MXPA99001354A - Resistance against nematodes and/or aphids - Google Patents
Resistance against nematodes and/or aphidsInfo
- Publication number
- MXPA99001354A MXPA99001354A MXPA/A/1999/001354A MX9901354A MXPA99001354A MX PA99001354 A MXPA99001354 A MX PA99001354A MX 9901354 A MX9901354 A MX 9901354A MX PA99001354 A MXPA99001354 A MX PA99001354A
- Authority
- MX
- Mexico
- Prior art keywords
- plant
- dna
- sequence
- plants
- nucleic acid
- Prior art date
Links
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Abstract
The invention relates to genes capable of conferring resistance against nematodes and/or aphids. Preferred nucleic acids of the invention are DNA sequences which are at least part of the DNA sequence provided on figures or homologous thereto. The invention further relates to vectors, cells and seeds comprising said nucleic acids, as well as genetically transformed plants which are resistant to nematodes and/or aphids. The invention also relates to oligonucleotides, primers, diagnostic kit and polypeptides.
Description
RESISTANCE AGAINST NEMATODES AND / OR AFIDS
FIELD OF THE INVENTION
The present invention relates to resistance genes, DNA constructs, microorganisms, plant cells and plants comprising said resistance genes. In addition, the invention relates to genetically transformed plants, which are resistant against nematodes and / or aphids. In addition, the invention relates to probes, and primers for the identification of resistance genes and diagnostic equipment comprising said zones and / or primers. Finally, the invention relates to polypeptides encoded by said resistance genes and to the use of such polypeptides.
BACKGROUND OF THE INVENTION
Plant pathogens are responsible for substantial losses of plants and plant products due to infection of the plant. Diseases of the plant, as a result of infection through pathogens or plant pests, cause damage to plants and / or plant products, reduce production and yield, limit the type of plants that can develop in certain areas geographical areas and as a result they cause severe (financial) losses to the farmer.
Plant parasitic nematodes occur worldwide and most of them live for most of their lives in the upper layer of the earth. Although the losses caused by the direct feeding of nematodes in plant roots are considered of minor importance, several species, among them the root-knot nematodes belonging to the Meloidogyne species, nematode cysts that belong to the Heterodera species and the Globodera species. and other nematodes such as the Nacobbus species, cause severe damage and economic losses of the crop. Plant root nematodes also occur throughout the world but are found more frequently and in larger numbers in areas with warmer climates and in greenhouses. The most important species of Meloidogyne are M. incognita, M. arenaria, M. hapla and -Vi. javanica, of which M. hapla also occurs in more temperate climatic zones. There are different means to control plant pathogens, such as mechanical soil cultivation, chemical treatment with pesticides, including nematicides and insecticides, or crop rotation. However, for certain plant pathogens, especially nematodes, these control means are insufficient to protect the plants from infection and resulting diseases. The only effective means for control involves plant host resistance (Russell, 1978, Plant Breeding for pest and disease resistance, Butterworths ed., P 485). The development of crops resistant to pathogens of common plants is one of the major objectives of the plant producers nowadays, in order to reduce or finally eliminate the great need for pesticides. The environmental burden of large amounts of pesticides injected into the soil or sprayed on crops, trees, etc., worldwide each year becomes more severe. In addition, government regulations in Western countries restrict the use or even prohibit the use of certain pesticides. Therefore, the need for plants to be resistant to one or more of their pathogens, or to have a reduced susceptibility to their attackers, has more and more pressure. The development of resistant plants is one of the important objectives of the current plant production programs. The genotypes of plants susceptible to particular pathogens are crossed with resistant plant genotypes in order to introduce the phenotype resistant to the breeding line. The damage through plant root nematodes results mainly from the invasion of the roots of the plant by larvae, which in a compatible relationship with the plant develop in a female reproduction. After the invasion, the larvae develop root cells in giant cells where they feed. After the infection, gills or knots are formed in the roots and the roots of the plant are otherwise damaged, become thickened and impede growth. The root system in this way is damaged in the consumption of water and nutritional elements that damages the growth and development of the plant.
Frequently, damage to infected plants is increased by parasitic fungi that attack weakened root tissue. Infected plants show reduced growth and smaller pale leaves, with fruit of poor quality and tiny or fruitless, and tend to wilt in warmer climates (Agrios, 1988 in: Plant Pathology, Academic Press, Inc.). The damage and / or yield reduction caused by plant root nematodes is substantial in total world agricultural production. In individual production losses it can be as high as 25-50%, or even the crop can be annihilated. In nematodes of plant roots in greenhouses these can be controlled with steam sterilization of the soil or soil fumigation with nematicide. Under field conditions, control can be achieved through the use of nematicides. However, the use of these, in some cases, very persistent chemical products, is highly debated and in some countries the use of certain nematicides is still prohibited. The production of genetically resistant genotypes is the most reliable and effective way to control plant root disease. For a number of crop species, the availability of resistance within the related plasma germ has been reported, for example, potato, cotton, tobacco, wheat, soy, tomato, eggplant, common bean and alfalfa. The development of resistance is mainly hampered by the limited occurrence of resistance genes (known) in the available plasma germ, and secondly, in some plant species, the existence of crossing barriers between the species of cultivated grain and the resistance that carries the related species, and thirdly, classification tests for resistance against nematode susceptibility are laborious and usually unreliable. Therefore, the production of resistance is very difficult and is not obtained, and possibly a time consuming one. The successful introduction of resistance genes has been achieved in tomatoes. The resistance gene Mi (Meloidogyne incognita) has been introduced into cultivated tomatoes, Lycopersicon esculentum, after crossing with the related wild species L. peruvianum (Pl 128657), using an embryo culture. The Mi gene confers resistance to several Meloidogyne species. (Fassuliotis, 1991, in: Genetic Improvement of Tomato, Springer Verlag edit.). The Mi resistance gene is reported to be a monogenic dominant gene (Gilbert and McGuire, 1956, Proc.Am. Soc. Hortic.Sci. 68,437-442), and is located on chromosome 6 of tomato. It is also postulated that the introduced region comprising the Mi site is involved in conferring resistance to the potato aphid (Macrosiphum euphorbia) (Kaloshian et al, 1995, Proc.Nat.Accid.Sci.USA, 92, 622-625. They have developed a complex mechanism against attack and infection with pathogens.The exact mechanism of their defense system has not yet been determined.The resistance to nematodes in tomato is expressed after penetration.After juvenile larvae enter At the root and they establish themselves on a feeding site, a hypersensitive reaction (HR) adjacent to the head of the nematode is activated which results in the local death of the host cells.The nematode is also adversely affected by its HR and dies (Fassuliotis, 1991, in: Genetic Improvement of Tomato, Springer Verlag edit.) Whether or not there exists a sense of gene relationship for the Flor gene (1956, Adv. Gen. 8, 29-54) as it is frequently This is the case in other plant-pathogen relationships where the resistance relies on the incompatibility of HR, is unknown. The isolation of plant genes without knowing their gene products is very laborious and difficult, due to the enormous genome sizes of the plant species: for example, the tomato has a genome size of 1000 Mb (109 base pairs of Nuclear DNA), corn has a genome size of 3000 Mb and wheat has even more than 16 x 109 base pairs. The search for a specific gene among these billions of base pairs is only possible when, (i) there are enough molecular markers hermetically bound to the gene of interest and (ii) there is good genetic material available (Tanksley et al., 1995, Trends in Genetics, 11, pp. 63-68 However, the isolation of few resistance genes has been reported, none of these resistance genes are able to confer to the host plant resistant to nematodes or aphids. isolated are RPS2 from Arabidopsis (resistance to Pseudomonas syringae expressing avrRpt2), N from tobacco (resistance to tobacco mosaic virus), Cf-9 from tomato (resistance to the fungal pathogen of leaves Cladosporium fulvum carrying avr9) and L6 from flax (resistance to the corresponding leaf mold fungus) (Dangl, 1995, Cell 80, 363-366) The present invention provides the first isolated nematode resistance gene, and in addition, provides the rimer resistance gene to isolated aphids. In addition, the present invention relates to a double-function resistance gene that confers reduced susceptibility to nematodes as well as to aphids, and preferably to Meloidogyne incognita and Macrosiphum euphorbiae, respectively.
COMPENDIUM OF THE INVENTION
The present invention relates to a nucleic acid comprising the resistance gene Mi, which when present and expressed in a plant is capable of conferring to said plant resistance against nematodes and / or aphids. In addition, the invention relates to the resistance gene Mi of which the DNA sequence is described herein. The invention also relates to a gene product encoded by the resistance gene Mi. In addition, the present invention relates to constructs of DNA, cosmids, vectors, bacterial strains, yeast cells and plant cells comprising the resistance gene Mi. In another aspect, the present invention relates to a genetically transformed plant, which is resistant to a nematode, said nematode being capable of infecting the non-transformed plant. In addition, the invention relates to resistance genes which are homologous to the Mi resistance gene, and which, when present in a plant, are capable of conferring resistance to pathogen infection to said plant. In addition, the present invention relates to a nucleic acid comprising the resistance gene Meu-1, which when present in a plant is capable of conferring on said plant reduced susceptibility to aphids. In particular, the resistance gene Meu-1 responds to the resistance gene Mi. Especially, the resistance gene Meu-1 has the same nucleotide sequence as the resistance gene Mi. In this way, the present invention also relates to genetically transformed plants, which are of reduced susceptibility, and preferably resistant to aphids, in particular to potato aphids.
Finally, the invention relates to oligonucleotides corresponding to the sequence of the resistance gene or part thereof, and detection equipment comprising such oligonucleotides.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows a physical map of YAC 1/1172, YAC 2/1256 and YAC 1/1084, with a size of 570, 500 and 470 kb, respectively. The position of the restriction sites Sfi \ and SssHIl and the size of the restriction fragments are indicated. The location of the various AFLP markers in the restriction fragments are indicated. Figure 2 shows a schematic drawing of the binary cosmid vector pJJ04541 which is used to construct a cosmid collection of YAC 1/546. Plasmid pRK290 (size 20 kb) (Ditta et al., 1980, Proc.Nat.Acid.Sci.USA, 77, 7347-7351) was used as the starting vector. "Tet" refers to the gene that confers resistance to tetracycline. "LB" means the repetition sequence of the left boundary T-DNA, and "RB" means the repetition of the right boundary. The promoter sequence of 35S cauliflower mosaic virus is indicated by "p35S", and "ocs3" 'indicates the 3' end of octopine synthase. "NPT" indicates neomycin phosphotransferase, and "cos" refers to the cos site of bacteriophage lambda that allows in vitro packaging. "pDBS" indicates the polylinker of pBluescript (Stratagene, La Jolla, CA, USA). Figure 3A shows a schematic representation of the detailed position of the AFLP markers of YAC 1/1172, YAC 2/1256 and YAC 1/1084. The placement is based on the cosmid contig built for the various defined regions.
Figure 3B shows a schematic representation of the cosmid coting of the region comprising the resistance gene
My. The cosmids Mi-32, Mi-30, Mi-11, Mi-18, Mi-01 and Mi-14 are represented by horizontal lines. The location of the AFLP, PM14 and PM25 markers is indicated. Figure 4 shows a physical fine map of the cosmids Mi-32, Mi-30, Mi-11, Mi-18, Mi-01 and Mi-14 for the restriction enzyme Psíl. The size of the Psíl fragments is indicated (in kb). The Ml phenotype, as identified in an in vitro disease assay, of the R0 plants comprising the various cosmids is indicated in the far right part of the drawing. The segment of DNA from which the nucleotide sequence was determined, is indicated by a double line in a bidirectional arrow. Figure 5 shows the nucleotide sequence of a DNA segment of approximately 9.9 kb around the AFLP marker, PM14, and an amino acid sequence deduced from the resistance gene Mi. The initiation codon (ATG position 3263-3265) is underlined and the termination codon (TAG position 7109-7111) is double underlined, showing an open reading frame (ORF1) encoding the 1257 amino acid polypeptide (Figure 7A). The resistance gene Mi comprises two intron sequences (shown in italics): an intron of 1306 nucleotides from position 1936 to position 3241 and an intron of 75 nucleotides from position 3305 to position 3379. A second initiation codon (ATG) position 3491-3493), which is in frame with the first initiation codon, results in a second open reading frame (ORF2) encoding a truncated polypeptide of 1206 amino acids (Figure 7B). The position of the AFLP marker, PM14 is from the position of nucleotide 6921 (5'-TGCAGGA-3 ') to the position of nucleotide 7034 (5'-AGATTA-3'). Figure 6 shows a physical map of the cosmids Mi-11 and Mi-18 and the determined nucleotide sequence of the cosmid Mi-11. The sequence is divided into four contigs: con25 (5618 bp), conlO (898 kb), con62 (2495 bp) and Mi (9870 bp). The lower part of the figure represents the presence ("+") or absence ("-") of several PCR fragments, which correspond to the parts of the DNA segment of Figure 5, which are represented as horizontal lines of different lengths on the right side of the frame, in the various genetic backgrounds (clone YAC 2/1256, E. coli containing the cosmid Mi-11, A. tumefaciens containing the cosmid Mi-11, E. coli containing the cosmid Mi-18, A. tumefaciens containing the cosmid Mi-18, line of tomato resistant E22, line of tomato susceptible 52201, plants R0 transformed with the cosmid Mi-11 and plants R0 transformed with the cosmid Mi-18). The nucleotide sequence of the cosmid Mi-11 and the cosmid Mi-18. Analysis of different contigs. Figure 7A shows the deduced amino acid sequence of the polypeptide encoded by ORF1. Figure 7B shows the deduced amino acid sequence of the truncated polypeptide encoded by ORF2. Figure 8 illustrates a schematic representation of the structure of the resistance gene Mi. Figure 9 illustrates a schematic representation of the family of the resistance gene Mi.
