CA2188562A1 - Method of introducing pathogen resistance in plants - Google Patents

Abstract

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

Description

WO 95131564 2 1 8 8 5 6 2 . ~ s 6 1075 ' 1 MET~IOD OF L~CUlIUCll~L~ PAT~IOGEN RESISTANCE IN PIANTS
The present invention relates to a method of introducins pathogen resistance in plants, particularly broad spectrum pathogen resistance, and plants which 5 may be obtained by said method and which show resistance to at least one but pref erably more than one pathogen .
Plants are constantly ~-hRll~nged by pot~nt;711y pathogenic mi~:Luuly~.-isms. Crop plants are lC particularly vulnerable, because they are usually grown as genetically uniform monocultures; when disease strikes, losses can be severe. ~owever, most plants are resistant to most plant p7thr-g~nc. To defend themselves, plants have evolved an array of both 15 preexisting and; n~llri h] e defences which include barriers to pathogen entry such as thickened or chemically crosslinked cell wall c c or toxic chemicals derived from complex plant biosynthetic pathways. P7thf~.onc must specialize to CiluullLvt:~lt the 20 defence -h7n; 1 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 25 interaction i8 said to be ;n~o~n.pAtihle~
Induced resistance is strongly correlated with the hypersensitive response (HR), an induced response Wo9S/31564 2 1 88562 PCIIGs95/0l075 assoc~ated with localized cell death at sites o~ =
attempted pathogen ingress. It is llypothPRi ~P~ that by HR the plant deprives the pathogen of living host cells but there is no certai~ty about whether localised cell 5 death results from or induces plant defence ~h~n; om~
Many plant defence -~-h~n; ~ are strongly induced in response to a challenge by an llnRllrrpRRful pathogen. Such an induction of enhanced resistance can be systemic (hereiIIafter referred to as systemic 10 acquired resistance (SAR) ~ (Ross, 1961; Ryals et al., 992). 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 est~hl i RhPd. For example, 15 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 wil not suffice. (Ryals et al., 1992; Enyedi et al., 1992). In addition, SAR is correlated with the 20 induction of a large array of pa~hn ~PnPR;R-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 rh~ l l Pn~ing of tobacco carrying the N gene 25 for resistance to tobacco mosaic virus (TMV) with TMV
(Ross, 1961) and challenging cucumber seP~l ;n~s with .-tobacco recrosis virus or ColletotrichzLm largenarium..

~ WO95/31564 21 88562 r~ 5 1075 Results show that a challenge with one pathogen leads to Pnh~nr~l resistance to a wide variety of other pathogens ( Ryal s e t al ., 1 9 9 2 ) .
SAR has also been r rrrPl ~te~i with increased 5 levels of salicylic acid in plants which have been rh~llPn~ed by pathogens (Malamy et al., l990; Metraux et al., 1990) which has been rnnfi 1 by studies that show that a supply of exogenous salicylic acid to unchallenged plants Gan result in SAR (nard et al., 1991; Hennig et al ., 1993 ) . Transgenic plants designed so that salicylic acid ~ t ir,n is prevented by expression of a salicylate hydroxylase gene show reduced SAR compared to non-transgenic plants . where salicylic acid ~ tion is not prevented (Gaffney 15 et al ., 1993 ) . SAR can also be induced by many chemicals m-nllfAct~red by Ciba-Geigy such as 2,6-dichloroisonicotinic acid (INA) (Uknes et al., 1992).
SAR is an attractive method by which broad spectrum disease control can be achieved. However, two 20 major drawbacks hinder its ~ .ial exploitation: SAR
is not a heritable trait and so the rhP- ~n 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 25 intervals.
,, Although the - - -h~n i omq causing SAR are not fully understood, it is believed that when a plant is ~V095131564 21 88562 challenged by a pathogen to which it is reslstant, it undergoes an HR at the site of attempted ingress of ~he ;n~ -t;hle pathogen. The induced HR leads to a systemic enhancement and acquisition of plant 5 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) cre~
by 1~ n~nt genes (R genes)~ Flor showed that when lO pathogens mutate to UV~:L~ - such R genes, these ' ::lt; nnq are recessive. Flor c~ncl 1~ d that for ~a R
gene to function, there must also be a ~uLLe~ uu~l~ing gene in the pathogen, an "avirulence gene" (Avr gene).
To become virulent, p~th~ nc must thus s~op making a 15 product that activates R gene-dependent defence mech~n; ~ (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 20 pathogens carry the corr~p~n~ii n~ AVR gene (Gabriel and Rolfe, l990). This recognition is then t~n~ red 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 25 currently widely used in plant breeding. Lines that are homozygous for the recessive allele of this gene ., actlvate the defence response (comprising formation of ~ WO g5/3l564 2 1 8 8 5 6 2 cell wall appositions ~ even in the absence of the pathogen (Wolter et al, 1993) . Thus the mlo - lt~tinn causes a defence mimic phenotype, also known as a necrotic or disease lesion mimic phenotype, and appears 5 to deregulate the def ence 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 10 have been sought in ArAh; ~npSi c . The characterization of one such mutant, acdl, has been re~orted (Greenberg and Ausubel, 1993 ) . Further mutants of this type have been reported at scientific ~t;n~q (the Arabidopsis acd2 ~tinn by F.M. Ausubel at a meeting at Rutgers 15 University, New Jersey, IJSA, April 1993; Arabidopsis ~ t;nnq now known as lsd (for lesions si l~t;n5 defence response) mutations by R. Dietrich and J. Dangl at the ARaPANET ( (Ar~hi~npsic Pathology Network) workshop in Wye College, ~Cent, UK in April 1993).
20 Manuscripts describing the acd2 and lsd I ~tinnc are Dietrich et al. and Greenberg et al. (1994). It is highly likely that the recessive, ~tinnc identified in such mutant screens that leave the defence response more constitutively on, or more rapidly activated, or 25 less easily inactivated, are in genes that normally ,, dampen down the defence response to prevent it bc~ n~
so severe that it is deleterious to the plant.

