WO1996032007A1 - Transgenic tomato plants containing a fusarium resitance gene - Google Patents

Abstract

The invention provides genes from the I2 Fusarium resistance locus of tomato belonging to a multigene family herein designated I2C. The DNA molecules of the invention are useful as a tomato resistance gene to plant vascular diseases caused by Fusarium pathogens, particularly Fusarium oxysporum f.sp. lycopersici race 2, or as probes for breeding Fusarium-resistant tomato lines or for screening of new diseases in plants of the Solanaceae family. Further provided are Fusarium-resistant tomato lines transformed by an I2C resistance gene of the invention.

Description

Transgenic Tomato Plants Containing a Fusarium Resistance Gene

FIELD OF THE INVENTION

The present invention relates to genes from the 12 Fusarium resistance locus of tomato belonging to a multigene family herein designated I2C, useful either as a tomato resistance gene to plant vascular diseases caused by Fusarium pathogens, or as probes for breeding Fusarium resistant tomatoes or for screening of new diseases in related plants of the Solanaceae family, and to transformed plants, particularly Fusarium resistant tomatoes.

BACKGROUND OF THE INVENTION Resistance to pathogens is thought to involve a specific recognition between a resistant plant and the pathogen, which triggers a set of responses that act to confine the pathogen. The specificity of this process is considered to involve a recognition between the products of a plant resistance (R) gene and a cognate pathogen avirulence gene (Dangl, 1995; Staskawicz et al., 1995). The characterization of resistance genes is of major importance for elucidating the initiation of the cascade of events that leads to specific resistance responses, as well as for more efficient introduction of resistance to pathogens into important crops.

Several resistance genes have been cloned recently by positional cloning or by transposon tagging. These genes include: the HM1 gene of maize (Johal and Briggs, 1992), the Flo gene of tomato (Martin et al, 1993), the Cf-9 gene of tomato (Jones et al., 1994), the RPS2 (Bent et al., 1994; Mindrinos et al., 1994) and the RPM1 (Grant, 1995) genes from Arabidopsis , the N gene from tobacco (Whitham et al., 1994), and the L6 96/32007 ₂ PCMJS96/05272

gene from flax (Ellis et al., 1995; Lawrence et al, 1995) These resistance genes show diverse biological characteristics. The HM1 gene is the only example to date where the gene product acts directly to inactivate a component of the pathogen attack, or a compatibility factor (Briggs and Johal, 1994). The other genes belong to a different genetic category, that of incompatibility (or gene for gene) interaction, based on the recognition by the resistance gene product of an avirulence (or incompatibility) component of the pathogen, which does not necessarily participate in the compatibility or in the infection processes (Briggs and Johal, 1994). These genes are all involved in resistance processes characterized by hypersensitive response (HR). In spite of their origin from different plant species, and their divergent specificity to viral, fungal or bacterial pathogens, a group of these R genes share several structural features. A nucleotide-binding domain (P-loop) and five additional amino-acid stretches of unknown function are conserved in their N-terminal region. A region of leucine-rich repeats (LRR) is present in their C terminus, though the consensus sequence and the length of the repeats are different among them. LRR were shown to be involved in protein-protein interactions in other proteins (Kobe and Deisenhofer, 1994; Kobe and Deisenhofer,

1 95), and may have similar role in resistance genes. The N gene, the L6 gene and the

Cf-9 gene were shown to belong to large gene families, partially clustered with the resistance gene. The detailed genomic distribution of these multigene families is yet unknown.

The soil-born fungus Fusarium oxysporum is the causative agent of severe wilt diseases in a large variety of plant species world-wide. It is an imperfect fungus for which no sexual cycle is known. The tomato-specific pathogen Fusarium oxysporum f sp. lycopersici (F.o.l) causes the disease Fusarium wilt. The fungus penetrates the vascular system of roots from both resistant and susceptible varieties, mainly through wounds. During a compatible interaction, which leads to disease, the fungus proceeds through the vascular system which eventually collapses. This leads to wilt and often to death of the plant. During an incompatible interaction, resulting in resistance, the fungus is confined to the lower part of the roots, and further symptoms do not develop. Several mechanisms, not including HR, were suggested to be involved in this resistance. They include: the production of inhibitory secondary metabolites, and structural barriers such as vascular gelation, callose deposition, and abnormal membrane outgrowths of vascular parenchyma cells, termed tyloses. Most of these processes, thought to be involved in resistance to vascular diseases, are detectabe also in compatible interactions, though to a lesser extent. Therefore the exact sequence of events that leads to resistance is still unknown. Three races of F.o.l. and their cognate R genes have been identified in tomato.

The classification of different F.o.l. isolates into races does not correlate with their general genetic resemblance, as established by restriction fragment length polymorphism (RFLP) analysis and distribution into vegetative compatibility groups (VCG; Elias et al., 1993). The / locus, introgressed from L. pimpine/lifolitim, confers resistance to F.o.l race 1, and is located on the short arm of chromosome 1 1, between the RFLP markers TG523 and CP58 (Eshed and Ori, unpublished). The /5 locus from chromosome 7 of L. pennellii confers resistance to races 1, 2 and 3 of F.o.l. (Bournival et al., 1990). This locus appears to be composed of three separate but linked genes (Scott and Jones, 1991). The 12 locus, introgressed from L pimpinellifolium, confers resistance to race 2 of the pathogen. We previously mapped 12 to the long arm of chromosome 1 1, between the RFLP markers TG105 and TG36, very close to TG105 (Segal et al., 1992; Ori et al., 1994). In previous studies we utilized recombinant inbred (Rl) lines for mapping 12 (Ori et al., 1994). However this population turned to be problematic for mapping of this region because of a very high recombination rate, including double recombinations, especially in the region of/2.

SUMMARY OF THE INVENTION

It has now been found in accordance with the present invention that high resolution genetic and physical mapping of the 12 region, using a large and conclusive F2 population (3200 meiotic gametes), show complete cosegregation between 12 and a cluster of genes on chromosome 1 1 belonging to a new multigene family, herein designated I2C.

