaaatgtc tccccgaatt ggaaacaaac 1020 ggaaaaagca tagccaggaa gtgtaaagga ctaccccttg ctattgtggt gattgcagga 1080 gctctaattg ggaaaagcaa aacaataaag gaatgggagc aagtggatca gagtgtgggc 1140 gaacatttca taaatagaga tcagccaaat agttgtgata aattggtacg gatgagttat 1200 gatgttttgc cttatgactg gaaagcttgc tttttatact tcggtacatt ccccagaggc 1260 tatttaatcc ctgccaggaa attgatccgc ttatggatcg cggaagggtt tatccagtac 1320 agaggggact tatcccctga gtgtaaagca gaggagtact tgaatgaact cgtaaataga 1380 aacttagtga tggtaatgca aaggacggtt gatggacaaa tcaaaacttg tcgtgttcat 1440 gacatgttgt atgagttttg ctggcaagag gctacgacag aggaaaatct tttccatgaa 1500 gtaaaattcg gtggtgagca atctgttcgt gaagtatcca ctcatcgtcg cttgtgcatt 1560 cattcctctg ttgtggagtt catttctaag aagccctctg gtgagcatgt taggtcgttc 1620 ctatgttttt ctccagaaaa aattgacact cccccaactg tcagtgcaaa catatcaaaa 1680 gcctttccat tgctaagggt gtttgatact gaatccatca aaatcaatcg cttttgcaag 1740 gagttctttc aattgtatca tctgaggtat attgctttct catttgactc gattaaagtc 1800 attccgaaac atgttgggga actttggaac gtacaaaccc tcattgtcaa cacacaacag 1860 atcaaccttg atattcaagc agacatattg aacatgcccc ggctgaggca tctgctcacc 1920 aacacgtctg ctaaattgcc tgcgcttgct aaccccaaaa caagtaagac taccttggta 1980 aatcaaagcc tgcaaaccct ctccacaatt gcaccagaaa gctgcactga gtatgttctc 2040 tcgagggctc caaacttgaa aaaactgggc attcgtggaa aaatagctaa gctaatggaa 2100 ccaagtcagt ctgtattgtt gaacaatgtt aagaggctgc aatttcttga gaacttgaag 2160 ctgataaatg ttggtcagat tgatcagaca caattacgcc ttcctccagc atctatattt 2220 ccaacaaagt tgaggaagct gactttatta gatacctggt tggagtggga tgatatgtct 2280 gtattgaaac agctggagaa ccttcaagtc ttgaagctga aggacaatgc atttaaggga 2340 gagaactggg aactaaatga tggaggtttt cctttcctac aagtgttatg cattgaaagg 2400 gcaaacttag tttcttggaa tgcttcaggt gatcacttcc cgagacttaa acatcttcac 2460 atatcatgtg ataaacttga gaagatcccc attggcctgg ctgatatatg cagcctccaa 2520 gtgatggatt tgcgaaattc cactaaatca gcagcaaaat ctgccagaga gatacaagcc 2580 aaaaaaaaca agctgcaacc tgctaaatcc cagaagttcg agctttctgt attccctcct 2640 gattctgatg tacagacagc ttct 2664 <210> 2 <211> 888 <212> PRT <213> unknown <220> <223> Tomato NRCl wild type protein <400> 2 Met val Asp val Gly val Glu Phe Leu Leu Glu Asn Leu Lys Gin Leu val Leu Asp Asn val Glu Leu lie Gly Gly Ala Lys Asp Glu lie Glu 25 30 Asn Leu Arg Asp Asp Leu Ser Glu Phe Asn Ala Phe Leu Lys Gin Ala 40 45 Ala Met val Arg Ser Glu Asn Pro val Leu Lys Glu Leu val Arq ser 50 55 60 lie Arg Lys val Val Asn Arg Ala Glu Asp Ala val Asp Lys Phe val 65 70 75 80 lie Glu Ala Lys Val His Lys Asp Lys Gly Phe Lys Gly Val Phe Asp Lys Pro Gly His Tyr Arg Arg val Arg Asp Ala Ala Val Glu lie Lys 100 105 110 Gly lie Arg Asp Lys Met Arg Glu lie Arg Gin Asn Lys Ala His Gly 115 120 125 Leu Gin Ala Leu Leu Gin Asp His Asp Asp Ser lie ser Arg Gly Gly 130 135 140 Glu Glu Arg Gin Pro Pro val val Glu Glu Asp Asp val val Gly Phe 145 150 155 160 Asp Asp Glu Ala Gin Thr val lie Asp Arg Leu Leu Glu Gly Ser Gly 165 170 175 Asp Leu Glu val lie Pro val val Gly Met Pro Gly Leu Gly Lys Thr 180 185 190 Thr Leu Ala Thr Lys lie Phe Lys His Pro Lys lie Glu Tyr Glu