Patent Description:
Late blight, caused by the oomycete Phytophthora infestans, is one of the most serious diseases in worldwide potato production. It was responsible for the Irish potato famine of the mid-19th century, resulting in the death of one million people. Although a lot of effort has been invested in controlling the pathogen, chemical control of P. infestans is still the main crop management strategy, but environmental safety is becoming more important and the pathogen is sometimes able to evolve resistance to the fungicide treatment. Therefore, introduction of resistance into modern potato varieties is the most durable strategy to control the disease.

In the last century, Solanum demissum, which is a hexaploid Mexican species, was extensively used in breeding for late-blight resistance in potato. Initially, a series of <NUM> R genes derived from S. demissum was described. Of these, R1, R2, R3a/b, R6, and R7 have been localized on the genetic maps of potato (Solanum tuberosum). However, these R genes confer pathovar-specific resistance and those that were introgressed into potato varieties, mainly R1, R2, R3, R4, and R10, were quickly overcome by the pathogen. Hence, new sources for resistance are required, and currently, several other wild Solanum species have been reported as being potential sources of resistance, many of which have been genetically characterized (Table <NUM>).

Recent efforts to identify late blight resistance have focused on major R genes conferring broad-spectrum resistance derived from diverse wild Solanum species. demissum, other wild Solanum species such as S. chacoense, S. berthaultii, S. brevidens, S. bulbocastanum, S. microdontum, S. sparsipilum, S. spegazzinii, S. , stoloniferum, S. sucrense, S. toralapanum, S. vernei and S. verrucosum have been reported as new sources for resistance to late blight (reviewed by (Jansky, <NUM>)).

chacoense, is a self-incompatible diploid species from South America, and is thought to be a source for late-blight resistance. A recent taxonomic rearrangement of the section Petota revealed its relationship with species like S. berthaultii and S. Several accessions of S. chacoense (CHC543-<NUM>), S. berthaultii (BER481-<NUM>, BER94-<NUM>) and S. tarijense (TAR852-<NUM>) have been tested in detached leaf assays (DLA) with multiple isolates (Table <NUM>) and in repeated field trials with isolate IPO-C. In all tests CHC543-<NUM>, BER94-<NUM>, BER481-<NUM> and TAR852-<NUM> remained unaffected, underscoring the relevance of the expressed R genes for resistance breeding. Tetraploid S. chacoense and hybrids have been shown in <NPL>.

Molecular cloning of the genes responsible for resistance and subsequent introduction of the genes into potato varieties is a third method that circumvents many of the problems encountered in the previous two strategies.

To date, multiple late blight R-genes have been cloned, like the allelic genes RB and Rpi-blb1 on chromosome <NUM> and Rpi-blb2 on chromosome <NUM> (Table <NUM>). Recently, also an Rpi-blb3 resistance gene has been isolated (<CIT>). Although the initial results obtained with RB and Rpi-blb1, -<NUM> and -<NUM> are promising, there is a further need for additional R-genes.

The invention now relates to a method for increasing resistance in a plant against Phytophthora infestans infection as set forward in the claims. Further, the invention also relates to a marker for marker assisted selection in plant breeding to obtain resistance against oomycetes, wherein said marker is chosen from the markers presented in Table <NUM>.

In another embodiment, the invention also relates to an isolated or recombinant nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: <NUM> or a fragment thereof, wherein said fragment comprises at least the LRR domain depicted as amino acids <NUM>-<NUM> of <FIG> of the amino acid sequence, which is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection , or a nucleic acid encoding the amino acid sequence of SEQ ID NOs: <NUM>-<NUM>, which is capable of increasing resistance in a plant of the Solanaceae family against an oomycete infection.

The invention further relates to a transgenic or tetraploid Solanum tuberosum, Solanum lycopersicon, Solanum melononga or Capsicum spp. cell comprising a nucleic acid according to any of claims <NUM>-<NUM>.

Also part of the invention is a vector comprising a nucleic acid sequence according to the invention. Preferably said vector further comprises the promoter and/or terminator to which the gene is naturally associated, more preferably a truncated promoter having less than <NUM> nucleotides upstream of the gene sequence.

The invention also is related to a transgenic or tetraploid Solanum tuberosum, Solanum lycopersicon, Solanum melononga, or Capsicum spp. host cell comprising a heterologous nucleic acid according to any of claims <NUM>-<NUM> or a vector according to claim <NUM>, preferably wherein such a host cell is an Agrobacterium cell or a plant cell.

The invention also relates to a transgenic or tetraploid plant cell comprising a nucleic acid according to the invention or a vector according to the invention, preferably wherein said plant cell is a cell from a Solanaceae, more preferably Solanum tuberosum, more preferably a tetraploid Solanum tuberosum. In a further embodiment the invention comprises a transgenic or tetraploid plant comprising such a cell and also a part derived from such a plant, preferably wherein said part is a tuber.

Also comprised in the current invention is a protein encoded by an isolated or recombinant nucleic acid according to the invention or a fragment thereof, preferably wherein said wherein said fragment comprises at least the LRR domain depicted as amino acids <NUM>-<NUM> of <FIG> of the amino acid sequence, which is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection, preferably wherein the protein has the amino acid sequence of Rpi-chc1 as depicted in <FIG>. The invention also relates to an antibody that (specifically) binds to the protein with the amino acid sequence of Rpi-chc1 as depicted in <FIG>.

As used herein, the term "plant or part thereof" means any complete or partial plant, single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which potato plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, tubers, including potato tubers for consumption or 'seed tubers' for cultivation or clonal propagation, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like.

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

As used herein, the term "variety" is as defined in the UPOV treaty and refers to any plant grouping within a single botanical taxon of the lowest known rank, which grouping can be: (a) defined by the expression of the characteristics that results from a given genotype or combination of genotypes, (b) distinguished from any other plant grouping by the expression of at least one of the said characteristics, and (c) considered as a unit with regard to its suitability for being propagated unchanged.

The term "cultivar" (for cultivated variety) as used herein is defined as a variety that is not normally found in nature but that has been cultivated by humans, i.e. having a biological status other than a "wild" status, which "wild" status indicates the original non-cultivated, or natural state of a plant or accession. The term "cultivar"specifically relates to a potatoplant having a ploidy level that is tetraploid. The term "cultivar" further includes, but is not limited to, semi-natural, semi-wild, weedy, traditional cultivar, landrace, breeding material, research material, breeder's line, synthetic population, hybrid, founder stock/base population, inbred line (parent of hybrid cultivar), segregating population, mutant/genetic stock, and advanced/improved cultivar.

As used herein, "crossing" means the fertilization of female plants (or gametes) by male plants (or gametes). The term "gamete" refers to the haploid or diploid reproductive cell (egg or sperm) produced in plants by meiosis, or by first or second restitution, or double reduction from a gametophyte and involved in sexual reproduction, during which two gametes of opposite sex fuse to form a diploid or polyploid zygote. The term generally includes reference to a pollen (including the sperm cell) and an ovule (including the ovum). "Crossing" therefore generally refers to the fertilization of ovules of one individual with pollen from another individual, whereas "selfing" refers to the fertilization of ovules of an individual with pollen from genetically the same individual.

The term "backcrossing" as used herein means the process wherein the plant resulting from a cross between two parental lines is crossed with one of its parental lines, wherein the parental line used in the backcross is referred to as the recurrent parent. Repeated backcrossing results in the genome becoming more and more similar to the recurrent parent, as far as this can be achieved given the level of homo- or heterozygosity of said parent.

As used herein, "selfing" is defined as refers to the process of self-fertilization wherein an individual is pollinated or fertilized with its own pollen.

The term "marker" as used herein means any indicator that is used in methods for inferring differences in characteristics of genomic sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.

As used herein, "locus" is defined as the genetic or physical position that a given gene occupies on a chromosome of a plant.

The term "allele(s)" as used herein means any of one or more alternative forms of a gene, all of which alleles relate to the presence or absence of a particular phenotypic trait or characteristic in a plant. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. It is in some instance more accurate to refer to "haplotypes" (i.e. an allele of a chromosomal segment) in stead of "allele", however, in these instances, the term "allele" should be understood to comprise the term "haplotype".

The term "heterozygous" as used herein, and confined to diploids, means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.

As used herein, and confined to diploids, "homozygous" is defined as a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.

As used herein, and confined to tetraploids, the term "nulliplex", "simplex", "duplex", "triplex" and "quadruplex", is defined as a genetic condition existing when a specific allele at a corresponding locus on corresponding homologous chromosomes is present <NUM>, <NUM>, <NUM>, <NUM> or <NUM> times, respectively. At the tetraploid level the phenotypic effect associated with a recessive allele is only observed when the allele is present in quadruplex condition, whereas the phenotypic effect associated with a dominant allele is already observed when the allele is present in a simplex or higher condition.