DETAILED DESCRIPTION OF THE INVENTION
In the description and examples that follow, a number of terms are used herein. In order to provide a clear and consistent understanding of the specification and claims, including the scope of such terms, the following definitions are provided. nucleic acid: DNA molecule of double chain structure. The nucleic acid can be genomic DNA, cDNA, synthetic DNA or any other DNA; oligonucleotide: a DNA molecule of short single chain structure; initiators: in general, the term initiator refers to a DNA molecule of individual chain structure, which can initiate DNA synthesis; nucleic acid hybridization: a method to detect related DNA sequences through the hybridization of
DNA of individual chain structure on supports such as a nylon membrane or nitrocellulose filter papers. Nucleic acid molecules having complementary base sequences will reform the double-stranded structure if they are mixed in solution under appropriate conditions. The double chain structure will be formed between two complementary nucleic acids of individual chain structure even if one is immobilized on a support. In a Southern hybridization procedure, this last situation occurs; hybridization probe: to detect a particular DNA sequence in the Southern hybridization method, a labeled DNA molecule or a hybridization probe is reacted with the fractionated DNA attached to a support such as a nylon membrane or filter paper. nitrocellulose. Areas on the filter that carry complementary DNA sequences to the labeled DNA probe are themselves marked as a consequence of the annealing reaction. Filter areas that exhibit such marking can then be detected according to the type of brand used. The hybridization probe is generally produced by molecular cloning of a specific DNA sequence or by synthesizing a synthetic oligonucleotide; homologous sequence: a sequence having at least 50%, preferably 60%, preferably 70%, most preferably 80% or even 90% sequence identity with the particular sequence, whereby the length of sequences to be compared for nucleic acids is generally at least 120 nucleotides, preferably 200 nucleotides and most preferably 300 nucleotides, and the length of the sequences to be compared for polypeptides is generally at least 40 amino acid residues, preferably 65 amino acid residues and most preferably 100 amino acid residues.
Alternatively, a homologous sequence refers to a sequence that can hybridize under severe conditions to a particular sequence, and / or a DNA sequence encoding a polypeptide having substantially the same properties as the polypeptide encoded by the particular DNA sequence, and / or a DNA sequence encoding a polypeptide having the same amino acid sequence as the polypeptide encoded by the particular DNA sequence and / or an amino acid sequence wherein some amino acid residues have been changed with respect to the sequence of amino acid of the particular polypeptide without substantially affecting the major properties of said polypeptide; "Severe conditions" refers to hybridization conditions which allow a nucleic acid sequence to hybridize to a particular sequence. In general, highly stringent conditions refer to the hybridization conditions that allow a nucleic acid sequence of at least 50 nucleotides and preferably about 200 or more nucleotides, to hybridize to a particular sequence at about 65 ° C in a solution comprising about 1M salt, preferably 6x SSC or any other solution having a comparable ionic strength, and washing at 65 ° C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. These conditions allow the detection of sequences that have around 90% or more of sequence identity. In general, minor stringent conditions refer to hybridization conditions that allow a nucleic acid sequence of at least 50 nucleotides and preferably around 200 or more nucleotides to hybridize to a particular sequence at about 45 ° C in a solution that comprises about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1M salt, preferably 6 x SSC or any other solution having a strength Comparable ionic. These conditions allow the detection of sequences that have up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions in order to identify sequences that have an identity ranging from 50% to 90%;
promoter: a transcription regulatory region upstream from the coding sequence containing the regulatory sequences required for transcription of the adjacent coding sequence and includes the 5 'untranslated region or the so-called mRNA leader sequence; terminator: a region downstream of the coding sequence that directs the termination of transcription, also termed the 3 'untranslated region which includes the poly-adenylation signal; resistance gene: a nucleic acid comprising a coding sequence as depicted in Figure 5, or part thereof, or any homologous or corresponding sequence; nematode (s): spp Meloidogyne, such as Meloidogyne incognita, M. arenarla or M. javanica, any other genotype that is not capable of infecting a host having a resistance gene according to the invention, such as but not limited to other plant root nematodes, such as M. hapla, cymato nematodes such as the Heterodera species, or the Globodera species, or other nematodes such as the Nacobbus species, insects such as potato aphids or any other pathogen or plant pest; Resistance gene product: a polypeptide having an amino acid sequence as presented in Figure 5, or part thereof, or any homologous amino acid sequence; R0 plant: primary regenerator from a transformation experiment, also denoted as a transformed plant or transgenic plant; - line R ^ the progeny of the own R0 plant. line R2: the progeny of the plant Ri own. line R ^ C: the progeny of a cross between a plant R ^ and a plant of the genotype that was originally used for the transformation experiment. In the present invention, it has been possible to identify and isolate the unknown Meloidogyne resistance gene (Mi). The gene was cloned from a tomato genotype, which is resistant to
Meloidogyne incognita. The isolated My resistance gene according to the invention can be transferred to a susceptible host plant using Agrobacterium-mediated transformation or any other known transformation method, and is involved in conferring resistance to the host plant against plant pathogens, especially nematodes. The host plant may be a tomato or any other genotype that is infected by the plant pathogen. The present invention also provides a nucleic acid sequence comprising a resistance gene Mi, which is presented in Figure 5. With the resistance gene Mi according to the invention, effective means are available for control against pathogens and / or plant pests, since the gene can be used to transform genotypes of susceptible plants thus producing genetically transformed plants having a reduced susceptibility or preferably being resistant to a pathogen or plant pest. In particular, a plant that is genetically transformed with the resistance gene Mi according to the invention has a reduced susceptibility to plant root nematodes. In a preferred embodiment, the resistance gene Mi comprises the coding sequence provided in Figure 5 or any corresponding sequence or homologous or cDNA sequence, preceded by a promoter region and followed by a region of terminator. The promoter region must be functional in plant cells and preferably corresponds to the natural promoter region of the resistance gene Mi. However, it should be recognized that any heterologous promoter region can be used in conjunction with the coding sequences, so long as it is functional in plant cells. Preferably, a constitutive promoter is used, such as the CaMV 35 S promoter or the T-DNA promoters all well known to those skilled in the art. In addition, a suitable terminator region must be functional in plant cells all known to those skilled in the art. further, the invention relates to a gene product of resistance Mi, which is encoded by the resistance gene Mi according to the invention and which has a deduced amino acid sequence provided in Figure 5 and Figure 7A or which is homologous to the deduced amino acid sequence or part thereof. In addition, the Mi resistance gene product or a truncated polypeptide as provided in Figure 7B, can be used to develop antibodies against it, said antibodies can be used for the detection of the presence of the Ml resistance gene product. In another aspect of the invention, the resistance gene Mi can be used for the design of oligonucleotides, which are complementary to a structure of the DNA sequence as described in Figure 5, or part thereof, which can be used as hybridization probes, therefore being labeled to allow detection, for the classification of genomic DNA or cDNA collections for homologous genes. Homologous sequences that can hybridize to the probe under severe hybridization conditions, and that encode a gene product that is involved to confer reduced susceptibility or resistance to a plant against a plant pathogen that normally infects said plant, fall within the scope of the invention. scope of the present invention. In another aspect of the invention, oligonucleotides based on the sequence of the Mi-resistance gene are designed, so that they can be used as hybridization probes in the Southern analysis. These probes can be used as molecular markers to distinguish plant genotypes that have the resistance gene and plant genotypes that lack the resistance gene. This probe can be used as an additional tool in the selection. In a preferred embodiment of the invention, the oligonucleotides are designed based on the sequence of the Mi-resistance gene, so that they can be used as primers in an amplification reaction, such as a polymerase chain reaction (PCR), by what the formation of an amplification product indicates the presence of a Mi resistance gene in a certain plant genotype. In a particular embodiment of the invention, said primers direct the amplification of polymorphic fragments, so-called molecular markers, which are tightly bound to the resistance gene Mi. In a preferred embodiment, said primers are used in the amplification of selective restriction fragments to identify AFLP markers, which are tightly linked to the resistance gene Mi. The invention also relates to diagnostic equipment, comprising oligonucleotides according to the invention, for the detection of the presence or absence of the resistance gene Mi within a genotype under study. Said diagnostic equipment surrounds the use of a laborious disease trial to classify genotypes having the resistance gene or not. In addition, the invention relates to DNA constructs comprising a DNA sequence corresponding to the coding sequence of the resistance gene Mi and functional regulatory sequences in plant cells, said DNA sequence can be genomic DNA, cDNA, synthetic DNA or DNA from any other origin.
Said regulatory sequences are either homologous or heterologous to the coding sequences of the resistance gene Mi.
Preferably, the ADM construct comprises a nucleic acid whose sequence is provided in Figure 5, or part thereof. The invention also relates to DNA constructs comprising the regulatory sequences, and most preferably the promoter region of the Mi resistance gene together with a structural gene sequence heterologous to the regulatory sequences. The invention also relates to a DNA vector comprising a DNA construct according to the invention.
