AU703644B2 - Method of introducing pathogen resistance in plants - Google Patents

Description

WO 95/31564 PCT/GB95/01075 1 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 an induced response WO 95/31564 PCT/GB95/01075 2 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.

l'i i~lasraa~ rm~ss~- WO95/31564 PCT/GB95/01075 3 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 2,6dichloroisonicotinic 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 WO 95/31564 PCTI/GB95101075 4 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

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WO 95/31564 PCT/GB95/01075 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 Isd (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 Isd 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.

IIIQgp~ssPIPl~lll~aaa~lR a~l lr~n~ WO95/31564 PCT/GB95/01075 6 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 lii~88~e~X9Panararaaa~la~- ~s WO95/31564 PCT/GB95/01075 7 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; K6hm 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

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WO 95/31564 PCTIGB95/01075 8 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 cysteinerich peptide with a final processed size of 28 amino acids. However, its role in compatible interactions is not clear. The T 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 WO 95/31564 PCT/GB95/01075 9 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 I- B ~YBIIIIRPUllsWOiRLear(~sRllb~r PCT/GB9501075 WO 95/31564 gene.

Various pathogen races that overcome these Cfgenes 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 and C. fulvum race 2.4.5.9 can overcome Cf-2, Cf-4, Cfand 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 Avr9 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 r I i' WO 95/31564 PCT/GB95/01075 11 pathogens, the method would not be viable unless the promoter is only induced by the appearance of a compatible pathogen. It the defence response provides further induction of the promoter the plant might experience spreading necrosis.

The present invention has reFulted from experiments involving transposon tac':ing 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

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WO 95/31564 PCT/GB95/01075 12 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: 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: (i) lu8i~Blianar~-nrr~---~ WO 95131564 PCTIGB95/01075 13 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/orplant 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 WO 95/31564 PCT/GB95/01075 14 pathogen resistance gene an avirulence gene (Avr) or other elicitor or ligand gene of an R gene, or both and R gene and an L gene.

The inactivation of the nucleotide sequence or sequences wiich 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. followiLg 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 WO 95/31564 PCT/GB95/01075 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 P IL~B~UIR~YUI~ WO 95/31564 PCT/GB95/01075 16 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 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 resistarce The activation of, for example, aplant 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

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~a8~eslsaR~ewsarsrusoleslrm~oYo~a~~ WO 95/31564 PCT/GB95/01075 17 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 defencerelated 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, Altenaria solani, Rhizoctonia solani) and viral pathogens (for example, I WO 95/31564 PCT/GB95101075 18 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 I I Ill~sl~llllarrr;u~ WO 95/31564 PCT/GB95/01075 19 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 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 I 1 II ~B~IPZSIUISI~ YBRB~yla~ s WO 95131564 PCTIGB95101075 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.

I i I ~il~ll~sr~l~- osr~ulUr^-- WO 95/31564 PC'rIGB95/01075 21 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 a vector) comprising WO95/31564 CI'IGCB9501075 22 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 may be accompanied, preceded or followed by introduction of nucleic acid 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 WO 95/315641 PCT/'GB95/01075 23 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 F1 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 nonautonomous inactivation system, such as the transposon or recombinase-recognition sequences. After making a cross between parents of this genetic constitution, on 1Il~B" UL~ Y1B~ launai~lanauuarrs~a WO 95/31564 PCTIGB9501075 24 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: represents a nucleotide sequence with one or more genes of type X 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) WO 95/31564 PCT/GB95/01075 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: Any combination of: 1. and 2. and (IA); 3. and or 4. and (RI);

(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: Any combination of: 1. and 2. (LI) and (A) 3. (LA) and (I) 4. (IA) and (L) 5. (IR) and (A) GMVEAAMNW WO 95/31564 PCT/GB95/01075 26 6. (AR) and (I) 7. (LR) anc (A) 8. and (LIA) 9. and (IAR) 10. and (ARL); or 11. and (kLT); 12. (RLIA) If genetic insert is coupled with either the R or the L gene, the number of possible combinations will then be (RI) and or

(RIA)

(RI) and (A) (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

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WO 95/31564 PCT/GB95/01075 27 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 ~rBS(g(~ilsRP~aa~o~ssma~w~ WO95/31564 PCT/GB95/01075 28 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 et al., 1992; Santa-Cruz et al., 1993; Kbhm 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 avrRPt2 (Bent et al., Mindrinos et 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 WO 95/31564 PCT/GB95/01075 29 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/Avr9 gene system. This can involve the matching of a transposon ina A-;ivated 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 li~li~8~Rap~R&n~raps*Psrw~ WO 95/31564 PCT/GB95/01075 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 plane 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 c WO 95/31564 PCT/GB95/01075 31 somatic excision and restoration of Avr9 gene expression, or vice versa.

Nucleotide sequences employed in the present invention may encode a wild-type sequence 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 w' 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 WO 95/31564 PCT/GB95/01075 32 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 mutator (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

DIII~~I-I-

WO 95/31564 PCT/GB95/01075 33 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 WO 95/31564 PCT/GB95/01075 34 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 WO 95/31564 PCT/GB95/01075 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 WO 95/31564 PCT/GB95/01075 36 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 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 WO 95/31564 PCT/GB95/01075 37 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 WO 95/31564 PCTIGB95/01075 38 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 nonlimiting 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 WO 95/31564 PCT/GB95/01075 39 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 s- res. 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: WO 95/31564 PCT/GB95/01075 mean number of lesions given at 7, 10, 14, 21, 24 and days after inoculation. C shows equivalent results for Mutant Figure 6 shows the appearance of tomato fruits on GAR (sAc, Cf-9*Ds right-hand) and GAR- Ac, Cf-9*Ds, Avr-9 left-hand) plants from mutant line M2i at 2, 3, 4, 5, 6 and 7 weeks after flower pollination. Dark green sectors formed on the GAR but not GAR- fruits by weeks. These dark green sectors were not visible on 1 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 P-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 WO 95/31564 PCT/GB95/01075 41 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.

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 maj'rity 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.

~pli~Bll~sl~nrra~lRrmspnans~ PC'T/GB95/01075 WO 95/31564 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 comprising a Cf-9 gene, inactivated by insertion of a Ds transposon, and an Avr-9 gene and 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 Ti/Pi: line comprising in its genome at least one of each of the four genes, R, L,I or A line comprising in its genome at least one of each of two of the four genes R, L, I or A 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 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 line comprising in its genome at T1, 2, 3/pl,2,3:

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WO 95/31564 PCT/GB95/01075 43 least one of each of three of the four genes R,L,I or A T4/P 4 line comprising in its genome at least one of each of the four genes R,L,I or A not present in T 1 ,2,3 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 Avr9 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 WO 95/31564 PCT/GB95/01075 44 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 noncapitalised 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 R Illlll~-0~ I~ 1 13~ ul l~ sl WO 95/31564 PJCTGB95/01075 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 ClaI/SalI 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 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 4~1~1_ WO 95/31564 PCT/GB95/01075 46 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 beta-glucuronidase (GUS) gene (Jefferson et al 1987) to monitor T-DNA segregation and 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: 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

I

gbP~ilBBil8A~atassP~na~aarar~l WO 95/31564 PCT/GB95/01075 47 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.

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 50% 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 I I~B~F ABIBF~I~I B sa~na~F WO 95/31564 PCT/GB95/01075 48 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 (Dbring, 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 Avr9carrying 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. 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

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Is~upza~arnn~-- I-a~i~ ll- WO 95/31564 PCT/GB95/01075 49 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 Cf0 tomato lines that expressed this gene.

(iii) Crossing AVR9 expressing stock with 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 Cf0 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.

j 1B~OIDII II~LP IP~I~ IVI1(~/~ WO 95/31564 PCT/GB95/01075 (iv) Tagging and inactivating Cf-9 Individuals that were homozygous for the Avr9 gene (section 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 BglII 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 BglII 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 thxt does not cut within the Ds (such as BglII), the digestion products -~pl LI~P~YILP UQII~ erUerrrsrPsasrua~- ~DIII WO 95/31564 PCT/GB95/01075 51 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,

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ptli~Llle~8I i~ur'nr~r~-rr~~ WO 95/31564 PCT/GB95/01075 52 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).

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 ;prapsrurnaaoa~ ~sasI~ UaBUI BY P~ WO 95/31564 CT/GB95/01075 53 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 tive 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 C' amino acids, 7 and 8 28 and 29 and 48 56 and 57 (>M31) and 789 and 790 (>M30) The arrows or indicates the direction of transcription-of the Ds element. F, 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 I- r l-sp WO 95/31564 PCT/GB95/01075 54 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 5 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 nonvariegated 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. 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 I WO 95/31564 PCT/GB95/01075 Cf-9*Ds #31, respectively. These individuals segregate from the Cf-9*Ds and for sAc. Cf0 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 nonvariegated 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 Ill~iB~YI~1Ps~i~ srarr~l~l~ ruac~~ WO 95/31564 PCT/GB95/01075 56 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 Avr9 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 Colletotrichum largenarium (which causes leaf and fruit spot).

M

B*RIBIIIU YBIWI~P~asrrr~rr~r~~ WO 95/31564 PCT/GB95/01075 57 For the P. infestans experiments, sibling backcross progeny from the mutatnt Cf-9* Ds lines M31 and M50 that were either variegating for necrosis or not and control plants lacking a Cf-gene (Cf0) 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 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 minimum of 18 plants were used for each genotype/spore.

For the Oidium lycopersici experiments the r Ip~ISDS~Nursr~-ns WO 95/31564 PCT/GB95/01075 58 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 0

C

during the 16 h photoperiod and at 18 0 C during the dark period. The RH was maintained at 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 By day 14 post inoculation more than 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 Cf0 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 a^ WO 95/31564 PCT/GB95/01075 59 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 1002l 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 1 WO 95/31564 PCT/GB95/01075 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 pedigrees using a basic tomato 0-1,3 glucanase probe and a tomato anionic peroxidase probe (pTAP 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

~RBI~B*O~IUY~U~

WO 95/31564 PCT/GB95/01075 61 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 Heterologous 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 WO 95/31564 PCT/GB95/01075 62 express Avr9 peptide constitutively, the F1 seedlings die within 2 days of seed germination (Figure 9).

When transgenic Arabidopsis expressing Cf-9 is crossed to Avr9 expressing transgenic Arabidopsis the F1 seedlings die 10 days after seed germination (Figure 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 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 ~Q$llyB~Wasrsrr~Ra~aa~arrsre~~ WO95/31564 PCT/GB95/01075 63 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 Avr4 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

I

WO 95/31564 PCT/GB95/01075 64 signal peptide of the tobacco PRIa 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 ClaI/SalI 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 vitro transcription. All nucleic acid manipulations were performed using standard techniques well known to those skilled in the art.

Tomato 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 in contrast to CfO controls which only showed signs of slight mechanical damage at the site of virus inoculation. Cf0 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 WO 95/31564 PCT/GB95/01075 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. Cf0 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 I I I

IW*I~S~W~RI(ISP~

WO95/31564 PCT/GB95/01075 66 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 1 I I-ld ~B~nBWPig~u;ss;saweoluEle~in~ WO95/31564 PCT/GB95/01075 67 plants (Cf0) but not on incompatible control plants (Cf2).

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

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PCT/G]395/01075 WO 95/31564PCG19/07 SEQ ID NO.

CATAGTCTTT

GAAAATTTTG

AATATTTTCC

AACATGCCAT

ATTGGTGGAA

CACTGTTATA

ATATTGTCAC

CATTCTGATG

ATTACGCTTA

TAAAGGTTTT

ATGGTGACCT

TAGACCAAAC

TCTTGTGGAA

TTTGCTTAAT

CATTTCTTGA

1

GCATATTTGG

GACCAAGCTA

GTTGAATGAA

GTCTGGACTC

GGTAGACGGT

CAACAACAAC

GAGTTTTTTT

CTTCGTACAA

CTATGATAGC

TCCCCGTTCT

ATTTGGATGG

TGAGAAGGAC

AATTAGCTCG

TTGTGCTATA

TTTCTTCTCT

ATTAAACAGG GGCATTATTG TTGACAACAC GAACATTTTT

TAAGGTAACT

CTGCACTATC

ACAAATTGAA

AAACTACGCT

TAGAGTATGT

ATTTATTGAA

GGTCTTTTTT

ATACACAAAC

TAACATTATT

ATGTCTGGAC

AGGTGGCGCA

AGTAGTAAAT

TTCCATCAAC

TTATATTAAA

TCAGCCCCAA

TGCATATATT

TTTTCAACTT

GATTAATCAA

TAAGAAAAAT

GGACCAAACT

TCCTGCTCCG

CTATGTGAGG

AACCAAACTA

AGACCAAACT

TTTTAGACCA

AGGTCAATTC

AGACAAGCTC

ACAATAGTGA

ATACTCAACT

TAAAGGTTTA

ACTTATTGAA

TTAAATTATA

ATTGATAACG

TCTTCCATCA

TAACTAGTAC

GAATAGTCAC

TCATTTTTAG

TTAGATGTAT

ATTAACTCAG

AACTATGAAG

TCTCAACTCT

ACCTGAGCAT

CCCGAATCAT

TAGGGTTTGT

TGAACCAP.AT

TTTTCAACTT

TAGTCTTTGG

CGGACATTGT

GCAGGTCGAT

TAAATTTTTC

ACAAAGCAAA

TGCAGAA

120 180 240 300 360 420 480 540 600 660 720 780 840 897

TATACCTCATCTAAATTATT

ATCAACATAA CAAGTTTTGA ATG GAT TGT GTA AAA CTT GTA TTC Met Asp Cys Val Lys Leu Val Phe