The terms "haploid", "diploid" and "tetraploid" as used herein are defined as having respectively one, two and four pairs of each chromosome in each cell (excluding reproductive cells).

The term "haplotype" as used herein means a combination of alleles at multiple loci that are transmitted together on the same chromosome. This includes haplotypes referring to as few as two loci, and haplotypes referring to an entire chromosome depending on the number of recombination events that have occurred between a given set of loci.

As used herein, the term "infer" or "inferring", when used in reference to assessing the presence of the fungal resistance as related to the expression of the Rpi-chc1 gene, means drawing a conclusion about the presence of said gene in a plant or part thereof using a process of analyzing individually or in combination nucleotide occurrence(s) of said gene in a nucleic acid sample of the plant or part thereof. As disclosed herein, the nucleotide occurrence(s) can be identified directly by examining the qualitative differences or quantitative differences in expression levels of nucleic acid molecules, or indirectly by examining (the expression level of) a the Rpi-chc1 protein.

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. A "pair of bi-directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

As used herein, the term "probe" means a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.

The terms "stringency" or "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least <NUM>-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about <NUM> lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which <NUM>% of a complementary target sequence hybridises to a perfectly matched probe or primer.

Typically, stringent conditions will be those in which the salt concentration is less than about <NUM> Na+ ion, typically about <NUM> to <NUM> Na+ ion concentration (or other salts) at pH <NUM> to <NUM> and the temperature is at least about <NUM> for short probes or primers (e.g. <NUM> to <NUM> nucleotides) and at least about <NUM> for long probes or primers (e.g. greater than <NUM> nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of <NUM>% formamide, <NUM> NaCl, <NUM>% SDS at <NUM> and a wash in 2x SSC at <NUM>. Exemplary high stringency conditions include hybridization in <NUM>% formamide, <NUM> NaCl, <NUM>% SDS at <NUM>, and a wash in <NUM>. 1x SSC at <NUM>. Hybridization procedures are well known in the art and are described in e.g. <NPL>.

The present invention describes the cloning of the Rpi-chc1 gene. Rpi-chc1 was mapped to a new R gene locus on chromosome <NUM> using a S. chacoense mapping population. Markers highly linked to Rpi-chc1 were used to generate a physical map of the R locus. Three R gene analogs (RGA) present on one of two BAC clones that encompassed the Rpi-chc1 locus were targeted for complementation analysis, one of which turned out to be the functional Rpi-chc1 gene. Outside the R-gene clusters described in this invention, Rpi-chc1 shares the highest amino acid sequence identity (<NUM>%) to a protein encoded by a gene with unknown function, designated ABF81421, from poplar (Populus trichocarpa). Lower percentages of homology (<<NUM>%) were found with R proteins previously identified within the Solanaceae (Table <NUM>).

The present description shows an isolated or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence Rpi-chc1 (=CHC_B2-<NUM>) as presented in <FIG> or a functional fragment or a functional homologue thereof, i.e. a functional fragment or a functional homologue of the amino sequence as shown in <FIG>.

The term "nucleic acid" means a single or double stranded DNA or RNA molecule.

Also included are the complementary sequences of the herein described nucleotide sequences.

The term "functional fragment thereof" is typically used to refer to a fragment of the Rpi-chc1 protein that is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete infection. Such a fragment is a truncated version of the Rpi-chc1 protein as presented in <FIG>. A truncated version/fragment of the Rpi-chc1 protein is a fragment that is smaller than <NUM> amino acids and comprises at least part of the LRR domain (i.e. part of the leucine-rich repeats domain which stretches from about amino acid <NUM> to amino acid <NUM> of Rpi-chc1) and/or the N-terminal parts of the Rpi-chc1 protein.

The term "functional homologue" is typically used to refer to a protein sequence that is highly homologous to or has a high identity with the herein described Rpi-chc1 protein, which protein is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete infection. Included are artificial changes or amino acid residue substitutions that at least partly maintain the effect of the Rpi-chc1 protein. For example, certain amino acid residues can conventionally be replaced by others of comparable nature, e.g. a basic residue by another basic residue, an acidic residue by another acidic residue, a hydrophobic residue by another hydrophobic residue, and so on. Examples of hydrophobic amino acids are valine, leucine and isoleucine. Phenylalanine, tyrosine and tryptophan are examples of amino acids with an aromatic side chain and cysteine as well as methionine are examples of amino acids with sulphur-containing side chains. Serine and threonine contain aliphatic hydroxyl groups and are considered to be hydrophilic. Aspartic acid and glutamic acid are examples of amino acids with an acidic side chain. In short, the term "functional homologue thereof" includes variants of the Rpi-chc1 protein in which amino acids have been inserted, replaced or deleted and which at least partly maintain the effect of the Rpi-chc1 protein (i.e. at least partly providing or increasing resistance in a plant of the Solanaceae family against an oomycete infection). Preferred variants are variants which only contain conventional amino acid replacements as described above. A high identity in the definition as mentioned above means an identity of at least <NUM>, <NUM> or <NUM>%. Even more preferred are amino acids that have an identity of <NUM>, <NUM>, <NUM>, <NUM> or <NUM>%. Most preferred are amino acids that have an identity of <NUM>, <NUM>, <NUM> or <NUM>% with the amino acid sequence of Rpi-chc1. Homologous proteins are for example the sequences aligned with CHC_B2-<NUM> in <FIG> and with the Rpi-chc1 ORF in <FIG>.

A functional homologous nucleic acid sequence is a nucleic acid sequence that encodes a functional homologous protein as described above.

Homology and/or identity percentages can for example be determined by using computer programs such as BLAST, ClustalW or ClustalX.

Many nucleic acid sequences code for a protein that is <NUM>% identical to the Rpi-chc1 protein as presented in <FIG>. This is because nucleotides in a nucleotide triplet may vary without changing the corresponding amino acid (wobble in the nucleotide triplets). Thus, without having an effect on the amino acid sequence of a protein the nucleotide sequence coding for this protein can be varied. However, in a preferred embodiment, the invention provides an isolated or recombinant nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: <NUM> or a fragment thereof, wherein said fragment comprises at least the LRR domain depicted as amino acids <NUM>-<NUM> of <FIG> of the amino acid sequence, which is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection. In a preferred embodiment, the invention provides an isolated, synthetic, or recombinant nucleic acid that represents the coding sequence (CDS) of the Rpi-chc1 protein, i.e. nucleotides <NUM>-<NUM> of <FIG> (shaded) as represented in SEQ ID NO: <NUM> The nucleotide sequences of homologues with a high identity are represented in <FIG> and SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>, and the corresponding amino acid sequences are given in the alignment of <FIG> and SEQ ID Nos: <NUM>-<NUM>.

Fragments as well as homologues of the herein described Rpi-chc1 gene and protein can for example be tested for their functionality by using an Agrobacterium tumefaciens transient transformation assays (agro-infiltration) and/or by using a detached leaf assay.

The experimental part for example describes a functional screen for testing candidate genes using agro-infiltration, whereby <NUM> week old wild type Nicotiana benthamiana plants are infiltrated with Agrobacterium strains containing the candidate Rpi-chc1 homologues. The infiltrated leaves are subsequently challenged one day after infiltration with a P. infestans strain that is virulent on N. benthamiana, for example IPO-C or <NUM>, in detached leaf assays. This system is equally suitable for testing candidate homologous fragments of Rpi-chc1. A person skilled in the art thus can easily determine whether or not an Rpi-chc1 homolog or fragment can be considered to be a functional homolog or fragment.

Transient gene expression, as is achieved through agro-infiltration, is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic RNA expression ceases after <NUM>-<NUM> days. It is shown that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. A system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced <NUM>-fold or more in the presence of p19 so that protein purification could be achieved from as little as <NUM> of infiltrated leaf material. Although it is clear that the use of p19 has advantages, an agroinfiltration without p19 can also be used to test the functionality of candidate fragments and functional homologues.

Alternatively, each candidate gene (for example being a fragment or homologue) construct is targeted for transformation to a susceptible potato cultivar, for example Desiree. Primary transformants are challenged in detached leaf assays using for example isolates IPO-<NUM>, IPO-C or <NUM>. Transformants that are resistant to these isolates harbour for example functional fragments or homologues of Rpi-chc1.

In yet another embodiment, the invention provides a vector comprising a nucleic acid as provided herein, i.e. a nucleic acid capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection. More particularly, the invention provides a vector comprising an isolated, synthetic or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence Rpi-chc1 of <FIG> or a functional fragment o. The invention also provides a vector comprising a nucleic acid sequence as depicted in <FIG>.

Examples of a suitable vector are pBeloBACII, pBINplus, pKGW-MG or any commercially available cloning vector.