Suitable vectors can be cloning vectors, transformation vectors, expression vectors, etc., which are well known to those skilled in the art. In addition, cells carrying a vector comprising a DNA sequence corresponding to the sequence described in
Figure 5 or part thereof, or homologous to it, are within the scope of the invention. In addition, cells carrying a DNA construct according to the invention are within the scope of this invention. In a preferred embodiment of the invention, a genetically transformed plant is obtained by introducing the Mi resistance gene into the genome of the plant, being susceptible to nematodes, using standard transformation techniques, wherein the genetically transformed plant is resistant to nematodes. In another embodiment of the invention, the resistance gene Mi can be transformed, using generally known transformation techniques, to a heterologous system, such as, but not limited to, melon, tobacco, Arabidopsis thaliana, potato, beet, seed rapeseed, cucumber, pepper, eggplant. A heterologous system refers to a plant species that is different from the plant species from which the resistance gene was isolated. In another embodiment of the invention, the resistance gene Mi corresponds to the resistance gene Macrosiphum euphorbiae (Meu-1), and is involved in conferring on the plants, transformed with the gene according to the invention, resistance to insects and in particular to aphids. The DNA sequence comprising the resistance gene Mi as provided in the present invention has numerous applications of which some are described herein, but which do not limit the scope of the invention. The present invention will be further described in detail in view of the isolation of the resistance gene Mi present in tomato lines, which are resistant to root-knot nematodes. For the isolation of the Mi resistance gene, a map-based cloning strategy (positional cloning) has been used, which comprises the following steps: (1) identification of molecular markers linked to the resistance gene Mi, (2) construction of a genomic YAC collection of high molecular weight, (3) form physical maps of the molecular markers in the YAC clones and the contig construct, (4) the construction of a cosmid collection of the YAC clones carrying the bound molecular markers, ( 5) formation of a fine physical map and construction of the contiguous cosmid, (6) genetic characterization of mutants of tomatoes susceptible to plant root nematodes, (7) transformation of susceptible plants with the cosmids forming the contig, (8) complementation analysis . For the identification of molecular markers, selective restriction fragment amplification technology, hereinafter also referred to as AFLP ™ technology, has been used, which randomly amplifies a subset of DNA fragments out of a complex mixture of many fragments of DNA. DNA and said amplified fragments generate traces that can be analyzed. In general, the total DNA of different genotypes of the same plant species is subject to the AFLP technology and to the different AFLP traces obtained from the different genotypes., and they were compared. Fragments that are present in one genotype and absent in another genotype are polymorphic fragments and are referred to as AFLP markers. The selectivity of AFLP fractions is obtained by using randomly selected selective nucleotides at the 3 'end of the PCR primers immediately adjacent to the nucleotides of the restriction site above. In a classification of AFLP, the DNA that will be studied is subjected to different combinations of initiator. The total amount of the different primers that can be used is determined by the number of selective nucleotides that are added to the 3 'end (4 primers with a selective nucleotide, 16 primers with 2 selective nucleotides, 64 primers with 3 selective nucleotides). If two different restriction enzymes are used, there is twice the number of primers. Those initiators can be used in a different combination. If all possible combinations are used in an AFLP classification, all fragments present must have been amplified with one of the initiator combinations (Zabeau and Vos, EP 0534858). For the identification of AFLP markers linked to the resistance gene Mi, different tomato lines were subjected to an AFLP classification. In a first step, two absolutely isogenic lines for resistance of nematodes against susceptibility were analyzed through the formation of AFLP traces using the following primers:
Initiators Psil 5'-GACTGCGTACATGCAGNN-3 'Initiators Mse \ 5'-GATGAGTCCTGAGTAANNN-3' The N's indicate the variable selective nucleotides. In the AFLP classification, all 16 possible initiators for the Psil initiator and the 64 possible initiators for the Mse \ primer were used in the two groups of absolutely isogenic lines, giving a total of 16 x 64 = 1024 of proven primer combinations. After analysis of all AFLP fingerprints, a total of 30 candidate AFLP markers linked to the resistance gene Mi were identified. These candidate markers were frequently tested on a panel of tomato lines resistant to nematodes and susceptible to nematodes for confirmation and distance from the Mi site link. The resistance gene Mi was introduced into the tomato grown in 1944 from Lycopersicon peruvianum. The tomato lines resistant to modern nematode were subjected to numerous crossing cycles hoping to obtain a result in a small introduced region of Lycopersicon peruvianum with the resistance gene Mi. The testing of candidate AFLP markers in these modern tomato genotypes is expected to be a good test to determine the close link to the Mi site. A panel of 7 resistant tomato genotypes and 11 susceptible tomato genotypes was tested with candidate AFLP markers. A total of 20 AFLP markers appeared to be present in all resistant lines and absent in all susceptible lines and are referred to as AFLP markers linked to Mi. Then, four of the AFLP markers were classified into a high molecular weight genomic library. The cloning of very large segments of DNA such as large artificial chromosomes in yeast became an essential step to isolate genes through positional cloning. The cloning capacity of the YAC vector allows isolation of DNA fragments up to one million base pairs in length. The Lycopersicon esculentum E22 tomato line, homozygous for the Mi site, was used as a source of DNA to build a YAC collection. A YAC collection containing 3840 clones with an average insert size of 520 kb was obtained, representing approximately 2.2 genome equivalents of the tomato genome. Three positive YAC genomes were obtained after the AFLP classification with the AFLP markers linked to Mi: 1/1172 and 2/1256. Subsequently, the presence of all AFLP markers linked to Mi in 3 YAC clones was determined. All the markers appeared to be present in one or more of the 3 YAC clones, which allowed a first placement of the various AFLP markers linked to Mi. The AFLP data indicated that the three YAC clones constituted an overlapping contig of approximately 1.4 Mb (see Figure 1). To determine the physical size of the Mi site comprising the AFLP markers linked to Mi and included in YAC clones 1/1084, 1/1172 and / or 2/1256, a large scale restriction map of the YAC contig was constructed. This defined a DNA segment comprising the Mi site of approximately 700 kb in which all the AFLP markers linked to Mi were located (see Figure 1). A size of 700 kb is still too large for direct localization of the Mi resistance gene. Such large inserts can not be transformed into plant cells directly. Therefore, a cosmid collection of the yeast strain containing YAC 1/1172 and a reconstructed cosmid collection of the yeast strain containing YAC 2/1256 were constructed using cosmid vectors which are suitable for Agrobacterium-mediated transformation. The size of this binary cosmid vector represents 29 kb and is shown schematically in Figure 2. The cloning capacity of this binary cosmid vector, using a paid lambda package extract is within the range of 9 to 24 kb . Two banks of approximately 250 were obtained, 000 cosmid clones each, of the yeast DNA fractionated in size. The cosmid banks were classified through colony hybridization using restriction fragments labeled with the YACs. In addition, positive cosmid clones were identified and, in addition, the cosmids were grouped into seven defined regions covering the Mi region. In the next step, the group of cosmids from the seven defined regions were printed using restriction fragment amplification to determine their relative order. A cosmid contig covering a DNA segment of approximately 700 kb could be constructed. Subsequently, the presence of AFLP markers linked to Mi in this cosmid contig was determined. A physical map of the DNA fragment comprising the resistance gene Mi was obtained with the positions of the various AFLP markers linked to Mi (see Figure 3). A total of 96 overlapping cosmids together constituted the DNA segment comprising the resistance gene Mi. The complementation analysis to identify the resistance gene Mi with said large group of cosmids is a very laborious task. Therefore, the position of the Mi resistance gene in the cosmid contig was determined using mutant tomato lines. These mutant lines are members of a family that originates from a common ancestor and contains a wild-type Mi genotype (resistant to nematodes) but a phenotype susceptible to mutant nematode. After analysis with the group of AFLP markers linked to Mi in a large number of these AFLP markers linked to Mi of three mutant lines appeared to be mutants in most of the mutants. These AFLP markers, therefore, showed a good correlation between the Mi genotype of AFLP and the Mi phenotype, in contrast to all the other 17 AFLP markers. Two of these AFLP markers, PM14 and PM25, were adjacent, and the region around these markers was measured as the very likely position for the resistance gene Mi. A group of 6 overlapping cosmids defining a DNA segment of approximately 50 kb around the AFLP, PM14 and PM25 markers was selected for complementation analysis (see Figure 4). The final step in the identification of the resistance gene Mi through positional cloning is the complementation of the corresponding susceptible phenotype. The 6 cosmids of the My candidate region were introduced into Agrobacterium tumefaciens through conjugation transfer in a coincidence in three relatives. The presence of the cosmid in the strains of A. tumefaciens was determined by comparing several restriction enzyme patterns, as well as DNA traces of A. tumefaciens strains with the E. coli strain containing the cosmid. Only those cultures of A. tumefaciens that carry a cosmid with the same DNA pattern as the corresponding E. coli culture were used to transform a susceptible tomato line. A susceptible tomato line was transformed with the cosmids Mi-32, Mi-30, Mi-11, Mi-18, Mi-01 and Mi-14 using standard transformation methods. The roots of R0 plants transformed from in vitro growth were tested for disease symptoms in order to identify cosmids with the resistance gene. The root explants were transferred in a solidified medium in plates of Petri dishes and inoculated with 10 gills of an axenic nematode culture of the plant root nematode Meloidogyne incognita. Disease symptoms were classified 6 weeks after inoculation. A transgenic plant was considered resistant when no gills were visible in its root crop. A transgenic plant was considered susceptible when at least two gills were induced in the root crop. Observations in the in vitro disease trial revealed that two cosmids were able to complement the susceptible phenotype. The presence of the AFLP marker, PM14, in resistant R0 plants indicated that the genomic insert present in the cosmids Mi-11 and Mi-18 is also present in the R0 plants and is involved to confer to the R0 plants resistance to Meloidogyne incognita. The primary regenerators (R0 plants) of the transformation experiments were developed in the greenhouse to fix seed to obtain Ri lines, which were tested for disease symptoms. The disease trial was carried out in plantings. For this, seeds were planted or small-rooted seedlings were transferred to soil infected with Meloidogyne incognita and disease symptoms were classified from 4 to 8 weeks after inoculation. The plants were considered resistant when 3 or less galls were visible on the roots. Plants are considered susceptible when more than 3 galls are formed in the roots. Observations of the in vivo disease assay revealed that the resistant R0 plants are corresponding to cosmid Mi-11 transformants.
In order to confirm the stable integration of the Mi resistance gene into the genome of the transgenic R0 plants, the resistant plants of the R lines were matched and developed in the greenhouse for a seed fixation to obtain R2 lines. Plantings of the R2 lines were subjected to an in vivo nematode disease assay. The results obtained indicated the stable inheritance of the resistance gene Mi. Finally, the inserts of the cosmids Mi-11 and Mi-18 were further characterized. The sequence analysis revealed a large open reading frame (ORF2) of 3621 nucleotides. The DNA sequence is listed in Figure 5. The DNA sequence comprising the resistance gene Mi was further subjected to transcription map formation studies in order to determine the existence of intron sequences. These map formation studies of transcripts were performed according to generally known methods, by which the genomic DNA sequences were compared with cDNA sequences. Comparison of cDNA sequences and genomic sequences revealed the existence of two intron sequences in the resistance gene Mi. An intron of 1306 nucleotides was located from nucleotide position 1936 to 3241 and a second intron of 75 nucleotides was located from nucleotide position 3305 to 3379, as presented in Figure 5. The position of the transcription initiation site was postulated in or upstream of nucleotide 1880. The first ATG start codon is located at the position of nucleotide 3263, which is 52 nucleotides upstream of the second intron, giving a large open reading frame (ORF1) encoding a polypeptide of 1257 amino acids (Figure 7A). Homology searches have shown that the polypeptides according to the invention belong to the LRR class of plant resistance proteins (Staskawicz et al., 1995, Science, 268, 661-667). In addition, the protein can be divided into four regions designated A to D: region A comprises a large amount of leucine residues, region B comprises a nucleotide binding site motif, region C is an LRR region comprising repeats with the following consensus sequence: a - a - NL - The a - a / S (Jones and Jones, 1997, Advances in Botanical Research, 24, 89-167) and region D reveals no homology to any known protein. For the identification and isolation of homologous sequences that fall within the scope of the present invention, genomic and cDNA libraries were classified with the coding sequence of the resistance gene Mi as a probe under severe hybridization conditions. The positive clones were isolated and used for complementation analysis. Hybridizations of Southern staining were performed in the YAC contig with an internal PstI fragment of the coding sequence of the resistance gene Mi. Three additional homologous regions could be identified: two in YAC 1/1172 and one in YAC 1/1084. Each region comprises from 2 to 3 homologues Mi indicative of the fact that the family of the Mi gene is composed of approximately 10 to 12 members. Surprisingly, the aphid disease assays revealed that the R0 plants, transformed with the cosmid Mi-11, are resistant to Meloidogyne incognita as well as resistant to Macrosiphum euphorbiae, indicating that the genome insert present in the cosmid Mi-11 is involved for confer on the plants R0 resistance to nematodes, just as it is involved to confer on the plants R0 resistance to aphids. In particular, a plant that is transformed with the resistance gene according to the invention has at least one reduced susceptibility to one or more pathogens, especially to plant root nematodes and / or aphids. In order to confirm the inheritance of aphid resistance, (i) the tomato lines R ^ previously obtained which were derived from transformants of the cosmid Mi-11 resistant to nematodes(Ii) the R2 lines derived from R4 plants resistant to matched nematodes, and (iii) the R., BC lines obtained from R- plants, resistant to cross nematodes with the susceptible tomato line 52201, were also tested for the resistance against M. euphorbiae. The results obtained indicated the inheritance of aphid resistance. The cosmid Mi-11 was used for the transformation of genotypes susceptible to tobacco and potato nematodes, according to the general known transformation methods. The roots of the transformed R0 plants developed in vitro from tobacco and potatoes were tested for disease symptoms as previously described. Observations of the disease trial on the root crops of the transformed plants indicated that the cosmid is involved to confer on the transformed plants a reduced susceptibility to nematodes. The resistance gene according to the invention has an effect for reducing the susceptibility of a heterologous plant species to nematodes, preferably to the species Meloidogyne especially Meloidogyne incognita. In addition, tobacco transformants were also tested for resistance to aphids, and the resistant R0 plants could be identified. The resistance gene according to the invention has a double function and has an effect on heterologous systems. The cosmid Mi-11 was deposited on August 5, 1996 as the pKGMi-11 plasmid in Centraalbureau voor Schimmelcultures at Baarn, Holland, under deposit number CBS 822.96. The cosmid Mi-18 was deposited on August 5, 1996 as the pKGMi-18 plasmid in Centraalbureau voor Schimmelcultures at Baarn, Holland, under deposit number CBS 821.96. The following examples will provide an additional illustration of the present invention, which however is not limited to these examples.
EXAMPLES
EXAMPLE 1 DISEASE ANALYSIS
An axigenic culture of Meloldogyne incognito of the plant root nematode remained in sterile roots of the tomato crop
Moneymaker Root cultures were grown on a solidified B5 medium (Gamborg et al. 1968, Experimental Cell Research 50:
151-158) with 2% sucrose and without hormones. The root explants (1-5 cm), derived from transgenic tomato plants grown in vitro, were transferred into the solidified B5 medium mentioned above to initiate the root cultures. At the same time, each root explant was inoculated with 10 galls of the axenic nematode culture. The gills were placed a few centimeters from the root explant. The Petri dishes with roots and gills were incubated in the dark at 25 ° C. After 4 to 6 weeks, the level of infection was determined by counting the number of gills formed in the root crops. The evaluation for resistance / susceptibility to M. incognita is as follows: A transgenic plant was considered resistant when no or less than 2 galls were visible in its root crop. A transgenic plant was considered susceptible when at least 2 galls were induced in its root crop.
