CA2227524A1 - Resistance against wilt inducing fungi - Google Patents

Abstract

The invention relates to a nucleic acid comprising the I-2 resistance gene which, when present and expressed in a plant, is capable of conferring said plant resistance against wilt inducing fungi. The DNA sequence is at least part of the DNA sequence provided in the figure or any DNA sequence homologous thereto. The invention also relates to a gene product encoded by the I-2 gene.

Description

W O 97/06259 PCTtEP96/03480 RESISTANCE AGAINST WILT INDUCING FUNGI

FIELD OF THE INVENTION
The present invention relates to ~esislance genes, DNA constructs, micro-oryar,is",!i, plant cells and plants co",prising said resistb"ce genes. Furthermore the invenl:ion relates to ye"etically lransrc"",ed plants which are resistant against wilt inducing fungi. In acldition, the invention relates to probes, and pri,~,er~ for the icJe,lliric;3~ion of the resislance genes and cliagnoslic kits c~",l~risi"g said probes and/or pril"er~. Finally, the invention relates to polypeptides encoded by said resistance! genes and the use of said poly,ueplides.

BACKGRC)UND OF THE INVENTION
Plant pdU,oye"s such as fungi are ,es~nsil)le for subslanlially losses of plants andl plant products due to i~l~ction of the plant. Plant ~i~e~ses, as a result of i"fection by plant pdU ,ogens, cause ddl "aye to the plants and~or plant products, reduce prod~ction and yield, limit the kind of plants that can grow in certain geoyrdph.:: areas and as a result cause severe (f,nancial) losses to the grower.Diffi rent means for control of the plant pdthcjgens exist~ such as ",echan:cal cultivation of the soil, chemical treatment with peslic;cles, including fungicides and inse~ti~;de.s, or crop ~~"ation. However, for certain plant pathogens~ especially soil bom fungi, these means of control are insufficient to protect the plants from inrectio" and resulting d i;ca-ses. The only effective means of control involves plant host r~sisl,3"ce (Russell, 1978, Plant Br*eclin~ for pest and dise~se resisla"ce, Butterworths edit., 485 pp). The dcv~lo~r"ent of cultivars r~sislaut to co"""on plant pathogens is one of the major goals of plant breeders today, in order to reduce or ulli",a~ely eliminate the extensive need for peslic;des. The burden for the environment of the large amounts of pesli~:des sprayed on crops, trees etc.
worldwide each year beco",es too severe. Morecwer, gove"""ental re~ 'ions in Western countries restrict the use or even forbid the use of certain peslic;des.Ther~fore, the need for plants which are resi~lanl to one or more of their ,catncgells, or which have a reduced susoeptibility to their attackers beco",es more and more pressing.

CONFIRMATION COPY

Plants have developed a complex defense mechanism against attack and infection by palhogel)s. In generai, their defense system is twofold, at the one hand it compriises a general resistance which is effective against dirrerenl pathogenspecies, and at the other hand, it consisls of a strong resistance against specific pathogen species. This latter resislance is generally based on a hypersensitivity reaction ~(HR). Although the exact defense rlle~nalliSIllS of the plant have still to be el~ ~cid~ted, it is assumed that when a p~ll ,os~en comes into contact with a host cell, an early event takes place that l~ er~ a rapid response that impedes further growth ol the pathogen and subsequent develop~enl of the disease. Frequently within the pathogen popu'~ion genotypes exist which can overcome this HR-resistance, so called races. If a host is resistant to a specific pall ,ogen race, but suscepli~lle to another race, one then speaks of rac~speciric r~sislance. It is furthermore believed that such a defense system in plant-pathogen interactions is based on a gene-for-gene relalionsl,i~ (Flor, 1956, Adv.Gen. 8, 29-54). In the gene-for~gene model it is posh ~ ed that for each gene conre~ g ~sblal~ce to thehost, there is a co"espond,"g gene in the patl)ogen that ~.,rer~ avirulence, andvice versa. Very recently, evidence on molec~ r level for the gene-for-gene cGncept nas been found with the cloning of a few plant resialance genes. The ,~sislance gene of a plant encodes for a product, a (ece~,lor mo'ecu'e, that can2 o recognize a product of the ~dtl ,ogen, an elicitor, encoded by a avirulence gene. If the reoeplor in~erd~ with the elicitor mcle~'e a h~er~ensiti~/e response is l,iggered, resulting into the destruction of the i,~ected plant cells and surrounding cells, and so preventing the multir~ tion and spread of the paU,ogen within the plant. This; postu'ated l l leol ,anis", has recently been confi" "ecl by the isol ~t or~ of a 2 5 few avirulence genes of the pathogen with the co"espor,di"g resislance genes of the host plant. Examples of such resistance genes are: RPS2 from Arabidopsis (resisldnce to Pseudomonas syringae ex~,ressing avrRpt2), N from lob~c~
(resislance to tol~Acw mosaic virus), Cf-9 from tomato (r~sis~anc~ to the leaf fungal patl ,ogen Cl~clDsponum fulvum carrying avr9) and L6 from flax (resistance to 3 o the co"esponding leaf rust fungal race) (Dangl, 1995, Cell 80, 36~366).

WO 97/06259 PCT~P96/03480 As stipu'~ted before the development of resistant plants is one of the important objectives of wrrent plant breeding plO9ldlllS. Plant genotypes susceptible for particular pdlhogens are crossed with resistd"l plant genotypes in order to introduce the resislant phenotype into the breecJing line. tlowever the5 breeding of resistance genes is resl,i~1ed due to several factors: (i) the limited occurrenc~ of (known) resi~lance genes in the available gerrnplasm (ii) in~i"palit,ility of uossing bet~veon difrere,)l speGies and (iii) the limited availability of reproducible and reliable d~5e~se: assays for certain ~,atl,ogens said assaysbeing a prerequisite in s~le~ion breeding.
Anongst the pdthogens causing serious dc",ages to plants one finds the group of soilbome cortical rots and vascular wilt inducing fungi such as Fusarium and Verticillium (Tollssolln 1981 in: Fusarium Dise~,es Biology and Ta~olloilly Nelson Toussoun & Cook edit. Penn. State Univ. Press 4~7 pp.). Said wilt inducing fungi infect the plants through the roots via direct penetlalion or via15 wounds after which the xylem vascular tissue of the plant is coloni~ed and sy",~lo,ns of i,l~ction with said fungi are wilting browning and dying of leavesfollowed Iby plant death. Entire plants or plant parts above the point of vascular invasion of the patl,ogen may die within a period of some weeks after i,lfection.
The fungi usually spread i"le"lally through the xylem vessels as mycelium or 2 o conidia until the entire plant is killed. Because of the fact that those fungi are able to survive! in the soil sap(ophytically they beco",e eslablished forever once they are intro~llced in the field. They are distributed more or less worldwide causing l~e",endolJs losses on most species of vegetables and flowers field crops fruit trees etc. Rec~u5e those fungi are so widesp~ead and so per~islenl in soils the 25 only effec:tive way of controlling said wilt inducing fungi is using ,esi~lant plant genotypes.
Most Fusarium species belong to the family of i",pe~ re~t fungi. This class of fungi is ~ Idrd~ri~e~ by the fact that only a vegela~i~e stage of the fungus is known. The generalive stage of those fungi has not been discovered yet. Due to 30 the overall classil,cation or ~onon,~ of fungi upon their ,no"~ gical ~;1 ,andl te~islics of the gene~dli~/e phase one should bear in mind that fungi belonging to the class of i"~l~el r~l fungi can be classified into another class once pCT~P96103480 their genera~i\/e stage is discovered and, subsequently, a change of clzssir,cation and name can follow (Gerlac~, 1981, in: Fusarium, Diseases, Biology and Taxonorny, Nelson, Toussoun 8~ Cook edit., Penn. State Univ. Press, p. 413426).
Moreover, it has been observed that some Fusarium species can "mutate" into 5 another species depending on the plant infe~led andlor the environment (Bolton &
Davidson, 1972, Can. J. Plant Sci, 52, 18g-196). Up to now most of the wilt inducing Fusarium belong to the species fusarium oxysporum. Di~rer~nt plant species are attacked by dirrei-~nt races or iso!-~es of Fusarium (Arll~sllung Al " ,slror,g, 1981, in: Fusarium, Dise~se~, Biology and Ta~or,on ,y, Nelson, Toocsolln & Cook edit., Penn. State Univ. Press, p. 391-399). However, some Fusarium isol-~es can infect ~itr~:renl plant speci~s Known Fusarium isol~~es L,re for example: Fusarium oxyspor~Jm f.sp. Iycopersid (tomato), F. oxysporum f.sp.
melonis (melon), F. oxysporum f.sp. bat?~t~s (sweet potato, lob~c~), f. oxyspoNmf.sp. cepae (onion), F. oxysporum f.sp. conglutinans (r~hL~ge, radish), F.
oxyspon~m f.sp. cubense (banana), F. oxysporum f.sp. l~as,r,~clu~m (cotton, alfalfa, soybean, ~Qh~C.CO) F. oxysporum f.sp. dianthii (ca",dlion), F. oxysporum f.sp.
chrysanthemi (chrysanthemum), F. oxysporum f.sp. tuber~si (potato), F.
oxysporum f.sp. cJ,-,la",i",s (cycla~"en), F. oxysporum f.sp. nicotianae (tob~cc~).
In the light of the prese"t invention it should be recoy,)i~ed that the name of 2 0 the wilt inducing fungi can cl ,ange in the future, but this will not effect the scope of the invention.
The isolation of plant genes without knowing their gene products is like looking for a needle in a haystack, bec~ Ise of the eno",~ous genome sizes of plant .speGes: e.g. tomato has a genon,e size of 1000 Mb (109 base pairs of nuclear DNA), maize has a genome size of 3000 Mb and wheat has even more than 16 x 109 base pairs. Sea~;t,ing for a specilic gene among these billions of base pairs is only feasibl~ when (i) there are enough moleall~r markers tightly linked to the gene of i,lteresl and (ii) there is good genetic male,ial available (Tanksley et al., 1995, Trends in Genetics, 11, p. 63-68).
SUMMARY OF THE INVENTION

W O 97/06259 PCT~P96/03480 The present invention relates to a nucleic acid co",p~isi"g the l-2 resistance gene which when p,esent and ex~,essed in a plant is capable of ~,)re"ing said plant resistant aga,"sl wilt inducing fungi. Fu,ll-en"ore the invention relates to the l-2 resislance gene of which the DNA sequence is disclosed herein. The inventions also relates to a gene product encoded by the l-2 resi~lanoe gene which is capable of l,iggering a hypersensitive response in the plant when it comes into contact with a gene product encoded by a co"esponcli"g avirulence gene of the plant pall-ogen. In addition the present invention relates to DNA constructs cosmids vectors bacterial strains yeast cells and plant cells c~,nprising the l-2 resistance gene. In another aspect the presenl invention relates to a genetically l~arIsrom~ed plant which is resistant to a wilt inducing fungus said fungus being capable of inr~ctir,~ the untransrolllled plant. Fu,ll,en"ore the invention relates to resisla"ce genes which are homo'~gQus to the l-2 r~sisLance gene and which when present in a plant are able of co"re"i"g said plant resislant to i"re~;lion by 1 5 pathogens.
Finally the invention relates to oligonucleotides col, esponding to the sequence of the l-2 r~sisla"ce gene or part thereof and detection kits comprisi"g said oligonucleotides.

2 o DES- ~lr I ION OF THE FIGURES
Fiqure 1 shows a sche",alic representalion of YAC 11546 with a size of 750 kb and the position of the BssHII Rsrl, Sfil and SgrA1 resl,i~lion sites (indicated by small lines). The l,atched bar represents the 255 kb SgrA1 r,ag",enl co",~,isingthe l-2 reci~lance gene. The most lower line repr~sents the size bar (in kb). The circle/arrowhead combination r~pr~senls the left arm of pYAC4 direction of the ce,,~,ulller Fiqure 2 shows a s~;l,e",alic drawing of the binary cosmid vector pJJ04541 whichis used to construct a cosmid library of YAC 1/546. Plasmid pRK290 (20 kb large)3 0 (Ditta et al 1980 Proc. Natl. Acad. Sci. USA 77 7347-7351 ) was used as ~lal lil ,9 vector. ~et refers to the gene con~" in~ r~sislance to tetracyclin. LB s,yn;lies T-DNA leR border repeat sequence and "RB" signihes the right border repeat. The cauliflower mosaic virus 35S ~r~moter sequence is indicated by "p35S" and "ocs3"' indicAtes the octopine synthase 3' end. "NPr' indicates neomycin phosphotransferase and "cos" refers to the bac leriophage lambda cos site 5 enabling in ~ro packaging. "pDBS" indic4tes the polylinker of pBluescript (SL,dlayene LaJolla CA USA).

Fiqure 3 shows part of a 4 ~% denaturing polyacrylamide gel with DNA r")~e(~li"ls of 24 cosmids using Re~ lion F(dg,nenl ~"~liricalio" with the enzyme 10 co",bi,-alion EcoRI/Msel. The templates used are depicted in the right part of the figure.

