EP3149201A1

EP3149201A1 - Targeted screening for novel disease resistance in plants

Info

Publication number: EP3149201A1
Application number: EP15730867.7A
Authority: EP
Inventors: Jaap Bakker; Johannes Helder
Original assignee: Wageningen Universiteit
Current assignee: Wageningen Universiteit
Priority date: 2014-05-30
Filing date: 2015-05-29
Publication date: 2017-04-05
Also published as: WO2015183096A1

Abstract

The present invention relates to a screening method for the identification of non-genetically modified plants with a specific resistance response to a pathogen, the method involving the transgenic expression of a putative R gene construct, the identification of polymorphic markers in the putative R gene by comparing its sequence with known R gene sequences, and screening of non-genetically modified plant populations for the presence of these polymorphic markers.

Description

Title: Targeted screening for novel disease resistance in plants FIELD OF THE INVENTION

The present invention relates to targeted screening methods for the identification of disease resistance in non-genetically modified plants. In particular, the invention relates to the design of screening methods that enable the detection of resistance genes present in low frequencies in non-genetically modified plant populations. The invention further pertains to the construction by in vitro techniques of artificial resistance gene constructs by recombination of domains or parts of different resistance genes to form new resistance gene assemblages. Such constructs when introduced and expressed in a target plant of interest by genetic modification techniques result in a transgenic plant with a pest or disease resistance spectrum not previously identified in said plant of interest. Following the selection of constructs that confer to said transgenic plant a pest or disease resistance of interest and the identification of unique sequence motifs for the new assemblage, non-genetically modified plant populations can efficiently be screened for the presence of the unique sequence motifs using methods of screening as disclosed to thereby provide individual non-genetically modified plants carrying said novel pest or disease resistance spectrum. Such non-genetically modified plants are then used in further breeding programs. The invention therefore also pertains to methods for screening of non-genetically modified plant populations for plants that exhibit the novel pest or disease resistance spectrum due to the fact that they harbor the new assemblages of genes or parts of genes although they are not transgenic, and to non-transgenic plants exhibiting the novel pest or disease resistance spectra identified or selected by using the screening method of the present invention. BACKGROUND OF THE INVENTION

In plants, immune receptors encoded by disease resistance (R) genes confer resistance to a broad spectrum of (mostly biotrophic) organisms including bacteria, fungi, oomycetes, viruses, nematodes and arthropods. The most numerous types of these R genes encode intracellular proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains, collectively referred to as NB-LRR proteins. Two structurally different classes of NB-LRR proteins exist that encode N-terminal domains which either share homology with the Toll/Interleukin-1 Receptor (TIR) cytoplasmic domain (TIR-NB-LRR class) or have a less conserved domain with a predicted coiled-coil (CC) structure (CC -NB-LRR class).

NB-LRR proteins recognize specific pathogen proteins, i.e. proteins that are specific to a particular pathogen or pathogen isolate. Traditionally, these pathogen proteins are known as avirulence (Avr) proteins as they render the pathogen unable to infect a host expressing the corresponding NB-LRR protein. The interaction between host and pathogen genotypes is therefore referred to as gene-for-gene resistance, with characteristic R gene/Avr gene combinations. Recognition of the Avr protein by its corresponding NB-LRR protein results in the activation of a defense response in the plant, often in the form of a hypersensitive response (HR) causing local necrosis and cell death to restrict the growth of a pathogen.

A large number of pathogen-encoded Avr proteins that elicit NB- LRR-mediated resistance from bacterial, viral, fungal and oomycete plant pathogens have been identified. Avr proteins recognized by NB-LRR proteins show little structural commonality. Many Avr proteins contribute to pathogen virulence on plants lacking the cognate R gene. Avr proteins are now

considered to be part of a larger repertoire of pathogen-secreted proteins that are called effectors to stress their presumed intrinsic virulence function.

Hence, R gene mediated resistance is often referred to as effector-triggered immunity (ETI). Avr genes from microbial pathogens have traditionally been identified by genetic approaches. Genetic identification of Avr genes from metazoan parasites such as nematodes has been challenging however, owing to the complexity of the parasite genome and life cycle, and a lack of genetically tractable model organisms. However, alternate approaches to identifying Avr proteins in nematodes have been successful.

Cyst nematodes of the genus Globodera are obligate plant parasites, spending the majority of their life cycle within roots. These nematodes induce the development of a complex feeding site structure, known as the syncytium, in the vascular cylinder of the roots of the host plant. Cyst nematodes produce an assortment of parasitism proteins, which are analogous to effector proteins of microbial pathogens. These proteins are synthesized in the oesophageal glands and some of these are injected by the nematode into the host cytoplasm. Both host range specificity and suppression of host plant resistance are considered to be controlled by nematode effector proteins. Many putative nematode effector proteins have been identified by virtue of their possession of a protein-sorting signal for extracellular secretion and expression in the esophageal gland. These proteins can be recognized by NB-LRR proteins.

Use of plant nematode resistance genes is an effective and environmentally safe method for managing these parasites. At least four nematode R genes encoding NB-LRR proteins have been identified in

Solanaceous species. Gpa2 is a potato gene that encodes a CC-NB-LRR protein and confers resistance against two field populations (D383 and D372) of the cyst nematode G. pallida. In pa^-expressing potatoes, nematodes penetrate roots, start the initiation of their feeding site and become sedentary. However, the tissue surrounding the developing feeding site subsequently becomes necrotic and collapses, suggesting the elicitation of an HR.

Gpa2 is closely related to the Rx and Rx2 genes, which confer resistance to Potato Virus X (PVX), through recognition of the viral coat protein (CP). Rx function is dependent on Ran GTPase-activating protein 2 (RanGAP2), a protein shown to interact with the N-terminal CC domains of the Rx, Rx2 and Gpa2 proteins. Domain swap experiments indicate that the N- terminal halves of the Rx and Gpa2 proteins are interchangeable for mediating HR responses in response to the PVX CP whereas the LRR domain appears to determine recognition specificity.

In general, plant genomes encode several hundred NB-LRR genes that are dispersed over various chromosomes. Many NB-LRR genes are located in clusters but may also occur as single loci. For example, tetraploid potato with 12 different chromosomes, harbors hundreds of NB-LRR genes per haploid set of chromosomes, distributed over dozens of clusters each containing several gene homologues. It is noted that each chromosome has its own set of homologous NB-LRR genes which may vary in sequence and often also in numbers. Despite the gene-for-gene resistance specificity of R genes, it is noteworthy that R genes, are responsible for resistance to a broad range of pathogens, including viruses, bacteria, fungi, nematodes and insects. In fact, R genes are highly polymorphic and are among the most rapidly evolving genes in plant genomes. Although mutations are a major source of variation, much of the diversity within resistance gene families appears to arise from sequence exchanges that shuffle polymorphic sites between R gene homologues.

Reshuffling of sequences plays a central role in generating novel resistance specificities. The gene pools of natural plant populations, such as Solanum, are thought to contain thousands of different R gene specificities, of which only a few are known to breeders.

In producing plants with novel resistance specificities, previous methods relied on the random identification of resistant plants to serve as sources or donors for introducing the novel resistances in ornamentals or crops. A problem with this method of improving pathogen resistance is that the outcome relies heavily on chance occurrences, both with respect to finding a novel source of resistance, and with respect to finding a resistance against a novel pathogen. The identification of a novel resistant plant without any knowledge of what to look for requires such extensive screening of available germplasms that progress in developing new resistant plant varieties is very slow and with no guarantee of success. Moreover, the introgression of a new resistance trait into the parental breeding lines of resistant hybrids may take many years to complete. When using a closely related variety as donor the procedures are already lengthy, and introduction across the species barrier may be even more cumbersome. Hence, there is a need for a more

straightforward and predictable manner of producing or selecting plants with novel pathogen resistance traits, such as resistance against newly identified pathogens or against novel strains of known pathogens. Because transgenic crops still face challenges with respect to consumer acceptance, breeders are looking for non-transgenic methods to produce or select non-transgenic plants with novel traits. The present invention aims to provide such methods. SUMMARY OF THE INVENTION

The present invention now provides non-transgenic methods to produce or select non-transgenic plants with novel traits. The present invention relates to methods involving the preparation of gene expression constructs comprising novel sequences of putative resistance (R) gene, preferably prepared by in vitro techniques, including genetic engineering techniques, wherein said putative R gene may confer to a plant a novel type of pest and/or disease resistance.

Alternatively, the putative R gene may be one that exists in nature, such as an allelic mutant, but that has not yet been identified as being beneficial to the breeding of plants of a species, variety, accession or cultivar of interest. The putative R gene construct, when expressed in a test plant and showing to confer to the plant a novel pathogen resistance phenotype (i.e. a pathogen resistance trait of interest), is said to comprises a novel R gene variant of interest, and non-genetically modified plants, preferably of a species, variety, accession or cultivar of interest, can be screened for harboring that novel R gene variant of interest in non-GMO form. The first step in a method of the invention encompasses the preparation of gene expression constructs comprising the putative R gene. This step comprises the (re)combination of sequence stretches or sequence portions (hereinafter indicated by the general term "domains") from different

(homologous) R genes (in particular NB-LRR gene sequences) to produce new R gene sequence combinations. This may be done, among others, by natural recombination, by chemical or biological synthesis including genetic

engineering, and/or by mutation.

The sequence of the putative R gene, or, after it has shown to confer a novel resistance type, of the R gene variant of interest, is thus prepared by assembling domains of different R gene homologous, and these domains may be individually recognized by characteristic polymorphisms, i.e. nucleotides in positions characteristic for that domain which are not shared by homologous domains in other R genes or R gene homologues. The (re)combination of domains from different (homologous) R gene sequences therefore results in a unique combination of polymorphisms in the putative R gene or the R gene variant of interest and they may then be used as a target for screening populations of non-GMO plants in order to detect the presence of a similar or matching R gene sequence in a non-GMO plant.

In non-GMO plants the novel R gene variant of interest will generally be the result of rare genetic events wherein domains from different homologous R genes (in particular NB-LRR genes) are (re)combined to produce novel R gene sequence-combinations that confer novel resistances. Such rare genetic events may comprise, among others, equal and non-equal recombination, gene conversion, and/or transposon activity. Such rare events may produce chimeric genes and said chimeric variants are good candidates for conferring novel resistance specificities. The chance of finding a plant having the R gene variant of interest by screening of random populations is, however, extremely small. For that reason, methods of the present invention comprise steps to increase the chance that the R gene variant of interest, or at least the unique combination of polymorphisms characterizing the recombination event, is present in the target population for screening. This may, inter alia, be achieved by providing a parent plant wherein the various domains are present as part of separate homologous R genes, and generating offspring from said parent plant, wherein the required combination of polymorphisms in a single R gene is the result of by recombination between homologous genes, to thereby provide the plant harboring the R gene variant of interest.

The present invention in a first aspect provides a screening method for identifying a non- genetically modified plant of a species, variety, accession or cultivar of interest that exhibits a pathogen resistance response novel to said species, variety, accession or cultivar of interest, the screening method comprises the steps of:

(a) providing a gene expression construct comprising a promoter that is functional in plants operably linked to a putative R gene wherein said putative R gene is:

(al) a chimeric R gene prepared by exchanging gene sequences between at least two different parental R genes selected from

R genes and R gene homologues;

(a2) a mutated R gene prepared by targeted and/or random

mutagenesis of an R gene and/or R gene homologue;

(a3) a transgenic R gene or R gene homologue from a plant of a species, variety, accession or cultivar other than said species, variety, accession or cultivar of interest;

(a4) a synthetic R gene made by de novo synthesis of a DNA

sequence based on information from existing R genes and/or

R gene homologues, or

(a5) a combination of (al)-(a4);

(b) transforming a test plant or part thereof with said gene expression construct, and permitting the expression of said putative R gene sequence in said test plant or part thereof while exposing said test plant or part thereof to a pathogen effector;

(c) determining the resistance response of said test plant or part thereof to said pathogen effector;

(d) selecting the putative R gene as an R gene variant of interest in case said putative R gene confers to said test plant or part thereof a pathogen resistance response of interest, and determining at least a part of the nucleotide sequence of said R gene variant of interest;

(e) comparing at least a part of the nucleotide sequence of said R gene variant of interest to the nucleotide sequence of at least a part of at least two R genes and/or R gene homologues of said species, variety, accession or cultivar of interest;

(f) identifying from said nucleotide sequence comparison a combination of at least two polymorphic markers unique to said R gene variant of interest;

(g) providing a non- genetically modified plant of said species, variety, accession or cultivar of interest as a parental plant amenable to selfing or outcrossing, wherein said plant harbours parental R genes and/or R gene homologues that are different from the R gene variant of interest but that together contain stretches of codons that, when combined in a single gene sequence, comprise the unique combination of at least two polymorphic markers of said R gene variant of interest;

(h) providing an offspring population of non- genetically modified plants of a species, variety, accession or cultivar of interest using the plant provided in step g) as a parent plant in a selfing or out-crossing step, while allowing recombination to occur between said at least two separate R genes and/or R gene homologues in said parent plant in the production of said offspring population, (to thereby provide a screening population having an enhanced chance of comprising a non- genetically modified plant having the said combination of at least two polymorphic markers in a single R gene or R gene homologue (as a natural recombinant)); (i) screening the genomes of the plants of said offspring population for the presence of a plant having said combination of polymorphic markers present in a single R gene or R gene homologue, to thereby provide a non- genetically modified plant comprising the R gene variant of interest, and

j) optionally confirming that the non-transgenic plant provided in step

(i) exhibits the pathogen resistance response of interest.

It should be clear that the offspring population of non-genetically modified plants of a species, variety, accession or cultivar of interest in step (h), above, is thus preferably a population that is obtained by a process wherein the plant provided in step g) is used as a parent plant in a selfing or out-crossing step, while, during said selfing or out-crossing, recombination is allowed to occur between said at least two separate R genes and/or R gene homologues in said parent plant in the production of said offspring population. This provides a screening population having an enhanced chance of comprising a non-genetically modified plant having the said combination of at least two polymorphic markers in a single R gene or R gene homologue (as a natural recombinant).

The novel pathogen resistance response in the non-genetically modified plant is herein conferred by a resistance (R) gene variant novel to or not previously encountered in a plant of said species, accession variety or cultivar.

The construct in embodiments of this invention comprises a promoter sequence functional in plants, and preferably a terminator sequence. The transcription of the constructs may suitably be controlled by a CAMV 35S promoter and Tnos terminator sequences. The gene expression construct provided in step a) comprises all regulatory sequence elements to ensure that the putative R gene can be expressed in a test plant. Yet, the expression of the putative R gene in a test plant may, in instances, not result in a resistance phenotype of interest. However, in the case that it confers to the test plant a desired pathogen resistance response, it may be selected as an R gene variant of interest.

When exchanging gene sequences in step al in order to prepare a chimeric R gene, one may exchange individual nucleotides, but more

preferably gene fragments or even whole domains between at least two different parental R genes

In preferred embodiments of the invention the parental R genes used in constructing the putative R gene in the form of a chimeric R gene, belong to the same class of R genes. In such instances, each of the parental R genes encodes a different protein product.