EXAMPLE 2 IDENTIFICATION OF AFLP MARKERS LINKED TO A
DNA SEGMENT UNDERSTANDING THE GENE
OF RESISTANCE My
Tomato Lines (Lycopersicon esculentum) A total of 9 Tomato lines resistant to Meloidogyne incognita and 13 Tomato lines susceptible to M. incognita were used to identify AFLP markers. Initially, the AFLP classification was performed in two groups of absolutely isogenic lines 83M-R (resistant) and 83M-S (susceptible), and Motelle (resistant) and Mobox (susceptible). The candidate markers that resulted from this first classification were confirmed through a second classification in resistant lines 7 M. unknown and susceptible lines 11. unknown. Two groups of absolutely isogenic lines: 1. 83M-R resistant Ruiter Zonen C.V. Bergschenhoek Holland (hereinafter "De Ruiter") 2. 83M-S susceptible De Ruiter 3. Sturdy motelle INRA, Montfavet, France 4. Mobox susceptible INRA, Montfavet, France Sturdy lines 7 M. incognito and susceptible lines M incognita for confirmation: 5. Resistant DR30 De Ruiter 6. DR17 resistant De Ruiter 7. E22 resistant Enza Zaden, Enkhuizer Zaadhandel BV Enkhuizen, Holland (hereinafter "Enza Zaden") 8. E1 resistant Enza Zaden 9. DR6 resistant of Ruiter 10. DR10 resistant of Ruiter 11. 1872 resistant Royal Sluis BV, Enkhuizen, Holland (hereinafter "Royal Sluis") 12. Moneymaker susceptible Agricultural University Wageninge
13. DR12 susceptible De Ruiter 14. DR23 susceptible De Ruiter 15. GT susceptible of Ruiter 16. RZ3 susceptible Rijk Zwaan Zaadteelt in Zaadhandel B. V., De Lier, The Netherlands (hereafter "Rijk Zwaan")
17. RZ5 susceptible Rijk Zwaan 18. E3 susceptible Enza Zaden 19. E7 susceptible Enza Zaden 20. E16 susceptible Enza Zaden 21. RS1 susceptible Royal Sluis 22. RS2 susceptible Royal Sluis
Isolation and Modification of DNA The total tomato DNA of the 22 lines described above was isolated from young leaves as described by Bernatzki and Tanksley (Theor.Appl. Genet, 72, 314-321). The typical yield was 50-100 μg of DNA per gram of fresh leaf material. The template DNA for the AFLP analysis with the Psíl-Msel enzyme combination as described by Zabeau and Vos (European patent application, EP 0534858), and is briefly described below: 0.5 μg of tomato DNA was incubated for 1 hour at 37 ° C with 5 units of Psil and 5 units of Mse \ in 40 μl of 10 mM Tris.HAc pH 7.5, 10 mM MgAc, 50 mM KAc, 5 mM DTT, 50 ng / μl BSA. Then, 10 μl of a solution containing 5 pMol Psyl-adapters, 50 pMol? Fsel-adapters, 1 unit T4 DNA ligase, 1 mM ATP in 10 mM Tris.HAc pH 7.5, 10 mM MgAc, 50 mM KAc, was added. 5 mM DTT, 50 ng / μl BSA, and incubation was continued for 3 hours at 37 ° C. The adapters are represented below:
The structure of the Psil adapter was: 5'-CTCGTAGACTGCGTACATGCA-3 '3'CATCTGACGCATGT-5' The structure of the Mse adapter was: 5'-GACGATGAGTCCTGAG-3 '3'-TACTCAGGACTCAT-5'
The adapters were prepared by adding equimolar amounts of both structures; the adapters were not phosphorylated. After ligation, the reaction mixture was diluted to 500 μl with 10 mM Tris. HCl, 0.1 mM EDTA pH 8.0, and stored at -20 ° C. The diluted reaction mixture is further named with template DNA.
AFLP Reactions The primers used for the AFLP classification are presented below: Initiators Psil: 5'-GACTGCGTACATGCAGNN-3 'Msel primers: 5'-GATGAGTCCTGAGTAANNN-3' The N's in the primers indicate that this part of the primers was variable. In the AFLP classification all 16 possible initiators were used for the Psíl initiator and the 64 possible initiators were used for the Msel initiator. This gave a total of 16 x 64 initiator combinations Psíl and Msel, and is 1024 initiator combinations. All 1024 primer combinations were used in the AFLP classification for AFLP markers linked to Mi. The AFLP reactions were carried out as follows:
AFLP reactions employed a radiolabelled Psil initiator and an unlabeled Msel initiator. The Psil primers were labeled at their terminus using (? -33P) ATP and T4 polynucleotide kinase. The labeling reactions were performed in 50 μl 25 mM Tris. HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, 0.5 mM spermidine.3HCI, using 500 ng of the oligonucleotide primer, (? -33P) ATP and 10 units of T4 polynucleotide kinase. For the AFLP analysis, a 20 μl reaction mixture containing 5 ng of the labeled Psil initiator (0.5 μl of the labeled reaction mixture), 30 ng of the Msel initiator, 5 μl of the template DNA, 0.4 units of Taq- polymerase, 10 mM Tris. HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of all 4 dNTPs. AFLP reactions were performed using the following cycle profile: a 30-second DNA denaturation step at 94 ° C, an annealing step of 30 seconds (see below), an extension step of one minute at 72 ° C . The annealing temperature in the first cycle was 65 ° C, it was subsequently reduced each cycle by 0.7 ° C for the next 12 cycles, and it was continued at 56 ° C for the remaining 23 cycles. All amplification reactions were performed in an EE-9600 thermocycler (Perkin Elmer Corp., Norwalk, CT, USA).
Gel Analysis of AFLP Reaction Products After amplification, the reaction products were mixed with an equal volume (20 μl) of formamide dye, 10 mM EDTA pH 8.0, and bromophenol blue and xylennol as trace dyes). The resulting mixtures were heated for 3 minutes at 90 ° C, and then cooled rapidly on ice. 2 μl of each sample was loaded on a denaturing polyacrylamide gel (sequencing) at 5% (Maxam and Gilbert, Methods in Enzymology 65, 499-560). The gel matrix was prepared using 5% acrylamide, 0.25% methylene bisacryl. 7.5 M urea in 50 mM Tris / 50 mM boric acid / 1 mM EDTA. To 100 ml of the gel solution was added 500 μl of 10% APS and 100 μl of TEMED and the gels were cast using a 38 x 50 cm SequiGen gel apparatus (Biorad Laboratories Inc., Hercules, CA, USA). ). Shark tooth combs were used to give 97 lanes in the SequiGen gel units. 100 mM Tris / 100 mM boric acid / 2 mM EDTA was used as the pH regulator of operation. Electrophoresis was performed at a constant energy, 110 Watts, for approximately 2 hours. After electrophoresis, the gels were fixed for 30 minutes in 10% acetic acid, dried on glass plates and exposed to Fuji phosphor image screens for 16 hours. The fingerprint patterns were visualized using a Fuji BAS-2000 phosphor image analysis system (Fuji Photo Film Company Ltd, Japan).
AFLP Classification for Linked Markers An AFLP classification was made using all possible 1024 combinations of the Psíl-Msel primers in the two groups of absolutely isogenic lines. The objective was to identify AFLP markers present both resistant and absent lines in both susceptible lines. The AFLP gels contained the AFLP traces of 24 initiator combinations of the 4 isogenic lines, giving a total of 43 gels. A total of 30 AFLP markers were identified present in both resistant and absent lines in both susceptible lines. These markers are referred to as AFLP markers linked to My candidates. Afterwards, AFLP reactions were performed to determine the presence of the 30 candidates in the 7 resistant tomato lines and 11 susceptible tomato lines. Of the 30 candidate markers, 20 markers appeared to be present in the 7 resistant and absent lines in the 11 susceptible lines. These 20 markers were used in additional studies to map the resistance gene Mi. The initiator combinations required identifying the Psíl-Msel markers and are presented in Table 1. In the column with the initiator combinations, "Psíl" refers to the sequence 5'- GACTGCGTACATGCAG-2 'and "Msel" is refers to the sequence 5'-GATGAGTCCTGAGTAA-3 '. For example, the PM14 marker can be identified using the Psil initiator having the following sequence 5'-GACTGCGTACATGCAGGA-3 ', and the Msel primer having the following sequence: 5'-GATGAGTCCTGAGTAATCT-3 \ TABLE 1
Marker Initiator combination with selective extensions (NN / NNN)
PM02 Pstl-AT / Msel-AAA PM07 Pstl-AA / Msel-TAC PM08 Pstl-CT / Msel-ACT PM10 Pstl-CA / Msel-TCT PM11 Pstl-TA / Msel-TGA PM13 Pstl-GA / Msel-ATC PM14 Pstl -GA / Msel-TCT PM15 Pstl-GT / Msel-GAC PM16 Pstl-GT / Msel-TCT PM17 Pstl-AT / Msel-AAG PM18 Pstl-AT / Msel-TAG PM19 Pstl-GG / Msel-ATT PM20 Pstl-TG / Msel-AAT PM21 Pstl-TG / Msel-TTT PM22 Pstl-TG / Msel-GCT PM23 Pstl-GT / Msel-GAA PM24 Pstl-AA / Msel-CTG PM25 Pstl-AC / Msel-GTG PM27 Pstl-AA / Msel -CTA PM29 Pstl-TA / Msel-GGA EXAMPLE 3 CONSTRUCTION AND CLASSIFICATION OF A YAC COLLECTION
OF JITOMATE
Material A line of tomato Lycopersicon esculentum E22 (Enza Zaden) homozygous for the site Mi was used as the source of DNA to build a YAC collection. Protoplasts were isolated from in vitro shoot leaves, which were aged 2 to 3 weeks as described by Van Daelen et al. (Plant Mol. Biol. 12, 342-352). Viable protoplasts (concentration of 50 million protoplasts per ml) were collected and mixed with an equal volume of agarose (1%, Seaplaque, FMC Bioproducts, Rockland, Maine, USA) to form a stopper. The protoplasts embedded in the plugs were lysed with a lysis mixture (0.5 M EDTA, 1% sarcosinate N-lauryl and 1 mg / ml proteinase K, pH = 8.0). After lysis, the stoppers were stored at 4 ° C in storage pH buffer (fresh lysis mixture) until used. Approximately 3 million protoplasts per plug, to obtain approximately 4.5 μg of chromosomal DNA that were used for further studies. Plasmid pYAC4 containing a single EcoRI cloning site was used as the cloning vector and yeast strain AB1380 was used as a host (Burke et al., Science 236, 806-812).
Construction of the YAC Collection Isolation of high molecular weight DNA, partial digestion with EcoRI in the presence of EcoRI methylase, ligation of vector arms to genomic DNA, size selection through electrophoresis in the field .gel was performed pulsed and yeast host transformation, as described by Burke et al., (Science 236, 806-812) and Larin et al., (Proc Nati, Acad Sci USA 88, 4123-4127). All standard manipulations were performed as described in Molecular cloning: a laboratory manual by Sambrook et al., (Cold Spring Harbor Laboratory Press). Finally, 3840 clones with an average insert size of 520 kb were obtained, which corresponds to 2.2 genome equivalents and the individual clones were stored in 40 96-well microtiter plates containing a 75 μl YPD solution (1% extract). of yeast, 2% peptone and 2% dextrose).
YAC Collection Classification
To reduce the number of samples handled, the cells of a 96-well microtiter plate were combined (a combination of plates) and used for DNA isolation as described by Ross et al (Nucleic Acids Res., 19, 6053). . The tomato YAC collection of 2.2 genome equivalents consisted of 40 96-well microtitre plates and as a result of the DNA of the 40 plate combinations were classified with the AFLP, PM10, PM13, PM21, and PM25 markers using the AFLP protocol as described in example 2. PM10, PM13, PM21, and PM25 were selected to classify the YAC plate combinations since these markers do not interfere with the background bands of the yeast strain AB1380. Three positive plate combinations of the 40 were identified with these four AFLP markers as shown in Table 2. Subsequently, a secondary classification was used with the four AFLP markers (PM10, PM13, PM21, and PM25) of the 96 clones of Individual YACs of each plate to find the correct direction of the YAC clones. Three individual YAC clones were identified, designated 1/1084, 1/1172 and 2/1256 (Table 2). Subsequently, the three individual YAC clones were analyzed with the remaining AFLP markers. All the markers identified PM02 to PM29 were presented to one or more of these YAC clones (Table 3). The size of the YAC clone was determined through electrophoretic analysis in pulse field gel (PFGE) using a homogeneous electric field of bound contour (CHEF, Chu et al Science, 235, 1582-1585) and it seemed to be 470 kb (1/1084), 570 kb (1/1172), and 500 kb (2/1256), respectively.
