FIELD OF THE INVENTION

The present invention relates to resistance genes, DNA constructs, micro- organisms, plant cells and plants comprising said resistance genes. Furthermore 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 the resistance genes and diagnostic kits comprising said probes and/or primers. Finally, the invention relates to polypeptides encoded by said resistance genes and the use of said polypeptides.

BACKGROUND OF THE INVENTION

Plant pathogens are responsible for substantially losses of plants and plant products due to infection of the plant. Plant diseases, as a result of infection by plant pathogens or pests, cause damage to the plants and/or plant products, reduce production and yield, limit the kind of plants that can grow in certain geographic areas and as a result cause severe (financial) losses to the grower.

Plant parasitic nematodes occur worldwide and most of them live most of their life in the topsoil layer. Although losses caused by direct feeding of nematodes on plant roots is considered to be of minor importance, several species, among them the root-knot nematodes belonging to the Meloidogyne species, the cyst nematodes belonging to the Heterodera species and Globodera species and other nematodes such as the Nacobbus species, cause severe damage and economic crop losses. Root-knot nematodes also occur throughout the world but are found more frequently and in greater numbers in areas with warmer climates and in greenhouses. The most important Meloidogyne species are M. incognita, M. arenaha, M. hapla and M. javanica, of which M. hapla also occurs in more temperate climatic zones.

Different means for control of the plant pathogens exist, such as mechanical cultivation of the soil, chemical treatment with pesticides, including nematicides and insecticides, or crop rotation. However, for certain plant pathogens, especially nematodes, these means of control are insufficient to protect the plants from

infection and resulting diseases The only effective means of control involves plant host resistance (Russell, 1978, Plant Breeding for pest and disease resistance, Butterworths edit , 485 pp) The development of cultivars resistant to common plant pathogens is one of the major goals of plant breeders today, in order to reduce or ultimately eliminate the extensive need for pesticides The burden for the environment of the large amounts of pesticides injected into the soil or sprayed on crops, trees etc worldwide each year becomes too severe Moreover, governmental regulations in Western countries restrict the use or even forbid the use of certain pesticides Therefore, the need for plants which are resistant to one or more of their pathogens, or which have a reduced susceptibility to their attackers becomes more and more pressing The development of resistant plants is one of the important objectives of current plant breeding programs Plant genotypes susceptible for particular pathogens are crossed with resistant plant genotypes in order to introduce the resistant phenotype into the breeding line Damage by root-knot nematodes results primarily from the invasion of the plant roots by larvae which in a compatible relationship with the plant develop into a reproducing female After invasion the larvae cause root cells to develop into giant cells on which they feed Upon infection galls or knots are formed on the roots and the plant roots become otherwise disturbed, thickened and stunted The root system thus disfunctions in the uptake of water and nutritional elements which damages the plant growth and development Frequently damage to infected plants is increased by parasitic fungi attacking the weakened root tissue Infected plants show reduced growth and smaller pale coloured leaves, with dwarf poor quality fruits or even without fruits, and tend to wilt in warmer climates (Agπos, 1988 in Plant Pathology, Academic Press, Inc ) The damage and/or yield reduction caused by root-knot nematodes is substantial on the total agricultural production worldwide In individual stand yield losses can be as high as 25-50 %, or even a crop may be killed

In greenhouses root-knot nematodes can be controlled with steam sterilization of the soil or soil fumigation with nematicides Under field conditions control can be achieved by the use of nematicides However, the use of such, in

some cases very persistent, chemicals is increasingly debated and in some countries the use of certain nematicides is even forbidden.

Breeding genetically resistant genotypes is the most reliable and effective way of controlling root-knot disease. For a number of crop species the availability of resistance within the related germplasm has been reported, e.g. potato, cotton, tobacco, wheat, soybean, tomato, eggplant, common bean and alfalfa Resistance breeding is hampered by firstly the limited occurrence of (known) resistance genes in the available germplasm, secondly, in some plant species the existence of crossing barriers between the cultivated crop species and the resistance bearing related species, and thirdly, screening tests for resistance versus susceptibility to nematodes are laborious and often not reliable Therefore, resistance breeding is very difficult or not to achieve, or if possible time consuming

Successful introduction of resistance genes has been realized in tomato. The resistance gene Mi (Meloidogyne incognita) has been introduced into cultivated tomato, Lycopersicon esculentυm, after crossing with the related wild species L peruvianum (PI 128657), using embryo culture. The Mi gene confers resistance to various Meloidogyne spp. (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 tomato chromosome 6 It is also postulated that the introgressed region comprising the Mi locus is involved in conferring resistance to potato aphid (Macrosiphυm euphorbia) (Kaloshian et al, 1995, Proc Natl. Acad. Sci. USA, 92, 622-625)

Plants have developed a complex defense mechanism against attack and infection by pathogens To date, the exact mechanism of their defense system is not yet elucidated.

Nematode resistance in tomato is expressed after penetration. After the juvenile larva enters the root and establishes itself at a feeding site, a hypersensitive reaction (HR) adjacent to the head of the nematode is triggered that results in local death of the host cells The nematode is also adversely affected by this HR and dies (Fassuliotis, 1991 , in ^' Genetic Improvement of Tomato, Springer Verlag edit.).

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Wether or not there exists a gene-for-gene relationship sensu Flor (1956, Adv.Gen 8, 29-54) as is frequently the case in other plant-pathogen relationships where resistance is based on HR-incompatibi ty is unknown

The isolation of plant genes without knowing their gene products is very laborious and difficult, because of the enormous genome sizes of plant species: e.g tomato has a genome size of 1000 Mb (10 ⁹ base pairs of nuclear DNA), maize has a genome size of 3000 Mb and wheat has even more than 16 x 10 ⁹ base pairs Searching for a specific gene among these billions of base pairs is only feasible when (i) there are enough molecular markers tightly linked to the gene of interest and (n) there is good genetic mateπal available (Tanksley et al., 1995, Trends in Genetics, 11, p 63-68)

Although, the isolation of a few resistance genes has been reported, none of these resistance genes are able to confer the host plant resistant to nematodes or to aphids Examples of such isolated resistance genes are RPS2 from Arabidopsis (resistance to Pseudomonas synngae expressing avrRpt2), N from tobacco (resistance to tobacco mosaic virus), Cf-9 from tomato (resistance to the leaf fungal pathogen Cladosponum fulvum carrying avr9) and L ⁶ from flax (resistance to the corresponding leaf rust fungal race) (Dangl, 1995, Cell 80, 363-366).

The present invention provides the first isolated nematode resistance gene, and furthermore, provides the first isolated aphid resistance gene Moreover, the present invention relates to a dual function resistance gene conferring reduced susceptibility to nematodes as well as aphids, and preferably to Meloidogyne incognita and Macrosiphum euphorbiae respectively

SUMMARY OF THE INVENTION

The present invention relates to a nucleic acid comprising the Mi resistance gene which when present and expressed in a plant is capable of conferring to said plant resistance against nematodes and/or aphids Furthermore, the invention relates to the Mi resistance gene of which the DNA sequence is disclosed herein. The invention also relates to a gene product encoded by the Mi resistance gene. In addition, the present invention relates to DNA constructs, cosmids, vectors, bacterial

s- strains, yeast cells and plant cells comprising the Mi resistance gene 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 untransformed plant Furthermore, the invention relates to resistance genes which are homologous to the Mi resistance gene, and which, when present in a plant, are able of conferring said plant resistance to infection by pathogens

Moreover, the present invention relates to a nucleic acid comprising the Meu-1 resistance gene which when present in a plant is capable of conferring to said plant reduced susceptibility to aphids In particular the Meu-1 resistance gene corresponds to the Mi resistance gene Especially the Meu-1 resistance gene has the same nucleotide sequence as the Mi resistance gene Thus, the present invention also relates to genetically transformed plants, which are reduced susceptible, and preferably resistant to aphids, in particular to potato aphids Finally, the invention relates to oligonucleotides corresponding to the sequence of the said resistance gene or part thereof, and detection kits comprising said oligonucleotides

DESCRIPTION OF THE FIGURES 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 S 7I and SssHII restriction sites and the size of the restriction fragments are indicated The location of the various AFLP markers on the restriction fragments are indicated

Figure 2 shows a schematic drawing of the binary cosmid vector pJJ04541 which is used to construct a cosmid library of YAC 1/546 Plasmid pRK290 (20 kb large) (Ditta et al, 1980, Proc Natl Acad Sci USA, 77, 7347-7351 ) was used as starting vector "Tet" refers to the gene conferring resistance to tetracyclin "LB" signifies T-DNA left border repeat sequence, and "RB" signifies the right border repeat The cauliflower mosaic virus 35S promoter sequence is indicated by "p35S", and "ocs3"' indicates the octopine synthase 3" end "NPT" indicates neomycin phσsphotransferase, and "cos" refers to the bacteπophage lambda cos site enabling

έ in vitro packaging "pDBS" indicates the poly nker of pBluescπpt (Stratagene, La Jolla, CA, USA)

Figure 3A shows a schematic representation of the detailed position of the AFLP markers on YAC 1/1172, YAC 2/1256 and YAC 1/1084 Positioning is based on the cosmid contig constructed for the various defined regions

Figure 3B shows a schematic representation of the cosmid contig of the region comprising the Mi resistance gene The cosmids Mι-32, Mι-30, Mι-11 , Mι-18, Mι-01 and Mι-14 are represented by horizontal lines The location of the AFLP markers PM14 and PM25 is indicated Figure 4 shows a physical fine map of the cosmids Mι-32, Mι-30, Mι-11 , Mi-

18, Mι-01 and Mι-14 for the restriction enzyme Pst\ The size of the Pst\ fragments is indicated (in kb) The Mi phenotype, as identified in an in vitro disease assay, of the Ro plants comprising the various cosmids is indicated in the right end part of the figure The DNA segment of which the nucleotide sequence was determined is indicated by a double line with a bidirectional arrow

Figure 5 shows the nucleotide sequence of a DNA segment of approximately 99 kb around the AFLP marker PM14, and a deduced ammo acid sequence of the Mi resistance gene 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 a polypeptide of 1257 ammo acids (fig 7A) The Mi resistance gene comprises two intron sequences (shown in italics) one intron of 1306 nucleotides from position 1936 to position 3241 and one 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 into a second open reading frame (ORF2) encoding a truncated polypeptide of 1206 am o acids (figure 7B)

The position of the AFLP marker PM14 is from nucleotide position 6921 (5'- TGCAGGA-3') to nucleotide position 7034 (5'-AGATTA-3')

Figure 6 shows a physical map of cosmids Mι-11 and Mι-18 and the determined nucleotide sequence of cosmid Mι-11 The sequence is divided in four contigs con25 (5618 bp), con10 (898 kb), coπ62 (2495 bp) and Mi (9870 bp).