As will be outlined below there are multiple ways in which a nucleic acid of the invention can be transferred to a plant. One suitable means of transfer is mediated by Agrobacterium in which the nucleic acid to be transferred is part of a binary vector and hence it is preferred that the above described vector is a binary vector. Another suitable means is by crossing a plant which contains the gene encoding Rpi-chc1 to a plant that does not contain the gene and to identify those progeny of the cross that have inherited the Rpi-chc1 gene.

The invention further provides a host cell comprising a nucleic acid as described herein or a vector as described herein. Examples of a preferred host cell are an E. coli cell suitable for BAC clones (e.g. DH10B) or an Agrobacterium (host) cell. In another embodiment, said host cell comprises a plant cell. A preferred plant cell is a cell derived from a member of the Solanaceae family and even more preferred said plant cell comprises a cell from Solanum tuberosum, Solanum lycopersicum, formerly known as Lycopersicon esculentum, pepper and eggplant. From such a cell, a transgenic or genetically modified plant (for example a potato or tomato plant) can be obtained by methods known by the skilled person (for example regeneration protocols).

The invention further provides a leaf, tuber, fruit or seed or part or progeny of a genetically modified plant as described herein.

In yet another embodiment, the invention provides a protein encoded by the herein described isolated or recombinant nucleic acid or a functional fragment or a functional homologue thereof. In a preferred embodiment, the invention provides a protein encoded by a nucleic acid sequence as depicted in <FIG>. In yet another preferred embodiment, the invention provides a protein comprising the amino acid sequence of <FIG> or a functional fragment or a functional homologue thereof. Further preferred are the functional (active) proteins depicted in <FIG>, more specifically the proteins designated as <NUM>-7_G12, <NUM>-5_C2, <NUM>-1_M8_M18_M20, <NUM>-1_I4_I6_I8, <NUM>-2031_L4_L7_l8, <NUM>-2_K4_K14_K22, <NUM>-2_J1_J3_J8, <NUM>-5_E14_E23, <NUM>-5_E28, <NUM>-9_H5_H30, <NUM>-7_G14_G22, <NUM>-2_K6_K30_K31 and <NUM>-7_G21 which are represented in SEQ ID NO: <NUM> - <NUM>.

The herein described Rpi-chc1 protein comprises <NUM> amino acids and the LRR domains of Rpi-chc1 consist of <NUM> imperfect repeats (<FIG>). Interestingly Rpi-chc1 shares the highest homology (<NUM>-<NUM>%) with other RGAs from the Rpi-chc1 gene cluster from S. chacoense and with genes from synthenic clusters on chromosome <NUM> from S. tuberosum (Table <NUM>). A lower (<NUM>%), but significant, extent of homology was found with a protein encoded by a gene with unknown function from poplar (accession number ABF81421, Table <NUM>). The different domains of Rpi-chc1 share varying degrees of homology with corresponding domains of the poplar protein encoded by ABF81421. The NBS domain is most conserved (<NUM>% aa identity), followed by the CC domain (<NUM>% aa identity). The LRR domain is least conserved (<NUM>% aa identity). Overall homologies of lower than <NUM>% are found with the FOM2 protein from cucumber, which confers resistance to fungal pathogen Fusarium oxysporum, Rpi-blb1 from S. bulbocastanum, R3a from S. demissum, and RPS1 from soybean (Glycine max), which confer resistance to Phytophthora sp. These sequence homologies show that Rpi-chc1 is a member of an ancient R-gene family that has not been characterised before in Solanaceae.

As already described, a functional fragment or a functional homologue thereof of Rpi-chc1 is a fragment or homologue that is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection.

Means to test the functionality of a functional fragment or a functional homologue of Rpi-chc1 have been provided above.

Based on the herein described nucleic acid sequences, the specification also describes probes and primers (i.e. oligonucleotide sequences complementary to one of the (complementary) DNA strands as described herein). Probes are for example useful in Southern or northern analysis and primers are for example useful in PCR analysis. Primers based on the herein described nucleic acid sequences are very useful to assist plant breeders active in the field of classical breeding and/or breeding by genetic modification of the nucleic acid content of a plant (preferably said plant is a Solanum tuberosum, Solanum lycopersicum, formerly known as Lycopersicon esculentum), pepper or eggplant in selecting a plant that is capable of expressing for example Rpi-chc1 or a functional fragment or functional homolog thereof.

Hence, the description provides a binding molecule capable of binding to a nucleic acid encoding Rpi-chc1 or a functional fragment or functional homolog thereof as described herein or its complementary nucleic acid. In a preferred embodiment, said binding molecule is a primer or a probe. As mentioned, such a binding molecule is very useful for plant breeders and hence the description further provides a method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to an oomycete infection. Preferably, the nucleic acid of a plant to be tested is isolated from said plant and the obtained isolated nucleic acid is brought in contact with one or multiple (preferably different) binding molecule(s). One can for example use a PCR analysis to test plants for the presence of absence of Rpi-chc1 in the plant genome. Such a method would be especially preferable in marker-free transformation protocols, such as described in <CIT>.

The herein described Rpi-chc1 protein can also be used to elicit antibodies by means known to the skilled person. The invention thus also provides an antibody that (specifically) binds to the protein encoded by the herein described isolated or recombinant nucleic acid (for example the nucleic acid sequence of <FIG>) or an antibody that (specifically) binds to a protein as depicted in <FIG> or a functional fragment or a functional homolog thereof. Such an antibody is for example useful in protein analysis methods such as Western blotting or ELISA, and hence can be used in selecting plants that successfully express the Rpi-chc1 gene.

Based on the herein provided nucleic acid sequences, the invention also provides the means to introduce or increase resistance against Phytophthora infestans infection in a plant. The invention therefore also provides a method for increasing resistance in a plant against Phytophthora infestans infection comprising transforming a plant or a part thereof with a nucleic acid encoding the amino acid sequence of SEQ ID NO: <NUM> or a functional fragment thereof, wherein said functional fragment comprises at least the LRR domain of the amino acid sequence, depicted as amino acids <NUM>-<NUM> of <FIG>, capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection or a homologue thereof, wherein said homologue has an identity of at least <NUM>% with the amino acid sequence of SEQ ID NO: <NUM>, which homologue is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection. - a vector comprising the herein described nucleic acid sequences, or - a host cell as described herein.

Such a method for increasing resistance in a plant against an oomycete infection may be based on classical breeding, departing from a parent plant that already contains the Rp1-chc1 gene or a functional homolog thereof, or it involves the transfer of DNA into a plant, i.e., involves a method for transforming a plant cell comprising providing said plant cell with a nucleic acid as described herein or a vector as described herein or a host cell as described herein.

There are multiple ways in which a recombinant nucleic acid can be transferred to a plant cell, for example Agrobacterium mediated transformation. However, besides by Agrobacterium infection, there are other means to effectively deliver DNA to recipient plant cells when one wishes to practice the invention. Suitable methods for delivering DNA to plant cells are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts, by desiccation/inhibition-mediated DNA uptake (<NPL>), by electroporation (<CIT>), by agitation with silicon carbide fibers (Kaeppler et al. , <NUM>; <CIT>; and <CIT>), and by acceleration of DNA coated particles (<CIT>; <CIT>; and <CIT>). Through the application of techniques such as these, cells from virtually any plant species may be stably transformed, and these cells may be developed into transgenic plants.

In case Agrobacterium mediated transfer is used, it is preferred to use a substantially virulent Agrobacterium such as A. tumefaciens, as exemplified by strain A281 or a strain derived thereof or another virulent strain available in the art. These Agrobacterium strains carry a DNA region originating from the virulence region of the Ti plasmid pTiBo542, which coordinates the processing of the T-DNA and its transfer into plant cells. Agrobacterium-based plant transformation is well known in the art (as e.g. described in, for example by <NPL>). Preferably a marker-free transformation protocol is used, such as described in <CIT>.

Alternatively, the nucleic acid of the Rpi-chc1 gene or a functional homolog thereof may be introduced into a plant by crossing. Such a crossing scheme starts off with the selection of a suitable parent plant. This may for instance be an original Solanum chacoense variety (such as accession CHC543-<NUM>), an original S. tarijense variety (such as accession TAR852-<NUM>), an original S. sucrense variety (such as accession SUC849-<NUM>) or an original S. berthaultii variety (such as accession BER481-<NUM> or BER94-<NUM>) or a plant that has obtained the desired nucleic acid by genetic engineering as described above.

Any suitable method known in the art for crossing selected plants may be applied. This includes both in vivo and in vitro methods. A person skilled in the art will appreciate that in vitro techniques such as protoplast fusion or embryo rescue may be applied when deemed suitable.

Selected plants that are used for crossing purposes may have any type of ploidy. For example, selected plants may be haploid, diploid or tetraploid. However, crossing diploid plants, such as S. chacoense, S. tarijense and S. berthaultii, will only provide diploid offspring. Crossing a diploid plant with a tetraploid plant will result in triploid offspring that is sterile.