Additional multigene family members are dispersed between four different loci, on three different chromosomes, either in clusters or as single genes. Two I2C genomic clones were isolated from the locus completely linked to 12 and sequenced, and were herein designated 12C-J and 12C-2. Their sequences show striking structural similarity with a group of recently isolated resistance (R) genes, which includes the above- mentioned RPS2 and the liPMl genes from Arabidopsts, the N gene from tobacco and the L6 gene from flax These genes confer resistances to specific pathogens of viral, bacterial and fungal origin, and share common features They contain a conserved nucleotide binding domain, termed P-loop, in their Ν terminus, and five other conserved domains of unknown function At least half of their C terminus is composed of leucine rich repeats (LRR)

A few partial cDΝA clones from the I2C family were further examined, such as the herein designated 12C-3 and I2C-4 cDΝA clones, and show that family members differ from each other mainly by insertions or deletions The deduced amino acid sequence encoded by members of this gene family reveals a region of LRRs, as well as a P-loop and other motifs in common with the above-mentioned recently characterized plant resistance genes

Thus, in one aspect, the present invention provides a D A molecule selected from the group comprising (l) a DΝA molecule having a nucleotide sequence derived from the coding region of the clone herein designated 12C- 1 (SEQ ID NO 1 ),

(ii) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated I2C-2 (SEQ ED NO 2),

(in) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated 12C-3 (SEQ ID NO 3),

(iv) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated I2C-4 (SEQ ID NO 4),

(v) a DNA molecule characterized by containing a coding sequence representing at least 60% similarity with the encoded open reading frame in the DNA sequence of at least one of the DNA molecules (I) and (n),

(vi) a DNA molecule capable of hybridization with any one of the DNA molecules (ι)- (v) under moderately stringent conditions,

(vπ) a DNA molecule that differs, by insertion, deletion or as a result of the degenerative nature of the genetic code, from the DNA sequences (ι)-(vι), and (viu) a fragment of any of the DNA molecules (ι)- (vn)

The DNA molecule defined in (v) above contains preferably a coding sequence representing 70-80% similarity with the encoded open reading frame in the DNA sequence of at least one of the DNA molecules (i) and (ii). The moderately stringent conditions required in (vi) above are such as those conditions described in Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, New York, 1989. One of the members of the multigene family I2C consisting of a DNA molecule as defined in (i)-(vii) above will confer resistance to Fusarium oxysporum f.sp lycopersici race 2 in tomato plants. In another aspect, the invention relates to a gene construct comprising such DNA as a genomic clone including regulatory sequences that flank the coding region thereof, and to a cosmid, into which said gene construct has been subcloned, for direct transformation of tomato plants.

In another embodiment, a DNA molecule according to the invention may be subcloned into a plant transformation vector under the control of regulatory elements capable of enabling the expression of said DNA molecule in plant cells. Said DNA regulatory sequences comprise, for example, a plant promoter, a DNA sequence that enhances translation of the mRNA transcribed from said DNA molecule and a po 1 y adeny 1 ation/term inator sequence .

In a further embodiment the invention provides a tomato cell line or a tomato plant line transformed with a cosmid or with an expression vector of the invention, and to tomato plants regenerated from said transformed cells In another aspect of the invention, a DNA molecule or fragment thereof as defined in (i)-(viii) above may be used as a direct RFLP probe employing standard protocols for breeding tomatoes resistant to Fusarium oxysporum f.sp lycopersici race 2, or to examine the homologous multigene family in related plants of the Solanaceae family, e.g. potato, pepper, petunia, eggplant, preferably plants which have colinear genomic maps with tomatoes, for finding new species-specific disease linkages with said probes. The thus bred tomato plants and related plants of the Solanaceae family are also encompassed by the present invention.

Thus the invention provides a method of selective breeding of Fusarium resistant tomatoes employing a DNA molecule according to the invention as a direct restriction fragment length polymorphism (RFLP) probe, which comprises:

(i) marking said DNA molecule with a suitable marker; and (ii) reacting said probe of (i) with DNA extract of a tomato plant under hybridization conditions; thus obtaining restriction-length polymorphism that is indicative of a resistance-type gene, which facilitates the selection of progeny that contains said resistance gene.

The invention further provides a method of screening new diseases in plants of the Solanaceae family employing a DNA molecule according to the invention as a direct RFLP probe which comprises:

(i) marking said DNA molecule with a suitable marker; and (ii) reacting said probe of (i) with DNA extracts from said Solanaceae species, thus identifying the homologous gene family in those species which can be linked to known resistance genes in those species.

DESCRIPTION OF THE DRAWINGS

Figs. 1A-C depict genetic and physical maps of the tomato gene 12 region. (1A) Genetic linkage map of chromosome 11, adopted from Eshed et al. (1995). The / and 12 Fusarium resistance loci were positioned according to Eshed (unpublished) and Segal et al. (1992), respectively; (IB) High resolution mapping of the genetic region spanning RFLP markers TG105A and TG36, as revealed from analysis of 1600 F2 and F3 individuals; (1C) Physical map of YAC 340-63, with relevant markers indicated. The total length of YAC 340-63 is 350 kb.

Fig. 2 shows Southern blot analysis of genomic tomato DNA of resistant and susceptible parental types and of the fixed recombinant F2 plant BR 30(5). DNA samples were digested with Taql, and the blot hybridized with SL8 probe. R and S indicate F.o.l race 2 resistant and susceptible individuals, respectively R lanes, parental types which are a nonrecombinant resistant type F2 individual from the F2 population initiated from

Br5577; S lanes, parental lines which are the sensitive tomato inbreds L. escuientum var.

