Patent Grant
9856494
This application is the U.S. National Phase of, and Applicants claim priority from, International Application Number PCT/NL2011/050386 filed May 31, 2011 and European Patent Application No. EP 10164531.5 filed May 31, 2010, each of which are incorporated herein by reference.
The invention relates to a resistance gene isolated from S. x edinense. Moreover, the invention relates to the use of said resistance gene, for example to clone functional homologues, and the use of said resistance gene(s) in a method to increase or confer at least partial resistance to an oomycete infection in a plant. More in specific the invention provides a resistance gene that is capable of increasing or conferring at least partial resistance to Phytophthora sp. (for example Phytophthora infestans) through genetic engineering techniques or through marker assisted breeding techniques.
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 11 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 8).
Recent efforts to identify late blight resistance have focused on major R genes conferring broad-spectrum resistance derived from diverse wild Solanum species. Beside S. demissum, other wild Solanum species such as S. acaule, 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, 2000)).
S. x edinense P. Berthault, a pentaploid (2n=5x=60) potato species from Mexico, is a natural hybrid between the Mexican Solanum demissum and the South American S. tuberosum spp. andigena. The pentaploid S. x edinense had been identified as an interesting source of resistance to P. infestans already in 1908 by Salaman and was included in breeding programs by Brioli in 1914 (Pavek et al. 2001; Toxopeus 1964). It was named after the Edinburgh Botanic Garden (Glendinning 1983), where its hybrid characteristic was first described. It has been used in breeding programs and has revealed good field resistance to P. infestans (Van Soest et al. 1984). Two functional R genes have been cloned from one S. x edinense genotype (edn151-3): Rpi-edn1.1 and Rpi-edn1.2 also known as R2-like (Champouret 2010). They were identified by allele mining of the R2 family. Both are located in the R2 cluster on chromosome 4. Both R genes recognize AVR2 (Champouret 2010; Lokossou et al. 2009) and their resistance is not effective against all P. infestans isolates, including IPO-C (Lokossou et al. 2009).
To date, not only from this species, but also from other Solanum species late blight R-genes have been cloned, like the allelic genes RB and Rpi-blb1 on chromosome 8 and Rpi-blb2 on chromosome 6 (Table 6) of S. bulbocastanum. Recently, also an Rpi-blb3 resistance gene has been isolated (WO 2008/091153). Also a resistance gene of S. chacoense has been characterized (EP 09170769.5). Although the initial results obtained with RB and Rpi-blb1, -2 and -3 are promising, there is a further need for additional R-genes, especially because allele mining of these genes in S. bulbocastanum genotypes revealed that natural stacking of Rpi-blb1, and -3 in a single genotype occurs at relatively high frequency (Lokossou 2010). S. venturii is another example of the presence of several R genes with different specificities in a single genotype (Pel 2010). Stacking several R genes in a single genotype appears to be a feasible strategy to achieve high level and durable protection against potential pathogens. Pyramiding of R genes is still controversial and it is not known whether it is a durable approach (McDowell et al. 2003; Pink et al. 1999; Pink 2002). The pyramiding of Rpi-ber1 (Rauscher et al. 2006), an R gene with a strong effect, and Rpi-mcd1 (Tan et al. 2008), an R gene with a weak effect, revealed an additive effect on the resistance level (Tan et al. 2010). Observing natural pyramiding of R genes strengthens the idea that plants can benefit from combining individual R genes, even including some with weaker effect (Pink 2002).
The invention now relates to an isolated or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence Rpi-edn2 of
In another embodiment, the invention relates to a vector comprising a nucleic acid sequence according to the invention. Further comprised in the invention is a host cell comprising a nucleic acid according to the invention or a vector according to the invention, wherein said host cell preferably is an Agrobacterium cell or a plant cell.
In another embodiment, the invention comprises a plant cell comprising a nucleic acid or a vector according to the invention. Said plant cell preferably is a cell from a Solanaceae plant, more preferably Solanum tuberosum, more preferably a tetraploid Solanum tuberosum. Also the invention relates to a transgenic plant comprising such a cell and a part derived from such a plant, more preferably wherein said part is a tuber.
Also part of the invention is a protein encoded by an isolated or recombinant nucleic acid according to the invention or a functional fragment or a functional homologue thereof, preferably wherein said protein has the amino acid sequence of Rpi-edn2 as depicted in
Further disclosed in the invention is an antibody that (specifically) binds to such a protein.
In yet another embodiment, the invention relates to a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection comprising providing a plant or a part thereof with a nucleic acid or a vector or a host cell or a protein according to the invention. In such a method said plant preferably is a plant from the Solanaceae family, more preferably Solanum tuberosum. Also preferred is such a method wherein said oomycete comprises Phytophthora, preferably Phytophthora infestans.
In still a further embodiment, the invention relates to a binding molecule capable of specifically binding to a nucleic acid according to the invention or its complementary nucleic acid, preferably wherein said binding molecule is a primer or a probe.
In yet a further embodiment the invention comprises a method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to an oomycete infection, said method comprising the steps of testing at least part of said plant or plant material or progeny thereof for the presence of absence of a nucleic acid according to the invention, preferably wherein said testing is performed with a primer or a probe that specifically binds to said nucleic acid, or where the testing involves detecting the presence of one or more of the markers of Table 5 and 7, or wherein the marker comprises part of the sequence of the Rpi-edn2 gene as depicted in
Further, the invention relates to a method for breeding a resistant tetraploid plant, comprising
In another embodiment, the invention comprises 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 5 and 7, or wherein the marker comprises part of the sequence of the Rpi-edn2 gene as depicted in
Two days after agro-infiltration with either pDEST32:edn2 or empty binary vector, the leaves were challenged by the inoculation with a zoospore suspension of P. infestans isolate IPO_C (left leaf half) and H30PO4 (right leaf half). Resistance to both isolates co-segregated with the chromosome 9 gene in the F1 population. Typical disease phenotypes developed 6 days after inoculation of control plants that had been agro-infiltrated with binary vector without. Resistance was visible as a HR or XR (eXtreme Resistance) in plants agro-infiltrated with Rpi-edn2.
Effector candidates were agroinfiltrated into the right leaf half of N. benthamiana at OD600=0.5 (spot2=Avr3a, 4=PITG_09616, 6=PITG_10540, and 8=PITG_15039). In the left leaf half the same effectors are co-infiltrated with R3a (spot1=Avr3a) or with Rpi-edn2 (spots 3=PITG_09616, 5=PITG_10540, and 7=PITG_15039). Pictures were taken six days after agro-infiltration.
Genes were predicted using FGENESH algorithm. A yellow arrow shows the presence of a mutator transposable element (gene a). Red arrows show the presence of Rpi-edn2 and Rpi-edn2-like sequences (genes b and c). The box in the first red arrow shows the location of the single exon encoding Rpi-edn2 protein. Positions in the BAC insert as depicted in
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 potato plant 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 0, 1, 2, 3 or 4 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”, “tetraploid” and “pentaploid” as used herein are defined as having respectively one, two, four and five 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-edn2 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 the Rpi-edn2 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” or “primer pair” 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 2-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 5° C. 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 50% 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 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes or primers (e.g. greater than 50 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 30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures are well known in the art and are described in e.g. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. eds. (1998) Current protocols in molecular biology. V. B. Chanda, series ed. New York: John Wiley & Sons.