TABLE 2 Combination PM10 PM13 PM21 PM25 YAC detected plate nr (size in kb) 2 - - + - YAC 1/1172 (570 kb)
16 + + - + YAC 2/1256 (500 kb)
4 - . 4 - + - - YAC 1/1084 (470 kb)
TABLE 3
Marker 1/1172 2/1256 1/1084 PM02 - - + PM07 - + - PM08 - + + PM10 - + - PM11 - + - PM13 - + + PM14 + + - PM15 - + - PM16 + - - PM17 - + - PM18 - + + PM19 - + - PM20 - + - PM21 + - - PM22 - + + PM23 - + - PM24 - + - PM25 - + - PM27 - + - PM29 - + - EXAMPLE 4 CONSTRUCTION OF A PHYSICAL MAP OF GREAT SCALE OF THE
CONTIG DE YAC DE Mi AND THE LOCATION OF THE AFLP MARKERS
The three clones of YAC 1/1172, 2/1256 and 1/1084 were subjected to partial digestion with an increased concentration of restriction enzymes Sfi \ and SssHil. Samples were fractionated through PFGE, transferred to a more membrane gene sorting (DuPont NEN, Boston, MA, USA) and analyzed through hybridization using adjacent end sequence probes according to the protocol to form brand maps of indirect end as described by Burke et al (Science 236, 806-812). A physical map of YAC 1/1172, 2/1256 and 1/1084 for the Sf / 'and BssHil enzymes could be constructed as shown in Figure 1. The overlap between the various YAC clones was determined through analysis of Southern tinsion using the restriction fragments obtained as a probe in the digestion of the three YAC clones. A YAC contig with a size of 1.4 Mb could be built. In order to isolate the YAC fragments, the digested products were operated on PFGE. Digestion of YAC 1/1172 with Sf / 'l resulted in two fragments (200 kb and 370 kb). Digestion of YAC 2/1256 with SssHil resulted in four fragments (40 kb, 90 kb, 110 kb and 260 kb), while digestion of YAC 1/1084 with SssHil gave two fragments with a size of 70 and 400 kb . As a result, the YAC contig of 1.4 Mb could be separated into 8 regions corresponding to the 8 restriction fragments obtained from the three YAC clones, converting the complete Mi region and adjacent sequences. To place the various AFLP markers within these 8 regions on the physical map, the AFLP markers were used as hybridization probes in the partial and complete digestions Sf / 'l and Filsil of YAC clones 1/1172, 2/1256 and 1/1084. Therefore, each AFLP marker fragment was excised from the dried gel and eluted through diffusion in a pH buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH = 8.0), 0.1 % SDS, re-applied with the corresponding unlabeled AFLP primers and, subsequently, marked with 32P according to the random primer method Feinberg and Vogelstein (Anal. Biochem., 132, 6-10). Each AFLP marker could be assigned to one or more of the eight regions as presented in Table 4 and Figure 1.
TABLE 4
Fragment YAC AFLP markers linked to Me detected through hybridization
200 kb Sfi \ - fragment 1/1172 370 kb Sfi \ - fragment 1/1172 PM14, PM16, PM21 260 kb SssHil- fragment PM10, PM11, PM17, PM19, PM23,
2/1256 PM24, PM29 90 kb ßssHil- fragment 2/1256 PM07, PM27 110 kb SssHil- fragment PM08, PM13, PM14, PM15, PM20,
2/1256 PM22, PM25 40 kb BssHil- fragment 2/1256 PM18 70kb SssHil- fragment 1/1084 PM08, PM13, PM22 400 kb SssHil- fragment PM02, PM18 1/1084
EXAMPLE 5 CONSTRUCTION OF A COLLECTION OF CLONE CONSUMPTION
YAC 1/1172 and 2/1256
Material The binary cosmid vector pJJ04541 is a derivative of pJJ1881 (Jones et al, Transgenic Research 1, 285-297) and was based on plasmid pRK290 containing the tetracycline resistance gene for selection in Escherichla coli and Agrobacterium tumefaciens. In the unique EcoRI site of pRK290, the sequences bearing T-DNA (LB, repetition of the left boundary, RB means the repetition of the right boundary) that flank: the cos site of lambda of bacteriophage. the neomycin phosphonotransferanase gene (Beck et al, Gene 19, 327-336) whose expression is activated by the 35S promoter sequence of the cauliflower mosaic virus (Odell et al, Mol Gen Genet 223, 369-378), and the poly linker sequence of pBluescript (Stratagene, La Jolla, California, USA). The size of pJJ04541 represents 29 kb and is shown schematically in Figure 2. The cloning capacity of this binary cosmid vector, using extracts of phage lambda package is within the range of 9 to 24 kb.
Construction of the Collection The total DNA of the strain Saccharomyces cerevisae AB1380 containing YAC 1/1172 and total DNA of the strain Saccharomyces cerevisae AB1380 containing YAC 2/1256 was isolated using zymoxysa to make protoplasts according to Green and Olsen (Proc Nati Acad Sci USA 87, 1213-1217). An aliquot of both DNAs was analyzed in PFGE. Both DNA isolates appeared to have a size of > 100 kb. Approximately 15 ug of each DNA was partially digested with Sau3A generating molecules with an average size of 15-25 kb. Subsequently, the samples were centrifuged through a gradient of 10-35% sucrose for 22 hours, 22,000 rpm at 20 ° C in a Beckman SW41 rotor. Fractions of 0.5 ml were collected using a needle punched through the bottom of the centrifuge tube. An aliquot of these fractions was analyzed on a 0.7% agarose gel. The fractions containing the DNA molecules with a size of «20 kb were combined and concentrated through precipitation with ethanol. Subsequently, the cohesive ends were partially filled with dATP and dGTP using the 5'-partial extension strategy of DNA produced by restriction endonuclease type II as described by Korch (Nucleic Acids Res. 15, 3199-3220) and Loftus et al (Biotechniques 12, 172-176). The binary cosmid vector pJJ04541 was completely digested with Xho \ and the linear fragment was partially filled with dTTP and dCTP as described by Korch (Nucleic Acids Res. 15, 3199-3220). The 20 kb fragments were ligated to the cosmid vector and translated into the E. coli strain XL1-Blue MR (Stratagene, La Jolla, California, USA) using packaging extracts of phage lambda Gigapack II XL (Stratagene, La Jolla , California, USA) as recommended by the manufacturers. The selection was made on LB agar plates (1% bactotriptone, 0.5% bacto-yeast extract and 1% NaCl, pH 7.5) containing 10 mg / l of tetracycline. Two banks of approximately 250,000 cosmid clones per bank were made from 2-3 μg of fractionated yeast DNA from clones YAC 1/1172 and 2/1256, respectively.
Subsequently, these transformants were stored in the microtitre plate cavities (96 wells, 100 μl of LB medium containing 10 mg / L of tetracycline). The replicas of the 96-well grid of cosmid clones in the microtiter plates were stamped on membrane filters Gene
Screen Plus (NEN DuPont) and allowed to develop in colonies on the medium. Colony hybridization, as described by
Sambrook et al (in: Molecular cloning: a laboratory manual, 1989,
Cold Spring Harbor Laboratory Press) using YAC clones labeled with 32P 1/1172 and 2/1256 revealed positive cosmids. From approximately 10,000 YAC colonies 1/1172 to approximately 200 positive cosmid clones were identified. From approximately 20,000 colonies of YAC 2/1256, 300 positive cosmid clones were identified.
EXAMPLE 6 FORMATION OF STRENGTH GEN SEGMENT MAP AND PLACING THE AFLP MARKERS
Division of the Cosmids in Defined Regions
In order to divide the cosmids into seven defined regions, the 200 positive cosmid clones of YAC 1/1172 and the 300 positive cosmid clones of YAC 2/1256 of they were hybridized with 7 of the 8 restriction fragments (YAC fragments) as was presented in example 4 (See Table 4 and Figure 1). The positive cosmids for each of the 7 YAC fragments were identified. In addition, cosmids that reacted positively with the overlapping restriction fragments of the two different YAC clones could be identified.
Construction of a Cosmic Contig of the Resistance Gene Segment Mi
In order to construct a cosmid contig of all the cosmids identified as positive in the various defined regions, restriction fragment amplification was used. Approximately 500 ng of each cosmid was used for template preparation and the primers in the amplification of restriction fragments were a primer 5'-GACTGCGTACCAATTC EcoRI-3 'having no selective nucleotide and MseI primer 5' -GATGAGTCCTGAGTAA-3 ' having no selective nucleotide according to the method described in Example 2. The EcoRI primer was labeled at the 5 'end and all 500 cosmids were amplified using the EcoRI / Msel initiator group. The DNA fingerprints contained approximately 8 to 20 amplified fragments. Groups cosmids containing amplified fragments of identical size were selected for each region and re-run on polyacrylamide gels as described in Example 2 until it could build an adjoining disposition of all the amplified fragments throughout the defined regions . In addition, the cosmid contig of a region was ligated with the adjacent regions in order to construct a cosmid contig of the Mi site. In this way, a cosmid contig of 96 cosmids was constructed by extending the Mi site of approximately 800 kb.
Detailed Positioning of AFLP Markers Linked to Me in Contig of Cosmido
In order to place the 20 AFLP markers linked to Mi in the cosmid contig, the 96 cosmids were digested with Psil followed by Southern tinsion analysis according to Southern, J. Mol. Biol. 98, 503-515. The AFLP markers were used as hybridization probes as described in Example 4 in the Southern staining of the 96 Psil digestions of the cosmids. The exact position of the AFLP markers linked to Mi, except marker PM02, is presented in Figure 3A.
EXAMPLE 7 GENETIC ANALYSIS OF MUTANTS
A family of mutant tomato lines was made available through Enza Zaden. These lines were derived from a hybrid heterozygous F ^ for the Mi resistance gene and heterozygous for the Aps-1 gene (phosphatase-1 acid coding), which is closely linked to Mi (Stevens and Rick, 1986, in: The Tomato Crop, Atherton &Rudich edit., Chapman and Hall, pp. 35-109). We could determine different alleles of the > 4ps-1 through isozyme analysis (Vallejos, 1983, in: Isozymes n plant genetics and breeding, Tanksley and Orton edit., Part A, Elsevier, Amsterdam, 469-515). The allele > 4ps-11 originates from L. peruvianum and has been introduced in several tomato genotypes resistant to nematodes through co-segregation with the resistance gene Mi. An outline of these mutant lines is presented below: Fi-hybrid (heterozygous >); Aps-1, phenotype resistant to Mi) combined F2-lines (segregate /.ps-1 1: 2: 1, segregate resistance Mi 3: 1) combination of heterozygous plants (> Aps-1) F2, F3-lines (segregate? ps-1 1: 2: 1, secrete resistance Mi 3: 1) i combination of heterozygous plants (4ps-1) F3, F4-lines (segregate Aps- 1: 2: 1, segregate resistance Mi 3: 1 ) l combination of heterozygous plants (> Aps-1) F4, F5-lines (segregate Aps-1 1: 2: 1, susceptible Mi) combination of heterozygous plants (> Aps-1) F5, F6-lines (segregate > 4ps-1 1: 2: 1, susceptible Mi) combination of heterozygous plants (? ps-11) F6, F7-lines (all \ ps-11, susceptible Mi) - heterozygous plant combination (> Aps-11 ) F7, F8-lines (all> 4ps-11, susceptible Mi) In the F, F2, F3 and F4 lines of this family the presence of the Aps-11 allele correlates with the Mi-resistant phenotype, whereas the absence of the allele /.ps-11 correlates with the susceptible phenotype to Mi. In F5 and the subsequent progenies, this correlation is lost: all plants are susceptible to nematodes without considering the Aps-11 alleles. Twenty individuals of each generation of F2, F3, F4? F5, F6, F7 and F8 were tested for resistance to nematodes, for the presence of the Aps-1 allele and the presence of AFLP markers linked to Mi. The crop nematode test was performed on soil contaminated with root galls of M. incognita. The results of resistance to nematodes were as indicated in the previous scheme: 3: 1 segregation in plants F2, F3, and F4 and susceptibility in F5 and populations of progeny. Most of the AFLP markers linked to Mi indicated an identical Mi genotype as the isozyme marker -4ps-1. However, 3 of the AFLP markers PM14, PM16 and PM25 appeared to segregate with the Mi: phenotype in most plants F5, F6, F7 and F8, the susceptibility to Mi was indicated by the absence of these markers. The AFLP markers PM14, PM16 and PM25 showed a correlation between the Mi genotype of AFLP and the Mi phenotype in the mutants. The markers PM14 and PM25 are adjacent to the physical map as shown in Figure 3B, and, therefore, it was postulated that the region surrounding these AFLP markers was a good candidate to understand the resistance gene Mi.