In the embodiment in step a) wherein the putative R gene is a transgenic R gene, the gene may be obtained by isolating said gene from a plant of a related genotype, cultivar or wild accession belonging to the plant species of interest, or from another species of plant than the species of interest.

In highly preferred embodiments of a method of the invention, the putative R gene is a chimeric R gene as defined under step (al).

In other highly preferred embodiments of a method of the invention, the construct provided in step (a) comprises as the putative R gene a

transgenic R gene as defined under (a3), and wherein the test plant is a plant of said species, variety, accession or cultivar of interest.

In step (b) the step of transforming a test plant or part thereof with the gene expression construct may involve expression through stable

integration or by transient expression of the putative R gene. Preferably, the transformation in step (b) is performed by agro-infiltration of a test plant or part thereof with Agrobacterium cells comprising a gene expression construct for expression of the putative R gene in said test plant. In an alternative preferred embodiment, the transformation and exposure in step (b) are performed simultaneously by agro-infiltration of a test plant or part thereof with Agrobacterium cells comprising a gene expression construct for simultaneous expression of the putative R gene and the pathogen effector in said test plant.

Step (c) of determining the resistance response of said test plant or part thereof to said pathogen effector may be performed by any technique available, including a conventional resistance bioassay.

In step (d), the step determining at least a part of the nucleotide sequence of the R gene variant of interest may not always be needed, such as in cases where the putative R gene is the result if site directed or targeted mutagenesis of a gene with a known sequence. Hence sequencing occurs in such instances only when needed.

In preferred embodiments of step (d), the pathogen resistance response of interest is a resistance specificity and/or a resistance spectrum that is desirable and not exhibited by said test plant or part thereof before its transformation.

In preferred embodiments of step (e), at least part of the nucleotide sequence of said R gene variant of interest is compared to the known nucleotide sequence of at least one related R gene or R gene homologue, such as an R gene or R gene homologue from the same class of R genes.

In preferred embodiments of the method of this invention, the polymorphic marker(s) identified in step (f) represent amino-acid changing mutation(s) in the protein product(s) of said R gene or R gene homologue.

In yet other preferred embodiments of the method of this invention, the step (g) of providing a non- genetically modified parental plant amenable to selfing or out-crossing, comprises the steps of:

(gl) providing a first optionally mutated non- genetically modified parental plant of said species, variety, accession or cultivar of interest, said plant comprising an R gene or R gene homologue comprising at least a first polymorphic marker of said combination of at least two polymorphic markers unique to said R gene variant of interest, (g2) providing a second optionally mutated non- genetically modified parental plant of said species, variety, accession or cultivar of interest, said plant comprising an R gene or R gene homologue comprising at least a second polymorphic marker of said combination of at least two polymorphic markers unique to said R gene variant of interest,

(g3) crossing said first and second parental plants to thereby provide an offspring population of non-genetically modified plants of interest and optionally further modifying the sequence of R genes or R gene homologues in plants or plant parts of said offspring population using methods that do not involve the use of recombinant nucleic acid molecules or genetically modified plant cells, and

g(4) screening said offspring population for a single non-genetically modified plant of interest having the combination of said first and second polymorphic markers present in at least two separate R genes or R gene homologues, wherein a first R gene or R gene homologue originates from said first parental plant, and wherein a second R gene or R gene homologue originates from said second parental plant;

(g5) selecting said single non-genetically modified plant of step (g4) and optionally further modifying the sequence of R genes or R gene

homologues in said plant using methods that do not involve the use of recombinant nucleic acid molecules or genetically modified plant cells, to thereby providie a non-genetically modified plant of interest that harbours parental R genes and/or R gene homologues that are different from the R gene variant of interest but that together contain stretches of codons that, when combined in a single gene sequence, characterize the unique combination of at least two polymorphic markers of said R gene variant of interest.

The above steps of controlled crossing or selfing providing a parental plant that is used in step (h) as a parental plant for producing a screening population of non-genetically modified plants of interest. The controlled crossing or selfing steps provide for a plant in which, ultimately, the occurrence of intragenic recombination between parental R genes and/or R gene homologues may result in the formation of chimeric R genes that contain the unique combination of at least two polymorphic markers that characterize the R gene variant of interest. The advantage of this approach is that this significantly increases the chance that in the offspring population of the parental plant a chimeric R gene is created that contains the unique

combination of polymorphisms observed for the R gene variant of interest, such as a combination of at least two SNPs each of which is derived from a different parental R gene, wherein said chimeric R gene is the result of genetic recombination between homologous R genes due to unequal or equal crossing over, gene conversion, and/or transposon activity, and/or other natural processes of genomic rearrangement in which sequence stretches are

exchanged, added or deleted.

In another preferred embodiment of a method of the invention, the step (gl) of providing the optionally mutated non-genetically modified parental plant(s) is preceded by a step (gO) of genotyping plants of a species, variety, accession or cultivar of interest, wherein said plants are subjected to a genotyping assay using one or more polymorphic markers unique to said R gene variant of interest.

A method of the invention as described above may further comprising a step (k) of subjecting the R gene variant of interest in said non- transgenic plant provided in step (i) to further mutagenesis using methods that do not involve the use of recombinant nucleic acid molecules or genetically modified plant cells to provide a non-transgenic plant exhibiting the pathogen resistance response of interest.

A method of the invention as described above may further comprising a step (1) of using the non-transgenic plant provided in step (i) in a plant breeding program as the breeding source of a novel pathogen resistance response. In another preferred embodiment of a method of the invention, the putative R gene and said R gene variant of interest belong to the class of R genes selected from the group consisting of NB-LRR genes, cytoplasmic

Ser/Thr kinases, Receptor-like proteins (e.g. Cf -genes), or receptor-like kinases.

In yet another preferred embodiment of a method of the invention, the combination of at least two polymorphisms unique to said R gene variant of interest is a unique combination of SNPs and/or mutations resulting from chimeric recombination of domains of at least two different R genes, domains of at least two different R gene homologues or domains of an R gene and an R gene homologue.

In still another preferred embodiment of a method of the invention, the pathogen is a virus, insect, mite, bacterium, fungus, oomycete or nematode.

In still another preferred embodiment of a method of the invention, the pathogen resistance response of interest is an enhanced resistance against a known pathogen or a resistance against a novel pathogen.

In another aspect, the present invention provides a plant obtained by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphic representation of the genomic organization of the pa^/Rxl homologues in the diploid potato clone SH83-92-488 (Bakker et al. 2003, Theoretical Applied Genetics 106: 1524-1531). The Rxl and Gpa2 genes are located in the same R gene cluster in potato (Solanum tuberosum), and confer resistance to potato virus X (PVX) and the potato cyst nematode Globodera pallida, respectively. In the diploid potato clone SH83-92-488, Rxl and Gpa2 are in coupling phase together with the resistance homologue SH- RGH1 and the pseudogene SH-RGH3. In SH83-92-488 at the same complex locus, but in repulsion phase four other Rx/Gpa2 homologues are located: the full length homologues SH-RGH5, SH-RGH6 and SH-RG7, and the pseudogene SH-RGH8.

Figure 2 gives an overview of the Gpa2/Rxl domains, secondary structure, sequence divergence and exchanged fragments. The first row shows the domain architecture with the coiled coil (CC), nucleotide binding domain (NB), ARCl and ARC2 domain, the LRR domain and the C-terminal domain which is extended in Rxl with an acidic tail (AT). The second row shows the predicted secondary structure with a-helices in black, β-strands in grey. The P- loop, acidic loop and acidic tail (AT) are designated and shown in light grey. In the LRR the 6-strands of the LRR repeats are numbered (R1-R15). In the third row the amino acid positions differing between Rxl and Gpa2 are indicated in black. For the exchanged sequence fragments (see Figure 3 and 4) the break points in the sequence in Gpa2 and Rxl are shown below. If the numbering of the positions differed between Rxl and Gpa2 both are given. The numbering of the fragments (1-5; CC to AT) in combination with G (Gpa2) or R (Rxl) is used to name the constructs in Figure 3 and 4 (Slootweg, 2009, Thesis Wageningen University).

Figure 3 shows the phenotypes of sequence exchange constructs resulting in changes in recognition specificities, including gradual changes (e.g. 5, 6, 7, 13) , autoactivation (e.g., 5, 8, 9, 10, 11, 12, 13, 22, 23, 24, 25), and loss of function (e.g. 33, 34, 35). Rxl sequence is depicted in white and Gpa2 sequence in black. The exchanged segments in the constructs are named with G or R in combination with one or more of the following numbers: 1, 2, 3a, 3b, 3c, 4al, 4a2, 4a3, 4b, 4c, 5. On the right hand side the hypersensitive phenotype of these constructs is given upon transient expression in Nicotiana benthamiana leaves for combinations with Green Fluorescent Protein (GFP) (to detect elicitor-independent activity), CP 106 (the coat protein of the avirulent PVX strain), CP 105 (the coat protein of the PVX breaker strain), RBP D383-1 (an elicitor from the cyst nematode G. pallida) and RBP Rook-4 (a non-eliciting homolog of G. pallida). The strength of the hypersensitive response (HR) after 7 days are given by a scale from 0 (no HR) to 5 (full HR, as observed by bleaching of the leaf area's) which is further visualized on scale from white (0) to dark black (5) (Slootweg, 2009, Thesis Wageningen

University).

Figure 4 shows the phenotypes of some putative R gene constructs upon transient expression in leaves of Nicotiana benthamiana. Phenotypes of the wild-type (Fig. 3, no. l), and five chimeric putative R gene constructs (Fig. 3, no. 11, 12, 13 and 14), in which fragments of the ARC domain in the Rxl background are replaced by Gpa2 sequences. The ARC fragments (3a, 3b, 3c) are shown in white (Rxl) or black (Gpa2) to depict the composition of the chimeric putative R gene construct. Coexpression with GFP (top panel) shows autoactivation, as indicated by bleaching (HR or necrosis) of the infiltrated leaf area (see also arrows). Coexpression with PVX CP 106 shows the elicitor- dependent activation. The numbering corresponds to the numbering of the constructs in Figure 3. As shown in Figure 3 and 4, construct 13 results in hardly any autoactivion, but recognizes both the coat protein (CP 106) of the avirulent strain and the coat protein of the virulent strain (CP 105) (Figure 3 and 4) (Slootweg, 2009, Thesis Wageningen University).

Figure 5 shows that a broadened resistance specificity is observed when the CC-NB-ARC 1-ARC2 region of resistance gene homologue SH-RGH6 is combined with the LRR of Rxl. Both the coat protein (CP106) of the avirulent strain (left) and the coat protein of the virulent strain (CP 105) (middle) are recognized by the chimeric putative R gene construct

SHecc nbsRxlLRR upon transient expression in N. benthamiana as indicated by bleaching of the leaf. No bleaching is observed upon co-expression with GFP (right).

Figure 6 shows the accumulation of PVX in transgenic plants transformed with the putative R gene G12R35 (construct no.6 in Figure 3). Accumulation of the virus was measured with ELISA (Slootweg, 2009, Thesis Wageningen University). The relative virus concentrations are shown on the y- axis. ELISA plates were coated with a 1: 1000 dilution of a polyclonal antibody against PVX that was conjugated with alkaline phosphatase. The virus level of PVX105 in the transgenes 2.2 and 2.4 expressing Gl2G3aR3bcR45 was significantly lower than in the diploid potato clone SH83-92-488 (SH) and plants transformed with the empty vector (EV). In addition, it is shown that the putative R gene Gl2G3aR3bcR45 preserved its ability to inhibit the accumulation of PVX106.

Figure 7 gives an impression of the frequency of the occurrence of sequence exchanges under natural circumstances. The Rx/Gpa2 homologues were amplified from various Solanum species. The sequence exchange tracks were detected by the comparison of 77 Rx/Gpa2 homologues using the RDP software package (Butterbach, 2007, Thesis, Wageningen University).

Conversion tracks are shown as horizontal bars and the breakpoints are indicated as vertical bars linked to the nucleotide sequence and protein structure of Rxl. The intensity of the color, ranging from light grey to black, indicates the relative abundance of the conversion tracks. The lightest color reflects a frequency of one and the darkest color reflects a frequency of more than 20 homologues in which the indicated sequence track is observed. All other colors indicate frequencies between 2 and 20 (Butterbach, 2007, Thesis, Wageningen University). Below the structural domains (CC, NB, ARCl, ARC2 and LRR) are indicated (Butterbach, 2007, Thesis, Wageningen University).

Figure 8 shows a multiple nucleotide alignment of Rxl, Gpa2, their homologues and the chimeric putative R gene SH6CC/NBSRX1LRR, in which the Informative Polymorphic Sites (IPS) are shown. The Informative Polymorphic Sites are the nucleotide positions where two or more nucleotides are different from the other nucleotides at the same position (Bakker et al. 2003,

Theoretical Applied Genetics 106: 1524-1531). The positions without variation are not shown. The homologues SH-RGHl, SH-RGH5, SH-RGH6 and SH-RG7 and the pseudogene SH-RGH3 have been identified in the diploid potato clone SH83-92-488. The homologues RH-RGH2, RH-RGH3, RH-RGH4 and RH- RGH5 have been identified in the diploid potato clone RH89-039-16. The putative R gene SH6CC/NBSRX1LRR was constructed by fusing the CC, NB, ARCl and ARC2 region of the resistance gene homologue SH-RGH6 with the LRR region of the Rxl gene. The two domains are joined between nucleotide position 1461 and 1488.

Figure 9 illustrates the principle of the invention for targeted screening of novel disease resistance in non- genetically modified plants. The resistance genes and resistance gene homologues are represented by white bars. The nucleotides are designated with capitals and numbers. The capitals refer to corresponding positions in the nucleotide alignment and the numbers refer to similarities and dissimilarities at these positions. Thus identical capitals with different numbers represent single nucleotide polymorphisms (SNPs), but may also represent additions and deletions of nucleotide sequence stretches. The principle of the invention is explained by comparing eight corresponding positions (A-H) in the alignment. However, in reality hundreds of polymorphic and conserved sites may be involved (see section Examples).

The selection of rare chimers (recombinants) conferring the resistance response of interest in non- genetically modified plants involves the following steps.

Step I. In the first step putative novel R genes are constructed by in vitro techniques by :

i) exchanging gene sequences between different R genes and/or R gene homologues by genetic modification techniques (claim lal).

ii) targeted and/or random mutagenesis of an R gene or R gene homologue (claim la2)

hi) isolation of a known R gene from another plant species or from a related genotype with less optimal agronomic properties (e.g. low yield, slow growth) than the plant genotype of interest (claim la3) iv) in vitro ale novo synthesis of a DNA sequence based on information from existing R gene homologues and/or R genes (claim la4)

v) combinations of the previous techniques (claim la5)

Step II. The in vitro constructs are expressed in plants to identify putative R genes with the desired resistance response (claims lb and lc). This step may involve a test plant other than the genotype, cultivar or even species of interest. For example, Nicotiana benthamiana is suitable test plant for various solanaceous species. For this step various methods may be used.