Phe 195 200 205 Phe Thr Arg Leu Trp Leu Tyr Val Ser Gin Ser Tyr Lys Thr Arg Glu 210 215 220 Leu Tyr Leu Asn lie lie Ser Lys Phe Thr Gly Asn Thr Lys His Cys 225 230 235 240 Arg Asp Met Ser Glu Lys Asp Leu Ala Leu Lys Val Gin Glu lie Leu 245 250 255 Glu Glu Gly Gly Lys Tyr Leu lie val Leu Asp Asp val Trp Ser Thr 260 265 270 Asp Ala Trp Asp Arg lie Lys lie Ala Phe Pro Lys Asn Asp Lys Gly 275 280 285 Asn Arg Val Leu Leu Thr Thr Arg Asp His Arg val Ala Arg Tyr Cys 290 295 300 Asn Arg Ser Pro His Asp Leu Lys Phe Leu Thr Asp Glu Glu Ser Trp 305 310 315 320 lie Leu Leu Glu Lys Arg Ala Phe His Lys Ala Lys Cys Leu Pro Glu 325 330 335 Leu Glu Thr Asn Gly Lys ser lie Ala Arg Lys Cys Lys Gly Leu Pro 340 345 350 Leu Ala He Val Val lie Ala Gly Ala Leu lie Gly Lys Ser Lys Thr 355 360 365 lie Lys Glu Trp Glu Gin Val Asp G n Ser Val Gly Glu His Phe lie 370 375 380 Asn Arg Asp Gin Pro Asn Ser Cys Asp Lys Leu Val Arg Met Ser Tyr 385 390 395 400 Asp Val Leu Pro Tyr Asp Trp Lys Ala Cys Phe Leu Tyr Phe Gly Thr 405 410 415 Phe Pro Arg Gly Tyr Leu lie Pro Ala Arg Lys Leu lie Arg Leu Trp 420 425 430 lie Ala Glu Gly Phe lie Gin Tyr Arg Gly Asp Leu Ser Pro Glu Cys 435 440 445 Lys Ala Glu Glu Tyr Leu Asn Glu Leu val Asn Arg Asn Leu val Met 450 455 460 Val Met Gin Arg Thr Val Asp Gly Gin lie Lys Thr cys Arg val His 465 470 475 480 Asp Met Leu Tyr Glu Phe Cys Trp Gin Glu Ala Thr Thr Glu Glu Asn 485 490 495 Leu Phe His Glu Val Lys Phe Gly Gly Glu Gin Ser Val Arg Glu Val 500 505 510 Ser Thr His Arg Arg Leu Cys lie His Ser ser val val Glu Phe lie 515 520 525 Ser Lys Lys Pro Ser Gly Glu His val Arg ser Phe Leu cys Phe ser 530 535 540 Pro Glu Lys lie Asp Thr Pro Pro Thr val Ser Ala Asn lie Ser Lys 545 550 555 560 Ala Phe Pro Leu Leu Arg val Phe Asp Thr Glu Ser lie Lys lie Asn 565 570 575 Arg Phe Cys Lys Glu Phe Phe Gin Leu Tyr His Leu Arg Tyr lie Ala 580 585 590 he ser Phe Asp ser lie Lys val lie Pro Lys His Val Gly Glu Leu 595 600 605 Trp Asn val Gin Thr Leu lie val Asn Thr Gin Gin lie Asn Leu Asp 610 615 620 lie Gin Ala Asp lie Leu Asn Met Pro Arg Leu Arg His Leu Leu Thr 625 630 635 640 Asn Thr Ser Ala Lys Leu Pro Ala Leu Ala Asn Pro Lys Thr Ser Lys 645 650 655 Thr Thr Leu val Asn Gin Ser Leu Gin Thr Leu Ser Thr lie Ala Pro 660 665 670 Glu Ser cys Thr Glu Tyr al Leu Ser Arg Ala Pro Asn Leu Lys Lys 675 680 685 Leu Gly lie Arg Gly Lys lie Ala Lys Leu Met Glu Pro Ser Gin Ser 690 695 700 val Leu Leu Asn Asn Val Lys Arg Leu Gin Phe Leu Glu Asn Leu Lys 705 710 715 720 Leu lie Asn val Gly Gin lie Asp Gin Thr Gin Leu Arg Leu Pro Pro 725 730 735 Ala ser lie Phe Pro Thr Lys Leu Arg Lys Leu Thr Leu Leu Asp Thr 740 745 750 Trp Leu Glu Trp Asp Asp Met Ser val Leu Lys Gin Leu Glu Asn Leu 755 760 765 Gin val Leu Lys Leu Lys Asp Asn Ala Phe Lys Gly Glu Asn Trp Glu 770 775 780 Leu Asn Asp Gly Gly Phe Pro Phe Leu Gin val Leu Cys lie Glu Arg 785 790 795 800 Ala Asn Leu val ser Trp Asn Ala Ser Gly Asp His Phe Pro Arg Leu 805 810 815 Lys His Leu His lie ser cys Asp Lys Leu Glu Lys H e Pro lie Gly 820 825 830 Leu