The present invention describes the cloning of the Rpi-edn2 gene. Rpi-edn2 was mapped to an R gene cluster on chromosome 9 of S. x edinense. The gene contains three domains that are common to other resistance genes, the CC, NBS and LRR domain.
To date, five principal classes of R-genes have been identified, based upon conserved protein domains (for review see Martin G B, Bogdanove A J, Sessa G, Annu Rev Plant Biol 2003, 54:23-61). The most abundant class are the cytoplasmic nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins (Rommens C M, Kishore G M, Curr Opin Biotechnol 2000, 11:120-125). The other classes comprise proteins with extracytoplasmic LRRs (eLRRs) anchored to a transmembrane (TM) domain (receptor-like proteins [RLPs]), cytoplasmic serine-threonine (Ser/Thr) receptor-like kinases (RLKs) with extracellular LRRs (such as disclosed in WO 2004/007712), cytoplasmic Ser/Thr kinases without LRRs, and proteins with a membrane anchor fused to a coiled coil (CC) domain. The common NBS-LRR-encoding proteins currently include over 20 functionally proven R-genes from diverse plant species (Van Der Biezen E A, Freddie C T, Kahn K, Parker J E, Jones J D, Plant J 2002, 29:439-451). Studies have focused on this family because its only known function to date is in disease resistance (Meyers B C, Kaushik S, Nandety R S, Curr Opin Plant Biol 2005, 8:129-134). Gene products are composed of a conserved central NBS and variable length C-terminal LRR domain of 10 to 40 short LRR motifs (Cannon S B, Zhu H, Baumgarten A M, Spangler R, May G, Cook D R, Young N D, J Mol Evol 2002, 54:548-562). The NBS domain is important for ATP binding and hydrolysis and is believed to be involved in signal transduction, triggered by the presence of the pathogen (van Der Biezen E A, Jones, Curr Biol 1998, 8:R226-R227; Tameling W I, Elzing a S D, Darmin P S, Vossen J H, Takken F L, Haring M A, Cornelissen B J, Plant Cell 2002, 14:2929-2939). The LRR domain is likely to be involved in protein-protein interactions, recognizing pathogen elicitor molecules (Young N D, Curr Opin Plant Biol 2000, 3:285-290. A high mutation rate in the LRR contributes to genetic variability, necessary for specific recognition of diverse pathogens (Michelmore R W, Meyers B C, Genome Res 1998, 8:1113-1. Two subfamilies exist in NBS-LRR R proteins based upon N-terminal motifs. The TIR NBS subfamily R proteins display homology between the N-terminal amino acid motif and the receptor domain in Drosophila Toll and basal mammalian Interleukin (IL) 1 immunity factors in animals (Parker J E, Coleman M J, Szabo V, Frost L N, Schmidt R, van Der Biezen E A, Moores T, Dean C, Daniels M J, Jones J D, Plant Cell 1997, 9:879-894. Non-TIR NBS subfamily R proteins can contain an N-terminal coiled-coil (CC) motif, a subset of which code for a leucine zipper sequence (LZ). TIR subfamily NBS-LRR proteins appear to be restricted to dicotyledons.
A coiled-coil (CC) domain is located in the N-terminal parts of the Rpi-edn2 protein between amino acids 1 and 153 (amino acid sequence depicted in
At the protein level, Rpi-edn2 shares 80% amino acid identity with Rpi-mcq1.1, 77% with Rpi-mcq1.2. Lower percentage homology was found with Rpi-vnt1 (73%) and Tm-22, a tomato resistance gene against Tomato Mosaic virus, sharing 73% and 72% identity, respectively.
In a first embodiment, the invention provides an isolated or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence Rpi-edn2 as presented in
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-edn2 protein or the nucleic acid sequence encoding therefore, that is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete infection, more specifically against P. infestans, more specifically against isolate IPO-C. Such a fragment is, for example, a truncated version of the Rpi-edn2 protein. A truncated version/fragment of the Rpi-edn2 protein is a fragment that is smaller than 863 amino acids and preferably comprises (part of) the NB-ARC and the LRR domains and/or the N-terminal CC domain of the Rpi-edn2 protein.
The term “functional homologue” is typically used to refer to a protein sequence or the nucleic acid sequence encoding for such a protein that is highly homologous to or has a high identity with the herein described Rpi-edn2 protein or nucleic acids, which (encoded) protein is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete infection, more specifically against P. infestans, more specifically against isolate IPO-C. Included are artificial changes or amino acid residue substitutions that at least partly maintain the effect of the Rpi-edn2 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-edn2 protein in which amino acids have been inserted, replaced or deleted and which at least partly maintain the effect of the Rpi-edn2 protein (i.e. at least partly providing or increasing resistance in a plant of the Solanaceae family against an oomycete infection, more specifically against P. infestans, more specifically against isolate IPO-C). Preferred variants are variants which only contain conventional amino acid replacements as described above. Also included in the term “functional homologue thereof” are homologous sequences. Preferably, such a homologue has more than 80% identity on the amino acid level. More preferably, the amino acid has an identity of at least 85 or 90%. Even more preferred are amino acids that have an identity of 91, 92, 93, 94 or 95%. Most preferred are amino acids that have an identity of 96, 97, 98 or 99% with the amino acid sequence of Rpi-edn2. Homologous proteins according to the invention have a higher degree of identity with the Rpi-edn2 sequence as the sequences aligned with those proteins in
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 100% identical to the Rpi-edn2 protein as presented in
Fragments as well as homologues of the herein described Rpi-edn2 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.
Agro-infiltration forms a functional screen for testing candidate genes, whereby 4 week old wild type Nicotiana benthamiana plants are infiltrated with Agrobacterium strains containing the candidate Rpi-edn2 homologues or nucleotide sequences coding therefore. The infiltrated leaves are subsequently challenged one or several (maximum 3) days after infiltration with a P. infestans strain that is virulent on N. benthamiana, for example IPO-C or 90128, in detached leaf assays. This system is equally suitable for testing candidate homologous fragments of Rpi-edn2.
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 2-3 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 50-fold or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. Although it is clear that the use of p19 has advantages, an agro-infiltration 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 H30P04, IPO-C, CA65, USA618 or 90128. Transformants that are resistant to these isolates, especially against IPO-C, harbour for example functional fragments or homologues of Rpi-edn2.
In yet another embodiment, the invention provides a vector comprising a nucleic acid as provided herein, i.e. a nucleic acid capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete 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-edn2 of
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-edn2 to a plant that does not contain the gene and to identify those progeny of the cross that have inherited the Rpi-edn2 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
The herein described Rpi-edn2 protein comprises 863 amino acids. Rpi-edn2 shares the highest homology with Rpi-mcq1.1 (80%) and Rpi-mcq1.2 (77%), R genes from S. mochiquense. However, as is shown in Table 6, the Rpi-edn2 protein of the present invention differs from these highly homologous proteins by the fact that it provides resistance towards a different spectrum of Phytophthora isolates. Rpi-edn2, like Rpi-vnt1, Tm-22 and Rpi-mcq1.1, is a member of the large family of CC-NBS-LRR resistance genes. The reference genes Tm-22, Rpi-vnt1 and Rpi-mcq1.1 can be said to be grouped in a so-called Tm-22 family subgroup, of which Rpi-edn2 forms a member. However, on basis of the sequence homology, Rpi-edn2 can be considered to form a new subclass within this Tm-22 family.