The lower part of the figure depicts the presence ("+") or absence ("-") of several PCR fragments, corresponding to parts of the DNA segment of Figure 5, which are represented as horizontal lines of different lengths at the right hand side of the table, in the various genetic backgrounds (YAC clone 2/1256, E coli containing cosmid Mi-1 1 , A. tumefaciens containing cosmid Mi-1 1 , E coli containing cosmid Mi-18, A. tumefaciens containing cosmid Mi-18, resistant tomato line E22, susceptible tomato line 52201 , R ₀ plants transformed with cosmid Mi-1 1 and R ₀ plants transformed with cosmid Mi-18).

Nucleotide sequence of cosmid Mi-1 1 and cosmid Mi-18. Analysis of different contigs.

Figure 7 A: shows the deduced amino acid sequence of the polypeptide encoded by ORF1 .

B: shows the deduced amino acid sequence of the truncated polypeptide encoded by ORF2. Figure 8 depicts a schematic representation of the structure of the Mi- resistance gene.

Figure 9 depicts a schematic representation of the Mi-resistance gene family.

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 to be given such terms, the following definitions are provided. nucleic acid: a double-stranded DNA molecule. The nucleic acid can be genomic DNA, cDNA, synthetic DNA or any other DNA; oligonucleotide: a short single-stranded DNA molecule; primers: in general, the term primer refers to a single-stranded DNA molecule which can prime the synthesis of DNA; nucleic acid hybridization: a method for detecting related DNA sequences by hybridization of single-stranded DNA on supports such as nylon membrane or nitrocellulose filter papers. Nucleic acid molecules that have complementary

base sequences will reform the double-stranded structure if mixed in solution under the proper conditions The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a support In a Southern hybridization procedure, the latter situation occurs, hybridization probe to detect a particular DNA sequence in the Southern hybridization procedure, a labelled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to a support such as nylon membrane or nitrocellulose filter paper The areas on the filter that carry DNA sequences complementary to the labelled DNA probe become labelled themselves as a consequence of the reanneahng reaction The areas of the filter that exhibit such labelling can then be detected according to the type of label used The hybridization probe is generally produced by molecular cloning of a specific DNA sequence or by synthesizing a synthetic ohgonucleotide, homologous sequence a sequence which has at least 50 %, preferably 60 %, more 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 more preferably 300 nucleotides and the length of sequences to be compared for polypeptides is generally at least 40 ammo acid residues, preferably 65 ammo acid residues and more preferably 100 ammo acid residues Alternatively, a homologous sequence refers to a sequence which can hybridize under stringent conditions to a particular sequence, and/or a DNA sequence coding for a polypeptide which has substantially the same properties as the polypeptide encoded by the particular DNA sequence, and/or a DNA sequence coding for a polypeptide having the same am o acid sequence as the polypeptide encoded by the particular DNA sequence and/or an ammo acid sequence in which some ammo acid residues have been changed with respect to the ammo acid sequence of the particular polypeptide without substantially affecting the major properties of said polypeptide,

3 stringent conditions refer to hybridization conditions which allow a nucleic acid sequence to hybridize to a particular sequence In general, high stringent conditions refer to the hybridization conditions which 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 1 M salt, preferably 6 x 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 having about 90 % or more sequence identity In general, lower stringent conditions refer to the hybridization conditions which 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 45 °C in a solution comprising 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 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength These conditions allow the detection of sequences having up to 50 % sequence identity The person skilled in the art will be able to modify these hybridization conditions in order to identify sequences varying in identity between 50 % and 90 %, promoter a transcription regulation region upstream from the coding sequence containing the regulatory sequences required for the transcription of the adjacent coding sequence and includes the 5' non-translated region or so called leader sequence of mRNA - terminator a region downstream of the coding sequence which directs the termination of the transcription, also called the 3' non-translated 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 corresponding or homologous sequence, - nematode(s) Meloidogyne spp such as Meloidogyne incognita, M arenaπa or M javamca or any other genotype which is not able to infect a host having

a resistance gene according to the invention, such as but not limited to other root-knot nematodes, such as M hapla, cyst nematodes such as Heterodera spp or Globodera spp , or other nematodes such as Nacobbus spp , insects, such as potato aphid or any other plant pathogen or pest, resistance gene product a polypeptide having an ammo acid sequence as depicted in Figure 5, or part thereof, or any homologous ammo acid sequence,

Ro plant primary regenerant from a transformation experiment, also denoted as transformed plant or transgenic plant,

R-i line the progeny of a selfed R ₀ plant

R ₂ line the progeny of a selfed R- plant

- R _T BC line the progeny of a backcross between a R- plant and a plant of the genotype which was originally used for the transformation experiment

In the present invention we have been able to identify and isolate the Meloidogyne incognita (Mi) resistance gene The gene was cloned from a tomato genotype which is resistant to Meloidogyne incognita The isolated Mi resistance gene according to the invention can be transferred to a susceptible host plant using Agrobacteπum mediated transformation or any other known transformation method, and is involved in conferring to the host plant resistance against plant pathogens, especially to nematodes The host plant can be tomato or any other genotype that is infected by said plant pathogen

The present invention provides also a nucleic acid sequence comprising the Mi resistance gene, which is depicted in Figure 5

With the Mi resistance gene according to the invention, one has an effective means of control against plant pathogens and/or pests, since the gene can be used for transforming susceptible plant genotypes thereby producing genetically transformed plants having a reduced susceptibility or being preferably resistant to a plant pathogen or pest In particular, a plant which is genetically transformed with the

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Mi resistance gene according to the invention has a reduced susceptibility to root- knot nematodes.

In a preferred embodiment the Mi resistance gene comprises the coding sequence provided in Figure 5 or any corresponding or homologous sequence or cDNA sequence, preceded by a promoter region and followed by a terminator region. The promoter region should be functional in plant cells and, preferably, corresponds to the native promoter region of the Mi resistance gene. However, it should be recognized that any heterologous promoter region can be used in conjunction with the coding sequences, as long as it is functional in plant cells. Preferably, a constitutive promoter is used, such as the CaMV 35 S promoter or T- DNA promoters, all well known to those skilled in the art. Furthermore, a suitable terminator region should be functional in plant cells all well known to those skilled in the art.

In addition the invention relates to the Mi resistance gene product which is encoded by the Mi resistance gene 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. Furthermore, the Mi resistance gene product or a truncated polypeptide as provided in figure 7B can be used for raising antibodies against it, which antibodies can be used for the detection of the presence of the Mi resistance gene product.

In another aspect of the invention, the Mi resistance gene can be used for the design of oligonucleotides which are complementary to one strand of the DNA sequence as described in Figure 5, or part thereof, which can be used as hybridization probes, being accordingly labelled to allow detection, for the screening of genomic DNA or cDNA libraries for homologous genes. Homologous sequences which can hybridize to the probe under stringent hybridization conditions, and which encode for a gene product that is involved in conferring reduced susceptibility or resistance to a plant against a plant pathogen which normally infects said plant, are comprised within the scope of the present invention. In another aspect o ^f the invention oligonucleotides are designed based on the

Mi resistance gene sequence, such that they can be used as hybridization probes in

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Southern analysis These probes can be used as molecular markers to distinguish plant genotypes having the resistance gene and plant genotypes lacking the resistance gene Such a probe can be used as an additional tool in selection In a preferred embodiment of the invention, oligonucleotides are designed based on the Mi resistance gene sequence, such that they can be used as primers in an amplification reaction, such as polymerase chain reaction (PCR), whereby the formation of an amplification product indicates the presence of the 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 closely linked to the Mi resistance gene In a preferred embodiment said primers are used in selective restriction fragment amplification to identify AFLP markers, which are. closely linked to the Mi resistance gene The invention also relates to diagnostic kits, comprising oligonucleotides according to the invention, for the detection of the presence or absence of the Mi resistance gene within a genotype under study Such a diagnostic kit circumvents the use of a laborious disease assay to screen for genotypes having the resistance gene or not

Furthermore the invention relates to DNA constructs comprising a DNA sequence corresponding to the coding sequence of the Mi resistance gene and regulatory sequences functional in plant cells, said DNA sequence can be genomic DNA, cDNA, synthetic DNA or DNA of any other origin Said regulatory sequences are either homologous or heterologous to the coding sequences of the Mi resistance gene Preferably, said DNA construct comprises a nucleic acid whose sequence is provided in Figure 5, or part thereof

The invention relates also to DNA constructs comprising the regulatory sequences, and more preferably the promoter region of the Mi resistance gene in conjunction with a structural gene sequence heterologous to said regulatory sequences

The invention relates also 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

Furthermore, cells harbouring a vector comprising a DNA sequence corresponding to the sequence as described in Figure 5 or part thereof, or homologous thereto, are within the scope of the invention. Moreover, cells carrying a DNA construct according to the invention, are within the scope of this invention In one preferred embodiment of the invention, a genetically transformed plant is obtained by introducing the Mi resistance gene within the genome of said plant, being susceptible to nematodes, using standard transformation techniques, wherein said genetically transformed plant is resistant to nematodes

In another embodiment of the invention, the Mi resistance gene can be transferred, using generally known transformation techniques, to a heterologous systems, such as but not limited to melon, tobacco, Arabidopsis thaliana, potato, sugarbeet, rapeseed cucumber, pepper, eggplant A heterologous system refers to a plant species which is different from the plant species from which the resistance gene was isolated In yet another embodiment of the invention, the Mi resistance gene corresponds to the Macrosiphum euphorbiae (Meυ-1) resistance gene, and is involved in conferring to plants, transformed with the gene according to the invention, resistance to insects and in particular to aphids

The DNA sequence comprising the Mi resistance gene as provided in the present invention has numerous applications of which some are described herein but which are not limiting the scope of the invention

The present invention will be further described in detail in view of the isolation of the Mi resistance gene present in tomato lines which are resistant to root-knot nematodes For the isolation of the Mi resistance gene we have used a map-based cloning (positional cloning) strategy, comprising the following steps

(1 ) identification of molecular markers linked to the Mi resistance gene,

(2) construction of a high molecular weight genomic YAC library,

(3) physical mapping of the molecular markers on the YAC clones and YAC contig building,

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(4) construction of a cosmid library of the YAC clones harbouring the linked molecular markers,

(5) physical fine mapping and cosmid contig building,

(6) genetic characterization of tomato mutants susceptible to root-knot nematodes,

