EP0759086A1 - Method of introducing pathogen resistance in plants - Google Patents

Abstract

Variegated plants have increased pathogen resistance: cells of the plant express a phenotype, which may comprise necrosis and/or a plant defence response, and other cells not expressing this phenotype have increased pathogen resistance. Embodiments of the invention employ various genes, including Cladosporium fulvum pathogen resistance genes, which are inactivated, for example as a result of insertion of a transposable genetic element, and then reactivated in plant cells to result in necrosis and/or a plant defence response, leading to increased pathogen resistance. Cells, plants and other compositions of matter are provided comprising various combinations of genes involved in this system.

Description

METHOD OF INTRODUCING PATHOGEN RESISTANCE IN PLANTS

The present invention relates to a method of introducing pathogen resistance in plants, particularly broad spectrum pathogen resistance, and plants which may be obtained by said method and which show resistance to at least one but preferably more than one pathogen.

Plants are constantly challenged by potentially pathogenic microorganisms. Crop plants are particularly vulnerable, because they are usually grown as genetically uniform monocultures; when disease strikes, losses can be severe. However, most plants are resistant to most plant pathogens. To defend themselves, plants have evolved an array of both preexisting and inducible defences which include barriers to pathogen entry such as thickened or chemically crosslinked cell wall components or toxic chemicals derived from complex plant biosynthetic pathways. Pathogens must specialize to circumvent the defence mechanisms of the host, especially those biotrophic pathogens that derive their nutrition from an intimate association with living plant cells. If the pathogen can cause disease, the interaction is said to be compatible, but if the plant is resistant, the interaction is said to be incompatible.

Induced resistance is strongly correlated with the hypersensitive response (HR) , an induced response associated with localized cell death at sites of attempted pathogen ingress. It is hypothesized that by HR the plant deprives the pathogen of living host cells but there is no certainty about whether localised cell death results from or induces plant defence mechanisms. Many plant defence mechanisms are strongly induced in response to a challenge by an unsuccessful pathogen. Such an induction of enhanced resistance can be systemic (hereinafter referred to as systemic acquired resistance (SAR) ) (Ross, 1961; Ryals et al . , 1992) . Acquired resistance can also be local (hereinafter referred to as LAR) (Ryals et al . , 1992). Acquired resistance has been extensively researched and various facts have been established. For example, biotic stimuli are required to provoke the HR resulting in areas of dead plant cells on the leaf. Cell death resulting from wounding or other abiotic stresses will not suffice. (Ryals et al. , 1992; Enyedi et al . , 1992) . In addition, SAR is correlated with the induction of a large array of pathogenesis-related (PR) proteins, some of which have demonstrated anti-fungal activity (Ward et al . , 1991).

A variety of examples of SAR have been studied and include challenging of tobacco carrying the N gene for resistance to tobacco mosaic virus (TMV) with TMV (Ross, 1961) and challenging cucumber seedlings with tobacco necrosis virus or Colletotrichum largenarium. Results show that a challenge with one pathogen leads to enhanced resistance to a wide variety of other pathogens (Ryals et al . , 1992).

SAR has also been correlated with increased levels of salicylic acid in plants which have been challenged by pathogens (Malamy et al . , 1990; Metraux et al . , 1990) which has been confirmed by studies that show that a supply of exogenous salicylic acid to unchallenged plants can result in SAR (Ward et al . , 1991; Hennig et al . , 1993). Transgenic plants designed so that salicylic acid accumulation is prevented by expression of a salicylate hydroxylase gene show reduced SAR compared to non-transgenic plants.where salicylic acid accumulation is not prevented (Gaffney et al . , 1993) . SAR can also be induced by many chemicals manufactured by Ciba-Geigy such as 2,6- dichloroisonicotinic acid (INA) (Uknes et al . , 1992).

SAR is an attractive method by which broad spectrum disease control can be achieved. However, two major drawbacks hinder its commercial exploitation: SAR is not a heritable trait and so the phenomenon has to be successfully induced into every plant in the crop stand; to be effective throughout the crop's life, the SAR phenotype has to be re-boosted at regular intervals.

Although the mechanisms causing SAR are not fully understood, it is believed that when a plant is challenged by a pathogen to which it is resistant, it undergoes an HR at the site of attempted ingress of the incompatible pathogen. The induced HR leads to a systemic enhancement and acquisition of plant resistance to virulent pathogens that would normally cause disease in the unchallenged plant.

It has long been known that HR-associated disease resistance is often (though not exclusively) specified by dominant genes (R genes) . Flor showed that when pathogens mutate to overcome such R genes, these mutations are recessive. Flor concluded that for an R gene to function, there must also be a corresponding gene in the pathogen, an "avirulence gene" (Avr gene) . To become virulent, pathogens must thus stop making a product that activates R gene-dependent defence mechanisms (Flor, 1971) . A broadly accepted working hypothesis, often termed the elicitor/receptor model, is that R genes encode products that enable plants to detect the presence of pathogens, provided said pathogens carry the corresponding AVR gene (Gabriel and Rolfe, 1990) . This recognition is then transduced into the activation of a defence response.

The mlo allele of the Mlo gene of barley is the one example of a recessive disease resistance gene currently widely used in plant breeding. Lines that are homozygous for the recessive allele of this gene activate the defence response (comprising formation of cell wall appositions) even in the absence of the pathogen (Wolter et al , 1993) . Thus the mlo mutation causes a defence mimic phenotype, also known as a necrotic or disease lesion mimic phenotype, and appears to deregulate the defence response, so that it is activated precociously, or is regulated on more of a "hair trigger" . A number of examples of such disease lesion mimic mutants exist in maize (Johal et al , 1994, Pryor, 1987, Walbot, 1983) . Recently, such mutants have been sought in Arabidopsis. The characterization of one such mutant, acdl, has been reported (Greenberg and Ausubel, 1993) . Further mutants of this type have been reported at scientific meetings (the Arabidopsis acd2 mutation by F.M. Ausubel at a meeting at Rutgers University, New Jersey, USA, April 1993; Arabidopsis mutations now known as lsd (for lesions simulating defence response) mutations by R. Dietrich and J. Dangl at the ARAPANET ( (Arabidopsis Pathology Network) workshop in Wye College, Kent, UK in April 1993) . Manuscripts describing the acd2 and lsd mutations are Dietrich et al. and Greenberg et al. (1994) . It is highly likely that the recessive mutations identified in such mutant screens that leave the defence response more constitutively on, or more rapidly activated, or less easily inactivated, are in genes that normally dampen down the defence response to prevent it becoming so severe that it is deleterious to the plant. Conceivably, such gene could be cloned, expressed in an antisense or sense configuration to reduce expression of the corresponding gene (Hamilton, 1990, Napoli et al , 1989) . Pathogen avirulence genes are still poorly understood. Several bacterial Avr genes encode hydrophilic proteins with no homology to other classes of protein, while others carry repeating units whose number can be modified to change the range of plants on which they exhibit avirulence (Keen, 1992; Long and Staskawicz, 1993). Additional bacterial.genes (hrp genes) are required for bacterial Avr genes to induce HR, and also for pathogenicity (Keen, 1992; Long and Staskawicz, 1993) . It is not clear why pathogens make products that enable the plant to detect them. It is widely believed that certain easily discarded Avr genes contribute to but are not required for pathogenicity, whereas other Avr genes are less dispensable (Keen, 1992; Long and Staskawicz, 1993). The characterization of two fungal avirulence genes, Avr 4 and Avr 9 (De Wit et al . , 1992; Joosten et al . , 1994), has also been reported. Research is also being undertaken to clone rice blast avirulence genes from the causal organism Magnaporthe grisea and the avirulence genes (NIP proteins) of the barley pathogen Rhynchosporium secalis . Two viral avirulence genes have also previously been cloned. Culver and Dawson, 1991, have shown that tobacco mosaic virus coat protein is the avirulence determinant for the N' gene product. In addition, the potato virus X coat protein appears to be the avirulence determinant for the Rx and Nx genes (Kavanagh et al . , 1992; Santa-Cruz et al . , 1993; Kδhm et al . , 1993; Goulden et al . , 1993).

Recently the map based cloning of the tomato Pto gene that confers "gene-for-gene" resistance to the bacterial speck pathogen Pseudomonas syringae pv tomato (Pst) has been reported (Martin et al . , 1993). It has also been recently reported that the Arabidopsis Rps2 gene (which confers Pseudomonas syringae resistance) and the tobacco N gene (which confers virus resistance) have been cloned (Keystone Symposium, January 1994) . Even more recently, the Rps2 and features of the Cf-9 gene sequences have been revealed at the 13th Annual Symposium in Columbia, Missouri, April 13th-16th 1994, on the Biology of Communication in Plants. International Patent Application No: PCT/GB94/02812 describes a method for generally identifying and cloning plant resistance genes.

The technology for gene isolation based primarily on genetic criteria has improved dramatically in recent years, and many workers are currently attempting to clone a variety of R genes. Targets include (amongst others) rust resistance genes in maize, Antirrhinum and flax (by transposon tagging) ; downy mildew resistance genes in lettuce and Arabidopsis (by map based cloning and T-DNA tagging) ; Cladosporium fulvum (Cf) resistance genes in tomato (by tagging, map based cloning and affinity labelling with avirulence gene products) ; virus resistance genes in tomato and tobacco (by map based cloning and tagging) ; nematode resistance genes in tomato (by map based cloning) ; and genes for resistance to bacterial pathogens in Arabidopsis and tomato (by map based cloning) . Tomato {Lycopersicon esculentum) is susceptible to disease caused by the leaf mould fungal pathogen Cladosporium fulvum. According to De Wit, 1992, the Avr9 gene of C. fulvum, which confers avirulence on C. fulvum races that attempt to attack tomato varieties that carry the Cf-9 gene, encodes a secreted cysteine- rich peptide with a final processed size of 28 amino acids. However, its role in compatible interactions is not clear. The R genes (Cf-genes) that act against C. fulvum have been identified and bred into cultivated varieties, often from related species of tomato (Dickinson et al . , 1993; Jones et al . , 1993).

It has been shown that C. fulvum contains Avr genes that confer recognition by plants which contain the Cf-genes , leading to activation of host defence mechanisms to attack the disease (incompatibility) .

The Avr4 and Avr9 genes encode small peptides that are secreted by the pathogen into the intercellular spaces of infected leaves, from which they can be extracted. This has enabled the purification and sequencing of these peptides and the isolation of the genes that encode them (De Wit, 1992; Joosten et al . , 1994). Experiments have shown that when the Avr9 gene is transformed into a race of pathogen that lacks Avr9, then the race of pathogen becomes avirulent on plants which are carrying the Cf-9 gene. In addition, it has been shown that disruption of the Avr9 gene in a pathogen race which is avirulent on plants carrying Cf- 9 gene confers compatibility on the Cf-9 containing plants (Van Den Ackerveken et al . , 1992, Marmeisse et al . , 1993) .

In addition, De Wit and colleagues have further shown that the secreted peptide encoded by the Avr9 gene can be injected into Cf-9 containing tomato leaves to elicit a necrotic response in the injected area. The necrotic response is consistent with local and vigorous activation of a defence response (De Wit, 1992; WO 91/15585) . International Patent Application No. PCT/GB94/02812 describes the transgenic expression of the Avr9 gene using the strong cauliflower mosaic virus 355 plant promoter to cause lethality in Cf-9 plants. This transgenic expression has been used to select mutants in which the Cf-9 gene has been inactivated by transposon insertion in order to isolate the Cf-9 gene and perform DNA sequence analysis of this gene .

Various pathogen races that overcome these Cf- genes have emerged and are named after the Cf-gene which they can overcome. For example, C. fulvum race 4 can overcome Cf-4; C. fulvum race 5 can overcome Cf-5 and C. fulvum race 2.4.5.9 can overcome Cf-2 , Cf-4 , Cf- 5 and Cf-9.

WO 91/15585 describes a hypothetical method whereby if a Cf-9 gene and/or an Avr9 gene were expressed under the control of a promoter that is induced by a broad range of pathogens, then a general defence response could be induced. However, there is a lack of enabling disclosure regarding which polynucleotide sequences could be used either as the resistance gene or as an actual promoter which would be suitably affected by a broad range of pathogens. A further problem with this proposed method is that necrosis induced by the Cf-9 and AvrS gene combination could lead to further induction of Avr9 and/or Cf-9 leading to spreading of the necrosis and severe reduction in the yield of the plant. This problem may arise since promoters such as promoters for plant defence genes and other genes involved in the defence response such as PR genes (pathogenesis related genes) , are induced in both a compatible and an incompatible interaction. Therefore, even if a promoter exists which is'effectively induced by a broad range of pathogens, the method would not be viable unless the promoter is only induced by the appearance of a compatible pathogen. If the defence response provides further induction of the promoter the plant might experience spreading necrosis.

The present invention has resulted from experiments involving transposon tagging of resistance genes, the first one being Cf-9. Numerous alleles of the Cf-9 gene { Cf-9*Ds) were isolated that had been inactivated by the maize element Dissociation (Ds) . These inactive Cf-9*Ds genes did not give rise to a constitutive and lethal activation of defence mechanisms in response to constitutively expressed Avr9 transgene (35S:SP:Avr9) . On backcrossing plants that carried the Cf-9*Ds and 35S:SP:Avr9 genes to tomato plants carrying an Activator (Ac) transposase gene ( sAc) in the homozygous state but lacking Cf-9, a quarter of the resultant progeny carried sAc, 35S:SP:Avr9 and Cf-9*Ds . These plants showed somatic excision of Ds from the Cf-9*Ds gene, somatically restoring Cf-9 function and giving rise to localised activation in cells of plant defence responses due to recognition of the constitutively expressed Avr-9 peptide. These cells died and gave rise to small necrotic sectors, the plants phenotypically showing variegation for a defence-related necrosis, similar to somatic flecks of necrosis that are associated with the induction of SAR in plants challenged with necrotising pathogens. Further work showed that plants that variegate for somatic sectors of plant defence response in this way have increased resistance to a range of pathogens.

Thus, a first aspect of the present invention relates to a method of providing pathogen resistance, in particular broad spectrum pathogen resistance, in plants by induction of variegation in which genes are expressed or suppressed resulting in the activation of necrosis. A method according to the present invention comprises: (i) inactivating a nucleotide sequence which contributes to plant cell necrosis or inactivating one or more nucleotide sequences forming part of a combination of nucleotide sequences which contribute to plant cell necrosis; (ii) introducing said nucleotide sequence or sequences into the genome of a plant; and (iii) restoring said nucleotide sequence or sequences to a functional form to yield a level of necrosis resulting in pathogen resistance. The plant cell necrosis is preferably defence-related plant cell necrosis.

A second aspect of the present invention relates to a method of providing pathogen resistance in plants by induction of variegation in which genes are expressed or suppressed resulting in the activation of a plant defence response which comprises: (ij inactivating a nucleotide sequence which contributes to the plant defence response or inactivating one or more nucleotide sequences forming part of a combination of nucleotide sequences which contribute to the plant defence response; (ii) introducing said nucleotide sequence or sequences into the genome of a plant; and (iii) restoring said inactivated nucleotide sequence or sequences to a functional form to result in pathogen resistance. The variegation will generally be for somatic sectors. Pathogen resistance will generally be increased compared with wild-type.

The nucleotide sequence or sequences comprise one or more genes. The plant defence response and/or plant cell necrosis occurs on expression of the gene or genes. The defence response and/or. plant cell necrosis can be conditional or unconditional on the expression of one or more interacting genes. A substance or a combination of substances may result in increased pathogen resistance. Examples are discussed further below.

For example, the nucleotide sequence or sequences may comprise a gene encoding either a substance which leads to necrosis, e.g. through activation of the plant defence response, or a substance which leads to a plant defence response with no sign of necrosis. For example, the sequence or sequences may comprise a plant pathogen resistance gene (R) , an avirulence gene (Avr) or other elicitor or ligand gene (L) of an R gene, or both and R gene and an L gene.

The inactivation of the nucleotide sequence or sequences which contribute to the plant defence response and/or plant cell necrosis is preferably effected by insertion of a transposable genetic element into the nucleotide sequence or one or more of the nucleotide sequences forming a combination of nucleotide sequences. The transposable genetic element is preferably a transposon or a nucleotide sequence flanked by specific nucleotide sequences so that transposon excision gives rise to activation of the plant defence response and/or necrosis. Thus, insertion of a genetic lesion into the nucleotide sequence disrupts the gene to prevent expression of a product able to function in contributing to the plant defence response and/or plant cell necrosis. In the absence of the lesion, e.g. following excision of a transposable element such as a transposon, the gene may be expressed to produce a functional product, i.e. gene function is restored. The lesion may be inserted into the part of the gene coding for the expression product, or may be in a regulatory sequence such as a promoter required for expression of the product.

In this form of the invention, re-activation within the plant is preferably carried out by restoraration of the inactivated nucleotide sequence or sequences resulting in activation of a plant defence response and/or necrosis. Such restoration may be caused or allowed by culturing of the plant. Where the nucleotide sequence is inactivated by virtue of insertion of a transposable element therein, the plant genome should contain at least one nucleotide sequence coding for a corresponding transposon activation system (for example, comprising a transposase) . Alternatively, the inactive form could be flanked by recombinase recognition sequences that are acted on by a site specific recombination system (comprising a specific recombinase) so that recombination activates the inactive form of the gene. Hence, when the inactivated nucleotide sequence or sequences are introduced into the plant genome somatic excision of the transposon or recombination of the nucleotide sequence occurs in some cells leading to activation of the plant defence response and/or necrosis in specific clones of cells.

The number of cells in which restoration of function occurs may vary. As discussed further below, certain measures are available for optimising the system, e.g. by controlling the frequency of spontaneous excision of a transposable element which is caused or allowed upon cultivation of a plant with the requisite nucleotide sequence or sequences within its genome .

The present invention further provides transgenic plants having increased pathogen resistance obtainable by the method of the present invention, and any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Derivatives of plants are also provided by the present invention. A derivative is any functional unit derived therefrom howsowever achieved (e.g. functional allele of gene* made by mutagenesis, recombinant DNA, synthesis, or plant which could not have been produced without the use or manufacture of the plant from which it is derived. )

Transgenic plants in accordance with the present invention may demonstrate increased pathogen resistance since the induced plant defence response and/or necrosis of plant cells may cause other cells, such as adjacent cells, to acquire pathogen resistance. The activation of, for example, a- plant resistance gene in a plant cell is inherited by the progeny and descendants of that cell. The expression of this plant resistance gene leads to initiation of the defence response in cells which may eventually lead to the death of the participating plant cells resulting in an area of plant cell necrosis. So, plants may have variegation for small somatic sectors in which defence- related plant cell necrosis is activated. This response may induce pathogen resistance in other cells. In an alternative, operating on the same general principle, the expression of one or more plant pathogen resistance gene may either lead to initiation of the defence response only resulting in variegation for small somatic sectors in which the plant defence response is activated or of plant cell necrosis which is not related to the plant defence response resulting in variegation for small somatic sectors in which plant cell necrosis is activated. Hence, the plant may acquire resistance to a broad range of pathogens and not only to the pathogen associated with the gene or genes contributing to necrosis, for example, C. fulvum in the case of the Cf- 9 /Avr gene combination. For example, a transgenic tomato plant according to the present invention may demonstrate resistance against a broad range of pathogens such as one or more bacterial plant pathogens (for example, Xanthomonas campestris, Pseudomonas syringae) , fungal plant pathogens (for example, Phytophthora infestans, Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae, Al tenaria solani , Rhizoctonia solani) and viral pathogens (for example, TMV, PVX, PVY, TSWV) . Similarly, other transgenic plants such as transgenic tobacco, Arabidopsis and potato plants may display resistance to a large number of major diseases of important crop species such as, Peronospora, Phytophthora, Puccinia, Erysiphe and Botrytis.

Thus, according to a further aspect of the invention there is provided a plant, or any part thereof, which is phenotypically variegated, with clones of cells expressing a first phenotype and other cells expressing a second phenotype which is increased pathogen resistance compared with wild-type. The first phenotype is preferably necrosis and/or a plant defence response phenotype. As discussed, plants variegated by somatic sector for such a phenotype may have enhanced pathogen resistance as a result of a second phenotype in cells, which may be adjacent to the cells with the first phenotype which are necrotic and/or in which a plant defence response is .activated. The phenotypic variegation is likely to result from expression in cells with the first phenotype of a gene or gene, or nucleic acid comprising a gene or genes, which contributes to such phenotype, whereas other cells without such phenotype lack such gene expression. As discussed herein, this may result from reactivation of a previously inactivated gene, such as a resistance gene, for example by random excision of a transposable element such as a transposon.

In a further aspect, the present invention provides a host cell, such as a plant or microbial cell, or a plant comprising at least one such cell, containing (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of a transposable element such as a transposon, and (ii) nucleic acid encoding a molecule able to reverse the inactivation, such as, in the case of a transposon, a transposase. Thus, the cell may comprise a plant resistance gene or other gene involved in the plant defence response or able to kill a cell when expressed therein (either alone or incombination with one or more sequences, for example in the case of an R gene the corresponding elicitor) , the gene being inactivated by insertion therein of a transposon, and the cell further comprising a gene encoding a transposase.

In an exemplary embodiment, the genome of the cell comprises the gene Cf-9, or a mutant, derivative, variant or allele thereof which retains Cf-9 function, inactivated by insertion therein of a transposon, the genome also comprising the Avr- 9 gene, or a mutant, derivative, variant or allele thereof which retains Avr -9 function, and a gene encoding a transposase able to excise the transposon from the Cf-9 gene or functional equivalent. Other resistance genes may be employed, as may genes which do not require the presence of an elicitor molecule to cause cell necrosis, as discussed further elsewhere herein.

The cell may comprise the nucleic acid encoding the various genes by virtue of introduction into the cell or an ancestor thereof of the nucleic acid, e.g. by transformation, using any suitable technique available to those skilled in the art. Furthermore, plants which comprise such cells, and seed therefore, may be produced by crossing suitable parents to create a hybrid whose genome contains the required nucleic acid, in accordance with any available plant breeding technique. For example, a parent strain comprising within its genome a plant resistance gene containing a transposon or other inactivating lesion may be crossed with a second strain comprising within its genome a gene encoding the elicitor molecule for the plant resistance gene and a suitable transposase for excision of the transposon. At least a proportion of the hybrid progeny of the parents, i.e. seed or plants grown therefrom, will comprise the required nucleic acid for activation in the plant of, in this example, the plant resistance gene and, following interaction with the elicitor, the plant defence response and/or plant cell necrosis.' Plants according to this aspect of the present invention will be variegated genetically. Clones of cells will have one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis reactivated by removal of the inactivating lesion such as a transposon, so that a first phenotype such as necrosis is shown, while in other cells the sequence or sequences will remain inactivated so these cells will not show the first phenotype.