As already described, a functional fragment or a functional homologue thereof of Rpi-edn2 is a fragment or homologue that is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against an oomycete infection.
Means to test the functionality of a functional fragment or a functional homologue of Rpi-edn2 have been provided above.
Based on the herein described nucleic acid sequences, the invention also provides probes and primers (i.e. oligonucleotide sequences complementary to the (complementary) DNA strand as described in
Hence, in a further embodiment, the invention provides a binding molecule capable of binding to a nucleic acid 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 invention 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-edn2 in the plant genome. Such a method would be especially preferable in marker-free transformation protocols, such as described in WO 03/010319.
The herein described Rpi-edn2 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(s) encoded by the herein described isolated or recombinant nucleic acid (for example the nucleic acid sequence of
Based on the herein provided nucleic acid sequence, the invention also provides the means to introduce or increase resistance against an oomycete infection in a plant. The invention therefore also provides a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection comprising providing a plant or a part thereof with:
Such a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection may be based on classical breeding, departing from a parent plant that already contains the Rpi-edn2 gene, 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 one or more nucleic acid sequences 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 (Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985), by electroporation (U.S. Pat. No. 5,384,253), by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523; and U.S. Pat. No. 5,464,765), and by acceleration of DNA coated particles (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No. 5,538,880). 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 Komari, T. et al.: Plant Transformation Technology: Agrobacterium-Mediated Transformation, in: Handbook of Plant Biotechnology, Eds. Christou, P. and Klee, H., John Wiley & Sons, Ltd, Chichester, UK 2004, pp. 233-262). Preferably a marker-free transformation protocol is used, such as described in WO 03/010319.
In a preferred embodiment, the target plant is transformed with additional resistance genes, a phenomenon known under the name of “gene stacking”. As is explained and shown in the experimental part, the presence of multiple resistance genes can enhance the resistance of a plant against infection because firstly the genes can complement each other with respect to resistance to various isolates or pathotypes of the infectious agent, and secondly, triggering more than one resistance mechanism (that by itself would not lead to a full resistance) can lead to a substantial increase of the resistance reactions in the host plant, which could well be sufficient to reach full resistance.
Alternatively, the nucleic acid of the Rpi-edn2 gene, and optionally other resistance genes, like Rpi-mcq1.1, Rpi-mcq1.2, Rpi-vnt1, Rpi-chc1, Rpi-avl1.1, Rpi-avl1.2, Rpi-blb1, Rpi-blb2, Rpi-blb3, and many others, 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 x edinense genotype (such as accession GLKS 25492, GLKS 25493 and GLKS 25494), 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 in the method according to the invention. 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 in the methods according to the invention may have any type of ploidy. For example, selected plants may be haploid, diploid, triploid, tetraploid or pentaploid.
Methods for crossing a polyploid plant with a tetraploid plant are well known in the art and can be readily applied by a person skilled in the art. For example, S. x edinense has been used for a long time in breeding programs especially for its good field resistance to P. infestans (van Soest, 1984). Crosses of the pentaploid S. x edinense with a tetraploid variety (e.g. Concurrent) yield tetraploid progeny. 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 in a method according to the invention, 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, in the present invention the presence of such a marker is detected at DNA level. Alternatively, proper expression of the Rpi-edn2 protein can be assessed in plant parts by transforming an immunoassay with an antibody that specifically binds the protein. Next to the primers and probes according to the invention, 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 Tm2-like profiling markers as indicated in Table. 7. Highly preferred markers are Tm1900, Tm19F-Mse, Stm021, mcq-ATG1, mcq-c2-stop, EDN-F and EDN-R and primers that were used for the Tm2-like profiling as described in the experimental part and Table 5.
Even more highly preferred markers are derived from the nucleotide sequence presented in
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 EP131623 and the bacterial aphIV gene suggested in EP186425 conferring resistance to hygromycin. EP 275957 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 EP218571. Suitable examples of reporter genes are beta-glucuronidase (GUS), beta-galactosidase, luciferase and green fluorescent protein (GFP). However, preferably a marker-free approach, such as disclosed in WO 03/010319, is used, where the presence of the resistance gene(s) can be assayed with nucleotide sequence based assays.
In a preferred embodiment, the invention provides a method for providing at least partial resistance or increasing resistance in a plant against an oomycete infection comprising providing a plant or a part thereof with:
The invention also provides a plant that is obtainable by using a method for providing at least partial resistance or increasing resistance in a plant against an oomycete 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. annuum, C. baccatum, C. chinense, C. frutescens and C. pubescens. The invention thus also provides a plant that has been provided with a nucleic acid encoding a Rpi-edn2 protein or a functional fragment or a functional homologue thereof.
The invention further provides a transgenic plant part or progeny of a plant according to the invention comprising a nucleic acid encoding the Rpi-edn2 amino acid sequence(s) of
In a preferred embodiment, the herein described nucleic acid is transferred to a Solanum variety other than Solanum edinense, i.e. the herein described nucleic acid is preferably provided to a non-edinense background, preferably S. lycopersicon or S. tuberosum. 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 to an oomycete infection, wherein said plant is provided with a nucleic acid encoding a further resistance gene, such as Rpi-blb1, -2, -3, Rpi-vnt1, Rpi-chc1, Rpi-avl1-1, Rpi-avl1-2, Rpi-R1, Rpi-R2, Rpi-R3a, Rpi-R3b, Rpi-mcd1 or Rpi-mcq1.
The invention further provides use of an isolated or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the Rpi-edn2 amino acid sequences of
In yet another embodiment, the invention provides a method for producing an Rpi-edn2 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, the plants that harbour the resistance molecules of the present invention also show a specific pathogen profile, in the sense that said plants will show a hypersensitive reaction (ending in necrosis of the infected tissue) with a number of elicitor or effector molecules derived from different isolates of Phytophthora infestans. As can be seen in Table 9, several elicitors evoke this response in the av1478-2 plant (for more details see the experimental part), such as PITG_20336, PITG_14039, PITG_20301, PITG_20303, PITG_20300, PITG_22880, PITG_09616, PITG_10540, PITG_15039, PITG_04097, PITG_04169, PITG_16726, PITG_23131 and PITG_07550_9, while other elicitors, such as Avr3a, Avr-vnt1, Avr-blb1, PITG_00774 and PITG_10465, only show no or a minimal response. Thus, also parts of the invention are those nucleic acids that, when transformed and expressed in plants, show a responsiveness to pathogen effectors that resemble the profile as depicted in Table 7, more specifically that show a reaction to PITG_20336, PITG_14039, PITG_20301, PITG_20303, PITG_20300, PITG_22880, PITG_09616, PITG_10540, PITG_15039, PITG_04097, PITG_04169, PITG_16726, PITG_23131 and PITG_07550_9, which shows in the occurrence of a HR in more than 50% of the cases.
The invention will be explained in more detail in the following, non-limiting example.