CTT

Leu -15 ATG CTA TAT ACC Met Leu Tyr Thr TTA TCC Leu Ser TTT CTC TGT Phe Leu Cys GAA GAT CAA Giu Asp Gin CAA CTT GCT Gin Leu Ala GCT CTT TCT Ala Leu Ser GCT TCT GAT Ala Ser Asp TAT CCA AGA Tyr Pro Arg TCA TCC TTG CCT CAT TTG TGC CCC Ser Ser Leu Pro His Leu Cys Pro CTT CTA CAA Leu Leu Gin i5 TAT TGT TAC Tyr Cys Tyr TTC AAG AAC ATG Phe Lys Asn Met ACC ATT AAT Thr Ile Asn GTA GAC ATT Val Asp Ile CCT AAT Pro Asn CAG TCA Gin Ser GAC ATA AGA Asp Ile Arg CTT TCT TGG AAC Leu Ser Trp Asn AGC ACA AGT TGC Ser Thr Ser Cys TGC TCA TGG Cys Ser Trp, GCG CTT GAC Ala Leu Asp GAT GGC GTT CAT Asp Gly Val His CTC CGT TGC AGC Leu Arg Cys Ser TGT GAC GAG ACG ACA GGA CAA GTG Cys Asp Giu Thr Thr Gly Gin Vai 1041 1089 1137 1185 1233 1281i 1329 CAA CTT CAA GGC AAG TTT CAT Gin Leu Gin Gly Lys Phe His

TTT

Phe

ACT

Thr CAA CTC TCC AAT CTC AAA AGG CTT GAT Gin Leu Ser Asn Leu Lys Arg Leu Asp

TTG

Leu 100

GAG

Glu TCC AAT AGT AGC CTC Ser Asn Ser Ser Leu TCT TTT AAT AAT TTC Ser Phe Asn Asn Phe 105 TTT TCA AAT TTG ACG Phe Ser Asn Leu Thr 120 GGA TCA CTC Gly Ser Leu TCA CCA AAA TTT Ser Prc~ Lys Phe WO 95/315640 PCT/GB95/01075 CAT CTC GAT His Leu Asp ATC TGT CAC Ile Cys His 140 TTG TCG CAT TCT Leu Ser His Ser AGT TTT Ser Phe 130 ACA GGT CTA ATT Thr Gly Leu Ile CCT TCT GAA Pro Ser Glu 135 GAT CAA TAT Asp Gin Tyr CTT TCT AAA CTA CAC Leu Ser Lys Leu His 145 GTT CTT CGT ATA Val Leu Arg Ile GGG CTT Gly Leu 155 AGT CTT GTA CCT Ser Leu Val Pro

TAC

Tyr 160 AAT TTT GAA CTG Asn Phe Giu Leu CTT AAG AAC TTG Leu Lys Asn Leu CAA TTA AGA GAG Gin Leu Arg Glu AAC CTT GAA TCT Asn Leu Giu Ser AAC ATC TCT TCC Asn Ile Ser Ser ATT CCT TCA AAT Ile Pro Ser Asn TCT TCT CAT TTA ACA ACT CTA CAA CTT Ser Ser His Leu Thr Thr Leu Gin Leu 195 TCA GGC Ser Gly 200 ACA GAG TTA Thr Giu Leu TTA CAA TCC Leu Gin Ser 220 GGG ATA TTG CCC Gly Ile Leu Pro

GAA

Glu 210 AGA GTT TTT CAC CTT TCC AAC Arg Val Phe His Leu Ser Asn 215 CTT CAT TTA TCA Leu His Leu Ser AAT CCC CAG CTC Asn Pro Gin Leu GTT AGG TTT Vai Arg Phe CCC ACA Pro Thr 235 ACC AAA TGG AAT Thr Lys Trp Asn AGT GCA TCA CTC.ATG ACG TTA TAC GTC Ser Ala Ser Leu Met Thr Leu Tyr Vai 245

GAT

Asp 250 AGT GTG AAT ATT Ser Val Asn Ile GAT AGG ATA CCT Asp Arg Ile Pro TCA TTT AGC CAT Ser Phe Ser His 1377 1425 1473 1521 1569 1617 1665 1713 1761 1809 1857 1905 1953 2001 2049 2097 2145 ACT TCA CTT CAT Thr Ser Leu His TTG TAC ATG GGT Leu Tyr Met Gly

CGT

Arg 275 TGT AAT CTG TCA Cys Asn Leu Ser GGG CCT Gly Pro 280 ATT CCT AAA Ile Pro Lys GGT GAT AAC Gly Asp Asn 300 CTA TGG AAT CTC Leu Trp Asn Leu

ACC

Thr 290 AAC ATA GTG TTT Asn Ile Val Phe TTG CAC CTT Leu His Leu 295 ATA TTT GAA Ile Phe Glu CAT CTT GAA GGA His Leu Glu Gly

CCA

Pro 305 ATT TCC CAT TTC Ile Ser His Phe AAG CTC Lys Leu 315 AAG AGG TTA TCA Lys Arg Leu Ser

CTT

Leu 320 GTA AAT AAC AAC Val Asn Asn Asn GAT GGC GGA CTT Asp Gly Gly Leu TTC TTA TCC TTT Phe Leu Ser Phe

AAC

Asn 335 ACC CAA CTT Thr Gin Leu GAA CGG Glu Arg 340 CTA GAT TTA TCA Leu Asp Leu Ser AAT TCC CTA ACT Asn Ser Leu Thr

GGT

Gly 350 CCA ATT CCA TCC Pro Ile Pro Ser ATA AGC GGA CTT Ile Ser Gly Leu CAA PAC Gin Asn 360 CTA GAA TGT Leu Glu'Cys TCC TGG ATA Ser Trp Ile 380 CTC TAC TTG TCA TCA AAC CAC TTG PAT GGG AGT ATA CCT Leti Tyr Leu Ser Sei Asn His Leu Asn Gly Ser Ile Pro 365 370 375 TTC TCC CTT CCT Phe Ser Leu Pro CTG GTT GAG TTA Leu Val Glu Leu TTG AGC PAT Leu Ser Asn Ag ISIW~PI~111~ PCTIGB95 1075 WO 95/31564 AAC ACT Asn Thr 395 TTC AGT GGA AAA Phe Ser Giy Lys CAA GAG TTC AAG Gin Giu Phe Lys AAA ACA TTA AGT Lys Thr Leu Ser GTT ACT CTA AAA Val Thr Leu Lys AAT AAG CTG AAA Asn Lys Leu Lys CGT ATT CCG AAT Arg lie Pro Asn CTC CTA AAC CAG Leu Leu Asn Gin AAC CTA CAA TTA Asn Leu Gin Leu CTC CTT TCA CAC Leu Leu Ser His AAT AAT Asn Asn 440 ATC AGT GGA Ile Ser Gly TTG TTA GAC Leu Leu Asp 460 ATT TCT TCA GCT Ile Ser Ser Ala TGC AAT CTG AAA Cys Asn Leu Lys ACA TTG ATA Thr Leu Ile 455 CCA CAA TGC Pro Gin Cys TTG GGA AGT AAT Leu Giy Ser Asn

AAT

Asn 465 TTG GAG GGA ACA Leu Giu Gly Thr GTG GTT Vai Vai 475 GAG AGG AAC GAA Glu Arg Asn Glu CTT TCG CAT TTG Leu Ser His Leu GAT TTG AGC AAA AAC Asp Leu Ser Lys Asn 485 GTT GGA AAC ATT TTA Val Giy Asn Ile Leu CTT AGT GGG ACA Leu Ser Giy Thr AAT ACA ACT TTT Asn Thr Thr Phe AGG GTC ATT AGC Arg Val Ile Ser CAC GGG AAT AAG His Gly Asn Lys ACG GGG AAA GTC Thr Giy Lys Vai CCA CGA Pro Arg 520 TCT ATG ATC Ser Met Ile

AAT

Asn 525 TGC AAG TAT TTG Cys Lys Tyr Leu CTA CTT GAT CTA Leu Leu Asp Leu GGT AAC AAT Gly Asn Asn 535 TTT CAA TTG Phe Gin Leu 2193 2241 2289 2337 2385 2433 2481 2529 2577 2625 2673 2721 2769 2817 2865 2913 2961 ATG TTG AAT GAC ACA TTT CCA AAC TGG TTG GGA TAC CTA Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Giy Tyr Leu 540 545 550 AAG ATT TTA AGC TTG AGA Lys Ile Leu Ser Leu Arg AAT AAG TTG CAT Asn Lys Leu His

GGT

Gly 565 CCC ATC AAA TCT Pro Ile Lys Ser

TCA

Ser 570 GGG AAT ACA AAC Gly Asn Thr Asn TTT ATG GGT CTT Phe Met Gly Leu

CAA

Gin 580 ATT CTT GAT CTA Ile Leu Asp Leu TCT AAT GGA TTT Ser Asn Giy Phe GGG AAT TTA CCC Gly Asn Leu Pro

GAA

Glu 595 AGA ATT TTG GGG Arg Ile Leu Gly AAT TTG Asn Leu 600 CAA ACC ATG Gin Thr Met TCT GAT CCA Ser Asp Pro 620 GAA ATT GAT GAG Glu Ile Asp Glu

AGT

Ser 610 ACA GGA TTC CCA Thr Gly Phe Pro GAG TAT ATT Glu Tyr Ile 615 ATT TCT ACA Ile Ser Thr TAT GAT ATT TAT Tyr Asp Ile Tyr

TAC

Tyr 625 AAT TAT TTG ACG Asn Tyr Leu Thr

ACA

Thr 630 AAG GGA Lys Gly 635 CAA GAT TAT GAT Gin Asp Tyr Asp

TCT

Ser 640 GTT CGA ATT TTG Vai Arg Ile Leu

GAT

Asp 645 TCT AAC ATG ATT Ser Asn Met Ile AAT CTC TCA AAG AAC AGA TTT GAA GGT CAT ATT CCA AGC ATT Asn Leu Ser Lys Asn Arg Phe Glu Giy His Ile Pro Ser Ile I Er- PCTIGB95/0 1075 WO 95/31564 GGA GAT CTT GTT GGA CTT CGT ACG TTG Gly Asp Leu Val Gly 670 Leu Arg Thr Leu

AAC

Asn 675 TTG TCT CAC AAT Leu Ser His Asn GTC TTG Val Leu 680 GAA GGT CAT ATA CCG GCA TCA TTT Pro Ala Ser Phe AAT TTA TCA GTA Asn Leu Ser Val Giu Gly His TTG GAT CTC Leu Asp Leu 700 GCA TCC CTC Ala Ser Leu 71S Ile 685

TCA

Ser TCT AAT AA Ser Asn Lys AGC GGA GAA ATT Ser Gly Glu Ile CTC GAA TCT Leu Giu Ser 695 CAG CAG CTT Gin Gin Leu AAT CAT CTT Asn His Leu ACA TTC CTT Thr Phe Leu GTC TTA AAT CTC Vai Leu Asn Leu GGA TGC ATC CCC Giy Cys Ile Pro

AAA

Lys 735 GGA AAA CAA TTT Giy Lys Gin Phe TTC GGG AAC Phe Gly Asn 3009 3057 3105 3153 3201 3249 3297 3345 3393 TAC CAA GGG Tyr Gin Gly

AAT

Asn 750 GAT GGG TTA CGC Asp Gly Leu Arg CCA CTC TCA Pro Leu Ser TGT GGT GGT Cys Gly Gly GAG GAG GAA Giu Giu Giu 780 GGT TAC GGT Giy Tyr Giy 795

GAA

Giu 765

GAA

Giu GAT CAA GTG Asp Gin Val GAT TCA CCA Asp Ser Pro ACA ACT Thr Thr 770 ATG ATC Met Ile 785 ATT GGA Ile Giy GCT GAG CTA GAT CAA GAA Ala Giu Leu Asp Gin Giu 775 AGT TGG CAG Ser Trp Gin GGG GTT CTC GTG Gly Val Leu Val 790 ATA TAG ATA ATG Ile Tyr Ile Met TGT GGA CTT Cys Gly Leu CTG TCC Leu Ser

GTA

Val 805

ATG

Met

TGG

Trp 810 TCA ACT CAA TAT Ser Thr Gin Tyr TGG TTT TCG Trp, Phe Ser

AGG

Arg 820

CAC

His GAT TTA AAG Asp Leu Lys TTG 3441 Leif 825 TAGTGAGTAG 3496 GAA CAC ATA Giu His Ilie ATT ACT Ile Thr 830 AAA ATG AAA Lys Met Lys AAG AAA AGA Lys Lys Arg