(7) transformation of susceptible plants with the cosmids forming the contig,

(8) complementation analysis

For the identification of molecular markers, we have used the selective restriction fragment amplification technology, hereinafter also denoted as AFLP™ technology, which randomly amplifies a subset of DNA fragments out of a complex mixture of many DNA fragments and said amplified fragments generate fingerprints that can be analyzed In general, total DNA of different genotypes of the same plant species are subjected to the AFLP technology and the different AFLP fmgerpπnts obtained from the different genotypes are compared Fragments that are present in one genotype and absent in another genotype are polymorphic fragments and are denoted as AFLP markers The selectivity in AFLP reactions is obtained by using randomly chosen selective nucleotides at the 3' end of the PCR primers immediately adjacent to the nucleotides of the restriction enzyme site In an AFLP screening the DNA to be studied is subjected to different primer combinations The total amount of different primers that can be used is determined by the number of selective nucleotides that are added to the 3' end (4 primers with 1 selective nucleotides, 16 primers with 2 selective nucleotides, 64 primers with 3 selective nucleotides) If two different restriction enzymes are used than there are twice the amount of primers Those primers can be used in different combination If all possible combinations are used in an AFLP screening, than all the fragments present should have been amplified with one of the primer combinations (Zabeau and Vos, EP 0534858)

For the identification of AFLP markers linked to the Mi resistance gene different tomato lines were subjected to an AFLP screening In a first step, two sets of nearly isogenic lines for nematode resistance versus susceptibility were analyzed by AFLP fingerprinting using the following primers

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Pstf-pπmers 5'-GACTGCGTACATGCAGNN-3'

Mse/-pπmers 5'-GATGAGTCCTGAGTAANNN-3'

The N's indicate the variable selective nucleotides In the AFLP screening all

16 primers possible for the Psf/-pπmer and all 64 primers possible for the Mse\- pπmer were used on the two sets of nearly isogenic lines, giving a total of 16 x 64 = 1024 tested primer combinations Upon analysis of all the AFLP fingerprints a total of 30 candidate AFLP markers linked to the Mi resistance gene were identified These candidate markers were subsequently tested on a panel of nematode resistant and nematode susceptible tomato lines for confirmation and distance of linkage to the Mi locus The Mi resistance gene was introgressed in the cultivated tomato in 1944 from Lycopersicon peruvianum Modern nematode resistant tomato lines have been subjected to numerous cycles of crossing expected to result in a small introgressed region from Lycopersicon peruvianum with the Mi resistance gene Testing of the candidate AFLP markers on these modern tomato genotypes is expected to be a good test for assessing close linkage to the Mi locus A panel of 7 resistant and 11 susceptible tomato genotypes was tested with the 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 Mi linked AFLP markers

Next, four of the AFLP markers were screened on a high molecular weight genomic library The cloning of very large segments of DNA as large artificial chromosomes in yeast has become an essential step in isolating genes via positional cloning The cloning capacity of the YAC vector allows the isolation of DNA fragments up to one million base pairs in length The tomato line Lycopersicon esculentum E22, homozygous for the Mi locus, was used as source DNA to construct a YAC library We obtained a YAC library containing 3840 clones with an average insert size of 520 Kb, representing approximately 22 genome equivalents of the tomato genome Three positive YAC clones were obtained after the AFLP screening with the Mi linked AFLP markers 1/1084, 1/1172 and 2/1256. Subsequently, the presence of all Mi linked AFLP markers was determined in the 3

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YAC clones All markers appeared present in one or more of the 3 YAC clones, which allowed a first positioning of the various Mi linked AFLP markers The AFLP data indicated that the 3 YAC clones constituted an overlapping contig of approximately 1 4 Mb (see Figure 1 ) To determine the physical size of the Mi locus comprising the Mi linked AFLP markers and comprised in YAC clones 1/1084, 1/1172 and/or 2/1256 a long-range restriction map of the YAC contig was constructed This defined a DNA segment comprising the Mi locus of about 700 kb on which all the Mi linked AFLP markers 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 cannot be transformed into plant cells directly Therefore, a cosmid library was constructed of the yeast strain containing YAC 1/1172 and a cosmid library was constructed of the yeast strain containing YAC 2/1256 using cosmid vectors which are suitable for Agrobacteπum mediated transformation The size of this binary cosmid vector amounts 29 kb and is shown schematically in Figure 2. The cloning capacity of this binary cosmid vector, using phage lambda packaging extract is within the range of 9 to 24 kb Two banks of approximately 250,000 cosmid clones each were obtained from size fractionated yeast DNA The cosmid banks were screened by colony hybridization using as probes labelled restriction fragments of the YACs Positive cosmids clones were identified and in addition, the cosmids were grouped into seven defined regions covering the Mi region

In the following step the set of cosmids of the seven defined regions were fingerprinted 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 the Mi linked AFLP markers in this cosmid contig was determined A physical map of the DNA segment comprising the Mi resistance gene with the positions of the various Mi linked AFLP markers was obtained (see Figure 3)

A total of 96 overlapping cosmids together constituted the DNA segment comprising the Mi resistance gene Complementation analysis to identify the Mi resistance gene with such a large set of cosmids is a very laborious task Therefore,

the position of the Mi resistance gene on the cosmid contig was determined using mutant tomato lines These mutant lines are members from a family originating from a common ancestor and contained a wild-type (nematode resistant) Mi genotype but a mutant nematode susceptible phenotype Upon analysis with the set of Mi linked AFLP markers on a large number of these mutant lines three Mi linked AFLP markers appeared to be absent in most mutants These AFLP markers, therefore showed a good correlation between the AFLP Mi genotype and the Mi phenotype, in contrast to all other 17 AFLP markers Two of these AFLP markers, PM14 and PM25 were adjacent, and the region around these markers was assumed to be the most likely position for the Mi resistance gene A set of 6 overlapping cosmids defining a DNA segment of approximately 50 kb around AFLP markers PM14 and PM25 was selected for complementation analysis (see Figure 4)

The final step in the identification of the Mi resistance gene via positional cloning is the complementation of the corresponding susceptible phenotype The 6 cosmids from the candidate Mi region were introduced in Agrobacteπum tumefaciens through conjugative transfer in a tπ-parental mating The presence of the cosmid in the A tumefaciens strains was determined comparing various restriction enzyme patterns as well as DNA fingerprints from the A tumefaciens strains with the E.coli strain containing the cosmid Only those A tumefaciens cultures harbouring 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 cosmids Mι-32, Mι-30, Mι-11 Mι-18, Mι-01 and Mι-1 using standard transformation methods

Roots of in vitro grown transformed R ₀ plants were tested for disease symptoms in order to identify cosmids with the resistance gene Root explants were transferred onto solidified medium in petπ dishes and inoculated with ten galls from an axenic nematode culture of the root-knot nematode Meloidogyne incognita Disease symptoms are scored six weeks after inoculation A transgenic plant is considered resistant when no galls or one gall are visible on its root culture A transgenic plant is considered susceptible when at least two galls have been induced on its root culture The observations of the in vitro disease assay revealed

that 2 cosmids were able to complement the susceptible phenotype The presence of the AFLP marker PM14 in the resistant R ₀ plants indicated that the genomic insert present in cosmids Mι-11 and Mι-18 is also present in the R ₀ plants and is involved in conferring the R ₀ plants resistant to Meloidogyne incognita The primary regenerants (R ₀ plants) of the transformation experiments were grown in the greenhouse for seed set to obtain Ri lines which were tested for disease symptoms The disease assay is performed on seedlings Therefor, seeds are sown or small rooted plantlets are transferred into soil infected with Meloidogyne incognita and disease symptoms are scored 4 to 8 weeks after inoculation Plants are considered to be resistant when three or less galls are visible on the roots Plants are considered to be susceptible when more than three galls are formed on the roots The observations of the in vivo disease assay revealed that the resistant Ro plants are corresponding to cosmid Mι-11 transformants

In order to confirm the stable integration of the Mi resistance gene into the genome of the transgenic R ₀ plants, resistant plants of the R- lines were selfed and grown in the greenhouse for seed set to obtain R ₂ lines Seedlings of the R ₂ lines were subjected to an in vivo nematode disease assay The results obtained indicated the stable inheritance of the Mi resistance gene

Finally, the inserts in cosmids Mι-11 and Mι-18 were further characterized Sequencing analysis revealed a large open reading frame (ORF2) of 3621 nucleotides The DNA sequence is listed in Figure 5

The DNA sequence comprising the Mi resistance gene was further subjected to transcript mapping studies in order to determine the existence of intron sequences These transcripts mapping studies were performed according to generally known methods whereby genomic DNA sequences are compared with cDNA sequences The comparison of cDNA sequences and genomic sequences revealed the existence of two intron sequences in the Mi resistance gene One intron of 1306 nucleotides is located from nucleotide position 1936 to 3241 and a second intron of 75 nucleotides is located from nucleotide position 3305 to 3379, as is depicted in Figure 5 The position of the transcription initiation site is postulated at or upstream of nucleotide 1880 The first ATG initiation codon is located at nucleotide

position 3263 which is 52 nucleotides upstream of the second intron, giving a large open reading frame (ORF1 ) encoding a polypeptide of 1257 am o 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 high amount of leucme residues, region B comprises a nucleotide binding site motif, region C is the LRR region comprising 13 repeats with the following consensus sequence a-a-NL-L-a 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 falling within the scope of the present invention, genomic and cDNA libraries were screened with the coding sequence of the Mi resistance gene as a probe under stringent hybridization conditions Positive clones were isolated and used for complementation analysis Southern blot hybridizations on the YAC contig have been performed with an internal Psfl fragment of the coding sequence of the Mi resistance gene Three additional homologous regions could be identified two in YAC 1/1172 and one in YAC 1 /1084 Each region comprises 2 to 3 Mi homologues indicative of the fact that the Mi gene family is composed of about 10 to 12 members Surprisingly, aphid disease assays revealed that the R ₀ plants, transformed with cosmid Mι-1 1 , are resistant to Meloidogyne incognita as well as resistant to Macrosiphum euphorbiae, indicating that the genome insert present in cosmid Mi-11 is involved in conferring the R ₀ plants resistant to nematodes as well as involved in conferring the R ₀ plants resistant to aphids In particular, a plant which is transformed with the resistance gene according to the invention has at least a reduced susceptibility to one or more pathogens, especially to root-knot nematodes and/or aphids

In order to confirm the inheritance of the aphid resistance, (i) the previously obtained R- tomato lines which were derived from nematode resistant cosmid Mι-1 1 transformants, (n) the R ₂ lines derived from selfed nematode resistant R- plants and (in) R-BC lines obtained from nematode resistant R-

plants backcrossed with susceptible tomato line 52201 , were also tested for resistance against M euphorbiae The results obtained indicated the inheritance of the aphid resistance

Cosmid Mι-11 was used for the transformation of nematode susceptible genotypes of tobacco and potato, according to general known transformation methods Roots of in vitro grown transformed R ₀ plants of tobacco and potato were tested for disease symptoms as previously described herein The observations of the disease assay on the root cultures of the transformed plants indicated that the cosmid is involved in conferring to the transformed plants a reduced susceptibility to nematodes The resistance gene according to the invention has an effect in reducing the susceptibility of a heterologous plant species to nematodes, preferably to Meloidogyne spp , especially Meloidogyne incognita