In the present study, we intended to identify the mapping position of the R gene responsible for the high level of resistance to P. infestans in S. x edinense, for further map based cloning. Two segregating populations were produced from different S. x edinense genotypes (edn151-1 and edn150-4) crossed with cv. Concurrent. They were tested with different isolates and effectors that could discriminate between the different R genes (Champouret 2010; Oh et al. 2009; Vleeshouwers et al. 2008). SSR markers, NBS profiling (van der Linden et al. 2004) and CAPS markers were used to link the segregation of the resistance to a chromosomal position. Gene family directed profiling (GDFP) was developed for different R genes and successfully applied to obtain markers that are closely linked to those R genes.
Materials and Methods
Plant Material and Mapping Population
S. x edinense P. Berthault accessions were provided by the Potato Collection Gross Lüsewitz, Germany (GLKS). The accessions were collected from an area near Toluca de Lerdo in Mexico (SolRgene database, www.plantbreeding.wur.nl/phytophthora/). Fifteen genotypes from three S. x edinense accessions (GLKS 25492, GLKS 25493 and GLSK 25494) were screened for resistance to P. infestans. Two resistant genotypes were selected and crossed with the susceptible cv. Concurrent to generate F1 mapping populations. The recombinant F1 genotypes of interest were transferred to in vitro culture to be maintained and multiplied. Resistant individual Edn150-4-104 was crossed with cv Aveka (
Phytophthora Isolates and Disease Tests
Phytophthora isolates and their race specificities and origin are shown in Table 1. These isolates are obtainable from by Geert Kessel, Francine Govers (Wageningen University, The Netherlands) and Paul Birch (University of Dundee, Scotland, UK). Plants were tested for resistance by three different disease assays: an in vitro assay (Huang 2005), a detached leaf assay, (Vleeshouwers et al. 1999), and a field experiment. The in vitro assay was performed once on five plantlets with the P. infestans isolate 90128. In the detached leaf assay, one leaf between the third and the fifth fully developed leaves was collected from five weeks old plants, and inoculated with the two isolates 90128 and IPO-C. The leaves were scored after six days as resistant (R) due to a hypersensitive response (HR), susceptible (S) if a sporulating lesion appeared or as quantitative (Q) for a response not clearly resistant or susceptible. Two field trials, including S. x edinense genotypes, were performed in the summer of 2005 and 2007, in Wageningen, the Netherlands. Each field trial consisted of two randomized blocks, and within the blocks, genotypes were represented as four-plant subplots which were treated as single experimental unit as described by Colon and Budding (1988). For comparisons between years, standard cultivars Ostara, Bildtstar, Eersteling, Pimpernel, Robijn and Biogold were included. Spreader rows consisted of potato cultivar Bintje, the border rows consisted of potato cultivar Nicola. For the inoculum production, a large number of potato cultivar Bintje leaves were inoculated in detached leaf assay with isolate IPO-C. After 6 days, spores were washed off to prepare a spore suspension in large containers. Zoospore release was induced by incubating the containers at 10° C. At nightfall, the zoospore suspension was sprayed on the potato field using a tractor with two spraying arms. Disease assessments were made at weekly intervals. The percentage of leaf area covered with late blight lesions was estimated for each plot (Colon et al. 1988). From these readings the area under the disease progress curve (AUDPC) was calculated (Fry 1978) and subsequently, the AUDPC values were transformed to a 1 (susceptible)-9 (resistant) scale (SolRgene database).
Marker Development
Young leaf tissue was collected from plants grown in the greenhouse. Genomic DNA was isolated by following the CTAB protocol (Park et al. 2005) with the Retsch machine in a 96 well format. Several marker technologies were used in this study: CAPS markers, SSR markers, NBS profiling markers (van der Linden et al. 2004) and R gene family directed (GFDP) profiling markers that represent particular R gene families.
A set of approximately 80 SSR markers, covering the potato genome (Collins et al. 1999; Feingold et al. 2005; Ghislain et al. 2004), was applied to determine the chromosomal position of the segregating R gene. Parental genomic DNA and 11 resistant and 11 susceptible F1 individuals from the mapping populations were used for the SSR marker screen. PCR reactions for the SSR markers were performed using a single PCR program: an initial cycle at 95° C. for 2 min; then 30 cycles of 95° C. for 30 s sec, 56° C. for 30 sec, using a ramp of 1° C./min, and 72° C. for 45 sec, using a ramp of 1° C./min; and a final step at 72° C. for 3 min Subsequently the PCR products were run on acrylamide gels and visualized using the LI-COR technology (Lincoln, Nebr., USA).
To confirm the mapping position and obtain PCR markers linked to the R genes, known CAPS markers from the SGN database (solgenomics.net/), and the SH x RH genetic map (van Os et al. 2006), located close to the R gene clusters, on the identified chromosome arm were tested.
Gene family directed profiling was used to develop markers closely linked to the R gene. It was performed as NBS profiling previously described (van der Linden et al. 2004) by replacing the NBS primers by gene family specific primers. For three R gene families R2, Tm2 and N, sequences available from NCBI (www.ncbi.nlm.nih.gov/) and sequences from allele mining studies performed in our laboratory were collected and aligned. Primers were designed on conserved sequences for each family on the different domains of the gene: CC or TIR, NBS and LRR (Table 1). Some degenerate primers were designed especially for the N-like profiling. This analysis was combined with a bulk segregant analysis (BSA, Michelmore et al. 1991) on the F1 populations. Eight F1 individuals giving a resistant or susceptible phenotype were pooled and screened with the primer/enzyme combinations. The PCR products were visualized by electrophoresis on acrylamide gels. The fragments identified to be associated with the resistance were cut out of the gel and sequenced.
Effector Screening
A collection of approximately 250 RXLR encoding genes (also referred to as effectors) derived from the P. infestans genome (Haas et al. 2009) were cloned without their signal peptide into the binary vector pMDC32 under the control of a double 35S promoter. All the plasmids were introduced into A. tumefaciens strain AGL1 (Lazo et al. 1991) in combination with the helper plasmid pBBR1MCS-5.virGN54D (Van Der Fits et al. 2000). The agroinfiltrations were carried out on young edn150-4 plants three weeks after transplanting from in vitro multiplication. In total 24 replications per effector clone were made. R3a and Avr3a (Bos et al. 2006) were used as positive control, and empty pMDC32 as negative control. The agroinfiltration experiments of the recombinant A. tumefaciens were performed as described by van der Hoorn et al. (2000) with some adaptations.
Agrobacterium tumefaciens cultures were grown in 3 ml of LB medium supplemented with antibiotics to select for the A. tumefaciens strains (carbenicilin), the binary vector (kanamycin or spectinomycin) and the helper plasmid (chloramphenicol). The next day, the cultures were transferred to 15 ml of YEB medium supplemented with antibiotics to select for the vector and the helper plasmid. On the third day, the cells were harvested and re-suspended in MMA solution supplemented with acetosyringone to a final OD600 of 0.3. Responses were scored from 3 to 8 days post-infiltration, the number of replicates responding to infiltration by (hypersensitive) cell death was counted and the percentage of responsive infiltrations was calculated.
For Rpi-edn2 co-infiltration with effector clones, Agrobacterium suspensions in MMA are prepared at OD600=0.5. Successively, a 1:1 mixture of the respective Agrobacterium suspensions is made and infiltrated into the leaves of Nicotiana benthamiana. One week after infiltration the occurrence of hypersensitive cell death is assessed.