TAT

Tyr 840

CTATACCTCC

ATGAAATTAT

ATTTCAGGAT

TATCTTTCAT

TAGATATGTT

AAAGAACACT

ACACAACTGA

AGGTATTCCA

CGACCTCCTT

TCAAAGATTT

AGTTTCTTAT

CCGTCACTAA

TGACTTCAAT

CGTATCTTGA

CTTGATCATT

CATCCTCAAA

CCGAGTTCCC

CCTATGAATA

AAACATTGTA

TAAGTTACTG

GAAAGAGACT

ATCTTTCAGA

GCTCTTAACT

AGTTGCTTGG

AAGATTTTAT

TTTCTCTCAA

TAGTCTGCTA

ATGATCCCCC

AGATTATTTT

TTCACTCTTC

GATGCAGATA

TTTCATTTGT

CTCTTTCGTC

TTTTAATTTT

GGGCTGCAG

TTGTATATCG

ATTTTTGAAA

AAAGCCTTTT

CTATGGCACG

ACATGATATC

TTCCATTGAA

3556 3616 3676 3736 3796 3856 3905 SEQ ID NO. 2: Met Asp Cys Vai Lys Leu Vai Phe Leu Met Leu Tyr Thr Phe Leu Cys -23 -20 -15 Gin Leu Ala Leu Sex Ser Ser Leu Pro His Leu Cys Pro Giu Asp Gin 1 WO 95/31564 PTG9I)17 PCT/GB95/01075 Al a Ala Tyr Asp Leu Phe Thr His Ile Gly Thr 170 Ile Thr Leu Pro Asp 250 Thr Ile Gly Lys Glu 330 Asn Leu Ser Ser Asp Pro Arg Gly Val Arg Cys Gin Leu Gly Ser Leu Asp Cys His 140 Leu Ser 155 Gln Leu Pro Ser Giu Leu Gin Ser 220 Thr Thr 235 Ser Val Ser Leu Pro Lys Asp Asn 300 Leu Lys 315 Phe Leu Ser Leu Leu Tyr Thr His Ser Ser Leu Leu 125 Leu Leu Arg Asn His 205 Leu Lys Asn His Pro 285 His Arg Ser rhr Phe Asp Leu Ser Trp Asn Cys Gin Asn Ile 110 Ser S er Val Giu Phe 190 Gly His Trp Ile Glu 270 Lieu Aeu Leu Phe Gly 350 Asp Leu Leu 95 Ser His Lys Pro Leu 175 Ser Ile Leu Asn Ala 255 Leu Trp Giu Ser Asn 335 Pro Giu Gin 80 Lys Pro Ser Leu Tyr 160 Asn Ser Leu Ser Ser 240 Asp Tyr Asn Gly Leu 320 Thr Ile Thr 65 Gly Arg Lys S er His 145 Asn Leu His Pro Val 225 Ser Arg Met Leu Pro 305 Val Gin Pro Asn Arg Lys 50 Thr Lys Leu Phe Phe 130 Val Phe Giu Leu Giu 210 Asn Ala Ile Gly Thr 290 Ile Asn Leu Ser *Met Thr Ser Gly Phe Asp Gly 115 Thr Leu Giu Ser Thr 195 Arg Pro Ser Pro Arg 275 Asn Ser Asn Giu Asn 355 Phe 20 Tyr Thr Gin His Leu 100 Giu Giy Arg Leu Val 180 Thr Val Gin ELeu Lys 260 :ys Ile H~is Asn k.rg 340 Ile Thr Val Ser Val Ser Ser Phe Leu Ile Leu 1.65 Asn Leu Phe Leu Met 245 Ser Asn Val Phe Phe 325 Leu S er Ile Asp Cys Ile Asn Phe Ser Ile Cys 150 Leu Ile Gin His Thr 230 Thr Phe Leu Phe Thr 310 Asp Asp Giy Asn Ile Cys Ala Ser Asn Asn Pro 135 Asp Lys S er Leu Leu 215 Val Leu Ser Ser Leu 295 Ile Gly Leu Leu Pro *Gin Ser Leu S er Asn Leu 120 Ser Gin Asn Ser Ser 200 Ser Arg Tyr His Giy 280 His Phe Giy Ser Gin 360 Asn Ser Trp Asp Leu Phe 105 Thr Giu Tyr Leu Thr 185 Gly A.sn Phe VTal Leu 265 Pro Leu Glu Aeu 3 er 345 %sn WO 95/31564 77 Leu Giu Cys Leu Tyr Leu Ser Ser Asn His Leu Asn Gly Ser Ile Prc/G 1195( 1075 Pro 365 Ser Asn Ala 410 Leu Ile Leu Val Arg 490 Arg Ser Met Lys Ser 570 Ser Gin Ser Lys Ile 650 Gly Glu Leu Trp Thr 395 Val Leu Ser Leu Vai 475 Leu Vai Met Leu Ile 555 Gly Asn Thr I Asp Gly 635 Asn Asp I Gly I Asp I Ile 380 Phe Thr Asn Gly Asp 460 Glu Ser Ile Ile Asn 540 Leu Asn Gly Met Pro 520 Gln Leu Leu iis eu 700 Phe Ser Leu Gin His 445 Leu Arg Gly Ser Asn 525 Asp Ser Thr Phe Lys 605 Tyr Asp Ser Vai Ile 685 Ser Ser Gly Lys Lys 430 Ile Gly Asn Thr Leu 510 Cys Thr Leu Asn Ser 590 Glu Asp Tyr Lys ly 670 Pro Ser Leu Lys Gin 415 Asn Ser Ser Glu Ile 495 His Lys Phe Arg Leu 575 Gly Ile Ile Asp Asn 655 Leu Ala Asn Pro Ser Ile 400 Asn Leu Ser Asn Tyr 480 Asn Gly Tyr Pro Ser 560 Phe Asn Asp yr Ser 640 Arg Arg Ser Lys 385 Gin Lys Gin Ala Asn 465 Leu Thr Asn Leu Asn 545 Asn Met Leu Glu Tyr 625 Vai Phe Thr Phe Ile 705 370 Leu Vai Glu Glu Phe Lys Leu Lys Gly 420 Leu Leu Leu 435 Ile Cys Asn 450 Leu Glu Gly Ser His Leu Thr Phe Ser 500 Lys Leu Thr 515 Thr Leu Leu 530 Trp Leu Gly Lys Leu His Gly Leu Gln 580 Pro Giu Arg 595 Ser Thr Gly 610 Asn Tyr Leu Arg Ile Leu Glu Gly His 660 Leu Asn Leu 675 Gin Asn Leu 690 Ser Giy Glu Leu Ser 405 Arg Leu Leu Thr Asp 485 Val Gly Asp Tyr Gly 565 Ile Ile Phe Thr Asp 645 Ile Ser Ser Ile Asp 390 Lys Ile Ser Lys Ile 470 Leu Gly Lys Leu Leu 550 Pro Leu Leu Pro Thr 630 Ser Pro His Vai Pro 710 375 Leu Thr Pro His Thr 455 Pro Ser Asn Vai Gly 535 Phe Ile Asp Gly Glu 615 Ile Asn Ser Asn Leu 695 3ml Ser Leu Asn Asn 440 Leu Gin Lys Ile Pro 520 Asn Gin Lys Leu Asn 600 Tyr Ser M~et lie Val 680 Glu Gln Asn Ser Ser 425 Asn lie Cys Asn Leu 505 Arg Asn Leu Ser Ser 585 Leu Ile Thr Ile Ile 665 Leu Ser Leu

I-

WO 95/31564 WO 95/ 1564PCT/GB9 5/01075 Ala Ser Leu Thr Phe Leu Giu Val Leu Asn Leu Ser His Asn His Leu 715 Val Gly Cys Ile Pro Lys 730 735 720 Giy Lys Gin Ser Tyr Gin Gly 750 Asp Gin Vai Thr Cys Gly Gly Giu Giu Giu 760 Gly Tyr Gly 795 Trp, Ser Thr 810 Giu His Ilie Arg Thr 770 Ile Phe Asp 740 Gly Phe 755 Pro Ala Ser Trp Ser Phe Gly Pro Leu Ser Asn Thr 745 Lys Leu 760 Giu Leu Asp Gin Giu 775 Gin Giv Val Leu Val Giu Asp Ser Pro Met 785 790 Cys Gly Leu Gin Tyr Pro 815 Ile Thr Thr 830 Val 800 Ile Giy Leu Ser Ile Tyr Ile Met Ala Trp Phe Ser Arg 820 Met Asp Leu Lys Lys Arg Lys Leu 825 Tyr 840 Lys Met Lys Lys His 835 SEQ ID NO. 3:

CATTTCTTGA

GATTGTGTAA

TCATCCTTGC

ATGTTTACCA

ATTCAGTCAT

GGCGTTCATT

CTTCAAGGCA

GATTTGTCTT

AATTTGACGC

TGTCACCTTT

CCTTACAATT

TCTGTAAACA

CTTTCAGGCA

CAATCCCTTC

AATAGCAGTG

CCTAAATCAT

TCAGGGCCTA

GATAACCATC

TCACTTGTAA

TTTCTTCTCT

AACTTGTATT

CTCATTTGTG

TTAATCCTAA

ATCCAAGAAC

GTGACGAGAC

AGTTTCATTC

TTAATAATTT

ATCTCGATTT

CTAAACTACA

TTGAACTGCT

TCTCTTCCAC

CAGAGTTACA

ATTTATCAGT

CATCACTCAT

TTAGCCATCT

TTCCTAAACC

TTGAAGGACC

ATAACAACTT

ATCAACATAA

CCTTATGCTA

CCCCGAAGAT

TGCTTCTGAT

TCTTTCTTGG

GACAGGACAA

CAATAGTAGC

CACTGGATCA

GTCGCATTCT

CGTTCTTCGT

CCTTAAGAAC

TATTCCTTCA

TGGGATATTG

CAATCCCCAG

GACGTTATAC

AACTTCACTT

TCTATGGAT

AATTTCCCAT

TGATGGCGGA

CAAGTTTTGA TCATTTTTAG TGCAGAAATG TATACCTTTC TCTGTCAACT

CAAGCTCTTT

TATTGTTACG

AACAAAAGCA

GTGATTGCGC

CTCTTTCAAC

CTCATTTCAC

AGTTTTACAG

ATATGTGATC

TTGACCCAAT

AATTTCTCTT

CCCGAAAGAG

CTCACGGTTA

GTCGATAGTG

CATGAGTTGT

CTCACCAACA

TTCACGATAT

CTTGAGTTCT

CTCTTCTACA

ACATAAGAAC

CAAGTTGCTG

TTGACCTCCG

TCTCCAATCT

CAAAATTTGG

GTCTAATTCC

AATATGGGCT

TAAGAGAGCT

CTCATTTAAC

TTTTTCACCT

GGTTTCCCAC

TGAATATTGC

ACATGGGTCG

TAGTGTTTTT

TTGAAAAGCT

TATCCTTTAA

TGCTTTATCC

ATTCAAGAAC

ATACGTAGsAC

CTCATGGGAT

TTGCAGCCAA

CAAAAGGCTT

TGAGTTTTCA

TTCTGAAATC

TAGTCTTGTA

CAACCTTGAA

AACTCTACAA

TTCCAACTTA

AACCAAATGG

TGATAGGATA

TTGTAATCTG

GCACCTTGGT

CAAGAGGTTA

CACCCAACTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 GAACGGCTAG ATTTATCATC CAATTCCCTA ACTGGTCCAA TTCCATCCAA CATAAGCGGA WO 95/3 1564 CGB/I07 PCTIGB95/01075 CTTCAAAACC TAGAATGTCT CTACTTGTCA TGGATATTCT CCCTTCCTTC ACTGGTTGAG AAAATTCAAG AGTTCAAGTC CAAAACATTA AAAGGTCGTA TTCCGAATTC ACTCCTAAAC CACAATAATA TCAGTGGACA TATTTCTTCA TTAGACTTGG GAAGTAATAA TTTGGAGGGA GAATACCTTT CGCATTTGGA TTTGAGCAA TTTAGTGTTG GAAACATTTT AAGGGTCATT GTCCCACGAT CTATGATCAA TTGCAAGTAT TTGAATGACA CATTTCCAAA CTGGTTGGGA AGATCAAATA AGTTGCATGG TCCCATCAAA CTTCAAATTC TTGATCTATC ATCTAATGGA GGGAATTTGC AAACCATGAA GGAAATTGAT GATCCATATG ATATTTATTA CAATTATTTG GATTCTGTTC GAATTTTGGA TTCTAACATG GGTCATATTC CAAGCATTAT TGGAGATCTT AATGTCTTGG AAGGTCATAT ACCGGCATCA CAT CTCTCAT CTAATAAAAT CAGCGGAGAA CTTGAAGTCT TAAATCTCTC TCACAATCAT TTTGATTCGT TCGGGAACAC TTCGTACCAA TCAAAACTTT GTGGTGGTGA AGATCAAGTG GAGGAAGAAG ATTCACCAAT GATCAGTTGG CTTGTTATTG GACTGTCCGT AATATACATA TCGAGGATGG ATTTAAAGTT GGAACACATA AGATATTAGT GAGTAGCTAT ACCTCCAGGA GGATGCAGAT AAAAGCCTTT TTATCTTTCA TTTTCATTTG TCTATGGCAC GTAGATATGT ACTCTTTCGT CACATGATAT CAAAGAACAC