Genetically modified plants or parts thereof can be inoculated with pathogens or effectors by injection or expressing genes encoding the effectors. This step can involve stable or transient transformation. A well known method for transient assays is agro-infiltration (claim 4). For example, leaves of Nicotiana benthamiana can be infiltrated with Agrobacterium cells comprising the putative novel R gene and a pathogen gene encoding the effector of interest (claim 4). Instead of transient expression of the effector-encoding gene, the transformed tissue may also be exposed to the pathogen itself.

Step III. In case a putative R gene mediates the resistance response of interest, it is selected as an R gene variant of interest (claim Id). In case the sequence is not known yet or only partly known, the R gene variant will be sequenced.

Step IV. To identify unique combinations of nucleotides in the R gene variant of interest that are responsible or associated with the novel resistance specificity, the nucleotide sequence of the R gene variant of interest is compared with other R genes having different resistance specificities and/or R gene homologues with unknown specificities (claims le and If). In case the R gene variant of interest is a chimeric R gene obtained by combining sequence stretches from different R genes and/or R gene homologues, the parental homologues are also included in the alignment. Preferably, the most closely related R genes and R gene homologues are selected to narrow down the number of possible combinations associated with the novel resistance response. The multiple alignment will reveal numerous combinations of nucleotides that are unique for the R gene variant of interest. These unique combinations comprise both conserved and polymorphic sites. The conserved sites maintain the backbone and are essential for the functionality of the R gene of interest. In theory one SNP may be sufficient to generate a novel resistance specificity, but this will only occur in the appropriate backbone. For example, H2 in the R gene variant of interest may be responsible for the novel resistance specificity but will also require conserved sites such as Al, C l and/or El. Because such intra-molecular interactions play an important role in determining the appropriate resistance response, it is difficult to predict which combination of nucleotides is essential for the novel resistance response. However, in practice it will not be necessary to identify exactly which sequence combination of the numerous possible combinations of Al, Bl, Cl, Dl, E l, F2, Gl and H2 in the R gene variant of interest is essential for the novel resistance response. The targeted screening in step VI is based on identifying novel combinations of sequence stretches in the progeny that are identical or closely related to the R gene variant of interest. Besides the sequence A1B1C1D1E1F2G1H2 of the R gene variant of interest, closely related variants such as A1B1C1D1E1F2G1H1 or A1B1C1D2E1F2G1H2 will be detected as well in step VI, and will also be tested on the resistance response of interest. To narrow down the number of possible nucleotide combinations associated with resistance, it is recommend to include in the alignment the most closely related R genes and R gene homologues available. Another option is to test various mutants of the R gene variant of interest.

Step V. To identify the R gene variant interest in an off-spring population of non-genetically modified plants, a parental plant is selected that harbours stretches of nucleotides that when combined in the appropriate way resemble the sequence of the R gene variant of interest (claims lg and lh). The stretches of nucleotides are preferably present in two different genes that are located in the same R gene cluster but are in repulsion phase. In case the R gene variant of interest is a chimeric R gene obtained by combining sequence stretches from two genes, the selection of the parental plant is straightforward and will be based on the presence of the parental R genes and/or R gene homologues. In other cases, the parental plant is selected by sequencing R genes and R gene homologues and searching for sequence stretches that when combined resemble the R gene variant of interest or a part thereof. In case the required sequence stretches are not available in a single genotype, additional crosses can be made to select the desired parental plant, as is illustrated in this Figure 9. The genotypes Gl and G2 are crossed to select genotype G3, that is crossed with genotype G4 to select genotype PI having the sequences

A1B1C1D1E1F1G1H1 and A1B2C2D2E1F2G1H2 in repulsion phase. PI harbours sequence stretches that when combined in the appropriate way match the sequence of the R gene variant of interest: A1B1C1D1E1F2G1H2.

For clarity it is noted that the additional crosses in this example are aimed at selecting a resistance gene homologue with the nucleotide

combination A1B2C1D2E1F2G1H2. In this example, a resistance gene homologue with the nucleotide combination AlBlClDlElFlGlHl is already available in genotype G4. In genotype Gl

A1B2C1D2E2F3G2H1/A2B1C2D3E1F2G1H1 recombination may lead to a gamete with the nucleotide combination A1B2C1D2E1F2G1H1.

Recombination in G3 between A1B2C1D2E1F2G1H1 and

A2B1C2D1E2F3G2H2 may result in a gamete with the combination

A1B2C1D2E1F2G1H2.

The parental plant PI may be selfed or crossed with a related genotype to generate the off-spring population for targeted screening on recombination events leading to the desired combination of nucleotides.

In case some polymorphisms are present in the R gene variant of interest, but not present in the genotype of interest, it may be an option to use random and/or targeted mutagenesis to change a few nucleotides in the ancestors of the parental plant PI. Step VI. The R gene homologues in the off-spring population are screened on the presence of novel combinations of sequence stretches that resemble the sequence A1B1C1D1E1F2G1H2 or parts thereof (claim li). This step may include random and/or targeted mutagenesis of a few nucleotides in the selected chimers, either off-spring population of PI and P2, but also in the next generation.

Step VII. The genotypes comprising the sequence A1B1C1D1E1F2G1H2 or parts thereof are tested on the resistance response of interest (claim lj). DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term "screening method", refers to strategies to identify plants that have an increased likelihood of having a pathogen resistance trait of interest so that such plants can be selected for more direct phenotypical characterization methods to definitively determine if the plants have the sought after pathogen resistance trait of interest. The "screening methods" of the invention are generally not intended to definitively diagnose a plant as having or not having the pathogen resistance trait of interest. Rather, such methods are intended to identify plants having an increased likelihood of having the trait so that these plants may be definitively diagnosed using other methods (e.g., resistance assays).

The term "plant", as used herein, refers to any type of plant. As used herein, the term "plant" includes the whole plant or any parts or derivatives thereof, preferably having the same genetic makeup as the plant from which it is obtained, such as plant organs (e.g. harvested or non-harvested carrot root), plant cells, plant protoplasts, plant cell and/or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, plant

transplants, seedlings, hypocotyl, cotyledon, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants (e.g. harvested tissues or organs), such as plant cuttings, vegetative propagations, embryos, pollen, ovules, flowers, leaves, seeds, clonally propagated plants, roots, taproots, stems, root tips, grafts, parts of any of these and the like. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, mature plants, roots or leaves. Alternatively, plant part may also include a plant seed that comprises one or two sets of chromosomes derived from the parent plant.

Below is an exemplary description of some plants that may be used in aspects of the invention. However, the list is provided for illustrative purposes only and is not limiting, as other types of plants will be known to those of skill in the art and could be used with the invention. The term preferably refers to a cultivated plant, more preferably a breeding line, still more preferably an essentially homozygous breeding line.

A common class of plants exploited in agriculture are vegetable crops, including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, Chinese cabbage, peppers, collards, potatoes, cucumber plants (marrows, cucumbers), pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, garlic, spinach, green onions, squash, greens, beet (sugar beet and fodder beet), sweet potatoes, swiss-chard, horseradish, tomatoes, kale, turnips, and spices.

Other types of plants frequently finding commercial use include fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits, melon, mango, papaya, and lychee. Many of the most widely grown plants are field crop plants such as evening primrose, meadow foam, corn (field, sweet, popcorn), hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), fiber plants (cotton, flax, hemp, jute), Lauraceae

(cinnamon, camphor), or plants such as coffee, sugarcane, tea, and natural rubber plants.

Another economically important group of plants are ornamental plants. Examples of commonly grown ornamental plants include Alstroemeria (e.g., Alstoemeria brasiliensis ), aster, azalea (e.g., Rhododendron sp.), begonias (e.g., Begonia sp.), bellflower, bouganvillea, cactus (e.g., Cactaceae schlumbergera truncata ), camellia, carnation (e.g., Dianthus caryophyllus ), chrysanthemums (e.g., Chrysanthemum sp.), clematis (e.g., Clematis sp.), cockscomb, columbine, cyclamen (e.g., Cyclamen sp.), daffodils (e.g., Narcissus sp.), false cypress, freesia (e.g., Freesia refracta ), geraniums, gerberas, gladiolus (e.g., Gladiolus sp.), holly, hibiscus (e.g., Hibiscus rosasanensis ), hydrangea (e.g., Macrophylla hydrangea ), juniper, lilies (e.g., Lilium sp.), magnolia, miniroses, orchids (e.g., members of the family Orchidaceae ), petunias (e.g., Petunia hybrida ), poinsettia (e.g., Euphorbia pulcherima), primroses, rhododendron, roses (e.g., Rosa sp.), snapdragons (e.g., Antirrhinum sp.), shrubs, trees such as forest (broad-leaved trees and evergreens, such as conifers) and tulips (e.g., Tulipa sp.).

Plants useful in the methods of the invention may be plants amenable to transformation techniques. However, this is not necessary, since the non- genetically modified plants (non-GMO plant) to be identified by a method of the invention will not undergo any genetic modification that would involve the introduction therein of heterologous genes.

Genetically modified plants useful in aspects of this invention may be of the same or different species to that of the non-GMO plant. Suitable genetically modified plants include plants of any of the species mentioned above. Preferably genetically modified plants used in aspects of the invention are N. benthamiana plants.

The term "crop plant", as used herein, refers to a plant which is harvested or provides a harvestable product. Suitable plants for use in aspects of the invention are protected (greenhouse) crop plants.

As used herein, the term "genetically modified plant" refers to a plant whose genome has been changed using genetic modification techniques. The term includes reference to a transgenic plant, which itself denotes a plant comprising a transgene.

The term "non-genetically modified plant" in the context of the present invention refers in particular to plants that are not considered as plants that are genetically modified under Directive 2001/18/EC of the

European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms. Plants obtained through crossing and mutagenesis, and that are not obtained by methods that involve the use of recombinant nucleic acid molecules or genetically modified plant cells are considered non-genetically modified plants.

The term "species" includes any taxonomic group of organisms which can interbreed, and thereby includes sub-species, varieties, accessions and cultivars.

The term "variety" or "cultivar" means a plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of a breeder's right are fully met, can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, distinguished from any other plant grouping by the expression of at least one of the said characteristics and considered as a unit with regard to its suitability for being propagated unchanged. The term "accession" when used herein is associated with sources of plants and refers to a plant or group of similar plants or group of seeds from these plants received from a single source at a single time. Accessions are generally indicated by an "accession number", which number refers to a unique identifier for each accession and is assigned when an accession is entered into a plant collection. The terms "germplasm" and "accessions" are somewhat interchangeable, and use of the term "accession" is not meant to exclude from the method of the invention use of wild accessions of plants that are not uniquely identified or part of a germplasm collection.

The term "cultivar" refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.

As used herein, the terms "pathogen resistance response" and the advantageous or desired form "pathogen resistance response of interest", as used herein, refer to any pathogen resistance feature that constitutes an improvement over the known or available resistances, and includes any improvement in the resistance, such as a reduced rate of establishment of infection, a reduced rate of progression after infection, or a novel resistance specificity, both with respect to the quantitative effect as well as a qualitative effect conferred by a putative R gene or an R gene variant of interest that constitutes an alteration of the response over the "parental" versions of the R gene. Novel resistance specificity includes resistance to novel types of pathogens, or to new combinations of pathogens. The term refers to any response or feature that will produce a full or partial pathogen resistance trait in a plant organism. In particular the term "pathogen resistance response of interest" refers to a new or previously unknown pathogen resistance trait in plants in general or in specific plant varieties of interest. Thus, the pathogen resistance response of interest may be found to occur in a related genotype of the same species of low or no agronomical interest, but would be novel to the target plant of interest. The term "novel" as used herein may refer to "unknown" or "not previously associated with".

The term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being

transcribed. Generally, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.

As used herein, the term "gene" refers to a functional protein, polypeptide or peptide-encoding nucleic acid unit. As will be understood by those skilled in the art, this functional term includes genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been designed and/or altered. Purified or isolated genes, nucleic acids, proteins and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.

As used herein, the term "putative R gene" refers to an R gene potentially or supposedly encoding a recombinant R protein for resistance against pathogens in a plant, and includes reference to R genes that are the result of recombination events and that may have been assembled,

synthesized, or otherwise produced, preferably as a result of man-made efforts, and any genes that are replicated or otherwise derived therefrom. "Man-made efforts" include enzymatic, cellular, and other biological processes, if such processes occur under conditions that are caused, enhanced, or controlled by human effort or intervention. As used herein, the term putative R gene is also meant to encompass a mutated R gene, which term itself refers to an R gene that has been subjected to mutagenesis. As used herein, the term putative R gene is also meant to encompass a native R gene that is heterologously expressed in a species in which such native R gene is not natively expressed. For example, a native tomato R gene is a putative R gene when expressed in, for example, potato. Also, a native R gene in wild genotypes is a putative R gene when expressed in highly yielding plant varieties of the same species. The term also includes reference to sequence-manipulated genes that are

themselves the result of natural events of DNA genetic recombination, which occur for instance during meiotic division, such as gene conversion and chromosomal crossover. A putative R gene may be the result of:

(i) exchange of gene sequences between different R genes and/or R gene homologues by genetic modification techniques,

(ii) targeted and/or random mutagenesis of an R gene,

(iii) a natural process in which case the putative R gene may be isolated from other plant species or from related genotypes with poor agronomic properties (low yield, slow growth), or

(iv) in vitro de novo synthesis of a new DNA sequence encoding a novel R gene.

Methods for generating the putative R gene include recombinant DNA or genetic modification techniques, including genome editing, targeted and/or random mutagenesis and in vitro de novo synthesis of a DNA. Also combinations of the aforementioned methods may be applied to create the putative R gene. Thus, a putative R gene can for example be generated by producing a chimeric R gene by the exchange of sequences between different R genes and/or R gene homologues, in combination with the use of a mutated R gene obtained by targeted and/or random mutagenesis. The skilled person will appreciate that the putative R gene may first be produced in vitro by a variety of molecular methods that allow the generation of novel sequences or enable the manipulation of sequences of known and unknown R genes.

Certain embodiments of putative R genes may be banned from being spread into natural or cultural plant populations as they are prohibited under GMO regulations. The present invention is aimed at searching for an R gene variant, with the same sequence signature as that of the putative R gene, in a natural (including non-GMO regulated) plant population, which population has naturally produced this R gene variant. The natural R gene variant of the putative R gene is in a preferred embodiment chimeric (i.e. the result of gene fragment exchange) as a result of a natural process, such as the process of recombination during meiosis. The putative R gene may further have been subjected to mutagenesis.

As used herein, the term "chimeric R gene" includes reference to artificially constructed or naturally occurring R gene variants that contain at least two portions each originating from different and distinct, but optionally homologous, R genes. Preferably, such chimeric R genes contain sequence tracks (e.g. gene fragments) originating from at least two parental R genes/i? gene homologues and encode an amino acid sequence that differs in at least two amino acid residues from both parental R genes from which the chimer was derived. In theory, a large variety of different combinations may occur in a "chimeric R gene" as defined herein. The length of the sequence tracks may vary from 6 bp to more than 3000 bp. In selecting the R gene variants of interest of interest, it is not the length of the track, but the number of SNPs that is exchanged or, more in particular, the number of amino acid

substitutions in the final chimeric R protein as this will eventually determine the change in function.