WO9S~31564 2 1 8 8 ,62 ~ ;S.~1075 Conceivably, such gene could be cloned, expressed in an antisen5e or sen5e 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 Sta8kawicz, 1993 ) . Additional bacterial genes (hrp genes) are required for b~rtPr;ill Avr genes to induce ~, and also for r~thn~pn;city (Keen, 1992; Long and Staskawicz, 1993 ) . It is not clear why r~thnganq 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 p~thn~pnl~it whereas other Avr genes are less rl;qpPnq~hle (Keen, 1992; ~ong 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 ~agnaporthe grisea and the avirulence genes (NIP
proteins ) of the barley pathogen Rhynrhnspnrim secalis. Two viral avirulence genes have also previously been cloned. Culver and Dawson, 1991, have ~ W095131564 2 1 8 8 5 62 shown that tobacco mo3aic virus coat protein is the avirulence detPrm; n;: nt for the N' gene product . In addition, the potato virus X coat protein appears to be the avirulence det~ n~nt for the Rx and Nx genes (Kavanagh et al., 1992; Santa-Cruz et al., 1993; Etohm 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 PsPI~ .c syringae pv tomato (Pst) has been reported (Martin et al., 1993). It has al60 been recently reported that the Ari~hirlnr~iq Rps2 gene (which confers P~ syri~gae resistance) and the tobacco N gene (which confers virus resistance) have been cloned (Key8tone Sympo8ium, January 1994).
Even more recently, the Rps2 and features of the Cf-9 gene Re ~ have been revealed at the 13th An~ual Symposium in Columbia, Missouri, April 13th-16th 1994, on the Biology of Communication in Plants.
TntPrnslt;onal 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 dr~r t;c~lly in recent years, and many workers are currently attempting to clone a variety of R genes. Targets include (amongst others) rust resiRtance genes in maize, An~irrhin~ and flax (by transposon tagging); downy mildew resistance W0 95/31564 2 1 8 8 5 6 2 ~ 075 genes in lettuce an~ 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);
5 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 p~thr~-.nl:~ in Arabidopsis and tomato (by map based cloning).
Tomato (~ycopersicon esculentum) is susceptible to disease caused by the leaf mould fungal pathogen ~7;1r7n~rnrium 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 15 that carry the Cf-9 gene, encodes a secreted cysteine-rich peptide with a final processed size of 28 amino acids. However, its role in compatible interactions is not clear. The R genes ~Cf-genes) that act against C.
fulvum have been irl~nt;f;f~rl and bred into cultivated 20 varieties, often from related species of tomato (Dickinson et al., 1993; Jones et al., 1993).
It has been shown that C. fulvum rfmt::; n~l Avr genes that confer recognition by plants which contain the Cf-genes, leading to activation of host defence 25 me~h~n;~ ~ to attack the disea9e (;nl , t;hil;ty).
The Avr4 and Avr9 genes encode amall peptides that are secreted by the pathogen into the inter~ r spaces W095131564 .~,1,~,'. 1075 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 ' 10 r~thn~Pn race which is avirulent on plants carrying Cf-9 gene confers cl ,-t;h;l;ty on the Cf-9 -nnt;l;nin~
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 rnnt~;n;n~ tomato leaves to elicit a necrotic response in the inj ected area .
The necrotic response is consistent with local and vigorous activation of a defence response (De Wit, 1992; W3 91/15585) . TntPrn~tinn~l Patent Application No. PCT/GB94/02812 describes the tr InF~gPn; n expression of the Avr9 gene using the strong n~lll i fl ower mosaic virus 355 plant promoter to cause lethality in Cf-9 plants. This transgenic Pxpression 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 Wo 95/31564 2 1 8 8 5 6 2 PCr/GB95/~l075 '10 gene .
Various pathogen races that overcome these Cf-genes have emerged and are named af ter the Cf-gene which they can overcome. For example, C. fulvum race 4 5 can overcome Cf-4; C. fulv~m race 5 can .,v~ Cf-5 and C. fulvuzn race 2.4.5.9 can uvc:~, ~ Cf-2, Cf-4, Cf-5 and Cf- 9 .
Wû 91/15585 describes a hypothetical method whereby i f a Cf - 9 gene and/or an Avr9 gene were 0 expressed under the control of a promoter that is induced by a broad range ûf pathogens, then a general defence response could be induced. IIowever, there i8 a lack of Pn~hl; nrJ ~i ~rl r~l~re regarding which polynucleotide seguences could be used either as the 15 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 i n~lllrt i n of Avr9 and/or Cf -9 20 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 a5 PR genes (pathogenesis related genes ), 25 are induced in both a, ,~t;hle and an in~ ,-t;hle interaction . Theref ore, even if a promoter exists which is effectively induced by a broad range of ~ wo95131564 21 88562 r~ 075 pathogens, the method would not be viable unless the promoter is only induced by the appearance of a compatible pathogen. If t~e defence response provides further I n~ r~ i ~An of the promoter the plant might 5 experience spreading necrosis.
The present invention has reeulted from experiments involving tr~nRrosrn tagging of resistance genes, the first one being Cf-9. Numerous alleles of the Cf-9 gene (Cf-9*D8) were isolated that had been 10 inactivated by the maize element Dissociation (Ds).
These ~active Cf-9*Ds genes did not give rise to a constitutive and lethal activation of def ence Ah~n;~ in response to constitutively ~.~ ssed Avr9 transgene (35S:SP:Avr9). On backcrossing plants that carried the Cf-9*Ds and 355:SP:Avr9 genes to tomato plants carrying an Activator (Ac) transposase gene (sAc) in the h~ _yy~us state but lacking Cf-9, a quarter of the resultant progeny carried sAc, 355:5P:Avr9 and Cf-9*D6. These plants showed somatic 20 ~Yr; R; r,n of Dg from the Cf-9*Ds gene, 80matically 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 25 necrotic sectors, the plants phenotypically showing variegation for a defence-related necrosis, similar to somatic flecks of necrosis that are associated with the WO9S/31S64 21 88562 Pcr/Gs9sl0l07s induction oE SAR in plants rhAl 1 rngc~l 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 5 pathogens. -Thus, a f irst aspect of the pre~ent inventionrelates to a method of providing rAthnJ~n resistance, in particular broad spectrum pathogen resistance, in plants by induction of variegation in which genes are 10 expressed or suppressed resulting in the activation of necrosis. A method according to the present invention comprises: (i) inactivating a nucleotide sequence which contributes to plant cell necrosis or inactivating one or more nucleotide sequences forming part o a 15 ' ~ ;nAr;nn of nucleoti~Le sequences which contribute to plant cell necrosis; (ii) introducing said nucleotide sequence or serluences into the genome of a plant; and (iii) restoring said nucleotide sequence or sequences to a functional form to yield a level of necrosis 20 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 25 by induction of variegation in which genes are ~ ssed or suppressed resulting in the activation of a plant defence response which comprises: (i) W0 95~31564 2 1 8 8 5 6 2 i ~ 07s inactivating a nucleotide sequence which contribute3 to the plant defence response or ~nactivating 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 .
0 The variegation will generally be for somatic sectors. Pathogen re8istance will generally be increased compared with wild-type.
The nucleotide sequence or sequences comprise one or more genes . The plant def ence response and/or plant cell necrosis occurs on expression of the gene or genes. The defence response and/or plant cell necrosis can be conditional or unconditional on the expression of one or more interacting genes. A substance or a combination of substances nay result in increased pathogen resistance . Examples are ~li Rr'llR,RPd 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 95/3156~ 2 1 8 8 ~ 6 2 PCT/GB95/0107~
pathogen resistance gene (R), an avirulence gene ~Avr) or other elicitor or ligand gene (L) of an R gene, or both and R gene and an L gene.
The inactivation of the nucleotide sequence or 5 sequences which contribute to the plant defence response and/or plant cell necrosis is preferably effected by insertion of a tr~ncpoqAhl e genetic element into the nucleotide sequence or one or more of the nucleotide sequences forming-a, ' in;~tinn of lO nucleotide seguences. The transposable genetic element is preferably a tr~nC~nsnn or a nucleotide sequence flanked by specific nucleotide sequences 90 that tr~nRrosnn excision gives rise to activation of the plant defence response and/or necrosis. Thus, 15 insertion of a genetic lesion into the nllclect;~lP
sequence disrupts the gene to prevent expression of a product able to function in contributing to the plant defence response and/or plant cell necrosis. In the absence of the lesion, e.g. following Pl~n;c1nn of a 20 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 25 required for expression of the product.
In this form of the invention, re-activation within the plant is preferably carried out by ~ Wo 95131564 2 1 8 8 5 6 2 ~ 1075 restoraration o~ 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 5 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 corr~cpnn~; n~ transposon activation system ~for example, comprising a tranEposase).
0 Alternatively, the inactive form could be flanked by n:~ce recognition sequences that are acted on by a site specific re~ ' ;n~ti~n system (comprising a specific rP ' ;n~ce) 80 that L~_ ` ;nzlt;nn activates the inactive form of the gene. Hence, when the 15 inactivated nucleotide SP~lPnre or sPs~lpn-pc are introduced into the plant genome somatic P~ri c j the tr~nRposnn or recombination of the nucleotide sequence occurs in some cells leading to ~ctivation of the plant defence response and/or necrosis in specific

2 0 clones of cells .
The number of cells in which restoration o~
function occurs may vary. As discussed further below, certain measures are available for opt;m;C;n~ the system, e.g. by controlling the frequency of 25 crnnt:lnPnu5 excision of a transposable element which is caused or allowed upon cultivation of a plant with the requisite ~ucleotide sequence or SPql~Pn~~Pc within its W095/31S64 2 ~ 8 ~ 56~ //a 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, 1~ including cuttings, seed and so on. Derivatives of plants are also provided by the present invention. A
derivative is any fl~nct;nn~l unit derived therefrom h~ ver achieved (e.g. flln~ti nnAl allele of gene made by a~nf~R;R, re~ ` in~nt DNA, synthesis, or 15 plant which could not have been y ~ ~ -lu-_~d without the use or manufacture of the plant from which it is derived. ) Transgenic plants in accordance with the present invention may demonstrate increased p~thng~n rl~i Rt~n~e 20 since the induced plant defence response and/or necrosis of plant cells may cause other cells, such as adj acent cells, to acquire pathogen resistance . The activation of, for example, a plant resistance gene in a plant cell is inherited by the progeny and 25 dest f-nrl~nt R 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 o95/31'64 l,7 '~''' ~a death of the participating plant cells resulting in an area of plant cell necrosis. So, plants may haYe variegation for small somatic sectors in which defence-related plant cell necrosis is activated. This response may induce pathogen resistance in other cells.
In an alternative, operating on the same general principle, the expre6sion of one or more plant pathogen resistance gene may either lead to initiation of the defence response only resulting in variegation for 10 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.
1~ ~Ience, the plant may acquire resistance to a broad range of path~g~nT~ and not only to the pathogen associated with the gene or genes contributing to necrosis, for example, C. fulvu17 in the case of the Cf-9/Avr gene ~ ;n~t;on ~or example, a transgenic 20 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, Xanf7- f,T campegtris, Pse7T' C7 syringae), fungal plant pathogens (for example, 2T7 Phytophthora infestans, Fusariu27 U~y~l~OLIllll, Botrytis cinerea, Verticilliu~7 dahliae, Altenaria solani, Rhizoctonia solani) and viral pathogens (for example, WO95131564 2 1 88 5 ~2 i~ s/01075 TMV, PVX, PVY, TSWV). Similarly, other transgenic plants such as transgenic tobacco, Araoidopsi~ and potato plants may display resistance to a large number of ma~or diseases of important crop species such as, 5 Peronospora, Phytophthnra, 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 10 clones of cells expressing a first phenotype and other cell5 expressing a second phenotype which is increased pathogen resistance compared with wild-type. The first phenotype is preferably necrosis and/or a plant defence r e~ se phenotype . As ~ r-llq~d~ plants variegated by 15 somatic sector for such a phelluLy~e may have f~nhAnred 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 def ence response is activated . The phenotypic 20 variegation is likely to result from expression in cells with the f irst 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. A8 25 discussed herein, this may result from reactivation of a previously inactivated gene, such as a resistance gene, for example by random ~l-r; qinn of a transposable ~ wo95131564 2 1 88562 .~. . 1075 '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, 5 ~ nt~in1n~ (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant def ence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly ina~tivated, for example by insertion of a transposable 10 element such as a trAnqrnsr~n, and (ii) nucleic acid f~nrf~l;n~ a molecule able to reverse the inactivation, such as, in the case of a transposon, a tr~nqp~R~qe.
Thus, the cell may comprise a plant resistance gene or other gene involved in the plant defence response or lS able to kill a cell when expressed therein (either alone or ;n~ ' ;n~r;~n with one or more sequences, for example in the case of an R gene the corrF-qr~nrl;n~
elicitor), the gene being inactivated by insertion therein of a tr~nqp~snn, and the cell further 20 comprising a gene encoding a tr~nqrss~qe.
In an ~ ry: ' ~ '; ', 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 25 genome also comprising the Avr-9 gene, or a mutant, derivative, variant or allele thereof which retains Avr-9 function, and a gene ~n~o~l; n~ a tr~nqros~qe able Wo 95l3l564 2 1 8 8 5 6 2 PCT/GB95/01075 to exci~e the transposon ~rom 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 cau6e cell 5 necrosis, as discu9sed further elsewhere herein.
The cell may comprise the nucleic acid Pnco~i n~
the various genes by virtue of i~troduction into the cell or an ancestor thereof - of the nucleic acid, e.g.
by transformation, using any suitable technique 10 available to tho9e 9killed in the art. Furt~-~ e, plants which comprise such cells, and seed therefore, may be produced by crossing suitable parents to create a hybrid whose genome (-nntW;nR the required nucleic acid, in accordance with any available plant breeding 15 technique. For example, a parent strain comprising within its genome a plant resistance gene rnntw;n;n~ a trwnqposon or other inactivating lesion may be crossed with a second strain comprising within its genome a gene encoding the elicitor molecule for the plant 20 resistance gene a~d a suitable transposase for excision of the trwnRrnsnn. 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 25 resistance gene and, following interaction with the elicitor, the plant defence response a~d/or plant cell necrosis .