M82 and L. escuientum var. S-365. BR 30(5) is the single recombinant identified within the SL8 cluster, from the entire F2 population. TR1-TR8 indicate resistant-type polymorphic bands, as established by examination of this and additional gels. Sizes in kb are indicated on the right. Figs. 3A-B show distribution of SZ-S-homologues in the tomato genome. (1A) Linkage maps of chromosomes 8, 9 and 1 1. The linkage maps were adopted from Eshed et al. ( 1995). The relevant introgressed regions of the ILs are illustrated on the right of each tomato linkage map (solid lines). Asterisks indicate approximate map positions of known disease resistance genes. The mapped positions of the SL8 loci are indicated; (IB) Southern blot of TaqI digested DNA of representative ILs. L. pentie/lii fragments in the blot are designated A, B, C, D and E according to their genomic location, as indicated in panel A.

Fig. 4 depicts the nucleotide sequence and deduced amino acid sequence of the clone herein designated I2C-1 [SEQ. ID. NOS: l and 5, respectively]. The first translated nucleotide is no. 1. Sequences conserved between resistance genes are double underlined.

Leucine Rich Repeats (LRRs) region and other AA repeats are single underlined. A putative Leucine-Zipper domain is underlined with dots.

Fig. 5 depicts the nucleotide sequence and deduced amino acid sequence of the clone herein designated I2C-2 [SEQ. ID. NOS:2 and 6, respectively]. The first translated nucleotide is no. 1. Sequences conserved between resistance genes are double underlined.

Fig. 6 depicts the partial nucleotide sequence of the 3' of the cDNA clone herein designated I2C-3. [SEQ. ID. NO:3]

Fig. 7 depicts the partial nucleotide sequence of the 3' of the cDNA clone herein designated I2C-4. [SEQ. ID. NO:4]

Fig. 8 shows comparison of the deduced amino acid sequences of the genomic clones I2C-1, I2C-2 [SEQ. ID. NOS:5 and 6] and of the resistance genes RPS2 and RMP1 from Arabidopsis, N from tobacco and L6 from flax (Bent et al., 1994; Dangl, 1995; Grant, 1995; Jones et al., 1994; Mindrinos et al., 1994; Lawrence et al., 1995; Whitham et al., 1994) [SEQ. ID. NOS:7, 8 and 9]. Residues numbers are from the first translated methionine of each sequence. Consensus sequence in the N terminal region is indicated only when minimum number of gaps was needed for alignment of at least 5 out of 6 residues. Symbols are: con., consensus sequence; a, aliphatic residue; - and +, negatively or positively charged residues, respectively. Boxes containing stretches of conserved residues are indicated by a line above the sequences, and numbered from I to VI Fig 9 shows alignment of the leucine reach repeats (LRR) of I2C-1 [SEQ ID NO 5), and their consensus sequence (Top) Alignment in the region from residues 558 to 1220 of 12C-1 where alignment to consensus sequence is optimized, (Bottom) A comparison of the consensus sequences of the 12C-1 LRR with those of the resistance genes RPS2, N and Cf-9, and the T-LR SAG expression site associated leucine rich protein from Tiypano oma brucei α represents an aliphatic residue

Fig 10 shows comparison of the 3¹ end of four 12C family members 12C-1 and 12C-2 are the deduced ammo acid sequences [SEQ ID NOS 5 and 6] as in Fig 8 /2C-5 and 12C-4 are the deduced amino acid sequences [SEQ ID NOS 10 and 1 1 ] derived from partial cDNA clones from a λgt 10 library The sequence of 12C-4 is a chimera between three ORFs, originally separated by one base insertions which caused two frame shifts The junctions where separated ORFs were combined are indicated by arrows Con indicates consensus and is shown when a residue is present in all 4 sequences Numbers are from the first methionine in sequences I2C-1 and 12C-2 [SEQ ID NOS 5, 6] and from the first residue of the available sequence for 12C-3 and 12C-4 [SEQ ID NOS 10 and 1 1 ] Brackets indicate a repeat unit of 23 amino acids which appears in variable copy number in the 3' end of the cDNA clones

Fig 1 1 depicts cosmids 12-134 and 12-150, which contain the genes 12C- 1 and I2C-2, respectively, in the BamHl site of cosmid TDNA 04541 Fig 12 shows sense constructs from the 12C-134 cosmid, which contains the genomic clone 12C-1 prepared in the PGA492 binary vector

Fig 13 shows sense (2-1 and 5-1 ) and antisense (6-3 and 3 1-17) constructs comprising the genomic clone I2C-1 or the cDNA clone I2C-3 for transformation of Fusarium resistant plants

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a multigene family, I2C. which is dispersed on three different chromosomes of the tomato genome Two lines of evidence suggest that a member of this gene family is the 12 Fusarium resistance gene The first is the complete cosegregation of some of the genes from this family with the 12 gene, the second is the striking structural similarity between members of this family and a group of recently isolated plant resistance genes (Bent et al , 1994, Dangl, 1995, Grant et al . 1995, Jones et al., 1994; Mindrinos et al., 1994; Lawrence et al., 1995; Whitham et al., 1994). In addition, I2C genes from the SL8D locus of the family, which maps to 12. were shown here to be very highly polymorphic between F.o.l. resistant and susceptible varieties. In a similar fashion, the Pto resistance gene was also shown to be a gene family highly polymorphic between bacterial speck resistant and sensitive varieties (Martin et al., 1993). Which of the family members are responsible for resistance can be determined by extensive complementation tests with all members of the SL8D cluster.

The I2C gene family contains a few motifs that have been identified in plant resistance genes. The N terminus contains a P-loop and 5 additional conserved boxes of unknown function. Different classes of P-loop motifs are common to many but not all nucleotide binding proteins (Saraste et al., 1990) . The consensus of this motif varies significantly between different classes of nucleotide binding proteins, but is highly conserved within each class. The consensus sequence GMGGaGKTT, where a designates an aliphatic amino acid, is highly conserved among the P-loops of the genes 12C-1, 12C- 2, RPS2, RPMl, N and L6. No other protein in the gene bank were found to contain this consensus sequence. In contrast to the Cf-9 gene, and perhaps the RPS2 gene, the 12C-1 deduced protein sequence does not predict any membrane-spanning domain. Residues 623 to 645 of 12C-1, included in the LRR region, fit the consensus for a leucine zipper (Busch and Sassone-Corsi, 1990) .This motif is considered to be involved in dimerization of DNA binding proteins. However, sequences that fit this consensus are abundant in the databanks, and the existence of this consensus does not necessarily imply a function.