The term "R gene variant of interest" as used herein refers to the selected putative R gene upon its demonstration of exhibiting a desired resistance response. R gene variants of interest may comprise a contiguous fragment of the sequence of a first R gene or homologue and one or more fragments of the sequence of one or more other R genes or homologues, wherein all fragments have been combined in and/or incorporated into a single DNA sequence. It should be noted that when reference is made herein to an R gene variant of interest, this term also includes reference to mutant genes that comprise one or more amino acid deletions, additions, or substitutions not observed in either of the parental R genes and/or R gene homologues. Such mutant proteins can comprise one or more amino acid deletions, substitutions and/or additions compared to the protein encoded by the individual R gene fragments.

Preferably, the sequence fragments of the mutant proteins still have at least 50%, more preferably at least 70%, even more preferably more than 90% amino acid sequence similarity with the original sequence fragments that have been combined to form the putative R gene.

The term further includes reference to R genes that have been the result of natural events of DNA genetic recombination, which occur for instance during meiotic division, such as gene conversion and chromosomal crossover. An R gene variant of interest may also be transposon-induced, and may for instance be formed through retrotransposition where a

retrotransposon copies the transcript of a gene and inserts it into the genome in a new location. An R gene variant of interest may also be the result of ectopic recombination between non-homologous genes.

"R gene variants of interest" as referred to herein may have the form of "chimeric R genes" and in the context of the ultimate target for the screening method of this invention when produced in non-genetically modified plants may be any form or sequence variant of an R gene that is the result of a natural recombination event occurring or having occurred in planta and which has resulted in the formation of a chimeric R gene wherein regions or domains of at least two distinct R genes or (highly similar) R gene homologous have been combined or "swapped" to thereby result in an R gene sequence that is different to that of the original R genes or R gene homologous. In addition to being chimeric, the R gene variant of interest may comprise artificially introduced mutations resulting from exposure of the original R genes or R gene homologues or of the putative R gene to mutagens, including but not hmited to chemical agents such as EMS, or to ionizing radiation such as UV radiation. Other techniques that may be used to introduce mutations on the R gene (before or after in planta recombination) include targeted mutagenesis. The term thus encompasses allelic variants, and mutant versions of the R gene variants of interest. The introduction of mutations involving GMO techniques is preferably expressly excluded in the context of the R gene variants of interest.

R gene variants of interest have not the typical hallmarks of the R genes that are currently used in plant breeding. Current R genes used in breeding are in most cases present in the form of multiple alleles having different gene sequences due to naturally occurring mutations that accumulate in the gene pool. In contrast, the R gene variants of interest will have the hallmarks of recent and rare recombination events. Exact copies of the R gene variants of interest will not or rarely be found in natural populations. In case exact copies are encountered chances are small that they have a high frequency and are found in various accessions. Also is it unlikely that multiple alleles exist having slightly different gene sequences due to naturally occurring mutations that have gradually accumulated in the gene pool. Thus, while most R genes presently used in plant breeding programs are present in various allelic variants each conferring the same resistance specificity and which are "old" in evolutionary genetics perspective as manifested by their presence in different accessions of the same species, or even by their

occurrence in different species. Exact copies of the R gene variants of interest will be hard to find in natural populations, and when they are found, their presence will usually be confined to their presence in a few accessions and no synonymous substitutions, or other subtle differences will be encountered.

"Gene sequence exchange" refers to the exchange of nucleotide sequences between a target and a donor nucleic acid through recombination.

The terms "recombination" and "genetic recombination" refer to the exchange (e.g. by chromosomal crossover) of DNA fragments between two DNA molecules or chromatids of paired chromosomes in a region of similar or identical nucleotide sequences. Such an equal or non-equal crossover may naturally take place in the event of meiosis. As used herein, recombination includes reference to both the natural process of genetic recombination, as well as the artificial process of recombinant DNA or genetic modification, both of which processes result in the remixing of genetic information from two DNA molecules to result in the formation of genes with altered sequences. The term "recombination" includes reference to the process of gene conversion. A recombination may also be transposon-induced, and may for instance be the result of retrotransposition where a retrotransposon copies the transcript of a gene and inserts it into the genome in a new location, or may also be the result of ectopic recombination between non-homologous genes. Formation of recombinant DNA is an in vitro process that may involve a cloning vector that replicates within a living cell thereby introducing foreign or artificially prepared DNA into said cell. Vectors are generally derived from plasmids or viruses that contain necessary genetic signals for replication, elements for inserting foreign DNA, selectable markers, and elements for expressing the foreign DNA. The DNA segments in a cloning vector can be combined by using a variety of methods, such as restriction enzyme/ligase cloning. An additional molecular technique for recombinant DNA is PCR, which technique involves a set of bidirectional primers for amplifying specific stretches of DNA in a cyclic primer extension procedure under the control of a polymerase enzyme.

The terms "parental R gene" and "parental R gene homologue", as used herein, refer to R genes, respectively R gene homologues, occurring in the parents of a cross and further includes reference to the source of the R gene of which a portion was incorporated into the putative R gene or R gene variant of interest.

As used herein, the terms "R gene" and "R gene homologue" are interchangeably used. Generally, the term "R gene" refers to a resistance gene of which the pathogen specificity is known, whereas the term "R gene homologue" refers to an R gene sequence having a high level of sequence similarity (or sequence identity) with an R gene of known specificity, but having an unknown pathogen specificity or resistance response. "Classes of R genes" include the NB-LRR class of R genes, the cell surface PRR (pattern recognition receptors) class of R genes, and the

Pseudomonas tomato resistance gene (Pto). The NB-LRR class of R genes itself contains two classes, one having an amino-terminal Toll/Interleukin 1 receptor homology region (TIR) which includes the N resistance gene of tobacco against tobacco mosaic virus (TMV), and the other having a coiled-coil domain (CC), which includes Gpa2 and Rx. The PRR class of R genes include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that lack a kinase domain. The Arabidopsis immune receptor FLS2 that recognizes the flg22 peptide from flagellin is a well-known example of a receptor-like kinase. The Cf genes e.g. Cf2, Cf4, and Cf9 of tomato for resistance against Cladosporium fulvum are examples of receptor-like proteins without a kinase domain. Pto encodes a Ser/Thr kinase, has no LRR and requires the presence of a linked NB-LRR gene, prf.

As used herein, the term "at least two different R genes" refers to at least two R genes having a different gene sequence. In particular, the differences in gene sequence are such that the genes encode R proteins with different amino acid sequence, such that the recombinant (e.g. chimeric) product resulting from their recombination or sequence exchange encodes an R protein with an amino acid sequence distinct from the protein product of the at least two different R genes from which the gene was produced (i.e. the

"parental" R genes). The at least two different R genes may have a different origin (i.e. may originate from a different and distinct plant variety, plant species or plant genus). However, it is preferred that recombinant chimeric R genes are constructed from R genes that are closely related, preferably from homologous R genes of a single resistance-gene cluster. Chimeric putative R genes are preferably constructed through the combination or exchange of sequences from R genes (e.g. domains) that are in repulsion phase, i.e., located in the same resistance-gene cluster but on opposite chromosomes. The at least two different R genes may elicit a different resistance response. However, in many cases the resistance response of both parental R genes will be unknown. The reason is that a given plant genome may comprise hundreds of R genes of which the resistance response is unknown.

"Mutated", as used herein, refers to plants or gene sequences that have been subjected to or that are the result of mutagenesis.

The term "targeted mutagenesis", as used herein, refers to the technique also known as "site-directed mutagenesis" that is used to induce an altered form of one or more specific amino acids by changing one or more specific nucleotides in a cloned gene. The site-directed mutagenesis method is described in Ling et al, "Approaches to DNA mutagenesis: an overview", Anal Biochem., 254 (2): 157-178 (1997); Dale et al., "Ohgonucleotide-directed random mutagenesis using the phosphorothioate method", Methods Mol. Biol., 57: 369-374 (1996); Smith, "In vitro mutagenesis" Ann. Rev. Genet., 19: 423- 462 (1985); Botstein & Shortie, "Strategies and applications of in vitro mutagenesis", Science, 229: 1193-1201 (1985); Carter, "Site-directed

mutagenesis", Biochem. J., 237: 1-7 (1986); and Kunkel, "The efficiency of oligonucleotide directed mutagenesis", Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin (1987)), which are herein incorporated by references. It is also possible to carry out site- directed mutagenesis by the PCR method using primers comprising a specific mutation (Ausubel et al., Current Protocols in Molecular Biology,

Greene/Wiley Interscience (1987)).

The term "random mutagenesis" used herein refers to a technique to induce an altered form of one or more specific amino acids by randomly changing one or more specific nucleotides in a cloned gene. The introduction of random changes in a cloned gene with error-prone PCR methods is inter alia described by Xu et al. in BioTechniques Vol. 27: 1102-1108 (1999).

The term "transgenic" refers to an organism or cell having received genetic material from a different organism or cell, resulting in the introduction of foreign DNA into said organism or cell, either naturally, or by any of a number of genetic engineering techniques.

The term "isolated" as used herein mean a specific nucleic acid, or a fragment thereof, in which contaminants (i.e. substances that differ from the specific nucleic acid molecule) have been separated or substantially separated from the specific nucleic acid.

The term "transforming" or "transformation" refers to the process of introducing DNA into a recipient plant cell, and includes both the subsequent integration into the plant cell's chromosomal DNA, as well as the transient transformation whereby the DNA is expressed from extrachromosomal elements. A number of techniques are known in the art for transformation of plants or plant cells in general, including Agrobacterium-mediated

transformation, electroporation, microinjection, microprojectile or particle gun technology (biolistics), liposomes, polyethylene glycol (PEG) mediated transformation, wounding, vacuum infiltration, passive infiltration or pressurized infiltration, and reagents that increase free DNA uptake.

Identification of transformed cells or plants is generally accomplished by including a selectable marker in the-transforming vector. Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. A preferred embodiment in the present invention comprises the transformation of plant cells with a putative R gene by agroinfiltration. This method involves infection with Agrobacterium tumefaciens and is well known to one of skill in the art. The method is for instance described in detail in Van de Hoorn et al., MPMI Vol. 13, No. 4, 2000, pp. 439-446, and Sparkes et al. (2006) Nat Protoc 1:2019-2025.

"Expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation. "Transient expression" of a gene or nucleotide sequence or

"transiently expressed" refers to the expression of a gene or nucleotide sequence that is not integrated into the host chromosome but which can function either independently (e.g., by being a part of an autonomously replicating plasmid or an expression cassette) or as a part of another biological system, such as a virus, for example. Transient expression may be achieved by "transient transformation" of a host cell, which term refers to the introduction of foreign DNA or a nucleotide sequence of interest into the host cell (for example, by such methods as Agrobacterium- ediated transformation or biolistic bombardment) without integration of the foreign DNA or nucleotide sequence of interest into a host cell chromosome, thereby precluding stable maintenance of the foreign DNA or nucleotide sequence of interest in the progeny of the host cell.

In the context of the present invention, the term "pathogen effector" is to be understood as a general term that includes reference to avirulence factor, elicitor, PAMP and effector. The term "pathogen effector" is used here in the broadest sense and applies to any pathogen molecule that can be recognized by the plant to trigger a resistance response, including the pathogenic organism itself. As used herein, the terms "avirulence factor" and "elicitor" are interchangeably used, and are broadly drawn to a molecule of phytopatho genie origin that enhances the production of a resistance-response- inducing molecule in the plant. Without wishing to be bound by theory, in the context of the present invention, it is assumed that the inheritance of both resistance in the host and the pathogen's ability to cause disease is controlled by pairs of matching genes. One is a plant gene called the resistance (R) gene. The other is a parasite gene called the avirulence (Avr) gene. Plants producing a specific R gene product are resistant towards a pathogen that produces the corresponding Avr gene product (or avirulence factor). Generally, the term avirulence factor is used for fungi, bacteria, oomycetes en nematodes. In virology the term elicitor is generally used. The term "effector" is commonly used to indicate a potential Avr gene for which the cognate R gene is not yet known.

As used herein, the term "resistance response" includes reference to the display of a resistance phenotype following pathogenic challenge, or following Avr protein contact or Avr gene expression as indicated herein. Such phenotype may be, for example, the extent, rate of progress, or degree of occurrence of a necrotic reaction in the plant tissue.

As used herein, the term "pathogen" refers to any plant pathogen including a plant pathogenic virus, a plant pathogenic bacterium, a plant pathogenic fungus, a plant pathogenic oomycete, a plant pathogenic nematode and a plant pathogenic arthropod. Pathogens used in aspects of this invention may include, but are not limited to plant pathogenic viruses, such as Potato virus X, plant pathogenic oomycetes, such as Phytophthora infestans, P. sojae and P. ramorum, plant pathogenic bacteria such as Pseudomonas syringae, plant pathogenic fungi such as Cladosporium fulvum, and plant pathogenic nematodes, such as Globodera pallida and G. rostochiensis.

As used herein, the term "selecting" refers to the process of physically isolating or harvesting a plant from a heterogeneous population of plants in order to further propagate the selected plant.

"R gene variant of interest" refers to an R gene that is capable of conferring a desired pathogen resistance response to a plant of interest. Both the artificial form selected upon in vitro evaluation in a test plant, as well as the natural counterpart are included in the term. Although the resistance response of the "in vitro" form and the "in planta" form are expected to be the same or similar, they do not necessarily share identical gene sequence as will be explained in more detail herein.

As used herein, the term "determining the sequence" is used in a broad sense and refers to any technique known in the art that allows the order of at least some consecutive nucleotides in at least part of a nucleic acid to be identified, including without limitation at least part of an extension product or a vector insert. Exemplar sequencing techniques include direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole- genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, etc..

As used herein, the term "genetic engineering techniques" or "recombinant DNA techniques" refers to any and all methods of genetic engineering, transformation and genetic modification involving alterations in a gene sequence due to intervention by man as known to one of skill in the art, and the terms are all used herein as synonyms for the modification and transfer of isolated or artificially synthesized and optionally cloned genes into the DNA, usually the chromosomal DNA or genome, of another organism, generally, but not exclusively, by the use of vectors.

The term "nucleotide sequence comparison" or "comparing nucleic acid sequences" in the context of this invention refers to the process of aligning two or more DNA sequences and evaluating the similarities and differences between the sequences with the intent to identify unique differences.

As used herein, the term "polymorphic marker" refers to segments of DNA that exhibit variation in a DNA sequence between distinct genes. Such markers include, but are not limited to, single nucleotide polymorphisms (SNPs), restriction fragment length polymorphisms (RFLPs), short tandem repeats, such as di-, tri- or tetra-nucleotide repeats (STRs), unique mutations, deletions or additions, and the like. Polymorphic markers according to the present invention can be used to specifically differentiate between known or parental R gene sequences and novel sequences of R gene variants of interest, and include mutations. In essence, a polymorphic marker is suitable for detection purpose whereby the presence of the polymorphic marker is indicative of a specific nucleic acid variant. The term "nucleotide

polymorphism" is broadly drawn to sequence variation between two nucleotide sequences that encode different R genes, wherein the position of dissimilarity between the sequences denotes the polymorphic position. Very suitable, the nucleotide polymorphism can be a single nucleotide polymorphism.