~ Wo95/31564 21 885~2 r~. 1075 , Plants according to this aspect of the pre6ent - 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, 80 that a first phenotype such as necrosis is shown, while in other cells the sequence or sequences will remain inactivated 80 these cells will not show the first phenotype.
Within the cell or cells, the nucleic acid may be incorporated within the ~ - . A gene stably incorporated into the genome of a plant is pa8sed from generation to generation to descPnf~nt~ of the plant, 80 such ~lPcpnrl~nt~ should show the desired phenotypic variegation and so may have Pnl~nt~p~l p~thn~l~n resistance .
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hy~rid progeny and clP~rpnr~nt~ 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 prora~atinn, sexual or asexual, including cuttings, seed and so on.
A further aspect of the present invention provides a method of making such a cell involving introduction of nucleic acid (e.g. a vector) comprising .

WO 95/31564 2 1 8 8 5 S 2 P~ 7s ( i ) nucleic acid e~coding 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, 5 for example by insertion of a transposable element such as a tr~nApo~t~n~ and/or (ii) nucleic acid encoding a molecule able to reverse the inactivation, such as, in the case of a transposon, a transposase into a plant -cell. Introduction of nucleic acid (i) may be 0 A~ ; ed, preceded or followed by introduction of nucleic acid (ii). Such introduction may be followed by ret ' ;n~t;~n between the nucleic acid and the plant cell genome to introduce the seguence of nucleotides into the genome. Desc~n~AntA of cells into which 15 nucleic acid has been introduced are ; nrl ~ t~ within the scope of the present invention.
The level of the plant def ence response and/or plant cell necrosis in the small somatic sectors should be sufficient to result in the ;n~ tion of acquired 0 resistance or the ;nt~ tir~n of other defence n; I ~ . 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
5 is applicable to the present invention Methods and plants etc. according to the present invention are particularly beneficial since the WO 95/3 1S64 1 ~ , '. 1 07S
2~ 62 nucleotide sequence or seque~ces which contribute to the plant defence response and/or plant cell necrosis, for example the avirulence and plant resistance genes, may be under co~trol of any suitable promoter, such as 5 a constitutive promoter or, in the case of R genes, their own endogenous promoter, or a cell type specific promoter Furth,~ e, the restoration of the nucleotide sequence or sequences, for example by the somatic excision of a transposon, gives rise to 10 LeuuLLellL and widespread induction of the plant defence response in many small clones of cells tllLUlyllULlt 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 appl i~t;nnc and is suitable for deployment in Fl hybrid seed pro~-]ct; ~In system. In such a system, one of the parents should be 1- 7-yyuus, for example, for the tr~ncpocAce or recombinase gene. In addition, in a 20 system where two ~ ~ ~ntc are required for ;n~ r;n~
the necrosis such as in the Avr9/Cf-9 gene, ;ni~t;r)n for example, this parent should also be _yyu~s for the constitutively expressed genes. The other parent should be homozygous for the gene that encodes the non-25 autonomous inactivation system, such as the transposonor recombinase-recognition sequences. After making a cross between parents of this genetic constitution, on Wo 95UI564 2 ~ 8 8 5 6 2 PCT/GB9S/01075 somatic excision or recombination, the function of the gene or genes which give rise to the defence response and/or plant cell necrosis i8 restored in somatic sectors in the resulting progeny.
It will be clear to the person skilled in the art that any gene or ~ ' ini9t;nn of gene8 which contributes to variegation for the plant defence response and/or plant cell necrosis may be uGed in the method of the ~
present invention. Furthermore, any system which gives rise to inactivation of the nucleotide sequence or sequences and subsequent rest~-r~ti~ n of functional sequence or SP~lPnC'eR may be used.
The present invention also provides in further aspects various compositions of matter comprising , ' in~tir~nR of nucleotide sPquPnre~ encoding various substances employed herein. Such c ~ ;n~t;~nR of nucleotide sequences which may be introduced into cells in accordance with the present invention follow:
(X): represents a nucleotide sequence with one or more genes of type X
(XY): represents a nucleotide sequence with one or more genes of type X and one ore more genes of type Y etc.
R: receptor gene L: ligand gene (capable of interacting with the R
gene ) W(1 95131S64 21 885~2 I: genetic insert A: activator of transposition o~ genetic insert.

R may encode a substance whose presence in a plant results in a plant defence response, necrosis 5 and/or increased pathogen resistance, with I being a genetic insert able to inactivate R and A .-nrQ~l;nrJ a substance able to reactivate R inactivated by I:
(l) Any, ' ;n~ti~n of:
l . (R), ( I ) and (A);
2. (R) and (IA);

3 . ( I ) and ~AR); or

4. (A) and (RI);

5. (RIA) .

Alternatively, R and L may encode substances lS 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 ~nro(l; ng a substance able to reactivate R and/or L inactivated by I:
20 (2) Any combination of:
l. (R), (L), (I) and (A);
2. (R), (LI) and (A) 3. (R), (LA) and (I) ~ . (R), ( IA) and (L) 5 . (L), ( IR) and (A) Woss~v1s64 2 ~ 6 2 . ~ iv/a

6. (~), (AR) and (I)

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

8. (R) and (LIA)

9. (L) and (IAR) lO. (I), and (ARL); or ll. (A) and (RLI);
12. (RLIA) If genetic insert (I) is coupled with either the R or the L gene, the number of possible combinations lO will then be (l): (RI~ and ~A); or (RIA) (2): (RI) (L) and (A) (R), (LI) and (A) (RI) and (LA) (RA) . and (LI) (RLIA) also provided by the present invention is a method of producing a plant, or a part, propagule, 20 derivative or descendant thereof, cnn~;ninr, nucleic acid comprising a nucleotide sequence or nucleotide sequences onrr,~inJ R, I and A, wherein R encodes a substance whose presence in a-plant results in a plant ~ WO 95/31564 2 1 8 8 5 6 2 ~ 075 2~
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 ~ ' ;n~tions 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 ri~cr,on~Ant thereof, ~ nt~in;n~ nucleic acid comprising a nucleotide sequence or nucleotide se~uences ~ncoriin~ R, L, I and A, wherein R and L encode substances whose presence together in a plant results in a plant def ence response, necrosis and/or increaged r~qthr~n resi8tance, 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 ~ i - c of the present invention are WO95/31564 2 1 88562 .~1 ~ ~lu/a now described in more detail below, by way of example and not limitation.
Nucleotide Se~uence or ~e~PnrP,q contri~uting to the Plant Defence Response and/or lVecrosis The nucleotide sequence or combination of nucleotide sequences in which at least one of the sequences is inactivated are rLumerous and may include an ~ng-; nppred allele of a ubiquitin conjugating enzyme (Becker et al., 1993), the CaMV gene VI protein ~TAk~hA~-hi 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 DawsonL 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; Kohm et al., 1993; Goulden et al., 1993), Pto and avrPto ~see e.g. Rommens et al., 1995), ~PS2 of ArAhi~r~pqiq thA7 iAnA and the avirulence gene avr~Pt2 (Bent et al., Mindrinos et al. ), and genes of Arahidopsis 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 BA~SE (Mariani et al., 1990) or WO95i31564 2 1 8 8 5 6 2 PCT~GBg5~01075 ~iphthPria 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 def ence response . It is widely 5 believed amongst researchers in this field that cell death arises from local induction of the defence response and that this cell death can activate adj acent cells to give rise to the defence response. However, the precise cause and effect rela~ n~hi~ between these