TCIAAACCACT

TTAGACTTGA

AGTGCCGTTA

CAGAAGAACC

GCTATCTGCA

ACAATCCCAC

AACAGACTTA

AGCTTGCACG

TTGACACTAC

TACCTATTTC

TCTTCAGGGA

TTTAGTGGGA

GAGAGTACAG

ACGACAATTT

ATTATCAATC

GTTGGACTTC

TTTCAAAATT

ATTCCGCAGC

CTTGTTGGAT

GGGAATGATG

ACAACTCCAG

CAGGGGGTTC

ATGTGGTCAA

ATTACTACGA

TTCAAAGATT

TAGTTTCTTA

TCCGTCACTA

TTGACTTCAA

TGAATGGGAG

GCAATAACAC

CTCTAAAACA

TACAATTACT

ATCTGAAAAC

AATGCGTGGT

GTGGGACAAT

GGAATAAGCT

TTGATCTAGG

AATTGAAGAT

ATACAAACTT

ATTTACCCGA

GATTCCCAGA

CTACAAAGGG

TCTCAAAGAA

GTACGTTGAA

TATCAGTACT

AGCTTGCATC

GCATCCCCAA

GGTTACGCGG

CTGAGCTAGA

TCGTGGGTTA

CTCAATATCC

AAATGAAAAA

TCCGAGTTCC

TCCTATGAAT

AAAACATTGT

TTAAGTTAAA

TATACCTTCC

TTTCAGTGGA

AAATAAGCTG

TCTCCTTTCA

ATTGATATTG

TGAGAGGAAC

CAATACAACT

AACGGGGAAA

TAACAATATG

TTTAAGCTTG

GTTTATGGGT

AAGAATTTTG

GTATATTTCT

ACAAGATTAT

CAGATTTGAA

CTTGTCTCAC

CGAATCTTTG

CCTCACATTC

AGGAAAACAA

ATTTCCACTC

TCAAGAAGAG

CGGTTGTGGA

AGCATGGTTT

GCACAAGAAA

CAGTTGCTTG

AAAGATTTTA

ATTTCTCTCA

1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 SEQ ID NO. 4: ATG GGA TTT GTT CTC TTT TCA CAA TTG CCT TCA Met Gly Phe Val Leu Phe Ser Gin Leu Pro Ser 1 5 10 ACA CTT CTC TTA TTC CTA GTA ATA TCC CAC TCT Thr Leu Leu Leu Phe Leu Val Ile Ser His Ser 25 TTT CTT CTT GTC TCT Phe Leu Leu Val Ser TGC CGT GCC TAC TGT Cys Arg Ala Tyr Cys WO 95/31564 WO 9531564PCT/GB95/01075 AAC AGT TCT TGT ACA AGA GCT TTT GAC TGT CTT GGA CAA TGT GGA AGA Asn Ser Ser Cys Thr Arg Ala Phe Asp Cys Leu Gly Gin Cys Gly Arg 40 TGC GAC TTT CAT AAG CTT CAA TGT GTA CAT TGA Cys Asp Phc2 His Lys Leu Gin Cys Val His SEQ ID NO. 1 CTCGAGTTCG GAACCTAAAA S1 GGTATATCAA TTTTTATATT 101 GCGATTTCCT TCACCGGAAA 151 GCAAAATGTA ACTTTTTTTT 201 TATTTGAATT TCAAAAAAAA 251 TACGATTTTC TTTTTAAAAT 301 CGATTTCCAT TTTTAATTTT 351 AAAAAATTTT AAAAAAAATT 401 ATTTTTTTAA AAAATGTTAT 451 TTTGCTTTTT TTGGAGGAA 501 GGTTAATATA AAATTTTATA 551 TAACTTTTAG GCTCCGGACT 601 TGCATAGTCT GAATTTTGAA 651 TCAGACATGA AATCTTTAAA 701 ACTACAGAAA AAGTATTATA 751 CGGAGTGATC GCGAGTGAAG 801 TCCATCTTGA GAGGTTGAGA 851 TATTAATATC C.AATTTTCTT 901 CAAGATACTT CATCATATAA 951 TTTGGAAAAT CGATTTTAGA 1001 TGAGCATGAA AAATTTGAAA 1051 AAAACACGGC TTTATTGAGT 1101 ACTTATTAAT TGAATATAAA 1151 AATTCTGACC ATTATCTCTT 1201 TTCATTTTTC AAAAGTTCCA 1251 cCATAAAAAT AAAATACCCT 1301 GACTAACATT TTCTCAAAGA 1351 CGAATCATCA TAATCTCTGA

GGTATAAAAT

AACCAAAACG

AAGCAAAATC

AAAAAAATGC

TATTTGAAAA

TCTTTTTTTG

TTTTAAATAA

GAAAAAGTCG

ATTTTGCAAA

TCGCTGTTGT

TAACGTTTTG

CAAGATTACT

GAGCCAAATA

AAAGTTTAAA

ATTCACGATA

TGAAAGAATT

TATCTTAATC

GAAGGCCATT

AAAAATAATC

GTCATTGCAA

TGGAGGTGTC

TGACGATAGT

ACTTGCAAGA

GATATTCTTT

CGTCATAAGA

TCTCAACATG

CACA

GACTGAGAAT

ATTAATAAAA

TCAAAATCGC

GCTACTACTG

ATATTTTCTT

TCAATAAAAT

GAAAATCCCT

AAGGCAGCGA

CTGCCTAGGT

ATCGTTGCAG

TCCAGCGATT

AAATTTTTGT

CCCTCTATCT

GTTTAATTTT

TAAAATTTGT

ATTTATTCAC

GGAGTTTTTG

TATCTCCAAT

ACCTATTCCG

TCCGTGAAGA

TTTAATTTTA

ATAAAAATAA

TCAAGTAGGG

AAAAAGTGAT

GCTCTTCATT

CATCAAATAT

ACAAAGAAAG

ACATGTGAGA

TGTTAGATAT

ATTTTAAAAT

TGAAACAACA

CAGCGATTTT

ATAAGCTATA

TTGTTTTTCC

ACCTAGGCAG

TTTTCGAAAA

AGCGATTTGA

TAGCAACGAT

TTGCCGTTTT

TAATATTTTA

TAGTTTATAA

CGCCATAAAT

ATATGTTGAA

AAGCCATCGT

ATATCCAGAA

AAAAAAAAAC

ACAAATTCCA

AATTCTTTTA

TCAAAATATT

AATACCCTTT

AAAATAAATA

ATTCAAATTT

TATTTGAATA

CAAGTAGGTC

ATTGAAAAAT

GAAGACATTA

GGTCCACTAC

WO 95'31564 WO 9531564PCTIGB95IO 1075 81 1401 TGTA~GAGATG AGAATTTTGA ACCAAATGTA TTATACACTA AGAGTGGTCA 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051

TGATCATTGT

GGCTAAATTA

GACAACATTT

CGCGTGTCTT

TGAATTGGAA

GTCTCTTCAC

TGCTTCGACT

AGAACCAGAA

TGCAAGGACT

GAATATTACA

GTGATAACAA

GACCTCTAAC

TCTTCCCTGA

GTTGATCTTT

ACAATAA.ACT

TTCAGTTTTT

GAGGAGGCAA

TAATTCCTTT

GGTATGGAGT

AATGCTAGTG

CATCCCTCCC TTCTCTTGAA GGTACCATTC CACCTGAGAT

CTTGAACAAC

TAGCCAAGCT

ATTCCTAAAG

TATCAACTTT

ACTTGTCTTT

GAAGAAATAA

TGCTCTTAAT

CTTTTTGTT

ATATGTTACC

TAATGGCTCT

TGTTTCTTTA

TACCTAAGAT

CTCTATTCCT

TTGTTAATAA

AACAACTTGT

TCCTGCTTCA

ATAATCAGCT

TTGTCTAGGT

AGAAATAGGT

CCATTAATGG

TTTTTGTTTC

AATCAGATT

TCAGATCATC

AAATAGGTTA

CTTAGTGGTT

TTTGTATCTT

GTTACCTAAG

GGCTCTATTC

TCTTTATGGA

TAAGATCTCT

ATTCCTGCTT

TGGAAATCAG

CTCTTAATGT

GCTTCATTGG

TCAGCTTTCT

CTATGTTGTA

TTGGGGAATC

TTCTGGCTCT

TGTATCTCTA

TACTTGAGTT

ATTTATTCCT

AACTATTTTG

ACAAAACAAT

TAGCAACCAA

AATTAGTATA

CAAACTATGA

CACTCTTTTC

CTGCCCTCTT

TTGGCTTCAT

TGTATGCTTT

TCATTGGTAC

AATCTTGATC

TGGTAATCTC

CAGGAACAAT

CGCATATTTC

CCTAAGGTCT

CCATTCCTGC

TACAATAATC

ATCTCTTACT

CTGCTTCATT

AATCAGCTTT

TACTTACCTA

CATTGGGGAA

CTTTCTGGCT

CCTAGGTTTG

GGAATCTGAA

GGCTCTATTC

TCTTTACAAT

TGAACAACTT

ATTCCTGCTT

CAATAATCAG

CTCTTACTTA

GCTTCATTG

GCAACTTTGA

CCAAAAC-TTG

ATTAGCAAAT

AGTTACGTAC

TGATGGTTTC

TACCTCTTTA

GAAATGGAAA

GGATTCCAAG

AATGGTAGGG

ACTCTATGCT

TTAGCAAGAA

ACAAATCTTG

ACCACCACAA

ACAATCALATT

CTTACTAAGC

TTCAGTGGGG

AGCTTTCTGG

GAGCTAGATT

GGGGAATATG

CTGGCTCTAT

GATTTGAGTG

TTTGAACAAC

CTATTCCTGA

AGTGAGAATG

AAACTTGTCT

CTGCTTCATT

AACCAGCTTT

GTCTATGTTG

CATTGGGGAA

CTTTCTGGCT

TCTAGATTTG

GCAATATGAG

CTCAGTCCTT

ACTTGAGAAT

TTGGAAAAAA

AATATCCTAT

TAGAAAAGTA

CAGTTGCATT

GCAACTTTCA

TTCTAATGCA

TAAACACGTT

TTTCCATTTT

CAATATCTAXL

TCTATCTTGA

ATCGGTTTAC

AAATGGATTT

TATCTTTGGG

AATCTGAACA

CTCTATTCCT

TGAGTGATAA

AACAACTTGT

TCCTGAAGAA

AGAATGCTCT

TTGTCTTTTT

AGAAATAGGT

CTCTTAATGG

AGGTTGAATC

GGGGAATCTG

CTGGCTCTAT

TATCTTTACA

TCTGAACAAC

CTATTCCTGA

AGTAATAACT

CAACTTGGCT

TTTATGAAAA TCAGCTTGCT AGCTCTGTTC CTGAAGAAAT WO 95/31564 WO 95/ 1564PCT/GB95/O 1075 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901

AGGTTACCTA

ATGGCTCTAT

AATCTTGTTA

CCTAAGGTCT

CTATTCCTGC

GTTAATAATC

ATCTCTTAAT

CTGCTTCATT

AATCAGCTTT

TACTTATCTA

CATTTGGCAA

CTCATTGGGG

GTTGTATATG

GTAATATCAG

AGGTCTCTTA

TCCTGCTTCA

ATAATCAGCT

CTTAATGTCC

TTCATTCGGG

AGCTTTCTGG

GACCTAGGTT

GGGGAATCTG

CTGGCTCTAT

TCTTTGGGTA

TATGAGAAAT

AAATTCCTTC

CCGAGAAACA

TAACCTTCAG

ATGTCCTTGA

TTCGGGAATT

TTCTGGCTCT

TTGATTTGAG

AATTTGAACA

CTCTATTCCT

TGAGTGAGAA

AACAACTTGT

TCCTGAAGAA

ATAACTCTC'T

CTGCAAGCTC

ATCTGTGTGC

ATTTGAAGGG

GTTTTGTCGA

TTCCAATTTA

AGGGAGCAAT.

GATATGCAGA

TGGATGTTCA

GGAGAGCTCC CTTCATCTAT

TTTTGGCAGA

TTAGTAGCCT

AACAATCTGG

CGAGGTTTTT

ATTTTAGCAT

TTTGAGTGAG

TGAACAACTT

ATTCCTGAAG

TGAGAATGCT

ACTTGTCTAG

GAAGAAATAG

TGCTCTTAAT

CTATGTTGTA

ATAGGTTACT

TAATGGACTT

TGATTCTCAA

AATTTGACAT

AAAAGTTCCG

TGTCATCTAA

ACATCACTAC

ACCACAATGT

ACAACAAACT

CTGATAAGTC

GTCTTTGGAC

AACTCAACGA

GTTTTAAGGT

GGCTGAAATC

CATTCTCGCA

AGGACAGTTG

1'GACTCGGTG 1'TTTGTCTTT

CATATTCCTT

ATCTCATAAT

CTATACTGGA

CCACAACAAC

CAATTATCTC

AGAGCAATTC

A.AAGGTTGTG

AATGCTCTTA

GTCTAGGTTG

AAATAGGTTA

CTTAATGGCT

GTTGAATCTT

GTTACCTAAG

GGCTCTATTC

TCTTTACAAT

TGAGTTCTCT

ATTCCTGCTT

TGATAACAP.T

CACTGGP.AGT

CAATGTTTGG

TAGTTTCAGT

AAATACTTGA

TTTGGCAATA

TTCTGGGACT

TCAACTTGCA

A.ATTGCAAAA

CACATTTCCC

TGACATCGAA

ATGTTTCCTG

P.GACTTACCA

ATAAAACAAT

GTAGTTGTGA

GTACACAGTT

CTGTCCTGGG

GCATTGCAAG

DLTCACTAGAC

TTGCTTCTCT

CAAGGATGCA

%TATGAAGGT

3CAAAGATCC 3951 CTTCCAACAA 4001 TGGCAATGAA 4051 AGCTGCAAGT 4101 ATGTGGTTGG 4151 TAAATTGCAT 4201 ATCTTCGAAT 4251 ACGAGTCTAT 4301 GGAGGAACCA 4351 CAAAGGGATT 4401 ATCGATCTTT 4451 AGATCTCATT 4501 GCTATATACC 4551 CTTTCGTTTA 4601 TACGTTTCTT CTAGAGGATG AAATCCCTCG TCTTGATTTA GGAGACAATC

GAACTTTGCC

GGACCTATAA

CATAGATCTC

TTGAACATTT

AGTTATGAAA

GGAGCTTGAA

CAAGCAACAA

GCGATCCGTA

ATCATCACTT

ACCAACTTTC

GAATTCTTAA

ACCTCAATTC

TACGTGGATA

AAAAACTATA

AGAGCTGAGA

GATCATCAAG

TCTCGCAATG

GAAAGGGATG

GCTATTACGA

ATTGTGAGAA

ATTTGAAGGA

TACTTAATGT

GGAAGTTTAT

AGGAGAGATA

ATCTCTCCCA

CGTACCTTTG

TCCAGTTTCA

4651 4701 4751

TCCCTCAAGG

AATGATGGAT

TGTGTCAGAG CAGTGTCTGC GCTAGAAGAT CAAGAAAGCA WO 95/31564PCIB5O17 PCT/GB95/01075 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451