Ala Asp lie Cys ser Leu Gin val Met Asp Leu Arg Asn Ser Thr 835 840 845 Lys ser Ala Ala Lys Ser Ala Arg Glu lie Gin Ala Lys Lys Asn Lys 850 855 860 Leu Gin Pro Ala Lys Ser Gin Lys Phe Glu Leu ser val Phe Pro Pro 865 870 875 880 Asp Ser Asp Val Gin Thr Ala Ser 885 <210> 3 <211> 3168 <212> DNA <213> unknown <220> <223> Tomato nrcl full length CDNA <400> 3 gaattcggca cgaggcttct tctgagcata attctcttct tctccaagaa atcaatcgaa 60 aaaaaaaaaa aaaggaaaac aatatggttg atgtaggggt tgaatttctg ttagagaact 120 tgaagcaatt ggtactggac aatgtggagt taatcggagg agctaaagat gaaatcgaga 180 atctgcgtga tgatttgagt gaattcaatg cctttctcaa gcaagctgca atggtccgca 240 gcgaaaaccc agttctcaaa gaactagtga ggagtatcag aaaagtggtg aatcgtgctg 300 aagatgctgt tgataaattt gtaattgaag ctaaagttca taaagacaaa gggtttaaag 360 gggttttcga taaacctgga cattatagaa gagtgaggga tgcagctgtg gagattaaag 420 gtatcagaga taaaatgaga gaaattcggc aaaataaggc acatggcctt caggctctac 480 ttcaagatca tgatgattca atcagcagag gtggagaaga gagacagcct cctgtggttg 540 aggaagatga tgtggtgggc tttgacgatg aggcgcagac ggtaatcgac cgtcttcttg 600 aaggatcagg tgatttagag gttattccag tagttggaat gcctggtctt ggcaaaacta 660 cactagccac taagatcttc aagcatccga agattgagta cgagttcttt actagacttt 720 ggctttacgt ttcccaatca tacaagacaa gagaattata tcttaacatc atcagtaaat 780 tcaccggaaa caccaaacat tgccgtgata tgtctgaaaa ggatttagct cttaaggtac 840 aagagatttt ggaagaagga ggaaaatact tgattgtctt ggatgatgtc tggtcgacag 900 atgcttggga tcgtatcaag attgctttcc cgaaaaatga caagggcaat agagtattgt 960 tgactactcg agaccaccgt gttgcaagat attgcaatag gagtccacat gatttaaaat 1020 ttctgactga tgaagagagt tggattttac tggagaaaag agcttttcac aaagctaaat 1080 gtctccccga attggaaaca aacggaaaaa gcatagccag gaagtgtaaa ggactacccc 1140 ttgctattgt ggtgattgca ggagctctaa ttgggaaaag caaaacaata aaggaatggg 1200 agcaagtgga tcagagtgtg ggcgaacatt tcataaatag agatcagcca aatagttgtg 1260 ataaattggt acggatgagt tatgatgttt tgccttatga ctggaaagct tgctttttat 1320 acttcggtac attccccaga ggctatttaa tccctgccag gaaattgatc cgcttatgga 1380 tcgcggaagg gtttatccag tacagagggg acttatcccc tgagtgtaaa gcagaggagt 1440 acttgaatga actcgtaaat agaaacttag tgatggtaat gcaaaggacg gttgatggac 1500 aaatcaaaac ttgtcgtgtt catgacatgt tgtatgagtt ttgctggcaa gaggctacga 1560 cagaggaaaa tcttttccat gaagtaaaat tcggtggtga gcaatctgtt cgtgaagtat 1620 ccactcatcg tcgcttgtgc attcattcct ctgttgtgga gttcatttct aagaagccct 1680 ctggtgagca tgttaggtcg ttcctatgtt tttctccaga aaaaattgac actcccccaa 1740 ctgtcagtgc aaacatatca aaagcctttc cattgctaag ggtgtttgat actgaatcca 1800 tcaaaatcaa tcgcttttgc aaggagttct ttcaattgta tcatctgagg tatattgctt 1860 tctcatttga ctcgattaaa gtcattccga aacatgttgg ggaactttgg aacgtacaaa 1920 ccctcattgt caacacacaa cagatcaacc ttgatattca agcagacata ttgaacatgc 1980 cccggctgag gcatctgctc accaacacgt ctgctaaatt gcctgcgctt gctaacccca 2040 aaacaagtaa gactaccttg gtaaatcaaa gcctgcaaac cctctccaca attgcaccag 2100 aaagctgcac tgagtatgtt ctctcgaggg ctccaaactt gaaaaaactg ggcattcgtg 2160 gaaaaatagc taagctaatg gaaccaagtc agtctgtatt gttgaacaat gttaagaggc 2220 tgcaatttct tgagaacttg aagctgataa atgttggtca