10 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-;nf~llr;hle defence 15 L~ullse, such as that leading to the activation of protease inhibitors or alkaloid biosynthesis (Ryan lg90). 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 (19g5) in 20 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 inactivated allele of the Cf- 9 gene to constitutive expression of the Avr9 gene .
25 This system can be replaced by similar ~ '-;n~t;rn~ Of related genes for example the Avr4 and Cf-4 gene, sequence provided herein (cloning of Cf-4 ifi described WO9513156~ 2 ~ ~85~ PCJ/GBgS/0107~ ~

in a co-pending GB application ~iled simult;~n~ouRl y with the present application); the Avr2 and the Cf-2 gene, se~auence provided herein ~ cloning of Cf -2 is described in GB 9506658,5, priority from which is 5 claimed herein); the Avr5 and the Cf-5 gene, or by cloning resistance genes and corr~ocrnn-l;n~ 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 lO 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 ~v~le~ 55iOn .
It should also be noted that complete Avr or 15 other ~1; f~; tnr gene may not be required. Instead a L ~ may be employed, representing a part of the elicitor molecule which interacts to provoke a plant defence response and/or plant cell necrosis.
It is possible that the nucleotide sequence 20 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 ~r~n~nt on the expression of z5 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 expres~ion due to ~ro gsr3lsG4 2 1 8 8 ~ 6 2 F~
somatic exci9ion and restoration of Avr9 gene expression, or vice versa.
Nucleotide sequences employed in the present invention may encode a wild-type sequence (e.g. gene) 5 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 se~Iuence. An alteration to or difference in a nucleotide sequence may or may not be reflected in a 10 change in encoded amino acid sequence, rl~pon(1;n~ on the ~lp~Pn~r;q~y 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 15 contribute to a plant defence response and/or plant cell necrosis. Of course, changes to the nucleic acid which make no dif f erence to the encoded amino acid sequence are included.
Similarly, homologues of the various genes whose 2 0 use is disclosed herein from other species or races may be employed, as may mutants, variants and derivatives o~ such homologues.

Inactivation and Reactivation of the ~ucleotide Sequence or Sequences Contributin~ to the Pla~t Defence 25 Response and/or Necrosis A m~thod according to the present invention may W095/31564 2 1 8~ ~62 P . . 1 /~
employ any o~ a variety o~ transposon systems known to the skilled person, including the maize :~
Activator/Dissociation (hereinafter referred to Ac/Ds system) (Fedoroff, 1989); the maize ~nh~nrPr/suppressor 5 mutator (En/Spm) system (Fedoroff, 1989); and the ~nt;rrhin-lm 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 21, 1990) or the "FLP/FRT" system (Lloyd and Davis, 199~) .
It will be d~clLe~ to the skilled person that the particular choice of transposon, re ' ;n~t;nn or other system used to inactivate the nucleotide sequence or se~uences which encode substances leading to the 15 plant defence response and/or plant cell necrosis is not P~R~nt;~l to or a limitation of the present invention .
In some systems, a tr~n~rnsnn or rP~ ; nAt i nn system might be so active that an unacceptable level of 20 necrosis is seen. If encountered, this may be overcome by Pn~;nPPring alleles of the tr2nsposon or rP ' ;n~e recognition seS~uence in which the frequency at which activated nucleotide sequences arise is reduced, such as with Ac(Cla) (Keller et al., 1993) . Alternatively, 25 e hPm; c~l or site-directed mutagenesis may be used to recover alleles of the necrosis-; n~ ; ng genes which ar~ less active and therefore result in less severe Wo 9S/31S64 2 1 8 8 5 6 2 levels of plant cell necrosis (Hammond-Kosack et al., 19 94 ) .
In other systems, transposition or re~: ' n;
may be inefficient resulting in too few activated 5 nucleotide se~uences leading to an insufficient level of plant cell necrosis. This may be uv~u",e by constructing suitable promoter fusions to the tr~n~ro~Re or re~_ ' ;n~e gene in the plant gene (Swinburne et al., 1992) to increase the frequency of 10 excision or re~ ' in~t;o~n to efficient levels. The most suitable promoter might give rise ,Ul~'~ ;n~ntly to late small sectors of necrosis during organ development rather than early large sectors.
Many other variations are possible as ;r~
15 for activating the defence response and/or necrosis after tr~n~pn~nn excision or re~ ` in~inn. 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 20 (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 specie3, may lead to 25 activation of the defence response and/or necrosis even in the absence of an avirulence product. Bonneus, et al (1995). In an alternative, the original disease -resistance gene may be mutated so that it binds to a defined chemical such as an agrichemical and this chemical activates Cf-9 to initiate the defence response and/or necrosis. Hence, genotypic variegation 5 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 iæ applied and recognised by the resistance gene triggering the same 10 reaction as if the avirulence gene product were present .

rntroducing the Nucleotide Sequence or Seguences which Contribute to Variegation for the Plant Defence ~esponse an~/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 def ence response and/or leads to plant cell 20 necrosis resulting in broad 8pectrum rath~ n resistance .
The nucleic acid may be in the form of a r~, ' ;n~n~ vector, for example a plasmid or agrobacterium binary vector (Van den Elzen et al., 25 1985). The nucleic acid may be under the control of an appropriate promoter and regulatory elements for WO 9S/3IS64 2 1 8 8 5 6 2 r~, ~ ; /, 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 d~ L iate 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, c~n~;nin~ cLL,~,v~Liate regulatory sequences, including promoter sequences, tPrmir~t~r fr~,~ ' q, polyadenylation sP~ PnrPq~ Pnh~nmPr sequences, marker genes and other SPqllPn~pc as Liate. For further details see, for example, Molecular Cloning: a La~oratory ~anual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for ~-n;~lllAtion of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DN~ into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in ~olecula~ 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 ref erence .
When introducing a chosen gene or gene construct into a cell, certain considerations must be taken into account, well known co those skilled in the art. The Wo 95/31564 2 1 g 8 5 6 2 PCT/GB9S/01075 nucleic acid to be inserted may be assembled within a construct which rnnt~in~ effective rer~ulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell.
5 Once the construct is within the cell membrane, integration into the endogenous chromosomal material may or may not occur according to different: a; '.C
of ~the invention. In a preferred ~ t, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. I~tegration may be promoted by inclusion of sequences which promote rel ;n~tion with the genome, in accordance with atandard technir~ues. Finally, as far as plants are rnnr~rn~ the target cell type should be such that cells can be regenerated into whole plants.
Plants transformed with a DNA segment rnntil;n;nrJ
pre-sequence may be produced by standard techni~ues which are already known f or the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed ~i-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 ,~ t ~US 5100792, EP-A-444882, EP-A-434616) microinjection ~WO 92/09696, WO 94/00583, EP 331083, EP
175966), electroporation ~EP 290395, WO 87066~4) or other ~orms of direct DNA uptake ~DE 4005152, WO

-~ WO95/31564 21 88562 r~ JUJ~
9012096, US 4684611). Agrnh~rt,orium transformation is widely used by tho3e 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 D~A uptake are pref erred where Agrobacterium is inef f icient or ineffective. Alternatively, a combination of different techniS~ues may be employed to enhance the f~f f i ri ~n. y Of the transformation process, eg. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microproj ectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
The particular choice of a transformation technology will be ~ t~rm;n~d by its efficiency to ` transform certain plant species as well as the experience and preference of the person practising the invention with a particular ~ hr~r~lo~y of choice. It will be apparent to the skilled person that the 20 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 conf er selectable phenotypes 25 such as resi3tance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, srectin~ ycin, ;mi~1~7~1;n~nf~F~

WO 9513156~ . 2 ~ 8 8 5 6 2 1 .I~ l075 i~nd glyphosate (~errera-Est~ella et al, 1983; van den Elzen et al, 1985) .
The present invention is particularly b~n~f i c i f or use in crop and amenity plants . Examples of 5 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, cl~ , other orn~ 1 plants, turf 10 grass, poplar, eucalyptus and pine.