ATTCTGAATT

AGTGGACTGT

AAATCTAAGA

TCATGCP.AAG

AATAATCACT

CTTCAGGTAT

AACAACTAAT

ATACAAATTG

AAATTTATAG

CAAGAATTAG

GCTTATGATT

GGGAAAATGT

TTGAATGATG

TCTA-AGAGAG

ATTACAACCT

AAGGAAAGGC

TAAGGTTGTA

ATAGCAGAGA

AGGTCTTAAA

TCTTATAAGT

CATAAGAAAA

TTGCTGGTAA

GTACCC:AGTG

TTATTAAACA

GACGAGATGA

ACATTGTTTG

GATTGATGCA

CAGGAATCAT

TACTGAGATC

GATCCCCGAA

AGTTACTGAG

AACTTCATAC

GAAACCGAAC

TAACTAGTAT

TTTCAATGAT

GTATTGGCAT

TGGCTTGCAA

GAGAAAGAAG

TCTAGACAAG

TCACGCTAAG

ATATGGTTTT

CTATAATCAC

TTGTGTGACT

AAGACGCTGG

GTTGGATTTG

AATATTTGA

TGTATGCATT

TTAGCGCACG

TGGGCTTGGT

CAAGTTTTAT

GTGGATAAGG

TGTGTTTAPA

TTGAACAGAT

CAACTCTCAA

AACTGCAGTA

CCCCCTAAAC

AAATAATCTA

AAATCCACAC

AAGAAACTCA

TACCTTTTGG

TTACATCGTT

GTTTTGTGTG

AAGGATTTCT

CTGCCAGCAC

ATCAAAGAGC

TAATATGATA

CGCAACATAC

TTTTGCTCGA

TTTTGGAAAG

ATCCATGATA

GAATCATTGA

CAGCGAGGTC

TTACCAATAC

CTCTAACACT

TTTTTATCAA

TTGGAAGCTG

CACTTTCTTA

TGTAAAGGAT

ATTTTTAGTT

ATTTTGATGA

TCTCGGATCA

ATAGAAGATA

TATCTTACAC

TTTTAGATAT

TAACTTCTCC

ATTTCTGTTG

GCACATCTGT

GTTCTATAAA

TACTAAGGCG

AAC-ATACGTT

GGTTTGCATA

ACACTAGCAC

CGCCAAGATG

ACAACCATTT

CTTGGAACAA

TGGTAAAACT

AAGTGCAGCC

GAAAGCACAA

ATGAAAAAAG

CAATTATTTA

TTTATCTATT

G

CAGCTCTGAT

TATATCTTGA

AAAACTGGAA

AAAGAAATTA

AGAAAGATTT

TATCTTTTTT

CAAATACTTA

TGATATATAA

TTTTTCAGAT

TTGCTTCTTC

TTATP.AGGTT

TATATAATAA

ATAATACTCA

GAACATACAA.

CCCAAAGCTT

GGGGAGCCTT

TGTTAATGAA

TATTAGTTTG

TCGTGAAAGA

TATAAGGACT

TTGTTGGATC

ATATTGGTGG

GGTTGCTCTG

PTGAGAGTAA

GACTTTATCA

ATCACTCAAA

PATTATGTAC

CCATAAGGAC

A~ATCTCTTCT

CAACAAAATG

GCACTTCATA

CAGGAAGAAA2 kACGAGCAGT

GGGCTATGGA

TCTCGACTGG

CACAAAATTA

CAGAAGAAGA

GATTTCAGAA

AGTTTATTCT

TTAAGGCTTG

CAAAGCCTAA

TTTCAGGAGC

CTATGTTGCA

TTCTTCAGTT

ATGTTGTGTA

CCTCAAAGAA

AGAAGAATAC

GTTATTATGG

GGCGTGCTGG

TTGAATGATC

TAATATTTGG

GCATGACTAT

CCTAAAGTAG

CTGAAGGGAA

GGGGTAGAAG

CAAACAACAA

kLAAATTTAAT

ACAACAAAT

'AAGATCAAG

%TAAAACTTA

rAGTCCAAGA

=CGTTCATC

rACATGAGCG

TAATATGAT

kGAAGAATAG 3CACTCAAGA WO 95/31564 WO 9531564PCTGB95O 1075 SE.Q ID NO. 6: 1 51 101 151 201 251 301 351 451 501 551 601 651 701 751 801 851 901 951 1001 1051

NMMVSRKVVS

SWIPSSNACK

DLSKNNIYGT

FHNQLNGFIP

NQLSGSIPEE

LSGSIPEEIC

GSIPEEIGYL

I PASLGNLNN

ASLGNLNNLS

FGNMSNLAFL

NLNNLSRLNL

NNLSRLNLVN

LSMLYLYNNQ

ALILNDNNLI

SMSSNSFSGE

QNNXLSGTLP

NQIM~TFPMW

NAFSQDLPTS

RILSLYTVID

LSILESLDLS

FESNSY'EGND

KAALMGYGSG

SLQFFTLFYL

DWYGVVCFNG

IPPEIGNLTN

KE IGYLRSLT

ISYLRSLTEL

YLRSLTYLDL

RSLNVLGLSE

LSMLYLYNNQ

RLYLYNNQLS

FLYENQLASS

VNNQLSGS IP

NQLSGSIPEE

LSGSIPEEIG

GEIPSSVCNL

LPSSISNLTS

TNFSIGCSLI

LGTLPELRVIJ

LFEHLKGMRT

LSSNKFEGHI

FNQLSGEIPQ

GLRGYPVSKG

LCIGISMIYI

FTVAFAS TEE

RVNTLNITNA

LVYLDLNNNQ

KLSLGINFLS

DLSDNALNGS

SENALNGS IP

NALNGSIPAS

LSGSIPASLG

GS IPEEIGYL

VPEEIGYLRS

EEIGYLRSLN

IGYLRSLNDL

YLSSLTYI.SL

TSLEVLYMPR

LQILDFGRNN

SLNILHGNELE

RLTSNKLHGP

VDKTMEEPSY

PSVLGDLIAI

QLASLTFLEF

CGKDPVSEKN

LISTGN

4

RWL

ATALLKWYCAT

SVIGTLYAFP

ISGTIPPQIG

GS IPASVGNL

IPASLGNMN

ASLGNIJNNLS

LGNLIGNhSRL

NLNNLSMLYL

SSLTYLDLSN

LNVLDLSENA

VLDLSENALN

GLSENALNGS

GNNSLNGLIP

NNLKGKVPQC

LEGAIPQCFG

DEIPRSLDNC

IRSSRAEIMF

ESYYDDSVVV

RILNVSHNAL

LNLSHNYLQG

YTVSALEDQE

ARIIEKLEHK

FINQNNSFLA

FSSLPSLENL

LLAKLQI IRI

NNLSFLYLYN

LSFLFLYGNQ

FLFLYGNQLS

NLVNNQLSGS

YNNQLSGS IP NS INGFIPAS

LNGSIPASFG

GS IPASFGN L

IPASLGNLNN

ASFANMhRNLQ

LGNISNJQVL

NISSLEVFDM

KKLQVLDLGD

PDLRIIDLSR

VTKGLELEIV

QGYI PS SLGS

CIPQGPQFRT

SNSEFFNDFW

I IMQRRKKQR 1101 GQRNYRRRNN HF* SEQ ID NO. 7:

MMMVSRKVVS

SWIPSSNACK

DLSKNNIYGT

FHNQLNGFIP

NQLSGSIPEE

LSGSIPEEIC

SLQFFTIJFYL

DWYGVVCFNG

IPPEIGNLTN

KEIGYLRSLT

ISYLRSLTEL

YLRSLTYLDL

FTVAFASTEE

RVNTLNITNA

LVY.EDLNNNQ

KLSLGINFLS

DLSDNALNGS

SENAILNGSIP

ATALLKWKAT

SVIGTLYAFP

ISGTIPPQIG

GS IPASVGNL IPASLGNMU~l

ASLGNINNLS

F1GNQNNSFLA

FSSLPSLENL

LLAKLQIIRI

NNLSFLYLYN

LSFLFLYGNQ

FLFLYGNQLS

WO 95/31564 WO 9531564PCTIGB9SIOI 075 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051

GSIPEEIGYL

I PASLGNLNN

ASLGNLNNLS

FGNMSNLAFL

N'LNNLSRLNIL

N1N1ISRLNLVN

LSMLYLYNNQ

ALILNDNNLI

SMSSNSFSGE

QNNKLSGTLP

NQLNDTFPMW

NAFSQDLPTS

RILSLYTVID

LSILESLDLS

FESNSYEGND

KAALMGYGSG

RSLNVLGLSE NALNGSIPAS

LSMLYLYNJQ

RLYLYNNQLS

FLYENQLASS

VNNQLSGSIP

NQLSGSIPEE

LSGS IPEEIG

GEIPSSVCNL

LPSS ISNLTS TNFS IGCSLI

LGTLPELRVL

LFEHLKGMRT

LSSNYKFEGHI

FNQLSGEIPQ

GLRGYP VS KG

LSGSIPASLG

GSIPEEIGYL

VPEEIGYLRS

EEIGYLRSLN

IGYLRSLNDL

YLSSLTYLSL

TSLEVLYMPR

LQILDFGRNN

SLNIJHGNELE

RLTSNICLHGP

VDKTMEEPSY

PSVIJGDLIAI

QLASLTFLEF

CG1DPVSEKN

LGNLKNLSRL

NLN1NLSMLYL

SSLTYLDLSN

LNVLDLSENA

VLDLSENALN

GLSENALNGS

GNNSLNGLIP

NNLKGKVPQC

LEGAIPQCFG

DEIPRSLDNC

IRSSRAEIMF

ESYYDDSVVV

RIUTVSHNAL

INLSHYLQG

YTVSALEDQE

NLVNNQLSGS

YNNQLSGSIP

NSINGFIPAS

LNGSIPASFG

GSIPASFGNL

IPASLGNLNN

ASFANMRNLQ

LGNISNLQVL

NISSLEVFDM

KKLQVLDLGD

PDLRIIDLSR

VTKGLELEIV

QGYIPSSLGS

CIPQGPQFRT

SNSEFFNDFW

LCIGISIIYI LISTGNIJRWL ARIIEELEHK IIMQRRKXQR 1101 GQRNYRRRNN RF* SEQ ID NO. 8: 1 GGTTTCTAGA AAAGTAGTCT CTTCACTTCA GTTTTTCACT CTTTTCTACC TCTTTACAGT TGCATTTGCT TCGACTGAGG

TGGAAAGCAA

TCCAAGTTCT

GTAGGGTAAA

TATGCTTTTC

CAAGAACAAT

ATCTTGTCTA

CCACAAATCG

TCAATTAAAT

CTAAGCTATC

GTGGGGAATC

TTCTGGCTCT

TAGATTTGAG

CTTTCAAGAA

AATGCAT GCA

CACGTTGAAT

CATTTTCATC

ATCTATGGTA

TCTTGACTTG

GTTTACTAGC

GGATTTATTC

TTTGGGTATC

TGAACAACTT

ATTCCTGAAG

TGATAATGCT

CCAGAATAAT

AGGACTGGTA

ATTACAAATG

CCTCCCTTCT

CCATTCCACC

AACAACAATC

CAAGCTTCAG

CTAAAGAAAT

AACTTTCTTA

GTCTTTTTTG

AAATAAGTTA

CTTAATGGCT

AGGCA.ACTGC

TCCTTTTTGG

TGGAGTTGTA

CTAGTGTCAT

CTTGAAAATC

TGAGATTGGT

AGATTTCAGG

ATCATCCGCA

AGGTTACCTA

GTGGTTCCAT

TATCTTTACA

CCTAAGATCT

CTATTCCTGC

CCTCTTGAAA

CTTCATGGAT

TGCTTTAATG

TGGTACACTC

TTGATCTTAG

AATCTCACAA

AACAATACCA

TATTTCACAA

AGGTCTCTTA

TCCTGCTTCA

ATAATCAGCT

CTTACTGAGC

TTCATTGGGG

701 AATATGAACA ACTTGTCTTT TTTGTTTCTT TATGGAAATC AGCTTTCTGG WO 95/31564 WO 9531564PCTIGB95O 1075 86 751 CTCTATTCCT GAAGAAATAT GTTACCTAAG ATCTCTTACT TACCTAGATT 801 TGAGTGAGAA 851 AACAACTTGT 901 TCCTGAAGAA 951 AGAATGCTCT 1001 TTGTCTAGGT 1051 TTCATTGGGG 1101 AGCTTTCTGG 1151 ATGTTGTATC 1201 GGGGAATCTG 1251 CTGGCTCTAT 1301 GATTTGAGTA 1351 TATGAGCAAC 1401 CTGTTCCTGA 1451 AGTGAGAATG 1501 CAACTTGTCT 1551 CTGAAGAAAT 1601 AATGCTCTTA 1651 GTCTAGGTTG 1701 AAATAGGTTA 1751 CTTAATGGCT 1801 GTTGTATCTT 1851 GTTACTTGAG 1901 GGACTTATTC 1951 TCTCAATGAT 2001 TGACATCACT 2051 GTTCCGCAAT 2101 ATUTAATAGT 2151 CACTACAA.AT

TGCTCTTAAT

CTTTTTTGTT

ATAGGTTACC

TAATGGCTCT

TGAATCTTGT

AATCTGAACA

CTCTATTCCT

TTTACAATAA

AACAACTTGT

TCCTGAAGAA

ATAACTCCAT

TTGGCTTTTT

AGAAATAGGT

CTCTTAATGG

AGGTTGAATC

AGGTTACCTA

ATGGCTCTAT

AATCTTGTTA

CCTAAGATCT

CTATTCCTGC

TACAATAATC

TTCTCTTACT

CTGCTTCATT

AACAATCTCA

GGAAGTGTTG

G=TGGGTA7A

TTCAGTGGAG

ACTTGATTTT

GCAATATTAG

GGGACTCTTC

CTTGCATGGC

GCAAAAAGCT(

TTTCCCATGT

GGCTCTATTC

TCTTTATGGA

TAAGATCTCT

ATTCCTGCTT

TAATAATCAG

ACTTGTCTAT

GCTTCATTGG

TCAGCTTTCT

CTAGGTTGTA

ATAGGTTACT

TAATGGATTT

TGTTTCTTTA

TACCTAAGGT

CTCTATTCCT

TTGTTAATAA

AGGTCTCTTA.