The term "single nucleotide polymorphism (SNP)" refers to a DNA sequence variation based on a single nucleotide difference (i.e. A, T, C, or G).

The term "unique" as used herein with reference to an R gene variant of interest is to be understood as referring to distinctive for the purpose of specific detection.

The term "linkage disequihbrium" refers to any degree of non- random genetic association between one or more allele(s) of two different polymorphic DNA sequences and that is due to the physical proximity of the two loci. Linkage disequihbrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain

unseparated for several generations with the consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby. Hence, testing of one of the DNA polymorphism (or marker) will give almost the same information as testing for the other one that is in linkage disequilibrium. This situation is encountered throughout the plant genome when two DNA polymorphisms that are very close to each other are studied.

The term "crossing" refers to the mating of two parent plants by cross pollination, wherein "cross-pollination" refers to the fertilization by the union of two gametes from different plants.

The term "controlled cross" (and the corresponding term "controlled selfing"), as used herein, refers to the process of crossing (or selfing, respectively) of sequence-selected parent plants with the intent of increasing the chance of producing offspring plants harbouring specific sequence recombinations .

The term "selfing" refers to self-pollination of a plant, i.e., the transfer of pollen from the anther to the stigma of the same plant.

The term "genotyping" a sample or an individual plant for a polymorphic marker refers to determining the specific allele or the specific nucleotide carried by an individual at a polymorphic marker.

As used herein, the term "population" refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.

As used herein, the terms "population of candidate source plants" and "population of non- genetically modified plants" refers to any collection of plants or plant materials such as roots, shoots or seeds, which may be screened by a method of the present invention for the potential presence of a novel resistance response. The candidate source plants may be plant specimens or parts thereof collected or isolated from nature, obtained from plant germplasm collections, or may be plants under cultivation. Essentially they are non genetically modified. Description of preferred embodiments

Without wishing to be bound by any theory, it is believed that random sequence exchange between R genes or R gene homologues is a natural process that plays a crucial role in generating novel resistance specificities in nature. The present invention utilizes this natural mechanism. First, all available methods for the creation of novel gene sequences are exploited, including GMO techniques, to provide for a gene that may prove to exhibit a novel pathogen resistance profile. This gene is termed the "putative R gene" herein. Once such a putative R gene sequence has shown to confer a pathogen resistance profile of interest (e.g. novel resistance specificity or novel resistance level) in a test environment (e.g. in a N. benthamiana plant or in a test plant of the species of interest) a screening method is used to identify this particular R gene sequence of interest in the form of a similar or identical gene in a non-GMO plant. This non-GMO variant of the gene is herein also termed the "R gene variant of interest".

It is an aspect of this invention to greatly improve the chances of finding a plant of the species of interest that harbours the R gene variant of interest. For this purpose, a population of non-GMO plants for screening is provided in which the chance of occurrence of the R gene variant of interest is enhanced by providing a gene pool from which the R gene variant of interest may be newly created by recombination stretches or domains of individual R genes or R gene homologues. Preferably, these recombination events take place within the genome of a single plant of the species of interest. To provide a population of non-GMO plants for screening, any available method for the creation of novel gene sequences may be employed, with the exclusion of GMO techniques. Such non-GMO techniques may be used to support the formation of the R gene variant of interest as a recombination event in one or more plants of a population, and may include in particular natural crossing and selfing of plants, optionally in combination with random or targeted

mutagenesis. Crossing can in particular be used to collect different R genes or R gene homologous into a single genome.

In one embodiment, the present invention is directed to a screening method for identifying a non-transgenic plant having a resistance response of interest, e.g., a resistance to a novel pathogen or an improved resistance. The method of the invention comprises providing a putative R gene, preferably by recombination of existing R gene sequences and determining the resistance response to a pathogen of a plant heterologously expressing said putative R gene; selecting a putative R gene that provides a resistance response of interest; determining the sequence of the putative R gene; identifying the said sequence in a non-transgenic plant, wherein the putative R gene in a non- transgenic plant is referred to herein as an R gene variant of interest. The unique sequence pattern of the R gene variant of interest is subsequently identified in a non-transgenic plant. It may then be optionally confirmed whether the gene products of the putative R gene and of the R gene variant of interest have substantially the same amino acid sequence over at least a part of their sequence, in particular the part that is associated with resistance, and, thus, exhibit substantially the same resistance response as observed for the putative R gene product.

In preferred embodiments, the step of determining the resistance response of a plant carrying a putative R gene can be carried out in a plant that transiently expresses the putative R gene. A suitable transient expression system is for instance an agroinfiltration system. In an alternative or additional preferred embodiment, the step of determining the resistance response of a plant carrying a putative R gene can be carried out in a plant where the response is conferred on the plant by the heterologous expression in that plant of both a putative R gene and the pathogen effector (e.g., an avirulence gene). In an alternative or additional preferred embodiment, the determining step can be carried out in a plant that heterologously expresses the putative R gene and is infected with the pathogen. Generating the putative R gene

The present invention is directed to a method comprising a step (a) of providing a transgenic plant expressing a putative R gene. The putative R gene may be generated in different ways. In certain embodiments, the putative R gene is generated by the substitution of nucleotide sequences of a first R gene with the nucleotide sequences of at least a second R gene having a different nucleotide sequence. In other embodiments, the putative R gene is generated by random or targeted mutagenesis (insertion, deletion,

substitution) of the nucleotide sequence of a single R gene. Also, both methods may be combined. Targeted mutagenesis can be performed by any method known to one of skill in the art. Suitable methods include, but are not limited to Zinc Finger Nuclease technology (Zhang et al., 2010. Proc. Natl. Acad. Sci. USA 107: 12028-12033; Oligonucleotide directed mutagenesis (ODM);

Meganuclease technique (Gao et al., 2009. The Plant Journal 61 (1): 176-187); TALE technique (Transcription activator-like effector proteins) (Miller et al., 2011. Nature Biotechnology 29: 143-148).

The putative R gene is preferably a chimeric gene, preferably a chimeric NB-LRR gene. In an especially preferred embodiment, the putative R gene is obtained by substituting nucleotide sequences (or even fragments) from a first NB-LRR class R gene (or R gene homologue) with nucleotide sequences or fragments from a second, different, NB-LRR class R gene (or R gene homologue). Thus, in a preferred embodiment, step (a) of a method of the invention comprises the provision of constructs comprising a putative R gene that represents a recombination of at least two different R genes each encoding a different NB-LRR resistance protein.

In general, the putative R gene can be generated from, for instance, at least two known R genes using standard recombinant techniques; and the resulting putative R gene can be transiently or stably expressed in a plant to thereby provide a transgenic plant expressing a putative R gene.

As stated, putative R genes can be obtained using standard methods known in the art for recombining sequences (i.e. by recombinant DNA technology or by natural recombination resulting from meiosis), and the resistance specificities conferred by the putative R gene can be identified by in vitro or in vivo (in planta) tests. In particularly preferred embodiments, the putative R gene can be generated by swapping or exchanging distinct domains, subdomains or parts thereof found in different R protein molecules or from another part of the same molecule resulting in a new genetic combination by using standard techniques from recombinant DNA technology. Any two or more R genes can be recombined in embodiments of this invention to generate putative R genes. Preferably, R genes from a single class can be recombined. In one embodiment, a single NB-LRR gene from a cluster of NB-LRR genes with a known resistance response (for instance a known resistance specificity, indicating that the specificity is to a known pathogen), is selected for recombination with another NB-LRR R gene. The main advantage of using a NB-LRR gene with a known resistance response is that the specificity and changes thereto can be tested in vitro, e.g. by comparing the transient expression of the known R gene with the transient expression of the putative R gene in the same plant. This may for instance be done as described herein by expressing both the R gene and its cognate pathogen avirulence gene in leaves and comparing the necrotic response in both instances. Further, in cases where a putative R gene is developed for generating a specific resistance response to a novel strain of a particular pathogen, it is useful to choose as the first R gene (or parental R gene, that is the starting point for generating the putative R gene) an R gene with a known resistance response (preferably a known resistance response to a different strain of the particular pathogen), since it is more efficient to start with an R protein that already confers resistance to one or more other strains of that particular pathogen. Pathogens often produce avirulence proteins that are related in structure and/or function, which increases the chance (small evolutionary step) to identify a novel R gene with an altered specificity for a related avirulence protein.

Alternatively, one can start with an R gene conferring resistance to a taxonomically unrelated pathogen species. For example, Rxl and Gpa2, two resistance genes in potato, have a high sequence identity while conferring resistance to unrelated pathogens, i.e. to a virus and to a nematode,

respectively. Further, NB-LRR genes with unknown resistance specificities may be selected for recombination on the basis of prior knowledge, for example, specific scaffolds, certain motifs or the 3-D structure. Once a first R gene has been selected, a second R gene with which the first is to be (re)combined is selected. Like in the first selection, any R gene can be selected at this point; however, preferably the second R gene is in the same class of R gene as the first R gene. For example, where an R gene of the NB-LRR class is selected, the second R gene is also of the class of NB-LRR R genes.

In an alternative preferred embodiment, the first and second R gene are homologous genes from the same cluster on a single chromosome, most preferably located at complex loci harbouring several tandemly repeated NB- LRR homologs.

In yet another preferred embodiment, the second NB-LRR-class R gene may be selected from a homologous cluster of NB-LRR genes on another haploid chromosome (i.e. a NB-LRR gene in repulsion phase). In cases where randomly selected candidate source plants (e.g. wild accessions) will be screened for novel resistance responses, genes in coupling phase may be used as well because it is unlikely that this coupling phase is maintained in all genotypes. Current theory says that genes in repulsion phase (i.e. on different chromosomes), are more likely to produce chimeras via recombination, gene conversion, and unequal cross-over, thus such a chimeric gene is more likely to be found in a non-transgenic plant.

Construction of putative R genes can involve swapping small regions of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids or larger regions of 60, 70, 75, 70, 90, 100, 125, 150, 200 amino acids (or corresponding triplets of nucleic acid bases) from one R gene into another. Further, recombinants can be made by swapping whole domains or subdomains between the two R proteins. For example, R genes of the NB-LRR family contain a nucleotide-binding domain (NB) fused to the C-terminus of a leucine-rich repeat (LRR) domain. The NB is part of a larger domain that is called the NB-ARC, as the NB domain is also found in the apoptotic protease-activating factor 1 (APAF-1), resistance (R) proteins found in plants and the apoptotic protein CED-4 found in the nematode Caenorhabditis elegans. The N-termini of NB-LRRs are structurally diverse. Some carry a domain having homology to the Toll and human interleukin-1 receptor (TIR) domain and these R proteins are called TIR-NB- LRRs or TNLs. Non-TIR NB-LRR members are referred to as CC-NB-LRRs or CNLs, because many of them contain a predicted Coiled Coil region (CC). The CC region is sometimes extended by a DNA binding domain such as a

BEAF/DREAF zinc finger domain (BED) or by a Solanaceous Domain (SD). Any of the foregoing domains can be recombined with each other to make a putative R gene. For example, the LRR region from a first R gene can be swapped with the LRR region in a second R gene, wherein the first and second R gene are genes with a different nucleotide sequence, or the NB region from a first R gene (or homologue) and the LRR region of a second R gene (or homologue) can be swapped into the backbone of a third R gene (or homologue) by sequence exchange. Preferably, the parts of the LRR domain are swapped or exchanged, because this is considered to constitute the main pathogen- specificity-determining region; however, sequence exchanges in all domains may lead to novel resistance responses. The recombinants can be of any type of chimera; ranging from equal recombinants, unequal recombinants to chimeras containing stretches of variable length that are inserted from the NB-LRR gene into the R gene (and vice versa).

Exemplary R genes that can be used to create putative R genes include, but are not limited to Rx from Solanum, Gpa2 from Solanum, Rpl-A from Maize, Rpl-B from Maize, Rpl-C from Maize, Rpl-D from Maize, Rpl-E from Maize, Rpl-F from Maize, Rpl-G from Maize, Rpl-H from Maize, Rpl-I from Maize, Rpl-J from Maize, Rpl-K from Maize, Rpl-L from Maize, Rpl-M from Maize, Rpl-N from Maize, Rp5 from Maize, Rp6 from Maize, L from flax, LI from flax, L2 from flax, L3 from flax, L4 from flax, L5 from flax, L6 from flax, L 7 from flax, L8 from flax, L9 from flax, Lll from flax, Cf-2 from tomato, Cf-4 from tomato, and Cf-9 from tomato. The R genes used to make putative R genes can be from any plant species in which such R genes are expressed, for example, from any non- transgenic species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant.

Procedures for preparing putative R genes as described herein such as by domain swaps, sequence exchange or site-directed mutagenesis are described inter alia in Slootweg et al. 2013 Plant Physiol. Vol. 162, pp. 1510- 1528, in the Thesis of E.J. Slootweg, 2009, Thesis Wageningen University, The Netherlands, ISBN 978-90-8585-467-8, in particular Chapter 3 therein, and in the in the Thesis of K.B. Koropacka, 2010, Thesis Wageningen University, The Netherlands, ISBN 978-90-8585-606-1, in particular Chapter 3 therein.

Producing the transgenic plant comprising the putative R gene

Methods for producing a transgenic plant having a putative R gene will generally involve the use of plasmids maintained in strains of, for instance, E. coli bacteria or Agrobacterium species, and the putative R genes produced in such cells then need to be transferred into a suitable host plant in order to produce the transgenic plant comprising the putative R gene.

Transgenic methods of producing a transgenic plant that involve the transfer of a nucleic acid sequence comprising a putative R gene are well known in the art and may involve transgenic methods for plant

transformation, using for instance a vector, or in any other suitable transfer element, such as a bombardment with a particle coated with said nucleic acid sequence.

Plant transformation generally involves the construction of a vector with an expression cassette that will function in plant cells. In the present invention, such a vector consists of a nucleic acid sequence that comprises a putative R gene, which vector may comprise such a gene that is under control of or operatively linked to a regulatory element, such as a promoter. The expression vector may contain one or more such operably linked gene/regulatory element combinations, provided that at least one of the genes contained in the combinations is a putative R gene. The vector(s) may be in the form of a plasmid, and can be used, alone or in combination with other plasmids, to provide transgenic plants capable of expressing the putative R gene, using transformation methods known in the art, such as the

Agrobacterium transformation system.

Expression vectors can include at least one marker gene, operably linked to a regulatory element (such as a promoter) that allows transformed cells containing the marker to be either recovered by negative selection (by inhibiting the growth of cells that do not contain the selectable marker gene), or by positive selection (by screening for the product encoded by the marker gene). Many commonly used selectable marker genes for plant transformation are known in the art, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor. Several positive selection methods are known in the art, such as mannose selection. Alternatively, marker-less transformation can be used to obtain plants without mentioned marker genes, the techniques for which are known in the art.