Fiqure 4 shows a sche",alic represe"~alio,l of the 255 kb SgrA1 r,ay",en~ with the position of tne Mtul and Sa/l restriction sites (ir,~ Atecl with small lines) and the posilion of the 18 AFLP markers EM01 to EM18 (indicAted with arrows). The cosmid contig of the DNA segment flanked by markers 18 and 12 is inclic~ted withho, i,un~al lines. The cosmic~s marked with an aslerisk are used in the complementation analysis. The upper line represenls the size bar (in kb) and thesizes of the Mlul and Sall res~ictiol~ r,dy",en~s are indi~le~l Fiqure 5 shows a schen,a~ic representalion of the overlapping coslll,Js A52 and B22 and partly of A55 and CC16 (the open arrow head indicates that the cosmid iscontinued). The posilion of the various resl, ic~ion sites is indicated with small lines.
The posilion of the AFLP markers EM05 EM14 and EM06 is in~licAted with an arrow. The DNA seg",enl of which the nuclectide sequence was determined is indic~1ed with a line with a Lidi,ectional arrow.

Fiqure 6 shows the n~ Ic~eotide sequence of a DNA segment of almost the completeoverlap between cosmids A52 B22 and A5~ and the decl~c~d amino acid sequence of the /-2 resislance gene. The initiation codon (ATG position 1798-1800) is underlined and the termination codon (TM posilion 5596 5598) is double underlined.

WO 97/06259 PCT~P96/03480 The position of the AFLP marker EM06 is from nucleotide position 3470(5'-AATTCJ~ 3') to nucleotide posilion 3565 (5'-AGATTA-3').
The positions of three intron sequences are given in italics: one intron of 86 nucleotides located upstream of the ATG initiation codon from nucleotide position 1703 to 1788 and two introns of respectively 399 and 82 nucleotides located downstream of the TM te~",inaliol- codon from respectively nucleolide posilion ~628 to 6026 and 6093 to 6174. The l,dns~ Jtional iniliation site is ~redicted to be located at least 201 nuc,~olides u~sl~~a", of the ATG i,lilialion codon. The L,ansaiptional termination site is predicted to be loc;ated at least 893 nucleotides do~"sl,ed"l of the TM te""inalion codon. A putative poly-adenylation signal (MUMA) is located at nucleotide position 6406 6411 and is given in bold.

Fiqure 7 shows a schematic drawing of plasmid pKG6016. "smlsp adt~' refers to the ~ tomycin/specti,)G")~cin resisLdnce gene the origin of replic~tion of pBR322 is in~ic~ed by "OnV'' and "bla" refers to the ampicilin rt:sislance gene."LB" sig"i~,es T-DNA left border repeat sequence and "RB" siyll;-les the right border repeat. The nopaline synthase p~o",oter sequence is indicated by "nos p~'and "nos :3"' indic~es the nopaline synthase 3' end. "nptll" indic~es the kanamycin resislance gene. "12-upstream" refers to the 1.3 kb DNA seS~Illen~ upstream of the 2 o coding se~uence of the 1-2 ,esislance gene "Fus12" refers to the coding sequence of the 1-2 resislance gene from nuc'ectide posilion 1798 to nucleotide position 5598 and "3' untrans" refers to the 1.1 kb DNA seylllent downsl~ea", of the c~ding sequence of the 1-2 resistance gene. The relevant r~sl, i~tion sites are indic~te-~

DETAILED DESCRIPTION OF T~E INVENTION
In the des~iption and e~d"l~,es that follow a number of terms are used herein. In order to provide a clear and consislent undersldndi, 19 of the specificdlion and claims including the scope to be given such temms the following definitions are provided.
- nucleic acid: a double-sl~dnded DNA Illo'~cule;
- oligonuclaotide: a short single-stranded DNA molecu'e;

- pri, ners: in general the temm primer refers to a single-stranded DNA
molecule which can prime the sy"U ,esis of DNA;
- nuclei~ acid hybri~ lion: a ~ethod for detecting related DNA sequences by hyL" jrli ~ on of single-sl~ anded DNA on supports such as nyion me",b,ane 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-s~, anded structure will be formed between two co",~le."entary singl~stranded nucleic acids even if one is immobilized on a support. In a Southem hyL,ri~ lion procedure the latter S jtl l~tion occurs;
- hyL~ridi~alion probe: to detect a particular DNA sequence in the Southern hyb,idi~alio,) procedure a labelled DNA rnc'~ulle or hybridization probe is reacted to the fractionated DNA bound to a support such as nylon ,n0,nb,ane or nitrocellulose filter paper. The areas on the filter that carry DNA sequences complementary to the labelled DNA probe become labelled Ule"-sElves as a ~nsequence of the reannealing rt:a~ion. The areas of the filter that exhibit such labelling can then be detected according to the type of label used. The hyL,ridi~alion probe is generally produced by molecular cloning of a specir,c DNA sequence or by s~"llhesi~iny a 2 o synthetic oligonucleotide;
- homo'~go!J.s sequence: a sequence which can hybridize under sl,i,lgen~
conditions to a particular sequence andlor a DNA sequence coding for a polypeptide which has the same properties as the poly~ptide encoJed by the particular DNA sequence and/or a DNA sequence coding for a poh~ep~ide having the same amino acid sequence as the poly~ep~ide encbded by the particular DNA sequence andlor an amino acid sequence in which some amino acid residues have been cl,anyed with respec~ to the amino acid sequence of the particular poly~epli~le without su6sla,)~ial effect on the major p,ope, lies of said polypeptide andlor a sequence which has at 3 o least 50 % p.~fe~ably 60 % more preferably 70 % most p~e~r&bly 80 % or even 90 % sequence identity with the particular sequence whereby the length of sequenoes to be compared for nucleic acids is generally at least WO 97/06259 PCT~P96J03480 120 nucleotides preferably 200 nucleoti~les and more prererably 300 nucleotides and the length of sequences to be co" ,pared for polypeptides is generally at least 40 amino acid resid~ ~es prefer~bly 65 amino acid residues and more preferdbly 100 amino acid resid~ ~es;
- p, c " ,o~er. a 1, ans~ iplion re~ t~on region upstream from the coding sequence co,)1a,ning the regulatory sequences required for the ll a, 's~ i~tion of the acljacenl coding sequence and includes the 5 non-translated region or so called leader sequence of mRNA;
- terminator: a region do~l,ea~ of the coding sequence which directs the termination of the 1,dns~i~1ion also called the 3 non-translated region which includes the poly-adenylation signal;
- resis1ar,ce gene: a nucleic acid having a coding sequence as depicted in Figure 6 or part thereof or any co"~sponding or IIG",G'~gous coding sequence;
15 - sl,ingenl condiliGns refer to hybri~ ion condi1iG"s which allow a nucleic acid sequence to hybridize to a particular sequence. In general high ~1,inge"1 condilions refer to the hyb,id;~ion conditions which allow a nucleic acid sequence of at least 50 nucleoticles and prt:ferably about 200 or more nl~cleotides to hybridize to a particular sequence at about 65 ~C in a solution co",~,rising about 1 M salt prefardbly 6 x SSC or any other solution having a co",par;atle ionic s1len!Jtll and washing at 65 ~C in a solution co",p~isi"g about 0 1 M salt or less rireferdbly 0 2 x SSC or any other solution having a co",pa,~ble ionic sl,~n~tl,. These conditions allow the dete~ion of sequences having about 90 % or more sequence identity.
In gener~l lower stringent condi1ions refer to the hyl"i~ !ion conditi~s which allow a nucleic acid sequence of at least 50 n~cleotides and prere,dbly about 200 or more nucleotides to hybridize to a particular sequence at about 45 ~C in a solution con,crising about 1 M salt p~efe,dbly 6 x SSC or any other solution having a co",parable ionic s1,en~tl" and washing at room temperature in a solution co",p,ising about 1 M salt preferably 6 x SSC or any other solution having a con,pa~ble ionic W O 97/06259 PCT~EP96/03480 slrenyU, These condilions allow the detection of sequences having up to 50 % sequence identity. The person skilled in the art will be able to modify these hybrid,,~1ion conditions in order to identify sequences varying in identity between 50 % and 90 %.
Altematively sllingel1l co,)~itions refer to hyb~idi~alion conditions which allow a nucleic acid sequence to hybridize selectively to the l-2 resislance gene in its geno",ic env,rLn"~enl suL.sla,)lially to the exc~ ~sion of hyl,, icli~alion with other DNA sequences of said genomic envi, on" ,en~.
- Fusarium 2: Fusarium oxysporum f.sp. Iycopersici race 2 or any other genotype which is not able to infect a host having a resislance gene according to the invention; other genotypes are such as but not limited to wilt inducing fungi soil bom fungi or any other plant patl ~ogens.
- resistance gene product: a polypeptide having an amino acid sequence as dep.~ted in Figure 6 or part thereof or any homologous amino acid 1 5 sequence;
- Ro plant: primary regenerant from a l, an~rorl "alion experi" ,enl also de"oted as t, ansru" ,)ed plant or tr~nsS~enic plant;
- R, line: the p, uyeny of a selfed Ro plant.
- R2 line: the prugeny of a selfed R, plant.
2 o - R,BC line: the progeny of a backc~oss between a R, plant and a plant of the genotype which was o~ iginally used for the l~ dn~rol,, ,alion expeti" ,ent.
In the p,esen~ invention we have been able to identify and isolate the Immun~ty-2 (1-2) resiala"ce gene. The gene was cloned from a tomato genotype which is resi~la, d to Fusarium oxysporum f.sp. Iyr~oper~ia race 2. The isol ~tecl l-2 2 5 resislance gene according to the invention can be transre" t:d to a suscepti~le host plant using Agroba~e~ m me~i~ted l,a"~ro""alion or any other known ~,an~rullll&tioll ",~U,od and is able to confer the host plant ,~sislan~ againstFusarium 2. The host plant can be tomato or any other genotype that is infected by Fusarium 2.
The presen~ invention provides also the nucleic acid sequence of the l-2 resisla"ce gene which is depicted in Figure 6.

W 097106259 PCT~EP96/03480 With the l-2 resistance gene accord"lg to the invention one has an effective means for control against wilt inducing fungi since the gene can be used for 11 ar,src 1 ")ing susceplible plant genotypes thereby producing genetically transformed plants having a reduced susoel~libility or being preferdbly resistant to 5 i"recliGn by wilt inducing fungi. In a ~efe~ed embodiment the /-2 resistance gene comprises the coding sequence preceded by a p,o",oter region and followed by a terminator region. The p(o",oter region should be fur,~tional in plant cells andp,e~rabty co~esponds to the native pro""~ter region of the 1-2 r~sislance gene.
However it should be recognized that any heterGlogous p~")o1er region can be 10 used in conjunction with the coding sequences as long as it is h,rl~;tional in plant cells. F',eferably a constitutive pru,,,oler is used such as the Cal\llV 35 S pro",oter or T-DNA prol"oter~ all well known to those skilled in the art. Furthermore a suitable ~e~ ",i"ator region should be functional in plant cells all well known to those skilled in the art.
In auldition the invention relates to the /-2 ,esislance gene product which is encoded by the 1-2 ,esis1cnce gene accor~ing to the invention and which has an amino acid sequence provided in Figure 6 or which is horY. - I go~ Is to the deduced amino acid sequence or part thereof as listed in Figure 6. The /-2 resis~ance gene product can be used for the idenlir,cation andlor isol~'ion of the ~"esponding 20 gene product encoded by an avirulence gene of the ,cdthogen. The relationshipbetwccn the 1-2 resistance gene product which is assumed to be acting like a ~ceplor molecule and the gene product of the pa1hogen which is assumed to be acting like an elicitor molecule is chard~teri~ed by the occurrence of a de~ense",echanisl,l r~action in the plant. Furthemlore the 1-2 r~sislance gene product can 25 be used for raising antibodies against it which antibodies can be used for the det~ction of the presence of the J-2 resis1ance gene product.
In anoU ,er aspect of the invention the 1-2 resi~1ance gene can be used for the design of oligonucleo1ides which are comple",entary to one strand of the DNAsequence as descril,ed in Figure 6 or part thereof which can be used as 3 0 hybri~ ion probes being accordi,-gl~ I~he'led to allow detection for the screening of geno",:c DNA or cDNA libr~ries for l)o"~r~!cgo~s genes. I IOrYI~'D9~llC
sequences which can hybridize to the probe and which encode for a gene product CA 02227~24 1998-01-21 W O97/06259 PCT~P96/03480 that is able to confer resis~anc~ to a plant against a fungus which normally infects said plant or both are comprised within the scope of the present invention.
In another aspect of the invention oligonucleotides are designed based on the l-2 resislance gene sequence such that they can be used as hyblidi~alion probes in Southern analysis. These probes can be used as molecular markers to distinguish plant genotypes having the resislance gene and plant genotypes lacking the resistance gene. Such a probe can be used as an addilional tool in selection breeding. In a p,~re"~d e~nb~di"~enl of the invention oligonucleoticles are designed based on the l-2 resisla"ce gene sequence such that they can be used as primers in an amplir,cation rea-1ion such as polymerase chain ,~aclion (PCR) whereby the fo""aliGn of an a,),plir,calion product indicales the presence of the l-2 resislance gene in a certain plant genotype. In a particular e",bodi",enl of the invention said p,i,ners direct the a",plir,calion of polymorphic fldylllents so called molecular markers which are closely linked to the l-2 resislance gene. The invention also relates to diagnoslic kits cG",prisi"g oligonucleotides according to the invention for the dete~1ion of the presence or absence of the l-2 resislancegene within a genotype under study. Such a diagnoslic kit circumvents the use of a laborious ~lise~se assay to screen for genotypes having the resislance gene or not.
2 o F~l ll ,e""Gr~ the invention relates to DNA constructs (A) comprising a DNA
sequence co(l~sponding to the coding sequence of the l-2 resi~la,lce gene and reg~ ory sequences functional in plant cells. Said reg~ tory sequences are either llo".o'cgo~ ls or heterologous to the coding sequences of the l-2 resiala"ce gene. The invention relates also to DNA constructs (B) ccn,p-ising the reg~ torysequences and more pr~fe,dbly the prc;"loter region of the l-2 resisla"ce gene in conjunction with a structural gene sequence heterulQgQus to said reg~ tory sequenoes.
The invention relates also to a DNA vector c~",~risi"g a DNA construct (A) and/or a DNA construct (B). Suitable vectors can be cloning vectors 30 l,~nsro""dlion vectors ex~,~ssion vectors etcwhich are well known to the person skilled in the art. Fullllellllore cells harbouring a vector co",pri~i"y a DNA