gattgatcag acacaattac 2280 gccttcctcc agcatctata tttccaacaa agttgaggaa gctgacttta ttagatacct 2340 ggttggagtg ggatgatatg tctgtattga aacagctgga gaaccttcaa gtcttgaagc 2400 tgaaggacaa tgcatttaag ggagagaact gggaactaaa tgatggaggt tttcctttcc 2460 tacaagtgtt atgcattgaa agggcaaact tagtttcttg gaatgcttca ggtgatcact 2520 tcccgagact taaacatctt cacatatcat gtgataaact tgagaagatc cccattggcc 2580 tggctgatat atgcagcctc caagtgatgg atttgcgaaa ttccactaaa tcagcagcaa 2640 aatctgccag agagatacaa gccaaaaaaa acaagctgca acctgctaaa tcccagaagt 2700 tcgagctttc tgtattccct cctgattctg atgtacagac agcttcttag aaaggtctaa 2760 aataaccaca tgctgcaggt tcaaccagcc tggttggcgc gcatggtttt tgcttcattt 2820 ggatcactgc tttcacagtg aaacctattg catttcataa gtggaacgac tgatccacgg 2880 ttttgcaact ctttggtttc ttactgtatt tgatgtgagt tcatgtttta ttgattgtgg 2940 atgcaatgtg tcatcatagc caaggaataa gaaccaaatg taacgttcaa gaaatatcag 3000 acattgtttc ttataagtta taccactctg aattttctcc ttttagatac aacagcagaa 3060 caagtcactt gttttgattc atacacaatt tgatcatgtg ttattattta aacagcactt 3120 ttggtaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aactcgag 3168 <210> 4 <211> 888 <212> PRT <213> unknown <220> <223> constitutive NRCl protein (D481V mutant) <400> 4 Met val Asp val Gly val Glu Phe Leu Leu Glu Asn Leu Lys Gin Leu 1 5 10 15 Val Leu Asp Asn val Glu Leu lie Gly Gly Ala Lys Asp Glu lie Glu 25 30 Asn Leu Arg Asp Asp Leu ser Glu Phe Asn Ala Phe Leu Lys Gin Ala 40 45 Ala Met al Arg Ser Glu Asn Pro val Leu Lys Glu Leu val Arg Ser 50 55 60 lie Arg Lys val val Asn Arg Ala Glu Asp Ala val Asp Lys Phe val 65 70 75 80 lie Glu Ala Lys val His Lys Asp Lys Gly Phe Lys Gly val Phe Asp 85 90 95 Lys Pro Gly His Tyr Arg Arg val Arg Asp Ala Ala val Glu lie Lys 100 105 110 Gly lie Arg Asp Lys Met Arg Glu He Arg Gin Asn Lys Ala His Gly 115 120 125 Leu Gin Ala Leu Leu Gin Asp His Asp Asp Ser He Ser Arg Gly Gly 130 135 140 Glu Glu Arg Gin Pro Pro val Val Glu Glu Asp Asp Val val Gly Phe 145 150 155 160 Asp Asp Glu Ala Gin Thr Val He Asp Arg Leu Leu Glu Gly ser Gly 165 170 175 Asp Leu Glu Val lie Pro Val Val Gly Met Pro Gly Leu Gly Lys Thr 180 185 190 Thr Leu Ala Thr Lys lie Phe Lys His Pro Lys lie Glu Tyr Glu Phe 195 200 205 Phe Thr Arg Leu Trp Leu Tyr Val Ser Gin Ser Tyr Lys Thr Arg Glu 210 215 220 Leu Tyr Leu Asn lie lie Ser Lys Phe Thr Gly Asn Thr Lys His cys 225 230 235 240 Arg Asp Met Ser Glu Lys Asp Leu Ala Leu Lys val Gin Glu lie Leu 245 250 255 Glu Glu Gly Gly Lys Tyr Leu lie val Leu Asp Asp Val Trp ser Thr 260 265 270 Asp Ala Trp Asp Arg lie Lys lie Ala Phe Pro Lys Asn Asp Lys Gly 275 280 285 Asn Arg val Leu Leu Thr Thr Arg Asp His Arg val Ala Arg Tyr Cys 290 295 300 Asn Arg Ser Pro His Asp Leu Lys Phe Leu Thr Asp Glu Glu Ser Trp 305 310 315 320 lie Leu Leu Glu Lys Arg Ala Phe His Lys Ala Lys Cys Leu Pro Glu 325 330 335 Leu Glu Thr Asn Gly Lys Ser lie Ala Arg Lys Cys Lys Gly Leu Pro 340 345 350 Leu Ala lie val val lie Ala Gly Ala Leu lie Gly Lys ser Lys Thr 355 360 365 lie Lys Glu Trp Glu Gin Val Asp Gin Ser Val Gly Glu His Phe lie 370 375 380 Asn Arg