One method for introducing an expression vector into a plant is based on the natural transformation system of Agrobacterium (See e.g. Horsch et al., 1985). For this, the putative R gene can be placed in a binary vector system wherein the putative R gene is placed in the location of the transfer DNA (T- DNA) of a plant tumor-inducing plasmid of Agrobacterium tumefaciens that also contains a helper Ti plasmid containing a virulence (vir) region, or other plasmid suitable for expression of the putative R gene in a plant. In one preferred embodiment, the putative R gene can be transiently expressed in leaves of a plant, preferably together with an avirulence gene from a pathogen.

Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant cells with Agrobacterium tumefaciens. Descriptions of Agrobacterium vectors systems and methods for

Agrobacterium-mediated gene transfer are provided in US Pat. No. 5,591,616. General descriptions of plant expression vectors and reporter genes and transformation protocols and descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer can be found in Gruber and Crosby, 1993. General methods of culturing plant tissues are provided for example by Miki et al., 1993 and by Phillips, et al., 1988. A proper reference handbook for molecular cloning techniques and suitable expression vectors is Sambrook and Russell, 2001.

The transgenic plant expressing the putative R gene may thus be the result of a stable transformation or, alternatively, may be the result of a transient transformation of the plant. A highly preferred embodiment in aspects of this invention involves transient expression of the putative R gene using the Agrobacterium tumefaciens transient transformation assay

(agroinfiltration) as described in Van de Hoorn et al., MPMI Vol. 13, No. 4, 2000, pp. 439-446.

Another method for introducing an expression vector into a plant is based on microprojectile-mediated transformation (particle bombardment) wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Another method for introducing DNA to plants is via the sonication of target cells. Alternatively, liposome or

spheroplast fusion has been used to introduce expression vectors into plants. Direct uptake of DNA into protoplasts using CaC precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Electroporation of protoplasts and whole cells and tissues has also been described.

Other well known techniques such as the use of BACs, wherein parts of the genome are introduced into bacterial artificial chromosomes (BACs), i.e. vectors used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells, based on naturally occurring F-factor plasmid found in the bacterium E. coli may for instance be employed in combination with the BIBAC system to produce transgenic plants.

Following transformation of target tissues, expression of the above described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.

Determining the resistance response of the transgenic plant

The present invention is directed to a method comprising a step (b) of selecting a transgenic plant that exhibits a pathogen resistance response of interest. Preferably, a pathogen resistance response of interest refers to a resistance response that differs from that of the original non-transgenic plant prior to expressing the putative R gene. The transgenic plant may be a plant that transiently expresses the putative R gene, for instance as is the case in agroinfiltration experiments. The pathogen resistance response of interest may refer to a response wherein the plant exhibits higher levels of resistance, or wherein the plant exhibits a novel pathogen specificity, i.e. exhibits resistance to a novel pathogen. Alternatively, a pathogen resistance response of interest may refer to a response wherein the plant exhibits a different pathogen specificity from that of the non-transgenic plant.

In a preferred embodiment, the step (c) of determining the pathogen resistance response of said transgenic plant is performed by providing a test plant, for instance a N. benthamiana plant or a plant of the species, variety, accession or cultivar of interest, that (transiently) expresses the putative R gene and exposing the transgenic plant to a pathogen effector, such as inoculating said plant with the pathogen. The step of exposing the test plant to a pathogen effector includes reference to exposure to the pathogen. In an alternative preferred embodiment, the step (c) of determining the resistance response of said transgenic plant to said pathogen is performed in a transgenic plant which, in addition to (transiently) expressing a putative R gene, also expresses a heterologous pathogen effector. This provides for an efficient and rapid assay for determining whether the putative R gene confers a resistance response of interest. Such assays may optionally be replaced or supplemented by an assay involving the inoculation with the pathogen itself and the observation of the resistance response to that pathogen.

In preferred embodiments, the resistance response to a pathogen conferred by the putative R gene can be determined using in vitro or in vivo (in situ) tests.

The resistance response to a pathogen can for instance be determined by detecting a necrotic reaction in the transgenic plant (or a tissue thereof) when, under conditions that the putative R gene is present and expressed in said plant (or said tissue), said plant or tissue is contacted with an avirulence factor. The extent of the necrosis may be measured

quantitatively or qualitatively.

The pathogen can be of any type that infects a plant and elicits an immune/resistance response in conjunction with an R gene, such as a bacterial pathogen, a viral pathogen, a fungal pathogen, a parasite, a nematode, etc. An avirulence gene of a pathogen can be any gene expressed by the pathogen and the avirulence factor presented by the pathogen or heterologously expressed in the plant may interact with the R gene such that a resistance response is produced.

In order to evoke a notable resistance response in the transgenic plant or in the candidate source plant one may bring about a HR response in the plants containing the putative R gene or R gene variant of interest. A HR response may be brought about by contacting the plant with an avirulence factor.

As indicated, the avirulence factor can be contacted with the plant in the form of the pathogen itself. Preferably, in the case of the transgenic plant, it can be heterologously expressed in the transgenic plant by expressing in said plant a transgenic avirulence gene of the pathogen. The avirulence gene to be expressed in a transgenic plant used in aspects of the invention can be selected from known avirulence genes (also referred to as effectors or elicitors). The skilled person will understand that embodiments of the present invention include i) the introduction or reconstruction of existing R genes that exist in different species or genotypes into new target cultivars of interest, and ii) the finding of entirely new R genes with novel resistance specificities. In case of existing R genes, the avirulence gene will often be known. In case of novel R genes, however, this gene may encode an R protein that directly or indirectly recognizes any pathogen derived molecule or pathogen-induced disturbance of the plant metabolism. This can be avirulence factors and effectors in the classic sense or other pathogen (derived) molecules, that cause an effect in the plant.

The present invention is ultimately intended to be part of methods for producing plants with novel pathogen resistance traits, such as resistance against newly identified pathogens or novel strains of known pathogens.

Depending on the type of resistance that is sought after, the skilled person will select the suitable pathogen effector that is to be used in the resistance response assays in methods of the present invention. These pathogen effector may be viral coat proteins, the elicitors harpin, syringolin or other elicitors of phytopathogenic bacteria, RXLR effectors of plant pathogenic oomycetes, the elicitor proteins from plant pathogenic fungi and yeast, the RAN-binding proteins from plant pathogenic nematodes, and the avirulence proteins of insects, but other avirulence factors may also be used.

Determining the sequence of at least a part of the putative R gene

Once a transgenic plant with a putative R gene is identified that exhibits a desirable pathogen resistance response, one has concomitantly identified a putative R gene that is associated with said resistance response of interest. The nucleotide sequence of at least a part, but preferably the entire nucleotide sequence, of said putative R gene in said selected plant may then be determined in order to identify nucleotide polymorphisms that characterize the novel putative R gene. The skilled person will understand that when the putative R gene was obtained by recombination of at least 2 parental R genes a sequence comparison may be made between the putative R gene and its at least two parental R genes. The skilled person will also understand that when the putative R gene was obtained by mutagenesis or de novo synthesis, a sequence comparison may be made between the putative R gene and the original R gene from which it was obtained via mutagenesis, or between the sequence of an R gene exhibiting the highest level of sequence similarity to the putative R gene. Such a comparison may result in the identification of nucleotide polymorphisms that characterize the novel putative R gene, and such polymorphisms are thereby associated with the resistance response of interest. Thus, in a preferred embodiment of a method of the invention, the identified putative R gene is sequenced, and the sequence information is used to find putative R gene-specific polymorphisms, which polymorphisms, in turn, can be used as markers for the generation of putative R gene-specific primers or probes, which in turn can be used to screen for plants having the same polymorphisms and having the same or a sequence of high similarity, that is likely to harbour a native or naturally occurring form of the R gene associated with the resistance response of interest.

Sequencing and Primer Design

Once a putative R gene demonstrating a resistance response of interest has been selected, i.e., producing a novel resistance response against a certain pathogen or pathogen effector, the putative R gene is isolated from the plant and sequenced using methods well known in the art. Once the sequence has been determined, non-transgenic plants encoding and expressing an R gene variant of interest having substantially the same nucleotide or amino acid sequence and thus substantially the same activity can be obtained by using the sequence information. As used herein, substantially the same nucleotide or amino acid sequence means having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% nucleotide or amino acid sequence identity when comparing whole R gene (product) sequences, respectively, between the R gene variant of interest or the expressed R gene variant of interest product and the putative R gene or encoded putative R gene product, respectively. As used herein, substantially the same nucleotide or amino acid sequence may also mean having at least 50%, 60%, 90%, 95%, 97%, 98%, 99% or 100% nucleotide or, preferably, amino acid sequence identity of the specificity-determining domain when comparing fragments of R gene sequences, respectively, between the R gene variant of interest or the expressed R gene variant of interest product and the putative R gene or encoded putative R gene product, respectively. Such fragments may have a size of up to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the R gene (product) sequences. Substantially the same resistance response activity refers to the resistance response in the non-transgenic plant to a pathogen or pathogen effector that is 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 100%, 110%, 115%, 120%, 125%, 130%, 140%, 150% or more of the resistance response to the pathogen or pathogen effector in the plant

heterologously expressing the putative R gene, such activity being, for example, the necrotic reaction produced in the plant tissue.

As used herein, any term referring to "percent sequence identity", such as "amino acid identity" refers to the degree of identity between any given query sequence and a subject sequence.

Specifically, the following terms are used to describe the sequence relationships between two or more nucleic acids, polynucleotides or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence. As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. When reference is made to sequence identity or sequence similarity herein, the two terms are used interchangeable, unless otherwise indicated.

The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 99%, or 100% identity in pairwise comparison), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.

The phrase "substantially identical ", in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least about 85%, identity, at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid residue identity, when compared and aligned pairwise for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In an exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 50 nucleotides in length. In another exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 100 nucleotides in length. In still another exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 150 nucleotides or more, in length. In one exemplary embodiment, the sequences are substantially identical over the entire length of nucleic acid or protein sequence.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually 20 to 50, about 50 to about 100, about 100 to about 200, more usually about 100 to about 150, or of about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 3000 or even more in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Percent identity can be determined using methods of alignment of sequences for comparison and identification of identities, which are well- known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981, Adv. Appl. Math. 2: 482); by the homology alignment algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48: 443); by the search for similarity method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. 85: 2444); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL (in the PC/Gene program by Intelligenetics, Mountain View, Calif.) and GAP, BESTFIT, BLAST, FASTA, and TFASTA (in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA); the CLUSTAL program is well described by Higgins and Sharp, 1988 (Gene 73: 237-244); Higgins and Sharp, 1989 (CABIOS 5: 151-153); Corpet et al, 1988 (Nucleic Acids Research 16: 10881-90); Huang et al., 1992 (Computer Applications in the Biosciences 8: 155-65); and Pearson et al., 1994 (Methods in Molecular Biology 24: 307-331).

In order to identify the R gene variant of interest in a candidate source plant, the skilled person will appreciate that sequence-specific probes or amplification primers may be designed that are able to hybridize and

specifically bind under stringent, preferably high stringent, conditions to both the putative R gene and the R gene variant of interest and/or capable of selectively amplifying both the putative R gene and the R gene variant of interest. For this it is essential that unique nucleotide polymorphisms and/or unique combinations of nucleotide polymorphisms are identified within the putative R gene, by comparing the sequence of the putative R gene with known R genes, with the R genes of the plants from which the recombinant was prepared, and/or with R genes and/or R gene homologues in the candidate source population. Such a comparison can be performed using the methods of alignment of sequences and the methods for comparison of aligned sequences as described above. Such sequence comparison may reveal nucleotide

polymorphisms to which the sequence-specific primers or probes can be designed, such that the primers or probes are capable of hybridizing under stringent, preferably high stringent, conditions to the nucleotide

polymorphisms and capable of selectively amplifying at least a fragment of the R gene variant of interest. The skilled person is well aware how such design may occur, and very suitable methods include eyeball comparison.

The nucleic acid primers are designed such that the R gene variants of interest can be detected, for instance by using nucleic acid amplification techniques, preferably a PCR assay using a pair of bidirectional primers, and the skilled person knows how such primers or probes can be optimized depending on the protocol in which they are to be used, such as blot

hybridization with genomic or cDNA, or in SNP arrays or by the use of PCR methodology. Since the screening is based on nucleotide sequence, any plant tissue containing DNA of the plant can be used in the screening assays, such as leaves, seeds, as well as cultured plant cells.

Primer design for the detection of the R gene variants of interest is a routine procedure in molecular genetics, in particular if based on detection of SNPs.

The design of SNP-specific detection procedures to screen candidate source populations is preferably as follows. A multitude of chimeric

combinations of R gene homologues with known and/or unknown specificities from a single R gene cluster are tested in the form of putative R genes for their pathogen resistance response in a transient Agrobacterium infiltration assay with a certain pathogen effector of interest. Upon optional confirmation of the pathogen resistance response of a putative R gene of interest via production of a transgenic plant stably transformed with said putative R gene of interest and inoculation of said plant with the life pathogen, a sexual crossing is performed wherein one of the parents contains the above indicated R gene cluster. In the offspring population unique combinations of SNPs will be present. Such unique combinations are not present in the R gene homologues of the said parental R gene cluster. Hence, the primer design may in one embodiment be directed towards detecting the unique combinations of SNPs. These SNPs represent preferably amino acid-changing sequence alterations in the R protein.

The primers do not necessarily have to be directed to amino acid- changing sequence alterations in the R protein. It will be understood that a positive amplification indicates a new association, and the resultant linkage will dictate that also other SNPs, adjacent to the sequence-changing SNPs, are encountered that may be used as targets for detection of the R gene variant of interest. It is also possible to sequence the complete amplification product in order to gain insight into the length and nature of the sequence exchange.

It will be possible to create additional SNPs in the offspring of the candidate source plants by treating the seed of the primary cross with non- GMO mutation technology (random and/or targeted mutagenesis), which additional SNPs in the offspring that do not occur in the parental R gene homologues but which may result in an improved resistance response. In that case, the primer design or other screening method will be directed towards detecting the unique combinations of SNPs as well as detecting the additional unique SNP in the candidate source plants. It is also possible to create the additional SNP in a second crossing, such that the R gene variant of interest is selected from the offspring of a first cross, and the gene is subsequently subjected in a second cross to a non-GMO mutation technology in order to obtain the additional SNPs, e.g. via EMS treatment of seed followed by another round of selection.

For this reason, candidate source plant populations are preferably obtained by performing controlled crosses or selfings because the familiar genetic background provides basic knowledge about the sequence of the R genes and R gene homologues, thus facilitating the screening.

Providing candidate source plants comprising R gene variants of interest As indicated, a candidate source plant may include any plant that may serve as a donor of a natural variant of the R gene variant of interest, and hence, that is a potential carrier of pathogen resistance response of interest. Such plants may be found among similar species to those to which the transgenic plant belongs, or it may be a different species. The skilled person will appreciate that the candidate source plant is preferably of a species that is crossable with the species or variety into which the R gene variant of interest must be transferred by use of non-recombinant (non-GMO) techniques, e.g. by introgression (crossing), whereas the transgenic plant used in aspects of the invention is preferably of a species the genes of which can be easily

manipulated in order to easily produce the transgenic plants having the putative R gene(s). Hence, transgenic plants used in aspects of the invention are not necessarily of the same variety or even species as the candidate source plants.