CA 02227~24 1998-01-21 W097/062~9 PCT~P96/03480 sequence ~"asponding to the sequence as desuibed in Figure 6 or part thereof, DNA constructs (A) or DNA constructs (B), are within the scope of the invention.In one pre~"ad embodiment of the invention, a genetically t,ansro""ed plant is obtained by introducing the J-2 resistance gene within the genome of said 5 plant, having a susceptiL,le genotype to Fusarium 2, using s1andard l~ dl l~rOI 11 ,alion techniques, wherein said genetically l~"~ro""ed plant is resistant to Fusarium 2.
Moreover~ the l-2 resislance gene is inherited in following generations of the said genelically 1,ar,~ru""ed plant and is able to confer the next gene,~lions of said plant resistant to Fusarium 2.
In yet another embodiment of the invention part of the DNA sequence co"~p,ising the l-2 resi~lance gene, is used for llal)~rulllli,)9 a plant which is susceplible to Fusarium 2. Such part can be obtained by digesting the DNA
sequence co",p,ising the l-2 ~esis1ance gene, in one or more steps, with one or more appropriate resL, i~,lion enzymes, chosen on the basis of the presence of their 15 recognition site in the l-2 resistance gene according to the invention, or in the sequences flanking the l-2 resislance gene. The obtained DNA seg",enl can be l,ansr~"ed to a suscepli~le host plant and genetically tra"sru",)ecJ plants having a resistant ~uhenotype can be identified when ino~ ted with Fusarium 2.
We have found that the l-2 resistance gene according to the presenl 2 o invention, is functio"al in homologous systems and/or heterologous systems, such as but not limited to tomato, melon, tDb~cc~, Arabidopsis, egg plant, potato species, and is involved in reducing the susceplibility and/or is c~pAh'e of con~"i"53 these plant species resislance against h/sariLIm 2 as defined above, and es~ci~"y against one or more wilt inducing fungi. A homologous system 25 refers to a plant species which is the same plants species from which the ~esi~lance gene was isol~'ecl and a heterologous system refers to a plant species which is difrarsnl from the plant species from which the r~sislance gene was isol~t~-~
The DNA sequence cornprising the l-2 resis1ance gene as provided in the 30 presen~ invention has numerous applications of which some are desuibed herein but which are not limiting the scope of the invention.

CA 02227524 l998-0l-2l W 097/06259 PCT~P96/03480 The presenl invention will be further described in detail in view of the isol~ion of the /-2 resistance gene present in tomato lines which are resistant against fusarium oxysporum f.sp. Iycopersici race 2. For the isolation of the J-2 resistance gene we have used a ma~based cloning (positional cloning) strategy c~",prising the following steps:
(1 ) ide"lir,calion of ".-lcu ll~r markers linked to the l-2 resistance gene (2) genetic ~ ~ ~aF P ng of the l-2 locus using I l lol l~hel~ir~l markers (3) construction of a high molecular weight geno" ,ic YAC library (4) physical mapping of the molecular markers on the YAC clones and YAC
contig building (~) construction of a cosmid library of the YAC clone harbouring the linked n). ec~ markers (6) physical fine mapping and cosmid contig building (7) ll ~l ls~ul llldlion of susceptiL le plants with the cosmids rc(" ,i, ,9 the contig 15 (8) complementation analysis.
For the identir,calion of molecular ",a,he,~ we have used the selective resL, iclion fragment amplir,cdli~n technology hereinafter denoted as AFLPT~
technology which ~ndon~ly amplifies a subset of DNA r,a~",ent~ out of a complex mixture of many DNA f,a~""e"Ls and said amplified r,a~~",enLs generaLe ringe"uri,)t~
2 o that can be analyzed.
In general total DNA of dXrrt:r~nl genotypes of the same plant species are subjected to the AFLP techno ~yy and the clirfe~enl AFLP ~ingel~,,i"l~ ob~ained from the dirr~renl genctypes are co",pared. F,a~",enls that are presenL in one genotype and absent in anolller genotype are poly",~ r~a~",ent~ and are 2 5 denoted as AFLP, na, ker~.
The selectivity in AFLP re~.tio"s is obtained by using ,a"dol"ly chosen selective nucleotides at the 3 end of the PCR primers in"lledialely adjacenl to the nucleotides of the resL,iu1ion enzyme site. In an AFLP sc,ee,,,,)y the DNA to bestudied is subjected to different primer combinaLions. The total amount of di~re,ent 30 ,c,i",e,s that can be used is dete""ined by the number of selective nucleotides that are added to the 3 end (4 pri",er.~i with 1 selective n~oleclirles 16 p,i",ers with 2 selective nucleotides 64 pri~ers with 3 selective nucleotilJes). If two diff~,ent W O97106259 PCT~P96/03480 resl~ic~ion enzymes are used than there are twice the amount of primers. Those primers can be used in clilr~r~nt co",~..)alioll. If all possible co")bindlions are used in an AFLP sueening than all the r'a~~,,,enl~ present should have been amplifiedwith one of the primer combinations (7~ 1 and Vos EP 0~34858).
For the idenliricatioll of AFLP markers linked to the l-2 resislance gene dirreler it tomato lines were subjected to an AFLP screenina. The tomato lines were pooled into clirrerel,l pools: on the one hand pools of tomato lines being resisla,)l against Fusarium 2 and on the other hand pools of tomato lines being suscerlil,le to Fusarium 2.
The pools were subjected to an AFLP screening using the EcoRI/Msel enzyme combination.
The following pri" ,er~ are used for the AFLP scr~el ,;ng.
EcoRI-primers: 5'~ACTGCGTACCMTTCNNN-3' Msel-pri" ,ers: ~'-GATGAGTCCTGAGTMNNN-3' The N's indicate the variable selective n~ ~G,eotides In the AFLP sueening all 64 possible prime,~ were used for both the EcoRI- and Msel-primer giving a total of 64 x 64 = 4096 primer comb.nalions. The objective of the screening was to identify AFLP markers linked to the l-2 lesblar,ce gene i.e. presenl in the finge,~,ints of the resialanl pools and absent in the r,nge".ri"ls of susceptible 2 o pools. In the analysis of all the AFLP finge, ~, i, lla a total of 18 AFLP markers were identified which were pr~sent in the r~sislanl pools and absent in the sensitivepools: these markers are l~elled to as candidate l-2 linked AFLP markers.
The presence of these candidate l-2 linked AFLP markers were conr"",ed on the individual tomato lines. All n,arke,~ were presenl in the resislanl Iines and 2 5 absent in the suscept;hle lines.
However the posilion of the AFLP markers with re5 ~ Ct to the /-2 resislance gene and the dialance between the l-2 resialance gene and the ,es~cli~re markers has still to be detemmined. For that purpose a ~enelic map ofthe l-2 locus was made by making gene~ic ~usses between Fusarium 2 r~sistanl tomato lines and susceplible tomato lines having ",G,~hological markers followedby a sueening for recombinants in the segr~galing F2 populations and screening of the F2 reco,nbi,)anls with the AFLP ",a,hers. The results indicated that the l-2 PCT~P96/03480 locus is flanked by marker EM18 at one end of the DNA seg",ent co",prising the J-2 resistance gene and markers EM03 EM12 and EM16 at the other end. All the other AFLP markers co-segregated with the J-2 resistance gene.
Next the AFLP markers were screened on a high molecular weight 5 genomic library. The cloning of very large segments of DNA as large artiflcialchromosomes in yeast has become an esse,ltial step in isolating genes via positional cloning. The cloning capacity of the YAC vector allows the iso!~tion of DNA f, dy" ~ents up to one million base pairs in length. The tomato line Lycopersicon esculentum F~, homozygous for the l-2 locus was used as source 10 DNA to construct a YAC library. We obtained a YAC library conlai"ing 3840 clones with an average insert size of 520 Kb representing appru~i",ately 2.2 geno",e equivalents of the tomato genome. One positive clone was obtained after an AFLP
screening with the l-2 linked AFLP markers and all markers were prese,)t on thisindividual YAC clone designated as YAC 11546. The size of this YAC clone is 15 determined to be 750 kb.
Further analysis has dete"nined that all of the AFLP markers were located on a SgrAI r(dy, nenl at a distance of 2~5 kb of the left arm of the YAC until the first SgrAI site. A schematic represenldtion of the physical map of the left arm of YAC
11546 containing the l-2 locus including the localion of the 18 AFLP markers is 20 depicted in Figure 4. The exact location of the AFLP markers can be determined on the basis of ",acp,ng cosmids on the physical map.
The size of an insert in YAC 11546 is still too large for direct loc~ lio" of the l-2 gene. Such large inserts cannot be l,ansr..""ed into plant cells directly.
There~,e a cosmid library was constructed of the yeast strain containing YAC
11546 using cosmid vectors which are suitable for Agrob~fenum ."e~
ro" "dtion~ The size of this binary cosmid vector amounts 29 kb and is shown scl,e",alically in Figure 2. The cloning ca,t~acily of this binary cosmid vector using phage lambda packag,ng extract is within the range of 9 to 24 kb. A bank of approxi",ately 250 000 cosmid clones was obtained from size ~a~tionaled yeast DNA. The cosmid bank was screened by colony hyb,i~ tion using the ate e~
SgrAI fragment as probe. Of about 10000 colonies appruxir"ately 1~0 positive cosmid clones were identif,ed.