In common with recently isolated plant resistance genes, the C-terminal parts of the 12C genes are composed of leucine rich repeats. The LRR consensus comprising 23 amino acids, together with a lack of a membrane spanning domain in the gene, are consistent with an intracellular location of this gene family (Jones et al., 1994). The LRRs of four members of the I2C family show high homology to each other and differ from each other mainly by insertions or deletions. This may be indicative of evolutionary processes, and hint at mechanisms that generate new diversity of LRR. Interestingly, one of the cDNA analyzed, 12C-4, contains stop codons within the LRR region, and therefore may result in a truncated protein. This is reminiscent of phenomena described for the N and the L6 genes, where truncated transcripts were described, apparently arising from alternative splicing. In the case of 12C-4 this mechanism is less likely, since the genes analysed thus far from the family appear to lack introns. The 12C LRR-region consensus is homologous to the trypanosome variable surface glycoprotein (VSG) expression site associated gene T-LR This gene is thought to be involved in the regulation of adenylate cyclase function (Ross et al , 1991 ; Smiley et al, 1990). LRR were described recently in many proteins, and may be involved in protein-protein interactions (Colicelli et al., 1990, Kobe and Deisenhofer, 1994) Thus, the LRR region may be responsible for specificity of interaction, either with a protein component from the pathogen, or with downstream factors involved in signal transduction The crystal structure of an interaction of an LRR containing protein, an RNAase inhibitor (Rl), with RNAase, was recently described (Kobe and Deisenhofer, 1995). The Rl contains a horse-shoe like structure in which individual, 28-29 residues long repeats constitute b-a hairpin units which are aligned parallel to a common axis (Kobe and Deisenhofer, 1993) However, the LRR consensus of 12C and of the other plant resistance genes differ from that of Rl. In addition, the repeat length of I2C varies between 19 and 32 amino acids per repeat. Similar variation in repeat length can be observed in other R genes, which may imply a less organized or different structure than that found for Rl.

Using novel tomato genetic populations (Eshed et al., 1992; Eshed and Zamir,

1994; Eshed and Zamir, 1995), all members of the 12C family have been mapped according to the present invention. The 12C genes are distributed to five locations in the genome, two of which are clusters of several genes, both on chromosome 1 1. Some of the recently isolated resistance genes were also shown to be members of large, clustered, gene families, but complete mapping data for them is lacking. The complex pattern of distribution of I2C is remindfull of the case of L and the M rust resistance genes of flax

(Ellis et al., 1995) In that case, L appears to be a single multiallelic gene, whereas homologous sequences map to a more complex M locus containing a gene cluster. Both loci may contain resistance genes specific for different races of the same pathogen, or to different pathogens It is interesting to note in this respect, that the 12C copy from chromosome 9 (SL8B) maps with a resolution of 5 cM to both the Tm-2a TMV resistance gene (Young et al., 1988), and the Frl Fusarium oxysporum f sp. radicis lycopersici (F.o.r.l.. Laterrot and Moretti, 1995). The 12C cluster SLSC maps in the vicinity of the

S Slemphylium resistance gene, with a resolution of 10 cM (Behare et al., 1991 )

However, no member of the J2C family maps to the / locus on the short arm of chromosome 1 1 , or the 13 locus in chromosome 7 (Bournival et al., 1990; Scott and Jones, 1991 ), which confers resistance to races 1, 2 and 3 of F.o.l.

Considering that a member of the I2C family encodes for the 12 resistance gene, the present invention shows commonalties between a wilt disease resistance gene and other disease resistance genes. Despite the lack of HR in vascular disease resistance, the

12C family belongs to the superclass of resistance genes described for leaf HR. This raises questions concerning the role of the various functional domains of R genes in upstream and downstream events that result in different types of resistance mechanisms.

For transformation of tomato plants, a genomic 12C clone according to the invention may be subcloned into any suitable cosmid, such as cosmid TDNA 04541

(Jones et al., 1992) Such constructs contain more than a few kb of genomic DNA upstream and downstream from the gene coding region sufficient for regulated expression. These constructs can be used for direct DNA transfer into plant cells by electroporation (Dekeyser et al., 1990); by polyethyleneglycol (PEG) precipitation (Hayashimoto et al., 1990), by balistic bombardment (Gordon-Kahn et al., 1990), or by

Agrobacterium-mediated transformation (Jones et al., 1992).

Other engineered constructs according to the invention comprise a DNA molecule of the invention and DNA regulatory elements enabling the expression of said DNA molecule in plant cells . Said DNA regulatory sequences comprise, for example, a plant promoter, a DNA sequence that enhances translation of the mRNA transcribed from said

DNA molecule and a polyadenylation/terminator sequence.

The plant promoter used in the invention is selected from tissue specific and non- tissue specific plant promoters of different kinds, derived from both mono- and dicotyledoneous plants. The preferred promoter is the commercially available cauliflower mosaic virus (CaMV) 35S promoter that is generally expressed in most, if not all, plant tissues, including vascular tissues. Another example of promoter expressed in vascular tissues that can be used in the invention is PRB-lb (Eyal et al., 1993)

The promoter is to be found in the 5' region of the gene. At the 3' end of the promoter, a short DNA sequence for 5' mRNA non-translated sequence may be added to enhance translation of the mRNA transcribed from the gene, such as the omega sequence derived from the coat protein gene of the tobacco mosaic virus (Gallie et al., 1987). Downstream at the 3' end of the resistance gene DNA coding sequence a terminator DNA sequence containing the 3' transcription termination and polyadenylation signal of the mRNA from the resistance gene is installed. Terminator DNA sequences comprised within the 3 ' flanking DNA sequences of any cloned genes can be used, such as the 3' untranslated sequence of the octopine synthase gene of the Ti plasmid of Agrobacterium tumefaciens (Greve et al., 1983), or more preferably the 3' untranslated sequence of the nopaline synthase gene (Depicker et al., 1982).