The population of candidate source plants may consist of one or more plants, preferably more than two plants, most preferably several hundreds of thousands or even millions of plants.

In certain embodiments of the invention, the population of candidate source plants can be the progeny of a cross between plants of the species or lines that contain the original parental R genes used to make the (chimeric) putative R gene. In other embodiments, the population of candidate source plants can be the progeny of a selfing of one of the plants of the species or lines that contains the original parental R genes used to make the (chimeric) putative R gene. In yet other embodiments, following or preceding the production of the candidate source plants by controlled crosses or selfings, the candidate source plants, or the parents of the cross or selfing, are subjected to random mutagenesis, for instance by subjecting the plants, seeds or embryos to chemical mutagens (ethyl methanesulfonate, EMS) or ionizing radiation. Such techniques are well known in the art. One step of a process of the invention comprises optionally

subjecting the parental R gene homologues to random mutagenesis, either before or after stimulating the genetic recombination process via sexual reproduction. The step of random mutagenesis when performed after the genetic recombination process via sexual reproduction allows for the

optimization of the R gene variant of interest sequence in candidate plants. For instance, when it is found that the offspring plants of the cross for producing the population of candidate source plants contains a sequence that is almost identical to the putative R gene but does not provide for the resistance pattern of interest, the use of random mutagenesis may alter the sequences in the candidate source plants population such that the sequence of the R gene variant of interest is produced therein and the resistance pattern of interest is displayed in such plants.

Thus, in the process of selecting the plant comprising the R gene variant of interest with the resistance response of interest, artificial

mutagenesis may be used to produce that plant prior to its selection. Plants thus obtained are not considered as plants that are genetically modified under Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms, since "mutagenesis" is explicitly mentioned as an exemption to the Directive. Hence, candidate source plants obtained through crossing and mutagenesis are exempt from the regulatory provisions that exist for deliberate release of genetically modified (transgenic) plants in Europe.

When selecting the parents for production of the candidate source plant population, notice may be taken of the manner in which the putative R gene is produced or obtained. In case the putative R gene is produced by recombination through gene exchange resulting in production of chimeric R genes, then at least one of the parents will contain the clusters of the parental R genes/i? gene homologues. After all, the recombination event that will give rise to the chimeric recombinant is the result of a meiotic recombination in the creation of the gametes of one of the parents.

However, if the putative R gene is a genetically altered form of an existing R gene, for instance, from a different species or from a different genotype of the same species to that of the target genotype, but with inferior agronomical characteristics, or if it is a putative R gene conferring the resistance response of interest that is obtained through targeted and/or random mutagenesis, then an additional step is needed to select the proper parental R gene/i? gene homologues. This selection step suitably comprises the sequencing of the related R gene homologues and the selection of nucleotide stretches containing the SNPs of interest, preferably originating from at least two R genes/i? gene homologues that, together, produce the resistance of interest. In order to increase the chance of finding the nucleotide stretches containing the SNPs of interest which may not be present in a single parent, but which will be present in separate genotypes, one or more additional crossings may be required in order to collect the stretches of interest into a single parent, preferably in clusters on homologous positions on different haploid chromosomes.

In order to screen a population of candidate source plants for a nucleotide polymorphism associated with a resistance response of interest, the skilled person will appreciate that it is not necessary to provide whole plants or even plant tissues. It is sufficient to provide a collection of nucleic acid screening targets representing the parts of the candidate source plants genome in which the R gene variant of interest may be present. A suitable collection of nucleic acid screening targets is considered to be any carrier material of R gene variant of interest such as root material, shoot material or seeds of plants and the plants may be plant specimens or parts thereof collected or isolated from nature (a landrace or local variety), obtained from plant germplasm collections, or may be commercial cultivars. Detecting a plant having a nucleotide polymorphism associated with a pathogen resistance response of interest

Step (f) in a method of the invention comprises the step of detecting in a population of candidate source plants comprising R gene variants of interest a plant that contains the unique combination of SNPs or mutations associated with the resistance response of interest. This step is essentially a step of screening for R gene variants of interest that are equivalent to the putative R gene. Thus, once a putative R gene having a resistance response of interest is identified and a unique combination of SNPs or mutations associated therewith is found, a population of non-transgenic plants may be screened for that nucleotide polymorphism in order to identify non-transgenic plants that natively encode an R gene that is associated with that resistance response of interest.

Such screening may use, for example, methods known in the art for screening nucleotide polymorphisms, such as using PCR technology, or using standard nucleic acid hybridization techniques. This step can be easily adapted and performed as part of a high through-put assay, since a plurality of non- transgenic plants, or tissues obtained therefrom, can be screened for the presence of the unique combination of SNPs or mutations, and thus for the R gene variants of interest, using standard PCR techniques for the detection of nucleotide polymorphisms and mutations. By using standard genotyping techniques for detection of nucleotide polymorphisms in genes, a single plant from among 100 milhon plants can be readily singled out based on its genotype and tested for its resistance phenotype. Since the screening is based on detection of specific nucleic acid sequences, any plant tissue containing DNA of the plant can be used in the screening assays, such as leaves, seeds, as well as cultured plant cells.

In general, a very large number of plants need to be screened. It is estimated that tens of thousands to hundreds of thousands, even up to 10 million plants or more, such as 100 milhon plants may be screened in order to encounter the rare recombination event associated with the unique

combination of SNPs or mutations. This, however, poses no problem to one of skill in the art. Genotyping techniques are available that are able to screen hundreds of thousands and even millions of plants for a genetic signature. Given the reward of finding a novel resistance pattern in a natural plant, the level of skill of the artisan, and the advancements and ready availability of high throughput screening and high throughput sequencing techniques, the screening of millions of plants poses no undue burden to one of skill in the art.

Once the candidate source plants (or parts thereof) comprising the nucleotide polymorphism associated with the resistance response of interest have been identified, such plants can be sexually or asexually propagated and cultivated, and used as breeding parents in the breeding of plants having resistance traits of interest. For that is it preferred that the presence of the resistance response of interest associated with the R gene variant of interest that is equivalent to the putative R gene is confirmed in the plants selected by the genotypic screening.

The skilled person will appreciate that the full-length gene sequence of the R gene variant of interest is not necessarily identical to the putative R gene to confer the pathogen resistance of interest. The R gene variant of interest may comprise only a part of the unique combinations of SNPs and/or mutations of the putative R gene, and may comprise additional base

differences, and the corresponding protein may differ in one or more amino acids from the 'in vitro sequence' of the putative R gene and its protein product, respectively. Consequentially, the R gene variant of interest as detected in a candidate source plant may or may not confer to the detected plant the resistance response of interest. If it does, the plant may be selected as a source plant of a new pathogen resistance trait of interest. If it does not, the R gene variant of interest in said plant obviously has some additional sequence differences with the putative R gene, it may then be sequenced and its sequence may be adjusted in order to display the functionality of interest using mutagenesis.

Determining the resistance response of interest in the candidate plant

Preferably, the step (j) of confirming that the candidate source plant thus detected exhibits the pathogen resistance trait of interest and/or the R gene variant of interest conferring to said plant the pathogen resistance trait of interest includes the performance of a phenotypic resistance test wherein the resistance response of the candidate plant is compared to that of the transgenic plant. Said comparison can include a quantitative or qualitative comparison of said resistance response, and the result of the test may thus be a quantitative scale or qualitative gradation. In case of a measurement on a quantitative scale, the skilled person is able to determine whether indeed the resistance response as observed in the transgenic plant is indeed also present in the candidate source plant based on the fact that both exhibit a response on the same scale. The candidate source plant in any case exhibits the resistance response of interest when, following pathogenic challenge, Avr protein contact, or Avr gene expression as indicated herein, the resistance response of, or the resistance phenotype displayed by, the candidate source plant is at least substantially the same as that of the transgenic plant. The resistance response is at least substantially the same, when a quantitative measurement indicates that the resistance response in the non-transgenic plant is at least 50%, preferably 70%, more preferably even 80%, 90%, 95% or 99%, and at most 200%, preferably 150%, more preferably 125%, 110%, or 105% of the resistance response in the transgenic plant heterologously expressing the putative R gene.

Preferably, the resistance response test used in the transgenic plant is the same as that used in the candidate source plant.

Similar to the steps relating to the determination of the resistance response in the transgenic plant, the step of determining the resistance response of the candidate source plant to said pathogen may in a preferred embodiment be performed in a candidate source plant which in addition to expressing the R gene variant of interest also expresses an avirulence gene from the pathogen.

In an alternative and highly preferred embodiment, the step of determining the resistance response of said candidate source plant to said pathogen is performed in a candidate source plant which is contacted with the pathogen effector, for instance infected with the pathogen. Any part of the plant may be contacted with the pathogen effector, such as leave, stem or root tissue. Preferably the leaves of the candidate source plant are contacted with the pathogen effector.

In preferred embodiments, the resistance response to a pathogen conferred by the R gene variant of interest in the candidate source plant can be determined using in vitro or in vivo (in situ) tests.

A resistance response of interest to the pathogen can for instance be observed as a necrotic reaction in the candidate source plant (or a tissue thereof) when, under conditions that the R gene variant of interest is present and expressed in said plant (or said tissue), said plant or tissue is contacted with a pathogen effector or the pathogen. The extent of the necrosis may be measured quantitatively or qualitatively.

Producing a non-transgenic breeding line

The nucleic acid representing the R gene variant of interest may be transferred from the donor plant to a suitable recipient plant by any method available. For instance, the said nucleic acid sequence may be transferred by crossing a donor plant with a plant of selected breeding line which does not have the resistance trait of interest, e.g. of which the resistance is to be improved, i.e. by introgression. Alternatively, the R gene variant of interest may be transferred from donor to recipient plant by protoplast fusion, by a doubled haploid technique or by embryo rescue or by any other non-GMO nucleic acid transfer system, optionally followed by selection of offspring plants comprising the R gene variant of interest for the resistance trait of interest.

As should be understood herein, the transfer of the R gene variant of interest for the resistance trait of interest from any candidate source plant or donor plant as indicated herein to a recipient plant, may in any step in a method of the present invention be assessed by markers (the nucleotide polymorphisms associated with the resistance response of interest, or any other markers subsequently found) and/or by performing a phenotypic assay conforming that the recipient plant (for instance of an elite breeding line) exhibits the resistance trait of interest.

Improved plant varieties obtained through chemical or high-energy mutagenesis are used in many breeding programs. These varieties do not fall under the current umbrella of regulations monitoring GMOs. Enhancing the value of crops through gene targeting does not involve the insertion of foreign DNA and, therefore, should not be considered recombinant DNA technology. Instead, this is simply a mutagenesis approach, albeit a highly precise one, that can be appreciated as accelerating the normal evolutionary process (Oh & May, Current Opinion in Biotechnology 2001, 12:169-172). Suitable methods for genome editing include e.g. Zinc finger nuclease technology (Zhang et al., 2010. Proc. Natl. Acad. Sci. USA 107: 12028-12033), Oligonucleotide directed mutagenesis (ODM), the Meganuclease technique (Gao et al., 2009. The Plant Journal 61 (1): 176-187), and TALE technique (Transcription activator-like effector proteins) (Miller et al., 2011. Nature Biotechnology 29: 143-148). The plants identified by methods of the present invention are preferably obtained by techniques that escape GMO regulations. Hence, plants that harbor the chimeric R gene variant may have been subjected to targeted mutagenesis or genome editing in order to induce homologous recombination.

Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many

equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features and method steps described.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.

EXAMPLES Example 1. In vitro sequence exchange to produce putative R genes with novel resistance traits.

A series of novel putative R genes is generated by in vitro shuffling sequence elements of Rxl and Gpa2 (Figures 1, 2, 3, 4 and 6), and also by exchanging a sequence stretch between Rxl and an Rxl/Gpa2 homologue with unknown specificity (Figure 5). The Rxl and Gpa2 genes are located in the same R gene cluster in potato (Solanum tuberosum), and confer resistance to potato virus X (PVX) and the potato cyst nematode Globodera pallida, respectively. In the diploid potato clone SH83-92-488, Rxl and Gpa2 are in coupling phase together with the resistance gene homologue SH-RGHl and the pseudogene SH-RGH3 (see Figure 1, and Bakker et al. 2003, Theoretical Applied Genetics 106: 1524-1531). In SH83-92-488 at the same complex locus, but in repulsion phase four other Rx/Gpa2 homologues are located: the full length homologues SH-RGH5, SH-RGH6 and SH-RG7, and the pseudogene SH-RGH8 (Figure 1). Sequence exchange positions were randomly chosen or optimized by using the available structure -function data of the Rxl and Gpa2 proteins (Slootweg, 2009, "Structure, function and subcellular localization of the potato Resistance protein Rxl", Thesis Wageningen University). The transcription of the constructs was controlled by the CAMV 35S promoter and Tnos terminator sequences.

Functional analysis of the newly generated putative R genes is performed by the detection of a local hypersensitive response (HR) upon agroinfiltration in N. benthamiana leaves in the presence and absence of the Rxl elicitors CP106 (avirulent) and CP105 (virulent), the cap sid proteins (CPs) from the potexviruses Potato Virus X (PVX), or the Gpa2 elicitors (effectors) D383-1 (eliciting) and Rook4 (non-eliciting), the RA -binding proteins (RBPs) from G. pallida population D383 and population Rookmaker (Sacco et al. 2009, 5(8):el000564). Figure 3 shows that exchanging stretches of nucleic acids between Rxl and Gpa2 results in a variety of phenotypes. The degree of bleaching of the Nicotiana benthamiana leaves (Figure 4) was used as a measure on a scale of 0 to 5 for the strength of the defence response. Several sequence exchanges lead to loss of function (e.g. constructs no. 33, 34, 35), while other putative R genes lead to autoactivation (e.g. constructs no. 8, 9, 10, 11, 12, 13, 22, 23, 24, 25) (Figure 3) as observed by triggering a defence response when the chimeres (the putative R genes as referred to herein) are solely co-expressed with Green Fluorescence Protein (GFP).

As shown in Figure 3, various putative R genes thus obtained show a different recognition spectrum than the parental Rxl and Gpa2 genes.

Several putative R genes (e.g. construct 5, 6, 7, 13), when compared with the parental Rxl gene, show an enhanced hypersensitive response for CP 105, the coat protein of the virulent strain of PVX. For example, the putative R gene Gl2G3aR3bcR45 (construct 13) shows a broadened recognition specificity, i.e. this chimera recognizes both the coat protein of the avirulent (CP 104) and the virulent (CP 105) PVX strain. As shown in Figure 5, replacing the CC, NB, ARC 1 and ARC2 region of the Rxl gene by that of the homologue SH-RGH6 results in a putative R gene SH6CC/NBSRX1LRR that also recognizes both the coat protein of the avirulent (CP 106) and the virulent (CP 105) PVX strain.