W O 97/06259 PCT~P96/03480 In the following step the position of the AFLP markers on the 255 kb SgrAI
r,~",en~ was determined on the basis of a cosmid contig. The positive cosmid clones were sc ree"ed with the 18 AFLP markers and their position was dete" ,~ined. A schematic outline of the cosmid contig and the physical fine 5 mapping of the 18 AFLP markers is depicted in Figure 4.
The final step in the identificalion of the l-2 resistance gene via positional cloning is the complemenlation of the co"~:s~cJnding suscepli~le phenotype. The cosmid clones were introd~ ~ced in Agrobacterivm tL~I~erdG~ns through conjugative transfer in a tri-parenlal mating. The presence of the cosmid in the A. tun,er~c,ens 10 strains was determined co",pari,-g various ~esl~iution enzyme patle~s as well as DNA fin~e~pri~ls from the A. tu~"era~ ns strains with the Eschericniacoli straincontaining the cosmid. Only those A. t~llle~dGens cultures harbouring a cosmid with the same DNA pattem as the correspon(ling E cofl culture were used to l,dnsro"" a suscep~ible tomato line.
A susce~lible tomato line was llansrorllled with several cosmids forming the cosmid contig using slanda,d l,~l,sro""dlion ",eU,ouls.
The primary regenerants (Ro plants) of the transro""alion e~eri",enl~ were grown in the y,ee"house for seed set to obtain R, lines. These were tested for dise~se s~""pto",s in order to identify cosmids with the ~esisldnce gene. The 20 ~lisease assay is pe,rul,,,ed on seedlings. Their roots are i""~er~ed in a conidial suspension of Fvsarium oxysporum f.sp. Iyoopersici race 2 and dise~se s~",p~on,sare scored three to four weeks after inoclJ'~tion. Plants are scored resistant when they are healthy without wilting s)""pto",s and/or without browning of the stem tissue. Plants being dead or having yellow wilting leaves and having severe 25 browning of the stem tissue are scored susceptil~le The observations of the rl;se~se assay revealed that 3 cosmids were able to complement the susceptihle phenotype thereby providing definitive evidence that a functional l-2 resistdncegene is loc~3led on each of these 3 cosmids.
To der"Gn~llate that the resistant phenotype of the t,ansgenic R4 plantsj 30 which were l,an~ro",)ed with the ovetlapF.ng cosmids A52 BZ and A55 is deter""ned by the genomic insert presenl in the various cosmids the presence of the co,~sponding AFLP markers was invesligaled (see Figure 5). Selective CA 02227~24 1998-01-21 W097/06259 PCT~P96/03480 restriction fragment amplification was peifull,,ed with the primer ~mb..lalions identifying the markers EM14 and ~M06 forthe R4 plants trar,~fol",ed with cosmids A52 or B22 and with the primer combinations identifying the markers EM06 and EM04 for the Ro plants t~ansfoll"ed with cosmid A55. The DNA rl,-ge,~ri"l:, obtained showed in both cases and for both markers the presence of the martcers in the resistant plants and the absence of the markers in the susoeptible plantsindi~ling that the three identirled overlapping cosmids A52 B22 and A5 co" "~rise the 1-2 resis~ance gene.
In order to confirrn the stable integ~alion of the 1-2 resistance gene into the genol "e of the ll dnsgenic Ro plants resistant plants of the R, lines were setfed and grown in the greenhouse for seed set to obtain R2 lines. Seedlings of the R2 lines were subjected to a disease assay as desuibed above and were scored for disease sy",pto",s: Wilting plants were considered to be susceplible whereas plants showing no wilting were cor,sidered to be resistant. The resutts obtainedindir~ted the stable Mendelian inherilance of the 1-2 resislance gene. Addilionally resistant plants of the Rl lines were backcrossed with the susceptitule tomato genotype used for the ll al ,~fu""alion ex~eri,Y)ents to obtain R,BC lines. The results of the dise~se assay pe,rulll,ed on seedlings of the R,BC lines confi""ed both the i, Ihel ilance as well as the dominance of the J-2 resislance gene.
2 o Finally the inserts in CO51llids A52 B22 and A55 were further cha,a~eri~ed and the minimal DNA segment co~ ising the /-2 resistance gene defined by the left end of cosmid A55 and the right end of cosmid B22 was sequenced. Sequencing analysis revealed a large open reading frame of 3798 nuclaolides. The DNA sequence is listed in Figure 6.
2 5 The DNA sequence comprising the 1-2 resislance gene was further subjected to ll ansc~ipt mapping studies in order to identify the 11 al-s~ iption i"ilialion site and t,dnscri~J~ion termination site and to determine the exisle"ce of intron sequences. These lla,~sc,ipt Illapping studies were pelf~JIrlled according to generally known methods whereby genomic DNA sequences are co",pared with cDNA sequences. The cornparison of cDNA sequences and genG",;c sequences revealed the exislence of three intron sequences in the /-2 resislance gene outside the coding sequence: one intron of 86 n~cleotides is located u~sl~ea", of the ATG

WO 97/06259 PCT~P96103480 ini~ialion codon and two introns of 399 and 82 n~ ~cleotides r~:specli~ely are located dow"sl,eal" of the TM termination codon, as is depicted in Figure 6. The l,ans~iption iniliaLion site is located at or upstream of nucleotide 1~97. The trans~i~tion te""i,)alion site is located at or immediatly downstream of nucleotide 5 6491. The position of a putative poly-adenylation signal is ded~ ~oed at nucleotides 6406 6411 (MUMA).
A DNA segment correspondin~ to part of the DNA sequence, as provided in Figure 6, starting at nl ~cleotide ~osilion 464 and ending at nucl~olide posilion 6658 was used for ~dnsfo""il1g a sl~-sc~ptible tomato genotype. The DNA segment was 10 obtained by digesting cosmid B22 with restriction enzymes BamHI and Sall, providing a 3.8 kb r,ag",erll, and with Scal and BamHI, providing a 2.4kb fragment, resulting in a 6.2 kb fragment col"prisi, l~ the coding sequence of the l-2 resistance gene flanked upstream by a 1.3 kb DNA sequence and downstream by a 1.1 kb DNA sequence. The DNA seyl"enl was cloned into a sl ~iPhle ~~ eg,dle 15 type vector and sl~hse~uently introduc~d through AgrDbacterium tu",erd~ens me~ ted l,~"sru",~alion into a tomato plant which is suscepliLJle to Fusarium 2.The Ro plants were grown in the greenhouse for seed set to obtain R, lines and these R, lines were subjected to the ~I ie~ assay as descriL~d above. The observations i"d;c~lecl that the DNA seg",ent is involved in conr~:"ing to the 2 o ll ansrul ",ed plants a rectuced susceptil,ility to Fusarium 2.
It is u"derslood that those skilled in the art can choose other parts of the DNA sequence on the basis of the teaching provided herewith, or using any other well known method, to obtain parts of the DNA sequence provided in Figure 6, or to obtain parts of the genomic insert prt:senl in cosmid B22 or A~5, introducing2 5 said parts into an appr~.priale DNA construct which allow e~,ession in plant cells, I,~nsr~"ing said constructs to a host plant being s~sceplil~le to Fusarium 2 andtesting the l,an~ro""ed plants for dise~ce sy",p~oms after inoclJI~'ion with Fusarium 2.
Cosmid B22 was used for the l,d,)sru,l"alior, of susceptible genotypes of 30 melon, tob~c~ as well as Ar~l~i lopsis according to general known llal1sro""dtion IlletllGds. The Ro plants were grQwn in the greenhouse for seed set to obtain R1 lines. These were tested for disease symptoms in order to identify the functionality of the l-2 resistance gene. The dise~se assay was pe,ru,,,,ed on seedlings as described herein. Cosmid B22 was also used for the l,dnsfc.""alion of a sus~pt;ble genotype of potato. Vegetatively p~p~g~1e-J l,an~o""ed plants were obtained and were subjected to a r~ise~-se assay on cuttings. The observations of the ~ise~se assay on the l,d"sro,l"ecl plants rcvE~'~d the complemenldlion of the susceplible phenotype.
For the identifi,~lion and iso!~lion of holllG'ogol-s sequence falling within the scope of the present invention genomic and cDNA libraries were sueened with the coding sequence of the l-2 resistance gene as a probe under sl,ingenl hy~ lion condilions. Positive clones were isolated and were used for co",ple llentation analysis.
Cosmid B22 has been deposiled on .luly 14 1995 as plas",id pKGI2-B22 at Centraalbureau voor Schimmelcultures at Baam The N~tl ,e~ lands under deposit number CBS ~46.95.
Cosmid A55 has been deposited on August 5 1996 as plasmid pKGI2-A55 at Cent, adlL,ureau voor Scl)il ,)rnelc.~ltures at Baarn The Netherlands under deposit number CBS 820.96.
The following e~d",~,les will provide a further illusl,dlion of the presenl 2 o invention which is nevertheless not limited to these eAdlll~lcs.

EXAMPLES

FX~M~LE1:DISEASE ASSAY
2 5 Fusarium oxysporum forma specialis Iycopersici race 2 was maintained on Czapek Dox Agar (Difco Labo,a~olies Detroit Ml USA). Conidial suspensions were obtained by culturing the fungus in Czapek Dox Broth (Difco Labor~luries DetroitMl USA) on a rec"~local shaker for 4 to 7 days at 25~ Celsius. The conidia were separdled from mycelium rray",en~s by filtration through a stainless steel filter with a pore size of 50 ~m. The suspensio,-s were adjusted to a concenl~tion of 2 x 106 conidia per ml by diluting with water.

CA 02227~24 1998-01-21 W O 97/06259 PCT~P96/03~80 SeeJli"gs of tG,-.ato Seeds of tomato were ge~ninaled in soil in the greenhouse at 25~ Celsius. Ten to14 days-old seedlings were used for inoculation with the fungus. The seedlings 5 were carefully pulled out of the soil and the roots were dipped in water for removing most of the adhering soil 9Jhse~l lently the roots were immersed in theconidial suspension for two minutes and the plants were repotted in soil. The pldnllets were grown in the greenhouse at a temperature of 25~ C at daytime (16 hours) and 22~ C at night (8 hours). After three to four weeks the plants were 10 scored for dise~se sy",pto",s.
The plants were ev~u~ted as follows: Resislanl plants rese"lble non-inoculated control plants; they are large non-wilting and/or without browning of stem tissue.
Sus~p~il le plants are dead or show typical s~",plo",s: small plants with yellowwilting leaves and severe browning of stem tissue.
Cuttin~s of tomato Cuttings were prepared from greenhouse~rown Ro plants. Small sideshools were cut from the plants and put in soil under 100% humidity at 20~ Celsius. After one to two weeks the cuttings started rooting. Two to three weeks old cuttings were used 2 o for inoc~ ll~'ion with the fungus. The plantlets were carefully pulled out of the soil.
The roots were dipped in water for removing most of the ~-JI .e, ing soil.
S! ~hse~uently the roots were i~ er~ed in the conidial suspension for five minutes and the plants were r~potled in soil. The plantlets were grown in the greenhouse at a ~e",per~ re of 25 ~C at daytime (16 hours) and 22 ~C at night (8 hours). After~5 three to four weeks the plants were scored for dise~se s~",lplo",s.
Evaluation of the plants was as described for seedlings of tomato.

EXAMPLE 2: IDENTIFICATION OF AFLP MARKERS LINKED TO A DNA

Tomato lines (Lycoper~icon esculentum) PCT~P96/03480 W 097/062~9 A total of 10 F. oxysporum f.sp. tycot~rsici race 2 resistant and 10 F. oxysporum f.sp. Iycopersici race 2 susceptible tomato lines were used, and are depicted below:

~j 1 DR9 resistsnt Oe Rulter Zonen C V~ BlelswUI~ ~he lletherbnds Ih-reinsnu ~De Rultn~
2 B~5 ro~ist~nt Rilk Zwssn ~ssdt~elt ~n Zssdh~ndel BY~ De U~r lh~
lletherlsnds Olerd~ltcr ~9 n~
3 E22 re~ist~nt E~9 ZJd~n~ d- E~cr ~dhJnd~l ~Y~ ln~huinn, ~r ~etherlsndslherclnalter~~n~aZ~den~
4 n6 rcslstsnt ~9 Zsden DR4 rcslstsnt De Rulter 6 RD r slstsnt Illlk~9-n 7 U re~lstsnt EmaZaden E7 r sl~t~nt EnzsZsd~n 9 RU r~ t nt ~llkZw~sn DRt2 rcsl~t~nt De Ruller n tCR210 ~u c~PUble Instltute o1H-rUcultur~lResearch lltll-hsmPton~6reatBrlt ln t2 CCR508 ~usc~ptlble In~tnut- ~1 Hortlculturs1 Re~esrch UltlehsmPton~ Crest ~rltsln 2 0 13 52201 ~u~cepUble RllkZwssn 14 DR5 ~usceptlble De Runer n2 ~usc~pUble Ems Zsden 16 n susceptlble ~~z~bd-n n. ~ ~u~ccp~ble Rll~ Zwssn 2 5 1~ E6 su~e~pUbl~ 9 Zsd~n 1J R~0 ~u~cDpUble Rllk ZwD9n DRn ~usc~pUble De Rulter ~ol~ion and modification of the DNA
30 Total tomato DNA from the 20 lines des~ibed above was isol~'ed from young leaves as ~es~ibed by Bema~ki and Tanksley (1986, Theor. Appl. Genet. 72, 314-321). The typical yield was 50 - 100 1l9 DNA per gram of fresh leaf n,ale,ial.
Template DNA for AFLP analysis with the enzyme co",binaliG~- EcoRI-Msel was WO 97/06259 PCT~P96/03480 prepared as described by Zabeau and Vos (European Patent Application EP
0534858) and is described briefly below:

0.5 ~19 of tomato DNA was inc~ ~h~ted for 1 hour at 37 ~C with 5 units ~coRI and 5 units Msel in 40!11 10 mM Tris.HAc pH 7.5 10 mM MgAc 50 mM KAc 5 mM DTT
50 ng/~ll BSA. Next 10 ~1 of a solution containing 5 pMol EcoRl~dapter~ 50 pMol Msel-adaptera 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 ng/~l BSA was added and the incubation was continued for 3 hours at 37~C. The adapte, a are depicted below:

The structure of the EcoRI-adapter was:

5 '-CTCGTAl;F~CTGCGTACC
CATCTGA~ rAA-5 ' The structure of the Msel~d~rter was:

5 ' -GACG~TGA~
TACTC~STCAT-5 ' A-Ja~le,a were prepared by adding equimolar amounts of both sl,~ncJs; A~la~tela were not pl~osphorylated. After ligation the reaction mixture was diluted to ~00 1ll with 10 mM Tris.HCI 0.1 mM EDTA pH 8.0 and stored at -20~C. The diluted reaction mixture is further rare" acl to as te" ,plale DNA.