Within the cell or cells, the nucleic acid may be incorporated within the chromosome. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such decendants should show the desired phenotypic variegation and so may have enhanced pathogen resistance.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. A further aspect of the present invention provides a method of making such a cell involving introduction of nucleic acid (e.g. a vector) comprising (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of a transposable element such as a transposon, and/or (ii) nucleic acid encoding a molecule able to reverse the inactivation, such as, in the case of a transposon, a transposase into a plant cell. Introduction of nucleic acid (i) may be accompanied, preceded or followed by introduction of nucleic acid (ii) . Such introduction may be followed by recombination between the nucleic acid and the plant cell genome to introduce the sequence of nucleotides into the genome. Descendants of cells into which nucleic acid has been introduced are included within the scope of the present invention.

The level of the plant defence response and/or . plant cell necrosis in the small somatic sectors should be sufficient to result in the induction of acquired resistance or the induction of other defence mechanisms. Since this method leads to activation of acquired resistance but is inherited it is referred to as Genetic Acquired Resistance (GAR) . Hence, any system which gives rise to a variegation leading to GAR is applicable to the present invention.

Methods and plants etc. according to the present invention are particularly beneficial since the nucleotide sequence or sequences which contribute to the plant defence response and/or plant cell necrosis, for example the avirulence and plant resistance genes, may be under control of any suitable promoter, such as a constitutive promoter or, in the case of R genes, their own endogenous promoter, or a cell type specific promoter. Furthermore, the restoration of the nucleotide sequence or sequences, for example by the somatic excision of a transposon, gives rise to recurrent and widespread induction of the plant defence response in many small clones of cells throughout the plant, irrespective of whether or not there has been a challenge by pathogen. The resistance conferred on the plant is therefore constitutive and broad. The present invention may be used for many applications and is suitable for deployment in Fl hybrid seed production system. In such a system, one of the parents should be homozygous, for example, for the transposase or recombinase gene. In addition, in a system where two components are required for inducing the necrosis such as in the Avr9/Cf- 9 gene combination for example, this parent should also be homozygous for the constitutively expressed genes. The other parent should be homozygous for the gene that encodes the non- autonomous inactivation system, such as the transposon or recombinase-recognition sequences. After making a cross between parents of this genetic constitution, on somatic excision or recombination, the function of the gene or genes which give rise to the defence response and/or plant cell necrosis is restored in somatic sectors in the resulting progeny. It will be clear to the person skilled in the art that any gene or combination of genes which contributes to variegation for the plant defence response and/or plant cell necrosis may be used in the method of the present invention. Furthermore, any system which gives rise to inactivation of the nucleotide sequence or sequences and subsequent restoration of functional sequence or sequences may be used.

The present invention also provides in further aspects various compositions of matter comprising combinations of nucleotide sequences encoding various substances employed herein. Such combinations of nucleotide sequences which may be introduced into cells in accordance with the present invention follow:

(X) : represents a nucleotide sequence with one or more genes of type X

(XY) : represents a nucleotide sequence with one or more genes of type X and one ore more genes of type Y etc. R: receptor gene L: ligand gene (capable of interacting with the R gene) I: genetic insert A: activator of transposition of genetic insert.

R may encode a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, with I being a genetic insert able to inactivate R and A encoding a substance able to reactivate R inactivated by I: (1) Any combination of:

1. (R) , (I) and (A) ; 2. (R) and (IA) ;

3. (I) and (AR) ; or

4. (A) and (RI) ;

5. (RIA) .

Alternatively, R and L may encode substances whose presence together in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I being a genetic insert able to inactivate R and/or L and A encoding a substance able to reactivate R and/or L inactivated by I: (2) Any combination of:

1. (R) , (L) , (I) and (A) ;

2. (R) , (LI) and (A)

3. (R) , (LA) and (I)

4. (R) , (IA) and (L) 5. (L) , (IR) and (A) 6. (L) , (AR) and (I)

7. (I) , (LR) and (A)

8. (R) and (LIA)

9. (L) and (IAR) 10. (I) , and (ARL) ; or

11. (A) and (RLI) ;

12. (RLIA)

If genetic insert (I) is coupled with either the R or the L gene, the number of possible combinations will then be

(1) : (RI) and (A) ; or (RIA)

(2) : (RI) (L) and (A) (R) , (LI) and (A) (RI) and (LA) (RA) .and (LI) (RLIA)

Also provided by the present invention is a method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a nucleotide sequence or nucleotide sequences encoding R, I and A, wherein R encodes a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and A encodes a substance able to reactivate R inactivated by I, comprising crossing plant lines whose genomes comprise any of R, I, A and combinations thereof, to produce the plant or an ancestor thereof.

A further aspect provides a method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a nucleotide sequence or nucleotide sequences encoding R, L, I and A, wherein R and L encode substances whose presence together in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and/or L and A encodes a substance able to reactivate R and/or L inactivated by I, comprising crossing plant lines whose genomes comprise any of R, L, I, A and combinations thereof, to produce the plant or an ancestor thereof. Said plant lines may contain nucleic acid comprising any of R, L, I, A and combinations thereof as a result of transformation of cells of the plant or an ancestor thereof

Herein, unless context demands otherwise, a "receptor" is a product encoded by a gene capable of interacting with another product, the ligand.

Various embodiments of the present invention are now described in more detail below, by way of example and not limitation.

Nucleotide Sequence or Sequences contributing to the Plant Defence Response and/or Necrosis The nucleotide sequence or combination of nucleotide sequences in which at least one of the sequences is inactivated are numerous and may include an engineered allele of a ubiquitin conjugating enzyme (Becker et al . , 1993), the CaMV gene VI protein (Takashashi et al . , 1989), a viral coat protein in the presence of the appropriate viral resistance gene, for example Tobacco Mosaic Virus Elicitor Coat Protein and the gene N' (Culver and Dawson, 1991) , a bacterial harpin protein (Wei et al . , 1992; He et al . , 1993), the gene N (see e.g. Whitham et al (1994) and a ToMV-Ob gene cloned by Padgett and Beachy (1993) , the potato virus X coat protein and its avirulence determinant, (Kavanagh eϋ al . , 1992; Santa-Cruz et al . , 1993; Kόhm et al . , 1993; Goulden et al . , 1993), Pto and avrPto (see e.g. Rommens et al. , 1995), RPS2 of Arabidopsis thaliana and the avirulence gene avrRPtΣ (Bent et al., Mindrinos et al.), and genes of Arabidopsis such as those identified by Greenberg et al. (1994), Dietrich et al., (1994) and Bowling et al. , (1994). Genes coding for substances leading to rapid cell death, such as BARNASE (Mariani et al . , 1990) or diphtheria toxin (Thorsness et al . , 1993) may be usable to induce the changes that lead to GAR even though cell death in these latter examples is not caused by activation of the defence response. It is widely believed amongst researchers in this field that cell death arises from local induction of the defence response and that this cell death can activate adjacent cells to give rise to the defence response. However, the precise cause and effect relationship between these events is not clear at the present time. It is also not clear whether the defence response in plants is necessarily coupled to necrosis. Hence, cells may respond to for example the BARNASE-induced death of adjacent cells by activating a wound-inducible defence response, such as that leading to the activation of protease inhibitors or alkaloid biosynthesis (Ryan 1990) . Other genes which may be employed in this way include a proton pump such as a bacterial proton pump like the one expressed by Mittler et al (1995) in transgenic tobacco plants .

A preferred example of the present invention is the use of the Cf-9 /Avr 9 gene system. This can involve the matching of a transposon inactivated allele of the Cf-9 gene to constitutive expression of the Avr9 gene. This system can be replaced by similar combinations of related genes for example the Avr4 and Cf-4 gene, sequence provided herein (cloning of Cf-4 is described in a co-pending GB application filed simultaneously with the present application) ; the Avr2 and the Cf-2 gene, sequence provided herein (cloning of Cf-2 is described in GB 9506658.5, priority from which is claimed herein) ; the Avr5 and the Cf-5 gene, or by cloning resistance genes and corresponding avirulence genes from other systems, such as RPP5, sequence provided herein (cloning of RPP5 is described in GB 9507232.8, priority from which is claimed herein). It certain cases it may be possible to provoke a suitable response in plant cells expressing an R gene in the absence of corresponding Avr, for instance by overexpression.

It should also be noted that complete Avr or other elicitor gene may not be required. Instead a fragment may be employed, representing a part of the elicitor molecule which interacts to provoke a plant defence response and/or plant cell necrosis.

It is possible that the nucleotide sequence comprises the inactivated R gene, the inactivated Avr gene or both, or comprises both the R and Avr gene wherein one of the genes is inactivated. Depending of the genes used, the plant defence response and/or plant cell necrosis may be dependent on the expression of both genes and so one example would be that the R gene could be constitutively expressed and the Avr gene could exhibit somatic variegation for expression due to somatic excision and restoration of Avr9 gene expression, or vice versa.

Nucleotide sequences employed in the present invention may encode a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence. An alteration to or difference in a nucleotide sequence may or may not be reflected in a change in encoded amino acid sequence, depending on the degeneracy of the genetic code. Preferred mutants, derivatives and alleles are those which retain a functional characteristic of the protein encoded by the wild-type gene, in the present context the ability to contribute to a plant defence response and/or plant cell necrosis. Of course, changes to the nucleic acid which make no .difference to the encoded amino acid sequence are included.

Similarly, homologues of the various genes whose use is disclosed herein from other species or races may be employed, as may mutants, variants and derivatives of such homologues.

Inactivation and Reactivation of the nucleotide Sequence or Sequences Contributing to the Plant Defence Response and/or Necrosis

A method according to the present invention may employ any of a variety of transposon systems known to the skilled person, including the maize Activator/Dissociation (hereinafter referred to Ac/Ds system) (Fedoroff, 1989) ; the maize Enhancer/Suppressor imitator (En/Spm) system (Fedoroff, 1989) ; and the

Antirrhinum Taml and Tam3 systems (Coen et al . , 1989). In addition, any modified recombination systems which are engineered to yield the appropriate results may be employed, such as, the bacterial Cre-Loxp (Odell et al , 1990) or the "FLP/FRT" system (Lloyd and Davis, 1994) . It will be apparent to the skilled person that the particular choice of transposon, recombination or other system used to inactivate the nucleotide sequence or sequences which encode substances leading to the plant defence response and/or plant cell necrosis is not essential to or a limitation of the present invention.

In some systems, a transposon or recombination system might be so active that an unacceptable level of necrosis is seen. If encountered, this may be overcome by engineering alleles of the transposon or recombinase recognition sequence in which the frequency at which activated nucleotide sequences arise is reduced, such as with Ac(Cla) (Keller et al . , 1993). Alternatively, chemical or site-directed mutagenesis may be used to recover alleles of the necrosis-inducing genes which are less active and therefore result in less severe levels of plant cell necrosis (Hammond-Kosack et al . , 1994) .

In other systems, transposition or recombination may be inefficient resulting in too few activated nucleotide sequences leading to an insufficient level of plant cell necrosis. This may be overcome by constructing suitable promoter fusions to the transposase or recombinase gene in the plant gene (Swinburne et al . , 1992) to increase the frequency of excision or recombination to efficient levels. The most suitable promoter might give rise predominantly to late small sectors of necrosis during organ development rather than early large sectors.

Many other variations are possible as mechanisms for activating the defence response and/or necrosis after transposon excision or recombination. A form of the Cf-9 gene may be constructed so that it activates the defence response even in the absence of its ligand. For example, the Drosophila receptor sevenless (involved in eye development) can be mutated so that it is activated in the absence of its ligand (Basler et al , 1991) . For example, high level expression of a disease resistance gene, or expression of a disease resistance gene in another species, may lead to activation of the defence response and/or necrosis even in the absence of an avirulence product. Bonneus, et al (1995) . In an alternative, the original disease resistance gene may be mutated so that it binds to a defined chemical such as an agrichemical and this chemical activates Cf-9 to initiate the defence response and/or necrosis. Hence, genotypic variegation for excision activating the gene may occur, without initiation of the somatic necrotic reaction due to the defence response. The defence response would be initiated when the agrichemical is applied and recognised by the resistance gene triggering the same reaction as if the avirulence gene product were present.

Introducing the Nucleotide Sequence or Sequences which Contribute to Variegation for the Plant Defence Response and/or Necrosis into the Plant Genome The inactivated nucleotide sequence, or combination of nucleotide sequences at least one of which is inactivated, codes for a substance or substances which when expressed in the plant activates the defence response and/or leads to plant cell necrosis resulting in broad spectrum pathogen resistance.

The nucleic acid may be in the form of a recombinant vector, for example a plasmid or agrobacterium binary vector (Van den Elzen et al. , 1985) . The nucleic acid may be under the control of an appropriate promoter and regulatory elements for expression in a plant cell. In the case of genomic DNA, this may contain its own promoter and regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter and regulatory elements for expression in the host cell.

Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory * sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference. When introducing a chosen gene or gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted may be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material may or may not occur according to different embodiments of the invention. In a preferred embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. Finally, as far as plants are concerned the target cell type should be such that cells can be regenerated into whole plants.

Plants transformed with a DNA segment containing pre-sequence may be produced by standard techniques which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984) , particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966), electroporation (EP 290395, WO 8706614) or other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611) . Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Although Agrobacterium has been reported to be able to transform foreign DNA into some monocotyledonous species (WO 92/14828) , microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg. bombardment with

Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233) . The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention.

Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate (Herrera-Estrella et al , 1983; van den Elzen et al , 1985) .

The present invention is particularly beneficial for use in crop and amenity plants. Examples of suitable plants include tobacco, potato, pepper, cucurbits, carrot, vegetable brassicas, lettuce, strawberry, oil seed brassicas, sugar beet, wheat, barley, maize, rice, soybeans, peas, sunflower, carnation, chrysanthemum, other ornamental plants, turf grass, poplar, eucalyptus and pine.

Still further details of embodiments of the present invention are described in the following non- limiting examples, with reference to the accompanying drawings. In the drawings:

Figure 1 schematically depicts the Cf-9 gene, showing tagged alleles. X marks a probable promoter.

Figure 2 illustrates genetic acquired resistance to C. fulvum induced following necrotic sector formation caused by the excision of a Ds element from the Cf-9 resistance gene in an Avr9 expressing tomato plant. The number of C. fulvum pustules per leaf is indicated, 14 days after inoculation.

Figure 3 illustrates genetic acquired resistance to Phytophthora infestans (late blight of tomato and potato) .* GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and CfO plants spray inoculated with 10,000 sporangiospores/mL. In panel A the appearance of leaves from the mutant 50 experiment 7 days after inoculation is shown. In panel B the rate of leaf abscission (in days after inoculation) in the various genotypes inoculated is given.

Figure 4 illustrates genetic acquired resistance to Phytophthora infestans (late blight of tomato and potato) . GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and CfO plants were spray inoculated with 100 sporangiospores/mL. In panel A the appearance of leaves from the mutant 50 (GAR+ - right-hand) experiment 7 days after inoculation is shown, compared with GAR- (left-hand) . In panel B the rate of sporulating lesion formation on the various plant genotypes inoculated is given, with the mean number of sporulating lesions/leaflet given at 5, 7, 10, 13 and 16 days after inoculation.

Figure 5 shows genetic acquired resistance to Oidium lycopersici (powdery mildew disease) . GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and CfO plants were painted with equivalent numbers of spores. In panel A the appearance of leaves 14 days after inoculation is shown, GAR- on the left, GAR+ on the right. In B, the rate of chlorotic lesion (upper panel) and sporulating lesion (lower panel) formation on the various plant genotypes is given for Mutant 31: mean number of lesions given at 7, 10, 14, 21, 24 and 30 days after inoculation. C shows equivalent results for Mutant 50.

Figure 6 shows the appearance of tomato fruits on GAR⁺ ( sAc, Cf-9*Ds - right-hand) and GAR^" ( sAc, Cf-9*Ds, Avr-9 - left-hand) plants from mutant line M23 at 2, 3, 4, 5, 6 and 7 weeks after flower pollination. Dark green sectors formed on the GAR⁺ but not GAR^" fruits by 5 weeks. These dark green sectors were not visible on the red fruit.

Figure 7 shows levels of defence-related gene expression in GAR+ and GAR- plants from Cf-9*Ds mutant lines M23, M31 and M50 just prior to the pathogen inoculation experiments. Northern analysis shows in panel A the levels of a basic β-1,3 glucanase gene transcript and in panel B the levels of an anionic peroxidase gene transcript.

Figure 8 illustrates functional expression of the Cf-9 gene under the control of its own promoter in tobacco and potato. In panel A is shown a tobacco leaf that has been injected with intercellular fluid (IF) either containing the Avr9 peptide or lacking the Avr9 peptide. Avr9+ IF"was obtained from transgenic tobacco or a compatible C. fulvum - tomato interaction involving race 5. Avr9- IF was obtained from untransformed tobacco or a compatible C. fulvum - tomato interaction involving race 2,4,5,9. Grey necrosis was visible 3-4 h after injection only in the leaf panels that had received the Avr+ IF. In panel B four separate potato leaves are shown that have each been injected with a single type of IF. Only the two leaves that received the Avr9+IF developed grey necrosis by 24 h.

Figure 9 shows development of the necrotic lethal phenotype in seedlings from the tobacco cross cv. Petite Havana 6201A (35S;SP;Avr9,)homozygote x cos 34.1 (genomic Cf-9) heterozygote. A time course for the period 5-12 days after seed planting (dsp) is shown. 50% of the seedlings become chlorotic and die within 2 days of seed germination.

Figure 10 shows development of the necrotic lethal phenotype in seedlings from the Arabidopsis cross 6201B4 ( 35S :SP:Avr9)heterozygote x cos 138 (genomic Cf-9) heterozygote. Appearance of seedlings 19 days after the majority of seedlings had germinated. One seedling has died and another has necrotic cotyledons.

Figure 11 shows a single T-DNA construct systems to apply GAR to potato plants. The T-DNA contains a Cf-9 gene sequence under the control of its own promoter which has been inactivated by an autonomous Ac element that is only capable of a low level of excision, the Ac (Cla) element (Keller et al. 1993; Schofield et al. 1994) and the 35S:SP:Avr9 transgene. Figure 12 shows a photograph of three leaves, two of which are diseased with C. fulvum and one which is expressing GAR and is resistant to the same inoculum of C. fulvum. Figure 13 illustrates how GAR⁺ plants may be made by crossing stable lines (1) comprising a Cf-9 gene, inactivated by insertion of a Ds transposon, and an Avr- 9 gene and (2) an Ac transposase gene, as described in Example 1. Figure 14 illustrates basic simplified haploid crossing schemes to produce plants with increased disease resistance. T: transgenic line

P: offspring of transgenic line T₁/P_χ : line comprising in its genome at least one of each of the four genes, R, L,I or A ^τι ₂/^pι ₂ line comprising in its genome at least one of each of two of the four genes R, L, I or A

T₃/P₃: line comprising in its genome at least one of each of the four genes R,L,I or A not present in T_{1 2} ^T ₃ /^p _{3 4} ^: line comprising in its genome at least one of two of the four genes R,

L, I or A not present in T_{1 2} ^_{l 2 3}/^_{1 2 3} ^: line comprising in its genome at least one of each of three of the four genes R,L,I or A T₄/P₄ line comprising in its genome at least one of each of the four genes R,L,I or A not present in T-_ _{2 3}

SEQ ID NO. 1 shows the genomic DNA sequence of the Cf-9 gene. Features: Nucleic acid sequence - Translation start at nucleotide 898; translation stop at nucleotide 3487; polyadenylation signal (AATAAA) at nucleotide 3703-3708; polyadenylation site at nucleotide 3823; a 115 bp intron in the 3' non-coding sequence from nucleotide 3507/9 to nucleotide 3622/4. Predicted Protein Sequence - primary translation product 863 amino acids; signal peptide sequence amino acids 1-23; mature peptide amino acids 24-863.

SEQ ID NO. 2 shows Cf-9 protein amino acid sequence.

SEQ ID NO. 3 shows the sequence of one of the Cf- 9 cDNA clones. Translation initiates at the ATG at position +58.Cf-9 genomic sequence

SEQ ID NO. 4 shows the amino acid sequence and DNA sequence of the preferred form of the chimaeric Avr 9 gene used as described herein.

SEQ ID NO. 5 shows the genomic DNA sequence of the Cf-2. 1 gene. Features: Nucleic acid sequence -

Translation start at nucleotide 1677; translation stop at nucleotide 5012; no consensus polyadenylation signal (AATAAA) exists in the characterised sequence downstream of the translation stop. Predicted Protein Sequence - primary translation product 1112 amino acids; signal peptide sequence amino acids 1-26; mature peptide amino acids 27-1112.

SEQ ID NO. 6 shows Cf-2 protein amino acid sequence, designated Cf-2.1.

SEQ ID NO. 7 shows the amino acid sequence encoded by the Cf-2.2 gene. Amino acids which differ between the two Cf-2 genes are underlined.

SEQ ID NO. 8 shows the sequence of an almost full length cDNA clone which corresponds to the Cf2-2 gene. SEQ ID NO. 9 shows the genomic DNA sequence of the RPP5 gene. Anticipated introns are shown in non¬ capitalised letters. Features: Nucleic acid sequence - Translation start at nucleotide 966; translation stop at nucleotide 5512.

SEQ ID NO. 10 shows predicted RPP5 protein amino acid sequence.

SEQ ID NO. 11 shows genomic DNA sequence of Cf-4. Features of this sequence include: translation start site at nucleotide 201, translation stop beginning at nucleotide 2619, consensus polyadenylation sequence beginning at nucleotide 2835, splice donor sequence in 3' untranslated sequence at 2641, splice acceptor sequence ending at nucleotide 2755, proposed site of polyadenylation at nucleotide 2955.

SEQ ID NO. 12 shows the predicted Cf-4 amino acid sequence. The predicted protein sequence is composed of a primary translation product of 806 amino acids, signal peptide sequence amino acids 1-23, mature peptide amino acids 24-806.

SEQ ID NO. 13 shows double-stranded nucleic acid and deduced amino acid sequence of a Clal/Sall DNA fragment encoding the PRla signal peptide sequence fused to a sequence proposed to encode the mature processed form of C. fulvum AVR4. Translation initiation codon at nucleotide 5, termination codon beginning at nucleotide 413. Amino acids 1-30 represent the signal peptide and amino acids 31-136 the mature AVR4 peptide.