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

Figure 1 srh t;~ lly depicts the Cf-9 gene, showing tagged alleles. X marks a probable promoter.
Figllre 2 illustrates genetic acquired resistance to C. fulv~n induced following necrotic sector formation caused by the excision of a Ds element from 20 ~he Cf-9 resistance gene in an Avr9 expressing tomato plant. The number of C. fulvu~n pustules per leaf is indicated, 14 days after inoculation.
Figure 3 illustrates genetic acquired resistance to Phytophthora lnfestans (late blight of tomato and 25 potato~ . GAR+ and GAR- plants from Cf-9~Ds, mutant WO 95/3156~ 2 1 8 8 5 6 2 P.~ ' 1075 lines M31 and M50 and cfo plants 3pray inoculated with 10, oOO sporangiospores/mL. In panel A the appearance of leaves from the muta~t 50 experiment 7 days after inoculation is shown. In panel B the rate of leaf 5 abscission (in days after ;nnC-llAt;nn) in the various genotypes inoculated is given.
Fi~ure 4 illustrates genetic acquired resistance to Phyt~Fhthnra 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 innt~ tet~1 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 15 sporulating lesion formation on the various plant y~ uLy~es ;nnr~ t~1 is given, with the mean number of sporulating lesions/leaflet given at 5, 7, 10, 13 and 16 dayg af ter inonl - l ~ t i nn .
Figure 5 shows genetic acquired resistance to 20 Oidium lycopersici (powdery mildew disease). GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and Cf O plants were painted with equivalent numbers of spores. In panel A the appearance of leaves 14 days after inoculation is shown, GAR- on the left, GAR+ on 25 the right. In B, the rate of chlorotic lesion (upper panel) and sporulating lesion ~lower panel) formation on the various plant genotype5 is given for Mutant 31:

WO9SMl564 21 88562 1~1~ . . ,~ ~

mean number of lesions given at 7, 10, 14, 21, 24 and 30 days after inoculation. C shows equivalent results for Mutant 50.
Figure 6 shows the appearance of tomato fruits on 5 GAR+ (sAc, Cf-9*Ds - right-hand) and GAR- ~6Ac, Cf-9*D6, Avr-9 - left-hand) plants from mutant line M23 at 2, 3, 4, 5, 6 and 7 weeks after flower poll ;nAt;on Dark green sectors formed on the GAR+ but not GAR- fruits by 5 weeks. These dark green sectors were not visible on 10 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 15 panel A the levels of a basic ,~-1,3 glllr~n~ 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 20 tobacco and potato. In panel A is shown a tobacco leaf that has been injected with intercellular fluid (IF) either cnnt~;n;ng the Avr9 peptide or lacking the Avr9 peptide. Avr9+ IF~was obtained from transgenic tobacco or a compatible C. fulvl~n - tomato interaction 25 involving race 5. Avr9- IF was obtained from untransformed tobacco or a compatible C. fulvum -tomato interaction involving race 2, 4, 5, 9 . Grey ~ W09~131564 2 1 88562 P~ 075 necrosis was vi6ible 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 5 leaves that received the Avr9+IF developed grey necrosis by 24 h.
Fitgllre 9 shows development of the necrotic lethal phenotype in seedlings f rom the tobacco cross cv .
Petite Xavana 6201A (355:5P:Avr9)~t 7yyuLe x cos 34.1 10 (genomic Cf-9) heterozygote. A time course for the period 5-12 days after seed pl~nt;nt3 (dsp) is shown.
50~ of the 5t-t~tll; n~q become chlorotic and die within 2 days of seed germination.
Figure 10 shows development of the necrotic 15 lethal phenotype in st~tll ;n~c from the ~r;~hi~nrsis cross 6201B4 (355:5P:Avr9) hete- uzyyuLe x cos 138 (genomic Cf-9) heterozygote . Appearance of 5t~t~.11; n~q 19 days after the majority of st~t~t~1 ;n~q had ~t~rm;n~tt?d.
One st-e~l; n5 has died and another has necrotic 20 cotyledons.
Figure 11 shows a single T-DNA construct systems to apply GAR to potato plants . The T-DNA rt~nr~; nc a Cf-9 gene seS[uence under the control of its own promoter which has been inactivated by an ~-ltt nl us Ac 25 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.

Wo 95/31564 2 1 ~ ~ ~ 6 2 PCr/GB95101075 Figure 12 shows a photograph Df three leaves, two of which are diseased with C. fulvum and one which is expressing GAR and is resistant tD the same inoculum of C. fulvum.
Figure 13 illustrates how GAR+ plants may be made by crossing stable lines (1) compriaing a Cf-9 gene, inactivated by insertion of a Ds trAncpnsnn, and an ~vr-9 gene and (2) an Ac transposase gene, as described in Example 1.
Figure 14 illustrates basic simplified haploid crossing schemes to produce plants with increased disease resistance.
T: transgenic line P: offspring of trAna~n; e line T1/Pl: line comprising in its genome at least one of each of the four genes, R, L,I or A
Tl,2/Pl,2 line comprising in its genome at least one of each of two of the f our genes R, L, I or A :
T3~P3: line comprising in its genome at least one of each of the four genes R, L, I or A not present in T1 2 T3,4/P3,~,: line comprising in its genome at least one of two of the four genes R, L, I or A not present in Tl 2 Tl,2,3/P1,2,3 line comprising in its genome at w095~ 64 2 ~ 8 8 5 6 2 ~ 1~ cTIu~a least one of each of three of the four genes R,L, I or A
T4/P4 line comprising in its genome at least one of each of the four genes R,L, I or A not present in T1 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 (AATA~A) at nucleotide 3703-3708; polyadenylation site at nucleotide 3823; a 115 ~p intron in the 3' non-coding sequence from nucleotide 3507/9 to nucleotide 3622/4.
Predicted Protein Sequence - primary tr~nRl~ti~-n 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 N0. 3 shows the sequence of one of the Cf-9 cDNA clones. Tr~nRl~t;nn initiates at the ATG at 20 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 descri~ed herein SEQ ID NO. 5 shows the genomic DNA sequence of 25 the Cf-2. 1 gene Features: Nucleic acid sequence -Translation start at nucleotide 1677; translation stop WO95131564 21 8 ~ 5 62 ~ a at nucleotide 5012; no consensus polyadenylation signal ~AATA~A) exists in the characterised sequence downstream of the translation stop. Predicted Protein Seriuence - primary translation product 1112 amlno 5 acids; signal peptide ser~uence 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 10 encoded by the Cf-2.2 gene. Amino acids which dif~er 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 sequenee of 15 the RPP5 gene. ~ntiri~t~ introns are shown in non-capitalised letters. Features: Nucleic acid sequenee -Translation start at nueleotide 966; translation stop at nueleotide 5512.
SEQ ID NO. 10 shows predieted RPP5 protein amino 20 aeid sequenee.
SEQ ID NO. 11 shows genomic DNA sequence of Cf-4.
Features of this sequence include: tr~nR~t;rn start site at nucleotide 201, translation stop beginning at nueleotide Z619, rrnR~nRuR polyadenylation seriuenee 25 beginning at nueleotide 2835, spliee donor sequenee in 3 ~ untranslated sequenee at 2641, splice acceptor ser~uence ending at nucleoti e 2755, proposed site o~

wo 95/31561 2 1 ~ 8 5 6 2 P~ I, ...,, '; . 075 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
f~s ~ encoding the PRla signal peptide sequence fused to a sequence proposed to encode the mature processed form of C. fulvum AVR4. Tr;ln~lati~n initiation codon at nucleotide 5, t-~rrn;n:lt jr-n codon be~; nn; ng at nucleotide 413 . Amino acids 1-30 represent the signal peptide and amino acids 31-136 the mature AVR4 peptide.
l~PLE 1 GENETIC ACQrJIR 3D RESISTANCE (G~R) Z7SING Cf-9 (i) B8t~h7 i~2hin~ a stock from w~ich 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 Tnt~rn~tion~ Patent Application No. PCT/GB94/02812 for further details). This inactivated Cf-9~Ds gene did not give rise to a WO95/31564 21 885b2 .~1~ 1075 constitutive and lethal activation of defence~
.h~n; ! in response to the constitutively expressed 3 5S: SP: Avr9 gene We have est~h~ hP-l 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 ~ntsl;n~ (a) a beta-glucuronidase ~GUS) gene (Jefferson et al 1987) to monitor T-DNA segregation and (b) stable Ac (sAc) that expresses tr~n~pn~e and can trans-activate a Ds, but which will not transpose (Scofield et al 1992).
The line FT33 did not carry a cf-g gene. We had to obtain r~: ` in~nt'~ that placed Cf-9 in cis with the T-DNA in FT33 in order to carry out linked targeted tagging. Two strategies were pursued simultaneously:
(a) FT33 was crossed to Cf 9, a stock that carries the Cf- 9 gene . The resulting F1 was then back crossed to Co (a stock that carries no Cf- genes). Progeny that carry the FT33 T-DNA are kanamycin resistant.
Kanamycin resistant progeny were tested for the wo ss/31564 2 1 8 8 ~ 6 2 ~ J