AFLP reactions The prill~ra used for the AFLP sueening are depicted below:

EcoRI-pri" ,era: 5'-GACTGCGTACCMTTCNNN-3' Msel-pri",a,s: 5'-GATGAGTCCTGAGTMNNN-3' WO 97/062~9 PCT~P96/03480 The N's in the ~ri~"ers indicate that this part of the pri~er~ was variable In the AFLP screening all 64 possible ~,i",e,~ were used for both the EcoRI- and Msel-primer. This gave a total of 64 x 64 cc",ll.inat,ons of EcoRI- and Msel-p~ , is 4096 primer combina~io,)s. All 4096 primer combinations were used in the AFLP
screening for 1-2 linked AFLP markers. The AFLP reactions were pe,ro""ed in the following way:

AFLP reactions employed a radio-actively labelled EcoRI-primer and a non-labelled Msel-primer. The ~coRI-pri",er~ were end-l~he'lQd using (y-33P)ATP and T4 polynucleotide kinase. The labelling rsa~ions were performed in 50 ~l 25 mM
Tris.HCI pH 7.5, 10 mM MgCI2, 5 mM DTT, 0.5 mM spe""iJi,)e.3HCI using 500 ng oligonucleotide primer, 100 ~lCi (y-33P)ATP and 10 units T4 polynucleotide kinase.
For AFLP analysis 20 lul reaction mixture were pr~pared contai, ling 5 ng labelled EcoRI-primer (0.5 ~I from the labelling rea~ion mixture), 30 ng Msel-primer, 5 ~l template-DNA, 0.4 units Taq-polymerase, 10 mM Tris.HCI pH 8.3, 1.5 mM MgCI2, 50 mM KCI, 0.2 mM of all 4 dNTPs. AFLP re~c1ions were pe~run"ed 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 e~-te"sion step at 72 ~C. Theannealing temperature in the first cycle was 65 ~C, was s!~hseq~ently reduoed eacn cycle by 0.7 ~C for the next 12 cycles, and was continued at 56 ~C for the remaining 23 cycles. All a",pliricalion r~a~tions were performed in a PE-9600 themmocycler (Perkin Elmer Corp., Norwalk, CT, USA).

Gel analysis of AFLP reaction pro~ ctC
After a"~plirication~ ~ea~tion products were mixed with an equal volume (20 ~11) of fo""a"lide dye (98% ~o"~,a,n.de, 10 mM EDTA pH 8.0, and bromo phenol blue and xylene cyanol as l,achi"g dyes). The resulting mixtures were heated for 3 minutes at 90~C, and then quickly cooled on ice. 2 1ll of each sa",ple was loaded on a 5%
denaturing (sequencing) polyacrylamide gel (Maxam and Gilbert, 1980, MeU lods inEnzymology 65, 49~560). The gel matrix was prepared using 5% acrylamide, 0.25% methylene bisacryl, 7.5 M urea in 50 mM Trisl50 mM Boric acidt1 mM

W097/06259 PCT~P96/03480 EDTA. To 100 ml of gel solution 500 ~11 of 10% APS and 100 1ll TEMED was added and gels were cast using a SequiGen 38 x ~0 cm gel apparalus (Biorad Laboralories Inc. Hercules CA USA). Sharktooth combs were used to give 97 lanes on the SequiGen gel units. 100 mM Tris/100 mM Boric acidl2 mM EDTA was 5 used as running buffer. Ele, lrophoresis was performed at conslanl power 110 Watts for approximately 2 hours. After ele~1,uphoresis gels were fixed for 30 minutes in 10% acetic acid dried on the glass plates and exrosed to Fuji phosphoimage screens for 16 hrs. Finge,l,,inl pC-ltlt:llls were visu~li7~d using a Fuji BAS-2000 phospl-o image analysis system (Fuji Photo Film Co,npany Ltd Japan).

AFLP sc~e.~ for linked markers The template DNAs of the 20 tomato lines were pooled in the following way:

resistant pool 1: tomato lines 1 - ~
15 rBsislanl pool 2: tomato lines 6 - 10 susceptible pool 3: tomato lines 11 - 15 susceptible pool 4: tomato lines 16 - 20 An AFLP sueening was pelrù~llled using all possible 4096 EcoRI-Msel primer 20 comb,alions on the 4 pools. The aim was to identify AFLP- markers presen~ in both resislanl pools and absent in both sensitive pools. AFLP gels contained theAFLP ringe"~ri"ls of 24 primer co"lbinalions of the 4 pools giving a total of 171 gels. Achlilional gels were run to reanalyse unsuccessful AFLP rea-;tions and toconfirm candidate ma,l~,s. A total of 18 AFLP r"a~ker~ were identirled pr~senl in both r~sisla,lt pools and absent in both susceptil-!c pools: these markers were rere"~ to as candi~Ja~e l-2 linked markers.
Next AFLP r~actions were pe, ru,,,,ed to determine the presence of all 18 candidate markers on the 20 individual tomato lines. All markers appear~d to be pr~sei)l in the 10 r~sislanl lines and absent in the 10 susceplible lines. These 18 ",ark~:jwere named EM01 to EM18 and were used in further studies to map the l-2 Fusarium resi~lance gene. The primer comb nalions required to identify marke,~

WO 97/06259 PCT~P96/03480 EM01 to EM18 are depicted in Table 1. In the column with the primer c~,nb.naLions, "EcoRI-" refers to the sequence 5'-GACTGCGTACCMTTC-3' and "Msel-" refers to the sequence 5'-GATGAGTCCTGAGTM-3'. For example, marker EM06 can be identified using the EcoRI-primer having the following 5 sequence: 5'-GACTGCGTACCMTTCAGA-3', and the Msel-primer having the following sequence: 5'-GATGAGTCCTGAGTMTCT-3'.

WO 97106259 PCT~P96/03480 marker primer combination cor,~a").,~g selective extensions (NNN) EM01 EcoRI-MA I Msel-AGG
EM02 EcoRI-CCA / Msel-TCA
EM03 EcoRI-CTC / Msel-GCT
EM04 EcoRI-GAG / Msel-GTC
EMO~ EcoRI-TCT / Msel-MG
EM06 EcoRI-AGA / Msel-TCT
EM07 EcoRI-CTT / Msel-MG
EM08 EcoRI-CM / Msel-GCT
EMO9 EcoRI-GTC / Msel-GTC
EM10 EcoRI-TGT / Msel-MT
EM11 EcoRI-TAG / Msel-AGC
EM12 EcoRI-TGC / Msel-MG
EM13 EcoRI-TM / Msel-ACC
EM14 EcoRI-TGC / Msel-AGA
EM15 EcoRI-CTT / Msel-ATG
2 o EM16 EcoRI-CAT / Msel-AGT
EM17 EcoRI-CTC / Msel-AGC
EM18 EcoRI-CGG / Msel-CAC

2 5 EXAMPLE 3: GENETIC MAPPING OF THE TOMATO l-2 LOCUS

D¢v~lDrment of SjC~¢tiC ...at~.ial Two F. oxysporum f.sp. Iycopersia race 2 resisl~nl tomato (Lycop~
esculentvm) lines have both been ~ossed with the s(~s~pti~l!Q tomato lines GCR210 and GCR508. Both ~~sistant lines, DR9 and RZ5, are breeding lines from two seed cor,~panies, De Ruiter Zonen and Rijk Zwaan, respectively. The s~,sc~pt;ble tomato line GCR210 is homozygous for the ~ecessive r"G"~ h~'~gical W O 97/06259 PCT~P96/03480 marker gene a (anthocyaninless) The susceptible line GCR508 is homozygous for the recessive morphological marker gene sub (subtilis) (SteYens and Rick 1986 in: The Tomato Crop Atherton 8 Rudich edit. Chap~"an and Hall p. 3~109). Both susceptible lines were obtained from the Institute of Horticultural Research 5 (Littleha,npton United Kingdom).
The dominant l-2 gene (co,-re"ing resislance to F. oxysporvm f.sp. Iycopersici 2), and recessive genes a and sub have all been mapped to the long arm of ~,rL""osome 11 of tomato (Stevens and Rick 1986 in: The Tomato Crop Atherton & Rudich edit. Chapman and Hall p. 35-109): I-2 on position 85 of this c~,o",osome a on position 68 (17 ce"t;Mo~yans (cM) from l-2) and sub on position 89 (4 cM from l-2).
The crusses made are depic-ted below:

Cross 1: DR9 (+ ~ I-2 I-2) x GCR210 (a a i-2 i-2) Cross 2: RZ5 (+ + I-2 I-2) x GCR210 (a a i-2 i-2) Cross 3: DR9 (I-2 1-2 + +) x GCR508 (i-2 ~2 sub sub) Cross 4: RZ5 (I-2 1-2 + +) x GCR508 (~2 i-2 sub sub) F, plants from all c~osses were selfed for generating F2 seeds. The resulting F22 o popu~tions will seg,eyale for resislance to F. oxysporum f.sp. Iycopersici 2 and for the ",o"~l1ological markers.

Selection of ,~:cG,-~Li.~ants in the re~ion co"lai,.;..~ ~2 3000 F2 seeds from the ~usses 1 and 2 with GCR210 (containing a) were 25 germinated. After 10 to 14 days the seedlings were divided in two groups: onewith purple coloured hypocotyls (wild type seedlings) and the other with green hypocotyls (seedlings homozygous a). 330 wild type seedlings and ~65 anthocyaninless seedliflg~ were inoc~ ted with f. oxysporum f.sp. Iycopersici race 2. After three to four weeks the plants were scored for resistance/susceptibility.
3 o The results are shown below:

phe notype resi~lant susceptil,le wild type 278 ~2 anthocyaninless 169 3g6 The recombinants are suscepLible wild type plants and resistant anthocyaninless plants.

1~00 F2 seeds from the uosses 3 and 4 with GCR508 (conld"~ing sub) were ge"l,inaled. After 10 to 14 days the seedlings were irtoull~ted with F. oxysporum f.sp. Iycopersici rac~ 2. During the assay the plantlets were phenotypic~lly scored for growth habit normal or 'subtilis' and for resistance/susceplibility The results 15 are shown below:

pher,olype resi~lant susceptible 2 o wild type 913 38 subtilis 18 3~1 The rec~",binants are s~sceptii~'Q wild type plants and rssislant 'subtilis' plants.
F2 plants were grown in the g,ee,)house for seed set (by induced or spontaneous selfing). F3 seeds were obtained from most of the resislanl recombinant F2 plants and from a few of the susceplible rec~mbinants. These F3 lines were tested for resisld"c~/ s~l-sc~plil,ility in order to check the phenotype of the F2 plants. Twenty to 30 seedlings of each F3 line were inoc~ ted with F. oxysporum f.sp. Iycopersia 2 and evaluated as desc(ibed in Example 1. The F3 proge,l.~s of the resialant WO 97/06259 PCT~P96103480 recombinants did segregate 3:1 for resistance while those of the susceptible recombinants scored all susce~lible.

Screening of AFLP markers in F2 ~ecG,,ILinants cj An AFLP analysis with the 18ident~ledJ-2 markers EM01 to EM18 has been performed on 187 resistant recombinant plants 10 sus~ptible recomb,nant plants and 39 rontrol plants (12 resialanl and 27 suscepti~le) as desuibed in Example 2.
The following results were obtained:

- The markers EM01,EM02,EM~, EM05, EM~, EM07,EM08,EM09, EM10,EM11,EM13, EM14,EM15 and EM17 were present in all 199 resistant F2 plants and absent in all 37 susceptible plants; these markers are closely linked to the 1-2 gene.
- The markers EM03,EM12 and EM16 were present in all resislan~ plants and absent in the sl~sceptible plants with the exceplion of one anthocyaninless plant.
- The marker EM18is ~rese,)t in all resislant plants except for seven anthocya"inl~ss plants and absent in all susceptible plants.

20 From these data it could be concluded that the l-2 locus is flanked by marker EM18 at one end of the DNA sey",ent cc,nprising the l-2 ,esialal~ce gene and on the other end by the markers EM03,EM12 and EM16. All of the remaining ",arke,s completely c~seg,e~ale with the l-2 resislance gene based on the analysis of recombinants of crosses as described above.
EXAMPLE 4: CONSTRUCTION AND SCREENING OF A TOMATO YAC-LIBRARY

Material 30 The tomato line Lycopers~con esculen~um E22 (Enza Zaden) homozygous for the l-2 locus was used as source DNA to construct a YAC-library. Protopl-stc were W O 97106259 PCT~P96/03480 isolated from the leaves of In vffro shoots which were two to three weeks old asdescribed by Van Daelen et al. (1989, Plant Mol. Biol. 12, 341-352).
Viable protopl~ct.c (conce"traLion of 50 million protoplasts per ml) were colle~,1ed and mixed with an equal volume of agarose (1%, Se~pl-~ue, FMC BioProducts, 5 Rockland, Maine, USA) to form a plug. The protopl~stc embedded into the plugs were Iysed with Iysis mix (0.5 M EDTA, 1% N-Laurylsarcosinale and 1 mglml p,uleinase K, pH= 8.0). After Iysis, the plugs were stored at 4 ~C in slo~a~e buffer (fresh Iysis mix) until used. Approxi~alely 3 million prot~pl~sl~ per plug, to obtain about 4.5 ~9 of chromosomal DNA were used for further studies. Plasmid pYAC4 l O containing an unique ~coRI cloning site was used as cloning vector and the yeast strain AB1380 was used as a host (Burke eta/., 1987, Science 236, 806 812).