EXAMPLE 1

GENETIC ACQUIRED RESISTANCE (GAR) USING Cf-9

(i ) Establishing a stock from which gametes carrying a mutagenised Cf-9 gene may be obtained and identified During experiments to isolate the Cf-9 gene by transposon tagging, alleles of the Cf-9 gene ( Cf-9*Ds) were isolated that had been inactivated by insertion of the transposon Ds (See International Patent Application No. PCT/GB94/02812 for further details) . This inactivated Cf-9*Ds gene did not give rise to a constitutive and lethal activation of defence mechanisms in response to the constitutively expressed 35S:SP:Avr9 gene.

We have established the capacity to carry out transposon tagging in tomato using the maize transposon Activator (Ac) and its Dissociation (Ds) derivatives (Scofield et al 1992; Thomas et al 1993; Carroll et al 1993) . The strategy is founded on the fact that these transposons preferentially transpose to linked sites. Various lines that carry Dss at positions are useful, including FT33 (Rommens et al 1992) , carrying a Ds linked to Cf-9 , and lines that carry a construct SLJ10512 (Scofield et al 1992) which contains (a) a beta-glucuronidase (GUS) gene (Jefferson et al 1987) to monitor T-DNA segregation and (b) stable Ac (sAc) that expresses transposase and can trans-activate a Ds, but which will not transpose (Scofield et al 1992) .

The line FT33 did not carry a Cf-9 gene. We had to obtain recombinants that placed Cf-9 in cis with the T-DNA in FT33 in order to carry out linked targeted tagging. Two strategies were pursued simultaneously:

(a) FT33 was crossed to Cf9, a stock that carries the Cf-9 gene. The resulting Fl was then back crossed to CfO (a stock that carries no Cf- genes) . Progeny that carry the FT33 T-DNA are kanamycin resistant. Kanamycin resistant progeny were tested for the presence of Cf- 9 ; 5 C. fulvum resistant individuals were obtained among 180. We alsogenerated progeny that were homozygous for Cf-9 and carried that sAc T-DNA of SLJ10512. These were crossed to the recombinants in which Cf-9 and FT33 were in cis . In the FT33 T-DNA, a transposable Ds element is cloned into a hygromycin resistance gene, preventing its function. The somatic transactivation of this Ds element, which only occurs in the presence of transposase gene expression, results in activation of the hygromycin resistance. Thus from crossing the recombinants between Cf-9 and FT33, to the sAc-carrying Cf-9 homozygotes, hygromycin resistant individuals could be obtained which carry sAc and FT33, and are likely to be homozygous for Cf-9. 140 individuals of this genotype were thus obtained, (b) To accelerate obtaining individuals that carried sAc, FT33, and were Cf-9 homozygotes, the FT33/Cf-9 Fl was crossed to a line that was heterozygous for Cf-9 and. sAc. 25% of the resulting progeny carried both T-DNAs and were hygromycin resistant, and of those, slightly more than 5Q% were disease resistant because they carried at least one copy of the Cf-9 gene. An RFLP marker was available, designated CP46, that enabled us to distinguish between homozygotes and heterozygotes for the Cf-9 gene (Balint-kurti et al 1993) . In this manner two individuals that were Cf-9 homozygotes, and that carried both the FT33 T-DNA and sAc, were obtained. These two individuals were multiplied by taking cuttings so that more crosses could be made onto this genotype.

(ii) Establishing a tomato stock that expresses functional mature AVR9 protein

A likely frequency for obtaining any desired mutation in a gene tagging experiment is less than 1 in 1000, and often less than 1 in 10,000 (Dδring, 1989). To avoid screening many thousands of plants for mutations to disease sensitivity, we established a selection for such mutations based on expressing the fungal Avr9 gene in plants.

The sequence of the 28 amino acids of the mature Avr9 protein is known (van Kan et al 1991) . It is a secreted protein and can be extracted from intercellular fluid of leaves infected with Avr9- carrying races of C. fulvum. For secretion from plant cells, we designed oligonucleotides to assemble a gene that carried a 30 amino acid plant signal peptide, from the Prla gene (Cornelissen et al 1987) preceding the first amino acid of the mature Avr9 protein (see SEQ ID NO. 4) . The preferred Avr9 gene sequence depicted in SEQ ID NO. 4 shows a chimaeric gene engineered from the Pr-la signal peptide sequence (Cornelissen et al , 1987) and the Avr9 gene sequence (van Kan et al , 1991) . This reading frame was fused to the 355 promoter of cauliflower mosaic virus (Odell et al 1984), and the 3' terminator sequences of the octopine synthase gene (DeGreve et al 1983) , and introduced into binary plasmid vectors for plant transformation, using techniques well known to those skilled in the art, and readily available plasmids (Jones et al 1992) . We obtained transformed CfO tomato lines that expressed this gene.

(iii) Crossing AVR9 expressing stock wi th Cf-9 expressing stock

The transformed lines obtained in (ii) were crossed to plants that carried the Cf-9 gene. When the resulting progeny were germinated, 50% exhibited a necrotic phenotype, that culminated in seedling death. This outcome was only observed in seedlings that contained the Avr9 gene. When the same transformants were crossed to CfO plants, the resulting progeny were all fully viable. From selfing the primary transformants, individuals were identified that were homozygous for the Avr9 transgene. When Avr9 homozygotes were crossed to Cf- 9 , all progeny died. This system thus provides a powerful selection for individuals that carry mutations in the Cf-9 gene. (iv) Tagging and inactivating Cf-9

Individuals that were homozygous for the Avr 9 gene (section (iv) ) were used as male parents to pollinate individuals that were homozygous for Cf- 9 , and carried both sAc and the Ds in the FT33 T-DNA

(section (iiia) and (iiib) ) . Many thousands of progeny resulting from such a cross were germinated. Most died, but some survived.

DNA was obtained from survivors and subjected to Southern blot analysis using a Ds probe. It was observed that several independent mutations were correlated with insertions of the Ds into a Bglll fragment of a consistent size. This suggested that several independent mutations were a consequence of insertion of the Ds into the same DNA fragment.

Using primers to the Ds sequence, DNA adjacent to the Ds in transposed Ds-carrying mutant #18 was amplified using inverse PCR (Triglia et al 1988) . This DNA was used as a probe to other mutants, and proved that in independent mutations, the Ds had inserted into the same 6.7 kb Bglll fragment.

The Ds in FT33 contains a bacterial replicon and a chloramphenicol resistance gene as. a bacterial selectable marker (Rommens et al 1992) . This means that plant DNA carrying this transposed Ds can be digested with a restriction enzyme that does not cut within the Ds (such as Bglll) , the digestion products can be recircularized, and then used to transform E. coli . Chloramphenicol resistant clones can be obtained that carry the Ds and adjacent plant DNA. This procedure was used to obtain a clone that carried 1.8 kb of plant DNA on the 3' side of the Ds, and 4.9 kb of plant DNA on the 5' side of the Ds .

Our present understanding of the Cf-9 gene is depicted schematically in Figure 1. The Cf-9 gene sequence and the deduced amino acid sequence are shown in the sequence listing.

A series of primers (Fl, 2, 3, 4, 5, 6, 7, 12, 13, 10, 26, 27 and 25, indicated in Figure 1) was used to characterise a large number of independent mutations by PCR analysis in combination with primers based on the sequence of Ds. Therefore, these primers were used in polymerase chain reactions with primers based on the maize Ac/Ds transposon sequence, to characterise the locations of other mutations of Cf-9 that were caused by transposon insertion. Eighteen independent insertions have been characterized and are located as shown. Mutants E, #55, #74 and #100 gave incomplete survival and showed a necrotic phenotype, and based on the available sequence information, they are 5' to the actual reading frame and might permit enough Cf9 protein expression to activate an incomplete defence response.

Using the sequence obtained of the gene, oligonucleotide primers were designed that could be used in polymerase chain reactions in combination with primers based on the sequence of the Ds element, to characterize both the location and the orientation of other transposon insertions in the gene. These are shown on Figure 1. Based on the results of such experiments, the map positions of 17 other Ds insertions have been reliably assigned (as shown in Figure 1) . (v) Production of GAR plants

On backcrossing plants that carried the Cf- 9*Ds and 35S:SP:Avr9 gene to tomato plants that carried an Ac transposase gene (sAc that lacked the GUS gene) in the homozygous state, but lacked Cf-9, one quarter of the resulting progeny carried sAc, 35S:SP:Avr9 and Cf- 9*Ds (see Figure 13) plants showed somatic excision of Ds from the Cf- 9*Ds gene, somatically restoring Cf-9 function, and giving rise to necrotic somatic sectors in which the defence response was activated. Phenotypically, these plants thus showed a variegation for a defence-related necrosis, in the same manner that plants challenged with necrotizing pathogens show somatic flecks of HR that are associated with the induction of SAR. Necrotic sectors were visible on cotyledons, leaves, stems, petioles, sepals, and green fruits throughout plant development. Also, the necrotic sectors formed in both the lower and upper epidermis, in all mesophyll layers and in the cells surrounding the vascular tissue. The size of the necrotic sector and the frequency of their formation was determined by both the position of the Ds element in the Cf-9 sequence and the orientation of the Ds .

The plants that variegated for necrosis were tested to assess if they were more resistant to C. fulvum than their unvariegated siblings that either carried Cf-9*Ds or carried no Cf-9 gene. Plants from five independent Cf-9*Ds pedigrees were tested in which the Ds had independently inserted into five different locations in the Cf-9 gene. These five independent insertions were between Cf-9 amino acids, 7 and 8 (<M23), 28 and 29 (<M18) , 47 and 48 (>M50) , 56 and 57 (>M31) and 789 and 790 (>M30) The arrows (< or >). indicates the direction of transcription* of the Ds element. F₁ plants that developed somatic necrotic sectors were more resistant to C. fulvum than sibling offspring that did not develop necrotic sectors. On the plants with necrotic sectors an average of 1-2 small pustules per leaf developed, 14 days after inoculation with 5 x 10⁵ spores/ml. The plants lacking a Cf gene and the non variegating individuals all showed on average 38 large sporulating pustules per leaf. A example of this is shown in Figure 2.

Nine variegated Cf-9*Ds #20 plants, fifteen variegated Cf-9*Ds #23 plants, eighteen variegated Cf- 9*Ds #30 plants and twenty-eight variegated Cf-9*Ds #31 plants were tested, and compared to one hundred and ninety eight plants in total that did not variegate for necrosis. Plants were inoculated with C. fulvum (5 x 10⁵ spores/ml) when they were four weeks old and carried 2 expanded leaves. A similar result was obtained when variegated Cf-9*Ds #50 plants and non- variegated plants were inoculated with C. fulvum. On 18 variegated GAR⁺ #50 plants 1-3 pustules per leaf formed, whereas on 42 non-variegated GAR^" #50 plants over 35 pustules per leaf developed by 14 days after inoculation.

Sensitivity to the pathogen was measured by counting the number of sporulating pustules that were visible on each genotype 14 days and 21 days after inoculation. Samples were also taken for microscopic analysis. The results of the assay after 14 days are shown in Figure 2, and typical infections on each genotype after 21 days are shown in Figure 12. Figure 2 shows a histogram in which the sensitivity of different individual tomato plants is expressed on the y axis as the number of sporulating pustules per leaf. The Ds carried a GUS gene. M20, M23, M30 and M31 show C. fulvum growth on plants resulting from crosses between Cf-9*Ds and sAc, and derive from Cf-9*Ds #20, Cf-9*Ds #23, Cf-9*Ds #30 and Cf-9*Ds #31, respectively. These individuals segregate from the Cf-9*Ds and for sAc. CfO carries no R genes and M20, M23, M30 and M31 GUS- plants have lost by segregation both Cf- 9*Ds and sAc and are thus disease sensitive sibs, providing a good control for disease symptoms in sensitive individuals. If plants receive Ds without sAc they may be GUS+ without expressing the variegation for necrosis which requires both Cf-9*Ds and sAc. As can be seen, the necrotic individuals (which all carry the 35S:Avr9 gene) show distinctly fewer pustules per leaf than their disease sensitive sibs.

Figure 2 shows that in these experiments, CfO plants (lacking the Cf-9 gene) exhibited about 38 pustules per leaf and non-variegating individuals derived from Cf-9*Ds #20, Cf-9*Ds #23 or Cf-9*Ds #31 also showed about 38 pustules per leaf. The non- variegated individuals that carried Cf- 9*Ds #30 showed about 17 pustules per leaf indicating some residual action of the tagged Cf-9 allele. However, variegated individuals that carried Cf-9*Ds #20, Cf-9*Ds #23,. Cf- 9*Ds #30 or Cf-9*Ds #31 showed 1-3 pustules per leaf. In total seventy variegated individuals were assessed. These results demonstrate a very significant level of disease control by this method.

Figure 12 shows three leaves. Leaf 1 and Leaf 2 are infected with C. fulvum which confers the white fluffy appearance. Leaf 1 is CfO and Leaf 2 is a disease sensitive sib from Cf-9*Ds #23. Leaf 3 showing minimal sporulation is a necrotic individual (small sectors of necrosis are discernible) that carried Cf- 9*Ds #23, sAc and 35S:Avr9. Leaf 3 is therefore expressing GAR.

It is important to recognize that in this example regions of variegating plants that resist the C. fulvum pathogen do not contain a functional Cf-9 gene. Indeed all the cells that do carry a functional Cf-9 gene

(whose function was restored somatically by transposon excision) are killed as they turn on the defence response after recognition of the endogenously expressed AvrS peptide. Thus, non-resistant cells are being induced to resistance by necrosis being manifested in adjacent cells.

EXAMPLE 2

Pathogen resistance of variegated plants employing Cf-9

In addition to demonstrating that variegated plants produced in Example 1 have enhanced resistance to C. fulvum, we have established that the plants are also more resistant to three unrelated fungal pathogens, Phytophthora infestans (the causal agent of late blight disease of tomato and potato) and Oidium lycopersici (a powdery mildew) and Colleto richiun largenarium (which causes leaf and fruit spot) . For the P. infestans experiments, sibling backcross progeny from the utatnt Cf-9* Ds lines M31 and M50 that were either variegating for necrosis or not and control plants lacking a Cf-gene (CfO) were challenged by a spray application of sporangiosspores (10,000 or 100 spores/ml) of the highly virulent isolate DSSI (Al mating type) . After inoculation, the plants were kept in diffuse light conditions at a constant 100% RH and 16°C and a 12h photoperiod. Seven days after application of the high spore dose the leaves of the unvariegated plants and those of the CfO plants were completely destroyed by the spread of P. infestans lesions which had abundant sporangiospores at their margins. In contrast, the variegated plants were infected with P. infestans but the lesions were 3-5 mm in diameter and non-sporulating (Figure 3 A,B) . An additional 5-6 days were required before the entire green leaf tissue of the variegated plants was destroyed and fungal sporulation commenced. At the lower spore dose, by 7 days after inoculation, an average of 8-10 large sporulating lesions were present on each leaf of the unvariegated and CfO plants whereas on the plants variegating for necrosis there were 1-2 small non-sporulating lesions per 10 leaves (Figure 4 A,B) . A minimum of 18 plants were used for each genotype/spore.

For the Oidiu lycopersici experiments the identical plant genotypes were used. Each leaf was inoculated by brushing with an artist paintbrush the spores from a single 14 day old sporulating pustule over an entire upper surface. The inoculated plants were then kept under diffuse light conditions at 20°C during the 16 h photoperiod and at 18°C during the dark period. The RH was maintained at 70%.

By day 10 post inoculation 8-10 chlorotic lesions were evident on the leaves of the unvariegated and CfO plants and in 1-2 of these sporulation had commenced. By contrast on the variegated plants 1-2 smaller chlorotic non-sporulating lesions were present on each leaf (Figure 5) . By day 14 post inoculation more than 20 sporulating lesions per leaf were present on the unvariegated plants and these were accompanied by severe chlorotic symptoms on the remainder of the leaf. On the variegated plants 2-4 small sporulating lesions were present per leaf (Figure 5A) . An additional 7-10 days were required before a similar level of sporulation and chlorosis formed on the variegated leaves to that found on the unvariegated and CfO leaves at day 14 post-inoculation. (16 plants each) .

EXAMPLE 3

Variegation in fruit Dark green sectors formed on green tomato fruits of GAR plants, 5 weeks after flower pollination (Figure 6) . These sectors were not visible once the tomato fruit had turned red, which is encouraging for potential commercial exploitation. When mature red fruit taken from GAR⁺ and GAR^" plants were injected with lOOμl of spores of Colletotrichum laginarium (10⁴ spores/ml) only the GAR^" fruit exhibited the typical soft rot disease symptoms seven days later. Repeated inoculations of the GAR⁺ fruit failed to cause disease.

Collectively, the above results attest to a very significant level of disease control that can be achieved in the plants variegating for restoration of Cf-9 gene function whilst constitutively expressing the Avr9 gene. The data also indicate that the disease control achievable by this method is potentially broad spectrum because the four fungal pathogens controlled have very dissimilar modes of parasitism: C. fulvum is a biotroph that does not form haustoria and grows exclusively in the extracellular spaces of the leaf mesophyll layers; 0. Lycopersici is also a biotroph but colonises only the upper leaf epidermis and forms complex intracellular haustoria; P. infestans and C . largenarium are hemibiotroph that initially forms simple haustoria but later on kills host cells in both the epidermal and mesophyll layers. Homozygous Cf-9*Ds, 35S: SPAvr9 lines have been established for the tomato lines M31 and M50. The F, backcross progeny derived from crosses to a homozygous sAc source, may be assessed for their resistance to various pathogens, including:

Potato virus X, Pseudomonas syringae pv. tomato, Necrotrophic fungi - Botrytis spp, Colletotrichum spp, Nematodes - Meloidogyne incognata, Aphids - Green Peach Aphid, and fruit, pod, root or tuber attacking pathogens. Also, the effect of GAR on the establishedment of mycorrhizal associations may be tested.

The enhanced resistance exhibited in the plants variegating for necrosis has been termed Genetic Acquired Resistance (GAR) . It is distinct from SAR because it is a heritable trait and is active throughout the entire plants life.

When the expression of several defence-related genes were compared in the GAR^" and GAR⁺ plants, significantly higher levels of expression of each gene were found in the GAR⁺ plants. Examples of this are shown in Figure 7 for Cf-9*Ds lines from M23, M31 and M50 pedigrees using a basic tomato β-1,3 glucanase probe and a tomato anionic peroxidase probe (pTAP 4.5) .

The effectiveness of GAR in suppressing plant disease appears to be inversely related to sector size. The two independent Cf-9*Ds pedigrees that have the highest frequency of small necrotic sectors (lines M31 and M50) give the best GAR. This indicates that by carefully manipulating the frequency of somatic restoration of Cf-9 function even higher levels of plant protection be developed. Currently, there are two possible hypotheses to explain GAR. Either the initially activated host cells generate local and systemic signals whilst still alive, and the necrotic lesions are a by-product of the Cf-9- Avr9 mediated responses. Alternatively, the actual death and necrotic reactions, the final response of the activated host cells, generates specific local and systemic signals in a manner analogous to SAR. Exactly how GAR works does, not need to be known* for the present invention to be operated. Provided the required genetic components are present, GAR plants have enhanced pathogen resistance compared with wild-type.

EXAMPLE 4

Expression of Cf-9 in Heterologouε Plants Species and

Induction of Cell Necrosis We have shown that following the transfer of different genomic clones containing the Cf-9 gene into tobacco and potato, these sequences render the transgenic plants responsive to Avr9 elicitor (Figure 8) . Also when transgenic tobacco expression Cf-9 is crossed to transgenic tobacco plants engineered to express Avr9 peptide constitutively, the Fl seedlings die within 2 days of seed germination (Figure 9) .

When transgenic Arabidopsis expressing Cf-9 is crossed to Avr9 expressing transgenic Arabidopsis the Fl seedlings die 10 days after seed germination (Figure 10) .

Thus we have shown that in a variety of species, genes required for activation of plant defence, mediated by the Cf-9 protein, are present and functional.

EXAMPLE 5

Genetic Acquired Resistance Using Cf-9 in Potato

To apply GAR to potato plants a single T-DNA construct systems is used. The system is based around a single T-DNA construct (Figure 11) containing, a Cf-9 gene sequence under the control of its own promoter which has been inactivated by an autonomous Ac element that is only capable of a low level of excision (the Ac (Cla) element (Keller et al. 1993), and the 355:SP:Avr9 transgene) . The Ac element is inserted at various positions in the Cf-9 sequence and in both orientations in order to determine the best configuration to produce a high frequency of small somatic sectors where Cf-9 function has been restored.

Placing the Cf-9 sequence or other R gene sequence under the control of a cell-type specific promoter may enhance the GAR phenotype. Potential target cellular sites include the epidermis and the vascular parenchyma cells.

EXAMPLE 6

Expression of Cf-4 in transgenic plants and demonstration of increased pathogen resistance

The Cf-4 gene has been tested in transgenic plants in a number of ways: firstly by inoculation with a race of C. fulvum containing the corresponding avirulence gene Avr4 to test if that race gives an incompatible response on the transgenic plant; secondly by injecting leaves of a transformed plant with intercellular fluid isolated from a compatible interaction containing AVR4; thirdly, by delivering AVR4 in the form of recombinant potato virus X as described previously in studies of the Cf-9/AVR9 ^■interaction (Hammond-Kosack et al . , 1995).

The DNA sequence of the C. fulvum gene encoding AVR4 has been reported and the amino acid sequence of the mature processed polypeptide (Joosten et al . , 1994) . We amplified by PCR the Avr 4 gene from C. fulvum race 2,5 using primers to the published sequence and fused a sequence encoding the proposed mature polypeptide to a DNA sequence encoding the N-terminal signal peptide of the tobacco PRla protein. This would facilitate targeting of AVR4 to the intercellular space in transgenic plants where it is expressed. This chimeric gene (SPAvr4) was inserted into a cDNA copy of potato virus X, as a Clal/Sall DNA fragment (SEQ ID NO. 13) as described previously (Hammond-Kosack et al . ,1995) to generate PVX: SPAvr4. Infectious transcripts of the recombinant virus were generated by in vi tro transcription. All nucleic acid manipulations were performed using standard techniques well known to those skilled in the art.

Toma to

Experiments were designed to test the recombinant virus in 3 week old tomato seedlings. In Cf-4 containing plants inoculated cotyledons appeared desiccated and eventually abscised at 3 days post-inoculation (d.p.i.), in contrast to CfO controls which only showed signs of slight mechanical damage at the site of virus inoculation. CfO plants developed visible symptoms of virus infection at 7-10 d.p.i. comparable to symptoms observed with the wild type virus i.e. chlorotic mosaic symptoms. At 4-5 d.p.i. in plants containing Cf-4 necrotic lesions were observed in the younger leaves, presumably due to systemic spread of the virus as described previously in similar experiments with PVX containing Avr9 on Cf-9 containing plants (Hammond-Kosack et al . , 1995) . Other features included necrotic sectors on petioles and the stem. The necrotic phenotype was seen to spread systemically and at 14 d.p.i. the majority of Cf-4 containing seedlings had died. CfO control plants did not die but did show symptoms of chlorosis and vein-clearing.