Thus, when plants are selected that are diploid, their ploidy must be increased to tetraploid level before they can be crossed with another tetraploid plant. Methods for increasing the ploidy of a plant are well known in the art and can be readily applied by a person skilled in the art. For example, ploidy of a diploid plant for crossing purposes can be increased by using 2N gametes of said diploid plant. Ploidy can also be increased by inhibiting chromosome segregation during meiosis, for example by treating a diploid plant with colchicine. By applying such methods on a diploid plant, embryos or gametes are obtained that comprise double the usual number of chromosomes. Such embryos or gametes can then be used for crossing purposes. For potatoes a resistant tetraploid plant is preferred, since tetraploid plants are known to have higher yields of tubers.

Since the resistance characteristic has appeared to be a dominant trait, it is sufficient if only one allele with the functional gene is present.

Preferably, selected plants are crossed with each other using classical in vivo crossing methods that comprise one or more crossing steps including selfing. By applying such classical crossing steps characteristics of both the parents can be combined in the progeny. For example, a plant that provides a high yield can be crossed with a plant that contains large amounts of a certain nutrient. Such a crossing would provide progeny comprising both characteristics, i.e. plants that not only comprise large amounts of the nutrient but also provide high yields.

When applying backcrossing, F1 progeny is crossed with one of its high-yielding parents P to ensure that the characteristics of the F2 progeny resemble those of the high-yielding parent. For example, a selected diploid potato with oomycete resistance is made tetraploid by using colchicine and then crossed with a selected high-yielding tetraploid potato cultivar, with the purpose of ultimately providing a high-yielding tetraploid progeny having oomycete resistance. Also selfing may be applied. Selected plants, either parent or progeny, are then crossed with themselves to produce inbred varieties for breeding. For example, selected specimens from the above mentioned F1 progeny are crossed with themselves to provide an F2 progeny from which specimens can be selected that have an increased level of resistance.

After transfer of a nucleic acid into a plant or plant cell, it must be determined which plants or plant cells have been provided with said nucleic acid. When selecting and crossing a parental genotype, a marker is used to assist selection in at least one selection step. It is known in the art that markers, indicative for a certain trait or condition, can be found in vivo and in vitro at different biological levels. For example, markers can be found at peptide level or at gene level. At gene level, a marker can be detected at RNA level or DNA level. Preferably, the presence of such a marker is detected at DNA level, using the above described primers and/or probes. Alternatively, proper expression of the Rpi-chc1 protein or a functional homolog thereof can be assessed in plant parts by performing an immunoassay with an antibody that specifically binds the protein. Next to the primers and probes, use can also be made of specific markers that are to be found in the vicinity of the coding sequence. Such markers are indicated in the experimental part below and comprise the markers as indicated in Table. Markers are derived from accompanying BAC sequences.

In case of transgenic approaches selecting a transformed plant may be accomplished by using a selectable marker or a reporter gene. Among the selective markers or selection genes that are most widely used in plant transformation are the bacterial neomycin phosphotransferase genes (nptI, nptII and nptIII genes) conferring resistance to the selective agent kanamycin, suggested in <CIT> and the bacterial aphIV gene suggested in <CIT> conferring resistance to hygromycin. <CIT> discloses the use of an acetyl transferase gene from Streptomyces viridochromogenes that confers resistance to the herbicide phosphinotricin. Plant genes conferring relative resistance to the herbicide glyphosate are suggested in <CIT>. Suitable examples of reporter genes are beta-glucuronidase (GUS), beta-galactosidase, luciferase and green fluorescent protein (GFP).

In a preferred embodiment, the invention provides a method for increasing resistance in a plant against Phytophthora infestans infection comprising transforming a plant or a part thereof with:.

wherein said plant comprises a plant from the Solanaceae family, preferably a potato or tomato plant, more preferably a tetraploid potato plant.

The invention also provides a plant that is obtainable by using a method for increasing resistance in a plant against Phytophthora infestans infection as described above. A preferred plant is a plant from the Solanaceae family and even more preferred said plant is a Solanum tuberosum or a Solanum lycopersicum, formerly known as Lycopersicon esculentum, Solanum melononga, Capsicum spp. , such as C. baccatum, C. chinense, C. frutescens and C. The invention thus also provides a plant that has been provided with a nucleic acid encoding a Rpi-chc1 protein or a functional fragment or a functional homologue thereof.

The invention further provides a plant part or progeny of a plant according to the invention comprising a nucleic acid encoding the Rpi-chc1 amino acid sequence of <FIG> or a functional fragment or a functional homologue thereof.

In a preferred embodiment, the herein described nucleic acid is transferred to a Solanum variety other than Solanum chacoense, i.e. the herein described nucleic acid is preferably provided to a non-chacoense background, preferably S. lycopersicon or S. Of the latter most preferred is a tetraploid variety and more preferably to a commercial interesting variety such as Bintje, Desiree or Premiere, Spunta, Nicola, Favorit, Russet Burbank, Aveka or Lady Rosetta. It is also possible to provide the resistance according to the invention to a plant that is already partially resistant Phytophthora infestans infection, wherein said plant is provided with a nucleic acid encoding a further resistance gene, such as Rpi-blb1,-<NUM>, -<NUM>, Rpi-vnt1 or Rpi-mcq1.

In yet another embodiment, the description provides a method for producing Rpi-chc1 protein or a functional fragment or a functional homologue thereof comprising functionally linking a nucleic acid as described herein to a regulatory sequence and allowing said nucleic acid to be expressed in a host cell. Examples of a regulatory sequence are a promoter and/or terminator sequence. Further, as will become clear from Example <NUM>, it is preferred that the Rpi-chc1 sequence is expressed under control of its own promoter and terminator. Therefore, the description further provides the promoter and/or terminator sequences of Rpi-chc1 (<FIG> shows the nucleotide sequence of clone CHC B2-<NUM> (<NUM> bp) containing the Rpi-chc1 gene and regulatory sequences. The Rpi-chc1 coding region of <NUM> bp is highlighted in shading (nt <NUM>-<NUM>). The upstream <NUM> nucleotides (nt <NUM>-<NUM>) and the downstream <NUM> nucleotides (nt <NUM>-<NUM>) harbour the regulatory sequences that ensure correct expression of the gene. The skilled person is very well capable of cloning (part of) said regulatory sequences and testing their efficiency in transcription. It has further been found that even a better expression is obtained with a truncated promoter, i.e. a promoter containing less than <NUM>, preferably not more than <NUM> base pairs upstream of the gene sequence.

The invention will be explained in more detail in the following, nonlimiting examples.

A recent taxonomic regrouping of the Solanum section Petota revealed the lack of species structure in this section (Jacobs et al. In order to identify late blight resistance traits from the taxonomic group <NUM>-<NUM> (Jacobs et al. , <NUM>) we selected several accessions and tested their resistance levels to Phytophthora infestans in field trials. Five accessions, that were previously determined as S. tarijense (TAR), S. berthaultii (BER), and S. chacoense (CHC), with high resistance levels were selected (TAR852-<NUM>, BER94-<NUM>-<NUM>, BER481-<NUM>, BER493-<NUM>, CHC543-<NUM>). In order to study the genetic basis of these resistances, crosses were generated using BER493-<NUM>, CHC543-<NUM>, BER94-<NUM>-<NUM> as resistant parents. The resulting F1 populations were tested for the segregation of resistance to P. infestans in a detached leaf assay (Table <NUM>).

From literature it was known that a late blight resistance gene from S. berthaultii (Rpi-ber) was closely linked to TG63 on the long arm of chromosome <NUM> (Rauscher et al. , <NUM>), a region to which also the tomato Ph-<NUM> QTL from S. pimpenellifolium mapped (Moreau et al. We therefore developed CAPS markers in TG63 in the three populations. Using the polymorphism described in Table <NUM>, it was found that the resistances in <NUM>-<NUM> and <NUM> were closely linked to TG63 since one and two recombinants were found respectively. Also the resistance in <NUM> was linked to TG63 albeit a higher recombination frequency (<NUM> recombinations) was observed. It is concluded that this area on chromosome <NUM> is very important for resistance to late blight. Therefore, we set out to exploit the well characterised RH89-<NUM>-<NUM> physical map in order to generate a reference map of the TG63 locus. Using the polymorphism described in Table <NUM>, TG63 was mapped to RH10B41. At this mapposition the contig <NUM> was anchored. BAC end sequences in this contig were used to generate markers suitable for mapping in population <NUM>. RH199E15S (Table <NUM>) was found to co-segregate with resistance in <NUM> and <NUM>-<NUM>, indicating that <NUM> from RH89-<NUM>-<NUM> was in a locus synthenic with the Rpi-chc1 and Rpi ber locus.