The gene constructs of the invention can be subcloned into expression vectors, such as the Ti plasmids of Agrobacterium tumefaciens, the preferred plasmid being the pGA492 binary vector (An., 198^*6).

The expression vector comprising the resistance gene is then introduced into plant cells by a transformation protocol capable of transferring DNA to dicotyledoneous plant cells, preferably by infection of plant cells with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et al., 1985). For this purpose, tomato leaf explants are infected and the transformed tomato cells are cultured on a suitable medium, preferably a selectable growth medium. Tomato plants can then be regenerated from the resulting callus. Tissue cultures of transformed tomato cells are propagated to regenerate differentiated transformed whole plants. Transgenic plants are thereby obtained whose cells incorporate a Fusarium resistance gene in their genome, said gene being expressible in the cells. Seeds from the regenerated transgenic plants can be collected for future use. Transformed plants that are resistant to Fusarium oxysporum f.sp. lycopersici race 2 can be selected by incorporating a selectable marker such as resistance to kanamycin.

The DNA molecules of the invention can further be used as markers in selective plant breeding as direct RFLP probe as exemplified in Examples 2 and 3 and Figs. 2 and 3 herein, or small fragments thereof can be used for PCR-based technology in marker assisted breeding (review by Tanksley et al., 1995).

In the RFLP probe technology, the DNA from different varieties of tomatoes or related Solanaceae plants is digested by restriction enzymes and fractionated on an agarose gel. Digests are chosen such that the gene of interest will be polymorphic. The DNA fragments are transferred to a nitrocellulose or other similar blotting agent and hybridized to the I2C probe of the invention, preferably under conditions of 6xSSC, 0.5% SDS. at 65°C. The blots are washed, preferably at 2xSSC at room temperature, and subjected to autoradiography. Further washings at higher temperatures and lower SSC concentrations can be carried out for higher stringency.

The individual DNAs in the population that show the polymorphic signature of the resistance gene can then be further used in a breeding program for the desired traits. An advantage of using these DNA sequences as direct RFLP probes in selective plant breeding is that the presence of disease resistance in plants can then be examined without using phytopathological methods. In addition, by using a direct or tightly-linked DNA marker as probe (based on RFLP or PCR-based technologies), it is possible to select for the desirable trait, i.e. resistance gene without accompanying genetic drag, i.e. transfer of the desired trait by breeding without incorporation of flanking unwanted traits.

The DNA molecules of the invention can further be used as probes to identify homologous multigene families conferring resistance to different diseases in related plants of the Solanaceae family, such as those which have colinear genomic maps with tomatoes, e.g. potato, eggplant, pepper, petunia and the like, for example, by using a marker of the I2C-1 family as a DNA probe to clone the related gene family from said related Solanaceae species, and then using that clone or I2C members directly as DNA probes to analyze genetic lines of those species for RFLP linkage to resistances in said species. In the PCR-technology, a preferred approach uses specific oligonucleotides synthesized according to the sequences of selected regions of the I2C family such that the fragment generated in a PCR reaction will yield a polymorphic band for the resistance trait either by being specific for the resistance-type gene and therefore yielding a null PCR reaction in sensitive plants, or by yielding PCR fragments of different size or different restriction patterns upon using sensitive or resistant sources of the DNA.

The invention now will be illustrated according to the following non-limiting Examples and the drawings herein.

EXAMPLES In the Examples, the following Materials amd Methods are used. MATERIALS AND METHODS

(a) Plant material and genetic mapping

Four L. escuientum segregating F2 populations were used for genetic mapping. In each case, an initial cross was made between a parent resistant to F.o.l race 2 (R), and a susceptible parent (S) The resistant and susceptible parents for the first 3 populations were, respectively, c.v. Motelle and c v. Money Maker; c v Mogeor and c v. Vendor; and c.v. Motelle and LA 1 1 13 (chromosome 1 1 marker stock, kindly provided by Dr CM Rick (UC Davis). The fourth population was initiated from the commercial hybrid line Br5577 (AB Seeds, Ness Ziona, Israel), as FI . The results obtained from the 4 populations were pooled.

(b) Genomic and cDNA libraries, plasmids and probes

YAC8 (YAC 340-63, Cornell collection), which contains the RFLP marker TG105A, was generated from the F.o.l race 2 resistant tomato line Rio Grande - PtoR, and cloned in the vector pYAC 4 (Martin et al., 1992). Probes from YAC 340-63 that were used for the genetic and physical mapping are as follows. D2 is from a genomic lambda library of the tomato line VFNT cherry, selected during chromosome walking from TGI 05. SL8, SR8 and 6-16, are subclones of 1EMBL3 clones from a library of the yeast line that contains YAC 340-63. D 14 is a cDNA clone selected by YAC 340-63 from a cDNA library from roots of the F.o.l. race 2-resιstant tomato L. escuientum c.v Mogeor. Additional clones, previously described (Ori et al, 1 94), were not polymorphic or not informative in these populations cDNA clones which represent members of the 12C family were isolated from three different cDNA libraries: The cDNA libraries were all constructed in λgtlO, from roots or leaves of resistant-type L escuientum. Positive clones were equally abundant in the three different libraries While large (>3 kb sizes) clones have been previously isolated from these libraries, all the SL8 clones were partial and contained only the 3' end of the genes Cosmid clones were isolated from a genomic library of the F.o.l race 2 resistant variety L. escuientum var Mogeor. constructed in the cosmid TDNA 04541 (Jones et al.. 1992). (c) Sequence analysis

Cosmid clones were either subcloned into the Bluescript plasmid or sequenced directly. The cDNA clones were subcloned into the Bluescript vector and then sequenced. Sequencing was performed with an automated Applied Biosystems Sequencer. Sequence analysis was performed using the sequence analysis software package of the 'University Wisconsin, Genetics Computer Group' (Devereux et al, 1984).