These results confirm that Cf-4 is functional in transgenic tomato plants, resulting in a necrotic defence response in the presence of elicitor AVR4.

Tobacco

Using binary vector cosmids comprising Cf-4 , transgenic tobacco plants have also been produced (Fillatti et al .,1987; Horsch et al . , 1985) using techniques well known to those skilled in the art. Transgenic tobacco containing cosmids comprising Cf-4 were inoculated with PVX:SPAvr4. In most transformants necrotic lesions were observed at the site of virus inoculation 3-4 d.p.i. similar in appearance to lesions which appear in response to virus inoculation in some virus resistant varieties. In these individuals the necrosis was not strictly confined to local lesions which eventually coalesced and at 7-10 d.p.i. leaf necrosis was apparent over the entire region of virus inoculation. In several transformants the reaction to PVX. : SPAvr4 was more acute and the necrotic leaf sectors could be observed at 3-4 d.p.i. Neither of these phenotypes were observed in transgenic tobacco containing cosmids lacking Cf-4 or in non-transformed control plants challenged with PVX: SPAvr4 . Functional expression of Cf-4 in transgenic tobacco has thus also been shown, with activation of a necrotic defence response in the presence of elicitor AVR4.

Pathogen Resistance ^' Transgenic plants were propagated by cuttings so that Cf-4 activity could be detected by inoculation with PVX:SPAvr4 on 12 tomato transformants. Transgenic tomato plants containing Cf-4 exhibited leaf necrosis on inoculated leaves 3-4 d.p.i. This necrosis eventually spread systemically as previously observed in Cf-4 containing plants in the experiments described above. Transgenic plants exhibiting necrotic leaf sectors eventually died.

Cuttings of a number of transgenic plants obtained in the first round of transformation experiments were further assayed for Cf-4 function by inoculation with C. fulvum race 5. In 5 transgenic plants tested, a positive correlation was observed between plants exhibiting PVX : SPAvr4 dependent necrosis and resistance to the pathogen. In this experiment pathogen growth was observed on compatible control plants (CfO) but not on incompatible control plants (Cf2) .

All documents mentioned in the text are incorporated herein by reference.

REFERENCES :

Balint-Kurti, et al. (1994) Theor. App. Genet. 88:691-700.

Basler, et al. (1991) Cell, 64, 1069-1081.

Becker, et al. (1993) Plant Journal 3, 875-881. Bent, A.F., et al. (1994) Science 265, 1856.

Biffen, R.H. (1905) J. Ag. Sci. 1, 4-48.

Carroll B.J. et al. (1993) . Genetics (In Press) .

Chang, C, et al. (1992) The Plant Cell 4:1263-1271.

Christou, P. (1992) The Plant Journal, 2(3), 275-281. Coen, et al. (1989) In Mobile DNA. D.E. Berg and M.M.

Howe, eds. (Washington: ASM Pres) ,

Cornelissen, et al. (1987) Nucl.Acids.Res 15:6799-6811.

Culver, et al. (1991) Molecular Plant Microbe

Interactions 4, 458-463. De Wit, P.J.G.M. (1992) . Ann. Rev. Phytopathol. 30, 391-418.

DeGreve, et al. (1983) J. ol.Appl .Genet. 1:499-511.

Dickinson, et al . (1993) . Mol. Plant Mic. Int. 6, 341- 347. Dietrich; R.A. , et al . (1994) Cell 77, 565-577. Dόring H-P (1989) . An overview. Maydica 34:73-88. Enyedi, et al. (1992) Cell 70, 879-886. Fedoroff, N.V. (1989) In Mobile DNA. M. Howe and D. Berg, eds. (Washington: ASM Press) , pp. 375-411. Fillatti JJ, et al. (1987) . Bio/technol. 5:726-730. Flor, H.H. (1971) Ann. Rev. Phytopathol. 9, 275-296. Gabriel, et al. (1990) Ann.Rev.Phytopathol. 28:365-391. Gaffney, et al. (1993) Science 261, 754-756. Goulden, et al. (1993) The Plant Cell 5, 921-930. Greenberg, J.T., et al. (1994) Cell 77, 551-563. Greenberg, et al. (1993) Plant Journal 4,327-341. Hamilton, et al. (1990) Nature 346, 284-287. Hammond-Kosack, K.E., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 10445-10449. Hammond-Kosack, et al. (1994) The Plant Cell 6, 361- 374. Hammond-Kosack et al. (1995) Mol. Plant-Microbe

Interact. 8:181-185 He, et al. (1993) Cell 73, 1255-1266. • Hennig, et al. (1993) Plant Journal 4, 481-493.

Herrera - Estrella, et al. (1983) EMBO J. 2, 987-995. Horsch RB, et al. (1985) . Science (Wash.)

227:1229-1231. Jefferson, et al . (1987) EMBO.J. 6:3901-3907. Johal, et al. (1992) Science (Wash.) . 258:985-987. Johal, et al. (1994) Maydica 39, (in press) . Jones, et al. (1992) Transgen.Res. 1:285-297. Jones, D.A., et al . (1994) Science 266, 789.

Jones, et al. (1993) Mol. Plant Mic. Int. 6, 348-357.

Jones, et al . (1994) Curr. Biol. 4, 67-69.

Joosten, et al. (1994) . Nature 367, 384-386. Kavanagh, et al . (1992) Virology 189, 609-617.

Keen, N.T. (1992) Ann. Rev. Gen. 24, 447-463.

Keller, et al. (1993) Molec. Biol. 21, 159-170.

Kδhm, et al. (1993) The Plant Cell 5, 913-920.

Lloyd, et al. (1994) Mol Gen Genet 242, 653-657. Long, et al. (1993) Cell 73, 921-935.

Malamy, et al. (1990) Science (Wash.) . 250, 1002-1004.

Mariani, et al. (1990) Nature 347, 737-741.

Marmeisse, et al. (1993) MPMI 6, 412-417.

Martin, et al. (1993) Science 262, 1432-1436. Metraux, et al . (1990) Science (Wash.) . 250, 1004-1006.

Mindrinos, M. , et al. (1994) Cell 78, 1089-1099.

Mittler, R., et al. (1995) The Plant Cell 7, 29-42.

Napoli, et al . (1990) . The Plant Cell 2,279-289.

Odell, et al. (1984) Nature 313:810-812. Odell, et al. (1990) Mol. Gen. Genet. 223, 369-378.

Padgett, H.S., et al. (1993) The Plant Cell 5, 577-586.

Pryor, et al . (1983) Advances in Plant Pathology 10, 281-305.

Pryor, T. (1987) . Trends. Genet. 3,157-161. Rommens C.M.T., et al . (1992) Pl.Molec.Biol. 20:61-70.

Rommens, C.M.T., et al . (1995) The Plant Cell 7, 249- 257. Ross, A.F. (1961) Virology 14, 340-358. Ryals, et al. (1992) SEB Symposium 49, 205-229. Ryan, CA. (1990) Ann. Rev. Phytopathol. 28, 425-449. Santa Cruz, et al. (1993) Molecular Plant-microbe Interactions 6, 707-714.

Scofield, S., et al. (1992) The Plant Cell 4:573-582. Scott, A., et al. (1994) The Plant Cell 6, 1845-1857. Stein, J.C., et al. (1991) Proc.Natl.Acad.Sci.USA 88:8816-8820. Swinburne, et al (1992) The Plant Cell 4, 583-592.

Takahashi, et al (1989) Molecular & General Genetics

216, 188-194. Thomas, C ., et al. (1993) Molecular and General Genetics (In Press) . Thorsness, et al (1983) The Plant Cell 5, 253-261.

Triglia, T., et al. (1988) Nucleic Acids Res. 16:8186. Uknes, et al. (1992) The Plant Cell 4,645-656. Valon, C, et al. (1993) Pl.Molec.Biol. 23:415-421. van den Ackerveken, et al. (1993) Plant Physiol. van der Beek, et al. (1992) Theor.App.Genet. 84:106- 112. van den Elzen, et al. (1985) Plant. Mol. Biol. 5, 149-

154. van Kan J.A.L., et al. (1991) MPMI 4:52-59. van den Elzen, et al. (1985) Plant. Mol. Biol. 5, 299- 302. Van Den Ackerveken, et al. (1992) Plant Journal 2, 359- 366. Walbot, V., et al. (1983) T. Kosuge et al, eds. (Plenum

Press) , pp. 431-430. Walker, J.C, (1993) Plant Journal 3:451-456. Ward, et al (1991) The Plant Cell 3, 1085-1094. Wei, et al. (1992) Science (Wash.) . 257, 85-88. Whitham, S., et al . (1994) Cell 78, 1101-1115. Wolter, M. , et al . (1983) Molecular & General Genetics

239, 122-128.

SEQ ID NO . 1 :

CATAGTCTTT GCATATTTGG ATTAAACAGG GGCATTATTG AACCAAACTA TTAGATGTAT 60

GAAAATTTTG GACCAAGCTA TTGACAACAC GAACATTTTT AGACCAAACT ATTAACTCAG 120

AATATTTTCC GTTGAATGAA TAAGGTAACT AGTAGTAAAT TTTTAGACCA AACTATGAAG 180

AACATGCCAT GTCTGGACTC CTGCACTATC TTCCATCAAC AGGTCAATTC TCTCAACTCT 2 0

ATTGGTGGAA GGTAGACGGT ACAAATTGAA TTATATTAAA AGACAAGCTC ACCTGAGCAT 300

CACTGTTATA CAACAACAAC AAACTACGCT TCAGCCCCAA ACAATAGTGA CCCGAATCAT 360

ATATTGTCAC GAGTTTTTTT TAGAGTATGT TGCATATATT ATACTCAACT TAGGGTTTGT 420

CATTCTGATG CTTCGTACAA ATTTATTGAA TTTTCAACTT TAAAGGTTTA TGAACCAAAT 480

ATTACGCTTA CTATGATAGC GGTCTTTTTT GATTAATCAA ACTTATTGAA TTTTCAACTT 540

TAAAGGTTTT TCCCCGTTCT ATACACAAAC TAAGAAAAAT TTAAATTATA TAGTCTTTGG 600

ATGGTGACCT ATTTGGATGG TAACATTATT GGACCAAACT ATTGATAACG CGGACATTGT 660

TAGACCAAAC TGAGAAGGAC ATGTCTGGAC TCCTGCTCCG TCTTCCATCA GCAGGTCGAT 720

TCTTGTGGAA AATTAGCTCG AGGTGGCGCA CTATGTGAGG TAACTAGTAC TAAATTTTTC 780

TTTGCTTAAT TTGTGCTATA TATACCTCAT CTAAATTATT GAATAGTCAC ACAAAGCAAA 840

CATTTCTTGA TTTCTTCTCT ATCAACATAA CAAGTTTTGA TCATTTTTAG TGCAGAA 897

ATG GAT TGT GTA AAA CTT GTA TTC CTT ATG CTA TAT ACC TTT CTC TGT 945 Met Asp Cys Val Lys Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys -23 -20 -15 -10

CAA CTT GCT TTA TCC TCA TCC TTG CCT CAT TTG TGC CCC GAA GAT CAA 993 Gin Leu Ala Leu Ser Ser Ser Leu Pro His Leu Cys Pro Glu Asp Gin -5 1 5

GCT CTT TCT CTT CTA CAA TTC AAG AAC ATG TTT ACC ATT AAT CCT AAT 1041 Ala Leu Ser Leu Leu Gin Phe Lys Asn Met Phe Thr lie Asn Pro Asn 10 15 20 25

GCT TCT GAT TAT TGT TAC GAC ATA AGA ACA TAC GTA GAC ATT CAG TCA 1089 Ala Ser Asp Tyr Cys Tyr Asp lie Arg Thr Tyr Val Asp lie Gin Ser 30 35 40

TAT CCA AGA ACT CTT TCT TGG AAC AAA AGC ACA AGT TGC TGC TCA TGG 1137 Tyr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys Cys Ser Trp 45 50 55

GAT GGC GTT CAT TGT GAC GAG ACG ACA GGA CAA GTG ATT GCG CTT GAC 1185 Asp Gly Val His Cys Asp Glu Thr Thr Gly Gin Val He Ala Leu Asp 60 65 70

CTC CGT TGC AGC CAA CTT CAA GGC AAG TTT CAT TCC AAT AGT AGC CTC 1233 Leu Arg Cys Ser Gin Leu Gin Gly Lys Phe His Ser Asn Ser Ser Leu 75 80 85

TTT CAA CTC TCC AAT CTC AAA AGG CTT GAT TTG TCT TTT AAT AAT TTC 1281 Phe Gin Leu Ser Asn Leu Lys Arg Leu Asp Leu Ser Phe Asn Asn Phe 90 95 100 105

ACT GGA TCA CTC ATT TCA CCA AAA TTT GGT GAG TTT TCA AAT TTG ACG 1329 Thr Gly Ser Leu He Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr 110 115 120 CAT CTC GAT TTG TCG CAT TCT AGT TTT ACA GGT CTA ATT CCT TCT GAA 1377 His Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu He Pro Ser Glu 125 130 135

ATC TGT CAC CTT TCT AAA CTA CAC GTT CTT CGT ATA TGT GAT CAA TAT 1425 He Cys His Leu Ser Lys Leu His Val Leu Arg He Cys Asp Gin Tyr 140 145 150

GGG CTT AGT CTT GTA CCT TAC AAT TTT GAA CTG CTC CTT AAG AAC TTG 1473 Gly Leu Ser Leu Val Pro Tyr Asn Phe Glu Leu Leu Leu Lys Asn Leu 155 160 165

ACC CAA TTA AGA GAG CTC AAC CTT GAA TCT GTA AAC ATC TCT TCC ACT 1521 Thr Gin Leu Arg Glu Leu Asn Leu Glu Ser Val Asn He Ser Ser Thr 170 175 180 185

ATT CCT TCA AAT TTC TCT TCT CAT TTA ACA ACT CTA CAA CTT TCA GGC 1569 He Pro Ser Asn Phe Ser Ser His Leu Thr Thr Leu Gin Leu Ser Gly 190 195 200

ACA GAG TTA CAT GGG ATA TTG CCC GAA AGA GTT TTT CAC CTT TCC AAC 1617 Thr Glu Leu His Gly He Leu Pro Glu Arg Val Phe His Leu Ser Asn 205 210 215

TTA CAA TCC CTT CAT TTA TCA GTC AAT CCC CAG CTC ACG GTT AGG TTT 1665 Leu Gin Ser Leu His Leu Ser Val Asn Pro Gin Leu Thr Val Arg Phe 220 225 230

CCC ACA ACC AAA TGG AAT AGC AGT GCA TCA CTC ATG ACG TTA TAC GTC 1713 Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Tyr Val 235 240 245

GAT AGT GTG AAT ATT GCT GAT AGG ATA CCT AAA TCA TTT AGC CAT CTA 1761 Asp Ser Val Asn He Ala Asp Arg He Pro Lys Ser Phe Ser His Leu 250 255 260 265

ACT TCA CTT CAT GAG TTG TAC ATG GGT CGT TGT AAT CTG TCA GGG CCT 1809 Thr Ser Leu His Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro 270 275 280

ATT CCT AAA CCT CTA TGG AAT CTC ACC AAC ATA GTG TTT TTG CAC CTT 1857 He Pro Lys Pro Leu Trp Asn Leu Thr Asn He Val Phe Leu His Leu 285 290 295

GGT GAT AAC CAT CTT GAA GGA CCA ATT TCC CAT TTC ACG ATA TTT GAA 1905 Gly Asp Asn His Leu Glu Gly Pro He Ser His Phe Thr He Phe Glu 300 305 310

AAG CTC AAG AGG TTA TCA CTT GTA AAT AAC AAC TTT GAT GGC GGA CTT 1953 Lys Leu Lys Arg Leu Ser Leu Val Asn Asn Asn Phe Asp Gly Gly Leu 315 320 325

GAG TTC TTA TCC TTT AAC ACC CAA CTT GAA CGG CTA GAT TTA TCA TCC 2001 Glu Phe Leu Ser Phe Asn Thr Gin Leu Glu Arg Leu Asp Leu Ser Ser 330 335 340 345

AAT TCC CTA ACT GGT CCA ATT CCA TCC AAC ATA AGC GGA CTT CAA AAC 2049 Asn Ser Leu Thr Gly Pro He Pro Ser Asn He Ser Gly Leu Gin Asn 350 355 360

CTA GAA TGT CTC TAC TTG TCA TCA AAC CAC TTG AAT GGG AGT ATA CCT 2097 Leu Glu Cys Leu Tyr Leu Ser Ser Asn His Leu Asn Gly Ser He Pro 365 370 375

TCC TGG ATA TTC TCC CTT CCT TCA CTG GTT GAG TTA GAC TTG AGC AAT 2145 Ser Trp He Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn 380 385 390 AAC ACT TTC AGT GGA AAA ATT CAA GAG TTC AAG TCC AAA ACA TTA AGT 2193 Asn Thr Phe Ser Gly Lys He Gin Glu Phe Lys Ser Lys Thr Leu Ser 395 400 405

GCC GTT ACT CTA AAA CAA AAT AAG CTG AAA GGT CGT ATT CCG AAT TCA 2241 Ala Val Thr Leu Lys Gin Asn Lys Leu Lys Gly Arg He Pro Asn Ser 410 415 420 425

CTC CTA AAC CAG AAG AAC CTA CAA TTA CTT CTC CTT TCA CAC AAT AAT 2289 Leu Leu Asn Gin Lys Asn Leu Gin Leu Leu Leu Leu Ser His Asn Asn 430 435 440

ATC AGT GGA CAT ATT TCT TCA GCT ATC TGC AAT CTG AAA ACA TTG ATA 2337 He Ser Gly His He Ser Ser Ala He Cys Asn Leu Lys Thr Leu He 445 450 455

TTG TTA GAC TTG GGA AGT AAT AAT TTG GAG GGA ACA ATC CCA CAA TGC 2385 Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr He Pro Gin Cys 460 465 470

GTG GTT GAG AGG AAC GAA TAC CTT TCG CAT TTG GAT TTG AGC AAA AAC 2433 Val Val Glu Arg Asn Glu Tyr Leu Ser His Leu Asp Leu Ser Lys Asn 475 480 485

AGA CTT AGT GGG ACA ATC AAT ACA ACT TTT AGT GTT GGA AAC ATT TTA 2481 Arg Leu Ser Gly Thr He Asn Thr Thr Phe Ser Val Gly Asn He Leu 490 495 500 505

AGG GTC ATT AGC TTG CAC GGG AAT AAG CTA ACG GGG AAA GTC CCA CGA 2529 Arg Val He Ser Leu His Gly Asn Lys Leu Thr Gly Lys Val Pro Arg 510 515 520

TCT ATG ATC AAT TGC AAG TAT TTG ACA CTA CTT GAT CTA GGT AAC AAT 2577 Ser Met He Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn 525 530 535

ATG TTG AAT GAC ACA TTT CCA AAC TGG TTG GGA TAC CTA TTT CAA TTG 2625 Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gin Leu 540 545 550

AAG ATT TTA AGC TTG AGA TCA AAT AAG TTG CAT GGT CCC ATC AAA TCT 2673 Lys He Leu Ser Leu Arg Ser Asn Lys Leu His Gly Pro He Lys Ser 555 560 565

TCA GGG AAT ACA AAC TTG TTT ATG GGT CTT CAA ATT CTT GAT CTA TCA 2721 Ser Gly Asn Thr Asn Leu Phe Met Gly Leu Gin He Leu Asp Leu Ser 570 575 580 585

TCT AAT GGA TTT AGT GGG AAT TTA CCC GAA AGA ATT TTG GGG AAT TTG 2769 Ser Asn Gly Phe Ser Gly Asn Leu Pro Glu Arg He Leu Gly Asn Leu 590 595 600

CAA ACC ATG AAG GAA ATT GAT GAG AGT ACA GGA TTC CCA GAG TAT ATT 2817 Gin Thr Met Lys Glu He Asp Glu Ser Thr Gly Phe Pro Glu Tyr He 605 610 615

TCT GAT CCA TAT GAT ATT TAT TAC AAT TAT TTG ACG ACA ATT TCT ACA 2865 Ser Asp Pro Tyr Asp He Tyr Tyr Asn Tyr Leu Thr Thr He Ser Thr 620 625 630

AAG GGA CAA GAT TAT GAT TCT GTT CGA ATT TTG GAT TCT AAC ATG ATT 2913 Lys Gly Gin Asp Tyr Asp Ser Val Arg He Leu Asp Ser Asn Met He 635 640 645

ATC AAT CTC TCA AAG AAC AGA TTT GAA GGT CAT ATT CCA AGC ATT ATT 2961 He Asn Leu Ser Lys Asn Arg Phe Glu Gly His He Pro Ser He He 650 655 660 665 GGA GAT CTT GTT GGA CTT CGT ACG TTG AAC TTG TCT CAC AAT GTC TTG 3009 Gly Asp Leu Val Gly Leu Arg Thr Leu Asn Leu Ser His Asn Val Leu 670 675 680

GAA GGT CAT ATA CCG GCA TCA TTT CAA AAT TTA TCA GTA CTC GAA TCT 3057 Glu Gly His He Pro Ala Ser Phe Gin Asn Leu Ser Val Leu Glu Ser 685 690 695

TTG GAT CTC TCA TCT AAT AAA ATC AGC GGA GAA ATT CCG CAG CAG CTT 3105 Leu Asp Leu Ser Ser Asn Lys He Ser Gly Glu He Pro Gin Gin Leu 700 705 710

GCA TCC CTC ACA TTC CTT GAA GTC TTA AAT CTC TCT CAC AAT CAT CTT 3153 Ala Ser Leu Thr Phe Leu Glu Val Leu Asn Leu Ser His Asn His Leu 715 720 725

GTT GGA TGC ATC CCC AAA GGA AAA CAA TTT GAT TCG TTC GGG AAC ACT 3201 Val Gly Cys He Pro Lys Gly Lys Gin Phe Asp Ser Phe Gly Asn Thr 730 735 740 745

TCG TAC CAA GGG AAT GAT GGG TTA CGC GGA TTT CCA CTC TCA AAA CTT 3249 Ser Tyr Gin Gly Asn Asp Gly Leu Arg Gly Phe Pro Leu Ser Lys Leu 750 755 760

TGT GGT GGT GAA GAT CAA GTG ACA ACT CCA GCT GAG CTA GAT CAA GAA 3297 Cys Gly Gly Glu Asp Gin Val Thr Thr Pro Ala Glu Leu Asp Gin Glu 765 770 775