presence of Cf-9; 5 C. fulvll~n resistant individuals were obtained among 180. We alsogenerated progeny that were 1- _yy"us for Cf-9 and carried that sAc T-DNA of SLJ10512. These were crossed to the rPI~ ' ;nAn~S in 5 which Cf-9 and FT33 were ill cis. In the FT33 T-DNA, a tr~n~rr s~hle Ds element is cloned into a I~YYL' y~:in resistance gene, preventing its function. The somatic transactivation of this Ds element, which only occurs in the presence of trAn~p~ ce gene expression, results 10 in activation of the l1YYLL y~in resistance. Thus from crossing the recombinants between Cf-9 and FT33, to the sAc-carrying Cf-9 1- _yyuLe8, l1YYL~ yuin resistant individuals could be ~htA; r~Prl which carry sAc and FT33, and are likely to be ~ --yyuus for Cf-9.
15 140 individuals of this ye:~oLy~e were thus obtained.
(b) To accelerate ~hrA;n;ns individuals that carried sAc, FT33, and were Cf-9 hl -~yu~es, the FT33/Cf-9 F1 was crossed to a line that was heterozygous for Cf-9 and sAc. 2596 of the resulting 20 progeny carried both T-DNAs and were LyyL, y-:in resistant, and of those, slightly more than 5Q~ were disease resistant because they carried at least one copy o~ the Cf- 9 gene An RFLP marker was available, designated CP46, that enabled us to dist;n~liAh between 25 h~ yyutes and heterozygotes for the Cf-9 gene (3alint-kurti et al 1993). In this manner two individuals that were Cf-9 h~ yyutes~ and that W0 9Sl31564 2 1 8 8 5 6 2 carried both the FT33 T-DNA and sAc, were obtained.
These two individuals were multiplied by taking cutting6 80 that more crosses could be made onto this genotype .

5 (iiJ Establishing a tomato stock that expresse3 functional mature AV~9 protein A likely freguency for obtaining any desired mutation in a gene tagging experiment is less than 1 in lO00, and often less than 1 in 10, 000 (Doring, 1989) .
10 To avoid screening many thousands of plants for mutations to disease sensitivity, we est~hl; ahf~d a ~election for such mutations based on expressing the fungal Avr9 gene in plant8.
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 f luid of leaves inf ected with Avr9-carrying races of C. fulvum. For secretion from plant cells, we de8igned oligonucleotides to assemble a gene 20 that carried a 30 amino acid plant signal peptide, from the Prla gene (~r~rn~ sen et al 1987) preceding the first amino acid of the mature Avr9 protein (see SEQ ID
N0. 4). The preferred Avr9 gene sequence depicted in SEQ ID N0. 4 shows a r~;r~A~c gene ~n~;n~ed irom the 25 Pr-la signal peptide sequence (Corn~ s~n et al, 1987) and the Avr9 gene se~uence (van Kan et al, 1991). This .

-~ WO 95131564 2 1 8 8 5 6 2 r~ '01075 reading frame was fused to the 355 promoter of cauliflower mosaic viru8 (Odell et al 1984), and the 3 terminator sequences of the octopine synthase gene (DeGreve et al 1983), and introduced into binary 5 plasmid vectors for plant transformation, using techniquee well known to those skilled in the art, and readily available plasmids (Jones et al 1992). We obtained transformed CfO tomato lines that expressed this gene.

10 (iii) Crossing AV~Z9 expressiI~g 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~ l~h;h;tf~ a 15 necrotic phenotype, that mllm;n~ted in 9.o~l ;n~ death.
This outcome was only observed in soorll ;n~q that cnnt~;nPd the Avr9 gene. When the same tran~Lo,l..d~ts were crossed to Cf 0 plants, the resulting progeny were all fully viable.
From selfing the primary transformants, individuals were ;tl--nt;~;efl 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 ~tinnc ln :he Cf.9 gene.

Wo 95/31564 2 l 8 8 5 ~ ~ r ~ /a (iv) Tagging and inactivati~ Cf-9 Individuals that were 1- - yy~,us f or the Avr9 gene (section (iv) ) were used as male parents to pollinate individuals that were hc~u,J~yy~us for Cf-9, 5 and carried both sAc and the Ds in the FT33 T-DNA
(sectio~L (iiia) and (iiib) ) Many th~ n~l- of progeny resulting from such a cross were germinated. Most died, but some survived.
DNA was obtained f rom survivors and subj ected to 10 Southern blot analysis using a Ds probe. It was observed that several ; n~ t; nnq were correlated with insertions of the Ds into a BglII
rl _ - of a consistent size. This suggested that several independent _tinn~ were a consec~uence of 15 insertion of the Ds into the same DNA Ll _ ~ .
Using primers to the Ds se~Euence, DNA adjacent to the Ds in tL ~ sed 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 20 independent t~tinn_, the Ds had inserted i~to the same 6 . 7 kb ~glII ~
The Ds in FT33 contains a bacterial replicon and a chlc~l , ~; col resistance gene aa a bacterial selectable marker (Rommens et al 1992). This means 25 that plant DNA carrying this transposed Ds can be digested with a restriction enzyme that does not cut within the Ds (such as ~glII), the digestion products WO95/31564 2 l 8 8 5 6 2 1 ~1 . '0107~
can be r~ri rr~ rized, and then used to transform E~.
coli . ChluL , hrn i col resistant clones can be obtained that carry the Dr and adjacent plant DNA. This procedure was used to obtain a clone that carried 1. 8 5 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 10 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 char~rtf~ri ~e a large number of i n~ "t mutations by PCR analysis in combination with primers based on 15 the sequence of D8. Therefore, these primers were used in polymerase chain reactions with primers based on the maize Ac/Ds tr~nRpo~nn sequence, to characterise the locations of other ~ lt~tirn~ of Cf-9 that were caused by transposon insertion. Eighteen i~dependent 20 insertions have been characterized and are located as shown. Mutants E, #55, #74 and #100 gave in~ _ ,le~te 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 C~9 25 protein expression to activate an incomplete defence response.
Using the sequence obtained of the gene, WO95/~1564 2 1 ~ 5~2 P~
oli~r,nl-r1 ~ntide 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 ori rnt~t; rn Of 5 other transposon insertions in the gene. These are shown on Figure l Based on the results of such experiments, the map positions of 17 other DS
insertions have been reliably assigned (as shown in Figure l ) .
10 (v) Production of ~AF~ plants On backcrossing plants that carried the Cf-9*Ds and 35S:SP:Avr9 gene to tomato plant6 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, 355:5P:Avr9 and Cf-9*Ds (see Figure 13) plants showed somatic ~Tr;~r,n Of Ds from the Cf-9*Ds gene, somatically restoring Cf-9 function, and giving rise to necrotic somatic sectors in which the def ence 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 ~R that are associated with the induction of SAR.
Necrotic secto~s were visible on cotyledons, leaves, stems, petioies, sepals, and green fruits throughout plant development. Also, the necrotic sectors formed in both the lower and upper epidermis, in all mesophyll layers and in the cells surrounding the vascular tissue. The size of the necrotic sector and the frequency of their formation was determined by 5 both the position of the Ds element in the Cf-9 se~uence and the orien~t1c~n of the Ds.
The plants that variegated for necrosis were tested to assess if they were more resistant to C.
fulvum than their unvariegated ~ihl;n~q that either 10 carried Cf-9*Ds or carried no Cf-9 gene. Plants from five ;nf~PrPn~Pn~ Cf-g*Ds pedigrees were tested in which the Ds had ;n~PrPn~Pn~y inserted into five different locations in the Cf-9 gene. These five ;n~PrPn~Pnt insertions were between Cf-9 amino acids, 7 and 8 (<M23), 28 and 29 (~M18), 47 and 48 (>M50), 56 and 57 (~M31) and 789 and 790 (~M30) The arrows (~ or :.) indicates the direction of transcrlption of the Ds element. Fl plants that developed somatic necrotic sectors were more resistant to C. fulvum than sibling 20 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 25 showed on average 3 8 large sporulating pustules per - leaf. A example of this is shown in Figure 2.
Nine variegated Cf-9*Ds #20 plants, fifteen WO95131564 21 88562 1~11. 1 ,~