Asp Gin Pro Asn Ser Cys Asp Lys Leu val Arg Met Ser Tyr 385 390 395 400 Asp val Leu Pro Tyr Asp Trp Lys Ala cys Phe Leu Tyr Phe Gly Thr 405 410 415 Phe Pro Arg Gly Tyr Leu lie Pro Ala Arg Lys Leu lie Arg Leu Trp 420 425 430 lie Ala Glu Gly Phe lie Gin Tyr Arg Gly Asp Leu Ser Pro Glu Cys 435 440 445 Lys Ala Glu Glu Tyr Leu Asn Glu Leu Val Asn Arg Asn Leu val Met 450 455 460 val Met Gin Arg Thr val Asp Gly Gin lie Lys Thr cys Arg val His 465 470 475 480 val Met Leu Tyr Glu Phe Cys Trp Gin Glu Ala Thr Thr Glu Glu Asn 485 490 495 Leu Phe His Glu val Lys Phe Gly Gly Glu Gin ser val Arg Glu val 500 505 510 Ser Thr His Arg Arg Leu cys lie His Ser Ser val val Glu Phe lie 515 520 525 Ser Lys Lys Pro Ser Gly Glu His val Arg Ser Phe Leu Cys Phe Ser 530 535 S40 Pro Glu Lys lie Asp Thr Pro Pro Thr val Ser Ala Asn lie Ser Lys 545 550 555 560 Ala Phe Pro Leu Leu Arg Val Phe Asp Thr Glu Ser lie Lys lie Asn 565 570 575 Arg Phe Cys Lys Glu Phe Phe Gin Leu Tyr His Leu Arg Tyr He Ala 580 585 590 Phe Ser Phe Asp Ser lie Lys al lie Pro Lys His val Gly Glu Leu 595 600 605 Trp Asn val Gin Thr Leu lie Val Asn Thr Gin Gin lie Asn Leu Asp 610 615 620 lie Gin Ala Asp lie Leu Asn Met Pro Arg Leu Arg His Leu Leu Thr 625 630 635 640 Asn Thr ser Ala Lys Leu Pro Ala Leu Ala Asn Pro Lys Thr Ser Lys 645 650 655 Thr Thr Leu val Asn Gin Ser Leu Gin Thr Leu Ser Thr lie Ala Pro 660 665 670 Glu Ser Cys Thr Glu Tyr Val Leu Ser Arg Ala Pro Asn Leu Lys Lys 675 680 685 Leu Gly lie Arg Gly Lys lie Ala Lys Leu Met Glu Pro Ser Gin Ser 690 695 700 val Leu Leu Asn Asn val Lys Arg Leu Gin Phe Leu Glu Asn Leu Lys 705 710 715 720 Leu lie Asn val Gly Gin lie Asp Gin Thr Gin Leu Arg Leu Pro Pro 725 730 735 Ala ser lie Phe Pro Thr Lys Leu Arg Lys Leu Thr Leu Leu Asp Thr 740 745 750 Trp Leu Glu Trp Asp Asp Met 5er val Leu Lys Gin Leu Glu Asn Leu 755 760 765 Gin val Leu Lys Leu Lys Asp Asn Ala Phe Lys Gly Glu Asn Trp Glu 770 775 780 Leu Asn Asp Gly Gly Phe Pro Phe Leu Gin Val Leu Cys Ile Glu Arg 785 790 795 800 Ala Asn Leu Val Ser Trp Asn Ala Ser Gly Asp His Phe Pro Arg Leu 805 810 815 Lys His Leu His lie Ser cys Asp Lys Leu Glu Lys ile Pro ile Gly 820 825 830 Leu Ala Asp lie Cys Ser Leu Gin Val Met Asp Leu Arg Asn Ser Thr 835 840 845 Lys Ser Ala Ala Lys Ser Ala Arg Glu lie Gin Ala Lys Lys Asn Lys 850 855 860 Leu G n Pro Ala Lys Ser Gin Lys Phe Glu Leu Ser val Phe Pro Pro 865 870 875 880 Asp Ser Asp Val Gin Thr Ala Ser 885 <210> 5 <211> 421 <212> DNA <213> unknown <220> <223> 3'uTR of the tomato nrcl gene <400> 5 tagaaaggtc taaaataacc acatgctgca ggttcaacca gcctggttgg cgcgcatggt 60 ttttgcttca tttggatcac tgcttt aca gtgaaaccta ttgcatttca taagtggaac 120 gactgatcca cggttttgca actctttggt ttcttactgt atttgatgtg agttcatgtt 180 ttattgattg tggatgcaat gtgtcatcat agccaaggaa taagaaccaa atgtaacgtt 240 caagaaatat cagacattgt ttcttataag ttataccact ctgaattttc tccttttaga 300 tacaacagca gaacaagtca cttgttttga ttcatacaca atttgatcat gtgttattat 360 ttaaacagca cttttggtaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaactcga 420 g 421