TCCTGCTTCA

ATAATCAGCT

CTTAATGACC

TTCATTGGGG

AGCTTTCTGG

TATCTATCTT

TGGCAATATG

ITGGGGAAAT

rATATGCCGAC

TATCAGTAAC

kiGCTCCCTTC

GGCAGAAACA

TAGCCTCGAG C

CAACAAATTTI

nLATGAACTAG "CAAGTTCTT C 3GTTGGGAAC 'i

CTGCTTCATT

AATCAGCTTT

TAATGTCCTA

CATTGGGGAA

CTTTCTGGCT

GTTGTATCTT

GGAATCTGAA

GGCTCTATTC

TCTCTACAAT

TGAGTTCTCT

ATTCCTGCTT

TGAAAATCAG

CTCTTAATGT

GCTTCATTCG

TCAGCTTTCT

PTGTCCTTGA

ITCGGGAATT

TTCTGGCTCT

TAGGTTTGAG

A.ATCTGAACA

CTCTATTCCTC

TGGGTAATAAC

nLGAAATCTGC I'CCTTCATCT C 'AAACAATTT C 2TTCAGGTTT 9) kTCTATTTCC kTCTGGAGGG ;TTTTTGATA 9 EAGCATTGGA I GGATGAAAT C ATTTAGGAG 'TTGCCAGAG C

GGGGAATTTG

CTGGCTCTAT

GGTTTGAGTG

TCTGAAAAAC

CTATTCCTGC

TACAATAACC

CAACTTGTCT

CTGCTTCATT

AATCAGCTTT

TACTTATCTA

CATTTGGCAA

CTTGCTAGCT

CCTTGATTTG

GGAATTTGAA

GGCTCTATTC

TTTGAGTGAG

TGAACAACTT

!LTTCCTGAAG

E'GAGAATGCT

%CTTGTCTAT

3AAGAAATAG

'TCTCTTAAT

AGCTCTGAT

TGTGCAATT

AAGGGAAAA

'GTCGATGTC

~aTTTAACAT

GCAATACCA

~GCAGAACAA

~GTTCACTGA

~CCTCGGTCT

LCAATCAACT

TGAGAGTTT

2201 2251 2301 2351 2401

CAATGTTTTG

CAAACTTTCT

TAAGTCTCAA

TTGGACAATT

CAACGACACA

WO 95/31564 WO 95/ 1564PCT/GB95/0 1075 2451 TAAGGTTGAC 2501 GAAATCATGT 2551 CTCGCAAGAC 2601 CAGTTGATAA 2651 TCGGTGGTAG 2701 GTCTTTGTAC 2751 TTCCTTCTGT 2801 2851 2901 2951 3001 3051 3101 3151

CATAATGCAT

ACTGGAATCA

AACAACTTGC

TATCTCCAAG

CAATTCATAT

GTTGTGGCAA

GAAGATCAAG

TCTGATGGGC

ATCGAATAAA

TTCCTGATCT

TTACCAACGA

AACAATGGAG

TTGTGACAAA~

ACAGTTATCG

CCTGGGAGAT

TGCAAGGCTA

CTAGACCTTT

TTCTCTTACG

GATGCATCCC

GAAGGTAATG

AGATCCTGTG

AAAGCAATTC

TATGGAAGTG

GACTGGAAAT

AAATTATCAT

AGAAGAAATA

TCAGAACTTC

GGATTTGCTT

GTTTTATAAG

GATATACAAT

TTGCATGGAC

TCGAATCATA

GTCTATTTGA

GAACCAAGTT

GGGATTGGAG

ATCTTTCAAG

CTCATTGCGA

TATACCATCA

CGTTTAACCA

TTTCTTGAAT

TCAAGGACCT

ATGGATTACG

TCAGAGAAAA

TGAATTTTTC

GACTGTGTAT

CTAAGATGGC

GCAAAGGAGA*

ATCGCTTCTA

AGACTTTCAG

CTATAAGATC

GATCTCTCTC

ACATTTGAAA

ATGAAAGCTA

CTTGAAATTG

CAACAAATTT

TCCGTATACT

TCACTTGGAA

ACTTtCAGGA

TCTTAAATCT

CAATTCCGTA

TGGATATCCA

ACTATACAGT

AATGATTTTT

TGGCATATCC

TTGCAAGAAT

AAGAAGCAGC

GACAAGTTAC

GAGCCAAGAA

ATCAAGGGCT

GCAATGCATT

GGGATGAGGA

TTACGATGAC

TGAGAATTTT

GAAGGACATA

TAATGTATCT

GTTTATCTAT

GAGATACCAC

CTCCCACAAT

CCTTTGAGAG

GTTTCAAAAG

GTCTGCGCTA

GGAAAGCAGC

ATAATATATA

CATTGAAGAA

GAGGTCAAAG

CAATACCGAA

TAAGAAGACG

GATGTTGGAT

TGTAATATTA

3201 TCTTGATCTC 3251 CTGGAACACA 3301 AAATTACAGA 3351 AGATTTGATT 3401 CTGGTGTAAA 3451 TAGATTTTTA 3501 TGAATTTGAT CTTCCTGTGT TGCAGCTTAT CTTTTCTTCA GTTGGGAAAA AAATGTTGTG TTTATTGAAA 3551 AAAAAAAAAA AAAAAAAAAA AAA SEQ ID NTO. 9: 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 tatatatctt gtgagagatg cgagtcaagg acacaaaaag ctcgttgata ggatgctctc accaaatgct gctgacgcac aaaagagtaa gagagttaat attcttagtg ctcataatcg gaaacaagac tgtcttgtac atctgtgtag gaaatattat gacaaccaca aataatgtaa aaatcaggta tacttgaagg cagagagttt gatgtttgat aagcacgagt ccttaaacag taagcgaaac tttagcaaac ttaatttagc ttaatttagt atatacttat tttgatatta aaggacaact aggaaacgaa ttctgtcttg aaaattcaat attgatgaca gagttttgtg ggatggagtt ctagacgcaa agttaacccg tcttctatcc cagcaaatgc tttaaactaa tagagagtta gaactaatta attttcactt tctcctaatc aacaatcata gacacccaca tgtaagtttc tacaaagact ctctaaaaat aagtgattaa ttgttgtttc gagaaaatgg ttccacggcc aggcgacgaa agtacatgaa aatcgcagcc ttatacaatt attttcactt tattttcact atattatttg catgtgcatg agtacattct aaatatgtqt tgtctaattg aagacttatc atctttgtat atagatzgatc aggaattata ggcgaacgca gcttcttgaa tcagcgcaga a ccct tagaa tacagttgtt cttaaaaact tagcaaacta ttagtataca aattaaaatc tatgtattgg tacgataaaa gtttcaaaat cctagaactt ataattaagt gtagtgtaaa PCTIGB95O 1075 WO 95/31564 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 41S1 aaagctttcg tgtcttgctg gctctatcat

CGACGTTTTT

GCCATCTTCT

CATGGAATCG

TAGAGAAGCT

CAACGTGGTG

TTAGGTCAAA

TAGAAAACAG

TCAGCAAGGA

CTCACAGATA

tgttatgatt atttttgcat aaattgactt

ATCCAATGAT

ACTTCGTCGG

TTGGAATCCA

TGGTAAGAGT

TCCACCATCG

TCTGGC-ATGA

TCAAAAGGAC

ATCACAAGAA

CTTAAGACCT

AATTGTGATC

TTGTATATGA

TCCCAATATG

AGCATTTGAA

TCTTGGGTTC

ATGCCTAGGC

AGTCGGCTAC

TTGCATGTTT

GAAGATGATG

TACACCGGGT

GAGAAATTGA

CTGACGAATT

tttttcgcat tcgtaatttg taaaacgtag agattatatt

TTGGAATACG

ATAGATGAAA

TGGTTATTGG

GTAAACTCAA

TCTAATTTTA

GCTTGAGAAG

atttctaaac gtgtgtgctc

AGAAGATGGA

TCTTTAGCCA

GGAGACACTT

ATTGTTGGGG

AATCTCGAAT

GGGCATCGTT

GTCCATTGAA

CTCATAATGG

actaattcta tgtttaaaat t cagTCACTT

TAAAAGAALAT

CTTTTTGGAT

CACTAAACTG

TTCCAACCGT

TGCCCGAATT

TAGATTATCT

TAGAAGATTG

GACTGCCTTA

aggaaagtaa atttacttct ctcccATGGC

CCAAGCTTCA

CAAGGCTCTC

AGAGAAGCCG

AGGATCTCAA

CTTAAATGAA

TGGTGATTCC

ACCGGCGAAT

CAAACAACCA

TAG CAAATA7 ccaatatatc agacttcggt ttgttattag GTTTC.jAATA AAL I'GAAGCT

AGGAAGCTAG

ACCATCGGAA

CGCTTTCCTA

AGTTGAGTTG

ATAAAGATAG

AGTTCTTATC

TGGTGGGAAA

ACTCAAGATA

GGTGAAGCTG

CTTTTGGGAA

GTTGCCGAGC

ATCTTTAAAA

TTCGAAATGA

GATAGGTTAA

TTTCAATGGT

TTGGGCTTAC

GGATATATAG

TCGTGCAAAG

TTGAGGATAT

ctccttaaac gggattgata ctttgatgtg agttttcttc

TTTGCCACAC

AATCATTCAA

TCAGATGGGG

PAAGGCTATGG

AGGCTGAGTA

CTGTGGGATG

ataaaaacta tcttttcccc

TTTGTATAAT

TAAACCTCGA

CCTTCCTCGA

GGGGCTATTA

ATCTATCAGT

TATTTCCCTC

GCGTTTGCCT

AGTACAGTGA

t tagtgataa gttcattaac

GGAAGTCTCA

TCCAGATCTT

GCGTATCTTT

ATCTATTTAG

TTTCAACTTG

TGAGAAATTT

CGAACAAGAT

TTTCTGGAAC

TGAGATGTAT

gacgaagttt ctcctctctt tctcacacta cttaaaaatc ttcgtctctt ctctgagttc GGCTTCTTCT TCTTCTGGCA GACGGAGATA GTGGGGTTGA TGTTCGCA.AG ACGTTCCTCA GACGGCAAAT CAATCAATAC ATTCATCGAT CACAATCGCC CCTGAGCTTA TATCGGCGAT TCGTCATCTT CTCTAAGAAC TATGCTTCTT TTGGTTGAGA TCCACAAGTG CTTTAATGAT AGTTTTCTAC GACGTTGATC CTTCGGAAGT TTGGAAAGGT CTTTGAAAAG ACATGCGAGG GGGGATCAGA AACAAAGATG GGTGCAAGCT AGCCGGAGAG GATCTTCTGA ACGGgtacgt tgcttgcgtt ttcaattgtc tcagaactat tcttctttta ggggtgcttc ttaattgaca GCCTAATGAA GCGCATATGG TTGAAAAGAT AACTTATCAC TCGGTCAAAG TGTTTTGATG CATATTGAGG CAATAAAATC AGTATTGTGC AATGGTCGGG ATTTGGGGAC AGTCAGGGAT GAGCTCTTTT CAGTCAACTC TCTAGCCAGT ACTTATAAAA GCACCAGTGG TAGTGACGTC GCAAAAAGAG CTTCTCTCGG AAATCTTAGG AGCATTTTGG TGTGGTGGAG CAAAGGTTAA CTTCTTGATG ATGTGGATAA TCTAGAGTTT AGCTGAATGG TTTGGATCTG GAAGCAGAAT GGCAACTTCT CAAGGCTCAT GAGATTGACC CCATCTCAAG GTCTTGCTCT 'TAAGATGATA AGACTCTCCA CCTGATGATT TTAAGGAACT TTGTCGGTAG TCTTCCTTTG GGTCTCAGTG GGAAGGGACA AAGATGAGTG GGTGAAGATG TTCAGATGAT AAAATTGAGG AAACACTAAG ATAAAAAAAA TAGAGAGTTA TTTAAGTGCA TTTAAAGTCA GTAACGTCAA AGAATTACTT AATGTTGGCT*GAGAAGTCCC TCATACGTAT AGATGCACAA TTTGCTAGAG AAATTGGGTA TCCAAGGGTA.ATCCTGGAAA. ACGTCAATTT TCGAGAAGTA TTGACCGAGA AAACTgtaag gttgtaatgc atgactttat atcaatataa aacttaagca attgttgccc catgcgtaat tcagaaaaat aaaaagggtt gcgattgtta ggattttttt tcagGGGACC GAAACTCTTC CCGGGATATC TTACGACAAG GTCGTTCTTA AGGCATGCGT AA.TCTCCAAT ATCTAGAAAT TTCTACCTCA GAGCCTCGTT TATTTCCCTC TGGGATAATT GTCCATTGAA GCGTTTGCCT TCTGGTTGAA CTCAGAATGG TGAATAGTAA GAACTCAGGT actaattttt ttagtgatca aaaataaaaa tgtttaaaat gttcattaac tattttgttt tcagCCCCTT GGAAGTCTCA TCCTACAAAT TGAAAGAAAT TCCAGATCTT GGAATTAAAT CTTGAAGAAT GCGAATCTTT TTCAGAATGC CATTAAACTG AGGGAGTTAA ATAGATTTAA AATCATTAGA AGGCATGTGT TCCTAGTTGG TCAAGTAGGG AATGCACTCA GTAAACTCAA AAGTGTATTG TGGACTAATT TCTAATTTTA AGGCTGAGTA TCTGGTTGAA GCTTGAGAAG CTGTGGGATG GTACTCAGgt taaatatgtt agaaaaacta aaaataaaaa gtgtgtgctc tcttttcccc tattttgtta AGGAGATGAA TTTGAGGTAT TCCAACAATT TCTTTAGCCA TAAACCTCGA GGAATTAGAT GGTGACACTT CCTTCCTCGA TTCAGAATGC ATATGAGTGA ATGCGAAA.AT CTAGAGAGTT AAATCTCTCG AGTACCTCGA TCTCACTGGA CCCAGCAATC AAAATGGGAT GTGCCTGGAC TGTTTCCGGA AGGGAGAAAT GAGATCGTGG AAGAATCTCC CTGCTGGACT AGATTATCTC GCCTTGTGAA TTTCGCTCAG AACAACTCAC WO 95/31564 WO 9531564PCTGB95O 1075 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951