Furthermore tobacco transformants were also tested for aphid resistance, and resistant R ₀ plants could be identified

The resistance gene according to the invention has a dual function and has an effect in heterologous systems

Cosmid Mι-11 has been deposited on August 5, 1996 as plasmid pKGMι-11 at Centraalbureau voor Schimmelcultures at Baarn, The Netherlands, under deposit number CBS 822 96

Cosmid Mι-18 has been deposited on August 5, 1996 as plasmid pKGMι-18 at Centraalbureau voor Schimmelcultures at Baarn, The Netherlands, under deposit number CBS 821 96

The following examples will provide a further illustration of the present invention which is nevertheless not limited to these examples

EXAMPLES

EXAMPLE 1 : DISEASE ASSAY

An axenic culture of the root-knot nematode Meloidogyne incognita is maintained on sterile roots of the tomato cultivar Moneymaker. The root cultures are grown on solidified B5 medium (Gamborg et al 1968, Experimental Cell Research 50 151- 158) with 2% saccharose and without hormones

Root explants (1-5 cm), derived from in vitro grown transgenic tomato plants are transferred onto the solidified B5 medium mentioned above to start root cultures. At the same time each root explant is inoculated with ten galls from the axenic nematode culture The galls are placed a few centimetres from the root explant The Petπ dishes with the roots and galls are incubated in the dark at 25°C After four to six weeks the level of infection is determined by counting the number of galls formed on the root cultures The evaluation for resistance/susceptibility to M incognita is as follows

A transgenic plant is considered resistant when no or less than two galls are visible on its root culture A transgenic plant is considered susceptible when at least two galls have been induced on its root culture

EXAMPLE 2: IDENTIFICATION OF AFLP MARKERS LINKED TO A DNA

SEGMENT COMPRISING THE Mi RESISTANCE GENE

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 screening was performed on two sets of nearly isogenic lines 83M-R (resistant) and 83M-S (susceptible), and Motelle (resistant) and Mobox (susceptible). The candidate markers resulting from this first screening were confirmed by a second screening on 7 M incognita resistant and 11 M incognita susceptible lines.

Two sets of nearly isogenic lines

1. 83M-R resistant De Ruiter Zonen C.V., Bergschenhoek, The Netherlands (hereinafter "De Ruiter")

2. 83M-S susceptible De Ruiter

3. Motelle resistant INRA, Montfavet, France

4. Mobox susceptible INRA, Montfavet, France

The 7 M. incognita resistant lines and 11 M. incognita susceptible lines for confirmation :

5. DR30 resistant De Ruiter

6. DR17 resistant De Ruiter

7. E22 resistant Enza Zaden, de Enkhuizer Zaadhandel

B.V., Enkhuizeπ, The Netherlands

(hereinafter "Enza Zaden")

8. E1 resistant Enza Zaden

9. DR6 resistant De Ruiter

10. DR10 resistant De Ruiter

11. 1872 resistant Royal Sluis B.V., Enkhuizen, The

Netherlands (hereinafter "Royal Sluis")

12. Moneymaker susceptible Agricultural University Wageningen

13. DR12 susceptible De Ruiter

14. DR23 susceptible De Ruiter

15. GT susceptible De Ruiter

16. RZ3 susceptible Rijk Zwaan Zaadteelt en Zaadhandel

B.V., De Lier, The Netherlands

(hereinafter "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 the DNA

Total tomato DNA from the 22 lines described above was isolated from young leaves as described by Bematzki and Tanksley (Theor. Appl Genet 72, 314-321). The typical yield was 50 - 100 μg DNA per gram of fresh leaf material. Template DNA for AFLP analysis with the enzyme combination Pst\-Mse\ was prepared as described by Zabeau and Vos (European Patent Application, EP 0534858), and is described briefly below

0.5 μg of tomato DNA was incubated for 1 hour at 37°C with 5 units Psfl and 5 units Msel in 40μl 10 mM Tπs HAc pH 7 5, 10 mM MgAc, 50 mM KAc, 5 mM DTT, 50 ng/μl BSA Next 10 μl of a solution containing 5 pMol Psfl-adapters, 50 pMol Msel- adapters, 1 unit T4 DNA-ligase, 1 mM ATP in 10 mM Tris HAc pH 7 5, 10 mM MgAc, 50 mM KAc, 5 mM DTT, 50 πg/μl BSA was added, and the incubation was continued for 3 hours at 37°C The adapters are depicted below

The structure of the Pst l-adap.er was

δ'-CTCGTAGACTGCGTACATGCA-S ^* 3'-CATCTGACGCATGT-5'

The structure of the Msel-adapter was

5'-GACGATGAGTCCTGAG-3' S'-TACTCAGGACTCAT-δ'

zv

Adapters were prepared by adding equimoiar amounts of both strands; adapters were not phosphorylated After ligation, the reaction mixture was diluted to 500 μl with 10 mM Tris.HCI, 0 1 mM EDTA pH 8.0, and stored at -20°C. The diluted reaction mixture is further referred to as template DNA

AFLP reactions

The primers used for the AFLP screening are depicted below:

Psfl-pnmers 5'-GACTGCGTACATGCAGNN-3' Msel-pπmers 5'-GATGAGTCCTGAGTAANNN-3'

The N's in the primers indicate that this part of the primers was variable In the AFLP screening all 16 possible primers were used for the Psfl-pnmer and all 64 possible primers were used for the Msel-primer. This gave a total of 16 x 64 combinations of Psfl- and Msel-pπmers, is 1024 primer combinations. All 1024 primer combinations were used in the AFLP screening for Mi linked AFLP markers. The AFLP reactions were performed in the following way

AFLP reactions employed a radio-actively labelled Psfl-pnmer and a non-labelled Msel-primer The Psfl-pnmers were end-labelled using (γ- ³³ P)ATP and T4 polynucleotide kinase The labelling reactions were performed in 50 μl 25 mM

Tris.HCI pH 7.5, 10 mM MgCI ₂ , 5 mM DTT, 0.5 mM spermιdine.3HCI using 500 ng oligonucleotide primer, 100 μCi (γ- ³³ P)ATP and 10 units T4 polynucleotide kinase.

For AFLP analysis 20 μl reaction mixture were prepared containing 5 ng labelled Psfl-pnmer (0.5 μl from the labelling reaction mixture), 30 ng Msel-primer, 5 μl template-DNA, 0.4 units Taq-polymerase, 10 mM Tris.HCI pH 8.3, 1.5 mM MgCI ₂ , 50 mM KCl, 0.2 mM of all 4 dNTPs AFLP reactions were performed using the following cycle profile, a 30 seconds DNA denaturation step at 94°C, a 30 seconds annealing step (see below), and a 1 minute extension step at 72°C. The annealing temperature in the first cycle was 65°C, was subsequently reduced each cycle by 07°C for the next 12 cycles, and was continued at 56°C for the remaining 23 cycles. All

amplification reactions were performed in a PE-9600 thermocycler (Perkin Elmer Corp , Norwalk, CT, USA)

Gel analysis of AFLP reaction products After amplification, reaction products were mixed with an equal volume (20 μl) of formamide dye (98% formamide, 10 mM EDTA pH 8 0, and bromo phenol blue and xylene cyanol as tracking dyes) The resulting mixtures were heated for 3 minutes at 90°C, and then quickly cooled on ice 2 μl of each sample was loaded on a 5% denaturing (sequencing) polyacrylamide gel (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 Trιs/50 mM Boric acιd/1 mM EDTA To 100 ml of gel solution 500 μl of 10% APS and 100 μl TEMED was added and gels were cast using a SequiGen 38 x 50 cm gel apparatus (Biorad Laboratories Inc., Hercules, CA, USA) Sharktooth combs were used to give 97 lanes on the SequiGen gel units 100 mM Tπs/100 mM Boric acιd/2 mM EDTA was used as running buffer Electrophoresis was performed at constant power, 110 Watts, for approximately 2 hours After electrophoresis, gels were fixed for 30 minutes in 10% acetic acid dried on the glass plates and exposed to FUJI phospho image screens for 16 hours Fingerprint patterns were visualized using a FUJI BAS-2000 phospho image analysis system (FUJI Photo Film Company Ltd, Japan)

AFLP screening for linked markers

An AFLP screening was performed using all possible 1024 Psfl-Msel primer combinations on the two sets of nearly isogenic lines The aim was to identify AFLP markers present in both resistant lines and absent in both susceptible lines. AFLP gels contained the AFLP fingerprints of 24 primer combinations of the 4 isogenic lines, giving a total of 43 gels A total of 30 AFLP markers were identified present in both resistant lines and absent in both susceptible lines These markers are referred to as candidate Mi linked AFLP markers Next, AFLP reactions were performed to determine the presence of the 30 candidate markers on the 7 resistant and 11 susceptible tomato lines Of the 30 candidate

2 markers 20 markers appeared to be present in the 7 resistant lines and absent in the 11 susceptible lines. These 20 markers were used in further studies to map the Mi resistance gene The primer combinations required to identify the 20 Psfl-Msel markers are depicted in Table 1. In the column with the primer combinations, "Ps l-" refers to the sequence 5'-GACTGCGTACATGCAG-3' and "Msel-" refers to the sequence 5'-GATGAGTCCTGAGTAA-3'. For example, marker PM14 can be identified using the Psfl-pnmer having the following sequence: 5'- GACTGCGTACATGCAGGA-3', and the Msel-primer having the following sequence: 5'-GATGAGTCCTGAGTAATCT-3.'

TABLE 1

marker primer combination with selective extensions (NN/NNN)

PM02 Psfl-AT /Msel-AAA

PM07 Psfl-AA/Msel-TAC

PM08 Psfl-CT /Msel-ACT

PM10 Psfl-CA /Msel-TCT

PM11 Psfl-TA /Msel-TGA

PM13 Psfl-GA /Msel-ATC

PM14 Psfl-GA /Msel-TCT

PM15 Psfl-GT /Msel-GAC

PM16 Psfl-GT /Msel-TCT

PM17 Psfl-AT /Msel-AAG

PM18 Psfl-AT /Msel-TAG

PM19 Psfl-GG /Msel-ATT

PM20 Psfl-TG /Msel-AAT

PM21 Psfl-TG /Msel-TTT

PM22 Psfl-TG /Msel-GCT

PM23 Psfl-GT /Msel-GAA

PM24 Psfl-AA /Msel-CTG

2?