Besides anchoring TG63 genetically, it was also located in the physical map of RH89-<NUM>-<NUM> by PCR screening the RH BAC library. A positive contig, <NUM>, was found. Remarkably, contig <NUM> was anchored to RH10B38 using two independent markers (Jan de Boer, PGSC). CAPS markers were developed based on BAC end sequences in contig <NUM> and mapped in the <NUM>-<NUM> and <NUM> populations. Also these markers were closely linked to resistance, indicating that also this contig is in a locus synthenic with the Rpi-chc1 and Rpi-ber locus.

Using BAC-end sequences, three additional RH BAC contigs flanking contigs <NUM> and <NUM> were identified (<FIG>). In order to generate sufficient sequence information for finemapping two tiling paths consisting of <NUM> and <NUM> overlapping BACs (106G038, 137D014, 009D021 and 122B15, <NUM>, 04G12, 199E15) were composed and sequenced. Annotation of the RH BAC sequence (<FIG>) revealed the presence of two RGAs in the first tiling path (that mapped to RH10B38) and <NUM> RGAs in the second tiling path (that mapped to RH10B41, <NUM>), indicated as arrowheads in <FIG>. Several markers deriving from these and other chromosome <NUM> sequences were mapped in the S. chacoense population <NUM> (<FIG>) and in the S. berthaultii population <NUM>-<NUM> (<FIG>). The sizes of these populations were increased to <NUM> and <NUM> respectively. Recombinants in the relevant genomic area were screened for using markers RH099F09T and RH092A09S in population <NUM> en markers RH91C10T and RH199 E15 S in population <NUM>-<NUM>. Markers that were derived from the same RH BAC (RH137D14), 137D14-C37-<NUM> and 137D14-C37-<NUM> are only <NUM> kb apart in RH89-<NUM>-<NUM> and co-segregate in the <NUM> population (two recombinants) and in the <NUM>-<NUM> population (no recombinants), respectively. This strongly suggests that Rpi-chc1 and Rpi-ber are in synthenic gene clusters and that there might be an allelic relationship between the genes.

In order to clone Rpi-chc1, two BAC libraries were constructed using DNA derived from the resistant clone CHC543-<NUM>. The first library was constructed in the pCC1BAC BAC vector and contained approximately <NUM> clones with an average insert size of ~<NUM> Kbp, corresponding to <NUM> genome equivalent. A second library was constructed in the pIndigoBAC-<NUM> BAC vector and contained approximately <NUM> clones with an average insert size of ~<NUM> Kbp, corresponding to <NUM> genome equivalents. The first library was screened with marker RH106G03T (Table <NUM>, <FIG>), which cosegregated with resistance in the <NUM> population with only three recombination events. In this way BAC clones CHC B1 was identified. Both BAC ends of CHC B1 (B07_1_C15) were mapped and the RP end (marker B07_1_C15_RP'), which showed only one recombination event with the Rpi-chc1 resistance gene, was used to screen the second BAC library and identified CHC B2 (<NUM>-D06_3-D21) (<FIG>). CHC B2 turned out to contain the RH137D14 C37-<NUM> marker. Two recombination events were found with RH137D14 C37-<NUM>, on the other site of the Rpi-chc1 resistance gene. It was therefore concluded that the Rpi-chc1 locus was delimited to a <NUM> (<NUM>/<NUM> recombinants) interval that is physically spanned by the two partially overlapping BAC clones CHC B1 and CHC B2 (<FIG>).

By sequencing these two BACs, it was found that CHC B1 contained two RGAs and CHC B2 contained three RGAs, which were named CHC B1-<NUM>, CHC B1-<NUM>, CHC B2-<NUM>, CHC B2-<NUM>, and CHC B2-<NUM> respectively (<FIG>). The latter three RGAs were within mapping interval delimited by B07_1_C15_RP' and RH137D14 C37-<NUM>. Therefore, the three genes were subcloned into pBINplus vector under the control of their native regulatory elements by longrange PCR using the high fidelity polymerase Phusion®. The resulting subclones were completely sequenced and were found to be identical to their BAC template sequences.

Complementation analysis was carried out in Nicotiana benthamiana using the Agrobacterium tumefaciens transient assay (agroinfiltration) whereby <NUM>-week old wild type N. benthamiana plants were infiltrated with the Agrobacterium strain AGL1+virG containing pBINplus:CHC B2-<NUM>, pBINplus:CHC B2-<NUM>, and pBINplus:CHC B2-<NUM> respectively. As controls we used pBINplus without an insert and pBINplus:Rpi-blb1. Infiltrated leaves were challenged after two days with P. infestans strain <NUM> in detached leaf assays (DLA). Leaves infiltrated with pBINplus:CHC B2-<NUM> and pBINplus:Rpi-blb1 showed resistance to infection, while pBINplus:CHC B2-<NUM>, pBINplus:CHC B2-<NUM> and pBINplus without an insert were colonized by Phytophtora as was apparent from the sporulating lesions (<FIG>). This experiment clearly showed that CHC B2-<NUM> is an active resistance gene against P. Since none of the other genes present in the genetic mapping interval of Rpi-chc1 shows activity, it can be concluded that CHC B2-<NUM> is the Rpi-chc1 gene.

Interestingly, Rpi-chc1 shares the highest homology (<NUM>-<NUM>%) with other RGAs from the Rpi-chc1 gene cluster from S. chacoense and with genes from synthenic clusters on chromosome <NUM> from S. tuberosum clone RH89-<NUM>-<NUM> (Table <NUM>, <FIG>). A lower (<NUM>%), but significant, extent of homology was found with a protein encoded by a gene with unknown function from poplar (accession number ABF81421, Table <NUM>, <FIG>). The different domains of Rpi-chc1 protein share varying degrees of homology with corresponding domains of the poplar protein encoded by ABF81421. The NBS domain is most conserved (<NUM>% aa identity), followed by the CC domain (<NUM>% aa identity). The LRR domain is least conserved (<NUM>% aa identity). Overall homologies of lower than <NUM>% are found with the FOM2 protein from cucumber (Joobeur et al. , <NUM>), which confers resistance to fungal pathogen Fusarium oxysporum, Rpi-blb1 from S. bulbocastanum (Song et al. , <NUM>; van der Vossen et al. , <NUM>), R3a from S. demissum (Huang et al. , <NUM>), and RPS1-k from soybean (Glycine max)(Gao et al. , <NUM>), which confer resistance to Phytophthora sp.

Rpi-chc1 comprises an ORFs of <NUM> nucleotides (nt) that encode a protein of <NUM> amino acids harboring all sequences characteristic of a CC-NB-LRR R-proteins (<FIG>). In the N terminus <NUM> stretches of amino acids can be distinguished with the potential to fold into a coiled coil structure. The central NB-ARC domain contains stretches of amino acids which show similarity with the Kinase 1a, Kinase <NUM>, Kinase 3a, GLPL, RNBS-D and MHD subdomains (Bendahmane et al. , <NUM>; van der Biezen and Jones, <NUM>). In contrast to many other NB-LRR proteins, the Rpi-chc1 protein is characterized by the absence of an obvious RNBS-A sub-domain and the presence of a double MHD sub-domain. The C-terminal domain contains <NUM> imperfect leucine rich repeats (LRRs). Both LRR <NUM> and <NUM> contain the characteristic LDL signature, which often present in LRR3. Both the MHD and the LRR3 have been implicated in activity regulation and putative intra-molecular interactions (Bendahmane et al. , <NUM>; Tameling et al. Duplication of both of these subdomains might hint to a common regulatory mechanism.

In order to identify positions in the genome that contain Rpi-chc1 related nucleotide sequences a new technique was developed that is derived from the NBS profiling (Brugmans et al. , <NUM>; van der Linden et al. , <NUM>) and will be referred to as "locus directed profiling". Instead of the primers that were used previously, which target domains that are generally present in all R-genes, we now used primers that are conserved within the family of Rpi-chc1 sequences (Table <NUM>). This way only Rpi-chc1 related genes are expected to be targeted. Genomic DNA from parents and offspring from different populations (SHxRH, <NUM>-<NUM>) was digested with either RsaI, HaeIII, AluI or MseI. An adaptor was ligated to the digestion products and using an adaptor primer combined with the Rpi-chc1 family specific primer, multiple fragments of varying molecular weight were created in a PCR reaction. Polymorphic bands were detected in the two populations using the Licor polyacrylamide gelsystem. Polymorphic bands were.

scored in <NUM> offspring plants from the SHxRH population and successively the marker segregation patterns were fitted to the UHD map (van Os et al. <NUM> % of the markers mapped to the long arm of chromosome <NUM> where the Rpi-chc1 gene is located. Also sequence analysis of the isolated marker fragments showed strong homology to the Rpi-chc1 gene family (Table 4b). Altogether these data show that "locus directed profiling" was a successful approach to generate markers in a specified genomic area. On chromosome <NUM> three different loci were tagged with high frequency (Table 4A). Interestingly, the first two loci coincided with the map positions of contigs <NUM> and <NUM>, which map to RH10B38-<NUM> and RH10B41-<NUM> respectively. A third group of markers mapped to RH10B54. Interestingly, the Rpi-ber1 gene (Park et al. , <NUM>) is in the same marker interval as the RH10B54 cluster. In order to test whether the Rpi-ber gene was potentially a Rpi-chc1 homolog, in population <NUM>-<NUM>, <NUM> Rpi-chc1 locus directed profiling markers were developed. <NUM> of these markers derived from the resistant parent. <NUM> of them were linked to resistance (<NUM> in coupling phase, <NUM> in repulsion phase). <NUM> coupling phase markers and <NUM> repulsion phase markers were completely linked to resistance in the first <NUM> individuals of the population. This strongly suggests that Rpi-ber is a Rpi-chc1 homolog. Within the <NUM> linked Rpi-chc1 locus directed profiling markers, four groups of recombination patterns could be distinguished, each group is marked by the name of a representative marker in <FIG>. Three marker groups match the RH10B38-<NUM> cluster, one marker group matches the RH10B41-<NUM> cluster. This result confirms our finding from the SHxRH population, that the family of Rpi-chc1 related sequences on chromosome <NUM> is located in at least two closely linked clusters.