Screen for Resistance to P. Infestans in S. x Edinense Accessions
To identify a resistant genotype for R gene mapping and cloning, in total 15 genotypes from three S. x edinense accessions were tested for resistance to P. infestans. The 15 individuals were first tested by an in vitro assay with isolate 90128. Fourteen genotypes gave a high level of resistance and one genotype had a lower level of resistance (Table 2). From each accession two highly resistant genotypes per accession were selected. Their resistance to isolate 90128 was confirmed in a detached leaf assay, and inoculation with an additional isolate, IPO-C resulted also in resistant phenotypes. Two field experiments in 2005 and 2007 confirmed the strong resistance to IPO-C in all tested genotypes. Two resistant genotypes edn150-4 and edn151-1 were chosen to generate F1 populations.
Segregation of Resistance in the Mapping Populations
The genotypes edn150-4 and edn151-1 were crossed with cv. Concurrent to generate F1 mapping populations. The F1 individuals were phenotyped for their resistance to four different P. infestans isolates in a detached leaf assay. 159 individuals from the edn150-4 x cv. Concurrent population and 125 from the edn151-1 x cv. Concurrent population were tested. The resistance to each of the four isolates segregated in the two populations (Table 3).
The resistance to 90128 segregated in the two populations. The segregation pattern of the resistance to 90128 was different from the segregation pattern of the resistance to the other isolates. Champouret (2010) cloned two functional R2 homologues (Rpi-edn1.1 and R2-like) in S. x edinense genotype edn151-3. R2 confers resistance to 90128 and susceptibility to the other isolates tested on the population. Therefore, it was hypothesized that the R gene, Rpi-edn1, conferring resistance to 90128 is located in the R2 cluster.
The segregation of the resistance to IPO-C in both populations also followed a different pattern than the segregation of the resistance to isolates PIC99189 and UK7824 (Table 4). This suggested that at least three R-genes are responsible for the observed segregation patterns and that these three genes could be distinguished based on their isolate recognition spectrum.
In summary, the two F1 populations showed similar segregation ratios for resistance and susceptibility (Table 3) to three isolates (90128, 99189 and UK7824) that were independent between isolates (Table 4). The segregation of the resistance to IPO-C is slightly skewed in both populations. But the number of resistant F1 plants is higher for the population edn151-1 x cv. Concurrent whereas the number of susceptible F1 plants is higher for the population edn150-4 x cv. Concurrent. It can be speculated that the same set of three R genes is present in both S. x edinense parental genotypes. Therefore, the rest of the study focused only on one F1 population: edn150-4 x cv. Concurrent.
Marker Development
The population edn150-4 x cv. Concurrent was used to map the R genes segregating in the population. The mapping position of the two genes Rpi-edn1.1, which derived from edn150-4 and R10, which derived from parent Concurrent, are known. So we tested markers known to be present in the locus of interest. For Rpi-edn1.1, located in the R2 cluster on chromosome 4, R2 gene family profiling was performed. R10 maps on chromosome 11 in the R3 cluster (Bradshaw et al. 2006), so CAPS markers from the R3 cluster were tested. The mapping position of the other two genes was unknown, and a genome wide screen was performed. SSR screening and NBS profiling were carried out to determine the map position of the R gene giving resistance to IPO-C and the R gene giving resistance to PIC99189. A subset of F1 individuals resistant or susceptible to all isolates was selected for that purpose. The DNA of the F1 individuals was kept separate for the SSR screening and bulked for the NBS profiling.
Rpi-edn1 from the R2 Cluster is Present in edn150-4
The homologues R2-like and Rpi-edn1.1 have been cloned from the genotype edn151-3, which was derived from the same accession as edn151-1 (Champouret 2010). We investigated whether this gene would also occur in edn150-4. Seven R2 profiling primers (Table 1) were designed on several conserved regions of the R2 gene family. The primers were tested in combination with RsaI, which cuts frequently in the R2 sequence on the parental and F1 bulked DNA. Each primer revealed at least one fragment showing association with the resistance in the bulks. The primer (R2ch4F4) giving the largest number of polymorphic bands was tested on the individuals of the whole population. The resulting NBS marker R2ch4F4-Rsa (fragment of 400 bp) was linked to the resistance to 90128 with 10 recombinants out of 45 individuals (˜20 cM). Agro-infiltration assay with PiAvr2 was performed on a subset of the population and it was confirmed that the PiAvr2 response co-segregated with the resistance to 90128 in 40 F1 individuals (
Rpi-edn2 Maps on Chromosome 9
The screen of the set of approximately 80 SSRs applied on the parents and 24 F1 individuals resulted in one linked marker associated with the resistance to IPO-C. This marker, Stm021, (Table 5) is located on chromosome 9 (Bakker et al., manuscript in preparation). The linkage with resistance to IPO-C was confirmed with 17 recombinants out of 116 individuals (˜15 cM).
We propose to call this gene Rpi-edn2, the R gene conferring resistance to IPO-C, located on the long arm of chromosome 9 (
Cloning of Rpi-edn2 Using a Candidate Gene/Allele Mining Approach.
To date, cloning of R genes is typically done through a positional cloning strategy. Once a functional gene is cloned from a specific R locus, one can try to clone functional homologs from the same or different species in order to determine sequence diversification at a given locus. Here we demonstrate that based on a map position combined with a candidate gene mining approach allele specific markers can be generated which can form a starting point for the cloning of the functional R gene.
The inventors adopted a homology based candidate gene mining strategy to clone Rpi-edn2. The first step was to design primers incorporating the putative—start and stop codons of candidate mcq1 gene homologs i.e. mcq-ATG-1 5′-atggctgaaattcttcttac-3′, mcq-c1-stop 5′-tcatattctgagctttgcaag-3′, mcq-c2-stop 5′-tcatactctcagttttgcaagtc-3′ (table 5).
The primer mcq-ATG-1 combined with the primer reverse 2 amplifies the functional gene in mcq.
No amplicons of the expected size were generated with primer set mcq-ATG and mcq-c1-stop when tested on the parental genotypes of both mapping populations. However, when primers mcq-ATG-1 and mcq-c2-stop were combined, a single amplicon of approximately 2.4 kb was amplified in both the resistant as susceptible progeny. Subsequently, PCR products of both susceptible and resistant plant were subjected to restriction digestion using the restriction enzymes Msel, Haelll, NlaIII, HpaII, DpnII, AIuI, HhaI, HinfI, DdeI, HpychIV, Rsal or TaqI. After MseI-digestion a specific restriction fragment of approx. 600 bp was visible that was 100% linked in 60 genotypes segregating for Phytophthora resistance.
The undigested PCR products of a resistant plant were cloned into the pGEMR-T Easy vector and 24 individual clones were subjected to enzyme digestion with MseI. A total of 9 different classes could be distinguished based on the MseI digestion pattern. Clones of all 9 classes were sequenced.
The obtained sequences shared 80-90% similarity to each other. Based on MseI digestion pattern clone EDN61 was predicted to cause the polymorphism that co-segregated with Rpi-edn2
A specific SCAR marker was designed for EDN61: EDN F 5′-gcatcatgtctgcacctatg-3′ and EDN R 5′ctttgatgtggatggatggtg-3′ (table 5) in the initial mapping populations. When tested, the marker co-segregated with resistance, confirming that EDN61 was genetically very close to Rpi-edn2 and could potentially be a candidate for Rpi-edn2.