(d) Physical mapping

Yeast DNA for pulse field gel electrophoresis analysis was digested with limiting amounts of the restriction enzymes Mlul, Xhol and Sail, to obtain successive partial digestions. The digests were fractionated on counter clamped homogeneous electric field (CHEF) gels (BioRad), blotted and hybridized with probes. The maximal distance between a pair of markers was estimated according to the smallest partial band that contained both markers. Additionally, the DNA was digested with the rare cutters SgrAl and Pmel.

(e) Genetic mapping

RFLP analysis and F.o.l. inoculation were performed as previously described (Segal et al., 1992). F2 plants ( 1200) were screened for recombinants between TGI 05 and TG36. When necessary, F3 seedlings from the recombinant plants were screened in order to fix the recombination to a homozygous state. An additional 400 F3 plants were screened, and more recombinants were identified in the region. All the recombinants were analyzed for the different RFLP markers and for Fusarium resistance.

Example 1. High resolution mapping of 12 The 12 Fusarium resistance gene was previously mapped to chromosome 1 1 , between RFLP markers TG105A and TG36, 0.4 centimorgan from TG105A (Figure 1A, and Segal et al., 1992). To obtain higher resolution mapping of 12, we generated new markers in the region of TG105A, by chromosome walking from TGJ05A on lambda clones, and by subcloning a 350 kb YAC clone, YAC 340-63, that hybridized to TGI 05 A. Pulse Field Gel Electrophoresis (PFGE) of YAC 340-63 was used to physically position genetically informative markers, as shown in Figure lC. In order to localize the position of 12 relative to the new markers, a segregating population of 1600 plants (F2 and F3) was screened for recombinations between TG105A and TG36, and 57 recombination events were detected. The recombinant plants were then tested for F.o.l race 2 resistance, and for RFLP markers located between TG105A and TG36. According to the resulting map (Figure IB), 12 maps to the multi-copy marker SL8D, which represents the edge of YAC 340-63 (Figure 1C) and lies genetically between markers 6- 16 and TG36, 0.23 cM from 6-16 and 1.3 cM from TG36. In previous studies we utilized recombinant inbred (Rl) lines for mapping of 12 (Ori et al, 1994). Interestingly, in spite of some inconsistencies in linearity of markers between the Rl and the F2 population described here, SL8D completely cosegregated with 12 also when mapped using the R lines (data not shown). The SL8 marker showed a remarkably high rate of polymorphism between F.o.l race 2 resistant and susceptible lines. In comparison, flanking markers showed a much lower degree of polymorphism, as judged by the paucity of restriction digests that yielded polymorphic bands.

Example 2. SL8 is a member of a gene family cosegregating with 12

The complete genetic cosegregation of SL8D with the 12 resistance gene and the unique level of polymorphism between resistant and susceptible lines prompted us to further characterize the multicopy marker SL8. Sequence analysis revealed that SL8 contains an open reading frame with similarity to a group of recently isolated resistance genes (see below). This suggested that SL8 is a part of a gene that belongs to a family which includes the 12 resistance gene. The gene of which the SL8 probe was the 3' part was therefore designated I2C-1 (12 candidate 1 ).

We wished to further characterize the different SL8 family members as RFLP markers, draw criteria to distinguish between them, and analyze their genomic distribution. A comparison of the SL8 RFLP patterns of resistant and susceptible type lines, obtained with the restriction enzyme Taql, is shown in Figure 2. Approximately 17 different Taql bands hybridized to the SL8 probe, and many of them were polymorphic between resistant and sensitive lines. Resistant-type bands, consistent among all tested lines, were designated TR1-TR8. The rest of the bands were either nonpolymorphic or polymorphic between the susceptible lines, because of the different parental origins. As several polymorphic Taql bands were detected with the SL8 probe, direct allelism cannot be established. In the entire F2 population, a single recombinant plant was identified between all the polymorphic SL8-bands (BR 30(5) in Figure 2), which separated SL8D into two distinct loci, SL8D-1 and SL8D-2. (Figure IB). Except for this case, all polymorphic SL8 bands cosegregated. The recombinant individual BR30 (5) is sensitive to F.o.l race 2, and contains one resistant-type Taql band, TR7, but lacks the others (TR1-6 and TR8). Similar additional southern blots and progeny tests of BR 30(5) confirmed these results (data not shown). Therefore, bands TR1-TR6 and TR8 appear to completely cosegregate with each other and with 12, and are all candidates for the resistance gene. However, the possibility of a recombination within the gene should also be considered. This could result in a sensitive plant containing a part of the resistance gene, and consequently a polymoφhic band that belongs to the resistance gene. In addition, the possibility exists that a nonpolymorphic band represents the resistance gene. The latter is unlikely as will be shown below.