In a different population (<NUM>) deriving from S. berthaultii accession <NUM>-<NUM> an NBS profile marker generated with the previously described NBS5a6 primer was found to be closely linked to Phytophthora resistance in this population. It mapped to the telomeric site relative to TG403 on the long arm of chromosome <NUM> (<FIG>). Sequence analysis of this fragment revealed high homology to members of the Rpi-chc1 family. All together these results show that at least four, genetically different, Rpi-chc1 like clusters are present on chromosome <NUM>. This is similar to the situation on the long arm of chromosome <NUM>, where three different Tm2-<NUM> related clusters were identified (Foster et al. , <NUM>; Pel et al.

In this study we used four late blight resistant clones TAR852-<NUM> (deriving from CGN22729), BER94-<NUM>-<NUM> (deriving from PI473331), BER481-<NUM> (deriving from CGN18190) BER493-<NUM> (deriving from CGN17823), CHC543-<NUM> (deriving from BGRC63055). CHC543-<NUM> was crossed with CHC544-<NUM> to produce population <NUM>. BER94-<NUM>-<NUM> was crossed with the susceptible clone G254 to generate population <NUM>-<NUM>. BER493-<NUM> was crossed with RH89-<NUM>-<NUM> to produce population <NUM>. Potato cultivar Desiree was used for transformation. Wild-type Nicotiana benthamiana plants were used for transient complementation assays.

Characteristics and origin of P. infestans isolates used in this study are indicated in Table <NUM>.

Clone CHC543-<NUM> was used as a DNA source for the construction of the BAC libraries. High-molecular weight DNA preparation and BAC library construction were carried out as described by (Rouppe van der Voort et al. For the first library pCC1BAC backbone was used. For the second library pIndigoBAC-<NUM> was used, both from Epicenter. Approximately <NUM> clones with an average insert size of ~<NUM> Kbp , corresponding to <NUM> genome equivalents, were obtained for library <NUM>, and approximately <NUM> clones with an average insert size of ~<NUM> Kbp , corresponding to <NUM> genome equivalents, were obtained for library <NUM>. The BAC clones were stored as bacterial pools containing approximatively <NUM> to <NUM> white colonies. These were generated by scraping the colonies from the agar plates and successive resuspension into LB medium containing <NUM>% glycerol and <NUM>µg ml-<NUM> chloramphenicol using a sterile glass spreader. These so-called super pools were stored at -<NUM>. Marker screening of the BAC libraries was done, first by isolating plasmid DNA from each pool using the standard alkaline lysis protocol and PCR was carried out to identify positive pools. Bacteria corresponding to positive pools were diluted and plated on LB agar plate containing chloramphenicol (<NUM>µg ml-<NUM>). Individual white colonies were picked into <NUM>-well microtitre plates and single positive BAC clones were subsequently identified by marker screening as described by (Rouppe van der Voort et al. Names of BAC clones isolated from the super pools carry the prefix CHC and are extended with a number (B1 and B2), corresponding to the order in which they were identified.

Candidate RGAs were subcloned from BAC clone CHC B2 as follows. Primers were designed approximately <NUM> kb upstream of the predicted start codon and approximately <NUM> bp downstream of the predicted stop codon. (CHC B2-1F= MN459: tgaccctgcaggGGACCCCTTAACAAGTGATGTG,.

BAC clone sequencing was performed using a shotgun cloning strategy of 2kb and 6kb libraries and was carried out by Macrogen (South-Korea). Sequencing reactions were performed using the dye terminator principle. Sequence contigs were assembled by Macrogen. Gap closing was done using primer walking on shotgun clones or directly on the BAC.

The contig sequences were analyzed using the web-based application FGENESH (Softberry) in order to predict gene structure. RGAs and RGAs from publically accessible databases were aligned for homology and distance analysis using the DNA star software package (Lasergene). Conserved domains were identified using the web-based application SMART (EMBL).

Detached leaf assays were used to determine the resistance phenotypes of primary transformants and N. benthamiana leaves. For the phenotyping of the CHC population isolate <NUM> was used. For the phenotyping of the ber population, isolate IPO-C was used. The resistance spectra of the resistant parents was determined using the isolates described in Table <NUM>. Inoculum preparation and inoculation were performed as described by (Vleeshouwers et al. Six days after inoculation, plant phenotypes were determined. Leaves showing no symptoms or a localized necrosis at the point of inoculation were scored as resistant and those with clear sporulating lesions as susceptible.

Agrobacterium transient transformation assays (agro-infiltration) were carried out on N. benthamiana. Recombinant A. tumefaciens AGL1+ cultures were grown in LB medium (<NUM> gram bacteriological peptone, <NUM> gram NaCl and <NUM> gram yeast extract in <NUM> liter MQ water) supplemented with <NUM>/l Tetracycline and <NUM>/l Kanamycin for the pBINplus constructs. After one or two days a calculated amount of culture (according to OD <NUM> at <NUM>) was transferred to YEB medium (<NUM> gram beef extract, <NUM> gram bacteriological peptone, <NUM> gram sucrose, <NUM> gram yeast extract, <NUM> <NUM> MgSO4 in <NUM> liter MQ water) supplemented with Kanamycin for all strains. After <NUM> day overnight cells were centrifuged at <NUM> rpm and re-suspended in MMA medium (<NUM> gram sucrose, <NUM> gram MS salts and <NUM> gram MES) supplemented with <NUM> <NUM> acetosyringone to a final OD of <NUM> and infiltrated into <NUM> weeks old plants with a <NUM> syringe. Infiltrated leaves were subsequently challenged after two days with P. infestans strain <NUM> in detached leaf assays (DLA). Hypersensitive response (HR) or P. infestans sporulation were scored from <NUM> to <NUM> days post inoculation.

In this study we used <NUM> Solanum plants, their names as used in this study and accession numbers are listed in Table <NUM>. Nine late blight resistant plants were used for the isolation of functional homologs of Rpi-chc1 (tar852-<NUM>, ber94-<NUM>-<NUM> which derives from PI473331, ber481-<NUM>, ber493-<NUM>, -<NUM>, -<NUM>, chc543-<NUM>, ber324-<NUM>, ber487-<NUM>, ber561-<NUM>, and scr849-<NUM>). CHC543-<NUM> was crossed with CHC544-<NUM> to produce population <NUM>. BER94-<NUM>-<NUM> was crossed with the susceptible clone G254 to generate population <NUM>-<NUM>. BER493-<NUM> was crossed with RH89-<NUM>-<NUM> to produce population <NUM>. Potato cultivar Desiree was used for transformation. Wild-type Nicotiana benthamiana plants were used for transient complementation assays.

Rpi-chc1 homologs were PCR amplified using the long range high fidelity thermostable DNA polymerase Phusion® according to the manufacturer's instructions (New England Biolabs). Primers were designed, overlapping the start and stop codons of Rpi-chc1 and contained AttB1 and AttB2 extensions (MN595 and MN597, Table <NUM>). PCR products were recombined into pDONR221 using BP clonase® according to manufacturer's instructions (InVitroGen). DNA sequencing was performed at Baseclear (The Netherlands) using standard and custom primers (MN622-MN650, Table <NUM>). Sequences were analyzed and aligned for homology and phylogeny analysis using the DNA star software package (Lasergene).