Gene Structure of Rpi-edn2.
The open reading frames of Rpi-edn2 encode predicted peptide of 863 amino acids. The gene is intron-free.
The protein sequences of Rpi-edn2 harbours several conserved motifs of the CC-NBS-LRR class of R proteins (
At the protein level, Rpi-edn2 shares 80% amino acid identity with Rpi-mcq1.1, 77% with Rpi-mcq1.2. Lower percentage homology was found with Rpi-vnt1 (73%) and Tm-2-2 sharing 73% and 72% identity, respectively, showing that Rpi-edn2 defines a new subclass of the Tm2-2 gene family.
Calculated nucleotide identities were as follows:
Calculated amino acid identities were as follows:
The above multiple comparisons were performed, using AlignX (Vector NTI Suite Invitrogen) with an engine based on the CLUSTAL matix.
Introduction of Rpi-Vnt1, Rpi-Mcq1.1 and Rpi-edn2 into Potato Genotypes Susceptible to Phytophthora Infestans and into N. Benthamiana
The 2.6 kb fragment of EDN61 was cloned in between the Rpi-blb3 promoter and blb3 terminator, in the binary vector pDEST32 using the protocol described (Lokossou et al 2010). The resulting plasmid was named pDEST:edn2. It was introduced into Agrobacterium tumefaciens strain AGL1.
Binary vectors carrying the full-length Rpi-mcq1.1 and Rpi-vnt1 genes (WO2009/013468) are introduced into Agrobacterium tumefaciens strains AGL1. To ensure no rearrangements of the plasmids have occurred, plasmid is isolated from resulting transformants, and transformed back into E. coli strain DH5-α, digested and compared with digests of the original plasmid stocks.
Potato Transformation
Internodal cuttings from in vitro grown plants cv Desiree were used for transformation by Agrobacterium tumefaciens co-cultivation, according to the protocol described by Visser RGF (1991) In: K Lindsey (Ed) Plant Tissue Culture Manual, Kluwer Academic Publishers, Dordrecht/Boston/London, pp. B5: 1-9. Transformants were selected on MS20 medium (Murashige and Skoog, 1962 Physiol Plant 15: 473-497) with 20 g/L sucrose, containing 100 mg/L kanamycin.
Agrobacterium tumefaciens culture(s) with the appropriate antibiotic selection regime are set up and grown for 24 hours with shaking at 28° C. Stem internode sections (without nodes) are harvested from 4-6 week old potato cv. Desiree plants grown in aseptic culture on MS medium (2% sucrose). Stem internodes are cut into 2 to 5 mm lengths and placed on two layers of filterpaper on solid R3B media for 1 day before co-cultivation. The R3B medium used contained the salts and vitamins of MS medium (4.71 g/l) plus 3% saccharose, 2 mg/l NAA, 1 mg/l BAP and 0.8% agar, pH 5.8. The layers of filter paper was covered with 2 ml of PACM liquid media consisting of MS (4.71 g/l), 2.0 g/l casein hydrolysate, 3% saccharose, 1 mg/L 2,4 D and 0.5 mg/L kinetine, pH 6.5. 100 ul of overnight. Agrobacterium tumefaciens culture is added to stem sections and incubated for 20 minutes at 40 rpm in the dark at 24° C. The stem sections are removed from the Agrobacterium tumefaciens suspension, blotted dry and incubated for two days at 21° C. in a 16 hours photoperiod.
After two days the explants were transferred to Zcvh media consisting of 4.71 g/l MS, 2.0% saccharose, 0.8% agar, 200 mg/l cefotaxime, 200 mg/l vancomycine and 1 mg/l zeatine. Stem explants are subcultured onto fresh Zcvh media every 2 weeks for around 3-6 weeks or until the appearance of the first small calli. Once the calli have sufficiently developed the stem sections are transferred onto Zcvh media with selection antibiotics. Stem sections are subcultured every 7-10 days until shoots start to develop. Shoots appear within 2 months from the start of transformation. Shoots are removed with a sharp scalpel and planted into MS20 solid media with selection antibiotics. Transgenic plants harbouring appropriate antibiotic or herbicide resistance genes start to root normally within 2 weeks and are subsequently being transplanted to the glasshouse.
Transient Expression in Nicotiana benthamiana Transformation
pDEST32:edn2 containing the Rpi-edn2 open reading frame under the control of the Rpi-blb3 regulatory sequences, was transformed into Agrobacterium tumefaciens strain COR308. This bacterial strain and a strain containing an empty binary vector was agroinfiltrated into Nicotiana benthamiana in order to achieve transient expression. Two days after agroinfiltration the leaves were picked and, successively, challenged with P. infestans isolates IPO-C and H30PO4 in a detached leaf assay. Five days later, disease symptoms were observed. Leaves that were agroinfiltrated with empty vector showed sporulating lesions (
Effector Screening
In order to further support the unique recognition spectrum of Rpi-edn2 we set out to identify the component from P. infestans that is actively recognized by Rpi-edn2. Therefore the leaves of edn150-4 were infiltrated with the P. infestans effector collection of approximately 250 clones. As a positive control co-infiltration of R3a and Avr3a was performed and as a negative control the infiltration of pMDC32 alone was used (Table 9). No necrosis (0% of the infiltrated spots) was observed with the vast majority of the effector collection. We did observe recognition of Avr2 and Avr4 but this recognition was co-segregating in the F1 population with the Rpi-edn1 and Rpi-edn3 genes on chromosome 4 and 11 respectively (
Effector Recognition by Rpi-edn2.
In order to further define the recognition specificity of the cloned gene, Rpi-edn2 was expressed in leaves of N. benthamiana simultaneously with the Pi effector proteins listed in Table 9. These Pi effector proteins were found to induce hypersensitive cell death upon their expression in the leaves of edn150-4 from which Rpi-edn2 was cloned. By co-agroinfiltration it was shown that simultaneous expression of Rpi-edn2 and PITG_15039 resulted in a hypersensitive cell death. Infiltration of PITG_15039 alone did cause a slight cell death response but the co-expression with Rpi-edn2 clearly showed an enhanced cell death response, reminiscent of a HR (
Complementation Analysis of Rpi-edn2.
A total of 26 S. tuberosum cv. Desiree plants capable of growth on kanamycin were selected as putative Rpi-edn2 transformants. Following transfer to the glasshouse, leaves were excised and used in a detached leaf assay with P. infestans isolates 90128 and IPO-C to determine whether the transgene conferred blight resistance. Of the 26 transformants, 21 were confirmed as being resistant and did not show any signs of blight infection. Some plants exhibited signs of a hypersensitive response localised to the inoculation site. The remaining 5 plants were susceptible to both isolates, as was the control (non-transformed Desiree). The Rpi-edn2 transgene also conferred resistance to P. infestans isolates IPO-0 and EC 1, as detailed in Table 4.
Detached leaves of transgenic potato cv. Desiree carrying Rpi-vnt1 were inoculated with a range of P. infestans isolates (Table 5) to determine the range of isolates against which Rpi-vnt1 confers resistance. Of the isolates tested, only isolate EC1 from Ecuador was able to overcome Rpi-vnt1 and cause disease on the inoculated plants.