Example 3. Genomic distribution of I2C As only a subset of the SL8 copies showed polymoφhism between the parents of the F2 populations used for mapping, additional populations were incoφorated to map all the SL8 fragments. The first is an introgression-lines (IL) population, in which single chromosome segments from L. pennellii were introgressed into a L. escuientum background. Both parental species of the DL population carry the susceptible allele at the 12 locus (Eshed and Zamir, 1994). Figure 3 A illustrates the genomic segments introgressed from chromosomes 8, 9 and 11 in the IL lines, which proved relevant for the SL8 mapping. All SL8 copies appear to be polymoφhic between L. escuientum and L. pennellii (Figure 3B, lanes 1 and 2), a feature which facilitated their mapping. DNA digests from the IL lines were compared by southern blot hybridization with SL8 with that L. escuientum and L. pennellii (Figure 3B). L. pennellii bands that are contained in each IL, as well as their allelic L. escuientum bands that are absent from these lines, represent SL8 copies that originate from the region introgressed in the respective line. One SL8 copy mapped to the short arm of chromosome 8 (SL8A ), as one L. pennellii- type band is present, and one L. escuientum -type band is absent from IL 8-1. Similarly, one copy with weaker homology mapped near the centromere of chromosome 9 (SL8B, Figure 3A,B). More accurate location of these two copies was obtained using lines containing shorter introgressed segments of the region, derived by selection of recombinants from the F2 of the original IL crossed back to L. escuientum var. M82 as illustrated in Figure 3 A The rest of the SLS copies mapped to the long arm of chromosome 1 1. Two of the introgression lines, IL 1 1-3 and IL 1 1-4, contain L. pennellii segments from the long arm of chromosome 1 1 (Figure 3 A). By comparing these two lines, three genetically distinct groups of SL8 family members could be identified on chromosome 1 1. The first (SL8(^T) maps to the region exclusively introgressed in IL 1 1-3, the second (SL8D) to the region of overlap between the IL 1 1-3 and IL 1 1-4, and the third (SL8E) to the region exclusively introgressed in IL 1 1-4. As previously established, the 12 resistance gene maps to the region of overlap designated SL8D. Higher resolution mapping of the chromosome 1 1-based SLS loci was accomplished using an F2 population of 150 plants, generated from an initial cross between L. escuientum and an introgression line that contains the long arm of chromosome 1 1 (line 1 1 , Eshed et al., 1992). Analysis of the F2 population corroborated the division of SL8 markers into clusters. SL8C and SL8D cosegregated completely with the RFLP markers TG546 and 6-16, respectively, and SL8E mapped between markers TG26 and TG105, 0.25 cM from TG26 (Figure 3). The susceptible-type L. escuientum is a common parent between the IL population and the F2 population. A comparison of the SL8 RFLP patterns of the ILs (Figure 3) with those of resistant and susceptible L. escuientum plants (Figure 2), shows clearly that nearly all fragments that belong to group SL8D are polymorphic between the resistant and susceptible F2 parents (compare Figure 2 and Figure 3). The nonpolymorphic bands in Figure 2 belong mostly to the other groups. This indicates that the region containing cluster SL8D is the region which was originally introgressed from L. pimpinellifolium into L. escuientum.

Example 4. Heterogeneity in recombination rates in the 12 region

The locus SL8-D. containing at least 4 clustered members of the I2C family, spans a 0.03 cM region in the 12 locus. Two cosmid clones of approximately 20 kb insert from this cluster, I2C- 134 and I2C-150 (Fig. 1 1 ), contain only one copy of SL8. Hence, 0 03 cM genetic interval spans at least 20 kb, which assigns an estimation of at least 670 kb/cM in the region containing this group. This ratio is similar to the average of 550 kb/cM over the entire genome. In contrast, in proximal regions the ratio is <150 kb/cM between D2 and SL8 (12C-1; Figure 1 ) and approximately 43 kb/cM between TG105A and TG26 (Segal et al., 1992). High variability in the physical to genetic ratio is common in the context of different regions of the chromosome, and is shown here to fluctuate regionally as well.

Example 5. I2C Genes share structural similarity with a family of plant resistance genes

Candidate members of the 12C gene family were isolated from genomic libraries of F.o.l race 2 resistant tomatoes utilizing SL8 as a probe. The isolated clones were compared to the genomic DNA on southern blots, and clones that contain resistant-type polymorphic bands from the SL8D locus were further characterized. The cosmid clone I2C-134 (Figs. 1 1, 12) contains the 12C-1 gene, that includes in its 3" region the SL8 marker (Fig. 1C). It was found to contain the polymorphic. bands TR1 and TR5 (Fig. 2). 12C-134 also exhibits resistant-type polymorphic bands after digestion with other endonucleases, such as Hindlll, Dral and EcoRI (data not shown). Cosmid 12C-150 (Fig. 1 1 ) contains another gene, 12C-2, represented by the polymoφhic bands TR4 and TR7 (Fig. 2). As the recombination in individual BR 30(5) has occurred between band TR7 and the other resistant-type bands, it would be expected that the gene which contains TR7 will not contain other resistant-type bands. The presence of both TR4 and TR7 bands in 12C-150 could be explained either by a recombination within the gene 12C-2 in the individual BR 30(5), or by comigration of bands of different origin. Other cosmids isolated contained non-polymorphic bands, and were not sequenced.

One continuous open reading frame was identified in each of the genes 12C-1 and 12C-2. Fig. 8 shows a comparison between the deduced amino acid sequences of the 12C- I and the 12C-2 genes and recently isolated plant resistance genes. Although the overall homology is rather low, an intriguing structural similarity is apparent. All genes contain in their N terminus a conserved nucleotide binding site, P-loop, and other conserved amino acid stretches of unknown function, which are shown in Fig. 8 as boxes I- VI. In their C terminus they all display a long region of leucine-rich-repeats (LRR), which spans at least half of the gene. The LRR of 12C-1 are aligned in Fig. 9. The N terminal parts of the 12C repeat segment are comparable to the consensus LRR found in the resistance genes RPS2, N gene and L6, and to the consensus of the T-LR VSG expression site associated gene from Trypatiosoma (Fig. 9, Ross et al., 1991; Smiley et al., 1990). The latter protein shares 52 % similarity and 25 % identity with the 3' part of 12C-1 The C-terminal parts of the repeat segments are not conserved, and are of variable length.

Example 6. Transcribed sequences from the I2C gene family contain insertion and frame-shifts.

In order to compare different resistant-type members of the 12C family, three different cDNA libraries of resistant tomato varieties were screened with the SL8 probe. Fifteen independent clones were isolated, and all of them were shorter than 12C-1, containing only the 3' ends of the genes. The reason for obtaining only partial clones is not known, as much longer inserts have been isolated from these libraries. Two of the longest clones, designated 12C-3 and 12C-4, of 1200 and 1600 bp long, respectivley, were sequenced. Interestingly, one of the cDNA clones, 12C-4, contains two frame shifts, and thus, if translated, would result in a truncated peptide. In Fig. 10, 12C-4 is artificially shown as a continuous chimera of the 3 different ORFs, and the junctions are indicated with arrows. The comparison of the 3' region of the two genomic and two cDNA clones is shown in Fig. 10. Striking insertions or deletions, chiefly in the C-terminal region, can be observed. Most insertions are shared by at least two different genes, though the combinations are different for each insertion. Interestingly, close to the C-terminus the different genes contain between 2 and 6 repeats of an almost identical sequence. These repeats are indicated in Fig. 10. The largest insertions in genes I2C-3 and I2C-4 are made up exclusively of these repeats.