In order to produce clones containing the promoter and terminator of Rpi-chc1 for construction of triple point gateway application mediated expression constructs, specific primers were designed (MN598, MN599, MN600, MN601, MN670; Table <NUM>) matching the Rpi-chc1 promoter and terminator sequences, to which AttB4, AttB1 and AttB2, AttB3 recombination sites were added, respectively. PCR products were generated using the long range high fidelity thermostable DNA polymerase Phusion® according to the manufacturer's instructions. PCR products were recombined using BP clonase®. The occurrence of PCR errors was ruled out zzusing sequence analysis of the resulting clones using primers MN651 and <NUM> as listed in Table8. Triple point gateway reactions were performed using these constructs and ORF sequences in pDONR221 by LR clonase.

Detached leaf assays were used to determine the resistance phenotypes of primary transformants and N. benthamiana leaves. For the phenotyping of the CHC transgenics isolate <NUM> was used. For the phenotyping of the Rpi-chc1 homologs in N. benthamiana, isolate IPO-C was used. Inoculum preparation and inoculation were performed as described by Vleeshouwers et al. Six days after inoculation, plant phenotypes were determined. Leaves showing no symptoms or a localized necrosis at the point of inoculation were scored as resistant and those with clear sporulating lesions as susceptible.

Agrobacterium transient transformation assays (agro-infiltration) were carried out on N. benthamiana. Recombinant A. tumefaciens COR308 cultures were grown in LB medium (<NUM> gram bacteriological peptone, <NUM> gram NaCl and <NUM> gram yeast extract in <NUM> liter MQ water) supplemented with <NUM>/l tetracycline and <NUM>/l kanamycin for the pBINplus constructs. After one or two days a calculated amount of culture (according to OD <NUM> at <NUM>) was transferred to YEB medium (<NUM> gram beef extract, <NUM> gram bacteriological peptone, <NUM> gram sucrose, <NUM> gram yeast extract, <NUM> <NUM> MgSO<NUM> in <NUM> liter MQ water) supplemented with kanamycin for all strains. After <NUM> day overnight cells were centrifuged at <NUM> rpm and re-suspended in MMA medium (<NUM> gram sucrose, <NUM> gram MS salts and <NUM> gram MES) supplemented with <NUM> <NUM> acetosyringone to a final OD of <NUM> and infiltrated into <NUM> weeks old plants with a <NUM> syringe. Infiltrated leaves were subsequently challenged after two days with P. infestans strain <NUM> in detached leaf assays (DLA). Hypersensitive response (HR) or P. infestans sporulation were scored from <NUM> to <NUM> days post inoculation.

A set of <NUM> effectors was present in Agrobacterium tumefaciens COR308 in a PVX plasmid (PEX set). The binary plasmids contain an effector from Pi cloned inside the PVX genome. Upon agro-infiltration both effector and PVX will be expressed. Within the time course of the experiment PVX can not spread systemically and we are only interested in the local expression of the effector. Upon recognition of the encoded effector by the R-gene, an HR can be observed between <NUM> and <NUM> dpi. PVX symptoms are visible after <NUM> days and are generally first observed in non-infiltrated leaves.

As a positive control we used R3a and Avr3a-KI, an R-gene - Avr-gene combination which is known to give a strong response (Armstrong et al. Screening with the Rpi-chc1 candidate showed necrotic spots with two potential effectors genes RD12-<NUM> and RD12-<NUM> (<FIG>).

In the previous example we described the map based cloning of the Rpi-chc1 gene from Solanum chacoense accession <NUM>-<NUM>. Rpi-chc1 is the founder of a previously undescribed R gene family of the CC-NB-LRR class and is located on chromosome <NUM> near marker TG63. The gene was present in a gene cluster with five homologs. Genetic analysis revealed that only three of these homologs (CHC B2-<NUM>, CHC B2-<NUM>, and CHC B2-<NUM> could potentially encode Rpi-chc1. Transient complementation analysis in N. benthamiana suggested that CHC B2-<NUM> was the active copy.

In this experiment we show by stable transformation of the susceptible cv. Desiree that indeed CHC B2-<NUM> could complement the Phytophthora infestans (Pi) susceptibility (<FIG>). This result supports our previous suggestion that CHC B2-<NUM> is Rpi-chc1. Also this result shows that Rpi-chc1 can be functional in a broad spectrum of Solanaceous species, such as S. chacoense and N. benthamiana but also in S.

In order to understand the activity spectrum of Rpi-chc1, it was investigated which component of Pi was recognized. Until now all Pi components being recognized by host R-proteins are effectors of the RXLR class. Pi isolate T30-<NUM> is a-virulent on plants expressing Rpi-chc1 and therefore the cognate component must be expressed in this isolate. Recently the genome of T30-<NUM> was sequenced and its genome appears to encode hundreds of RXLR effectors (Haas et al. Sixty-five RXLR effectors comprising all known Avr's (Avr1, Avr2, Avr3a, Arv4, Avr-blb1, Avr-blb2) and also a few non RXLR effectors (Inf1, PiNIP) effectors were cloned into the plant expression vector pGR106 and are referred to as the PEX set (Vleeshouwers et al. The PEX set was screened by co-agro-infiltration with Rpi-chc1 in N. benthamiana. This way both the selected effector and the Rpi-chc1 gene are expressed in the same cells. In case the effector is recognized by Rpi-chc1 it will induce a hypersensitive response (HR) and will result in a necrotic lesion in the infiltrated area of the leaf. This phenomenon was well described for the co-infiltration of R3a and Avr3a (Armstrong et al. , <NUM>) which was included in our experiments as a positive control (<FIG>). Leaf areas that were agro-infiltrated with Rpi-chc1 alone remained green which showed that Rpi-chc1 in itself did not induce cell death. Also co-infiltration with the previously described Avr's (Avr1, Avr2, Avr3a, Arv4, Avr-blb1, Avr-blb2) did not induce HR, which showed that Rpi-chc1 recognizes a new component of Pi and that it has a unique way of inducing resistance. On the other hand some effectors in the PEX set produced an Rpi-chc1 independent hypersensitive response (<FIG> leaf C). There were, however also two clones in the PEX set that only showed an Rpi-chc1 dependent cell death (<FIG> leaf B). Both clones (RD12-<NUM> and RD12-<NUM>) were highly homologous to each other and in fact encoded identical proteins. RD31, that encodes a protein with <NUM>% identity to RD12 was not recognized (<FIG> leaf A), showing that recognition by Rpi-chc1 was quite specific. In order to test the specificity of recognition on the R-gene side, RD12 was co-infiltrated with Rpi-blb1, Rpi-blb3 and R3a. Also the Rpi-chc1 paralogs CHC B2-<NUM> and CHC B2-<NUM> (see Example <NUM>), which showed <NUM>% and <NUM>% identity, respectively, at the amino acid level to Rpi-chc1, were tested by co-infiltration. None of these R-genes or R-gene paralogs produced a hypersensitive response upon co-infiltration with RD12 (data not shown). These results clearly showed that Rpi-chc1 could specifically recognize Pi component RD12. RD12 (=PITG_16245 has several paralogs in the Pi genome (PITG_16418, PITG_16427, PITG_16233, PITG_16240, PITG_20934, PITG_20936, PITG_20336, and PITG_23230), of which the sequences are given below.

It can not be excluded that also these paralogs are recognized by Rpi-chc1 in the interaction with Pi. Neither can it be ruled out that additional unrelated Pi components can be recognized since dual specificity R-genes have been described (Jones and Dangl, <NUM>).

In order to determine which regulatory sequences were most suited to drive the expression of the open reading frames of Rpi-chc1, we used the strategy described before (Lokossou et al. , <NUM>) in which the candidate ORFs are cloned in between the desired promoters and terminators using a triple point gateway strategy. The Rpi-chc1 ORF was cloned in between its own 3kb promoter and <NUM> kb terminator (p-chc1-long) which were also present in the initial complementation analyses as presented in <FIG>. In addition, Rpi-chc1 ORF was cloned in between three alternative promoter/terminator combinations. A shorter version (<NUM> kb) of its own promoter and its own <NUM> kb terminator (p-chc1-short); the double <NUM> promoter in pMDC32, and the Rpi-blb3 promoter/terminator combination (Lokossou et al. In order to test which was the optimal promoter terminator combination, the four Rpi-chc1 constructs were transformed to AGL-<NUM>+virG, cultures were mixed <NUM>:<NUM> with A. tumefaciens COR308 containing PEX-RD12. Serial diltutions in MMA medium were infiltrated in the leaves of N. benthamiana (<FIG>). The p-chc1-long construct induced HR in mixtures with RD12 of OD<NUM> <NUM> and <NUM>. The p-chc1-short construct also expressed HR in a two fold lower concentration (OD<NUM>=<NUM>). Remarkably, the <NUM> and Rpi-blb3 promoter/terminator constructs were not suitable for functional expression of the Rpi-chc1 gene. These results show that the promoter of Rpi-chc1 is functionally distinct from the other promoters tested. Furthermore, it is concluded that sequences upstream (< -<NUM> bp) in the Rpi-chc1 promoter contain inhibitory elements for expression.