PM25 Psfl-AC /Msel-GTG

PM27 Psfl-AA /Msel-CTA

PM29 Psfl-TA /Msel-GGA

EXAMPLE 3: CONSTRUCTION AND SCREENING OF A TOMATO YAC

LIBRARY Material

The tomato line Lycopersicon esculentum E22 (Enza Zaden) homozygous for the Mi locus, was used as source DNA to construct a YAC library Protoplasts were isolated from the leaves of in vitro shoots which were two to three weeks old as described by Van Daelen et al (Plant Mol Biol 12, 341-352)

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 plug The protoplasts embedded into the plugs were lysed with lysis mix (05 M EDTA, 1 % N-Laurylsarcosmate and 1 mg/ml protemase K, pH= 8 0) After lysis, the plugs were stored at 4°C in storage buffer (fresh lysis mix) until used Approximately 3 million protoplasts per plug, to obtain about 4 5 μg of chromosomal DNA were used for further studies Plasmid pYAC4 containing an unique EcoRI cloning site was used as cloning vector and the yeast strain AB1380 was used as a host (Burke et al, Science 236, 806-812).

YAC library construction

High molecular weight DNA isolation, partial digestion with EcoRI in the presence of EcoRI methylase, ligation of vector arms to genomic DNA, size selection by pulsed field gel electrophoresis and transformation of the yeast host was performed as described by Burke et al, (Science 236, 806-812) and Laπn et al, (Proc Natl Acad Sci

USA 88, 4123-4127).

All standard manipulations were carried out as described in Molecular cloning: a laboratory manual by Sambrook et al, (Cold Spring Harbor Laboratory Press).

3840 clones with a average insert size of 520 kb, which corresponds to 2.2 genome equivalents were finally obtained and the individual clones were stored in 40 96- wells micrσtiter plates containing 75 μl YPD solution (1% yeast extract, 2% peptone and 2% dextrose)

Screening YAC library

To reduce the number of samples handled, the cells of one 96-well microtiter plate were pooled (a platepool) and used for DNA isolation as described by Ross et al (Nucleic Acids Res , 19, 6053) The 2.2 genome equivalent tomato YAC library consists of 40 96-wells microtiter wells and as a result DNA of the 40 platepools were screened with the AFLP markers PM10, PM13, PM21 and PM25 using the AFLP protocol as described in Example 2 PM10, PM13, PM21 and PM25 were selected to screen the YAC platepools because these markers do not interfere with the background bands of the yeast strain AB1380 Three positive platepools out of the 40 were identified with these four AFLP markers as shown in Table 2. Subsequently, a secondary screening with the four AFLP markers (PM10, PM13, PM21 and PM25) of the 96 individual YAC clones of each plate was employed to find the correct address 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 of the identified markers PM02 to PM29 were present on one or more these three YAC clones (Table 3) The size of the YAC clone was determined by Pulse-field gel electrophoretic (PFGE) analysis using contour-clamped homogeneous electric field (CHEF, Chu et al Science, 235, 1582-1585) and appeared to be 470 kb (1/1084), 570 kb (1/1172), and 500 kb (2/1256) respectively

2J

TABLE 2

Platepool nr PM10 PM13 PM21 PM25 YAC detected (size in kb)

2 YAC 1/1172 (570 kb)

16 + YAC 2/1256 (500 kb) 4 + YAC 1/1084 (470 kb)

TABLE 3

Marker 1 /1 172 2/1256 1/1084

PM02 PM07 + PM08 + PM10 + PM11 + PM13 + PM14 + PM15 + PM16 PM17 + PM18 + PM19 + PM20 + PM21 PM22 + PM23 + PM24 +

So

PM25 - +

PM27 - +

PM29 - +

EXAMPLE 4: CONSTRUCTION OF A LONG RANGE PHYSICAL MAP OF

THE Mi YAC CONTIG AND LOCATION OF THE AFLP MARKERS

The 3 YAC clones 1/1172, 2/1256 and 1/1084 were subjected to partial digestion with increasing concentration of the restriction enzymes Sfi\ and SssHII. The samples were fractionated by PFGE, transferred to a Gene Screen Plus membrane (DuPont NEN, Boston, MA, USA) and assayed by hybridization using end-adjacent sequence probes according to the protocol for indirect end-label mapping as described by Burke et al (Science 236, 806-812) A physical map of YAC 1/1172, 2/1256 and 1/1084 for the enzymes Sfι\ and SssHII could be constructed as shown in Figure 1 The overlap between the various YAC clones was determined by Southern blot analysis using the obtained restriction fragments as a probe on digest of the three YAC clones A YAC contig with a size of 1 4 Mb could be constructed. In order to isolate the YAC fragments the digests were run on PFGE Digestion of YAC 1/1172 with Sfι\ resulted in two fragments (200 Kb and 370 Kb) Digestion of YAC 2/1256 with SssHII resulted in four fragments (40 Kb, 90 Kb, 110 Kb and 260 Kb) whereas digestion of YAC 1/1084 with SssHII gave two fragments with a size of 70 and 400 kb As a result the 1 4 Mb YAC contig could be dissected into 8 regions corresponding to the 8 restriction fragments obtained from the three YAC clones, covering the complete Mi region and adjacent sequences

To position the various AFLP markers within these 8 regions on the physical map, the AFLP markers were used as hybridization probes on the partial and complete Sffl and SssHII digests of YAC clones 1/1172, 2/1256 and 1/1084 Therefore, each AFLP marker fragment was excised from the dried gel and eluted by means of diffusion in a buffer containing 05 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH=8 0), 0 1% SDS, re-amplified with the corresponding

unlabelled AFLP primers and, subsequently labelled with 32 r P according to the random primer method of Feinberg and Vogelstein (Anal. Biochem. 132, 6-10). Each AFLP marker could be assigned to one or more of the eight regions as outlined in Table 4 and Figure 1

TABLE 4

YAC fragment Mi linked AFLP markers detected by hybridization

200 kb Sffl-fragmeπt 1/1172 370 kb Sfil-fragment 1/1172 PM14, PM16, PM21 260 kb SssHII-fragment 2/1256 PM10, PM11 , PM17, PM19, PM23, PM24,

PM29

90 kb SssHII-fragment 2/1255 PM07, PM27 110 kb SssHII-fragment 2/1256 PM08, PM13, PM14, PM15, PM20, PM22,

PM25

40 kb SssHII-fragment 2/1256 PM18 70 kb SssHII-fragment 1/1084 PM08, PM13. PM22 400 kb SssHII-fragment 1/1084 PM02, PM18

EXAMPLE 5: CONSTRUCTION OF A COSMID LIBRARY OF YAC CLONES 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 is based on plasmid pRK290 containing the tetracyclin resistance gene for selection in Escherichia coli and Agrobacterium tumefaciens. Into the unique EcoRI site of pRK290, T-DNA carrying sequences (LB; left border repeat, RB signifies the right border repeat) that flank the cos site of bacteriophage lambda

the neomycin phosphotransferase gene (Beck et al, Gene 19, 327-336) whose expression is driven by the cauliflower mosaic virus 35S promoter sequence (Odell et al, Mol Gen Genet 223, 369-378), and the pBluescπpt (Stratagene, La Jolla, California, USA) polylmker sequence. The size of pJJ04541 amounts 29 kb and is shown schematically in Figure 2 The cloning capacity of this binary cosmid vector, using phage lambda packaging extracts is within the range of 9 to 24 kb

Library construction Total DNA of the Saccharomyces cerevisae strain AB1380 containing YAC 1/1172 and total DNA of the Saccharomyces cerevisae strain AB1380 containing YAC 2/1256 was isolated using zymolyase to make protoplasts according to Green and Olsen (Proc Natl Acad Sci USA 87, 1213-1217) An aliquot of both DNAs was analyzed on PFGE. Both DNA isolates appeared to

Approximately 15 μg of each DNA was partially digested with Sau3A generating molecules with an average size of 15-25 kb Subsequently, the samples were centnfugated through a 10-35% sucrose gradient for 22 hours, 22 000 rpm at 20°C in a Beckman SW41 rotor 0 5 ml fractions were collected using a needle pierced through the bottom of the centrifuge tube An aliquot of these fractions was analyzed on a 07% agarose gel The fractions containing DNA molecules with a size of *20 kb were pooled and concentrated by ethanol precipitation

Subsequently, the cohesive ends were partially filled- with dATP and dGTP using the strategy of partial filling of 5'-extensιons of DNA produced by type II restriction endonuclease as described by Korch (Nucleic Acids Res 15, 3199-3220) and Loftus et al (Biotechniques 12, 172-176)

The binary cosmid vector pJJ04541 was digested completely with Xho\ and the linear fragment was partially filled-in 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 transduced to E. co// strain XL1-Blue MR (Stratagene, La Jolla, California, USA) using phage lambda

Gigapack II XL packaging extracts (Stratagene, La Jolla, California, USA) as recommended by the manufacturers. Selection was performed on LB (1% bacto- tryptone, 0.5% bacto-yeast extract and 1% NaCl, pH 7.5) agar plates containing 10 mg/l of tetracyclin. Two banks of approximately 250.000 cosmid clones per bank were made from 2-3 μg of size fractionated yeast DNA of YAC clones 1/1172 and 2/1256 respectively.

Subsequently, these transformants were stored into the wells of microtiter plates (96- wells, 100 μl of LB medium containing 10 mg/l of tetracyclin). Replicas of the 96-well grid of cosmid clones in microtiter plates were stamped onto Gene Screen Plus membrane filters (NEN Dupont) and allowed to grow into colonies on media. Colony hybridization, as described by Sambrook et al (in: Molecular cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press), using ³² P-labelled YAC clones 1/1172 and 2/1256 revealed positive cosmids. Of about 10.000 colonies of YAC 1/1172 approximately 200 positive cosmid clones were identified. Of about 20.000 colonies of YAC 2/1256 300 positive cosmid clones were identified.

EXAMPLE 6: FINE MAPPING OF THE Mi RESISTANCE GENE SEGMENT

AND POSITIONING OF THE AFLP MARKERS

Dividing 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 were hybridized with 7 of the 8 restriction fragments (YAC fragments) as outlined in Example 4 (see Table 4 and Figure 1 ). Positive cosmids for each of the 7 YAC fragments were identified. In addition, cosmids could be identified which reacted positively with the overlapping restriction fragments of the two different YAC clones.