EXAMPLE 8 PHYSICAL MAP OF TRANSLATING COSMETIC CLONES
BUY N DENDING THE RESISTANCE GENE My
The identification of hybridizing cosmids with the AFLP markers linked to Mi PM14 and PM25 was performed in example 6. PM14 identifies the cosmids Mi-11, Mi-18 and Mi-01, while PM25 identifies the cosmids Mi-18 and Mi-01. Subsequently, a small array of cosmids around the cosmids Mi-11, Mi-18 and Mi-01 was selected for the cosmid contig described in example 6. A contig of 6 cosmids comprising the 3 identified cosmids and the adjacent cosmids, He was selected. These 6 cosmids are Mi-32, Mi-30, Mi-11, Mi-18, Mi-01 and Mi-14. In order to make a physical fine map of these 6 cosmids, the DNA samples from the cosmid contig were digested with Psil followed by electrophoresis on a 0.8% agarose gel. The physical overlap between the various cosmids could be determined. The combination of this data with the data obtained with respect to the detailed placement of the AFLP markers linked to Mi in contig of cosmid (see example 6) a physical fine map with the location of PM14 and PM25 could be constructed as shown in the Figure 4. The cosmid contig around the AFLP markers PM14 and PM25 was calculated to be approximately 50 kb.
EXAMPLE 9 TRANSFORMATION
Transfer of Cosmides to Agrobacterium Tumefaciens
The cosmid clones Mi-32, Mi-30, Mi-11, Mi-18, Mi-01, Mi-14 and the control cosmid pJJ04541 were introduced into Agrobacterium tumefaciens through conjugation transfer in a tri-parent mating with the auxiliary strain HB101 (pRK2013) essentially in accordance with Deblaere et al (Methods in Enzymology 153, 277-292). E. coli was developed in the LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl, pH 7.5) supplemented with 5 mg / l of tetracycline at 37 ° C. The HB101 helper strain (pRK2013) was grown under identical conditions in the LB medium supplemented with 100 mg / L kanamycin sulfate. The Agrobacterium tumefaciens strain AGL1 (Lazo et al, Bio / Technology, 9, 963-971, 1991) was grown in LB medium supplemented with 100 mg / l carbenicillin at 28 ° C. During the night, the cultures were diluted 1: 100 in the LB medium without any antibiotic and after 6 hours of development, 0.1 ml of each Agrobacterium culture, the auxiliary strain culture and a culture of the cosmid strain were mixed and placed in LB agar plates without antibiotic. After incubation overnight at 28 ° C, the mixtures were placed on agar plates with an LB medium containing 100 mg / l of carbenicillin and 10 mg / l of tetracycline to classify the transconjugates. The plates were incubated for 3-4 days at 28 ° C. Two serial passages were made through the selective agar plates to select the individual transconjugate Agrobacterium colonies.
Characterization of Transconjugates of A. Tumefaciens
Small-scale cultures were developed from selected colonies and grown in the LB medium containing 10 mg / L of tetracycline. The plasmid DNA was isolated through alaclin lysis using the method described by Ish-Horowicz et al (Nucí Acids Res. 9, 2989-2997, 1981), and was digested with BglU using standard techniques. In addition, restriction fragment amplification was performed on mini-DNA preparation of A. tumefaciens using the EcoRI / Msel enzyme combination and the primers having no selective nucleotide as described in Example 6. Subsequently, the enzyme pattern of Bkg / ll restriction as well as the DNA fingerprint of the transconjugate A. tumefaciens were compared with those of the mini-DNA preparation of the E. coli strain containing the cosmid. Only those transconjugados A. tumefaciens that carry a cosmid with the same DNA pattern as the corresponding E. coli culture were used to transform a susceptible tomato line.
Transformation of a Tomato Line Susceptible
The seeds of the susceptible tomato line 52201 (Rijk
Zwaan, De Lier, The Netherlands) were sterilized on the surface in 2% sodium hypochlorite for 10 minutes, rinsed three times in sterile distilled water and placed on a germination medium (consisting of medium medium MS medium in accordance with with Murashige and Skoog, Physiol., Plant 15, 473-497, with 1% (w / v) sucrose and 0.8% agar) in glass jars or polypropylene culture vessels. They were allowed to germinate for 8 days. The culture conditions were 25 ° C, a photon flux density of 30 μmoles. m "2.s" 1, and a photoperiod of 16/24 hours.
Tomato transformation was performed according to Koornneef et al (1986), in: Tomato Biotechnology, 169-178, Alan R. Liss, Inc., and is briefly described below. 8-day-old cotyledonie explants were precultured for 24 hours in Petri dishes containing a feeder layer of Petunia Hybrid suspension cells placed in plates on an MS20 medium (culture medium according to Murashige and Skoog, (1962) Physiol. Plant 15, 473-497, with 2% (w / v) of sucrose and 0.8% of agar) supplemented with 10.7 μM of α-naphthaleneacetic acid and 4.4 μM of 6-benzylaminopurine. The explants were then infected with the diluted overnight culture of Agrobacterium tumefaciens containing the cosmid clones Mi-32, Mi-30, Mi-11, Mi-18, Mi-01 and Mi-14 or the cosmid vector pJJ04541 for 5- 10 minutes, they were dried by distinction on a sterile filter paper and co-cultivated for 48 hours on the plates of the original feeder layer. The culture conditions were as described above. The overnight cultures of Agrobacterium tumefaciens were diluted in a liquid MS20 medium (medium according to Murashige and Skoog (1962) with 2% (w / v) of sucrose, pH 5.7 to OD60o of 0.8) After co-cultivation, the cotyledonary slurs were transferred to Petri dishes with a selective medium consisting of MS20 supplemented with 4.56 μM of zeatin, 67.3 μM of vancomycin, 418.9 μM of cefotaxime and 171.6 μM of camamicin sulfate, and cultured under the culture conditions described The explants were subcultured every three weeks on fresh medium, the outlets were separated from the underlying calluses transferred to glass jars with a selective medium without zeatin to form roots.The root formation in a medium containing camamicin sulfate was considered as an indication of the transgenic nature of the outbreak in question, the truly transgenic regenerators were propagated in vitro subc finalizing the apical meristem and auxiliary shoots in glass jars with fresh selective media without zeatin.
EXAMPLE 10 COMPLEMENTATION ANALYSIS
Identification of Cosmids with the Resistance Gene My Sorting for Resistance in Roots of Transformed Plants
Roots were subjected to transformed R0 plants grown in vitro to the disease assay as described in Example 1. From each transformant, two root explants were analyzed. In total, 72 R0 of 7 different transformations of cosmid were tested; 6 cosmids carrying DNA from tomato insert and 1 cosmid, pJJ04541, without DNA from tomato insert. The results are shown in Table 1. 63 transgenic R0 plants appeared susceptible, since the gills were formed in at least one of the two root crops. Nine R0 plants were classified as resistant, since no gills were found in the root crops. Seven resistant plants were derived from the transformation with the cosmid Mi-11, while two resistant plants were derived with the cosmid Mi-18, which overlaps a large part with the cosmid Mi-11. The cosmids Mi-11 and Mi-18 They were used for additional molecular analysis.
TABLE 1
Cosmic Plants R (Resistant Susceptible Mi -32 0 8 Mi -30 or 11 Mi -11 7 4 Mi -18 2 8 Mi -01 0 10 Mi -14 0 15 PJJ04541 0 7
Molecular Analysis of Transformed Plants with a Resistant Phenotype
To demonstrate that the resistant phenotype of transgenic R0 plants, which have had the overlapping cosmids Mi-11 and Mi-18, was determined through the genomic insert present in the various cosmids, an AFLP analysis was performed with the AFLP marker, PM14 . The amplification of selective restriction fragments was carried out with the primer combination identifying the marker PM14 for the R0 plants transformed with the cosmids Mi-11 and Mi-18. The DNA traces obtained showed the presence of the PM14 marker in the resistant plants indicating that the genomic insert present in the cosmids Mi-11 and Mi-18 is also present in the R0 plants and that the two overlapping cosmids Mi-11 and M -18 comprise the resistance gene to Mi.
The inserts of the cosmids Mi-11 and Mi-18 and the inserts in the adjacent cosmids M-32 and Mi-30 on one side and the cosmids Mi-01 and Mi-14 on the other side, were also characterized. The region of DNA comprising the resistance gene Mi based on the overlap between the cosmids Mi-11 and Mi-18, was estimated at approximately 16-18 kb. Based on the susceptibility of the R0 plants having the insert present in the cosmid Mi-30, this region could be narrowed to approximately 12 kb. A segment of DNA comprising the resistance gene Mi, which corresponds to the region flanked by the right ends of the cosmids Mi-30 and Mi-11, was segmented (see Figure 4).
EXAMPLE 11 NUCLEOTIDE SEQUENCE AND AMINO ACID SEQUENCE
DEDUCTIBLE OF THE RESISTANCE GENE Ml DEL JITOMATE
Subcloning of the Overlapping DNA Segment
To determine the sequence of the overlapping DNA segment in the cosmids Mi-11 and Mi-18 containing the resistance gene Mi, a group of random subclones with an insert size of approximately 2 kb was generated. Shear stress was applied to 7.5 μg of CsCl purified DNA from cosmids Mi-11 and Mi-18 for 10 seconds at 4 ° C at 15% probe energy (in 40 μl 10mM Tris-acetate, 10mM Mg- acetate and 50mM K-acetate) using a Misonix sound applicator (Misonix Inc., Farmingdale, NY, USA) (type XL2020) with a full-water horn (type 431A). Subsequently, the DNA was heated for 10 minutes at 60 ° C and cooled to room temperature. The ends of the DNA fragments were repaired by adding 10 μl of a repair mixture (10mM Tris-acetate, 10mM Mg-acetate, 50mM K-acetate, 10U of Klenow DNA polymerase, 10U of DNA-T4 polymerase and 2 mM of all 4 dNTP's) and was followed by incubation for 30 minutes at 20 ° C. The shear stress DNA was separated through electrophoresis on a 1% Seakem GTG agarose gel (FMC Bio Products, Rockland, ME, USA). The fraction with a size of 1.8-2.2 kb was separated from the gel and subsequently the gel slice was digested with β-agarase I according to the manufacturer's protocol (New England Biolabs Ine, Beverly, MA, USA) and the DNA was precipitated. A modified pUC19 vector (designated pStuc) was used to clone the 1.8-2.2 kb fraction. In this vector, the BamHI / Sall fragment from pUC19 was replaced by a DNA fragment containing a restriction site Stul, Spel and Salí using two oligonucleotide primers and standard cloning techniques as described by Sambrook et al. (in: Molecular Cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press). The 1.8-2.2 kb fraction was ligated at 16 ° C in a Stul digested vector and a dephosphorylated pStuc vector. The ligation mixture was consequently transformed into Epicurian Coli XL2-Blue MRF 'ultracompetent cells (Stratagene, La Jolla, CA, USA). Individual colonies were grown and stored in 384-well microtiter plates (100 μl of LB medium containing 100 mg / l of carbenicillin). To isolate clones represented the overlapping DNA region in the cosmids Mi-11 and Mi-18 containing the resistance gene Mi, the fragment of 8.6 and 4.5 kb Psil of the cosmid clone Mi-18 (see Figure 4) as well as the AFLP marker, PM14, were used as hybridization probes in colony hybridizations. Therefore, replicas of the 384 cavity grid of clones in microtitre plates were stamped onto Gene Screen Plus membrane filters (DuPont NEN, Boston, MA, USA) and allowed to develop in colonies on the media. 84 positive clones were used to isolate the plasmid DNA using the alkaline lysis method as described by Ish-Horowicz et al. 1981, Nucí. Acids Res. 9, 2989-2997.
Sequence Analysis
The reaction set was used for ABI PRISM dye terminator cycle sequencing to perform sequencing reactions in a Gene-Amp PCR model 9600 system (Perkin-Elmer, Foster City, CA, USA). Standard M13 forward and reverse initiators were used. The reaction products were analyzed on 48 cm gels of an ABI 377 prism. The DNA sequence of 84 selected clones was determined using the standard forward and reverse sequencing primers. The sequence and analysis assembly was made with the 1994 version of the sequence analysis program STADEN (Dear and Staden, 1991, Nucí Acids Res. 19, 3907-3911). A contiguous DNA sequence of about 9.9 kb nucleotides could be formed and is shown in Figure 5. A large open reading frame of 3621 nucleotides (ORF2) could be deduced by encoding a truncated polypeptide and 1206 amino acids (Figure 7B).
EXAMPLE 12 INFECTION WITH NEMATODES IN VEGETABLE ROOTS: SOIL INOCULATION
Soil infected with the plant root nematode
Meloidogyne incognito was prepared as follows: root systems were cut from highly infected tomato plants with a large number of gills (or vegetable roots), in pieces and mixed in the fresh soil. Seeds were planted or small-rooted seedlings were transfected into infected soil. The plants were grown in a greenhouse at a temperature of 25 ° C. After 4 to 8 weeks, the plants were carefully removed from the soil and the roots were rinsed with water in order to remove the adhering soil. The level of infection was determined by counting the number of galls formed. The plants were considered resistant when three or less galls were visible in the roots. The plants were considered susceptible when more than three galls were formed in the root system.