TTTTCTCAAT

AGgtacattg ctaattaagt ttatgtctta tataggtata aatgtttttt

TCTGAAA.ACC

GCTTTTATGT

TTGGGAATCT

CTGGAGGTTC

TCTCAGTGGT

TTGTATGTCT

TCAAAGGCCA

GGTGACACTT

ACATGAACAG

TCATCTCTCG

TCCTCTGATT

TTGAAGAAGT

CGGATGTATT

ACTGACTAGT

AGGCGTTGAG

TGTGTACCAT

TGCGTGTTCT

TTAGATTGTC

TCCATTACGA

AACAGAGCAC

GCCTTTTGCC

atttgtttta cgatcgtttg atcaacttac aaatacagag tagaggggga gatgaacaaa tatcattctg ataaatttat acacaagcca

GTGAGCGGCT

ttaatgctat atacccaaat catacataca aaattaaaat tcagTCGCTT

TGAAAGAACT

CTC-AGCGGGT

TCAAAATTTG

TTCCGACCGA

TGCTCAAGTT

CTATCTGGAA

CCAAiGCTCGA

CCTTCTACAA

ATGCACAGGG

AAACCCTCGA

TCAACTAGAA

TCCCTGCTGC

GTTGCCAGAG

CTTACGCTCG

TGATGCAACT

TATCTGAAAA

GATTATTACT

AACGATGACT

TCAAAGAATG

AACCAACAAA

Ataattagag taggatcaaa atatataatg c ctatggcca ctttcgttag agctttgtgt tacctatctt ttttgctctt aatgataatg ttttttctgc

GCAAGCTTGA

gctgattttt ttgtttttat taataatgtt gattatcatc

GGAAGTCTCG

TCCAGATCTT

GCAAAAGTTT

AGACGTTTGT

TGTCAACTTG

TGAGAACTTT

AACACCGCCA

GTCTTTGATA

TTGGGAATCT

CTGGAGCTTC

TCTCAGTGGT

TCGAATGTCT

ATTGAGGATT

GTTGAAAAAC

CCGACTTTAC

GTGGTAGCGA

CATTGAATAT

CTGATGACTT

GTCAACGATG

CGGTGTACGA

CTACGAGAAG

ctgaaacttg ataccatagc cagatgacat aattgtagcq ggcagggaga gttgatgatt gtatcctgtt ttttaggata acaaaacgat.

agatatagac

GAAGCTATGG

gtttaccttc ggcttgtggt taattataat gataatgat t

AAGAGATGGA

TCAAAGGCCA

GGTGACACTT

ACATGAACAG

TCATCTCTCG

TCCTCTGATT

TTGAAGAAAT

CTCAACAACT

TCAAAATTTG

TTCCGACCGA

TGCTCAAGTT

CTATCTAGAA

TCACGAGGCT

ATCTCCCCAA

AGACTGTAGA

CAATGGAAGA

ACATGTGAAC

TGAGGTAAAT

TGGAGTTTAA

CTCTTGTATG

CAAGAAGCGG

taaagcaatc gacagactat cggggacatc gacacaggat agcatcatca acatgataaa tatggtaa ct acttgggatc ttcataggtt gatgatatgt

GAAGGCATCC

tgttatataa cgatccacgg tttaaacata gaagcatacc

TCTGTCAGAA

CCAATCTGAA

CCTTCTACAA

ATGCACAGGG

AAACCCTCGA

TCAACTAATA

TCCAGATCTT

GCAAAAGTTT

AGACGTTTGT

TGTCAACTTG

TGAGAACTTT

AACACCGCCA

CACTGTACTA

ACATTTTCAG

GGTGTCATCA

TCACGTTTCT

GTTTCTGGGA

CGGAACCCAA

dTTTTGTTGC

TCTATCAAGA

ATGCGGGTAA

ttttgacttg ttgatagaat tgaagaagat tggccgctct tcaacatctc tgaagaacaa gaagcatctt gaccattatt ttgacttttg ggagatcatt SEQ ID NO. 1 MAASSSSGRR RYDVFPSFSG VDVRKTFLSF{ LLKALDGKSI NTFIDHGIER 51 SRTIAPELIS AIREAP.ISIV IFSKNYASST WCLNELVEIH KCFNDLGQMV 101 IPVFYDVDPS EVRKQTGEFG KVFEKTCEVS KDKQPGDQKQ RWVQALTDIA 151 NIAGEDLLNG PNEAHMVEKI SNDVSNKLIT RSKCFDDFVG IEAI{IEAIKS 201 VLCLESKEAR MIGIWGQSGI GKSTIGRALF SQLSSQFHHR AFLTYKSTSG 251 SDVSGMKLSW QKELLSEILG QIGJIKIEHFG VVEQRLNH-KK VLILLlDVDN 301 LEFLKTLVGK AEWFGSGSRI IVITQDRQLL KAI{EIDLVYE VKLPSQGLAL 351 KMISQYAFGK DSPPDDFKEL AFEVAELVGS LPLGLSVLGS SLKGRDKDEW 401 VKMMPRLRND SDDKIEETLR VGYDRLNKIGN RELFKCIACF FNGFKVSNVK 451 ELLEDDVGLT MLAEKSLIRI TPGGYIEMHN LLEKLGREID RAKSKGNPGK 501 RQFLTNFEDI REVLTEKTGT ETLLGIRLPH PGYLTTRSFL IDEKSFKGMR 551 NLQYLE IGYW SDGVLPQSLV YFPRKLKRLW WDNCPLKRLP SNFKAEYLVE 601 LRMVNSKLEK LWDGTQPLGS LKKMDLYNSY K1LKEIPDLSL AINLEELNLE 651 ECES'L'.ETLPS SIQNAIKLRE INCWGGLLID LKSLEGMCNL EYLSVPSWSS PCT/GB95/01075 WO 95/3 1564 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251

RECTQGIVYF

DGTQSLGSLK

QNATKLIYLD

AWTRLSRTRL

QLTFLNVSGC

LCLSGCKSLV

SGCSSLRTFP

TLPSTIGNLQ

LISTRIECLY

TSLTLAIJFTD

CSDYYSDDFE

EHNQQTTRSK

PRKLKSVLWT

EMNLRYSNNL

MSECENLESF

FPEGRNEIVV

KLEKLWEGIQ

TLPSTIGNIQ

LISTNIVCLY

NLRRLYMNRC

LENTAIEEVP

CRGVIKALSD

VNRNPIRLST

KRMRVSLLP

NCPLKRLPSN

KEIPDLSLAI

PTVFNLKSLE

EDCFWNKNLP

SLGSLEEMDL

NLRRLYMNRC

LENTAIEEIP

TGLELLPTDV

CCIEDFTRLT

ATVVATMEDH

MTVNDV'EFKF

FKAEYLVELI

NIJEELDLFGC

YLDLTGCPNL

AGLDYLDCLM

SESENLKELP

TGLEVLPTDV

DLSKATKLES

NLSSLETLDL

VLRMYCCQRL

VSCVPLSENI

CCSITIKECG

MEYSELEKLW

VSLVTLPSSI

RNFPAIKMGC

RCMPCEFRSE

DLSKATNLKL

NLSSLETLDL

LILLNNCKSLV

SGCSSLRTFP

KNISPNIFRJ

EYTCERFWDA

VRLLYVYQET

SEQ ID No.11: 1 GACCAAACTG GACTCCTGCT CCGTCTTCCA TCAGC-AGGTC AATTCTCGTG 51 GAAAATTAGC 101 TTATTGCGT 151 CACACAAAGC 201 ATGGGTTGTG 251 ACTTGTTTCC 301 TTGCTCTTCT 351 GATTATTGTT 401 CTGCTCATGG 451 AGCTTGACCT 501 AGCCTCTTTC 551 TTTCACTGGA 601 CGCATCTCGA 651 ATCTCTCATC 701 TACTTTTGGT 751 TAAAAGTGCT 801 AATTTCTCTT 851 TGGGATATTG

TCGAGGTGGC

AATTTGTGCT

AAACATCTCT

TAAAACTTGT

TCGTCATCCT

AGAPATTCAAG

ACGACAGAAG

GATGGCGTTC

CCGTTGCATC

AACTCTCCAA

TCGCCCATTT

TTTGTCGCAT

TTTCTAAACT

CCTCACAATT

CGACCTTGAA

CTCATTTAAC

CCCGAAAGAG

CAATCCCCAG

CATCACTCAT

GCACTATGTG

ATATATACCT

TAATTAGTTT

GTTTTTCATG

TACCTCATTT

AACATGTTTA

AACTCTTTCT

ATTGTGACGA

CAACTTCAAG

TCTCAAAAGG

CACCTAAATT

TCAAGTTTTA

ATACGTTCTT

TTGAATTGCT

TCTATCAACA

AAATCTATGG

TTTTCCACCT

CTCACGGTTA

GAAGTTATAT

AGGTAGCTAG

CATCTAAATT

TGATCATTTT

CTATATGTCT

GTGCCCCGAA

CCGTTAATCC

TGGAACAAAA

AACGACAGGA

GCAAGTTTCA

CTTGATTTGT

TGGTGAGTTT

GGGGTGTAAT

CGTATTAGTC

TCTTAAGAAC

TCTCTTCCAC

CTTCCATACA

TTCCGACTTA

GGTTTCCCAC

CTrCTATAATG

TACTAAATGT

ATTGAATAGA

TAGTGCAGAA

TTCTCTTTCA

GATCAAGCTC

TAATGCTTCT

GCACAAGTTG

CAAGTGATTG

TTCCAATAGT

CTTATAATGA

TCAGATTTGA

CCCTTCTGAA

TAAATGAGCT

TTGACCCAAT

TATTCCTTTG

CAGAGTTACG

GAATTTCTCG

AACCAAATGG

TGAATATTGA

ATTTATCAAG

AATAGCAGTG

WO 95/31564 PCT/GB95/01 075 1001 TGATAGGATA CCTGAATCAT TTAGCCATCT 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601

ACATGAGTCG

CTCACCAACA

AATTCCATCC

CATCAAACAA

TCACTGATAG

AGAGTTCAAG

TAAAAGGTCC

CTTCTCCTTT

CAATCTGAAA

GAACAATCCC

GATTTGAGCA

TGGAAACATT

AAGTCCCACG

GGTAACAATA

TCAATTGAAG

AATCTTCAGG

TCATCTAATG

GCAAACCATG

CTGATCCATA

GGACAAGATT

TCTCTCAAAG

TTGTTGGACT

ATACCGGCA1%T

ATCTAATAAA

TCCTTGAAGT

AAAGGAAAAC

TGGGTTACGC

TGACAACTCC

ATGATCAGTT

TGGACTGTCC

TTTCGAGGAT

AAGCACAAGA

TTCTAATCTG

TAGTGTTTTT

AACGTAAGCG

CTTAAATGGG

GGTTAGACTT

TCCAAAACAT

TATTCCGAAT

CACACAATAA

ACATTGATAT

GCAATGCGTG

ACAACAGACT

TTAAGGGTCA

ATCTATGATC

TGTTGAATGA

ATTTTAAGCT

GAATACAAAC

GATTTAGTGG

AAGGAAATTG

TGATATTTAT

ATGATTCTGT

AACAGATTTG

TCGTACGTTG

CATTTCAAAA

ATCAGCGGAG

CTTAAATCTC

AATTTGATTC

GGATTTCCAC

AGCTGAGCTA

GGCAGGGGGT

GTAATATACA

GGATTTAAAG

AAAGATATTA

TCAGGGCCTA

GGACCTTAAT

GACTACGTAA

AGTATACCAT

GAGCAATAAC

TAAG'2ACCGT

TCACTCCTAA

TATCAGTGGA

TGTTAGACTT

GTTGAGAGGA

TAGTGGGACA

TTAGCTTGCA

AATTGCAAGT

CACATTTCCA

TGAGATCAAA

TTGTTTATGG

GAATTTACCC

ATGAGAGTAC

TACAATTATT

TCGAATTTTG

AAGGTCATAT

AACTTGTCTC

TTTATCAGTA

AAATTCCGCA

TCTCACAATC

GTTCGGGAAC

TCTCAAAACT

GATCAAGAAG

TCTCGTGGGT

TAATGTGGTC

TTGGAACACA

GTGAGTAGCT

AACTTCACTT

TTCCTATAACC

AATAACCATC

CCTACAAATA

CCTGGATATT

ACTTTCAGTG

TACTCTAAAP.