Construction of a cosmid contig of the Mi resistance gene segment

In order to construct a cosmid contig of alt the positive identified cosmids 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 Eco I-pπmer 5'- GACTGCGTACCAATTC-3' having no selective nucleotides and a Msel-primer 5'- GATGAGTCCTGAGTAA-3' having no selective nucleotides according to the method as described in Example 2 The EcoPI-pπmer was labelled at the 5' end and all the 500 cosmids were amplified using EcoRI/Msel-pπmer set The DNA fingerprints contained about 8 to 20 amplified fragments Sets of cosmids containing amplified fragments of identical size were selected from each region and were rerun on polyacrylamide gels as described in Example 2 until a contiguous array of all the amplified fragments throughout the defined regions could be constructed In addition, the cosmid contig of one region was aligned with the adjacent regions in order to construct a cosmid contig of the Mi locus In this way a cosmid contig of 96 cosmids was constructed spanning the Mi locus of approximately 800 kb

Detailed positioning of the Mi linked AFLP markers on the cosmid contig In order to position the 20 Mi linked AFLP markers on the cosmid contig, the 96 cosmids were digested with Psfl followed by Southern blot analysis according to Southern, J Mol Biol 98, 503-515

The AFLP markers were used as hybridization probes as described in Example 4 on the Southern blot of the 96 Psfl digests of the cosmids The exact position of the Mi linked AFLP markers, except marker PM02, is outlined in Figure 3A

EXAMPLE 7: GENETIC ANALYSIS OF Mi MUTANTS

A family of mutant tomato lines was made available through Enza Zaden These lines were derived from a F, hybrid heterozygous for the Mi resistance gene and heterozygous for the Aps-1 gene (encoding acid phosphatase-1 ), which is very closely linked to Mi (Stevens and Rick, 1986, in The Tomato Crop, Atherton & Rudich edit , Chapman and Hall, p 35-109) Different alleles of the Aps-1 gene can be determined by isozyme analysis (Vallejos, 1983, in Isozymes in plant genetics and breeding, Tanksley and Orton edit , part A, Elsevier, Amsterdam, 469-515) The Aps- allele originates from L peruvianum and has been introgressed into several

3r nematode resistant tomato genotypes by co-segregation with the Mi resistance gene A scheme of these mutant lines is depicted below

Fi - hybrid (heterozygous Aps-1, Mi resistant phenotype) -i- selfed

F ₂ - lines (segregating Aps-1 1 2 1 , segregating Mi resistance 3 1 )

I selfmg of heterozygous (Aps- 1) F ₂ plants F ₃ - lines (segregating Aps-1 1 2 1 , segregating Mi resistance 3 1)

I selfmg of heterozygous (Aps- 1) F ₃ plants F - lines (segregating Aps-1 1 2 1 , segregating Mi resistance 3 1 )

I selfmg of heterozygous (Aps- 1) F ₄ plants F ₅ - lines (segregating Aps-1 λ 2 λ , Mι susceptible)

I selfmg of heterozygous (Aps- 1) F _s plants F ₆ - lines (segregating Aps-1 1 2 1 , Mi susceptible) •! selfmg of homozygous (Aps- 1 ^λ ) F ₆ plants

F ₇ - lines (all Aps-1 Mi susceptible) i selfmg of homozygous (Aps- 1 ) F ₇ plants F ₈ - lines (all Aps- Mi susceptible)

In the F-, F ₂ , F ₃ and F ₄ lines of this family the presence of the Aps-7 ¹ allele correlates with the Mi resistant phenotype, whereas absence of the Aps- allele correlates with the Mi susceptible phenotype In the F ₅ and subsequent progenies this correlation is lost all plants are susceptible to nematodes regardless of the Aps- 1 alleles Twenty individuals from each F ₂ , F ₃ , F _4l F ₅ , F ₆ , F ₇ and F _β generation were tested for nematode resistance, for presence of the Aps-1 allele and presence of the Mi linked AFLP markers Nematode testing of seedlings was performed in soil contaminated with root galls of M incognita The nematode resistance results were as indicated in the above scheme 3 1 segregation in F , F ₃ and F ₄ plants and susceptibility in F ₅ and progeny populations Most of the Mi linked AFLP markers indicated an identical Mi genotype as the Aps-1 isozyme marker However, 3 of the AFLP markers PM14,

PM16 and PM25 appeared to segregate with the Mi phenotype In most F ₅ , F ₆ , F ₇ and F ₈ plants the Mt susceptibility was indicated by the absence of these markers The AFLP markers PM14, PM16 and PM25 showed a correlation between the AFLP Mi genotype and Mi phenotype in the mutants Markers PM14 and PM25 are adjacent on 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 comprise the Mi resistance gene

EXAMPLE 8: PHYSICAL MAP OF THE OVERLAPPING COSMID CLONES COMPRISING THE Mi RESISTANCE GENE

The identification of cosmids hybridizing with the Mi linked AFLP markers PM14 and PM25 was performed in Example 6 PM1 identifies cosmids Mι-11 , Mι-18 and Mι-01 whereas PM25 identifies cosmids Mι-18 and Mι-01 Subsequently, a small cosmid array around cosmids Mι-11 , Mι-18 and Mι-01 was selected from the cosmid contig described in Example 6 A contig of 6 cosmids comprising the 3 identified cosmids and the adjacent cosmids, was selected These 6 cosmids are Mι-32, Mι-30, Mι-11 , Mι-18, Mι-01 and Mι-1 In order to make a physical fine map of these 6 cosmids, the DNA samples of the cosmid contig were digested with Psfl followed by electrophoresis on a 08%-agarose gel The physical overlap between the various cosmids could be determined Combining these data with the data obtained about the detailed positioning of the Mi linked AFLP markers on the cosmid contig (see Example 6) a physical fine map with the location of PM14 and PM25 could be constructed as shown in Figure 4 The cosmid contig around the AFLP markers PM14 and PM25 was calculated to be approximately 50 kb

EXAMPLE 9: TRANSFORMATION

Transfer of cosmids to Agrobacterium tumefaciens

The cosmid clones Mι-32, Mι-30, Mι-11 , Mι-18, Mι-01 , Mι-14 and the control cosmid pJJ04541 were introduced in Agrobacterium tumefaciens through conjugative transfer in a tπ-parental mating with helper strain HB101 (pRK2013) essentially

according to Deblaere et al (Methods in Enzymology 153, 277-292) E.coli were grown in LB medium (1 % bacto-tryptone, 0 5% bacto-yeast extract and 1 % NaCl, pH 7.5) supplemented with 5 mg/l tetracyclin at 37°C The helper strain HB101 (pRK2013) was grown under identical conditions in LB medium supplemented with 100 mg/l kanamycm sulphate

Agrobactenum tumefaciens strain AGL1 (Lazo et al, Bio/Technology, 9, 963-971 , 1991) was grown in LB medium supplemented with 100 mg/l carbenicillm at 28°C Overnight cultures were diluted 1 100 in LB medium without any antibiotics and after 6 hours of growth, 0 1 ml each of the Agrobacterium culture, the helper strain culture and a cosmid strain culture were mixed and plated on LB agar plates without antibiotics After overnight incubation at 28°C, the mixture was plated on LB medium agar plates containing 100 mg/l carbenicillm and 10 mg/l tetracyclin to screen for transconjugants Plates were incubated for 3-4 days at 28°C Two serial passages through selective agar plates were performed to select for single transconjugant Agrobacterium colonies

Characterization of A. tumefaciens transconjugants

Small-scale cultures were grown from selected colonies and grown in LB medium containing 10 mg/l tetracyclin Plasmid DNA was isolated by alkaline lysis using the method as described by Ish-Horowicz et al (Nucl Acids Res 9, 2989-2997, 1981 ), and digested with Sg/ll using standard techniques In addition, restriction fragment amplification on mmiprep DNA of A tumefaciens was performed using the enzyme combination EcoRI/Msel and primers having no selective nucleotide as described in Example 6 Subsequently, the Sg/ll restriction enzyme pattern as well as the DNA fingerprint of the A tumefaciens transconjugant were compared with those of mimprep DNA of the E coli strain containing the cosmid Only those A tumefaciens transconjugants harbouring a cosmid with the same DNA pattern as the corresponding E coli culture were used to transform a susceptible tomato line

3β

Transformation of a susceptible tomato line

Seeds of the susceptible tomato line 52201 (Rijk Zwaan, De Lier, The Netherlands) were surface-sterilized in 2% sodium hypochloπte for 10 minutes, rinsed three times in sterile distilled water, and placed on germination medium (consisting of half- strength MS medium according to 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 left to germinate for 8 days. Culture conditions were 25°C, a photon flux density of 30 μmol.m ^'2 .s ^'1 and a photopeπod of 16 /24 h. Transformation of tomato was performed according to Koomneef et al (1986), In: Tomato Biotechnology, 169-178, Alan R. Liss, Inc., and is described briefly below. Eight day old cotyledon explants were precultured for 24 hours in Petπ dishes containing a feeder layer of Petunia hybπda suspension cells plated on MS20 medium (culture medium according to Murashige and Skoog, (1962) Physiol. Plant. 15, 473-497 with 2% (w/v) sucrose and 0.8% agar) supplemented with 10.7 μM α- naphthaleneacetic acid and 4 4 μM 6-beπzylamιnopurιne. 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 Mι-14 or the cosmid vector pJJ04541 for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates Culture conditions were as described above. Overnight cultures of Agrobactenum tumefaciens were diluted in liquid MS20 medium (medium according to Murashige and Skoog (1962) with 2% (w/v/) sucrose, pH 5.7) to an O.D. ₆ ∞ of 0.8.

Following the cocultivation, the cotyledon explants were transferred to Petri dishes with selective medium consisting of MS20 supplemented with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulphate, and cultured under the culture conditions described above The explants were subcultured every 3 weeks onto fresh medium. Emerging shoots were dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots The formation of roots in a medium containing kanamycin sulphate was regarded as an indication of the transgenic nature of the shoot in question. Truly transgenic regenerants were propagated in vitro by subculturing the

33 apical menstem and auxiliary buds into glass jars with fresh selective medium without zeatin

EXAMPLE 10: COMPLEMENTATION ANALYSIS

Identification of cosmids with the Mi resistance gene by screening for resistance in roots of transformed plants

Roots of in vitro grown transformed R ₀ plants have been subjected to the disease assay as described in Example 1 From each transformant two root explants have been assayed In total 72 R ₀ plants of 7 different cosmid transformations have been tested, 6 cosmids carrying tomato insert DNA and one cosmid, pJJ04541 , is without tomato insert DNA The results are shown in Table 1 Sixty three transgenic R ₀ plants appeared susceptible because galls had been formed on at least one of the two root cultures Nine R ₀ plants scored resistant, because no galls could be found on the root cultures Seven resistant plants had been derived from transformation with cosmid Mι-11 , while two resistant plants had been derived with cosmid Mι-18, that is overlapping for a great part with cosmid Mι-11 The cosmids Mι-11 and Mι-18 were used for further molecular analysis

TABLE 1

Cosmid Ro plants

Resistant Susceptible

Mι-32 0 8 Mι-30 0 11 Mi- 11 7 4 MM 8 2 8 Mι-01 0 10 Mι-14 0 15 PJJ04541 0 7

Ho Molecular analysis of the transformed plants with a resistant phenotype

To demonstrate that the resistant phenotype of transgenic R ₀ plants, which had been with the overlapping cosmids Mι-11 and Mι-18, is determined by the genomic insert present in the various cosmids, an AFLP analysis with the AFLP marker PM14 was performed. Selective restriction fragment amplification was performed with the primer combination identifying marker PM14 for the R ₀ plants transformed with cosmids Mi- 11 and Mi-18. The DNA fingerprints obtained showed the presence of the marker PM14 in the resistant plants indicating that the genomic insert present in cosmids Mi- 11 and Mi-18 is also present in the R ₀ plants and that the two identified overlapping cosmids Mi-11 and Mι-18 comprise the Mi resistance gene.