EXAMPLE 13 COMPLEMENTATION ANALYSIS
Identification of cosmids with the resistance gene Mi classifying the resistance in the combined progenies of the transformed plants. The primary regenerators (generation R0) of the transformation experiments were developed in the greenhouse for seed fixation. For each cosmid, 10 to 15 regenerators were developed and RT seeds were harvested. The R-Í lines of at least 7 R0 plants of each cosmid were tested for resistance against Meloidogyne incognita in order to identify cosmids with the resistance gene. 20 of 30 plantings or seedlings of each R-line were inoculated and evaluated as described in Example 12. In 63 Ri lines of 7 different transformations of cosmids were tested: 6 cosmids carrying the DNA of tomato insert and one cosmid, pJJ04541, without the tomato insert DNA. The results are shown in Table 2. Fifty-four transgenic R0 plants appeared to be susceptible, since galls were formed in the root systems of all tested R ^ plants. Nine R0 plants were considered resistant, since at least half of the plants of each Ri line had 3 or less galls. A R ^ line was completely resistant, 6 R ^ lines were segregated in a ratio of approximately 3: 1 (resistant to susceptible seedlings), and the progenies of two R0 plants segregated 1: 1. The nine resistant R0 plants were derived from transformations with the cosmid Mi-11. Additional genetic evidence for the presence of the resistance gene Mi in the cosmid Mi-11 was obtained in the next generation. The resistant R-plants were combined. Fourteen of the resulting R2 lines, which originated from 4 different R0 plants, were tested for resistance against M. incognita. From 20 to 30 sows of each R2 line were inoculated and evaluated as described in Example 12. The results are shown in Table 3. Five R2 lines were completely resistant, indicating that the parent Ri plants were homozygous for the resistance Mi. Eight R2 lines were segregated in a 3: 1 ratio, indicating that their R-parent plants were heterozygous for the resistance gene Mi. An R2 line segregated in a ratio of approximately 1: 1, and none of the tested lines appeared to be fully susceptible. These results prove that the selected R-selected plants, which are derived from several plants transformed with the cosmid Mi-11, contain the functional Mi functional resistance gene.
TABLE 2
TABLE 3
EXAMPLE 14 INFECTION ASSAY FOR PFPA
Small tomato plants (4 weeks old) were inoculated with the potato aphid (Macrosiphum euphorbiae) by placing 5 to 8 female aphids on the leaves. The plants were grown in the greenhouse at a temperature of 18 to 20 ° C. After one to two weeks, the level of resistance was determined by counting the number of newly emerged aphids. The plants were considered resistant when no live aphids were present on the stem or leaves. Plants were susceptible when newly born aphids were present.
EXAMPLE 15 COMPLEMENTATION ANALYSIS
Identification of cosmids with the eu-1 resistance gene classifying for resistance in the combined progenies * of transformed plants. A subset of Ri lines obtained in Example 13 was tested for resistance against Macrosiphum euphorbiae in order to identify cosmids with the Mezv-1 resistance gene. From 10 to 15 seedlings of each R ^ line were inoculated and evaluated as described in Example 14. In total, 41 R ^ lines of 7 different transformations of cosmid were tested: 6 cosmids carrying the tomato insert DNA and a cosmid , pJJ04541, without DNA of tomato insert. The results are shown in Table 4. Thirty-six transgenic R0 plants were considered susceptible, since dozens of aphids proliferated in all or almost all plants of each R line. Five R0 plants are resistant, since at least half of the plants of each R-line were without live aphids. These 5 resistant R0 plants were transformed with the cosmid Mi-11. The results obtained strongly indicate that the Ro plants that are derived from transformations with the cosmid Mi-11, contain a functional Meu-1 resistance gene.
TABLE 4
Additional genetic evidence for the presence of the Meu-1 resistance gene in the cosmid Mi-11 was obtained in the next generation. Twenty-four R2 lines which were obtained from combinations of R-resistant plants to the nematode (see Example 13), which originated from 9 different plants R0, were tested for resistance against M. euphorbiae. From 11 to 15 sows of each line R2 were inoculated and evaluated as described in Example 14. The results are shown in Table 5. A line R2 segregated in a ratio of 3: 1 and 8 lines of R2 segregated in a ratio of about 1: 1. In these nine lines, the potato aphid resistance phenotype is clearly visible. Five R2 lines seemed to be completely susceptible. The remaining 10 R2 lines classified in intermediate form: segregated in a ratio of approximately 1: 3. These results indicate that several plants R ^ which are resistant to Meloldogyne incognita and which are derived from R0 plants transformed with the cosmid Mi-11, have a Meu-functional resistance gene. In addition, 8 RTBC lines that were obtained from R4 resistant nematode plants were crossed with the line of susceptible tomatoes 52201 and were tested for resistance against M. euphorbiae, in order to confirm the inheritance of the Meu-? Resistance gene. inserted. From 12 to 15 plantings of each R? BC line were inoculated and evaluated as described in Example 14. The results are shown in Table 6. The segregation relationships shown in Table 5 and Table 6 only serve to illustrate the inheritance of the resistance phenotype.
TABLE 5
TABLE 6
EXAMPLE 16 TRAINING OF TRANSCRIPTION MAP
Transcription map formation studies were performed to map the 5 'and 3' ends of the resistance gene Mi and to determine if the resistance gene Mi contains any intron. The polymerase chain reaction to amplify parts of the transcripts from the resistance gene Mi was used for this purpose. The total RNA from the leaf tissue of the resistant tomato culture E22 was isolated according to the hot phenol method as described by Sambrook et al (in: Molecular Cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press). Poly A + RNA was isolated using biotinylated oligo (dT) bound to Dynabeads M-280 Streptavidin (DYNAL A.S., Oslo, Norway) according to the manufacturer's instructions. A cDNA library was constructed using the Superscrpt Rnase H reverse transcriptase cDNA kit from Life Technologies, Inc. Gaithersburg, MD, USA and the protocol supplied by the manufacturer. The 5 'and 3' RACE products were obtained using the Marathon cloning amplification equipment from Clontech (Paolo Alto, CA, USA). The primers used were designated based on the My genomic sequence, and especially at the 5 'end of the ORF2 coding sequence. Subsequently, the various 5 'and 3'-RACE fragments were cloned into the TA PCRII cloning vector (Invitrogen Corporation, San Diego, CA, USA) and sequenced using standard protocols. The nucleotide sequences obtained were aligned with the 9.9 kb genomic sequence and two intron sequences could be deduced for the 5 'end of the Mi resistance gene. An intron of 1306 nucleotides was located from nucleotide position 1936 to 3241 and the second from nucleotide position 3305 to 3379 (Figure 5). The M major transcript detected with the Marathon cDNA amplification equipment mapped at the position of nucleotide 1800. Therefore, it is concluded that the transcriptional initiation site of Mi is located at or upstream of nucleotide 1880. The first ATG codon which can be detected within the 5 'cDNA was located at the position of nucleotide 3263, 52 nucleotides upstream of the second intron, and a large open reading frame (ORF1) encoding a polypeptide of 1257 amino acids could be deduced and shown in Figure 7A. As a result, this second intron is located between amino acid 14 and 15 of the resistance gene product Mi.
EXAMPLE 17 PCR ANALYSIS OF TRANSFORMED PLANTS MI-11 AND MI-18
The data obtained from the analysis of complementation in roots of transformed plants (Example 10) indicated that the resistance gene Mi was located in a DNA segment overlapping between the cosmids Mi-11 and Mi-18, excluding the segment of DNA corresponding to the cosmid Mi-30, transformants of which all were susceptible. This region was estimated to be 12 kb. However, in the complementation analysis in the combined progenies of transformed plants, only the plants transformed with the cosmid Mi-11 classified in resistant form (Examples 13 and 15). To address the question of why transformed Mi-18 plants became susceptible, a PCR analysis was performed in the presence or absence of the putative Mi-ORF in the Mi-11 and Mi-18 transformed plants. The following DNA samples were analyzed: 1. Clone YAC 2/1256.
2-3. Cosmid Mi-11 in E. coli and in A. tumefaciens, respectively. 4-5. Cosmid Mi-18 in E. coli and in A. tumefaciens, respectively.
6. Tomato line E22 (resistant). 7. Tomato line 52201 (susceptible). 8-12. Five plants transformed with the cosmid Mi-11. 13-17. Five plants transformed with the cosmid Mi-18. The DNA was digested with Psil and the Psil adapters were ligated. Subsequently, a PCR analysis was performed with an initiator identifying the Psíl site and the 3 additional selective nucleotides or PM14 marker and several PCR primers located upstream of PM14 using the rTh polymerase enzyme (Gene Amp XL PCR equipment, Perkin Elmer). The products generated ranged in size from 443 to 6,110 bp and span the upstream region of the complete PM14 of the putative Mi-ORF (see Figure 6). It seems that all templates generated PCR products of the expected size with the exception of the 5 plants transformed with the cosmid Mi-18. Only the smallest PCR product (443 bp) was formed. These data indicate that almost the entire upstream region PM14 was not present in plants transformed with the cosmid Mi-18. These deletions do not occur with the cosmid Mi-18 present in E. coli or A. tumefaciens, but occur only in transformed plants. Therefore, it is concluded that these eliminations are responsible for the phenotype susceptible to Meloidogyne incognita and / or Macrosiphum euphorbiae of transformed plants Mi-18.
EXAMPLE 18 NUCLEOTIDE SEQUENCE OF COSMEm MI-11
The observation that only plants transformed with the cosmid Mi-11 that showed a resistant phenotype may indicate that the optional open reading frames present in Mi-11 may be candidates for coding resistance against nematodes and / or aphids. Therefore, the nucleotide sequence of the region upstream of the postulated ORF1 was determined to identify additional open reading frames. A group of random subclones with an insert size of 2 kb were isolated using the fragment of 2.1, 4.7 and 2.9 kb Psil of the cosmid clone Mi-11 as hybridization probes in the colony hybridization essentially as described in Example 11 Forty-nine positive clones were used to determine the DNA sequence using standard forward and reverse sequence primers. Sequence assembly and analysis were performed as described in Example 11. Three stretches of contiguous DNA with sizes of 5618 bp
(con25), 898 bp (conlO) and 2495 bp (con62) could be detected. The gaps between these stretches of DNA and the DNA sequence of 9870 bp containing putative Mi-ORF (Figure 6) was calculated using PCR and varied between 50-200 bp. The three determined contigs (con25, con10 and con62) were analyzed for the distribution of stop codons in the 6 possible frames. No significant ORF framework with a size of or greater than 120 amino acids could be postulated. In addition, no DNA homology was detected with the putative ORF1. In this way, the only significant ORF present in the cosmid Mi-11 was ORF1 as described in Figure 5. Based on these results, it is concluded that the polynucleotide encoded by ORF1 confers resistance to nematodes, as well as to aphids and, therefore, that the resistance gene Mi and the resistance gene Meu-? they are named with the same coding sequence as presented in Figure 5.
EXAMPLE 19 TRANSFORMATION OF TOBACCO AND ANALYSIS OF
COMPLEMENTATION
Tobacco Transformation
The Petit Havana tobacco culture, type SR1, was transformed with the cosmid Mi-11 or the cosmid vector pJJ04541, using the protocol described by Horsh et al. (Science 227, 1229-1231, 1985).
Complementation Analysis: Classification for Nematode Resistance in Root Crops of Transformed Tobacco Plants
Roots of transformed R0 plants grown in vitro from tobacco were subjected to disease testing as described in Example 1. From each of the 31 transformants, two or more root explants were analyzed. In addition, the 17 Mi-11 transformants were analyzed by PCR for the presence of the putative MÍ-ORF1 by classifying an internal fragment with a size of 823 base pairs (varying from nucleotide position 4824 to 5646, see Figure 5). The simple PCR primers for the fragment were deduced from the sequence shown in Figure 5. The primers used had the following sequences: primer S21: 5'-CCAAGGACAGAGGTCTAATCG-3 'primer S22: 5'-TTGAGGTGATGTGGTAAATGG-3' initiator S21 has target in the sequence of nucleotide position 4824 A 4844 and initiator S22 has target in the sequence of nucleotide position 5626 A 5646 (see Figure 5).
The results of the in vitro disease test and the analysis
PCR (presence "+" or absence "-" of the internal PCR fragment) are shown in Table 7. "Mi-11" represents transformed plants comprising the putative MÍ-ORF1 and "MÍ-11?" represents those transformed plants that have a putative Mi-ORFI deletion, as determined by the PCR analysis (described above). Twenty-nine R0 transformants were susceptible, since the gills were formed in at least one of the proven root crops. Generally, the rate of gill formation in tobacco roots is slightly lower than in susceptible tomato roots. Two R0 plants were classified as resistant to Meloidogyne incognita, since no gills were found in the root crops. Both resistant plants were transformed with the cosmid Mi-11 comprising the internal PCR fragment indicating the presence of the resistance gene Mi.