S. tuberosum cv Desiree complemented with pSLJ21153 Rpi-mcq1 (WO2009/013468) were subjected to detached leaf assays using P. infestans isolates 90128, EC1, Hica and IPO-complex. For construct pSLJ21153, 12 transgenic lines were shown to be resistant to isolates 90128 and EC1, but susceptible to IPO-complex (Table 5).
These results demonstrate a broad spectrum resistance of Rpi-edn2 to P. infestans isolates, substantially different from its homologs Rpi-vnt1 and Rpi-mcq1.
Stacking with Other R Genes
In the past, single R genes were quickly overcome after introgression in potato, necessitating for the future a strategy with multiple R genes that need to be combined simultaneously. Since extensive resistance screenings in our laboratory are providing a continuous inflow of novel R genes from a diversity of Solanum species, we have a collection of R genes to choose from. The challenge now is to prioritize which R gene should be cloned, and also, which combinations of R genes should be made for application. The main criteria are to achieve a broad spectrum of resistance (acting against many isolates), a high level of the resistance (combining two different weak R genes can still achieve satisfactory level in the field), and enhanced durability (combination of R genes interacting with different effectors may be less easy to break). To select the best R genes, available candidates should be classified. Up till now, R genes can only be classified based on the donor species and their genetic localization (at least before cloning). Here we disclose new ways for classification, mainly based on functionality. R genes can be categorized based on the effector they interact with. An elicitor is typically a pathogen molecule that triggers defense responses resulting in enhanced resistance to an invading pathogen. Examples of elicitors are ATR1, ATR13, Avr1b, AVR3a, IPI-O, Avr-chc1 and elicitors depicted in Table 9.
In yet another embodiment, the invention provides a method for determining whether an R-gene from Solanum provides resistance to a variety of Phytophthora isolates, comprising providing a plant with said R-gene and testing said plant for a reaction to a defined set of effectors, representing said variety of isolates. This method avoids the use of multiple Phytophthora isolates. Such a method provides a better resolution compared to use of a Phytophthora isolate. In a preferred embodiment, the invention provides a method for determining whether an R-gene from Solanum provides resistance to a variety of Phytophthora isolates, comprising providing a plant with said R-gene and testing said plant for a reaction to a defined set of effectors, representing said variety of isolates, wherein at least one said effectors is an elicitor.
Such a method is also very useful in respect of classical breeding in that it can be used to more easily select a suitable variety for crossings.
The invention further provides a method for determining whether a set of R-genes from Solanum provides resistance to a variety of Phytophthora pathotypes, comprising providing a plant with said set of R-genes and testing said plant for a reaction to a defined set of effectors, representing said variety of pathotypes. This method is very useful in testing the effect of stacked R-genes.
The invention further provides a plant obtainable by the method according to the methods as described above, i.e.
(i) a method for providing a potato plant with a combination of resistance genes which provides resistance against a variety of pathotypes (or strains or isolates) of Phytophthora, comprising providing said plant with at least two R-genes that together provide resistance against said variety of pathotypes, said R-genes being identified by a method as described in application, i.e. a method for determining whether an R-gene from Solanum provides resistance to a variety of Phytophthora pathotypes or
(ii) a method for providing a potato plant having acquired resistance to a variety of Phytophthora pathotypes through the introduction of an R-gene with additional resistance against at least one additional pathotype of Phytophthora, comprising providing said plant with at least one further R-gene obtained by a method as described herein, i.e. a method for identifying a resistance gene (R-gene) in Solanum comprising
Preferably, said at least one gene of said combination of resistance genes is Rpi-edn2 or said further R-gene is provided with a nucleic acid encoding a further resistance gene, such as Rpi-blb1, -2, -3, Rpi-vnt1, Rpi-chc1, Rpi-avl1-1, Rpi-avl1-2, Rpi-R1, Rpi-R2, Rpi-R3a, Rpi-R3b, Rpi-mcd1 or Rpi-mcq1.
Discussion
Many R gene clusters in Solanum have already been identified. A fruitful approach to map novel R genes is to search for association with known R gene clusters. This simplifies R gene mapping, since they should lie in one of the R gene clusters listed in several reviews (Gebhardt et al. 2001; Grube et al. 2000; Pan et al. 2000). The identification of the mapping position of a new potato R gene is still not a routine task, especially in tetraploid (or in general, polyploid) plants. The first step is to identify a susceptible plant for the cross with the resistant genotype. The second step is to choose a P. infestans strain that will allow the characterization of the resistance in the F1 individuals. The next step is to confirm that the resistance phenotype is distinct in the population, that the resistance is segregating and that it follows a 1:1 ratio. Once all the conditions are met, the identification of the mapping position can begin. To be successful this step requires a sufficient level of heterozygosity from the resistant parent and an optimal level of polymorphism in the population. For most of these aspects, little can be done to increase the chance of mapping a new R gene. To improve the scoring of the phenotype or the development of linked markers, new approaches have shown to be successful in this study. The response to effectors can be used instead of the resistance phenotype to P. infestans to score the population. SSR markers and general or specific profiling approaches can also be useful for the development of diagnostic markers and the identification of the R genes chromosomal position.
A single P. infestans strain can contain several effectors and a single genotype can contain several R genes. A good example of this complexity is the isolate UK7824 that contains at least two known Avrs (Avr4 and Avr10), and the two S. x edinense genotypes that contain at least three R genes. This complexity can make R gene mapping a difficult task. For mapping, the gene of interest should be dominant and occur in simplex, or at least be scored as single trait. The use of effectors to score the segregation of the R genes in the F1 population allows the scoring of a single R gene recognizing a specific effector. The responses to Avr2 and Avr4 segregating in the F1 population were associated with resistance to 90128 and PIC99189, respectively. It shows that effector responses can be used to phenotype an F1 population and map an R gene, which can be very useful in the presence of multiple R genes in a single genotype. Having the avirulence gene to R10 (Avr10) would allow the scoring of the plants for the presence of R10 and simplify its mapping.
SSR marker screening and NBS profiling are the two approaches used in this study to identify R genes map position. NBS profiling identified the mapping position of Rpi-edn3 on chromosome 11 and SSR marker the one of Rpi-edn2 on chromosome 9. Both marker technologies are suitable for polyploids and are complementary to each other. SSR marker screening is an addition to the NBS profiling to identify R gene map position. A large set of SSR markers that cover the potato genome is now available (Collins et al. 1999; Feingold et al. 2005; Ghislain et al. 2004). Bakker et al., (manuscript in preparation) have developed the largest and most useful set of SSR markers for R gene mapping purposes because the primers were designed from BAC sequences selected with R gene analogue (RGA) probes and mapped in R gene clusters in the SH x RH UHD genetic map (van Os et al. 2006). These SSR markers allow the identification of a position of a gene for novel resistance on a chromosomal arm. For mapping purposes, the SSR marker screen approach has several technical advantages. The presence of polymorphism and its segregation in the population is determined directly with one PCR. One primer combination shows several alleles, which means that the probability to determine which allele is associated with the resistance, especially for polyploid populations, is higher than with other marker approaches. Another advantage of the SSR marker approach for R genes mapping is that the mapping position of each marker is already known. So the identification of a marker associated with a novel resistance will directly assign the R gene to a particular chromosome arm, and hence a probable R gene cluster.