GAG GAG GAA GAA GAT TCA CCA ATG ATC AGT TGG CAG GGG GTT CTC GTG 3345 Glu Glu Glu Glu Asp Ser Pro Met He Ser Trp Gin Gly Val Leu Val 780 785 790

GGT TAC GGT TGT GGA CTT GTT ATT GGA CTG TCC GTA ATA TAC ATA ATG 3393 Gly Tyr Gly Cys Gly Leu Val He Gly Leu Ser Val He Tyr He Met 795 800 805

TGG TCA ACT CAA TAT CCA GCA TGG TTT TCG AGG ATG GAT TTA AAG TTG 3441 Trp Ser Thr Gin Tyr Pro Ala Trp Phe Ser Arg Met Asp Leu Lys Leu 810 815 820 825

GAA CAC ATA ATT ACT ACG AAA ATG AAA AAG CAC AAG AAA AGA TAT TAGTGAGTAG 3496 Glu His He He Thr Thr Lys Met Lys Lys His Lys Lys Arg Tyr 830 835 840

CTATACCTCC AGGTATTCCA CTTGATCATT ATCTTTCAGA AGATTATTTT TTGTATATCG 3556

ATGAAATTAT CGACCTCCTT CATCCTCAAA GCTCTTAACT TTCACTCTTC ATTTTTGAAA 3616

ATTTCAGGAT TCAAAGATTT CCGAGTTCCC AGTTGCTTGG GATGCAGATA AAAGCCTTT.T 3676

TATCTTTCAT AGTTTCTTAT CCTATGAATA AAGATTTTAT TTTCATTTGT CTATGGCACG 3736

TAGATATGTT CCGTCACTAA AAACATTGTA TTTCTCTCAA CTCTTTCGTC ACATGATATC 3796

AAAGAACACT TGACTTCAAT TAAGTTACTG TAGTCTGCTA TTTTAATTTT TTCCATTGAA 3856

ACACAACTGA CGTATCTTGA GAAAGAGACT ATGATCCCCC GGGCTGCAG 3905

SEQ ID NO. 2:

Met Asp Cys Val Lys Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys -23 -20 -15 -10

Gin Leu Ala Leu Ser Ser Ser Leu Pro His Leu Cys Pro Glu Asp Gin

-5 1 5 Ala Leu Ser Leu Leu Gin Phe Lys Asn Met Phe Thr He Asn Pro Asn 10 15 20 25

Ala Ser Asp Tyr Cys Tyr Asp He Arg Thr Tyr Val Asp He Gin Ser 30 35 40

Tyr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys Cys Ser Trp 45 50 55

Asp Gly Val His Cys Asp Glu Thr Thr Gly Gin Val He Ala Leu Asp 60 65 70

Leu Arg Cys Ser Gin Leu Gin Gly Lys Phe His Ser Asn Ser Ser Leu 75 80 85

Phe Gin Leu Ser Asn Leu Lys Arg Leu Asp Leu Ser Phe Asn Asn Phe 90 95 100 105

Thr Gly Ser Leu He Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr 110 115 120

His Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu He Pro Ser Glu 125 130 135

He Cys His Leu Ser Lys Leu His Val Leu Arg He Cys Asp Gin Tyr 140 145 150

Gly Leu Ser Leu Val Pro Tyr Asn Phe Glu Leu Leu Leu Lys Asn Leu 155 160 165

Thr Gin Leu Arg Glu Leu Asn Leu Glu Ser Val Asn He Ser Ser Thr 170 175 180 185

He Pro Ser Asn Phe Ser Ser His Leu Thr Thr Leu Gin Leu Ser Gly 190 195 200

Thr Glu Leu His Gly He Leu Pro Glu Arg Val Phe His Leu Ser Asn 205 210 215

Leu Gin Ser Leu His Leu Ser Val Asn Pro Gin Leu Thr Val Arg Phe 220 225 230

Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Tyr Val 235 240 245

Asp Ser Val Asn He Ala Asp Arg He Pro Lys Ser Phe Ser His Leu 250 255 260 265

Thr Ser Leu His Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro 270 275 280

He Pro Lys Pro Leu Trp Asn Leu Thr Asn He Val Phe Leu His Leu 285 290 295

Gly Asp Asn His Leu Glu Gly Pro He Ser His Phe Thr He Phe Glu 300 305 310

Lys Leu Lys Arg Leu Ser Leu Val Asn Asn Asn Phe Asp Gly Gly Leu 315 320 325

Glu Phe Leu Ser Phe Asn Thr Gin Leu Glu Arg Leu Asp Leu Ser Ser 330 335 340 345

Asn Ser Leu Thr Gly Pro He Pro Ser Asn He Ser Gly Leu Gin Asn 350 355 360 Leu Glu Cys Leu Tyr Leu Ser Ser Asn His Leu Asn Gly Ser He Pro 365 370 375

Ser Trp He Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn 380 385 390

Asn Thr Phe Ser Gly Lys He Gin Glu Phe Lys Ser Lys Thr Leu Ser 395 400 405

Ala Val Thr Leu Lys Gin Asn Lys Leu Lys Gly Arg He Pro Asn Ser 410 415 420 425

Leu Leu Asn Gin Lys Asn Leu Gin Leu Leu Leu Leu Ser His Asn Asn 430 435 440

He Ser Gly His He Ser Ser Ala He Cys Asn Leu Lys Thr Leu He 445 450 455

Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr He Pro Gin Cys 460 465 470

Val Val Glu Arg Asn Glu Tyr Leu Ser His Leu Asp Leu Ser Lys Asn 475 480 485

Arg Leu Ser Gly Thr He Asn Thr Thr Phe Ser Val Gly Asn He Leu 490 495 500 505

Arg Val He Ser Leu His Gly Asn Lys Leu Thr Gly Lys Val Pro Arg 510 515 520

Ser Met He Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn 525 530 535

Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gin Leu 540 545 550

Lys He Leu Ser Leu Arg Ser Asn Lys Leu His Gly Pro He Lys Ser 555 560 565

Ser Gly Asn Thr Asn Leu Phe Met Gly Leu Gin He Leu Asp Leu Ser 570 575 580 585

Ser Asn Gly Phe Ser Gly Asn Leu Pro Glu Arg He Leu Gly Asn Leu 590 595 600

Gin Thr Met Lys Glu He Asp Glu Ser Thr Gly Phe Pro Glu Tyr He 605 610 615

Ser Asp Pro Tyr Asp He Tyr Tyr Asn Tyr Leu Thr Thr He Ser Thr 620 625 630

Lys Gly Gin Asp Tyr Asp Ser Val Arg He Leu Asp Ser Asn Met He 635 640 645

He Asn Leu Ser Lys Asn Arg Phe Glu Gly His He Pro Ser He He 650 655 660 665

Gly Asp Leu Val Gly Leu Arg Thr Leu Asn Leu Ser His Asn Val Leu 670 675 680

Glu Gly His He Pro Ala Ser Phe Gin Asn Leu Ser Val Leu Glu Ser 685 690 695

Leu Asp Leu Ser Ser Asn Lys He Ser Gly Glu He Pro Gin Gin Leu 700 705 710 OOZT VODODWXVO wooxvooxx wooxooxov vxoooxxwo oxvoxvxxxv ovxoooowo

0*TI XXOWOOOVO WXXXOOXVX XOXXOVOXXO VOOOOOXVOX XOWOWXV WXDXXOVOX

080T VXXODVOWO XOOWWOXX XVXVOOVOXX VOOOXXXW OOVOOWOXX OXVOOWXVO oεoτ OOXXOOVOO xxxxxoxovx vowoovoxo xwooxvxox oovwxooxx vxooooovox

096 OXOXWXOXX OOXDOOXVOV XOXXDVOXVO XOVOXXOW OXVOODVXX xvoxvwxoo 006 VXVOOVXVOX OOXXVXWOX OXOVXVOOXO OVXVXXOOVO VOXOVOXVO OXOVODVXW

0*8 OOXVWOOW OVOOOXXXDD vxxooovoxo ovooooxwo ovoxvxxxv oxxoooxwo

08t. VXXOWOOXX XOOVOXXXXX OVOVWOOOO OXXVXVOOOX VOVXXOVOVO VODOVOXXXO

OZL wovxoxow owxxxvoxo xxoxoxxxw voxxooxxvx ovooxxoxox vovwxoxox

099 woxxoowo xoovovowx xwooovoxx wowxxoo xooxowoxx xxwovxxoo

009 vxoxxoxovx xooooxvxw oxvoxoxvxv xooxxoxxoo vovxovwxo xxxoovoxox

0*S oxvwoxoxx ooxxwxoxo ovovxxxxov xoxxvoooxo xxvooxoxv ooovoxxxw

08* voxxxxovox ooxxxwwo ovoxxxvoxo voxvooxovo xxwxwxx xxoxoxxxvo

O Z* XOOOWWO XOXWOOXOX OWOXXXOXO ODVXDVXWO OXXVOXXXOV VODDWOXXO

09ε wooovooxx oooxoovoxx ooooxxvoxo wovoovovo ovovoovoxo xxvoxxoooo ooε xvoooxvoxo oxooxxowo voowwow ooxxoxxxox owowooxv xvoxovoxxv 0*3 OVOVXOOVXV OWOWXVOV OOVXXOXXVX XVDXOXXOOX wxooxwxx voovxxxoxv

081 owowoxxv vovxoxxoxo xxxoxoowo xvowooooo oxoxxxvoxo ooxxooxvox ozτ ooxvxxxoox xowoxoxox oxxxoovxvx vxooxvxxoo xxvxoxxow wxoxoxxvo 09 DXWVOVODX OVXXXXXVOX VOXXXXDWO wxvowoxv xoxoxxoxxx voxxoxxxvo

• £ O αi Oas

sλ^~ι xv_j_ x^~- _ sχι sχι STH nχo

SZ8 0Z8 ST8 018 si sΛ-i ns-x dsv 3SW S^V -Z3S 3Id d j, BXV oxa xλj. uχζ> -iHX χ-~S

S08 008 S6t.

33W 3X1 fa 3X1 X*Λ J3S ns-x Λχo 3X1 X^Λ ns λχo sΛo Aχo Λx Λχo

06t. S8t. 08t.

XBΛ ns-i Έ χo uχo dj:χ as sχi }3W o^d *Z3S ds nχø nχo nχo nχo

St.t. Ot.t. S9t. nχo UXD sv nsq nχo ιsχv o^d *zqx qx χτ=Λ χo dsv nχo λ.χo Aχo sλo

09t. SSt. O St. na^~ι sA-i ss ns*ι oαd 3iϊ *^XO B-^V πsq λχ dsv usv *Λχo ^~~XO XΛJ, X^S s*t. o*t. sε. oεt.

.njl, usv XD 3tϊd 23S dsv 3tjd uχo s L~ι λ.χ s-^T O-id 3X1 s*^0 ^XD X^Λ

^<≡_* ZL O ZL SXL na~ι STH usv STH -iss nsq usv ns χι=Λ nχo ns*χ aqd -mx ns*i as BXY

8

St.0I0/S6a9/X3«I f9SI£/S6 OΛV oε sz oε sΛo Λ BXV β s o ss STH ΛSS sχi X^_Λ πa*χ θtjd πs ns*τ ns αqx 96 XDX OVX OOO XOO ODX XOX OVO OOX VXV VXD VXO OXX VXX DJ.D XXO VOV sτ oτ s τ

•-I3S X^Λ ns-x nsq 3t{d -33S o^d ns-i uχo -I3S 3^d nsq I^Λ 3^d -* 033W 8* XOX 0X0 XXO XXO XXX VOX XOO OXX WO VOX XXX 0X0 XXO XXX VDD OXV

0883 WWWVWV VWXXDWXX WOXXOVOXX OVOWOVWO XVXVDXVOVO XDOXXXOXOV 0383 VOXOXOXXXV XOXXVOWW VXOVOXOOOX XOXVXVOVXD ovoooxvxox oxxxvoxxxx 09t.3 VXXXXVDVW xwoxvxoox vxxoxxxovx voxxxoxvxx xxxoooww xvovooxvoo

OOt.3 OXXODXXOVO OOXXOVOOOX XXVOVWOXX VOOVOOXOOV XVXOOVXOVO XDVXXVXVOV

0*93 VWOWOVOO VWWOXVW VOOVXOVXXV VXVOVOWOO XXDWVXXXV ooxvoovoox

08S3 XXXDDXVODV OOXVXWOXO WOXDDXOXV VXVOVXVXW XDOOXOXOVD DXXVXXOXXO

03S3 VOOXOXXOOO VXXOOOXDOX OXXDOODOVO DDXXOVOXVO XWOOVOXXV OWOWDOVO

09*3 OVOWOWOX VOVXOOVOXO OVOOXOWOV OXOWOXVOV iQLΩ€>^DΩJ.S XXXOWWOX

00*3 OXOVOOXXXV OOOOOVXXOO DXVDXWODO WOOVXOOXX OVOWOOOOX xooxxvoxxx

0*€3 WOWWOOV WOOOOXVOO XVOOXXOXXO XVOXWOVOX OXOXOXVWX XOXOWDXXO

0833 OXXVOVOXOO OXVOOXXOOV ODVODOOXXV WDVODOOVO XWWXWXO XVOXOXOXVO

0333 OXXXOXWDO XOVXOVOXVX XXWWOXXX VOXVOOOOOV XVXVOXOOW OOXXOXDXW

0913 OVOXOXOXXO WDXXDOVXO OXXOVOOXXO XXOXVOVDOX XVXXVODWO OXXVXVOXDO

00T3 WOXXXVDVO WOVWOXOX OXWOXVXXV DXVOWXOXX VOOXXXXWO OXXOXOXXVO

0*03 XVXXVDWOV DDOVWOVXO XXWOVOOV OXXXVXXWO XXVXXXVXV DXVXVOOXVO

086X XOXXXVXVXD VOVOOOXXVO DVOVXOVOVO XVDXXWVOD WOXVOOVW OOXXXWOOD

0361 DXXXXWOW VOOOOVXXXV VDDDXDVXXX VDDXWXOXV OXVXOXVDXX OXXVWOXXO

098X XODOXVXXXD XXOVWOVXV VDOOVOXXOX VWOXVOOOX OOXVODXXDV VXVWOXVOV

0081 OXXODWXXX XVOWDXXW OXXXVXOOVX VODDXXDDXO VWOOXXXVO VOVDXWOXX

0*t.X OXVXWOWX ODVXOXVOXX OVXOVOVOXX XVXOWODXX WOXVDXVXO XVDOVOOOXD

089T V ODDOOW XOOWXWDO OOV0DXX0DV XXVDXODDW XXXXVOVWO DXXOXDVXXX

039T XOWOVXWO XWOVOODXD VXXOVDVOW VWOOVDXXX VDDXXXVOOO XXXOOVXWO

09ST OWDDVDVOX XODXOODXW OVOOOXWOV VODDVDDXXX WXWXDWD OOXXOVOVXX

OOST DXXVXVOXXV OWWOXOXV VOOXOXVXOD VOXXOXXXVX VOVODXOVOX vxwxwovo

0**1 VOXXXOOXOX OVXXWOVX OOWOWOVO OWVXOOXOV OXXWDOOXX vxooxoowv

08 i OXODWXV vowwxoxo VXXDOODXOV VXXVOVWVO OXDWOXXDV DWOXXWW

03 τ VODXDVOXXX OVOWXWOO VOXXOVOVXX DVOXXODXOV oxxooxxooo oxxvxvoox

0931 OOXXOOVXVX DVDODXWOX ovoowvox voxoxxovxo oxoxwovx oowwoxxo

6 L

SZ_0l0/S6a9/XD«I f9SΪ€/S6OΛV AAC AGT TCT TGT ACA AGA GCT TTT GAC TGT CTT GGA CAA TGT GGA AGA 144 Asn Ser Ser Cys Thr Arg Ala Phe Asp Cys Leu Gly Gin Cys Gly Arg 35 40 45

TGC GAC TTT CAT AAG CTT CAA TGT GTA CAT TGA 177

Cys Asp Phe His Lys Leu Gin Cys Val His 50 55

SEQ ID NO. 5:

1 CTCGAGTTCG GAACCTAAAA GGTATAAAAT ATTAATAAAA ATTTTAAAAT

51 GGTATATCAA TTTTTATATT AACCAAAACG TCAAAATCGC TGAAACAACA

101 GCGATTTCCT TCACCGGAAA AAGCAAAATC GCTACTACTG CAGCGATTTT

151 GCAAAATGTA ACTTTTTTTT AAAAAAATGC ATATTTTCTT ATAAGCTATA

201 TATTTGAATT TCAAAAAAAA TATTTGAAAA TCAATAAAAT TTGTTTTTCC

251 TACGATTTTC TTTTTAAAAT TCTTTTTTTG GAAAATCCCT ACCTAGGCAG

301 CGATTTCCAT TTTTAATTTT TTTTAAATAA AAGGCAGCGA TTTTCGAAAA

351 AAAAAATTTT AAAAAAAATT GAAAAAGTCG CTGCCTAGGT AGCGATTTGA

401 ATTTTTTTAA AAAATGTTAT ATTTTGCAAA ATCGTTGCAG TAGCAACGAT

451 TTTGCTTTTT TTGGAGGAAA TCGCTGTTGT TCCAGCGATT TTGCCGTTTT

501 GGTTAATATA AAATTTTATA TAACGTTTTG AAATTTTTGT TAATATTTTA

551 TAACTTTTAG GCTCCGGACT CAAGATTACT CCCTCTATCT TAGTTTATAA

601 TGCATAGTCT GAATTTTGAA GAGCCAAATA GTTTAATTTT CGCCATAAAT

651 TCAGACATGA AATCTTTAAA AAAGTTTAAA TAAAATTTGT ATATGTTGAA

701 ACTACAGAAA AAGTATTATA ATTCACGATA ATTTATTCAC AAGCCATCGT

751 CGGAGTGATC GCGAGTGAAG TGAAAGAATT GGAGTTTTTG ATATCCAGAA

801 TCCATCTTGA GAGGTTGAGA TATCTTAATC TATCTCCAAT AAAAAAAAAC

851 TATTAATATC CAATTTTCTT GAAGGCCATT ACCTATTCCG ACAAATTCCA

901 CAAGATACTT CATCATATAA AAAAATAATC TCCGTGAAGA AATTCTTTTA

951 TTTGGAAAAT CGATTTTAGA GTCATTGCAA TTTAATTTTA TCAAAATATT

1001 TGAGCATGAA AAATTTGAAA TGGAGGTGTC ATAAAAATAA AATACCCTTT

1051 AAAACACGGC TTTATTGAGT TGACGATAGT TCAAGTAGGG AAAATAAATA

1101 ACTTATTAAT TGAATATAAA ACTTGCAAGA AAAAAGTGAT ATTCAAATTT

1151 AATTCTGACC ATTATCTCTT GATATTCTTT GCTCTTCATT TATTTGAATA

1201 TTCATTTTTC AAAAGTTCCA CGTCATAAGA CATCAAATAT CAAGTAGGTC

1251 CCATAAAAAT AAAATACCCT TCTCAACATG ACAAAGAAAG ATTGAAAAAT

1301 GACTAACATT TTCTCAAAGA CAAAAACAAA ACATGTGAGA GAAGACATTA

1351 CGAATCATCA TAATCTCTGA GACTGAGAAT TGTTAGATAT GGTCCACTAC 1401 TGTAGAGATG AGAATTTTGA ACCAAATGTA TTATACACTA AGAGTGGTCA

1451 TGATCATTGT GTGATAACAA AACTATTTTG GCAACTTTGA CTCAGTCCTT

1501 GGCTAAATTA GACCTCTAAC ACAAAACAAT CCAAAAGTTG ACTTGAGAAT

1551 GACAACATTT TCTTCCCTGA TAGCAACCAA ATTAGCAAAT TTGGAAAAAA

1601 CGCGTGTCTT GTTGATCTTT AATTAGTATA AGTTACGTAC AATATCCTAT

1651 TGAATTGGAA ACAATAAACT CAAACTATGA TGATGGTTTC TAGAAAAGTA

1701 GTCTCTTCAC TTCAGTTTTT CACTCTTTTC TACCTCTTTA CAGTTGCATT

1751 TGCTTCGACT GAGGAGGCAA CTGCCCTCTT GAAATGGAAA GCAACTTTCA

1801 AGAACCAGAA TAATTCCTTT TTGGCTTCAT GGATTCCAAG TTCTAATGCA

1851 TGCAAGGACT GGTATGGAGT TGTATGCTTT AATGGTAGGG TAAACACGTT

1901 GAATATTACA AATGCTAGTG TCATTGGTAC ACTCTATGCT TTTCCATTTT

1951 CATCCCTCCC TTCTCTTGAA AATCTTGATC TTAGCAAGAA CAATATCTAT

2001 GGTACCATTC CACCTGAGAT TGGTAATCTC ACAAATCTTG TCTATCTTGA

2051 CTTGAACAAC AATCAGATTT CAGGAACAAT ACCACCACAA ATCGGTTTAC

2101 TAGCCAAGCT TCAGATCATC CGCATATTTC ACAATCAATT AAATGGATTT

2151 ATTCCTAAAG AAATAGGTTA CCTAAGGTCT CTTACTAAGC TATCTTTGGG

2201 TATCAACTTT CTTAGTGGTT CCATTCCTGC TTCAGTGGGG AATCTGAACA

2251 ACTTGTCTTT TTTGTATCTT TACAATAATC AGCTTTCTGG CTCTATTCCT

2301 GAAGAAATAA GTTACCTAAG ATCTCTTACT GAGCTAGATT TGAGTGATAA

2351 TGCTCTTAAT GGCTCTATTC CTGCTTCATT GGGGAATATG AACAACTTGT

2401 CTTTTTTGTT TCTTTATGGA AATCAGCTTT CTGGCTCTAT TCCTGAAGAA

2451 ATATGTTACC TAAGATCTCT TACTTACCTA GATTTGAGTG AGAATGCTCT

2501 TAATGGCTCT ATTCCTGCTT CATTGGGGAA TTTGAACAAC TTGTCTTTTT

2551 TGTTTCTTTA TGGAAATCAG CTTTCTGGCT CTATTCCTGA AGAAATAGGT

2601 TACCTAAGAT CTCTTAATGT CCTAGGTTTG AGTGAGAATG CTCTTAATGG

2651 CTCTATTCCT GCTTCATTGG GGAATCTGAA AAACTTGTCT AGGTTGAATC

2701 TTGTTAATAA TCAGCTTTCT GGCTCTATTC CTGCTTCATT GGGGAATCTG

2751 AACAACTTGT CTATGTTGTA TCTTTACAAT AACCAGCTTT CTGGCTCTAT

2801 TCCTGCTTCA TTGGGGAATC TGAACAACTT GTCTATGTTG TATCTTTACA

2851 ATAATCAGCT TTCTGGCTCT ATTCCTGCTT CATTGGGGAA TCTGAACAAC

2901 TTGTCTAGGT TGTATCTCTA CAATAATCAG CTTTCTGGCT CTATTCCTGA

2951 AGAAATAGGT TACTTGAGTT CTCTTACTTA TCTAGATTTG AGTAATAACT

3001 CCATTAATGG ATTTATTCCT GCTTCATTTG GCAATATGAG CAACTTGGCT

3051 TTTTTGTTTC TTTATGAAAA TCAGCTTGCT AGCTCTGTTC CTGAAGAAAT 3101 AGGTTACCTA AGGTCTCTTA ATGTCCTTGA TTTGAGTGAG AATGCTCTTA

3151 ATGGCTCTAT TCCTGCTTCA TTCGGGAATT TGAACAACTT GTCTAGGTTG

3201 AATCTTGTTA ATAATCAGCT TTCTGGCTCT ATTCCTGAAG AAATAGGTTA

3251 CCTAAGGTCT CTTAATGTCC TTGATTTGAG TGAGAATGCT CTTAATGGCT

3301 CTATTCCTGC TTCATTCGGG AATTTGAACA ACTTGTCTAG GTTGAATCTT

3351 GTTAATAATC AGCTTTCTGG CTCTATTCCT GAAGAAATAG GTTACCTAAG

3401 ATCTCTTAAT GACCTAGGTT TGAGTGAGAA TGCTCTTAAT GGCTCTATTC

3451 CTGCTTCATT GGGGAATCTG AACAACTTGT CTATGTTGTA TCTTTACAAT

3501 AATCAGCTTT CTGGCTCTAT TCCTGAAGAA ATAGGTTACT TGAGTTCTCT

3551 TACTTATCTA TCTTTGGGTA ATAACTCTCT TAATGGACTT ATTCCTGCTT

3601 CATTTGGCAA TATGAGAAAT CTGCAAGCTC TGATTCTCAA TGATAACAAT

3651 CTCATTGGGG AAATTCCTTC ATCTGTGTGC AATTTGACAT CACTGGAAGT

3701 GTTGTATATG CCGAGAAACA ATTTGAAGGG AAAAGTTCCG CAATGTTTGG

3751 GTAATATCAG TAACCTTCAG GTTTTGTCGA TGTCATCTAA TAGTTTCAGT

3801 GGAGAGCTCC CTTCATCTAT TTCCAATTTA ACATCACTAC AAATACTTGA

3851 TTTTGGCAGA AACAATCTGG AGGGAGCAAT. ACCACAATGT TTTGGCAATA

3901 TTAGTAGCCT CGAGGTTTTT GATATGCAGA ACAACAAACT TTCTGGGACT

3951 CTTCCAACAA ATTTTAGCAT TGGATGTTCA CTGATAAGTC TCAACTTGCA

4001 TGGCAATGAA CTAGAGGATG AAATCCCTCG GTCTTTGGAC AATTGCAAAA

4051 AGCTGCAAGT TCTTGATTTA GGAGACAATC AACTCAACGA CACATTTCCC

4101 ATGTGGTTGG GAACTTTGCC AGAGCTGAGA GTTTTAAGGT TGACATCGAA

4151 TAAATTGCAT GGACCTATAA GATCATCAAG GGCTGAAATC ATGTTTCCTG

4201 ATCTTCGAAT CATAGATCTC TCTCGCAATG CATTCTCGCA AGACTTACCA

4251 ACGAGTCTAT TTGAACATTT GAAAGGGATG AGGACAGTTG ATAAAACAAT

4301 GGAGGAACCA AGTTATGAAA GCTATTACGA TGACTCGGTG GTAGTTGTGA

4351 CAAAGGGATT GGAGCTTGAA ATTGTGAGAA TTTTGTCTTT GTACACAGTT

4401 ATCGATCTTT CAAGCAACAA ATTTGAAGGA CATATTCCTT CTGTCCTGGG

4451 AGATCTCATT GCGATCCGTA TACTTAATGT ATCTCATAAT GCATTGCAAG

4501 GCTATATACC ATCATCACTT GGAAGTTTAT CTATACTGGA ATCACTAGAC

4551 CTTTCGTTTA ACCAACTTTC AGGAGAGATA CCACAACAAC TTGCTTCTCT

4601 TACGTTTCTT GAATTCTTAA ATCTCTCCCA CAATTATCTC CAAGGATGCA

4651 TCCCTCAAGG ACCTCAATTC CGTACCTTTG AGAGCAATTC ATATGAAGGT

4701 AATGATGGAT TACGTGGATA TCCAGTTTCA AAAGGTTGTG GCAAAGATCC

4751 TGTGTCAGAG AAAAACTATA CAGTGTCTGC GCTAGAAGAT CAAGAAAGCA 4801 ATTCTGAATT TTTCAATGAT TTTTGGAAAG CAGCTCTGAT GGGCTATGGA

4851 AGTGGACTGT GTATTGGCAT ATCCATGATA TATATCTTGA TCTCGACTGG

4901 AAATCTAAGA TGGCTTGCAA GAATCATTGA AAAACTGGAA CACAAAATTA

4951 TCATGCAAAG GAGAAAGAAG CAGCGAGGTC AAAGAAATTA CAGAAGAAGA

5001 AATAATCACT TCTAGACAAG TTACCAATAC AGAAAGATTT GATTTCAGAA

5051 CTTCAGGTAT TCACGCTAAG CTCTAACACT TATCTTTTTT AGTTTATTCT

5101 AACAACTAAT ATATGGTTTT TTTTTATCAA CAAATACTTA TTAAGGCTTG

5151 ATACAAATTG CTATAATCAC TTGGAAGCTG TGATATATAA CAAAGCCTAA

5201 AAATTTATAG TTGTGTGACT CACTTTCTTA TTTTTCAGAT TTTCAGGAGC

5251 CAAGAATTAG AAGACGCTGG TGTAAAGGAT TTGCTTCTTC CTATGTTGCA

5301 GCTTATGATT GTTGGATTTG ATTTTTAGTT TTATAAGGTT TTCTTCAGTT

5351 GGGAAAATGT AATATTTTGA ATTTTGATGA TATATAATAA ATGTTGTGTA

5401 TTGAATGATG TGTATGCATT TCTCGGATCA ATAATACTCA CCTCAAAGAA

5451 TCTAAGAGAG TTAGCGCACG ATAGAAGATA GAACATACAA AGAAGAATAC

5501 ATTACAACCT TGGGCTTGGT TATCTTACAC CCCAAAGCTT GTTATTATGG

5551 AAGGAAAGGC CAAGTTTTAT TTTTAGATAT GGGGAGCCTT GGCGTGCTGG

5601 TAAGGTTGTA GTGGATAAGG TAACTTCTCC TGTTAATGAA TTGAATGATC

5651 ATAGCAGAGA TGTGTTTAAA ATTTCTGTTG TATTAGTTTG TAATATTTGG

5701 AGGTCTTAAA TTGAACAGAT GCACATCTGT TCGTGAAAGA GCATGACTAT

5751 TCTTATAAGT CAACTCTCAA GTTCTATAAA TATAAGGACT CCTAAAGTAG

5801 CATAAGAAAA AACTGCAGTA TACTAAGGCG TTGTTGGATC CTGAAGGGAA

5851 TTGCTGGTAA CCCCCTAAAC AACATACGTT ATATTGGTGG GGGGTAGAAG

5901 GTACCCAGTG AAATAATCTA GGTTTGCATA GGTTGCTCTG CAAACAACAA

5951 TTATTAAACA AAATCCACAC ACACTAGCAC ATGAGAGTAA AAAATTTAAT

6001 GACGAGATGA AAGAAACTCA CGCCAAGATG GACTTTATCA AACAACAAAT

6051 ACATTGTTTG TACCTTTTGG ACAACCATTT ATCACTCAAA GAAGATCAAG

6101 GATTGATGCA TTACATCGTT CTTGGAACAA AATTATGTAC ATAAAACTTA

6151 CAGGAATCAT GTTTTGTGTG TGGTAAAACT CCATAAGGAC TAGTCCAAGA

6201 TACTGAGATC AAGGATTTCT AAGTGCAGCC AATCTCTTCT CCAGTTCATC

6251 GATCCCCGAA CTGCCAGCAC GAAAGCACAA CAACAAAATG TACATGAGCG

6301 AGTTACTGAG ATCAAAGAGC ATGAAAAAAG GCACTTCATA CTAATATGAT

6351 AACTTCATAC TAATATGATA CAATTATTTA CAGGAAGAAA AGAAGAATAG

6401 GAAACCGAAC CGCAACATAC TTTATCTATT AACGAGCAGT GCACTCAAGA

6451 TAACTAGTAT TTTTGCTCGA G SEQ ID NO . 6 :

1 MMMVSRKWS SLQFFTLFYL FTVAFASTEE ATALLKWKAT FKNQNNSFLA

51 SWIPSSNACK DWYGWCFNG RVNTLNITNA SVIGTLYAFP FSSLPSLENL

101 DLSKNNIYGT IPPEIGNLTN LVYLDLNNNQ ISGTIPPQIG LLAILQIIRI

151 FHNQLNGFIP KEIGYLRSLT KLSLGINFLS GSIPASVGNL NNLSFLYLYN

201 NQLSGSIPEE ISYLRSLTEL DLSDNALNGS IPASLGNMNN LSFLFLYGNQ

251 LSGSIPEEIC YLRSLTYLDL SENALNGSIP ASLGNLNNLS FLFLYGNQLS

301 GSIPEEIGYL RSLNVLGLSE NALNGSIPAS LGNLKNLSRL NLVNNQLSGS

351 IPASLGNLNN LSMLYLYNNQ LSGSIPASLG NLNNLSMLYL YNNQLSGSIP

401 ASLGNLNNLS RLYLYNNQLS GSIPEEIGYL SSLTYLDLSN NSINGFIPAS

451 FGNMSNLAFL FLYENQLASS VPEEIGYLRS LNVLDLSENA LNGSIPASFG

501 NLNNLSRLNL VNNQLSGSIP EEIGYLRSLN VLDLSENALN GSIPASFGNL

551 NNLSRLNLVN NQLSGSIPEE IGYLRSLNDL GLSENALNGS IPASLGNLNN

601 LSMLYLYNNQ LSGSIPEEIG YLSSLTYLSL GNNSLNGLIP ASFANMRNLQ

651 ALILNDNNLI GEIPSSVCNL TSLEVLYMPR NNLKGKVPQC LGNISNLQVL

701 SMSSNSFSGE LPSSISNLTS LQILDFGRNN LEGAIPQCFG NISSLEVFDM

751 QNNKLSGTLP TNFSIGCSLI SLNLHGNELE DEIPRSLDNC KKLQVLDLGD

801 NQLNDTFPMW LGTLPELRVL RLTSNKLHGP IRSSRAEIMF PDLRIIDLSR

851 NAFSQDLPTS LFEHLKGMRT VDKTMEEPSY ESYYDDSVW VTKGLELEIV

901 RILSLYTVID LSSNKFEGHI PSVLGDLIAI RILNVSHNAL QGYIPSSLGS

951 LSILESLDLS FNQLSGEIPQ QLASLTFLEF LNLSHNYLQG CIPQGPQFRT

1001 FESNSYEGND GLRGYPVSKG CGKDPVSEKN YTVSALEDQE SNSEFFNDFW

1051 KAALMGYGSG LCIGISMIYI LISTGNLRWL ARIIEKLEHK IIMQRRKKQR

1101 GQRNYRRRNN HF*

SEQ ID NO. 7:

1 MMMVSRKWS SLQFFTLFYL FTVAFASTEE ATALLKWKAT FKNQNNSFLA

51 SWIPSSNACK DWYGWCFNG RVNTLNITNA SVIGTLYAFP FSSLPSLENL

101 DLSKNNIYGT IPPEIGNLTN LVYLDLNNNQ ISGTIPPQIG LLAKLQIIRI

151 FHNQLNGFIP KEIGYLRSLT KLSLGINFLS GSIPASVGNL NNLSFLYLYN

201 NQLSGSIPEE ISYLRSLTEL DLSDNALNGS IPASLGNMNN LSFLFLYGNQ

251 LSGSIPEEIC YLRSLTYLDL SENALNGSIP ASLGNLNNLS FLFLYGNQLS 301 GSIPEEIGYL RSLNVLGLSE NALNGSIPAS LGNLKNLSRL NLVNNQLSGS

351 IPASLGNLNN LSMLYLYNNQ LSGSIPASLG NLNNLSMLYL YNNQLSGSIP

401 ASLGNLNNLS RLYLYNNQLS GSIPEEIGYL SSLTYLDLSN NSINGFIPAS

451 FGNMSNLAFL FLYENQLASS VPEEIGYLRS LNVLDLSENA LNGSIPASFG

501 NLNNLSRLNL VNNQLSGSIP EEIGYLRSLN VLDLSENALN GSIPASFGNL

551 NNLSRLNLVN NQLSGSIPEE IGYLRSLNDL GLSENALNGS IPASLGNLNN

601 LSMLYLYNNQ LSGSIPEEIG YLSSLTYLSL GNNSLNGLIP ASFANMRNLQ

651 ALILNDNNLI GEIPSSVCNL TSLEVLYMPR NNLKGKVPQC LGNISNLQVL

701 SMSSNSFSGE LPSSISNLTS LQILDFGRNN LEGAIPQCFG NISSLEVFDM

751 QNNKLSGTLP TNFSIGCSLI SLNLHGNELE DEIPRSLDNC KKLQVLDLGD

801 NQLNDTFPMW LGTLPELRVL RLTSNKLHGP IRSSRAEIMF PDLRIIDLSR

851 NAFSQDLPTS LFEHLKGMRT VDKTMEEPSY ESYYDDSWV VTKGLELEIV

901 RILSLYTVID LSSNKFEGHI PSVLGDLIAI RILNVSHNAL QGYIPSSLGS

951 LSILESLDLS FNQLSGEIPQ QLASLTFLEF LNLSHNYLQG CIPQGPQFRT

1001 FESNSYEGND GLRGYPVSKG CGKDPVSEKN YTVSALEDQE SNSEFFNDFW

1051 KAALMGYGSG LCIGISIIYI LISTGNLRWL ARIIEELEHK IIMQRRKKQR

1101 GQRNYRRRNN RF*

SEQ ID NO. 8:

1 GGTTTCTAGA AAAGTAGTCT CTTCACTTCA GTTTTTCACT CTTTTCTACC

51 TCTTTACAGT TGCATTTGCT TCGACTGAGG AGGCAACTGC CCTCTTGAAA

101 TGGAAAGCAA CTTTCAAGAA CCAGAATAAT TCCTTTTTGG CTTCATGGAT

151 TCCAAGTTCT AATGCAT.GCA AGGACTGGTA TGGAGTTGTA TGCTTTAATG

201 GTAGGGTAAA CACGTTGAAT ATTACAAATG CTAGTGTCAT TGGTACACTC

251 TATGCTTTTC CATTTTCATC CCTCCCTTCT CTTGAAAATC TTGATCTTAG

301 CAAGAACAAT ATCTATGGTA CCATTCCACC TGAGATTGGT AATCTCACAA

351 ATCTTGTCTA TCTTGACTTG AACAACAATC AGATTTCAGG AACAATACCA

401 CCACAAATCG GTTTACTAGC CAAGCTTCAG ATCATCCGCA TATTTCACAA

451 TCAATTAAAT GGATTTATTC CTAAAGAAAT AGGTTACCTA AGGTCTCTTA

501 CTAAGCTATC TTTGGGTATC AACTTTCTTA GTGGTTCCAT TCCTGCTTCA

551 GTGGGGAATC TGAACAACTT GTCTTTTTTG TATCTTTACA ATAATCAGCT

601 TTCTGGCTCT ATTCCTGAAG AAATAAGTTA CCTAAGATCT CTTACTGAGC

651 TAGATTTGAG TGATAATGCT CTTAATGGCT CTATTCCTGC TTCATTGGGG

701 AATATGAACA ACTTGTCTTT TTTGTTTCTT TATGGAAATC AGCTTTCTGG 751 CTCTATTCCT GAAGAAATAT GTTACCTAAG ATCTCTTACT TACCTAGATT

801 TGAGTGAGAA TGCTCTTAAT GGCTCTATTC CTGCTTCATT GGGGAATTTG

851 AACAACTTGT CTTTTTTGTT TCTTTATGGA AATCAGCTTT CTGGCTCTAT

901 TCCTGAAGAA ATAGGTTACC TAAGATCTCT TAATGTCCTA GGTTTGAGTG

951 AGAATGCTCT TAATGGCTCT ATTCCTGCTT CATTGGGGAA TCTGAAAAAC

1001 TTGTCTAGGT TGAATCTTGT TAATAATCAG CTTTCTGGCT CTATTCCTGC

1051 TTCATTGGGG AATCTGAACA ACTTGTCTAT GTTGTATCTT TACAATAACC

1101 AGCTTTCTGG CTCTATTCCT GCTTCATTGG GGAATCTGAA CAACTTGTCT

1151 ATGTTGTATC TTTACAATAA TCAGCTTTCT GGCTCTATTC CTGCTTCATT

1201 GGGGAATCTG AACAACTTGT CTAGGTTGTA TCTCTACAAT AATCAGCTTT

^' 1251 CTGGCTCTAT TCCTGAAGAA ATAGGTTACT TGAGTTCTCT TACTTATCTA

1301 GATTTGAGTA ATAACTCCAT TAATGGATTT ATTCCTGCTT CATTTGGCAA

1351 TATGAGCAAC TTGGCTTTTT TGTTTCTTTA TGAAAATCAG CTTGCTAGCT

1401 CTGTTCCTGA AGAAATAGGT TACCTAAGGT CTCTTAATGT CCTTGATTTG

1451 AGTGAGAATG CTCTTAATGG CTCTATTCCT GCTTCATTCG GGAATTTGAA

1501 CAACTTGTCT AGGTTGAATC TTGTTAATAA TCAGCTTTCT GGCTCTATTC

1551 CTGAAGAAAT AGGTTACCTA AGGTCTCTTA ATGTCCTTGA TTTGAGTGAG

1601 AATGCTCTTA ATGGCTCTAT TCCTGCTTCA TTCGGGAATT TGAACAACTT

1651 GTCTAGGTTG AATCTTGTTA ATAATCAGCT TTCTGGCTCT ATTCCTGAAG

1701 AAATAGGTTA CCTAAGATCT CTTAATGACC TAGGTTTGAG TGAGAATGCT

1751 CTTAATGGCT CTATTCCTGC TTCATTGGGG AATCTGAACA ACTTGTCTAT

1801 GTTGTATCTT TACAATAATC AGCTTTCTGG CTCTATTCCT GAAGAAATAG

1851 GTTACTTGAG TTCTCTTACT TATCTATCTT TGGGTAATAA CTCTCTTAAT

1901 GGACTTATTC CTGCTTCATT TGGCAATATG AGAAATCTGC AAGCTCTGAT

1951 TCTCAATGAT AACAATCTCA TTGGGGAAAT TCCTTCATCT GTGTGCAATT

2001 TGACATCACT GGAAGTGTTG TATATGCCGA GAAACAATTT GAAGGGAAAA

2051 GTTCCGCAAT GTTTGGGTAA TATCAGTAAC CTTCAGGTTT TGTCGATGTC

2101 ATCTAATAGT TTCAGTGGAG AGCTCCCTTC ATCTATTTCC AATTTAACAT

2151 CACTACAAAT ACTTGATTTT GGCAGAAACA ATCTGGAGGG AGCAATACCA

2201 CAATGTTTTG GCAATATTAG TAGCCTCGAG GTTTTTGATA TGCAGAACAA

2251 CAAACTTTCT GGGACTCTTC CAACAAATTT TAGCATTGGA TGTTCACTGA

2301 TAAGTCTCAA CTTGCATGGC AATGAACTAG AGGATGAAAT CCCTCGGTCT

2351 TTGGACAATT GCAAAAAGCT GCAAGTTCTT GATTTAGGAG ACAATCAACT

2401 CAACGACACA TTTCCCATGT GGTTGGGAAC TTTGCCAGAG CTGAGAGTTT 2451 TAAGGTTGAC ATCGAATAAA TTGCATGGAC CTATAAGATC ATCAAGGGCT

2501 GAAATCATGT TTCCTGATCT TCGAATCATA GATCTCTCTC GCAATGCATT

2551 CTCGCAAGAC TTACCAACGA GTCTATTTGA ACATTTGAAA GGGATGAGGA

2601 CAGTTGATAA AACAATGGAG GAACCAAGTT ATGAAAGCTA TTACGATGAC

2651 TCGGTGGTAG TTGTGACAAA GGGATTGGAG CTTGAAATTG TGAGAATTTT

2701 GTCTTTGTAC ACAGTTATCG ATCTTTCAAG CAACAAATTT GAAGGACATA

2751 TTCCTTCTGT CCTGGGAGAT CTCATTGCGA TCCGTATACT TAATGTATCT

2801 CATAATGCAT TGCAAGGCTA TATACCATCA TCACTTGGAA GTTTATCTAT

2851 ACTGGAATCA CTAGACCTTT CGTTTAACCA ACTTTCAGGA GAGATACCAC

2901 AACAACTTGC TTCTCTTACG TTTCTTGAAT TCTTAAATCT CTCCCACAAT

2951 TATCTCCAAG GATGCATCCC TCAAGGACCT CAATTCCGTA CCTTTGAGAG

3001 CAATTCATAT GAAGGTAATG ATGGATTACG TGGATATCCA GTTTCAAAAG

3051 GTTGTGGCAA AGATCCTGTG TCAGAGAAAA ACTATACAGT GTCTGCGCTA

3101 GAAGATCAAG AAAGCAATTC TGAATTTTTC AATGATTTTT GGAAAGCAGC

3151 TCTGATGGGC TATGGAAGTG GACTGTGTAT TGGCATATCC ATAATATATA

3201 TCTTGATCTC GACTGGAAAT CTAAGATGGC TTGCAAGAAT CATTGAAGAA

3251 CTGGAACACA AAATTATCAT GCAAAGGAGA AAGAAGCAGC GAGGTCAAAG

3301 AAATTACAGA AGAAGAAATA ATCGCTTCTA GACAAGTTAC CAATACCGAA

3351 AGATTTGATT TCAGAACTTC AGACTTTCAG GAGCCAAGAA TAAGAAGACG

3401 CTGGTGTAAA GGATTTGCTT CTTCCTGTGT TGCAGCTTAT GATGTTGGAT

3451 TAGATTTTTA GTTTTATAAG CTTTTCTTCA GTTGGGAAAA TGTAATATTA

3501 TGAATTTGAT GATATACAAT AAATGTTGTG TTTATTGAAA AAAAAAAAAA

3551 AAAAAAAAAA AAAAAAAAAA AAA

SEQ ID NO. 9:

1 tatatatctt aataatgtaa attgatgaca aagtgattaa atagatgatc

51 gtgagagatg aaatcaggta gagttttgtg ttgttgtttc aggaattata

101 cgagtcaagg tacttgaagg ggatggagtt gagaaaatgg ggcgaacgca

151 acacaaaaag cagagagttt ctagacgcaa ttccacggcc gcttcttgaa

201 ctcgttgata gatgtttgat agttaacccg aggcgacgaa tcagcgcaga

251 ggatgctctc aagcacgagt tcttctatcc agtacatgaa acccttagaa

301 accaaatgct ccttaaacag cagcaaatgc aatcgcagcc tacagttgtt

351 gctgacgcac taagcgaaac tttaaactaa ttatacaatt cttaaaaact

401 aaaagagtaa tttagcaaac tagagagtta attttcactt tagcaaacta

451 gagagttaat ttaatttagc gaactaatta tattttcact ttagtataca

501 attcttagtg ttaatttagt attttcactt atattatttg aattaaaatc

551 ctcataatcg atatacttat tctcctaatc catgtgcatg tatgtattgg

601 gaaacaagac tttgatatta aacaatcata agtacattct tacgataaaa

651 tgtcttgtac aaggacaact gacacccaca aaatatgtgt gtttcaaaat

701 atctgtgtag aggaaacgaa tgtaagtttc tgtctaattg cctagaactt

751 gaaatattat ttctgtcttg tacaaagact aagacttatc ataattaagt

801 gacaaccaca aaaattcaat ctctaaaaat atctttgtat gtagtgtaaa 851 aaagctttcg aggaaagtaa gacgaagttt ctcctctctt tctcacacta 901 tgtcttgctg atttacttct cttaaaaatc ttcgtctctt ctctgagttc 951 gctctatcat ctcccATGGC GGCTTCTTCT TCTTCTGGCA GACGGAGATA 1001 CGACGTTTTT CCAAGCTTCA GTGGGGTTGA TGTTCGCAAG ACGTTCCTCA 1051 GCCATCTTCT CAAGGCTCTC GACGGCAAAT CAATCAATAC ATTCATCGAT 1101 CATGGAATCG AGAGAAGCCG CACAATCGCC CCTGAGCTTA TATCGGCGAT 1151 TAGAGAAGCT AGGATCTCAA TCGTCATCTT CTCTAAGAAC TATGCTTCTT 1201 CAACGTGGTG CTTAAATGAA TTGGTTGAGA TCCACAAGTG CTTTAATGAT 1251 TTAGGTCAAA TGGTGATTCC AGTTTTCTAC GACGTTGATC CTTCGGAAGT 1301 TAGAAAACAG ACCGGCGAAT TTGGAAAGGT CTTTGAAAAG ACATGCGAGG 1351 TCAGCAAGGA CAAACAACCA GGGGATCAGA AACAAAGATG GGTGCAAGCT 1401 CTCACAGATA TAGCAAATAT AGCCGGAGAG GATCTTCTGA ACGGgtacgt 1451 tgttatgatt ccaatatatc tgcttgcgtt ttcaattgtc tcagaactat 1501 atttttgcat agacttcggt tcttctttta ggggtgcttc ttaattgaca 1551 aaattgactt ttgttattag GCCTAATGAA GCGCATATGG TTGAAAAGAT 1601 ATCCAATGAT GTTTCGAATA AACTTATCAC TCGGTCAAAG TGTTTTGATG 1651 ACTTCGTCGG AATTGAAGCT CATATTGAGG. CAATAAAATC AGTATTGTGC 1701 TTGGAATCCA AGGAAGCTAG AATGGTCGGG ATTTGGGGAC AGTCAGGGAT 1751 TGGTAAGAGT ACCATCGGAA GAGCTCTTTT CAGTCAACTC TCTAGCCAGT 1801 TCCACCATCG CGCTTTCCTA ACTTATAAAA GCACCAGTGG TAGTGACGTC 1851 TCTGGCATGA AGTTGAGTTG GCAAAAAGAG CTTCTCTCGG AAATCTTAGG 1901 TCAAAAGGAC ATAAAGATAG AGCATTTTGG TGTGGTGGAG CAAAGGTTAA 1951 ATCACAAGAA AGTTCTTATC CTTCTTGATG ATGTGGATAA TCTAGAGTTT 2001 CTTAAGACCT TGGTGGGAAA AGCTGAATGG TTTGGATCTG GAAGCAGAAT 2051 AATTGTGATC ACTCAAGATA GGCAACTTCT CAAGGCTCAT GAGATTGACC 2101 TTGTATATGA GGTGAAGCTG CCATCTCAAG GTCTTGCTCT TAAGATGATA 2151 TCCCAATATG CTTTTGGGAA AGACTCTCCA CCTGATGATT TTAAGGAACT 2201 AGCATTTGAA GTTGCCGAGC TTGTCGGTAG TCTTCCTTTG GGTCTCAGTG 2251 TCTTGGGTTC ATCTTTAAAA GGAAGGGACA AAGATGAGTG GGTGAAGATG 2301 ATGCCTAGGC TTCGAAATGA TTCAGATGAT AAAATTGAGG AAACACTAAG 2351 AGTCGGCTAC GATAGGTTAA ATAAAAAAAA TAGAGAGTTA TTTAAGTGCA 2401 TTGCATGTTT TTTCAATGGT TTTAAAGTCA GTAACGTCAA AGAATTACTT 2451 GAAGATGATG TTGGGCTTAC AATGTTGGCT GAGAAGTCCC TCATACGTAT 2501 TACACCGGGT GGATATATAG AGATGCACAA TTTGCTAGAG AAATTGGGTA 2551 GAGAAATTGA TCGTGCAAAG TCCAAGGGTA ATCCTGGAAA. ACGTCAATTT 2601 CTGACGAATT TTGAGGATAT TCGAGAAGTA TTGACCGAGA AAACTgtaag 2651 tttttcgcat ctccttaaac gttgtaatgc atgactttat atcaatataa 2701 tcgtaatttg gggattgata aacttaagca attgttgccc catgcgtaat 2751 taaaacgtag ctttgatgtg tcagaaaaat aaaaagggtt gcgattgtta 2801 agattatatt agttttcttc ggattttttt tcagGGGACC GAAACTCTTC 2851 TTGGAATACG TTTGCCACAC CCGGGATATC TTACGACAAG GTCGTTCTTA 2901 ATAGATGAAA AATCATTCAA AGGCATGCGT AATCTCCAAT ATCTAGAAAT 2951 TGGTTATTGG TCAGATGGGG TTCTACCTCA GAGCCTCGTT TATTTCCCTC 3001 GTAAACTCAA AAGGCTATGG TGGGATAATT GTCCATTGAA GCGTTTGCCT 3051 TCTAATTTTA AGGCTGAGTA TCTGGTTGAA CTCAGAATGG TGAATAGTAA 3101 GCTTGAGAAG CTGTGGGATG GAACTCAGGT actaattttt ttagtgatca 3151 atttctaaac ataaaaacta aaaataaaaa tgtttaaaat gttcattaac 3201 gtgtgtgctc tcttttcccc tattttgttt tcagCCCCTT GGAAGTCTCA 3251 AGAAGATGGA TTTGTATAAT TCCTACAAAT TGAAAGAAAT TCCAGATCTT 3301 TCTTTAGCCA TAAACCTCGA GGAATTAAAT CTTGAAGAAT GCGAATCTTT 3351 GGAGACACTT CCTTCCTCGA TTCAGAATGC CATTAAACTG AGGGAGTTAA 3401 ATTGTTGGGG GGGGCTATTA ATAGATTTAA AATCATTAGA AGGCATGTGT 3451 AATCTCGAAT ATCTATCAGT TCCTAGTTGG TCAAGTAGGG AATGCACTCA 3501 GGGCATCGTT TATTTCCCTC GTAAACTCAA AAGTGTATTG TGGACTAATT 3551 GTCCATTGAA GCGTTTGCCT TCTAATTTTA AGGCTGAGTA TCTGGTTGAA 3601 CTCATAATGG AGTACAGTGA GCTTGAGAAG CTGTGGGATG GTACTCAGgt 3651 actaattcta ttagtgataa taaatatgtt agaaaaacta aaaataaaaa 3701 tgtttaaaat gttcattaac gtgtgtgctc tcttttcccc tattttgtta 3751 tcagTCACTT GGAAGTCTCA AGGAGATGAA TTTGAGGTAT TCCAACAATT 3801 TAAAAGAAAT TCCAGATCTT TCTTTAGCCA TAAACCTCGA GGAATTAGAT 3851 CTTTTTGGAT GCGTATCTTT GGTGACACTT CCTTCCTCGA TTCAGAATGC 3901 CACTAAACTG ATCTATTTAG ATATGAGTGA ATGCGAAAAT CTAGAGAGTT 3951 TTCCAACCGT TTTCAACTTG AAATCTCTCG AGTACCTCGA TCTCACTGGA 4001 TGCCCGAATT TGAGAAATTT CCCAGCAATC AAAATGGGAT GTGCCTGGAC 4051 TAGATTATCT CGAACAAGAT TGTTTCCGGA AGGGAGAAAT GAGATCGTGG 4101 TAGAAGATTG TTTCTGGAAC AAGAATCTCC CTGCTGGACT AGATTATCTC 4151 GACTGCCTTA TGAGATGTAT GCCTTGTGAA TTTCGCTCAG AACAACTCAC 4201 TTTTCTCAAT GTGAGCGGCT GCAAGCTTGA GAAGCTATGG GAAGGCATCC

4251 AGgtacattg ttaatgctat gctgattttt gtttaccttc tgttatataa

4301 ctaattaagt atacccaaat ttgtttttat ggcttgtggt cgatccacgg

4351 ttatgtctta catacataca taataatgtt taattataat tttaaacata

4401 tataggtata aaattaaaat gattatcatc gataatgatt gaagcatacc

4451 aatgtttttt tcagTCGCTT GGAAGTCTCG AAGAGATGGA TCTGTCAGAA

4501 TCTGAAAACC TGAAAGAACT TCCAGATCTT TCAAAGGCCA CCAATCTGAA -

4551 GCTTTTATGT CTCAGCGGGT GCAAAAGTTT GGTGACACTT CCTTCTACAA

4601 TTGGGAATCT TCAAAATTTG AGACGTTTGT ACATGAACAG ATGCACAGGG

4651 CTGGAGGTTC TTCCGACCGA TGTCAACTTG TCATCTCTCG AAACCCTCGA

4701 TCTCAGTGGT TGCTCAAGTT TGAGAACTTT TCCTCTGATT TCAACTAATA

4751 TTGTATGTCT CTATCTGGAA AACACCGCCA TTGAAGAAAT TCCAGATCTT

4801 TCAAAGGCCA CCAAGCTCGA GTCTTTGATA CTCAACAACT GCAAAAGTTT

4851 GGTGACACTT CCTTCTACAA TTGGGAATCT TCAAAATTTG AGACGTTTGT

4901 ACATGAACAG ATGCACAGGG CTGGAGCTTC TTCCGACCGA TGTCAACTTG

4951 TCATCTCTCG AAACCCTCGA TCTCAGTGGT TGCTCAAGTT TGAGAACTTT

5001 TCCTCTGATT TCAACTAGAA TCGAATGTCT CTATCTAGAA AACACCGCCA

5051 TTGAAGAAGT TCCCTGCTGC ATTGAGGATT TCACGAGGCT CACTGTACTA

5101 CGGATGTATT GTTGCCAGAG GTTGAAAAAC ATCTCCCCAA ACATTTTCAG

5151 ACTGACTAGT CTTACGCTCG CCGACTTTAC AGACTGTAGA GGTGTCATCA

5201 AGGCGTTGAG TGATGCAACT GTGGTAGCGA CAATGGAAGA TCACGTTTCT

5251 TGTGTACCAT TATCTGAAAA CATTGAATAT ACATGTGAAC GTTTCTGGGA

5301 TGCGTGTTCT GATTATTACT CTGATGACTT TGAGGTAAAT CGGAACCCAA

5351 TTAGATTGTC AACGATGACT GTCAACGATG TGGAGTTTAA GTTTTGTTGC

5401 TCCATTACGA TCAAAGAATG CGGTGTACGA CTCTTGTATG TCTATCAAGA

5451 AACAGAGCAC AACCAACAAA CTACGAGAAG CAAGAAGCGG ATGCGGGTAA

5501 GCCTTTTGCC Ataattagag ctgaaacttg taaagcaatc ttttgacttg

5551 atttgtttta taggatcaaa ataccatagc gacagactat ttgatagaat

5601 cgatcgtttg atatataatg cagatgacat cggggacatc tgaagaagat

5651 atcaacttac cctatggcca aattgtagcg gacacaggat tggccgctct

5701 aaatacagag ctttcgttag ggcagggaga agcatcatca tcaacatctc

5751 tagaggggga agctttgtgt gttgatgatt acatgataaa tgaagaacaa

5801 gatgaacaaa tacctatctt gtatcctgtt tatggtaact gaagcatctt

5851 tatcattctg ttttgctctt ttttaggata acttgggatc gaccattatt

5901 ataaatttat aatgataatg acaaaacgat ttcataggtt ttgacttttg

5951 acacaagcca ttttttctgc agatatagac gatgatatgt ggagatcatt

SEQ ID NO. 10:

1 MAASSSSGRR RYDVFPSFSG VDVRKTFLSH LLKALDGKSI NTFIDHGIER

51 SRTIAPELIS AIREARISIV IFSKNYASST WCLNELVEIH KCFNDLGQMV

101 IPVFYDVDPS EVRKQTGEFG KVFEKTCEVS KDKQPGDQKQ RWVQALTDIA

151 NIAGEDLLNG PNEAHMVEKI SNDVSNKLIT RSKCFDDFVG IEAHIEAIKS

201 VLCLESKEAR MVGIWGQSGI GKSTIGRALF SQLSSQFHHR AFLTYKSTSG

251 SDVSGMKLSW QKELLSEILG QKDIKIEHFG WEQRLNHKK VLILLDDVDN

301 LEFLKTLVGK AEWFGSGSRI IVITQDRQLL KAHEIDLVYE VKLPSQGLAL

351 KMISQYAFGK DSPPDDFKEL AFEVAELVGS LPLGLSVLGS SLKGRDKDEW

401 VKMMPRLRND SDDKIEETLR VGYDRLNKKN RELFKCIACF FNGFKVSNVK

451 ELLEDDVGLT MLAEKSLIRI TPGGYIEMHN LLEKLGREID RAKSKGNPGK

501 RQFLTNFEDI REVLTEKTGT ETLLGIRLPH PGYLTTRSFL IDEKSFKGMR

551 NLQYLEIGYW SDGVLPQSLV YFPRKLKRLW WDNCPLKRLP SNFKAEYLVE

601 LRMVNSKLEK LWDGTQPLGS LKKMDLYNSY KLKEIPDLSL AINLEELNLE

651 ECESLETLPS SIQNAIKLRE LNCWGGLLID LKSLEGMCNL EYLSVPSWSS 701 RECTQGIVYF PRKLKSVLWT NCPLKRLPSN FKAEYLVELI MEYSELEKLW

751 DGTQSLGSLK EMNLRYSNNL KEIPDLSLAI NLEELDLFGC VSLVTLPSSI

801 QNATKLIYLD MSECENLESF PTVFNLKSLE YLDLTGCPNL RNFPAIKMGC

851 AWTRLSRTRL FPEGRNEIW EDCFWNKNLP AGLDYLDCLM RCMPCEFRSE

901 QLTFLNVSGC KLEKLWEGIQ SLGSLEEMDL SESENLKELP DLSKATNLKL

951 LCLSGCKSLV TLPSTIGNLQ NLRRLYMNRC TGLEVLPTDV NLSSLETLDL

1001 SGCSSLRTFP LISTNIVCLY LENTAIEEIP DLSKATKLES LILNNCKSLV

1051 TLPSTIGNLQ NLRRLYMNRC TGLELLPTDV NLSSLETLDL SGCSSLRTFP

1101 LISTRIECLY LENTAIEEVP CCIEDFTRLT VLRMYCCQRL KNISPNIFRL

1151 TSLTLADFTD CRGVIKALSD ATWATMEDH VSCVPLSENI EYTCERFWDA

1201 CSDYYSDDFE VNRNPIRLST MTVNDVEFKF CCSITIKECG VRLLYVYQET

1251 EHNQQTTRSK KR RVSLLP

SEQ ID No.11:

1 GACCAAACTG GACTCCTGCT CCGTCTTCCA TCAGCAGGTC AATTCTCGTG

51 GAAAATTAGC TCGAGGTGGC GCACTATGTG AGGTAGCTAG TACTAAATGT

101 TTATTTGCGT AATTTGTGCT ATATATACCT CATCTAAATT ATTGAATAGA

151 CACACAAAGC AAACATCTCT TAATTAGTTT TGATCATTTT TAGTGCAGAA

201 ATGGGTTGTG TAAAACTTGT GTTTTTCATG CTATATGTCT TTCTCTTTCA

251 ACTTGTTTCC TCGTCATCCT TACCTCATTT GTGCCCCGAA GATCAAGCTC

301 TTGCTCTTCT AGAATTCAAG AACATGTTTA CCGTTAATCC TAATGCTTCT

351 GATTATTGTT ACGACAGAAG AACTCTTTCT TGGAACAAAA GCACAAGTTG

401 CTGCTCATGG GATGGCGTTC ATTGTGACGA AACGACAGGA CAAGTGATTG

451 AGCTTGACCT CCGTTGCATC CAACTTCAAG GCAAGTTTCA TTCCAATAGT

501 AGCCTCTTTC AACTCTCCAA TCTCAAAAGG CTTGATTTGT CTTATAATGA

551 TTTCACTGGA TCGCCCATTT CACCTAAATT TGGTGAGTTT TCAGATTTGA

601 CGCATCTCGA TTTGTCGCAT TCAAGTTTTA GGGGTGTAAT CCCTTCTGAA

651 ATCTCTCATC TTTCTAAACT ATACGTTCTT CGTATTAGTC TAAATGAGCT

701 TACTTTTGGT CCTCACAATT TTGAATTGCT TCTTAAGAAC TTGACCCAAT

751 TAAAAGTGCT CGACCTTGAA TCTATCAACA TCTCTTCCAC TATTCCTTTG

801 AATTTCTCTT CTCATTTAAC AAATCTATGG CTTCCATACA CAGAGTTACG

851 TGGGATATTG CCCGAAAGAG TTTTCCACCT TTCCGACTTA GAATTTCTCG

901 ATTTATCAAG CAATCCCCAG CTCACGGTTA GGTTTCCCAC AACCAAATGG

951 AATAGCAGTG CATCACTCAT GAAGTTATAT CTCTATAATG TGAATATTGA 1001 TGATAGGATA CCTGAATCAT TTAGCCATCT AACTTCACTT CATAAGTTGT

1051 ACATGAGTCG TTCTAATCTG TCAGGGCCTA TTCCTAAACC TCTATGGAAT

1101 CTCACCAACA TAGTGTTTTT GGACCTTAAT AATAACCATC TTGAAGGACC

1151 AATTCCATCC AACGTAAGCG GACTACGTAA CCTACAAATA CTTTGGTTGT

1201 CATCAAACAA CTTAAATGGG AGTATACCAT CCTGGATATT CTCCCTTCCA

1251 TCACTGATAG GGTTAGACTT GAGCAATAAC ACTTTCAGTG GAAAAATTCA

1301 AGAGTTCAAG TCCAAAACAT TAAGTACCGT TACTCTAAAA CAAAATAAGC

1351 TAAAAGGTCC TATTCCGAAT TCACTCCTAA ACCAGAAGAA CCTACAATTC

1401 CTTCTCCTTT CACACAATAA TATCAGTGGA CATATTTCTT CAGCTATCTG

1451 CAATCTGAAA ACATTGATAT TGTTAGACTT GGGAAGTAAT AATTTGGAGG

1501 GAACAATCCC GCAATGCGTG GTTGAGAGGA ACGAATACCT TTCGCATTTG

1551 GATTTGAGCA ACAACAGACT TAGTGGGACA ATCAATACAA CTTTTAGTGT

1601 TGGAAACATT TTAAGGGTCA TTAGCTTGCA CGGGAATAAG CTAACGGGGA

1651 AAGTCCCACG ATCTATGATC AATTGCAAGT ATTTGACACT ACTTGATCTA

1701 GGTAACAATA TGTTGAATGA CACATTTCCA AACTGGTTGG GATACCTATT

1751 TCAATTGAAG ATTTTAAGCT TGAGATCAAA TAAGTTGCAT GGTCCCATCA

1801 AATCTTCAGG GAATACAAAC TTGTTTATGG GTCTTCAAAT TCTTGATCTA

1851 TCATCTAATG GATTTAGTGG GAATTTACCC GAAAGAATTT TGGGGAATTT

1901 GCAAACCATG AAGGAAATTG ATGAGAGTAC AGGATTCCCA GAGTATATTT

1951 CTGATCCATA TGATATTTAT TACAATTATT TGACGACAAT TTCTACAAAG

2001 GGACAAGATT ATGATTCTGT TCGAATTTTG GATTCTAACA TGATTATCAA

2051 TCTCTCAAAG AACAGATTTG AAGGTCATAT TCCAAGCATT ATTGGAGATC

2101 TTGTTGGACT TCGTACGTTG AACTTGTCTC ACAATGTCTT GGAAGGTCAT

2151 ATACCGGCAT CATTTCAAAA TTTATCAGTA CTCGAATCAT TGGATCTCTC

2201 ATCTAATAAA ATCAGCGGAG AAATTCCGCA GCAGCTTGCA TCCCTCACAT

2251 TCCTTGAAGT CTTAAATCTC TCTCACAATC ATCTTGTTGG ATGCATCCCC

2301 AAAGGAAAAC AATTTGATTC GTTCGGGAAC ACTTCGTACC AAGGGAATGA

2351 TGGGTTACGC GGATTTCCAC TCTCAAAACT TTGTGGTGGT GAAGATCAAG

2401 TGACAACTCC AGCTGAGCTA GATCAAGAAG AGGAGGAAGA AGATTCACCA

2451 ATGATCAGTT GGCAGGGGGT TCTCGTGGGT TACGGTTGTG GACTTGTTAT

2501 TGGACTGTCC GTAATATACA TAATGTGGTC AACTCAATAT CCAGCATGGT

2551 TTTCGAGGAT GGATTTAAAG TTGGAACACA TAATTACTAC GAAAATGAAA

2601 AAGCACAAGA AAAGATATTA GTGAGTAGCT ATACCTCCAG GTATTCCACT

2651 TGATCATTAT CTTTCAGAAG ATTATTTTTT GTATATCGAT GAAATTATCG 2701 ACCTCCTTCA TCCTCAAAGC TCTTAACTTT CACTCTTCAT TTTTGAAAAT

2751 TTCAGGATTC AAAGATTTCC GAGTTCCCAG TTGCTTGGGA TGCAGATAAA

2801 AGCCTTTTTA TCTTTCATAG TTTCTTATCC TATGAATAAA GATTTTATTT

2851 TCATTTGTCT ATGGCACGTA GATATGTTCC GTCACTAAAA ACATTGTATT

2901 TCTCTCAACT CTTTCGTCAC ATGATATCAA AGAACACTTG ACTTCAATTA

2951 AGTTACTGTA GTCTGCTATT TTAATTTCTT CCATTGAAAC ACAACTGACG

3001 TATCTTGAGA AAGAGACTAT GATCTCAGAA ATGGGAATCT CCCAATCCAA

SEQ ID No. 12:

1 MGCVKLVFFM LYVFLFQLVS SSSLPHLCPE DQALALLEFK NMFTVNPNAS

51 DYCYDRRTLS WNKSTSCCSW DGVHCDETTG QVIELDLRCI QLQGKFHSNS

101 SLFQLSNLKR LDLSYNDFTG SPISPKFGEF SDLTHLDLSH SSFRGVIPSE

151 ISHLSKLYVL RISLNELTFG PHNFELLLKN LTQLKVLDLE SINISSTIPL

201 NFSSHLTNLW LPYTELRGIL PERVFHLSDL EFLDLSSNPQ LTVRFPTTKW . .

251 NSSASLMKLY LYNVNIDDRI PESFSHLTSL HKLYMSRSNL SGPIPKPLWN

301 LTNIVFLDLN NNHLEGPIPS NVSGLRNLQI LWLSSNNLNG SIPSWIFSLP

351 SLIGLDLSNN TFSGKIQEFK SKTLSTVTLK QNKLKGPIPN SLLNQKNLQF

401 LLLSHNNISG HISSAICNLK TLILLDLGSN NLEGTIPQCV VERNEYLSHL

451 DLSNNRLSGT INTTFSVGNI LRVISLHGNK LTGKVPRSMI NCKYLTLLDL

501 GNNMLNDTFP NWLGYLFQLK ILSLRSNKLH GPIKSSGNTN LFMGLQILDL

551 SSNGFSGNLP ERILGNLQTM KEIDESTGFP EYISDPYDIY YNYLTTISTK

601 GQDYDSVRIL DSNMIINLSK NRFEGHIPSI IGDLVGLRTL NLSHNVLEGH

651 IPASFQNLSV LESLDLSSNK ISGEIPQQLA SLTFLEVLNL SHNHLVGCIP

701 KGKQFDSFGN TSYQGNDGLR GFPLSKLCGG EDQVTTPAEL DQEEEEEDSP

751 MISWQGVLVG YGCGLVIGLS VIYIMWSTQY PAWFSRMDLK LEHIITTKMK

801 KHKKRY ATCGΛTGGG TTTGTTCTCTTTTCACAATTGCC'TTCATTTC'PTCTTGTCTCTACACTTCT ₍-- + ■ 4 1 i 1 1- 60

TAGqTACCCTAAACAAGAGAAAAGTGTTAACGGAAGTAAAGAAGAACAGAGATGTGAAGA

M G F V L F S Q L P S F L L V S T L L CTTATTCCTAGTAATATCCCACTCTTGCCGTGCdAAAGCCCCCAAAACTCAACCATACAA

120

GAATAAGGATCATTATAGGGTGAGAACGGCACGqTTTCGGGGGTTTTGAGTTGGTATGTT L F L V I S H S C R A K A P K T Q P Y N

CCCATGCAAGCCCCAAGAAGTCATCGACACCAAGTGTATGGGTCCCAAGGATTGTCTCTA -i- + + + + + 180

GGGTACGTTCGGGGTTCTTCAGTAGCTGTGGTTCACATACCCAGGGTTCCTAACAGAGAT

P C K P Q E V I D T K C M G P K D C L Y

CCCGAACCCCGACAGTTGTACAACCTACATACAGTGTGTACCGCTCGACGAAGTTGGCAA + + + + + + 240

GGGCTTGGGGCTGTCAACATGTTGGATGTATGTCACACATGGCGAGCTGCTTCAACCGTT

P N P D S C T T Y I Q C V P L D E V G N

TGCGAAGCCTGTGGTTAAGCCATGTCCAAAAGGACTGCAGTGGAACGATAACGTTGGCAA + + + •+• + + 300

ACGCTTCGGACACCAATTCGGTACAGGTTTTCCTGACGTCACCTTGCTATTGCAACCGTT

A K P V V K P C P K G L Q W N D N V G K

GAAGTGGTGCGACTATCCAAACCTGAGTACGTGTCCGGTAAAGACGCCGCAACCGAAGCC + 1- + +• +^■ + 360.

CTTCACCACGCTGATAGGTTTGGACTCATGCACAGGCCATTTCTGCGGCGTTGGCTTCGG W C D Y P N L S T C P V K T P Q P K P

K K G G V G G K K A S V G H P G Y

ACAAGAAAGGGGATGGCTGTAACAGTTCTGGTACCAGAGCTATCGTGCTAGGGGATCCGT + -t- + + + + 480

TGTTCTTTCCCCTACCGACATTGTCAAGACCATGGTCTCGATAGCACGATCCCCTAGGCA

CGAC

GCTG

Claims

CLAIMS :

1. A method of providing increased pathogen

resistance in a plant, or a part or propagule of a plant, by induction of variegation in which a gene is expressed or suppressed in cells resulting in the activation of a plant defence response, which

comprises:

(i) inactivating a nucleotide sequence which

contributes to a plant defence, response or inactivating one or more nucleotide sequences forming a part of a combination of nucleotide sequences which contributes to a plant defence response;

(ii) introducing said nucleotide sequence or sequences into the genome of a plant; and

(iii) restoring said nucleotide sequence or sequences to a functional form in cells of the plant or a

descendant thereof, or a part or propagule of the plant or descendant, to result in increased pathogen' resistance.

2. A method of providing increased pathogen

resistance in a plant, or a part or propagule thereof, by induction of variegation in which a gene is

expressed or suppressed resulting in necrosis, which comprises:

(i) inactivating a nucleotide sequence which

contributes to necrosis or inactivating one or more nucleotide sequences forming part of a combination of nucleotide sequences which contributes to necrosis; (ii) introducing said nucleotide sequence or

sequences into the genome of a plant; and

(iii) restoring said inactivated nucleotide sequence or sequences to a functional form in cells of the plant or a descendant thereof, or a part or propagule of the plant or descendant, to result in necrosis.

3. A method according to claim 1 or claim 2 wherein said nucleotide sequence encodes or sequences encode a substance or a combination of substances which result in increased pathogen resistance.

4. A method according to any one of the preceding claims wherein said nucleotide sequence or sequences comprises a gene and activation of the plant defence response and/or necrosis due to the expression of said nucleotide sequence or sequences is not dependent on the expression of any other gene comprised in said nucleotide sequence or sequences.

5. A method according to any one of claims 1 to 3 wherein said nucleotide sequence or combination of nucleotide sequences comprises one or more genes and wherein activation of the plant defence response and/or necrosis due to the expression of said nucleotide sequence or sequences is conditional on the expression of one or more interacting genes.

6. A method according to claim 5 wherein said nucleotide sequences encodes or nucleotide sequences encode one or more substances which are or together are capable of inducing the plant defence response and/or necrosis, and at least one of said nucleotide sequences is inactivated in step (i).

7. A method according to claim 6 wherein said nucleotide sequence comprises a plant pathogen

resistance gene (R) or a mutant, variant or derivative thereof, or a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof, or another R gene elicitor (E), or both (i) an R gene or a mutant, variant, or derivative thereof and (ii) a corresponding Avr gene, or a mutant, variant or derivative thereof, or another R gene elicitor (E).

8. A method according to claim 7 wherein said plant pathogen resistance gene (R) is a tomato Cf-9 gene or a mutant, variant, derivative or homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-9 gene or a mutant, variant, derivative or homologue thereof, or encodes another Cf-9 elicitor.

9. A method according to claim 7 wherein said plant pathogen resistance gene (R) is a tomato Cf-2 gene or a mutant, variant, derivative or homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-2 gene or a mutant, variant, derivative or homologue thereof, or encodes another Cf-2 elictor; or wherein said plant pathogene resistance gene (R) is a tomato Cf-4 gene or a mutant, variant, derivative or homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-4 gene or a mutant, variant, derivative or homologue thereof, or encodes another Cf-4 elictor; or wherein said plant pathogen resistance gene (R) is the tobacco N' gene or a mutant, variant, derivative or homologue thereof, and the avirulence gene is a suitable Tobacco Mosaic Virus coat protein, or a mutant, variant, derivative or homologue thereof or encodes another N' elicitor; or wherein said plant pathogen resistance gene (R) is the potato Rx gene or a mutant, variant, derivative or homologue thereof and the avirulence gene is a suitable PVX coat protein or a mutant, variant, derivative or homologue thereof or another Rx elicitor; or wherein said plant pathogen resistance gene is another viral resistance gene and the avirulence gene encodes a corresponding viral coat protein or other elicitor of the viral resistance gene.

10. A method according to claim 5 wherein said nucleotide sequence encodes a Cauliflower Mosaic Virus gene VI protein, a bacterial harpin gene protein, an Arabidopsis RPP5 gene protein, a ubiquitin conjugating enzyme, an RNase such as Barnase, a mutant, variant, derivative or homologue of any of these, or other toxic polypeptide or peptide such as diphtheria toxin or a mutant, variant, derivative or homologue thereof.

11. A method according to claim 4 in which the plant defence response or necrosis is dependent on the expression from a nucleotide sequence leading to the reduction of expression of a gene that negatively regulates the plant defence response, resulting in the plant defence response and/or necrosis.

12. A method according to claim 4 in which the plant defence response or necrosis is dependent on the expression of an allele of a gene from a nucleotide sequence which activates the plant defence response in the absence of a ligand that is capable of interacting with the product of said gene, resulting in the plant defence response and/or necrosis.

13. A method according to claim 5 in which the plant defence response or necrosis is dependent on the expression of a mutant allele of a gene from a

nucleotide sequence which is capable of activating the plant defence response and the expression of an

enfeebled negative regulator of the defence response, leading to the plant defence response and/or necrosis.

14. A method according to any of the preceding claims wherein the inactivation of said nucleotide sequence or of one or more of said nucleotide sequences is effected by the insertion therein of a transposable genetic element.

15. A method according to claim 14 wherein said transposable genetic element is a transposon or a nucleotide sequence bordered by specific nucleotide sequences that can be recognised by a site specific recombination system.

16. A method according to any of the preceding claims wherein said plant genome comprises at least one nucleotide sequence encoding a substance capable of restoring said inactivated nucleotide sequence or sequences to a functional form to result in increased pathogen resistance.

17. A method according to claim 16 which comprises restoring said inactivated nucleotide sequence or sequences to a functional form by excision or

rearrangement of said transposable genetic element.

18. A method according to claim 17 wherein when said transposable element is a transposon, said plant genome comprises at least one nucleotide sequence coding for a corresponding transposon activation system to effect somatic excision of said transposon.

19. A method according to claim 18 wherein the genes encoding the transposon and transposase are derived from the Activator/Dissociation transposable element family (Ac/Ds) or from the Enhancer/Suppressor mutator transposon family (En/Spm).

20. A method according to claim 17 wherein when said inactive form of said nucleotide sequence or sequences is flanked by recombinase recognition sequences, said recombinase recognition sequences are acted on by a site specific recombination system which comprises a specific recombinase to result in recombination.

21. A transgenic plant, or descendant thereof, or part or propagule of the plant or descendant,

obtainable using a method of any of the preceding claims with increased pathogen resistance compared with wild-type.

22. A plant, or a descendant thereof, or a part or propagule of the plant or descendant, or a derivative of any of these, which is phenotypically variegated, comprising a cell or clone expressing a first phenotype and other cells expressing a second phenotype

comprising increased pathogen resistance compared with wild-type.

23. A plant, descendant, derivative, part or propagule according to claim 22 wherein the first phenotype is necrosis and/or a plant defence response phenotype.

24. A plant, descendant, derivative, part or

propagule according to claim 22 or claim 23 wherein the phenotypic variegation results from expression in cells with the first phenotype from a nucleotide sequence or sequences which contribute to such phenotype, said expression from said nucleotide sequence or sequences being inactivated in cells not having said first phenotype.

25. A plant, descendant, derivative, part or

propagule according to claim 24 wherein said expression results from reactivation of a previously inactivated gene.

26. A plant, descendant, derivative, part or

propagule according to claim 24 or claim 25 wherein said inactivation results from insertion of a

transposable genetic element into said nucleotide sequence or one or more of said nucleotide sequences.

27. A plant, descendant, derivative, part or

propagule according to any one of claims 24 to 26, wherein said nucleotide sequence or sequences

comprises: a gene (R) which is a plant pathogen resistance gene or a mutant, variant or derivative thereof; or a gene (L) which is a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof, or another elicitor or ligand gene the product of which can interact with the product of a R-gene; or both an R gene and an L gene.

28. A plant, descendant, derivative, part or

propagule according to claim 27 wherein the R gene is a tomato Cf-9 gene or a mutant, variant, derivative or homologue thereof and the L gene is a Cladosporium fulvum Avr- 9 gene or a mutant, variant, derivative or homologue thereof, or encodes another Cf-9 elicitor.

29. A plant, descendant, derivative, part or

propagule according to claim 27 wherein said R gene is: (i) a pathogen resistance gene from tomato;

(ii) a pathogen resistance gene from tobacco;

(iii) a pathogen resistance gene from potato;

(iv) a pathogen resistance gene from Arabidopsis;

(v) a pathogen resistance gene from flax;

(vi) a nucleotide sequence encoding a CaMV gene VI protein;

(vii) a nucleotide sequence encoding a bacterial

harpin gene protein;

(viii) a nucleotide sequence encoding a ubiquitin

conjugating enzyme;

(ix) a nucleotide sequence encoding an RNase; (x) a nucleotide sequence encoding a toxic peptide; (xi) a mutant, variant, derivative or homologue of any of (i) to (x);

30. A plant, descendant, derivative, part or

propagule according to claim 29 wherein said pathogen resistance gene from tomato is selected from

Cladosporium fulvum resistance genes including Cf-2, Cf-4 , Cf-5 and Cf-9; said pathogen resistance gene from tobacco is N'; said pathogen resistance gene from potato is Nx; said pathogen resistance gene from

Arabidopsis is RPP5 or RP52 ; said pathogen resistance gene from flax is L6; said RNase is Barnase; or said toxic peptide is diphtheria toxin.

31. A plant, descendant, derivative, part or

propagule according to claim 27 wherein said L gene is: (i) a Cladosporium fulvum avirulence gene or another elicitor of a resistance gene for a Cladosporium fulvum avirulence gene;

(ii) a suitable TMV coat protein or another N'

elicitor;

(iii) a suitable PVX coat protein or another Rx

elicitor; or

(iv) a mutant, variant, derivative or homologue of any of (i) to (iii).

32. A plant, descendant, derivative, part or propagule according to claim 31 wherein said

Cladosporium fulvum avirulence gene is Avr2, Avr4 , Avr5 or Avr9.

33. A cell containing (i) nucleic acid encoding one or more than one nucleotide sequence which causes or contributes to the plant defence response and/or cell necrosis, at least one said nucleotide sequence being reversibly inactivated and (ii) nucleic acid encoding a molecule or molecules able to reverse the inactivation.

34. A cell according to claim 33 wherein the

inactivation results from insertion of a transposable genetic element into one or more of said nucleotide sequences.

35. A cell according to claim 34 wherein said

transposable genetic element is a transposon and said molecule or molecules provide a corresponding

transposon activation system to effect excision of said transposon.

36. A cell according to any one of claims 33 to 35 wherein said nucleotide sequence or sequences

comprises: a gene (R) which is a plant pathogen

resistance gene or a mutant, variant or derivative thereof; or a gene (L) which is a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof, or another elicitor or ligand gene the product of which can interact with the product of a R-gene; or both an R gene and an L gene.

37. A cell according to claim 36 wherein the R gene is a tomato Cf-9 gene or a mutant, variant, derivative or homologue thereof and the L gene is a Cladosporium fulvum Avr-9 gene or a mutant, variant, derivative or homologue thereof, or encodes another Cf-9 elicitor.

38. A cell according to claim 37 wherein said R gene is:

(i) a pathogen resistance gene from tomato;

(ii) a pathogen resistance gene from tobacco;

(iii) a pathogen resistance gene from potato;

(iv) a pathogen resistance gene from Arabidopsis; (v) a pathogen resistance gene from flax;

(vi) a nucleotide sequence encoding a CaMV gene VI protein;

(vii) a nucleotide sequence encoding a bacterial

harpin gene protein;

(viii) a nucleotide sequence encoding a ubiquitin

conjugating enzyme;

(ix) a nucleotide sequence encoding an RNase;

(x) a nucleotide sequence encoding a toxic peptide;

(xi) a mutant, variant, derivative or homologue of any of (i) to (x);

39. A cell according to claim 38 wherein said

pathogen resistance gene from tomato is selected from Cladosporium fulvum resistance genes including Cf-2, Cf-4 , Cf-5 and Cf-9; said pathogen resistance gene from tobacco is N'; said pathogen resistance gene from potato is Nx; said pathogen resistance gene from

Arabidopsis is RPP5 or RP52; said pathogen resistance gene from flax is L6; said RNase is Barnase; or said toxic peptide is diphtheria toxin.

40. A cell according to claim 36 wherein said L gene is:

(i) a Cladosporium fulvum avirulence gene or another elicitor of a resistance gene for a Cladosporium fulvum avirulence gene;

(ii) a suitable TMV coat protein or another N'

elicitor;

(iii) a suitable PVX coat protein or another Rx

elicitor; or

(iv) a mutant, variant, derivative or homologue of any of (i) to (iii).

41. A cell according to claim 40 wherein said

Cladosporium fulvum avirulence gene is Avr2, Avr4 , Avr5 or Avr9.

42. A cell according to any one of claims 33 to 41 which is a microbial cell.

43. A cell according to any one of claims 33 to 41 which is a plant cell.

44. A plant or any part or propagule or derivative thereof comprising a cell according to claim 43.

45. A plant, part, propagule or derivative according to claim 44 which is variegated for cells wherein said nucleotide sequence is inactivated or activated.

46. A method of producing a cell according to any one of claims 33 to 45 comprising introduction of nucleic acid (i) and/or (ii) into the cell or an ancestor thereof.

47. A composition of matter comprising any of the following combinations of nucleotide sequences:

(i) a nucleotide sequence comprising R, a nucleotide sequence comprising I and a nucleotide sequence

comprising A;

(ii) a nucleotide sequence comprising R, and a

nucleotide sequence comprising I and A;

(iii) a nucleotide sequence comprising I, and a

nucleotide sequence comprising A and R;

(iv) a nucleotide sequence comprising A, and a

nucleotide sequence comprising R and I; and

(v) a nucleotide sequence comprising R, I and A;

wherein R encodes a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and A encodes a substance able to reactivate R inactivated by I.

48. A composition of matter comprising any of the following combinations of nucleotide sequences:

(i) a nucleotide sequence comprising R, a nucleotide sequence comprising L, a nucleotide sequence comprising I, and a nucleotide sequence comprising A;

(ii) a nucleotide sequence comprising R, a nucleotide sequence comprising L and I, and a nucleotide sequence comprising (A);

(iii) a nucleotide sequence comprising R, a nucleotide sequence comprising L and A, and a nucleotide sequence comprising I;

(iv) a nucleotide sequence comprising R, a nucleotide sequence comprising I and A, and a nucleotide sequence comprising L;

(v) a nucleotide sequence comprising L, a nucleotide sequence comprising I and R, and a nucleotide sequence comprising A;

(vi) a nucleotide sequence comprising L, a nucleotide sequence comprising A and R, and a nucleotide sequence comprising I;

(vii) a nucleotide sequence comprising I, a nucleotide sequence comprising L and R, and a nucleotide sequence comprising A; (viii) a nucleotide sequence comprising R, and a nucleotide sequence comprising L, I and A;

(ix) a nucleotide sequence comprising L, and a

nucleotide sequence comprising I, A and R;

(x) a nucleotide sequence comprising I, and a

nucleotide sequence comprising A, R and L;

(xi) a nucleotide sequence comprising A and a

nucleotide sequence comprising A, R and I; and

(xii) a nucleotide sequence comprising R, L, I and A; wherein R and L encode substances whose presence together in a plant results in a plant defence

response, necrosis and/or increased pathogen

resistance, I is a genetic insert able to inactivate R and/or L and A encodes a substance able to reactivate R and/or L inactivated by I.

49. A composition of matter according to claim 47 or 48 which is one or more nucleic acid vectors.

50. A composition of matter according to any one of claims 47 to 49 wherein a cell contains any of said combinations of nucleotide sequences.

51. A plant, or a part, propagule, derivative or descendant thereof, comprising a cell according to the composition of claim 50.

52. A method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a nucleotide sequence or nucleotide sequences encoding R, I and A, wherein R encodes a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and A encodes a substance able to

reactivate R inactivated by I, comprising crossing plant lines whose genomes comprise any of R, I, A and combinations thereof, to produce the plant or an ancestor thereof.

53, A method according to claim 52 wherein one or more of said plant lines contains nucleic acid

comprising any of R, I, A and combinations thereof as a result of transformation of cells of the plant or an ancestor thereof.

54. A method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a nucleotide sequence or nucleotide sequences encoding R, L, I and A, wherein R and L encode substances whose presence together in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and/or L and A encodes a substance able to reactivate R and/or L inactivated by I, comprising crossing plant lines whose genomes comprise any of R, L, I, A and combinations thereof, to produce the plant or an ancestor thereof.

55, A method according to claim 54 wherein one or more of said plant lines contains nucleic acid

comprising any of R, L, I, A and combinations thereof as a result of transformation of cells of the plant or an ancestor thereof.

56. A plant, or a part, propagule, derivative or descendant thereof, obtainable using a method according to any one of claims 52 to 55.