YAC library cG"s~ ction High .r,~le~ weight DNA isol~tion, partial ~ Jestion with EcoRI in the prt:se"ce15 of Ec~RI methylase, ligation of vector arms to genon"c DNA, size selection by pulsed field gel elect.ophur~sis and ~ sro~ 2lion of the yeast host was pe,ro,--,ed as desc,ibecJ by Burke ef a/. (1987, Science 236, 806 812) and Larin et a/. (1g91, Proc. Natl. Acad. Sci. USA 88, 4123-4127).
All slar,da,d manirl ~'~tions were carried out as desuibed in Molecular cloning: a 20 labo,atory manual by Sambroo'~ et a/., (1989, Cold Spring Harbor Labordlory Press).
3840 clones with a average insert size of 520 kb, which co--espol,-is to 2.2 ~enome equivalents were finally obtained and the individual clones were stored in 40 9~wells miuotiter plates containing 75 1ll Y~D solution (1% yeast extract, 2%25 peptone and 2% de~rose).

Screening YAC library To reduce the number of samples handled, the cells of one 96 well ."iuutiter plate was pooled (a plate pool) and used for DNA isol~tion as desuil,ed by Ross et al.(1991, Nucleic Acids Res., 19, 6053). The 2.2 geno.. ,e equivalent tomato YAC
library cGnsis~s of 40 96-wells microtiter wells and as a result DNA of the 40 plate W 097/062~9 PCT~P96/03480 pools were sueened with the AFLP-markers EM01 EM12 and EM18 (see Example 2) using the AFLP-prolocol as described in Example 2. One positive platepool out of the 40 was identifed with all three AFLP-markers. Subsequently a seconda~ sueening of the 96 individual YAC clones was employed to find the 5 correct address of the YAC or YACs. One individual YAC clone was identified designated 1/546 and subsequently analyzed with the remaining AFLP markers.
As expecte~i all of identified markers EM01 to EM18 were pr~sent on this YAC-clone since the flanking markers and one co-segregating marker were used in the screening. The size of the YAC-clone was ~leter" ,i,)ed by Pulse-field gel 10 elect,opl,or~tic (PFGE) analysis using contour-clamped homogeneous electric field (CHEF; Chu et a/. 1986 Science 235 1~82-158~) and appeared to be 750 kb.

EICAMPLE5: CONSTRUCTION OF A PHYSICAL MAP OF YAC 1/546 AND
LOCATION OF THE AFLP MARKERS
YAC 11546 was subjected to partial digeslior, with i~ easi"g concenl,alio" of the restriction enzymes SgrA1 Rslll, Sfil and BssHII. The s~ les were fractionated by PFGE lldl~srell e:d to a Gene Sueen Plus ",e",brd,)e (DuPont NEN Boston MA USA) and assayed by hy6,i~ A~ion using end-acljacent sequence probes 20 according to the plotocol for inJ;r~ct end-label ",apping as desuibed by Burke et a/. (1987 Science 236 806 812).
In Figure 1 a sc'ne"~atic represe"lalion is given of the physical map of YAC 1/546.
To detemmine the position of the various AFLP markers on the physical map the AFLP markers were used as hybridization probes on the partial digests of YAC
25 1/546. Tller~fure the AFLP-marker fragment was excised from the dried gel andeluted by means of dfflusion in a buffer containing 0.5 M ammonium ace~a~e 10 mM magnesium acet~ 1 mM EDTA (pl~=8.0) 0.1% SDS re-amplified with the PCR p,imer~ used in the AFLP reaction and l~h~ d with 32p accordin~ to the ranclo,n primer n~tl,od of Feinberg and VG9el;l~ jn (1983 Anal. Biochem. 132 10).

W O 97/062~9 PCT~P96/03480 It appeared that all of the AFLP-markers were located on a SgrA1 fragment, at a distar)ce of 255 kb of the left arm of the YAC until the first SgrA1 site.

EXAMPLE 6: CONSTRUCTION OF A ~osr~l~ LIBRARY OF YAC 11546 Matenal The binary cosmid vector pJJ04541 is a derivative of pJJ1881 (Jones et a/., 1~92, T~ansge,1ic Researcl- 1, 285-297) and is based on plasmid pRK290 containing the tetracyclin resislance gene for selection in Eschenchia cofi and Agrobac~erium 10 tL",e~aci~ns. Into the unique ~coRI site of pRK290, T-DNA carrying sequences (LB; left border repeat, RB signifies the right border repeat) that flank - the cos site of bacteriophage larnbda - the neomycin phospholrar,~reldse gene (Beck et a/., 1982, Gene 19, 327-336) whose expression is driven by the cauliflower mosaic virus 35S
promoter sequence (Odell et a/., 1984, Mol. Gen. Genet. 223, 36~378), and - the pBluescript (St, dlagene, La Jolla, CA, USA) polylinker sequence.
The size of pJJ04541 amounts 29 kb and is shown sche",atically in Figure 2. The cloning capacily of this binary cosmid vector, using phage lambda packaging extracts is within the range of 9 to 24 kb.

Library co. .st. ~Jction Total DNA of the Sacchar~myces cere~Jtsae strain AB1380 containing YAC 1/546 was isol ~~c' using zymolyase to make prolopl~slc according to Green and Olsen (1990, Proc. Natl. Acad. Sci. USA 87, 1213-1217).
An aliquot was analyzed on PFGE and appeared to have a size of 2100 kb.
Appro,ci",dlely 15 ~9 of this DNA was partially digested with Sau3A gene,ali.,g molecules with an average size of 1~25 kb. Sllhseqllentlyl this sample was oentrifugated through a 10-35% sucrose gradient for 22 hours, 22.000 rpm at 20 ~C in a Beck")an SW41 rotor. 0.5 ml ~,a~lions were collected using a needle pierced through the bottom of the centrifuge tube. An aliquot of these r~c~lions was PCT~P96103480 W O 97/062~9 analyzed on a 0.7% agarose gel. The fractions containing DNA molecules with a size of ~20 kb were pooled and co"cenl,a~ed by ethanol prec,pita~ion.
Sl Ihsec~l lently the cohesive ends were partially filled-in with dATP and dGTP using the strategy of partial filling of 5-extensiGns of DNA produced by type ll resl,iction endonuclease as described by Korch (1987 Nucleic Acids Res. 15 319g-3220) andLoftusetal.(1992 Biotechniques12 172-176).
The binary cosmid vector pJJ04541 was .Jiges~ad completely with Xhol and the linear fragment was partially filled-in with drrP and dCTP as described by Korch(1987 NucleicAcidsRes.15 319g-3220).
The 2~kb fragments were ligated to the cosmid vector and transduoed to ~. coli strain XL1 -Blue MR (Stratagene La Jolla CA USA) using phage la" ,bda t igal-~ck 11 XL packaging e~l,a.;ts (S~raLagene La Jolla CA USA) as recommended by the manufacturers. Selection was performed on LB (1% bacto-tryptone 0.5% bacto-yeast extract and 1% NaCI pH 7.~) agar plates containing 10 l 5 mg/l of tetracyclin. A bank of appro~imalely 250.0~0 cosmid clones was made from 2-3 ~19 of size r, actionaled yeast DNA.
Sl~hsequently these l,dnsfo""anls were stored into the wells of microtiter plates (96 wells 100 ~l of LB medium containing 10 mg/l of tetracyclin). Replic~c of the 96 well grid of cosmid clones in microtiter plates were stamped onto Gene SareenPlus me",Lra,-e filters (DuPont NEN Boston MA USA) and allowed to grow into colonies on media. Colony hyb, idi~alion using 32P~ hcllE!c! SgrA1 r, ~9" ,~nl revealed positive cosmids. Of about 10.000 colonies ap,cro~i",~lely 1~0 positivecosmid clones were identified.

2 5 E)tAMPLE 7: DETAILED PHYSICAL MAP OF THE 255 SgrA1 FRI~r/1FI~lT AND
LOCATION OF THE AFLP MARKERS

ConstnJction of a cosmid contig of the 255 kb SgrA1 f.~n.enl Stancla~ tecl"~ Jes for growth and manipulation of ~sm;ds in E coli were followed (Sa",broGI; et a/. in Mole~ cloning: a labo(dtory manual 1989 Cold Spring Harbor Laboralory Press). Cosmid DNA was isolated by alkaline Iysis using WO 97/06259 PCT~P96/03480 the method as described by Ish-Horowicz et at. (1981 Nucl. Acids Res. 9 2989-2997). Approximately 500 ng was used for te",plale preparation and the pri",er:, in the amplification of re~l~ iction ~ ayl "enls were the EcoRI-primer 5 -GACTGCGTACCMTTC-3 having no selective nuclcotide and the Msel-primer 5-GATGAGTCCTGAGTM-3 having no selective n~ ~leotir~ as described in Example 2. The EcoRI-primer was labelled at the 5 end and each of the 150 DNAs was amplified using the described primer set. The DNA finge,,uri,,ts co,lta,ned about 8 to 20 amplified r,ag,nenls. Sets of DNA samples containing amplified r,c~""enls of iJe"tical size were selected and were rerun on polyacrylamide gelsas described in Example 2 until a conti~uous array of all the amplified r,ay",ents throughout the SgrA1 fragment was obtah~ed. The final r",ge,~u,int of the cosmidcontig is shown in Figure 3.
Resl, ic~ion r~ ay~nenl amplir~calion is an exr~llent tool for contig building of cos",ids however the level of overlap between two adjacenl cos",:~s cannot be determined using the enzyme con~b;r,ation as des~ ed above. The,e~re the DNA sdlllples of the cosmid contig were digested with EcoRI and Hindlll followed by Southem blot anaiysis aocord,"g to Southem J. Mol. Biol. 98 503-515. ~P-Iabelled SgrAI
r, dy" ,ent was used as a probe and the sizes of the overlapping r, dyments bet~cn two adjacent cosmid DNAs was dete~ ,"ed making use of size markers. Adjacent cosmids having an overlap of at least 5 kb were used for co",ple",enldlion analysis.

Fine mapping of the 18 AFLP .--~.k~r~ on the 255 kb SgrAI r,~ ~."e.)l The indirect end-label ",apping technique as des~ibed by Burke et at. (1987 Science 236 806 812) forthe restriction enzymes Mlul and Sall and using the left-end probe (see desaibed in example 5) was pe,fc"",ed to construct a more precise physical map of the 255 kB SgrAI r,dy",enl containing the l-2 r~sisl~ncegene. Pulse field gel eleclrophor~sis was pelrulllled under condiliuns that "~a~i",ise resolution in the range up to 100 kb.
To posilion the AFLP markers EM01 to EM18 on the physical map of the 255 kb SgrA1 fldy~llent two types of hy~idi~ation analysis were pelro,ll,ecl. First the W 097/06259 PCT~P96/03480 AFLP markers were used as probes on the Mlul and Sa/l partial digests and secondly the AFLP markers were used as probes on DNAs of the cosmid contig using slandard hybridization techniques as desuibed by Sambrook ef al. (in Molecular cloning: a laboralory manual, 1989, Cold Spring Harbor Labordtory c, Press).
A physical fine map for the enzymes Mlul and Sall of the SgrA1 fragment and the posilion of the 18 AFLP markers EM01 to EM18 is shown in Figure 4.

EXAMPLE 8: TRANSFORMATION

T.~ rer of cosmids to Agr~ ct~ri.Jm tL.n,efacie.,s The cosmid clones were introduoed in Agrobacterium tu",efac,ens through conjugative l,ansrer in a tri-parental mating with helper strain HB101 (pRK2013)essentially according to Deblaere et al. (1987, Metllocls in Enzymology 153, 277-292). E.coii were grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCI, pH 7.5) suppleme~,led with 5 mgll tetracyclin at 37~C. The helper strain HB101 (pRK2013) was grown under idenlical conditions in LB
medium supplemented with 100 mg/l kanamycin sulphate. Agrobacterium tu",erd~ ns strain C58C1 RifR (pGV3101) (Van Larebeke et al., 1974, Nature, 252,169-170) was grown in LB medium supple",entecJ with 100 mgll rifampicin at 28~C.Ove~"i~l ,t cultures were collected by oentrifugation at 4000 r.p.m. for 5 min and resuspended in LB medium without any supFlen,ent~, and 0.5 ml each of the Agr~bacterium culture, the helper strain culture and a cosmid strain culture were mixed and plated on LB agar plates without antibiotics. After ove" ,:;l ,t in~ ~h~tion at 28~C, the mixture was plated on LB agar plates containing 100 mgll rifampicinand ~ mg/l tetracyclin to select for single ll ansconjugant Agr~bacterrum colonies in serial p~-ss~ges through selective agar plates.