variegated Cf-9*Ds #23 plants, eighteen variegated Cf-9*D8 #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 5 necrosis. Plants were inoculated with C. fulvum (5 x 105 spores/ml) when they were four weeks old and carried 2 ~An~l,od leaves. A similar result was obtained when variegated Cf-9*Ds #50 plants and non-variegated plants were; nnrlll A~ed with C. fulvuzn. On 18 variegated GARt #50 plants 1-3 pustules per lea~
formed, whereas on 42 non-variegated GAR- #50 plants over 35 pustules per leaf developed by 14 days after ;nnClllAt;rn.
Sensitivity to the pathogen was measured by rclln~;n~ the number of sporulating pus~ules 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 ex~oressed on the y axis as the number of sporulating pustules per leaf. The Ds carried a GUS gene. M20, M23, M30 and M31 show C. fulvum growth on plants resulting from crosses between Cf-9*Ds and sAc, and derive from Cf-9*Ds #20, Cf-9*Ds #23, Cf-9*Ds #30 and Wo9s/3ls64 2 1 88562 ,~ L~ "
Cf-9*Ds #31, respectively. These individuals segregate from the Cf-9*Ds and for sAc. CfO carries no R genes - and M20, M23, M30 and M31 GUS- plants have lost by segregation both Cf-9*Ds and sAc and are thus 5 disea6e sensitive sibs, providing a good control for disease symptoms in sensitive individualæ. 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 10 individuals (which all carry the 35S:~vr9 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 15 pustules per leaf and non-vari~t; n~ individuals derived from Cf-9*Ds #20, Cf-9*Ds #23 or cf-g*Ds #3 also showed about 3 8 pustules per leaf . The non-variegated individuals that carried Cf-9*Ds #30 showed about 17 pustules per lea~ i~dicating some residual 20 action of the tagged Cf-9 allele. ~lowever, variegated individuals that carried Cf-9~Ds #20, Cf-9*Ds #23, Cf-g*D8 #30 or Cf-9*Ds #31 showed 1-3 pustules per leaf.
In total seventy variegated individuals were assessed.
These results demonstrate a very si~n i f i l-~nt level of 25 disease control by this method.
- Figure 12 shows three leaves. Leaf 1 and Leaf 2 are infected with C. fulvum which confers the white Wo 95/31564 2 ~ 8 8 5 6 2 PCT1Gsg5/01075 fluffy appearance. Leaf l 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 355:Avr9. Leaf 3 is therefore expressing GAR.
It is important to recosnize that in this example regions of variegating plants that resist the C fulvurn pathogen do not contain a functional Cf-9 gene. Indeed all the cells that do carry a fllnrt j r~n~l Cf-9 gene (whose function was restored somatically by transposon f.Yr; R; nn) are killed as they turn on the defence response after recognition of the ~nrlrg~nrl1cly expressed Avr9 peptide. Thus, non resistant cells are being induced to resistance by necrosis being manifested in adjacent cells.
E:XA~PLE: 2 Pathogen reBi6t~nce of v~riegated pl ntB employlng Cf-9 In addition to demonstrating that variegated plants produced in Example l have ~nh~nr~d resistance to C. fulvum, we have est~hl; F~h~ that the plants are also more resistant to three unrelated fungal pathogens, Phytophthora infestan~ (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).

WO 9S/31564 2 1 ~ ~ ~ 6 2 PcT/Gs95/01075 For the P. infestans experiments, sibling backcross progeny from the mutatnt Cf-g* Ds li~es M31 and M50 that were either variegating for necrosis or not and control plants lacking a Cf-gene (CfO) were 5 challenged by a spray application of sporangiosspores (lO,OOO or 100 spores/ml) of the highly virulent isolate DSSI (Al mating type). After ;nrr~ t;on, the plants ~qere kept in diffuse light conditions at a constant 1009~ 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 Cf O plants were completely destroyed by the spread of P.infestans lesions which had i~hlln~n~
sporangiospores at their margins. In contrast, the 15 variegated plants were infected with P. infestans but the lesions were 3-5 mm in ~;: ~t~r and non-sporulating (Figure 3 A, B) . An additional 5-6 days were re~uired before the enti~e green leaf tissue of the variegated plants was destroyed and fungal sporulation ~ r~d.
20 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 Cf O plants whereas on the plants variegating for necrosis there were 1-2 small non-sporulating lesions per 10 leaves 25 (Figure 4 A, B) . A minimum of 18 plants were used for - each genotype/spore.
For the Oidium lycope~sici experiments the WO95/31564 21 88562 1~1~ 5 .~75 .

i~nt i ~ l plant genotypes werë 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 surf ace . The inoculated plants were then kept under diffuse ~ight conditions at 20 C
during the 16 h photoperiod and at 18 C during the dark period. The RH was ~n~int;~;n~tl at 7096.
By day 10 post inor~ ti~n 8-10 chlorotic lesions were evident on the leaves of the unvariegated and Cf 0 plants and in 1-2 of these sporulation had ~ e~l.
By contrast on the variegated plants 1-2 smaller chlorotic non- sporulating lesions were present on each leaf (Figure 5). By day 14 post inor~ t;~n more than 20 sporulating lesions per leaf were present on the unvariegated plants and these were ~: _ i ed by severe chlorotic symptoms on the l. ; n~-~ of the leaf .
On the variegated plants 2-4 small sporulating lesions were present per leaf (Figure 5A). An additional 7-10 days were reSEuired before a similar level of sporulation and chlorosis formed on the variegated leaves to that f olmd on the unYariegated and Cf 0 leaves at day 14 post-inoculation. (16 plants each).
E~PLE 3 Variegation in fruit Dark green sectors formed on green tomato fruits of GAR plants, 5 weeks after flower po11 in~ti~n (Figure .

~ Wo 95/31564 2 1 8 8 5 6 2 ~ 075 6~ . These sectors were not visible once the tomato fruit had turned red, which is encouraging for potential commercial exploitation. When mature red fruit taken from GAR+ and GAR- plants were injected with lO0f~1 of spores of Colletotrichum laginariu~ (104 spores/ml) only the GAR- fruit exhibited the typical soft rot disease symptoms seven day6 later. Repeated ino~ t;~nc of the GAR+ fruit failed to cau3e 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 f-lnct;on whilst constitutively expressing the Avr9 gene. The data also indicate that the disease control achievable by this method is pot~nt ~ y broad spectrum because the four fungal pathogens controlled have very dissimilar modes of parasitism: C. fulvu~r~ is a biotroph that does not form haustoria and grows exclusively in the extr~ r 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. infe6tans and C.largenarium are hemibiotroph that initially forms simple haustoria but later on kills host cells in both the epidermal and mesophyll layers.
- 25 ~1~ y~cJus Cf-9*Ds, 35S:SPAvr9 lines have been established for the tomato lines M31 and M50. The W095/3l564 2 1 8 ~ 5 62 .~. ~ 5 ~1075 backcros3 progeny derived from crosses to a h _yyuus sAc source, may be assessed for their resistance to various pathogens, including:
Potato virus X, Ps~ syringae pv. tomato, Necrotrophic fungi - Botrytis spp, Colletotrichum spp, N: to~f~c - Meloidogyne incognata, Aphids - Green Peach Aphid, and fruit, pod, root or tuber attacking pathogens. Also, the effect of GAR on the est~hl;~h~ of mycorrhizal assor;~ti~n~ may be tested.
The ~nh~n--cl 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 lif e .
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 GARt plants. Examples of this are shown in Figure 7 for C~-9*Ds lines from M23, M31 and M50 pedigrees using a basic tomato ~-1,3 glucanase probe and a tomato anionic peroxidase probe (pTAP ~ . 5 ) .
The ef f ectiveness of GAR in suppressing plant disease appears to be inversely related to sector size.
The two; n-l~r~n~nt Cf -9*Ds pedigrees that have the highest frequency of small necrotic sectors (lines M31 wo ss/31s64 r~ 07~

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 specif ic 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 ~p~^ated. Provided the re~uired genetic, _ ^-lt~ are present, GAR plants have ~nl~n~ed pathogen resistance ,~ ed with wild-type.
E~A~PI,E: 4 Expre/33ion of Cf-9 in Heterologous Pl~nt~ Species and Induction of Cell Necro~i~
We have shown that following the transfer of different genomic clones rnnt~;nin~ the Cf-9 gene into tobacco and potato, these sequences render the ~Ld~S~ ic 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 2 1 ~ 8 5 6 2 express Avr9 peptide constitutively, the Fl seedlings die within 2 days of seed g~rm;n~t;rm (Figure 9).
When transgenic Ar~hi~ p~is expressing Cf-9 is crossed to Avr9 expressing transgenic Ar~hi~f~rs;~ the Fl seedlings die lO days after seed germination (Figure 10) .
Thus we have shown that in a variety of species, genes required for activation of plant defence, mediated by the Cf-9 protein, are present and functional.
EXA~P~E 5 Genetic Ac$~uired liesist~nce Using Cf-9 in Potato To apply GAR to potato plants a single T-DNA
construct systems is used.
The system i8 based around a single T-DNA
construct (Figure ll) rnnt~;n;ng~ a Cf-g gene seqn~nr~
under the control of its own promoter which has been inactivated by an ~ltnr ~ Ac element that is only capable of a low level of ~ n (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 or;!~nt~ti-~n~:
in order to determine the best conf iguration to produce a high fre~uency of small somatic sectors where Cf-9 function has been restored.
Placing the C~-9 sequence or other R gene ~ W0 95~31564 2 1 8 ~ 5 6 2 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.
5 E~PLE 6 Expres~ion o Cf-g in trancgenic plants and demonstration of increased pathogen res~tance The Cf-4 gene has been tested in transgenic plants in a number of ways: firstly by inocl~lAtinn with 10 a race of C. .fulvum -,mt:-;n;n~ the corr~qp~n~l;n~
avirulence gene Avr4 to test if that race gives an ; n. t; hle response on the transgenic plant; secondly by injecting leaves of a transformed plant with intercellular fluid; Aol~ted from a compatible 15 interaction r~nt~;n;n~ AVR4; thirdly, by delivering AVR4 in the form of r~__ ` ;n~nt potato virus X as described previously in studies of the Cf-9/AVR9 interaction (TT i-Kosack et al., 1995).
The DNA sequence of the C. fulvurn gene ,~nr~n~; n~
20 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 pllhl i Ch~-:l sequence and fused a sequence encoding the proposed mature 25 polypeptide to a DNA sequence encoaing the N-t,~nini, Wo95/31564 21 8 8 5 6 2 PCT1GB95101075 signal peptide of the tobacco PRla protein. This would f acilitate targeting of AVR4 ~o the intercellular space in transgenic plants where it is expressed. This chimeric gene (SPAvr4~ was inserted into a cDNA copy of 5 potato virus X, as a ClaI/Sa~I DNA fragment ~SEQ ID NO.
13) as described previously (Hammond-Kosack et al.,l995) to generate PVX:SPAvr4. Infectious transcripts of the rerr-~i n~nt virus were generated by in vitro tran8cription. All nucleic acid r~~n;r~ tions lO were performed using standard techniques well known to those skilled in the art.