NBS profiling was designed to specifically target R genes but it can easily be adapted to target other conserved gene families. It was adapted for peroxidase profiling in barley to map peroxidase clusters on the genome and correlate them with resistance QTL map position (González et al. 2010). In this study, we adapted the NBS profiling to specific R gene families and showed its success for three R gene families: R2, Tm2 and the N gene family. R genes from the same cluster usually have similarities in their sequences not shared with other R genes (McDowell et al. 2006; Meyers et al. 2005) so it is possible to design specific primers for a particular R gene cluster. Sequence information on R genes is largely available and more sequences will become available with potato genome sequencing. This approach could be developed for each R gene cluster and could be an addition to the standard NBS profiling or SSR marker screen for R gene mapping purposes.
S. x Edinense, a Lesson from Nature on R Gene Stacking
S. x edinense shows a high level of resistance in the different assays and the resistance seems well established in the natural population and effective to a wide range of Phytophthora infestans isolates. Breeders would very much like to introduce such a level of wide spectrum resistance in their varieties. This study revealed that the resistance observed in two S. x edinense genotypes is explained by the presence of at least three genes that each has been overcome by some P. infestans strains. Each Rpi-edn gene causes resistance to an isolate to which none of the other Rpi-edn genes confer resistance. This suggests a natural stacking of R genes that may be caused by selection pressure to keep all three R genes together in most genotypes of the species. The second aspect that could explain the level of resistance in S. x edinense is the provenance of the stacked R genes. S. x edinense is a natural hybrid between S. demissum and S. tuberosum ssp. andigena (Serquen et al. 2002). S. demissum originates from Mexico (Watanabe et al. 1991) and S. tuberosum ssp. andigena from Bolivia where it was domesticated (Van Soest et al. 1984). The centre of origin of P. infestans still has not been determined. Some studies brought evidence favoring a Mexican origin, and more recent studies suggest a South American origin (Gomez-Alpizar et al. 2007). Presently, Mexico and South America are both considered as centers of diversity of P. infestans. This implies a co-evolution between the pathogen and the plant host in both regions, so R genes have evolved in both places, and may have evolved differently. Wild Solanum species originating from a center of diversity should be a valuable source for resistance and for stacking (Goodwin 1994). As presented in
In conclusion, the investigation of inheritance of stacked R genes in potato genotypes can be made easier by using Avr genes from the complementing R genes and R gene cluster specific markers. If that is not possible, more generations of backcrossing are needed to unravel the nature of the different R genes involved. The natural stacking of broken R genes located on different clusters and originating from geographically distinct centers of diversity of P. infestans confers a strong level of resistance in S. x edinense. This study shows that stacking of R genes does occur in nature and seems to be a successful strategy to fend off the pathogen. It may be taken as a natural proof of principle, and applied in an agricultural context as a strategy to achieve durable resistance.
The coding sequence from Rpi-edn2 was isolated by a PCR approach. In order to isolate the Rpi-edn2 gene, including the promotor and terminator sequences, a bacterial artificial chromosome (BAC) library was constructed. Chromosome sized genomic DNA from edn140-5 was isolated and mechanically sheared. Fragments of around 80 kb were ligated into pCC1BAC. A library consisting of 200.000 clones, providing a 10× coverage of the genome, was divided into 600 pools of over 300 colonies each. The pools were screened using Rpi-edn2 specific PCR marker that was listed in the initial application. Eight colonies proved to be positive in this PCR screen and one pool was selected to identify the individual BAC clone containing the Rpi-edn2 gene. Individual colonies from the positive BAC pool were screened and identified a clone with an insert of around 26 kb. Sequence analysis of the entire BAC clone (
Methods:
Plant material. Clone edn150-4 was maintained in the laboratory of Plant Breeding by in vitro culture. BAC library construction was performed at RXbioscience (Rockville, USA).
PCR screening with the Rpi-edn2 specific marker was performed as described in the initial application.
Sequence analysis of the BAC clone was performed at Macrogen (Seoul, South Korea) using a 454 sequencer (Roche).
Gene prediction was performed using the FGENESH algorythm (linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind).
Phytophthora
infestans isolates used to phenotype the two segregating
edn
150-4
9
9
9
9
9
edn
151-1
9
9
9
9
9
Phytophthora infestans isolates in detached leaf assay.
170 individuals in total.
254 individuals in total (Excluding the unclear phenotype Q).
S.
berthaultii
S.
bulbocastanum
S.
caripense
S.
demissum
S.
microdontum
S.
mochiquense
S.
papita
S.
paucissectum
S.
phureja
S.
pinnatisectum
S.
stoloniferum
S.
venturii
S.
vernei
tuberosum
Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file entitled “Sequence_listing_294-417PCTUS.txt,” created Jan. 25, 2013. The sequence.txt file is 125 kilobyte size.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10164531 | May 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2011/050386 | 5/31/2011 | WO | 00 | 1/28/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2011/152722 | 12/8/2011 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO2009013468 | Jan 2009 | WO |
| WO 2009013468 | Jan 2009 | WO |
| WO 2009103960 | Aug 2009 | WO |
| Entry |
|---|
| ADB85624.1 —rpi-vnt1-like protein, partial [Solanum okadae]—Protein—NCBI, GenBank, Jan. 1, 2010. |
| Reverberi, Roberto, and Lorenzo Reverberi. “Factors affecting the antigen-antibody reaction.” Blood Transfusion 5.4 (2007): 227. |
| Foster, Simon J., et al. “Rpi-vnt1. 1, a Tm-22 homolog from Solanum venturii, confers resistance to potato late blight.” Molecular Plant-Microbe Interactions 22.5 (2009): 589-600. |
| Ugent, Donald. “Morphological variation in Solanum x edinense, a hybrid of the common potato.” Evolution (1967): 696-712. |
| Fry, William E. and Niklaus J. Grünwald. 2010. Introduction to Oomycetes. The Plant Health Instructor. DOI:10.1094/PHI-I-2010-1207-01. |
| Kamoun, Sophien. “Molecular genetics of pathogenic oomycetes.” Eukaryotic cell 2.2 (2003): 191-199. |
| van der Lee, Theo, et al. “Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10, and R11 potato lines.” Molecular plant-microbe interactions 14.12 (2001): 1444-1452. |
| Ellis, Jeff, Peter Dodds, and Tony Pryor. “Structure, function and evolution of plant disease resistance genes.” Current opinion in plant biology 3.4 (2000): 278-284. |
| Database EMBL., “Solanum okadae isolate oka970-3 rip-vnt1-like protein gene, partial cds.”, Jan. 28, 2010, XP002604522. |
| Collins, A, Et al., “QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour”, Molecular Breeding, vol. 5, No. 5, 1999, pp. 387-398, XP002604523. |
| Van Der Linden C Gerard, et al., “Efficient targeting of plant disease resistance loci using NBS profiling”, Theoretical and Applied Genetics, vol. 109, No. 2, Jul. 2004, pp. 384-393. |
| Number | Date | Country | |
|---|---|---|---|
| 20140041072 A1 | Feb 2014 | US |