The inserts in cosmids Mι-11 and Mι-18 and the inserts in the adjacent cosmids Mi- 32, Mι-30 on one side and cosmids Mι-01 and Mι-14 on the other side, were further characterized. The DNA region comprising the Mi resistance gene based on the overlap between the cosmids Mι-11 and Mi-18, was estimated at approximately 16- 18 kb. Based on the susceptibility of the R ₀ plants having the insert present in cosmid Mi-30, this region could be narrowed down to approximately 12 kb. A DNA segment comprising the Mi resistance gene, corresponding to the region flanked by the right ends of cosmids Mι-30 and Mι-11 , was sequenced (see Figure 4)

EXAMPLE 11: NUCLEOTIDE SEQUENCE AND DEDUCED AMINO ACID

SEQUENCE OF THE Mi RESISTANCE GENE FROM TOMATO

Subcloning of the overlapping DNA segment To determine the sequence of the overlapping DNA segment in cosmids Mi-11 and Mi-18 containing the Mi resistance gene, a set of random subclones with a insert size of approximately 2 kb were generated. 7.5 μg of CsCI purified DNA of cosmids Mi-11 and Mi-18 was sheared for 10 seconds at 4°C at 15% probe power (in 40 μl 10mM Tris-acetate, 10mM Mg-acetate and 50mM K-acetate) using a Misonix (Misonix Inc., Farmingdale, NY, USA) sonicator (type XL2020) with a water filled cup horn (type 431 A) 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 10μl of a repair mixture (10mM Tris-acetate, 10mM Mg-acetate, 50 mM K- acetate, 10U Klenow DNA polymerase, 10U T ₄ DNA polymerase and 2 mM of all 4 dNTP's) and followed by incubation for 30 minutes at 20°C. The sheared DNA was separated by electrophoresis on 1% Seakem GTG agarose gel (FMC Bio Products, Rockland, ME, USA). The fraction with a size of 1.8-2.2 kb was excised from the gel and subsequently the gel slice was digested with β-agarase I according to the protocol of the manufacturer (New England Biolabs Inc, 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 of pUC19 was replaced by a DNA fragment containing a StuI, Spel and Sail restriction site 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 the a StuI digested and dephosphorylated pStuc vector. The ligation mixture was subsequently transformed to Epicuriaπ Coli XL2-Blue MRF' ultracompetent cells (Stratagene, La Jolla, CA, USA). Individual colonies were grown and stored in 384-wells microtiter plates (100 μl of LB medium containing 100 mg/l of carbenicillin). To isolate clones representing the overlapping DNA region in cosmids Mi-11 and Mi- 18 containing the Mi resistance gene, the 8.6 and 4.5 kb Pstl fragment of 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-well grid of clones in microtiter plates were stamped onto Gene Screen Plus membrane filters (DuPoπt NEN, Boston, MA, USA) and allowed to grow into colonies on media.

Eighty four positive clones were used to isolate plasmid DNA using the alkaline lysis method as described by Ish-Horowicz et al. 1981 , Nucl. Acids Res. 9, 2989-2997.

Sequence analysis The ABI PRISM dye terminator cycle sequencing ready reaction kit was used to perform sequencing reactions in a Gene-Amp PCR system Model 9600 (Perkin-

Vz

Elmer, Foster City, CA USA) Standard M13 forward and reverse primers were used The reaction products were analyzed on 48 cm gels of an ABI Prism 377 The DNA sequence of 84 selected clones was determined using the standard forward and reverse sequencing primers Sequence assembly and analysis was done with the 1994 version of the STADEN sequence analysis program (Dear and Staden, 1991 , Nucl Acids Res 19, 3907-3911 ) A contiguous DNA sequence of approximately 99 kb nucleotides could be formed and is shown in Figure 5 A large open reading frame of 3621 nucleotides (ORF2) encoding a truncated polypeptide of 1206 ammo acids (figure 7B) could be deduced

EXAMPLE 12: ROOT-KNOT NEMATODE INFECTION: SOIL INOCULATION

Soil infected with the root-knot nematode Meloidogyne incognita had been prepared as follows root systems of heavily infected tomato plants with a high number of galls (or root-knots), were cut into pieces and mixed through fresh

Seeds were sown or small rooted plantlets were transferred into the infected soil

The plants were grown in a greenhouse at a temperature of 25°C After 4 to 8 weeks, the plants were carefully pulled out of the soil and the roots were rinsed in water in order to remove the adhering soil The level of infection was determined by counting the number of galls formed

Plants were considered to be resistant when three galls or less were visible on the roots Plants were considered susceptible when more than three galls were formed on the root system

EXAMPLE 13: COMPLEMENTATION ANALYSIS

Identification of cosmids with the Mi resistance gene by screening for resistance in the selfed progenies of transformed plants

The primary regeneraπts (R ₀ generation) of the transformation experiments were grown in the greenhouse for seed set For each cosmid, ten to fifteen regenerants were grown and Ri seeds were harvested Ri lines of at least seven Ro plants of each cosmid were tested for resistance against Meloidogyne incognita in order to identify cosmids with the resistance gene Twenty to 30 seedlings or plantlets of each R1 line were inoculated and evaluated as described in Example 12

In total 63 R- lines of 7 different cosmid transformations have been tested, 6 cosmids carrying tomato insert DNA and one cosmid, pJJ04541 , without tomato insert DNA The results are shown in Table 2 Fifty-four transgenic R ₀ plants appeared to be susceptible, because galls had been formed on the root systems of all tested R,-plants Nine R ₀ plants are considered resistant, because at least half of the plants of each R, line had three or less galls One R, line was completely resistant, six R, lines segregated in a ratio of about 3 1 (resistant to susceptible plantlets), and the progenies of two R ₀ plants segregated 1 1 All the nine resistant R ₀ plants had been derived from transformations with cosmid Mi- 11

Additional genetic evidence for the presence of the Mi resistance gene on cosmid Mι-1 1 was obtained in the next generation Resistant Ri plants were selfed Fourteen of the resulting R ₂ lines, which originated from four different R ₀ plants, were tested for resistance against M incognita Twenty to thirty seedlings of each R ₂ line were inoculated and evaluated as described in Example 12 The results are shown in Table 3 Five R ₂ lines were completely resistant, indicating that the parental Ri plants were homozygous for the Mi resistance gene Eight R lines segregated in a ratio of 3 1 , indicating that their parental Ri plants were heterozygous for the Mi resistance gene One R ₂ line was segregating in a ratio of about 1 1 , and none of the tested lines appeared to be completely susceptible These results prove that the selected R, plants, which are derived from several plants transformed with cosmid Mι-11 , contain the functional Mi resistance gene

^■ vv

TABLE 2

Cosmid Number of R, -lines of independent R ₀ plants tested

Total Segregation ratio R:S (resistant to susceptible)

1 :0 3:1 1:1 0:1

Mi-32 7 0 0 0 7

Mi-30 9 0 0 0 9

Mi-11 9 1 6 2 0

Mi-18 8 0 0 0 8

Mi-01 10 0 0 0 10

Mi-14 9 0 0 0 9 pKK04541 11 0 0 0 11

TABLE 3

Cosmid Number of R ₂ -lιnes of independent R- plants tested

Total Segregation ratio R:S (resistant to susceptible)

1 :0 3:1 1 :1 0:1

Mι-11 14 5 8 1 0

EXAMPLE 14: POTATO APHID INFECTION ASSAY

Small tomato plants (4 weeks old) were inoculated with the potato aphid (Macrosiphum euphorbiae) by placing five to eight 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 born aphids

Plants were considered to be resistant when no living 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 Meu-1 resistance gene by screening for resistance in the selfed progenies of transformed plants

A subset of the Ri lines obtained in Example 13 was tested for resistance against Macrosiphum euphorbiae in order to identify cosmids with the Meu-1 resistance gene Ten to fifteen plantlets of each Ri line were inoculated and evaluated as described in Example 14 In total 41 Ri lines of 7 different cosmid transformations have been tested, 6 cosmids carrying tomato insert DNA and one cosmid, pJJ04541 , without tomato insert DNA The results are shown in Table 4 Thirty-six transgenic R ₀ plants are considered susceptible, because dozens of aphids were proliferating on all or most plants of each Ri line Five R ₀ plants are resistant, because at least half of the plants of each Ri line were without living aphids All these five resistant R ₀ plants had been transformed with cosmid Mι-11

The obtained results strongly indicate that the R ₀ plants which are derived from transformations with cosmid Mι-11 , contain a functional Meu-1 resistance gene

TABLE 4 Cosmid Number of Rι-lιnes of independent R ₀ plants tested

Total Segregation ratio R S (resistant to susceptible)

1 0 3 1 1 -1 0 1

Mι-32 4 0 0 0 4

Mι-30 7 0 0 0 7

Mι-11 7 1 2 2 2

Mι-18 7 0 0 0 7

Mi-01 6 0 0 0 6

Mι-14 5 0 0 0 5 pJJ04541 5 0 0 0 5

Additional genetic evidence for the presence of the Meu-1 resistance gene on cosmid Mi-11 was obtained in the next generation. Twenty-four R ₂ lines that had been obtained from selfings of nematode resistant Ri plants (see Example 13), which originated from nine different R ₀ plants, were tested for resistance against M euphorbiae. Eleven to fifteen seedlings of each R ₂ line were inoculated and evaluated as described in Example 14. The results are shown in Table 5. One R ₂ line segregated in a ratio of 3:1 and eight R ₂ lines were segregating in a ratio of about 1 :1. In these nine lines the potato aphid resistance phenotype is clearly visible Five R ₂ lines appeared to be completely susceptible The remaining ten R ₂ lines scored intermediate- they segregated in a ratio of about 1.3 These results indicate that several Ri plants, which are resistant to Meloidogyne incognita and which are derived from R ₀ plants transformed with cosmid Mι-11 , have a functional Meu-1 resistance gene. In addition, eight RiBC lines that were obtained from nematode resistant

R _T plants backcrossed with susceptible tomato line 52201 were tested for resistance against M euphorbiae, in order to confirm inheritance of the introgressed Meu-1 resistance gene Twelve to fifteen seedlings of each R-iBC line were inoculated and evaluated as described in Example 14 The results are shown in Table 6

The segregation ratios shown in Table 5 and Table 6 only serve to illustrate the inheritance of the resistance phenotype.