To further support the suggestion that Rpi-chc1 can be active in a wide range of Solanum species and also study divergence of the Rpi-chc1 allele sequence and activity in the germplasm we screened <NUM> genotypes (Table <NUM>) from our germplasm collection for the presence of Rpi-chc1 related sequences using a sequence alignment of the active Rpi-chc1 and several related sequences identified in the initial application that were derived from RH89-<NUM>-<NUM> and from the inactive paralogs in chc543-<NUM>. Primer pairs (Table <NUM>) were designed in such a way that only the active copy was predicted to be amplified by PCR. As shown in <FIG>, primer combinations D and E were highly specific since PCR products were observed only in reactions that contained the Rpi-chc1 template and no amplification was found from the templates that contained closely related sequences. Primer combinations D and E were used to screen the recombinants in the finemapping population (n=<NUM>) of S. chacoense and S. berthaultii (n=<NUM>; Rpi-ber; accession PI265858; <NUM>-<NUM> * G254) in which Pi resistance is segregating. No recombinants were found between the marker and the resistance in either population (data not shown). This showed that both markers are highly specific. Also this showed that the Rpi-ber gene is related to Rpi-chc1 and that Rpi-chc1 derived molecular markers can be used to tag these resistance genes.

Genotype chc543-<NUM>, from which Rpi-chc1 was isolated, is located in taxonomic group10-<NUM> (Jacobs et al. In order to screen for other Rpi-chc1 homologous sequences, <NUM> genotypes in our germplasm collection (Table <NUM>) located in taxonomic groups <NUM>-<NUM> till <NUM>-<NUM> were selected. DNA integrity was confirmed using Ef1-a PCR (data not shown) and successively primer combination D was used to screen for Rpi-chc1 related sequences. Six genotypes were found to be positive in this screen (<FIG>). First of all chc543-<NUM> was found, which confirmed the robustness of the screen. Besides, five other genotypes were identified amongst which S. berthaultii plants <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>, confirming the previous suggestion that Rpi-chc1 and Rpi-ber are very related. Also two other species were tagged, S. tarijense (<NUM>-<NUM>) and S. sucrense (<NUM>-<NUM>).

In order to further characterize functional and sequence conservation or divergence of Rpi-chc1 we set out to clone the open reading frames from the plants that were positive in the germplasm screen and in addition from plants known to contain resistance genes on chromosome <NUM> (described in <FIG>). Primers overlapping the start- and the stopcodon of Rpi-chc1 were designed and attB1 and AttB2 extensions were added for BP cloning into pDONR221. PCR reactions using the proofreading polymerase Phusion® resulted in specific products for all selected genotypes. These PCR fragments were cloned and for each genotype six colonies were selected and end sequenced. Some genotypes produced only one sequence type and for those genotypes we concluded that only one target gene was amplified. For genotypes with two or more sequence types an additional <NUM> colonies were end sequenced and grouped. From each sequence group three clones were fully sequenced using Rpi-chc1 derived internal primers. This resulted in the identification of <NUM> new Rpi-chc1 like sequences (<FIG>). The encoded protein sequences were aligned using clustal-W together with previously identified Rpi-chc1 homologs (<FIG>). This resulted in the phylogenetic tree as presented in <FIG>. From chc543-<NUM> we isolated two sequence types. The first type was identical to Rpi-chc1. The second sequence type located in a different clade (clade <NUM> in <FIG>) with multiple sequences, all deriving from S. berthaultii plants, showing that this approach was successful in identifying Rpi-chc1 homologs. Four genotypes yielded only one sequence type <NUM>-<NUM>, RH89-<NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>-<NUM>. The first three located to the same clade (clade <NUM> in <FIG>). RH89-<NUM>-<NUM> sequences RH_D3, D4, and D7 were identical to each other and showed two nucleotide mismatches with RH137D14 c13-<NUM>, a sequence that was generated during construction of the RH physical map in the initial application. Both sequences located to clade <NUM> which also contained S. sucrense sequences <NUM>-1_M8, M18, and M20, and also S. berthaultii sequences <NUM>-<NUM>, I4, I6 andI8 was M20. In addition S. tarijense <NUM>-5_E3 was present in clade <NUM>. Because RH89-<NUM>-<NUM> is susceptible to Pi infection, it is suggested that these sequences represent inactive homologs. Two other sequences isolated from S. tarijense <NUM>-<NUM> located in clade <NUM> which also harboured the Rpi-chc1 gene. Furthermore, three sequences from S. berthaultii plants <NUM>-<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> were found in this clade which showed only minor sequence deviation and encoded identical aminoacid sequences. Clade <NUM> contained only sequences from S. berthaultii plants. Clade <NUM> contained only sequences that were identified before as also was the case in the remaining group, referred to as group <NUM>. Clades <NUM> till <NUM> had a <NUM> a. N-terminal extension of the encoded protein as compared to proteins in clade <NUM> and group <NUM>. Sequences in clade <NUM>, <NUM> and <NUM> mapped to the R-gene cluster within <NUM> to TG63. No sequences in clades <NUM> and <NUM> have been genetically mapped. Comparison with the newly available S. phureja genome sequence revealed that sequences from clade <NUM> till <NUM> had closest homologs in the TG63 cluster. Comparison to the tomato genome revealed that also here an Rpi-chc1 cluster near TG63 existed. As shown before, at this genetic location the Pi resistance gene Ph-<NUM> was mapped. Some tomato plants, that were sequenced did not carry the Ph-<NUM> resistance gene but a potential inactive allele could be present (<FIG>). Group <NUM> sequences had closest homology to a related R-gene cluster near TG403 on chromosome <NUM>, an area where we also mapped Pi resistance (see <FIG>), showing that also Rpi-chc1 homologous sequences from this cluster potentially encode Pi resistance.

Now we have identified <NUM> new Rpi-chc1 homologs and we have shown sequence diversification, the question arises if functionality is conserved or diversified among those sequences. All identified sequences, which are ORFs, were subcloned using triple point gateway recombination under the control of the Rpi-chc1-short promoter and the Rpi-chc1 terminator in the binary vector pDEST236. Based on the results in <FIG>, this was considered the best constellation to drive the expression of the mined Rpi-chc1 homologs. Successively, the constructs were transformed into A. tumefaciens strain COR308 for transient complementation assays in N. benthamiana. Alternatively, for co-expression with the cognate Pi effector RD12, the Rpi-chc1 homologs were transformed into A. tumefaciens strain AGL1+virG. Both experiments are complementary since the transient complementation assay could show whether a Rpi-chc1 could induce resistance, the co-infiltration could indicate the recognition specificity of the gene. All experiments were repeated at least twice and the results are summarized in Table <NUM>. Several combinations of RD12 responsiveness and IPO-C resistance can be observed. Two clear groups can be distinguished. A first group is not responsive to RD12 and is susceptible to IPO-C (group <NUM>; Table <NUM>). These sequences are inactive homologs and mainly locate in phylogenetic clade <NUM> (<FIG>). The second group (group <NUM>; Table <NUM>) are functional homologs of Rpi-chc1 since they are actively inducing resistance against Pi and they recognize the same Pi component (RD12). The sequences of this group are also clearly distinct from the other sequences since they all locate in clade <NUM> (<FIG>). tarijertse <NUM>-<NUM> clone E28 induces HR in the absence of RD12 and is in that sense unique in its activity pattern and constitutes activity group <NUM>. Since it does not induce resistance it is most likely an inactive allele. Another allele from the same plant (clone E14) does not recognize RD12 but does induce strong resistance. Activity group <NUM> is therefore distinct from group <NUM> because it most likely recognizes a different component from Pi. Activity group <NUM> is quite similar to group <NUM>; the only difference is that disease resistance is not that strong. This suggests that also group <NUM> recognizes different components from Pi and will have a different resistance spectrum. The last group (Group <NUM>) is distinct because RD12 is only weakly recognised and also resistance is weak. Summarising, these data show that the closest related Rpi-chc1 homologs have a conserved resistance mechanism, while less related sequences have a more diversified resistance mechanism. Altogether, these data show that multiple members of the Rpi-chc1 gene family, with different extents of similarity, are functional in providing resistance again Pi.

Claim 1:
A method for increasing resistance in a plant against Phytophthora infestans infection comprising transforming a plant or a part thereof with a nucleic acid encoding the amino acid sequence of SEQ ID NO: <NUM> or a functional fragment thereof, wherein said functional fragment comprises at least the LRR domain of the amino acid sequence, depicted as amino acids <NUM>-<NUM> of Fig. <NUM>, capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection or a homologue thereof, wherein said homologue has an identity of at least <NUM>% with the amino acid sequence of SEQ ID NO: <NUM>, which homologue is capable of increasing resistance in a plant of the Solanaceae family against Phytophthora infestans infection.