Chal~tt,.i~ation of A. tu.-.efaciens t,~.nscc,njugants Small-scale cultures were grown from selected colonies and grown in LB medium containing 10 mgll tetracyclin. rlas",id DNA was isol~ted by alkaline Iysis using WO 97/06259 PCT~P96103480 the method as described by Ish-Horowic~ et al. (1981, Nucl. Acids Res 9, 298 2997). and digested with Bglll using standard techniques. In adcJilion, resl,ic~ion rldylnent amplirica~ion on ",i.~iprep DNA of A. tumefaciens was performed using the enzyme combination EcoRI/Msel and pri",ers having no selective nucleotide 5 as described in Example 7. Subsequently, the ~39nl resl,i~1ion enzyme pattem as well as the DNA ringe,~rir~t of the A. tumefaciens transconjugant were co""~aredwith those of miniprep DNA of the E c~fi strain containing the cosmid. Only those A. tumefaciens t~dnsconjugants harbouring a cosmid with the same DNA pattem as the correspoudi"g E. c~li culture were used to llall~rul,ll a susceplible tomato 10 line.

Tra,.sfG.,..ation of a susceptible tomato line Seeds of the susceplible tomato line 52201 (Rijk Zwaan) were surface-slerili ed in 2% sodium hypochlorite for 10 min, rinsed three times in sterile ~istilled water, and placed on ge""i,)aliGn medium (consisling of half-sl,en~tl, MS medium according to Mu,dshige and Skoog (1962, Physiol. Plant. 15, 47~497), with 1% (wlv) suuose and 0.8% agar) in glass jars or polypropylene culture vessels. They were left to germinate for 8 days. Culture cond,tions were 25~C, a photon flux density of 30 ~mol.m 2.s-' and a photoperiod of 16 ~24 h.
T~"~ro""dlion of tomato was peiru""ed according to Koomneef et a/. (1987, In:
Tomato Biotecl " ,~'ogy, 169-178, Alan R. Liss, Inc.), and is des~ ibed briefly below.
Eight day old cotyledon e~lants were precuitured for 24 h in Petri dishes contai,)ing a feeder layer of Petunia hybnda suspension cells plated on MS20 medium (culture medium according to Mu,ashige and Skoog (1962, Physiol. Plant.
15, 47~497) with 2% (w/v) sucrose and 0.8% agar) supple",enled with 10.7 ~M ~-naphU ,alene~ ic acid and 4.4 ~M 6-benzylaminopurine. The ex~lants were then infected with the diluted ovemight culture of Agr~bactenum tL.",erdc,ens containing the cosmid clone M12, DD2, CC14, A52, B22, A55, CC16, A44, A29, CC3, AA2, M9, BB8 or the cosmid vector pJJ04541 for 5-10 min, blotted dry on sterile filter paper and coc~lhlred for 48 h on the original feeder layer plates. Culture WO 97/06259 PCT~EP96/03480 conditions were as desuibed above. Ove, I lighl cultures of AgrDbactenum tu",etdL,i~ns were diluted in liquid MS20 medium (medium according to Murashige and Skoog (1962 Physiol. Plant. 1~, 473 497) with 2% (wlvl) suaose pH 5.7) to an O.D.~oD of 0.8.

Following the cocultivation the cotyledon e~la~ ~ls were ll dnsr~ d to Petri dishes with selective medium consisting of MS20 supple~ented with 4.~6 ~M zeatin 67.3 ~M ~anw",ycin 418.9 ~M cefola~i"le and 171.6 ~lM kanamycin sulphate and cultured under the culture condilions des~ iL ed above. The explants were lO sllhcllltu~ed every 3 weeks onto fresh medium. Ellle~y;.lg shoots were ~l;ssected from the underlying callus and l,ansr~,ed to glass jars with selective medium without zeatin to forrn roots. The rulmalion of roots in a medium containing kanamycin sulphate was rt:garded as an inr~ic~tion of the l,d"sgenic nature of the shoot in question. Truly l,~nsgen~ regene~anls were p~upA~1ed in vitro by 15 s~lh~JI~llring the apical ",erislem and auxiliary buds into glass jars with fresh selective medium without zeatin.

Seed production Of every individual t,dnsrullllanl~ one or two clones were kept fn v~tro, while the 20 remaining shoots were potted in soil harclened off in a relative humidity >8~% at 20 ~C and a photoperiod of 16/24h for 7-10 days and l,d,~sre"ed to the y,eenhouse for seed prorl~ ~ction. Mature tomato plants were grown at 2~32 ~C ina ven~ilaled y~~nhouse with supplementary illumination from Son-T lamps (Philips Eindhoven The NeU,erlands). They were watered daily treated agai.,sl 25 mildew and Trialeurodes i"r~:,lalion and vibrated to ensure good seed set when flowering. Mature fruits were harvested the seeds e~ cted cleaned in 1% HCI for 1 h rinsed with water and dried.

W 097/06259 PCT~P96/03480 EXAMPLE 9: COMPLEMENTATION ANALYSIS

I.JG"urcaffon of cosmids ~nth the resistance gene by sc,~.l;..y for resistance in tra"srG..,.~J plants The l,d"s~",led plants (Ro plants) of the Irdr,sfo"nalion e~peri",ents were grown in the greenhouse for seed set as described in Example 8. For each of the cosmids M12 DD2 CC14 A52 B22 A55 CC16 A44 A29 CC3 M2 AA9 BB8 or pJJ04541 twenty Ro plants were grown. R, lines of at least nine Ro plants of each cosmid were tested for rJise~se s~""pton,s except for pJJ04541 in which case four plants were tested in order to identify cosmids with the /-2 resislance gene.
Twenty to 25 seecllinys of each R, line were inocu'~~ed and ev-'u~ted as described in Example 1. In total 144 R1 lines of the above mentiG,)ed 14 cli~,enl cosmid t(an~fo""ations have been tested; 13 cosmids contain tomato insert DNA
and one cosmid pJJ04541 is without insert DNA. The results are shown in Table 2. 128 trdnsge,)ic Ro plants appeared susoeptiL e bec~l~se their R, lines were completely or nearly completely (at least 80% of the seedlings) obviously rJiseA.se~l Sixteen Ro plants are resistant bec~use their R, lines were seg,egating.
Most of the R, lines sey,egaled in a ratio of about 3:1 ,esisl~nt and susc~ptil le seedlings. The remaining R, lines had a small number of susceplible seedlings (seg,egation ratios of 5:1 and 10:1) indicating the presence of more than one copy of the insert in the correspo"d"~g Ro plants. The 16 ,~sistanl Ro plants had been derived from lra"sro""ations with one of the following cosmids: A52 B22 and A55;these three cos,n.ls overlap with each other. These idenlified cosmids were usedforfurther",~ ec~ analysis.

PCT~P96103480 cosmid R4 plants resistant susoeptitlc CC16 o 15 pJJ04541 0 4 l~lolec!~laranalysis of the tra..ar~..-.~.J plants with a resistant phenotype To de",onsl,~le that the resislar,l phenotype of l,dnsgenic plants which were l(dn~ro""ecJ with one of the o~e, lapping cosmids A52 B22 and A55 is detemmined by the genomic insert presenl in the various cosmids an AFLP analysis with the AFLP r"a,ker~ EM04 EM06 and EM14 was ~e,ro""ed. The AFLP markers EM14 and EM06 were positio"ed on plasmids A52 and B22 wheress EM06 and EM04 were positioned on cosmid A55 (see Figure 5) DNA was isol-ted from young leaves of resistdnl as well as succ~plible R, plantsderived from a resi~lant Ro plant as desc i~ed by Bellldkki and Tanksley (1986 Theor. Appl. Genet. 72 314-321). Selective r~ tion fragment amplir,calion was W097/06259 PCT~P96/03480 performed with the primer combinations identifying the markers EM14 and EM06 for the R~o plants l,ansru,,,,ed with cosmids A52 and B22 and with the prime combinations identifying the markers EM06 and EM04 for the Ro plants Irar,sro""ed with cosmids A5~. The DNA finge"u,i"ls obtained showed in both 5 cases and for both markers the presence of the markers in the ,~sislanl plants and the absence of the markers in the suscepliLle plants inclicaling that the three iuJenlified overlapping cosmids A52, B22 and A55 contain the 1-2 resistance gene.

G,,~ ,atio,~ of the resistant ~I.e..Gtype in secor..l g~ rd~;~,..s of 10 l~ S~G~ plant~s Addilional genetic evidence for the presence of the 1-2 resislance gene on cosmids A52, B22 and A55 has been obtained in the next generalion of the R, plants.
Resistant plants of the Rl lines, I,dnsru,,,,ed with cosmid A52, BZ or A55 that seu,,egaled in a ratio of 3:1 (or higher) were grown in the greenhouse for seed set 15 as desuibed in Example 8. All plants were selfed and most of the plants were baclcuossed with the s--scepli61e line 52201, the originally l,dnsru,,,,ed tomato genotype in this invention, as the male parent. Of each resialanl Ro plant at least two R2 lines (resulting from selfing of R1 plants) and, if available, two R~BC lines (resulted from R~ backcrossed with 52201) were tested for Fusarium 2 resialancel20 in order to confil l" inherilance of the int, ugl t~ssed J-2 resislance gene. Seedlings of the R2 lines and R1BC lines were inocul-ted and evaluated as des~i~ed in Example 1.
At least half of the seedlings of eac~h tested line appeared to be resislant. Most R2 lines were segregating 3:1 or were completely r~sislanl, while most R1BC lines 25 were segregatillg 1:1 or were completely resislanl. The results indicate that R~
plants were either heterozygous for the /-2,~sislance gene (segr~galiGns of 3:1 in the R2 and 1:1 in the R,BC) or homozygous for the /-2 r~sblance gene (all seedlings in the R2 and R,BC were resialanl).

WO 97/06259 PCT~P96/03480 EXAMPLE 10: PHYSICAL MAP OF THE OVERLAPPING COSMID

Since the size of the cosmid inserts is within the range of 13-24 kb slanclard techniques such as single double and partial digestion analysis with reslliction5 enzymes which are present in the polylinker sequence of the cosmid vector werepe,ro""ed as desuibed by Sambrook et al. (in~ eu~~ cloning: a laboratory manual 1989 Cold Spring Harbor Laboralory Press). A physical map of cosmid A52 B22 and partially A~5 was constructed and the overlap between the three cosmids giving rise to a resislant phenolype (A52 B22 and A~) with respect to the 10 adjacent cosmid clone CC16 revealing a suscept;hle phenolype was dete,l"ined and is depicted in Figure 5.
The insert size in cosll,ids A52 and B22 could be c~lc-ul~ted and amount 23 and 17 kb respectively. It appeared that the plant insert of cosmid B22 complelely fits within cosmid A52. The minimal DNA segment contai". ~y the l-2 resislance gene 15 was def,ned by the lefl-end of cosmid A55 until the right-end of cosmid B22 e"co",passing a region of apprc.till~ ely 8 kb in size.

EXAMPLE 11: NUCLEOTIDE SEQUENCE AND DEDUCED AMINO ACID
SEQUENCE OF THE ~2 ~ l~ I ANCE GENE
20 Various r,ay",enls of the 8 kb DNA sey"~enl of cosmid B22 were subl_loned into the E. coli vector pBluescript (Sl,alagene La Jolla CA USA) using ~landa,.l techniques as des~ibed by Salllbruok et al. (in: Mol~u~'~ cloning: a laboratory manual 1989 Cold Spring ~larbor Laborat~ry Press) and used for sequence analysis making use of the rl ,a",~acia Autoread Sequencing Kit and the 25 rl,a""acia LKB AL.F. DNA Sequencer device (rl,a""acia LKB Uppsala Sweden). The n~lcleotide sequence of 6.~ kb ranging from appro~i",a~ely 600 nucleo~i.1es upstream of a Hindlll site until the 3 end of cosmid B22 was determined and is shown in Figure 6. A large open reading frame of 3798 nucleotides encoding a protein of 1266 amino acids could be cleduce~