TABLE 7
Genotype Fragment PCR Plants R Resistant Susceptible Mi-11 2 7 MÍ-11? 0 8 pJJ04541 0 14
Complementation Analysis: Classification for Resistance to Aphids in Cuts of Transformed Tobacco Plants
Root cuts from transformed R0 plants of tobacco were inoculated and evaluated as described in Example 14. From each of the 23 transformants, 2 or 3 cuts were analyzed for resistance against Macrosiphum euphorbiae. The results of the infection assay and the PCR analysis (as described above) are shown in Table 8. Twenty-one R0 plants were considered susceptible, since several live aphids were counted in at least one of the tested cuts. In general, the level of proliferation of aphids on tobacco is low compared with the proliferation in susceptible tomato plants. Two R0 plants were classified as resistant, since all the cuttings of these plants were without live aphids. The aphid-resistant plants were transformed with the cosmid Mi-11, comprising the resistance gene Mi, as indicated by the presence of the internal PCR fragment.
TABLE 8
Genotype Fragment PCR Plants R0 Resistant Susceptible Mi-11 2 3 MÍ-11? 0 6 pJJ04541 0 12
EXAMPLE 20 POTATO TRANSFORMATION AND COMPLEMENTATION ANALYSIS
Potato Transformation
The variety of potato Diamant (Cebeco Zaden B.V., Vlijmen, Holland) was used for the transformation. Explants between nodes of plants developed in vitro were transformed with the cosmid Mi-11 or the vector of cosmid pJJ04541 using the protocol described by Ooms et al. (Theor. Appl. Genet. 73, 744-750).
Complementation Analysis: Classification for Resistance of Nematodes in Root Crops of Transformed Plants
The roots of transformed R0 plants grown in vitro from potato were subjected to the disease assay as described in Example 1. From each of the 31 transformants, at least 2 root explants were analyzed. In addition, the 26 Mi-11 transformants were analyzed by PCR using the primers S21 and S22 as described in Example 19. The results of the in vitro disease assay and the PCR analysis (presence "+" or absence "-" of the internal PCR fragment) are shown in Table 9. "Mi-11" represents transformed plants comprising putative MÍ-ORF1 and "MÍ-11?" represents those transformed plants having a deletion in the putative MY-ORF1, as determined through PCR analysis (described above). Twenty-eight R0 transformants were susceptible, since galls were formed in at least one of the root crops. Generally, the rate of gall formation in potato roots is lower than in susceptible tomato roots. Three R0 plants were classified as resistant to Meloidogyne incognita, since no gills were found in the root crops. All these resistant plants were transformed with the cosmid Mi-1 comprising the internal PCR fragment indicating the presence of the resistance gene Mi.
TABLE 9
Genotype Fragment PCR Plants R (Resistant S susceptible Mi-11 3 17 MÍ-11? 0 6 pJJ04541 0 5
Complementation Analysis: Classification for Resistance of Nematodes in Cuts of Transformed Plants
Root cuts of transformed R0 plants were submitted
Mi-11 of potato to the disease trial as described in Example 12. For each of the 19 transformants, 1 to 3 cuts were analyzed for resistance against Meloidogyne incognita. The results are shown in Table 10. In addition, 36 root cuttings from untransformed potato plants (Diamant variety) were analyzed (as susceptible controls) and all were susceptible. A R0 plant was resistant to Meloidogyne incognita, since no gall was found in the root system.
TABLE 10
Genotype Fragment PCR Plants R0 Resistible Susceptible Mi-11 1 12 MÍ-11? 0 6 Control no-transf. 0 1
Claims (44)
1. A nucleic acid whose DNA sequence is the DNA of Figure 5 or part thereof or a DNA sequence homologous to the DNA sequence of Figure 5.
2. The nucleic acid according to claim 1, which is capable, when it is transferred to a host plant, which is susceptible to a plant pathogen, to render said host plant resistant to the plant pathogen.
3. A nucleic acid sequence, wherein it is a cDNA corresponding to a nucleic acid whose DNA sequence is at least part of the DNA sequence provided in Figure 5.
4. The nucleic acid according to claim 1 or 3, where, when transferred to a host plant, it is capable of making it resistant to nematodes.
5. The nucleic acid according to claim 1 or 3, wherein, when transferred to a host plant, it is capable of making it resistant to aphids.
6. The nucleic acid according to claim 1 or 3, wherein, when transferred to a host plant, it is capable of making it resistant to nematodes and aphids.
7. A nucleic acid wherein the DNA sequence corresponds to a sequence starting at nucleotide 3263 and ending at nucleotide 7111 of the sequence of Figure 5 or any DNA sequence homologous thereto.
8. A nucleic acid wherein the DNA sequence corresponds to a promoter sequence located 5 'upstream of nucleotide 3263 or any DNA sequence homologous thereto.
9. A nucleic acid according to claim 1 or 3, wherein the DNA sequence corresponds to at least part of the genomic insert present in the cosmid Mi-11, or any DNA sequence homologous thereto.
10. A recombinant DNA construct comprising a nucleic acid according to any of the claims 1-9.
11. A recombinant DNA construct according to claim 10, wherein the nucleic acid is under the control of a promoter that is functional in a plant cell, the promoter being either endogenous or exogenous to the plant cell, and effective to control the transcription of said DNA sequence in plant cells.
12. A recombinant DNA construct according to claim 11, wherein the promoter corresponds to a promoter sequence located upstream 5 'of nucleotide 3263 as provided in Figure 5, or any DNA sequence homologous thereto.
13. A suitable vector for transforming plant cells comprising a DNA construct according to any of claims 10-12.
14. Plasmid pKGMi-11 as deposited under the number CBS 822.96.
15. Plasmid pKGMi-18 as deposited under the number CBS 821.96.
16. Bacterial cells comprising a vector or plasmid according to any of claims 13-15.
17. The recombinant plant genome comprising, incorporated therein, a DNA construct according to any of claims 10-12.
18. Plant cells comprising a construction of DNA according to any of claims 10-12.
19. A plant comprising plant cells according to claim 18.
20. A plant according to claim 19, which has a reduced susceptibility to nematodes. The plant according to claim 20, wherein the nematode is a plant root nematode, especially Meloidogyne incognita. 22. The plant according to claim 19, wherein it has a susceptibility deduced to aphids, especially to Macrosiphum euphorbiae. 23. A seed comprising a DNA construct according to any of claims 10-12. 24. The recombinant plant genome according to claim 17, characterized in that it is in a plant cell environment. 25. A method for obtaining plants having a reduced susceptibility to a pathogen, comprising the following steps: (i) inserting into the genome of a plant cell or a DNA construct according to any of claims 10-12, (ii) obtain transformed plant cells, (iii) regenerate from the cells of genetically transformed plants, and (iv) optionally, propagate said plants. 26. The method according to claim 25, wherein the pathogen is a nematode, and preferably a plant root nematode, especially Meloidogyne incognita. 27. A process according to claim 25, wherein the pathogen is an aphid, and preferably Macrosiphum euphorbiae. 28. A method for protecting plants in the culture against infection by pathogens, which comprises: (i) providing the genome of plants with a DNA construct according to any of claims 10-12, and (ii) developing plants. 29. A method for isolating a nucleic acid according to claim 1-6, comprising the following steps: (i) classifying a genomic or cDNA library of a plant with a DNA sequence according to claim 1-9 , (ii) identify positive clones that hybridize to the DNA sequence, and (ii) isolate the positive clones. 30. The method according to claim 29, wherein said collection originates from a first plant and the DNA sequence belongs to a second plant. 31. A selective restriction fragment amplification method for identifying a nucleic acid according to claims 1-9, using primer combinations identifying at least one of the AFLP markers, PM02 to PM29, as described in Table 3 32. The method according to claim 31, wherein the initiator combination identifies the AFLP marker, PM14. 33. An oligonucleotide comprising a DNA sequence corresponding to at least a part of nucleic acid according to claims 1-9. 34. The oligonucleotide according to claim 33, characterized in that it is of sufficient size to selectively hybridize to the DNA sequence of any of claims 1 to 9 under severe hybridization conditions. 35. An oligonucleotide according to claim 34, wherein the DNA sequence corresponds to the sequence starting at nucleotide 6921 and ending at nucleotide 7034. 36. An oligonucleotide according to claim 35, wherein the sequence of DNA is located at the 3 'end, and preferably corresponds to the sequence 5'TGCAGGA-3', which can initiate DNA synthesis. 37. An oligonucleotide according to claim 35, wherein the DNA sequence is located at the 3 'end, and preferably corresponds to the 5'TAATCT-3' sequence, which can initiate DNA synthesis. 38. An initiator combination comprising a first oligonucleotide according to claim 36 and a second oligonucleotide according to claim 37. 39. A diagnostic kit comprising at least one oligonucleotide according to any of claims 33- 37 40. A diagnostic kit comprising an initiator combination according to claim 38. 41. A method for detecting the presence or absence of a DNA sequence according to claim 1-9, particularly in a plant DNA using a diagnostic kit according to claim 39 or 40. 42. A polypeptide which is the expression product of a recombinant DNA nucleic acid according to any of claims 1 to 13. 43. A polypeptide having a sequence of amino acid having the sequence provided in Figure 7A or encoded by the corresponding homologous sequence according to any of claims 1 to 3. 44. An RNA having a ribonucleic acid sequence of a transcription of part or all of the DNA sequence of any of claims 1 to 3.
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| EP96401764A EP0823481A1 (en) | 1996-08-09 | 1996-08-09 | Resistance against nematodes |
| EP97401101 | 1997-05-16 | ||
| EP97401101.7 | 1997-05-16 |
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| MXPA99001354A true MXPA99001354A (en) | 2000-02-02 |
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| EP (2) | EP0937155B1 (en) |
| JP (2) | JP4339404B2 (en) |
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| PT937155E (en) * | 1996-08-09 | 2005-03-31 | Keygene Nv | RESISTANCE AGAINST NEMATOIDS AND / OR AFIDS |
| WO1998058065A1 (en) * | 1997-06-18 | 1998-12-23 | Kirin Beer Kabushiki Kaisha | Mold-resistant plants and method of construction of same |
| GB9827152D0 (en) | 1998-07-03 | 1999-02-03 | Devgen Nv | Characterisation of gene function using double stranded rna inhibition |
| US7897843B2 (en) | 1999-03-23 | 2011-03-01 | Mendel Biotechnology, Inc. | Transcriptional regulation of plant biomass and abiotic stress tolerance |
| ES2226203T3 (en) | 1998-12-23 | 2005-03-16 | Idea Ag | IMPROVED FORMULATION FOR NON-INVASIVE TOPICAL APPLICATION. |
| FR2849863B1 (en) * | 2003-01-13 | 2008-02-22 | Genoplante Valor | GENE OF RESISTANCE TO APHIS GOSSYPII |
| PT1654276E (en) * | 2003-08-11 | 2013-03-25 | Kweek En Researchbed Agrico Bv | Fungus resistant plants and their uses |
| AR060563A1 (en) | 2006-04-24 | 2008-06-25 | Mendel Biotechnology Inc | DISEASE PROMOTERS FOR DISEASE |
| EP3574747A1 (en) | 2006-06-29 | 2019-12-04 | Mendel Biotechnology, Inc. | Improved yield and stress tolerance in transgenic plants |
| EP2471355A1 (en) | 2006-08-07 | 2012-07-04 | Mendel Biotechnology, Inc. | Plants with enhanced size and growth rate |
| WO2015002318A1 (en) * | 2013-07-05 | 2015-01-08 | タキイ種苗株式会社 | Root-knot nematode resistance marker for tomato plant, root-knot nematode resistant tomato plant, production method for root-knot nematode resistant tomato plant, and screening method for root-knot nematode resistant tomato plant |
| WO2022176971A1 (en) * | 2021-02-19 | 2022-08-25 | 国立大学法人 熊本大学 | Plant parasitic nematode controlling agent |
| CN113444740B (en) * | 2021-06-10 | 2022-08-09 | 上海交通大学 | Recombinant vector capable of improving soybean aphid resistance gene and application thereof |
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| GB9104617D0 (en) | 1991-03-05 | 1991-04-17 | Nickerson Int Seed | Pest control |
| ES2299229T3 (en) | 1991-09-24 | 2008-05-16 | Keygene N.V. | CREATORS, KITS AND SERIES OF RESTRICTION FRAGMENTS USED IN THE SELECTIVE AMPLIFICATION OF RESTRICTION FRAGMENTS. |
| GB9205474D0 (en) * | 1992-03-13 | 1992-04-29 | Cambridge Advanced Tech | Root knot nematode resistance |
| EP0736097A1 (en) | 1993-12-24 | 1996-10-09 | John Innes Centre Innovations Limited | Plant pathogen resistance genes and uses thereof |
| PT937155E (en) * | 1996-08-09 | 2005-03-31 | Keygene Nv | RESISTANCE AGAINST NEMATOIDS AND / OR AFIDS |
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