Example 7. Plant transformation procedures

In order to correlate the disease resistance capacity of the I2C genes, they have been transformed into tomato plants in a few different formats:

(a) Cosmid clones 12C-134 and 12C-150 containing the complete inserts of clones I2C-1 and I2C-2, respectively, in the BamHl site of cosmid TDNA 04541 (Jones et al., 1992) (Fig. 1 1 ), were directly transformed into sensitive tomato lines VF-36 and Money Maker (Jones et al., 1992). Another 10 cosmids have been isolated by homology to 12C and were similarly transformed. To this end the cosmids were transferred into Agrobacterium tumefaciens using standard transformation procedures. The binary vector A. tumefaciens LBA 4404 is suitable for the transformation procedure. Tomato explants are inoculated as described in Jones et al., 1992.

(b) Similarly, clone 12C-1 was introduced into the PGA492 binary vector supplemented with the B-domain of the 35S promoter (constructs 134 A and 134H) and were directly transformed into the same sensitive tomato lines as described above.

Fig. 12 depicts sense constructs from the I2C-134 cosmid containing the I2C-1 clone in the PGA492 binary vector. A depicts an Accl subclone, 134A, containing approximately 3kb upstream and 300 bp downstream to the open reading frame (ORF), cloned into the Clal site of the PGA492 vector; B. depicts a Hindi subclone, 134H, containing around 3kb upstream and 800 bp downstream to the translated region, cloned into the Hpal site of the PGA492 vector; D depicts a small subclone from the BS subclone of cosmid 134 depicted in C, containing 300 bp upstream and 800 bp downstream to the translated region, cloned downstream from the B domain (B Dom) of the 35S promoter, in the PGA492 vector, to create 134B. (c) Antisense and sense partial clones were constructed with partial sequences from I2C-1, I2C-2, and I2C-3, and subcloned into 35S omega expression vectors in the PGA492 binary vector. These constructs (2-1, 6-3, 5-1 and 31-17) were transformed into resistant tomato lines (Motelle) as described in section (a) above.

Fig. 13 depicts the antisense and sense constructs 2-1, 6-3, 5-1 and 31-17 for transformation of Fusarium resistant tomatoes. On the top is a map of SL8-134 (12); indicated is the Hindlll fragment used for the antisense and sense cloning in 2-1 and 6-3, which spans nucleotides 2540-3716 in the I2C-1 sequence shown in Fig. 4. Constructs 5- 1 and 31-17 contain the full-lenght insert of cDNA I2C-3. 35S is the cauliflower mosaic virus (CaMV) 35S promoter, Ω is a translation enhancer from tobacco mosaic virus, and nos 3' is a terminator, 3' untranslated sequence of the nopaline synthase gene. All the clones were prepared in the PAG492 binary vector using the unique Xbal site.

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Claims

CLAIMS:

1. A DNA molecule selected from the group comprising:

(i) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated 12C-1 (SEQ. ID. NO. : l ); (ii) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated I2C-2 (SEQ. ID. NO.:2);

(iii) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated 12C-3 (SEQ. ID. NO.:3);

(iv) a DNA molecule having a nucleotide sequence derived from the coding region of the clone herein designated 12C-4 (SEQ. ID. NO.:4);

(v) a DN A molecule characterized by containing a coding sequence representing at least 60% similarity with the encoded open reading frame in the DNA sequence of at least one of the DNA molecules (i) and (ii),

(vi) a DNA molecule capable of hybridization with any one of the DNA molecules (i)- (v) under moderately stringent conditions;

(vii) a DNA molecule that differs, by insertion, deletion or as a result of the degenerative nature of the genetic code, from the DNA sequences (i)-(vi); and (viii) a fragment of any of the DNA molecules (i)- (vii).

2. A DNA molecule according to claim 1 which is expressed in tomato plants and confers resistance to Fusarium oxysporum f.sp lycopersici race 2 in said tomato plants.

3. A gene construct containing a DNA molecule according to claim 2.

4. A gene construct according to claim 3 wherein said DNA molecule includes regulatory sequences that flank the coding region.

5. A cosmid into which is subcloned a gene construct according to claim 4.

6. A gene construct according to claim 3 which further comprises DNA regulatory sequences enabling the expression of said D A molecule in plant cells.

7. An expression vector comprising a gene construct according to claim 6 wherein said DNA regulatory sequences comprise a plant promoter, a DNA sequence that enhances translation of the mRNA transcribed from said DNA molecule and a polyadenylation/terminator sequence.

8. A tomato cell line or plant line transformed with a cosmid according to claim 5 or with an expression vector according to claim 6 or 7.

9. Tomato plants regenerated from transformed cells according to claim 8.

10. A method of selective breeding of Fusarium resistant tomatoes employing a DNA molecule according to claim 1 as a direct restriction fragment length polymoφhism (RFLP) probe, which comprises:

(i) marking said DNA molecule with a suitable marker; and (ii) reacting said probe of (i) with DNA extract of a tomato plant under hybridization conditions; thus obtaining restriction-length polymoφhism that is indicative of a resistance-type gene, which facilitates the selection of progeny that contains said resistance gene.

1 1. A method of screening new diseases in plants of the Solanaceae family employing a DNA molecule according to claim 1 as a direct RFLP probe which comprises:

(i) marking said DNA molecule with a suitable marker; and (ii) reacting said probe of (i) with DNA extracts from said Solanaceae species, thus identifying the homologous gene family in those species which can be linked to known resistance genes in those species.

12. Use of a DNA molecule according to claim 1 as a direct RFLP probe for selective breeding of Fusarium resistant tomatoes.

13. Use of a DNA molecule according to claim 1 as a direct RFLP probe for screening new diseases in plants of the Solanaceae family.