TABLE 5

Cosmid Number of R ₂ -lιnes of independent Ri plants tested

Total Segregation ratio R:S (resistant and susceptible)

1 0 3:1 1:1 1:3 0:1

Mi-11 24 0 1 8 10 5

TABLE 6

Cosmid Number of RιBC-lιnes of independent Ri plants tested

Total 1 0 3 1 1'1 1:3 0 1

Mi-11 8 0 1 5 2 0

EXAMPLE 16: TRANSCRIPT MAPPING

Transcript mapping studies were performed to map the 5' and 3' end of the Mi- resistance gene and to determine whether the Mi resistance gene contains any introns The polymerase chain reaction to amplify parts of the transcripts from the Mi resistance gene was used for this purpose

Total RNA from leaf tissue of the resistant tomato cultivar 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 olιgo(dT) bound to Dynabeads M-280 Streptavidin (DYNAL A S , Oslo, Norway) according to the instructions of the manufacturer A cDNA library was constructed using the Superscript Rnase H Reverse Transcπptase cDNA kit from Life technologies, Inc. Gaithersburg, MD, USA and the protocol supplied by the manufacturer 5' and 3' RACE products were obtained using the Marathon cDNA amplification kit from Clontech (Paolo Alto, CA, USA) The primers used were designed based on the genomic Mi-sequence, and especially on the 5' end of the coding sequence of ORF2 Subsequently, the various 5' and 3'-RACE fragments were cloned into the TA cloning vector pCRII (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 One intron of 1306 nucleotides was located from nucleotide position 1936 to 3241 and the second one from nucleotide position 3305 to 3379 (Figure 5)

18

The largest Mi-transcript detected with the Marathon cDNA amplification kit maps at nucleotide position 1880 Hence, we conclude that the Mi transcπptional initiation site is positioned at or upstream of nucleotide 1880 The first ATG codon that could be detected within the 5' cDNA was located at nucleotide position 3263, 52 nucleotides upstream of the second intron, and a large open reading frame (ORF1 ) encoding a polypeptide of 1257 am o acids could be deduced and is shown in Figure 7A As a result, this second intron is located between ammo acid 14 and 15 of the M/-resιstance gene product

EXAMPLE 17: PCR ANALYSIS OF MI-11 AND MI-18 TRANSFORMED

PLANTS

Data obtained from complementation analysis in roots of transformed plants (Example 10) indicated that the Mi resistance gene was located on a DNA segment overlapping between cosmids Mι-11 and Mι-18, excluding the DNA segment corresponding to cosmid Mι-30, transformants of which were all susceptible This region was estimated to be about 12 kb However, in complementation analysis on the selved progenies of transformed plants, only cosmid Mι-11 transformed plants scored resistant (Examples 13 and 15) To address the question why Mι-18 transformed plants scored susceptible, a PCR analysis on the presence or absence of the putative Mi-ORF in transformed Mi- 11 and Mι-18 plants was performed The following DNA samples have been analysed 1 YAC clone 2/1256 2-3 Cosmid Mι-11 in E coli and in A tumefaciens, respectively 4-5 Cosmid Mι-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 cosmid Mι-11 13-17 Five plants transformed with cosmid Mι-18

^■ V3

The DNA was digested with Psfl and Psfl-adaptors were ligated Subsequently, a PCR analysis was performed with a primer identifying the Psfl site and three additional selective nucleotides or marker PM14 and various PCR primers located upstream of PM14 using the enzym rTh polymerase (Gene Amp XL PCR kit, Perkin Elmer) The products generated varied in size from 443 to 6110 bp and encompass the complete PM14 upstream region of the putative Mi-ORF (see Figure 6)

It appeared that all templates generated PCR products of the expected size with the exception of the five plants transformed with cosmid Mι-18 Only the smallest PCR product (443 bp) was formed These data indicate that almost the complete PM14 upstream region was not present in plants transformed with cosmid Mι-18 These deletions do not occur with cosmid Mι-18 present in E coli or A tumefaciens but occur only in transformed plants Hence, we conclude that these deletions are responsible for the susceptible phenotype to Meloidogyne incognita and/or Macrosiphum euphorbiae of Mι-18 transformed plants

EXAMPLE 18: NUCLEOTIDE SEQUENCE OF COSMID MI-11

The observation that only plants transformed with cosmid Mι-1 1 showed a resistant phenotype might indicate that additional open reading frames present on Mι-11 could be candidates to encode for 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 set of random subclones with an insert size of 2 kb were isolated using the 2 1 , 4 7 and 2 9 kb PstI fragment of cosmid clone Mι-11 as hybridization probes in colony hybridization essentially as described in Example 11 Fourty nine positive clones were used to determine the DNA sequence using the standard forward and reverse sequencing primers Sequence assembly and analysis was performed as described in Example 11 Three contiguous DNA stretches with sizes of 5618 bp (con25), 898 bp (con10) and 2495 bp (con62) could be deduced The gaps between these DNA stretches

s-o and the 9870 bp DNA sequence containing the putative Mi-ORF (Figure 6) was calculated using PCR and varied between 50-200 bp.

The three determined contigs (con25, conIO and con62) were analysed for the distribution of stop codons in all six possible frames. No significant ORF frames with a size of or superior to 120 am o acids could be postulated. In addition, no DNA homology with the putative ORF1 was detected. Hence, the only significant ORF present on cosmid Mι-11 was ORF1 as described in Figure 5. Based on these results, it can be concluded that the polynucleotide encoded by ORF1 confers resistance to nematodes as well as to aphids and, hence, that the Mi- resistance gene and the Meu-1 resistance gene are referring to the same coding sequence as depicted in figure 5.

EXAMPLE 19: TRANSFORMATION OF TOBACCO AND

COMPLEMENTATION ANALYSES

Transformation of tobacco

The tobacco cultivar Petit Havana, type SR1 , was transformed with cosmid Mi-11 or the cosmid vector pJJ04541 using the protocol as described by Horsch et al. (Science 227, 1229-1231 , 1985)

Complementation analysis: screening for nematode resistance in root cultures of transformed tobacco plants

Roots of in vitro grown transformed R ₀ plants of tobacco have been subjected to the disease assay as described in Example 1. From each of the 31 transformants two or more root explants have been assayed. In addition, all 17 Mi-11 transformants have been analyzed by PCR for the presence of the putative Mi ORF1 by screening for an internal fragment with a size of 823 base pairs (ranging from nucleotide position 4824 to 5646, see Figure 5). Simple PCR primers for the fragment were deduced from the sequence as shown in Figure 5. The primers used have the following sequences:

S7 primer S21 5'-CCAAGGACAGAGGTCTAATCG-3" primer S22 5'-TTGAGGTGATGTGGTAAATGG-3'

Primer S21 targets the sequence from nucleotide position 4824 to 4844 and primer S22 targets the sequence from nucleotide position 5626 to 5646 (see

Figure 5).

The results of the in vitro disease assay and of the PCR analysis (presence "+" or absence "-" of the internal PCR fragment) are shown in Table 7 "Mi-11" represents transformed plants comprising the putative Mi ORF1 and "Mi-11Δ" represents those transformed plants having a deletion in the putative Mi ORF1 , as determined by the PCR analysis (described above) Twenty-nine R ₀ transformants were susceptible, because galls had been formed on at least one of the tested root cultures Generally, the rate of gall formation on tobacco roots is slightly lower than on susceptible tomato roots Two R ₀ plants scored resistant to Meloidogyne incognita, because no galls could be found on the root cultures. Both resistant plants were transformed with cosmid Mι-11 comprising the internal PCR fragment indicating the presence of the Mi resistance gene

TABLE 7

Genotype PCR R ₀ plants fragment

Resistant Susceptible

Mi- 11 2 7

Mι-11Δ 0 8

PJJ04541 0 14

Complementation analysis: screening for aphid resistance in cuttings of transformed tobacco plants

Rooted cuttings of transformed R ₀ plants of tobacco were inoculated and evaluated as described in Example 14 From each of the 23 transformants two or three cuttings have been assayed 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 R ₀ plants are considered susceptible, because several living aphids were counted on at least one of the tested cuttings In general, the level of proliferation of the aphids on tobacco is low compared with the proliferation on susceptible tomato plants Two R ₀ plants scored resistant, because all cuttings of these plants were without living aphids. The aphid resistant plants were transformed with cosmid Mι-11 , comprising the Mi resistance gene, as indicated by the presence of the internal PCR fragment.

TABLE 8

Genotype PCR Ro plants fragment

Resistant Susceptible

Mi-11 + 2 3

Mι-11Δ - 0 6

PJJ04541 - 0 12

EXAMPLE 20 TRANSFORMATION OF POTATO AND

COMPLEMENTATION ANALYSES

Transformation of potato The potato variety Diamant (Cebeco Zaden B V , V jmen, The Netherlands) was used for transformation Internode explants of in vitro grown plants were transformed with cosmid Mι-11 or the cosmid vector pJJ04541 using the protocol as described by Ooms et al (Theor Appl Genet 73, 744-750)

Complementation analysis: screening for nematode resistance in root cultures of transformed plants

Roots of in vitro grown transformed R ₀ plants of potato have been subjected to the disease assay as described in Example 1 From each of the 31 transformants at least two root explants have been assayed In addition, all 26 Mι-11 transformants have been analyzed by PCR using primers S21 and S22 as described in Example 19 The results of the in vitro disease assay and of the PCR analysis (presence "+" or absence "-" of the internal PCR fragment) are shown in Table 9 "Mι-11 " represents transformed plants comprising the putative Mi ORF1 and "Mι-11Δ" represents those transformed plants having a deletion in the putative Mi ORF1 , as determined by the PCR analysis (described above) Twenty-eight R ₀ transformants were susceptible, because galls had been formed on at least one of the root cultures Generally, the rate of gall formation on potato roots is lower than on susceptible tomato roots Three R ₀ plants scored resistant to Meloidogyne incognita, because no galls could be found on the root cultures All these resistant plants were transformed with cosmid Mι-11 comprising the internal PCR fragment indicating the presence of the Mi resistance gene

ry

TABLE 9

Genotype PCR Ro plants fragment

Resistant Susceptible

Mi- 11 3 17

MM 1Δ 0 6 pJJ04541 0 5

Complementation analysis: screening for nematode resistance in cuttings of transformed plants

Rooted cuttings of Mι-11 transformed R ₀ plants of potato have been subjected to the disease assay as described in Example 12. From each of the 19 transformants one to three cuttings have been assayed for resistance against Meloidogyne incognita. The results are shown in Table 10. In addition, 36 rooted cuttings of non-transformed potato plants (variety Diamant) were assayed (as susceptible controls) and were all susceptible. One R ₀ plant scored resistant to Meloidogyne incognita, because no galls could be found on the root system.

TABLE 10

Genotype PCR R ₀ plants fragment

Resistant Susceptible

Mi- 11 + 1 12 Mi-11Δ 0 6 Non-transf. control 0 1