ACCAGAAGAA

CATATTTCTT

GGGAAGTAAT

ACGAATACCT

ATCAATACAA

CGGGAATAAG

ATTTGACACT

AACTGGTTGG

TAAGTTGCAT

GTCTTCAAAT

GAAAGAATTT

AGGATTCCCA

TGACGACAAT

GATTCTAACA

TCCAAGCATT

ACAATGTCTT

CTCGAATCAT

GCAGCTTGCA

ATCTTGTTGG

ACTTCGTACC

TTGTGGTGGT

AGGAGGAAGA

TACGGTTGTG

AACTCAATAT

TAATTACTAC

ATACCTCCAG

CATAAGTTGT

TCTATGGAAT

TTGAAGGACC

CTTTGGTT43T

CTCCCTTCCA

GAAAAATTCA

CAAAATAAGC

CCTACAATTC

CAGCTATCTG

AATTTGGAGG

TTCGCATTTG

CTTTTAGTGT

CTAACGGGGA

ACTTGATCTA

GATACC1'ATT

GGTCCCATCA

TCTTGATCTA

TGGGGAATTT

GAGTATATTT

TTCTACAAAG

TGATTATCAA

ATTGGAGATC

GGAAGGTCAT

TGGATCTCTC

TCCCTCACAT

ATGCATCCCC

AAGGGAATGA

GAAGATCAAG

AGATTCACCA

GACTTGTTAT

CCAGCATGGT

GAAAATGAAA

GTATTCCACT

2651 TGATCATTAT CTTTCAGAAG ATTATTTTTT GTATATCGAT GAAATTATCG WO 95/31564 PCT/GB95/0 1075 2701 2751 2801 2851 2901

ACCTCCTTCA

TTCAGGATTC

AGCCTTTTTA

TCATTTGTCT

TCTCTCAACT

TCCTCAAAGC

AAAGATTTCC

TCTTTCATAG

ATGGCACGTA

CTTTCGTCAC

GTCTGCTATT

AAGAGACTAT

TCTTAACTTT

GAGTTCCCAG

TTTCTTATCC

GATATGTTCC

ATGATATCAA

TTAATTTCTT

GATCTCAGAA

CACTCTTCAT

TTGCTTGGGA

TATGAATAAA

GTCACTAAAA

AGAACACTTG

CCATTGAAAC

ATGGGAATCT

TTTTGAAAAT

TGCAGATAAA

GATTTTATTT

ACATTGTAT

ACTTCAATTA

ACAACTGACG

CCCAATCCAA

2951 AGTTACTGTA 3001 TATCTTGAGA SEQ ID No. 12:

MGCVKLVFFM

DYCYDRRTLS

SLFQLSNLKR.

ISHLSKLYVL

NFSSHLTNJW

LYVFLFQLVS

WNKSTSCCSW

LDLSYNDFTG

RISLNELTFG

LPYTELRGIL

SSSLPHLCPE

DGVHCDETTG

S PIS PKFGEF

PHFELLLKN

PERVFHLSDL

DQALALLEFK

QVIELDLRCI

S DLTHLDLS H

LTQLKVLDLE

EFLjDLSSNPQ

NMFTVNPNAS

QLQGKFHSNS

SSFRGVIPSE

SINISSTIPL

LTVRFPTTKW

251 NSSA.SLMKLY LYNVXIDDRT PESFSHLTSL HKLYMSRSNL SGPTPKPLWN

LTNIVFLDLN

SLIGLDLSNN

LLLSHNlNISG

DLSNNRLSGT

GNNMLNDTFP

S SNFS GULP

GQDYDSVRIL

IPASFQNLSV

KGKQFDSFGN

MI SWQGVLVG

KHKKRY

NNHIJEGPIPS

TFSGKIQEFK

HISSAICNLK

INTTFSVGNI

NWLGYLFQLK

ERILGNLQTM

DSNMI INTSK

LESLDLSSNK

TSYQGNDGLR

YGCGLVIGI

4

S

NVSGLP.NLQI

SKTLSTVTLK

TLILLDLGSN

LRVISLHGNX

ILSLRSNKLH

KEIDESTGFP

NRFEGHI PSI

ISGEIPQQLA

GFPLSKLCGG

VIYIMWSTQY

LWLSSNNLNG SIPSWIFSLP QNKLKGP IPN SLLNQKNLQF NLEGTIPQCV VERINEYLSHL LTGKVPRSMI NCKYLTLLDL GPIKSSGNTN LFMGLQILflL EYISDPYDIY YNYLTTISTK IGDLVGLRTL NLSHNVLEGH SLTFLEVLNL SHflNHLVGCIP EDQVTTPAEL DQEEEEEDSP PAWFSRMDLK LEHIITTKNI( WO 95/31564 PCT/GB95/01075 93 ATCG TGGGATW--GTTC-TCTTTTCACAAkTTGCCTTCATTTCTTCTTGTCTCTAkCACTTCT 1 -4

TA~TCCAAAGGAATTACGATAGAACGGTTAG

M G F V L F S QL P S F L L V S T L L CTTATTCCTAGTAATATCCCACTCTTGrCCGTGC4AAGCCCCCAAAACTCA ACCATACAA 120 GAATA7AGGATCATTATAGGGTGAGAACGGCACG TCGGGGGTTTTGAGTTGGTATGTT L F LV I S H S C R A K A P K T Q P Y N

CCAGAGCCAAGCTGCCCATTTGTCAGATTTT

121 180

GGTXGTGGTCTATGTTGTCCLACAGTCTAAAA

P C K P Q E VI D T X C M G P K D C L Y

CCCGAACCCCGACAGTTGTACAACCTACATACAGTGTGTACCGCTCGCGAGTTGGCAA

181 240 GGC TTGGGGCTGTCAACATGTTGGATGTATGTCACACATGACTGCTTACCGTT P N P D S C T T Y I Q C V P L D E V G N TGGACTTGTACAGCAMGCGAT-AC TAGTG- 300

ACGCTTCGGACACCATTCGGTACAGTTTCCTGACGTCACCTTGCTATTCACCGTT

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

GAGGTCATTCACTATCGGCGTAGCCGACGAC

360.

CTTCACCACGCTGATAGGTTTGGACTCATGCACAGGCCATTTCTGCGGCGTTGGCTTCGG

K WC D Y P NWL S T C P V KT P Q P K P

GAAGAGGGAGGTGTCGGAGGGAAGAAGGCGTCGGTTGGACATCCTGGCTAGAGTCGG

2

CTTCTTCCCTCCACAGCCTCCCTTCTTCCGCACCCA.CCTGTAGGACCGATAALCTCAGCC

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

ACAAGAAAGGGGATG-GCTCTAACAGTTCTCGTACCAGAGCTATCGTGCTAGGGGATCCGT

421 480

TGTTCTTTCCCCTACCGACATTGTCAGCCATGGTCTCGATAGACGATCCCCTAGC

CGAC

481

GCTG

Claims (43)

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 compri. ;s 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: inactivating a nucleotide sequence which contributes to necrosis or inactivating one or more nucleotide sequences forming part of a combination of WO 95/31564 PCT/GB95/01075 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. 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. 96

6. A method according to claim 5 wh Ain 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

7. A method according to claim 6 wherein said nucleotide sequence comprises a plant pathogen resistance gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides, or a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides, or another R gene elicitor or both an R gene or a said mutant, variant, or derivative thereof and (ii) a corresponding Avr gene, or a said mutant, variant or derivative thereof, or another R gene elicitor

8. A method according to claim 7 wherein said plant pathogen resistance gene is a tomato Cf-9 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-9 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or AMENDED SHEET more nucleotides or a homologue thereof, or encodes another Cf-9 elicitor.

9. A method according to claim 7 wherein said plant pathogen resistance gene is a tomato Cf-2 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-2 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof, or encodes another Cf-2 elictor; or wherein said plant pathogene resistance gene is a tomato Cf-4 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the avirulence gene is a Cladosporium fulvum Avr-4 gene or a mutant, vari nt or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof, or encodes another Cf-4 elictor; or wherein said plant pathogen resistance gene is the tobacco N' gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof, and the avirulence gene is a suitable Tobacco Mosaic Virus coat protein, or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof or encodes another N' elicitor; or wherein said plant pathogen resistance gene is the potato Rx gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the avirulence gene is a suitable PVX coat protein or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides 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. 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 or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides ora homologue of any of these, or other toxic polypeptide or peptide such as diphtheria toxin or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof. 99

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. 100 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) 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, the phenotypic variegation resulting from expression in cells with the first phenotype from a previously inactivated nucleotide sequence or sequences restored to a functional form and which contribute to such phenotype, said nucleotide sequence or sequences being in a non-functional form in cells not having said first phenotype. 102

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 said non-functional form results from insertion of a transposable genetic element into said nucleotide sequence or one or more of said nucleotide sequences.

25. A plant, descendant, derivative, part or propagule according to any one of claims 22 to 24, wherein said nucleotide sequence or sequences comprises: a gene which is a plant pathogen resistance gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides; or a gene (L) which is a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides, 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.

26. A plant, descendant, derivative, part or propagule according to claim 25 wherein the R gene is a tomato Cf-9 gene or a mutant, variant or derivative 103 thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the L gene is a Cladosporium fulvum Avr-9 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof, or encodes another Cf-9 elicitor.

27. A plant, descendant, derivative, part or propagule according to claim 25 wherein said R gene is: a tomato pathogen resistance gene; (ii) a tobacco pathogen resistance gene; (iii) a potato pathogen resistance gene; (iv) a Arabidopsis pathogen resistance gene; a flax pathogen resistance gene; (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; a nucleotide sequence encoding a toxic peptide; (xi) a mutant, variant or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or homologue of any of to

28. A plant, descendant, derivative, part or I 104 propagule according to claim 27 wherein said tomato pathogen resistance gene is selected from Cladosporium fulvum resistance genes including Cf-2, Cf-4,. Cf-5 and Cf-9; said tobacco pathogen resistance gene is said potato pathogen resistance gene is Nx; said Arabidopsis pathogen resistance gene is RPP5 or RP52; said flax pathogen resistance gene is L6; said RNase is Barnase; or said toxic peptide is diphtheria toxin.

29. A plant, descendant, derivative, part or propagule according to claim 25 wherein said L gene is: 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 or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or homologue of any of to (iii). A plant, descendant, derivative, part or propagule according to claim 29 wherein said Cladosporium fulvum avirulence gene is Avr2, Avr4, or Avr9. 105

31. A cell containing 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 in a non-functional form and (ii) nucleic acid encoding a molecule or molecules able to restore said nucleotide sequence or sequences to a functional form.

32. A cell according to claim 31 wherein said non- functional form results from insertion of a transposable genetic element into one or more of said nucleotide sequences.

33. A cell according to claim 32 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.

34. A cell according to any one of claims 31 to 33 wherein said nucleotide sequence or sequences comprises: a gene which is a plant pathogen resistance gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides; or a gene (L) which is a pathogen avirulence gene (Avr) or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more 106 nuclectides, 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. A cell according to claim 34 wherein the R gene is a tomato Cf-9 gene or a mutant, variant or derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue thereof and the L gene is a Cladosporium fulvum Avr-9 gene or a mutant, variant or derivati 'e thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or homologue thereof, or encodes another Cf-9 elicitor.

36. is: A cell according to claim 35 wherein said R gene a tomato pathogen resistance gene; (ii) a tobacco pathogen resistance gene; (iii) a potato pathogen resistance gene; (iv) a Arabidopsis pathogen resistance gene; a flax pathogen resistance gene; (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; 107 a nucleotide sequence encoding a toxic peptide; (xi) a mutant, variant, derivative or homologue of any of to

37. A cell according to claim 36 wherein said tomato pathogen resistance gene is selected from Cladosporium fulvum resistance genes including Cf-2, Cf-4, Cf-5 and Cf-9; said tobacco pathogen resistance gene is said potato pathogen resistance gene is Nx; said Arabidopsis pathogen resistance gene is RPP5 or RP52; said flax pathogen resistance gene is L6; said RNase is Barnase; or said toxic peptide is diphtheria toxin.

38. A cell according to claim 34 wherein said L gene is: 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 or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue of any of to (iii).

39. A cell according to claim 38 wherein said 108 Cladosporium fulvum avirulence gene is Avr2, Avr4, or Avr9. A cell according to any one of claims 31 to 39 which is a microbial cell.

41. A cell according to any one of claims 31 to 39 which is a plant cell.

42. A plant or any part or propagule or derivative thereof comprising a cell according to claim 41.

43. A plant, part, propagule or derivative according to claim 42 which is variegated for cells wherein said nucleotide sequence is inactivated or activated. 1 i A method of producing a cell according to any one of claims 31 to 43 comprising introduction of nucleic acid and/or (ii) into the cell or an ancestor thereof. A composition comprising any of the following combinations of nucleotide sequences: 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; 109 (iii) a nucleotide sequence comprising I, and a nucieotide sequence comprising A and R; (iv) a nucleotide sequence comprising A, and a nucleotide sequence comprising R and I; 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.

46. A composition comprising any of the following combinations of nucleotide sequences: 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 (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; a nucleotide sequence comprising L, a nucleotide sequence comprising I and R, and a nucleotide sequence comprising A; 110 (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; 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; (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.

47. A composition according to claim 45 or 46 which is one or more nucleic acid vectors.

48. A composition according to any one of claims to 47 wherein a cell contains any of said combinations of nucleotide sequences. ii

49. A plant, or a part, propagule, derivative or descendant thereof, comprising a cell according to the composition of claim 48. 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. 51, A method according to claim 50 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.

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, 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.

53. A method according to claim 52 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.

54. A plant, or a part, propagule, derivative or descendant thereof, obtainable using a method according to any one of claims 50 to 53.