WO 97/06259 PCT~P96/03480 EXAMPLE 12: TRANSCRIPT MAPPING
T,ansc(ip~ mapp~ng studies were pe,~u,,,,ed to map the 5 and 3 end of the l-2 resislance gene and to determine whether the l-2 ~sisla,)ce gene col,lp,ises anyintrons. The polymerase chain rea~ion to amplify parts of the l,anscripts from the 1-2 resistance gene was used for this purpose.
Total RNA from leaf tissue of the resistant tomato cultivar F~? was isol-ted using the hot phenol method as desuibed by SdlllbrOO'; et a/. (in: MoleuJ~~ cloning: alaboratory manual 1989 Cold Spring l~arbor Laboralory Press). Poly A+ RNA was isolated using biotinylated oligo(dT) bound to Dyn~be~s M-280 Streptavidin (DYNAL A.S. Oslo Norway) accord;,)g to the instructions of the manufacturer. A
cDNA library was constructed using the Super~ ipt RNase H Reverse T,a"s~iptase cDNA kit from Life tec~")olo~ies Inc. (Gait~e~burg MD USA) and the protocol supplied by the manufacturer.
The cDNA was used as template for oligonucleotide pri",er~ in various PCR
reactions. These rjri,ne~ were desiy,~ed based on the nuclaolide sequence provided in Figure 6 and six primer sets were used covering the coding sequenoe of the l-2 resislance gene. In addilion 5 and 3 RACE products were obtained using the Ma,dtl,ol- cDNA a",pl;rication kit from Clontech (Paolo Alto CA USA).
~S~ ~hsequently the various PCR f~ dyl "enls obtained with the 6 inte" ~al primer sets and the 5 and 3-RACE ~,dy",enls were cloned into the TA cloning vector pCRII
(Invitrogen Cw~o~a~ion San Diego CA, USA) and sequenced using ~lar,~ard pr~tocols The nuclaûtide sequences obtained were aligned with the 6.5 kb ge"o",.c sequence and three intron sequences could be ~e~luc~cl An intron of 86 nu s:~;des was located just upslredlll the ATG initidlion codon from nucleotide position 1703 to 1788 (Figure 6). The 5 end of the largest tr~ns~ ipt slelec1ed with the Ma,dU,on cDNA amplirication kit after analysis of 16 individual t,ansro""ants maps at nucleotide position 1597. Hence we conclude that the t(ans~iptional initiation site of the l-2 resislance gene is positioned at or u,u~ dlll of nucleoti~e 1597.
Two other intron sequences with a size of 399 and 82 nucleolicles were located in the 3 ut-l~anslated region at nucleotide posilion 5628 to 6026 and 6093 to 6174 respectively. The 3 end of the transcript was mapped at nucleotide position 6491based on the sequenoe the 3 end RACE product. Hence we conclude that the 1, dns~ iplional termination site of the l-2 resis~a, Ice gene is positioned at or immediatly downstream of nucleoticle 6491. A putative poly-adenylation signal (MUAAA) is located at nucleotide posilion 640~6411 appro~imalely 80 nucleotides upsl~ea,n of the t,ans~iptional tennination site and the stretch of U
resid~ ~es. The various introns and the ,t~osilio" of the l,a~s~iptional iniliation and te",~ination site are depicted in Figure 6.
The DNA sequence of the internal PCR products derived from cDNA as template indicated that the 1-2 resi~lance gene does not conlc.;n any introns within the coding region.

EXAMPLE 13: DNA SEGMENT COMPRISING THE ~2 Ktsl~lANcE
GENE.
Based on the physical map of the three overlapping cosmid clones cor"ui isi, ,9 the 12-r~sistance gene it could be deduced that the DNA sey",enl con~prising the l-2resislance gene was defined by the left end of cosmid A55 until the right end ofcosmid B22 encompassi"5a a region of appru~i"~alely 8 kb in size. One large openreading frame with a size of 3798 r u~lectirles encoding a protein of 1266 amino2 o acids could be deduced from this 8 kb region. Part of this 8 kb DNA sey",ent was ,et, ~"~ru" "ed into the susceptible tomato line 52201.
As sla,li"g vector for su~cloni"g of the DNA sey",ent the cointey,ate vector pKG1505 was used. This vector is a derivative of plasmid pGV1500 the prototype co.nlegr~ta vector ~les~ibed by Deblaer~ et a/. (1987 M~lhod~ in Enzymoloqy 153 277-292). The vector is based on the c~,l""o,- cloning vector pBR3Z. It contains a sl,~plo",ycin/spectinG",ycin resislance gene to be used as sel ction marker in Agrobacterium h""eÇdc~ns and r"..i"lana.,ce of the -c:nleg,al~
structure. The vector contains the left (LB) and right (RB) border repeat sequenoes of the oc~o~i. ,e TL-DNA. Vector pKG1505 differs from pGV1500 in that it contains 30 no residual T-DNA between the border repeat sequences but a nos-nptll-nos W O 97/06259 PCT~P96J03480 cassette for kanamycin resistance in plants instead and a synthetic polylinker sequence.
Using slancla~d techniques for the construction of recombinant DNA molecules as des~ibed by Sa~ look et al. (in: Molecular clon.ng: a laboratory manual 1989 Cold Spring tlarbor Laboratory Press) plas" ,. i pKG6016 (Figure 7) was constructed. Plasmid pKG6016 w"~prises a 6.2 kb ScallSan r,dg",ent from cosmid B22 comprising the coding sequence of the J-2 resisl~,~ce gene and a 1.3 kb upstream and 1.1 do\h, Is~ a~,, DNA region.
For this construction a 3.8 kb BamHllSatl f~ag,ne~l from cosmid B22 was cloned into pKG1505 resulting in plasmid pKG6014. This 3.8 kb DNA seg,nenl contains the second half of the l-2 resistance gene and the 1.1 kb 3 untranslated region.Moreover a 2.4 kb Scal/BamHI ~,ay"lent from cosmid B22 was cloned into the EcoRVlBamHI sites of pBluescript (St,dlagene La Jolla CA USA) resulting in plas" ~id pKG6015. S~ ~hsequently pKG6015 was cut with Xhol and BamHI and the 2.4 kb DNA segment containing the first half of the J-2 resislance gene including a 1.3 kb u,.)~llealll p(o~oter region was introcluced into pKG6014 resulting into plasmid pKG6016.

Plasmid pKG6016 was introd~ced into Agrobacterium tL",efdc,~ns strain C58C1RifR (pGV2260) (Deblaere et al., 1987 hllElllGcls in Enzymology 153 277-292) through conjugative t,ansrer in a tri-paren~al mating with helper strain HB101 (pRK2013) as described before. Selection was pel ru""ed on plates containing 300~ug/ml of spe~li"or"ycin and 100 ~lg/ml of slleptu",ycin.
The Agrobactenum tumefaciens l~ansconjugants were .;I,a,acteri~ed by Southem blot analysis of c~u~oso~al DNA using the Sm gene as a probe. Tl1ertf~,e chl-ul I IOSo, "al DNA was isol ~ed according to the , nethGcJ ~les~ i~J by Li~,lensl~ in and Draper (in: DNA cloning 1985 volume ll 67-119 D.M. Glover edit. IRL Press) digested with the l~sl,i~tion enzymes ~coRI or Hindlll and blotted onto a nitrocellulose me,~lL,rane (Gene Sueen Plus DuPont NEN Boston MA
USA). Those l,dnsconjugants containing one copy of the intey,dlive plas",;cJ were W O97/06259 PCT~P96103480 selected for lldnsru~ alion to the susce~tible tomato line 52201 using the ,urutocol as described before.

IndiYidual t,~r,sru""ar,ls were t~d"sre"ed to the greenhouse of which fifteen Ro5 plants were grown for seed production. R, lines of six Ro plants were tested for diseAce sy",pto",s. Twenty to 30 seedlings of each R, line were inoaJl~ed with Fusanum oxysporum f.sp. Iycopersici race 2 and evaluated: wilting plants were consider~d to be susceplible, whereas not wilting plants were considered to be ,t:sisla,)l. The observations indiGAted that the DNA segment is involved in the o resistance.

Claims (43)

1. A nucleic acid whose DNA sequence is at least part of the DNA
sequence provided in figure 6 or any DNA sequence homologous thereto.

2. The nucleic acid of claim 1 wherein said DNA sequence homologous to the DNA sequence of figure 6 is capable, when transferred to a host plant also liable of being rendered resistant by the DNA sequence of figure 6, of also rendering it resistant to Fusarium 2.

3. The nucleic acid of claim 1 which is capable when transferred to a host plant, which is susceptible to a plant pathogen, of rendering said host plant resistant to said plant pathogen.

4. A nucleic acid according to claim 1 wherein said DNA sequence corresponds to a coding sequence starting at nucleotide 1798 and ending at nucleotide 5598 or any DNA sequence homologous thereto.

5. A nucleic acid according to claim 1 wherein said DNA sequence corresponds to a promoter sequence located 5' upstream of nucleotide 1798 or any DNA sequence homologous thereto.

6. A nucleic acid according to claim 1 wherein said DNA sequence corresponds to a sequence starting at nucleotide 464 and ending at nucleotide 6658 or any DNA sequence homologous thereto.

7. A nucleic acid of claim 1 wherein said DNA sequence corresponds to at least part of the genomic insert present in cosmid B22, and preferably corresponds to the overlapping genomic DNA sequence between cosmid B22 and cosmid A55, or any DNA sequence homologous thereto.

8. A nucleic acid of claim 7 wherein said overlapping genomic DNA
sequence is defined by the left end of the genomic insert present in cosmid A55 and the right end of the genomic insert present in cosmid B22.

9. A recombinant DNA construct comprising a nucleic acid according to any of claims 1-8.

10. A recombinant DNA construct of claim 9 in which said nucleic acid is under control of a promoter which is functional in a plant cell, said promoter being either endogenous or exogenous to said plant cell, and effective to control the transcription of said DNA sequence in such plant cells.

11. A recombinant DNA construct of claim 10 in which said promoter corresponds to a promoter sequence located 5' upstream of nucleotide 1797 as provided in figure 6, or any DNA sequence homologous thereto.

12. A vector suitable for transforming plant cells comprising a DNA
construct according to any of claims 9-11.

13. Plasmid pKGI2-B22 as deposited under number CBS 546.95.

14. Plasmid pKGI2-A55 as deposited under number CBS 820.96.

15. Bacterial cells comprising a vector or plasmid according to any of claims 12-14.

16. Recombinant plant genome comprising, incorporated thereinto, a DNA construct according to any of claims 9-11.

17. Plant cells comprising a DNA construct according to any of claims 9-11.

18. Plant comprising plant cells according to claim 17.

19. Plant according to claim 18 which has a reduced susceptibility to Fusarium 2.

20. Plant according to claim 19 wherein said plant is tomato and wherein said Fusarium 2 is Fusarium oxysporum f.sp. lycopersici race 2.

21. Seed comprising a DNA construct according to any of claims 9-11.

22. The recombinant plant genome of claim 16, in a plant cellular environment.

23. Process for obtaining plants having reduced susceptibility to a fungus, comprising the following steps:
i) inserting into the genome of a plant cell a DNA construct according to any of claims 9-11, ii) obtaining transformed plant cells, iii) regenerating from said transformed plant cells genetically transformed plants, and iv) optionally, propagating said plants.

24. Process according to claim 23 further comprising selecting transformed plants having reduced susceptibility to said fungus.

25. Process according to claim 23 or 24 wherein said fungus is a soil bom fungus, and preferably a wilt inducing fungus.

26. Process according to any of claims 23-25 wherein said plant is tomato and wherein said fungus is Fusarium oxysporum f.sp. lycopersici race 2.

27. Process for protecting plants in cultivation against fungal infection which comprises:
i) providing the genome of plants with a DNA construct according to any of claims 9-11, and ii) growing said plants.

28. Process for isolating a nucleic acid according to claim 1-8, comprising the following steps:
i) screening a genomic or cDNA library of a plant with a DNA
sequence according to claim 1-8, ii) identifying positive clones which hybridize to said DNA sequence, iii) isolating said positive clones.

29. The process of claim 28 wherein said library originates from a first plant and the DNA sequence belongs to a second plant.

30. Process of selective restriction fragment amplification for identifying a nucleic acid according to claim 1-8 using primer combinations identifying at least one of the AFLP markers EM01 to EM18.

31. The process of claim 30 wherein said primer combination identifies AFLP marker EM06.

32. An oligonucleotide comprising a DNA sequence which corresponds to at least part of the nucleic acid according to claim 1-8.

33. The oligonucleotide of claim 32, which is of a size sufficient to hybridize selectively to the DNA sequence of any of claims 1 to 8 under stringent hybridization conditions.

34. An oligonucleotide according to claim 32 wherein said DNA
sequence corresponds to the sequence starting at nucleotide 3470 and ending at nucleotide 3565.

35. An oligonucleotide according to claim 32 wherein said DNA
sequence is located at the 3'end, and preferably corresponds to the sequence 5'-AATTCAGA-3', which can prime the synthesis of DNA.

36. An oligonucleotide according to claim 32 wherein said DNA
sequence is located at the 3'end, and preferably corresponds to the sequence 5'-TAATCT-3' which can prime the synthesis of DNA.

37. A primer combination comprising a first oligonucleotide according to claim 35 and a second oligonucleotide according to claim 36.

38. Diagnostic kit comprising at least one oligonucleotide according to any of claims 32-36.

39. Diagnostic kit comprising a primer combination according to claim 37.

40. Process for detecting the presence or absence of a DNA sequence according to claim 1-8, particularly in a plant DNA using a diagnostic kit according to claim 38 or 39.

41. A polypeptide having an amino acid sequence having the sequence provided in figure 6 or coded by the corresponding homologous sequence according to claim 1 or 2.

42. Process for the identification of elicitor molecules using the polypeptide according to claim 41 as a receptor molecule.

43. A RNA having a ribonucleic acid sequence of a transcript of part or all of the DNA sequence of claim 1 or 2.