Claims (12)

1. A method for producing a transgenic plant having enhanced hypersensitive response compared to a non-lransgenic control plant, said method comprising the steps of: (a) transforming a plant or plant cell with a nucleotide sequence encoding an NRC1 protein, said NRC1 protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70% amino acid identity to the amino acid sequence of SEQ ID NO: 2 over its entire length, operably linked to a promoter active in plant cells, (b) regenerating a plant.

2. The method according to claim 1 , wherein said nucleotide sequence is integrated into the genome of said plant.

3. The method according to claim 1 or 2, further comprising step (c) screening the regenerated plant, or a plant derived therefrom by selfing or crossing, for resistance to one or more plant pathogens and identifying a plant comprising enhanced resistance to one or more of said plant pathogens.

4. The method according to any one of the preceding claims, wherein said promoter is a pathogen inducible promoter.

5. The method according to any one of the preceding claims, wherein the plant belongs to the family Solanaceae,

6. The method according to claim 5, wherein the plant is of the genus Solcmum.

7. A transgenic plant, plant cell, seed or fruit, comprising a nucleotide sequence encoding an NRC1 protein, said NRC1 protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70% amino acid identity to the amino acid sequence of SEQ ID NO: 2 over its entire length, operably linked to a promoter active in plant cells, integrated into its genome.

8. An isolated protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70% amino acid sequence identity to SEQ ID NO: 2 over the entire length.

9. An isolated nucleic acid molecule encoding the protein according to claim 8.

10. A chimeric gene comprising a promoter active in plant cells operably linked to a nucleic acid molecule according to claim 9 and, optionally, further operably linked to a 3' untranslated nucleic acid molecule.

11. 1 1. A vector comprising the chimeric gene according to claim 10.

12. Use of a nucleic acid molecule encoding an N C 1 protein for the generation of plants having enhanced hypersensitivity response, wherein said NRC1 protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70% amino acid sequence identity to SEQ ID NO: 2 over the entire length. Patent Attorney G.E. Ehrlich (1995) Ltd. 11 Menachem Begin Road 52 521 Ramat Gan