To confirm the results of the transient expression in leaves of N. benthamiana, plants were transformed with the putative R gene G12R35 (construct no. 6). To mimic wild type expression levels, these newly generated R gene homologues were expressed under control of the native Rxl or Gpa2 promoter, which are virtually identical and can replace each other as shown in previous studies (Slootweg, 2009, Thesis Wageningen University). Inoculation of transgenic plants with the avirulent strain (PVX 106) and the virulent strain (PVX105) showed that the accumulation of the virus, as measured with ELISA (Slootweg, 2009, Thesis Wageningen University), is inhibited by the putative R gene G12R3R45. The virus level of PVX105 in the transgenes 2.2 and 2.4 expressing G12R3R45 was significantly lower than in the diploid potato clone SH83-92-488 (SH) and plants transformed with the empty vector (EV) (Figure 6). In addition, as shown in Figure 6, the putative R gene G12R3R45 preserved its ability to inhibit the accumulation of PVX106. The putative R genes, now being identified as providing R gene-type resistance, are hereinafter referred to as R gene variants of interest.

Example 2. Identifying unique combinations of SNPs and/ or mutations associated with novel resistance specificities.

Sequences of the novel R gene variant of interest as described herein are compared in a multiple alignment with the resistance genes Rxl and Gpa2, the Rxl/Gpa2 homologues SH-RGH1, SH-RGH5, SH-RGH6 and SH-RG7 and the pseudogene SH-RGH3. This comparison enables retrieving combinations of SNPs and other mutations that are unique for the R gene variant of interest SH6CC/NBS RXILRR when compared with the R genes and their homologues in the diploid potato clone SH83-92-488. To identify the R gene variant of interest with the desired broadened resistance specificity to PVX in an offspring population of a cross between SH83-92-488 and RH89-039- 16, a further selection of combinations of SNPs and/or mutations is made by adding in the alignment the Rx/Gpa2 homologues present in RH89-039-16. These are the homologues RH-RGH2, RH-RGH3, RH-RGH4 and RH-RGH5. (Bakker et al. 2003, Theoretical Applied Genetics 106: 1524-1531). This extended alignment enables identifying combinations of SNPs and/or mutations that are unique for the R gene variant of interest SH6CC/NBS RXILRR and that are associated with the novel resistance specificity. Figure 8 shows a multiple nucleotide

alignment in which the Informative Polymorphic Sites (IPS) are shown. The Informative Polymorphic Sites are the nucleotide positions where two or more nucleotides are different from the other nucleotides at the same position (Bakker et al. 2003, Theoretical Applied Genetics 106: 1524-1531). The novel broadened resistance specificity conferred by SH6CC/NBSRX1LRR is generated by the interaction between one or more polymorphisms seen in SH6CC/NBS between nucleotide position 14 and 1461 and one or more polymorphisms seen in RXILRR between nucleotide position 1488 and 3239. From the multiple alignments shown in Figure 8, it is not clear whether the novel resistance specificity is mediated by a single unique combination of SNPs, or by two or more unique combinations. However, the unique combinations of SNPs and/or mutations distilled from the multiple alignments all enable the identification of the R gene variant of interest that confers the desired resistance, because the sequence exchange tracks generated during sexual reproduction will span in general a series of polymorphisms (see Figure 7). This linkage between polymorphisms implies that many unique combinations of SNPs and/or mutations identified in the R gene variant of interest will be associated with the broadened PVX resistance specificity mediated by SH6CC/NBS RXILRR. Numerous unique combinations of SNPs and/mutations can be extracted from the multiple sequence alignment. Unique SNPs for Rxl in the LRR region are; 1495C, 1524A, 1535T, 1655A, 1662G, 1748A, 1859G, 2059C, 2133A, 2140A, 2140A, 2151A, 2261T, 2372G, 2427T, 2437G, 2512T, 2524A, 2567G, 2570A, 2585C, 2586G, 2670T, 2769C, 2835C, 3032T, 3188G, 3205G, 3219A and a unique deletion (TCC) at positions 2214, 2215, and 2216. No unique SNPs can be identified in the CC, NB, ARCl and ARC2 regions of SH- RGH6, demonstrating that no unique SNPs and /or mutations in both partners are required to generate novel resistance specificities and that the uniqueness of the combination of the SNPs and/or mutations is sufficient to confer the broadened resistance specificity to PVX. The unique SNPs for Rxl in the LRR region together with polymorphisms that are shared between two or more homologues can be used to identify unique combinations of SNPs and /or mutations that may be associated with the novel resistance conferred by the R gene variant of interest SH6CC/NBS RXILRR.

To identify R gene variants of interest with resistance to both PVX105 and PVX106 in plant populations with unknown parents, the alignment can be further extended to a collection of more than 77 Rxl/Gpa2 homologs previously obtained from 11 distinct Solanum species, including cultivated potato and tomato (Butterbach, 2007, Thesis, Wageningen

University). This alignment will enable to select combinations of SNPs and/or mutations that can be used to identify an R gene variant of interest with the desired resistance in plant populations with unknown parents or partly unknown parents, for example wild accessions and collections of not well- defined breeding material.

Example 3. Identification of rare R sene variants of interest conferring novel resistance in an offspring population with known parental R sene homolosues

The aforementioned alignments reveal unique combinations of SNPs and/or mutations that enable the identification of R gene variants of interest that potentially mediate resistance to PVX105 and PVX106. To select the desired R gene variant of interest, a population of non-genetically modified plants candidate source population is generated and screened. Hereto, an offspring population of a cross between SH83-92-488 and RH89-039-16 is used to retrieve the R gene variants of interest that are identical to or partly identical to the identified R gene variant of interest SH6CC/NBS RXILRR. The R gene variants of interest that potentially mediate the desired resistance are retrieved by identifying in the offspring seedlings combinations of SNPs and/or mutations that are unique for the identified R gene variant of interest

SH6CC/NBS RXILRR Preferably, the selected R gene variants of interest contain all polymorphisms observed in the CC, NB, ARCl and ARC2 region of the resistance gene homologue SH-RGH6 and all polymorphisms observed in the LRR region of Rxl. However, it is stressed that other closely related chimers that only contain a subset of the polymorphism observed in SH6CC/NBS RXILRR may also confer the desired resistance. It is expected that a variety of chimers will confer resistance to both PVX105 and PVX106, although they are only partly similar to SH6CC/NBS RXILRR.

The unique combinations of SNPs and/or mutations are used to design highly specific primer pairs, to amplify the naturally occurring R gene variants of interest resembling the identified R gene variant of interest

SH6CC/NBSRX1LRR with the PCR technique. Primer performance and specificity is tested on plasmid DNA harbouring the target sequence of R gene variant of interest SH6CC/NBS RXILRR A range of template concentrations is used to determine the minimal template concentration required for the specific amplification of the target sequence in genomic DNA isolated from the two parental potato genotypes SH83-92-488 and RH89-039-16.

Ten thousand (10,000) to one million (1,000,000) seeds from the offspring of a cross between the genotypes SH83-92-488 and RH89-039-16 are used to select R gene variants of interest that are similar to SH6CC/NBS RXILRR or contain a part of the polymorphisms observed in SH6CC/NBS RXILRR. TO obtain template DNA for high-throughput PCR screening of genotypes, DNA is extracted from leaves or other parts of the seedlings. In addition, it is possible to isolate genomic DNA from root cap cells released during seedling growth. A DNA pooling strategy is used to identify seedlings harbouring the desired R gene variant of interest. The amplification products are sequenced to search for stretches of sequences that are the result of in planta recombination events. Seedlings potentially harbouring the R gene variants of interest, i.e. that share combinations of SNPs and/or mutations that are unique for the identified and selected sequence of R gene variant of interest SH6CC/NBS RXILRR, are propagated for further analyses. The selected genotypes are inoculated with the avirulent strain (PV 106) and the virulent strain

(PVX105) and the accumulation of the virus is measured with ELISA

(Slootweg, 2009, Thesis Wageningen University). Resistant genotypes showing a novel recognition spectrum are propagated and used to produce non- genetically modified varieties with novel resistance properties.

The design of the primers can be further optimized by a mutational analysis of the R gene variant of interest SH6CC/NBS RXILRR to pinpoint combinations of SNPs and/or mutations that are required for mediating the broadened resistance specificity. It is expected that only a part of the observed polymorphisms in SH6CC/NBSRX1LRR is essential for conferring the broadened resistance specificity to PVX. This mutational analysis increases the

percentage of PCR selected seedlings that mediate both PVX105 and PVX106 resistance. The PCR based selection of seedlings containing R gene variants of interest can be further optimized by a multiplex PCR approach using two or more primer pairs. In addition, it is noted that various other recently developed high throughput methods are applicable as well to detect the desired R gene variants of interest in seedlings.

Example 4. Identification of rare R gene variants of interest conferring novel resistance in candidate source populations with unknown pedigrees. To identify R gene variants of interest in plant populations with unknown pedigrees, a similar strategy can be followed as described for the controlled cross between SH83-92-488 and RH89-039-16. The combinations of SNPs and/or mutations present in SH6CC/NBS RXILRR and that are absent in the 77 Rxl/Gpa2 homologs are used to design primer pairs and tested for an optimal design of the DNA pools. Plasmid DNA harbouring the target sequence SH6CC/NBSRX1LRR is mixed in varying concentrations with genomic DNA containing equal portions of DNA of the 11 wild Solanum species from which the 77 Rxl/Gpa2 homologs were isolated (Butterbach, 2007, Thesis,

Wageningen University). These primer pairs can, for example, be used to screen collections of breeding material, to screen collections of (wild) Solanum species present in gene banks ( e.g. CGN, Wageningen, The Netherlands) or wild accessions recently collected in the centre of origin. Here again the screening method can be optimized by a multiplex PCR approach using two or more primer pairs and /or using recently developed high throughput sequencing technologies.

Claims

1. A screening method for identifying a non-genetically modified plant of a species, variety, accession or cultivar of interest that exhibits a pathogen resistance response novel to said species, variety, accession or cultivar of interest, the screening method comprises the steps of:

R genes and R gene homologues;

(a2) a mutated R gene prepared by targeted and/or random

mutagenesis of an R gene and/or R gene homologue;

(a4) a synthetic R gene made by de novo synthesis of a DNA

sequence based on information from existing R genes and/or

R gene homologues, or

(a5) a combination of (al)-(a4);

(f) identifying from said nucleotide sequence comparison a combination of at least two polymorphic markers unique to said ? gene variant of interest;

(g) providing a non- genetically modified plant of said species, variety, accession or cultivar of interest as a parental plant amenable to selfing or outcrossing, wherein said plant harbours parental R genes and/or R gene homologues that are different from the R gene variant of interest but that together contain stretches of codons that, when combined in a single gene sequence, characterize the unique combination of at least two polymorphic markers of said R gene variant of interest;

(h) providing an offspring population of non- genetically modified plants of interest using the plant provided in step g) as a parent plant in a selfing or out-crossing step, while allowing recombination to occur between said at least two separate R genes and/or R gene homologues in said parent plant in the production of said offspring population;

(i) screening the genomes of the plants of said offspring population for the presence of a plant having said combination of polymorphic markers present in a single R gene or R gene homologue, to thereby provide a non- genetically modified plant comprising the R gene variant of interest, and

j) optionally confirming that the non-transgenic plant provided in step

(i) exhibits the pathogen resistance response of interest.

2. The method of claim 1, wherein the construct provided in step (a) comprises as the putative R gene a chimeric R gene as defined under (al).

3. The method of claim 1, wherein the construct provided in step (a) comprises as the putative R gene a transgenic R gene as defined under (a3), and wherein the test plant is a plant of said species, variety, accession or cultivar of interest.

4. The method of any one of the preceding claims, wherein the

transformation in step (b) is performed by agro-infiltration of a test plant or part thereof with Agrobacterium cells comprising a gene expression construct for expression of the putative R gene in said test plant, or, wherein the transformation and exposure in step (b) are performed simultaneously by agro- infiltration of a test plant or part thereof with Agrobacterium cells comprising a gene expression construct for simultaneous expression of the putative R gene and the pathogen effector in said test plant.

5. The method of any one of the preceding claims, wherein said

polymorphic marker(s) identified in step (f) represent amino-acid changing mutation(s) in the protein product(s) of said R gene or R gene homologue.

6. The method of any one of the preceding claims, wherein the step (g) of providing a non-genetically modified parental plant amenable to selfing or outcrossing, comprises the steps of:

(gl) providing a first optionally mutated non-genetically modified parental plant of said species, variety, accession or cultivar of interest, said plant comprising an R gene or R gene homologue comprising at least a first polymorphic marker of said combination of at least two polymorphic markers unique to said R gene variant of interest,

(g2) providing a second optionally mutated non-genetically modified parental plant of said species, variety, accession or cultivar of interest, said plant comprising an R gene or R gene homologue comprising at least a second polymorphic marker of said combination of at least two polymorphic markers unique to said R gene variant of interest,

(g4) screening said offspring population for a single non-genetically modified plant of interest having the combination of said first and second polymorphic markers present in at least two separate R genes or R gene homologues, wherein a first R gene or R gene homologue originates from said first parental plant, and wherein a second R gene or R gene homologue originates from said second parental plant;

homologues in said plant using methods that do not involve the use of recombinant nucleic acid molecules or genetically modified plant cells, to thereby provide a non-genetically modified plant of interest that harbours parental R genes and/or R gene homologues that are different from the R gene variant of interest but that together contain stretches of codons that, when combined in a single gene sequence, characterize the unique combination of at least two polymorphic markers of said R gene variant of interest.

7. The method of claim 6, wherein said step (gl) of providing the optionally mutated non-genetically modified parental plant(s) is preceded by a step (gO) of genotyping plants of a species, variety, accession or cultivar of interest, wherein said plants are subjected to a genotyping assay using one or more polymorphic markers present in said R gene variant of interest.

8. The method of any one of the preceding claims, further comprising the step of (k) subjecting the R gene variant of interest in said non-transgenic plant provided in step (i) to further mutagenesis using methods that do not involve the use of recombinant nucleic acid molecules or genetically modified plant cells to provide a non-transgenic plant exhibiting the pathogen

resistance response of interest.

9. The method of any one of the preceding claims, wherein said method further comprises the step (1) of using the non-transgenic plant provided in step (i) in a plant breeding program as the breeding source of a novel pathogen resistance response.

10. The method of any one of the preceding claims, wherein said putative R gene and said ? gene variant of interest belong to the class of R genes selected from the group consisting of NB-LRR genes, cytoplasmic Ser/Thr kinases, Receptor-like proteins (e.g. Cf -genes), or receptor-like kinases.

11. The method of any one of the preceding claims, wherein said

combination of at least two polymorphisms unique to said R gene variant of interest is a unique combination of SNPs and/or mutations resulting from chimeric recombination of domains of at least two different R genes, domains of at least two different R gene homologues or domains of an R gene and an R gene homologue.

12. The method of any one of the preceding claims, wherein said pathogen is a virus, insect, mite, bacterium, fungus, oomycete or nematode.

13. The method of any one of the preceding claims, wherein the pathogen resistance response of interest is an enhanced resistance against a known pathogen or a resistance against a novel pathogen. A plant obtained by the method of any of claims 1-13.