Toma to Experiments were designed to test the r/~c ' ;n:lnt virus in 3 week old tomato 8eedlings. In Cf-4 15 rnnt:~;n;n~ plants inoculated cotyledons appeared desiccated and eventually Ahsr; ~ed at 3 days post-inoculation Id.p.i.), in contrast to CfO controls which only showed signs of slight mechanical damage at the site of virus innC1~l~tir~n. CfO plants developed 20 visible symptoms of virus infection at 7-lO 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 rnnt;3;n;ng Cf-4 necrotic lesions were observed in the younger leaves, presumably due to systemic 25 spread of the virus as described previously in similar experiments with PVX -nnt~;n;ng Avr9 on Cf-9 rnnt~;nin~

WO 95131564 6 ~ /a 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 ~ntA;n1n~
5 seedlings had died . Cf O 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 f~ i tor AVR4 .

10 To~acco Using binary vector cosmids comprising cf-s, transgenic tobacco plants have also been produced (Fillatti et al . ,1987; Horsch et al., 1985) using techni5Iues well known to those skilled in the art.
Transgenic tobacco ~-~nt~;n;n~ cosmids comprising C-4 were inoculated with PVX:SPAvr4. In most transformants necrotic lesions were observed at the site of viru8 inO~1l7At;c n 3-4 d.p.i. similar in appearance to lesions which appear in response to virus 20 inocll1At;C~n in some virus resistant varieties. In these individuals the necrosis was not strictly nf;n~d to local lesions which eventually coalesced and at 7-10 d.p. i . leaf necrosis was apparent over the entire region of virus inoclllAt;~n. In several 25 transformants the reaction to PVX:SPAvr4 was more acute and the necrotic leaf sectors could be observed at 3-4 WO95131564 21 ~ 8 5 ~ i /a d.p . i . Neither o~ these phenotypes were observed in transgenic tobacco rnnt~;n;nr~ cosmids lacking Cf-4 or in non-transformed control plants rh~llPnrJP~ with PVX: SPAvr4 .
F-1nrt;rn~1 expression of cf-s in transgenic tobacco has thus also been shown, with activation of a necrotic defence response in the presence of elicitor AVR4 .
Pa thogen Resi s tan ce 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 rnnt~;n;nJ Cf-4 exhibited leaf necrosis on ;nn~ tPd leaves 3-4 d.p.i. This necrosis eventually spread systemioally as previously obsc:L v~d in Cf-4 rnnt~;n;n~ plants in the expP~i c 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-9 function by inoculation with C. fulvum race 5. In 5 transgenic plants tested, a positive correlation was observed between plants exhibiting PVX:SPAvr4 flPren~lPnt necrosis and resistance to the pathogen. In this P~Pri~^^nt pathogen growth was .bseLve-l on c -tihle control ~ Wo9Sl31564 2 1 ~ 8 5 6 2 ~ J

plants (CfO) but not on incompatible control plants (Cf2) .

All documents - ; ~n~-cl in the text are incorporated herein by reference.

5 ~r Balint-Kurti, et al. (1994~ Theor. App. Genet.
88: 691-700 .
Basler, et al. (1991) Cell, 64, 1069-1081.
Becker,' et al. (1993) Plant Journal 3, 875-881.
Bent, A.F., et al. (1994) Science 265, 1856.
Biffen, R.H. (1905) J. Ag. Sci. 1, 4-48.
Carroll B.J. et al. (1993). Genetics (In Press).
Chang, C., et al. (1992) The Plant Cell 4:1263-1271.
ChristoU, P. (1992) The Plant Journal, 2 (3), 275-281.
Coen, et al. (1989) In Mobile DNA. D.E. Berg and M.M.
Ho~e, eds. (Washington: ASM Pres), ~'r.rn~ s~n, et al. (1987) Nucl .Acids.Res 15 :6799-6811.
Culver, et al. (1991) Molecular Plant Microbe Interactions 4, 458-463.
De Wit, P.J.G.M. (1992) . Ann. Rev. Phytopathol. 30, 391-418 .
DeGreve, et al. (1983) J.Mol.Appl.Genet. 1:499-511.
Dickinson, et al. (1993) . Mol. Plant Mic. Int. 6, 341-- 347.
25 Dietrich,~ R.A., et al. (1994) Cell 77, 565-577.

Doring X-P ~1989) . An overview. Maydica 34: 73-88 .
Enyedi, et al. (1992) Cell 70, 879-886.
Fedoroff, N.V. (1989) In Mobile DNA. M. Xowe and D.
Berg, eds. (Washington: ASM Press), pp. 375-411.
Fillatti JJ, et al. (1987). Bio/technol. 5:726-730.
Flor, X.H. (1971) Ann. Rev. Phytopathol. 9, 275-296.
Gabriel, et al. (1990) Ann.Rev.Phytopathol. 28:365-391.
Gaffney, et al. (1993) Science 261, 754-756.
Goulden, et al. (1993) The Plant Cell 5, 921-930.
Greenberg, J.T., et al. (1994) Cell 77, 551-563.
Greenberg, et al . (1993 ) Plant Journal 4, 327-341.
Hamilton, et al. (1990) Nature 346, 284-287.
Hammond-Kosack, K.E., et al. (1994) Proc. Natl. Acad.
Sci. USA 91, 10445-10449.
Xammond-Kosack, et al. (1994) The Plant Cell 6, 361-374 .
Xammond-Kosack et al. (1995) Mol. Plant-Microbe Interact . 8 :181-185 Xe, et al. (1993) Cell 73, 1255-1266.
Hennig, et al. (1993) Plant Journal 4, 481-493.
Herrera - Estrella, et al. (1983) EM;30 J. 2, 987-995.
Xorsch RB, et al . (1985) . Science (Wash. ) 227: 1229-1231 .
Jefferson, et al. (1987) EM~30.J. 6 :3901-3907.
Johal, et al. (1992) Science (Wash.). 258:985-987.
Johal, et al. (1994) Maydica 39, (in press).
Jones, et al. (1992) Transgen.Res. 1:285-297.

~ WO95131564 21 88562 .~1~ .s~07~

Jones, D.A., et al. (1994) Science 266, 789.
Jones, et al. (1993) Mol. Plant Mic. Int. 6, 348-357.
Jones, et al. (1994) Curr. Biol. 4, 67-69.
Joosten, et al. (1994). Nature 367, 384-386.
Kavanagh, et al. (1992) Virology 189, 609-617.
Keen, N.T. (1992) Ann. Rev. Gen. 24, 447-463.
Keller, et al. (1993) Molec. Biol. 21, 159-170.
Kohm, et al. (1993) The Plant Cell 5, 913-920.
I,loyd, et al. (1994) Mol Gen Genet 242, 653-657.
I.ong, et al. (1993) Cell 73, 921-935.
Malamy, et al . (1990) Science (Wash. ) . 250, 1002-1004 .
Mariani, et al. (1990) Nature 347, 737-741.
M~ e, et al. (1993) MPMI 6, 412-417.
Martin, et al. (1993) Science 262, 1432-1436.
Metraux, et al . (1990) Science (Wash. ) . 250, 1004-1006 .
Mindrinos, M., et al. (1994) Cell 78, 1089-1099.
Mittler, R., et al. (1995) The Plant Cell 7, 29-42.
Napoli, et al. (1990). The Plant Cell 2,279-289.
Odell, et al. ~1984) Nature 313:810-812.
Odell, et al. (1990) Mol. Gen. Genet. 223, 369-378.
Padgett, H.S., et al. (1993) The Plant Cell 5, 577-586.
Pryor, et al. (1983) Advanceæ in Plant Pathology 10, 281-305 .
Pryor, T. (1987). Trends. Genet. 3,157-161.
Rommens C.M.T., et al. (1992) Pl.Molec.Biol. 20:61-70.
Rommens, C.M.T., et al. ~1995) The Plant Cell 7, 249-257 .

WO 95l31564 21 ~ 2 ~ ~ i /a Ross, A.F. (1961) Virology 14, 340-358.
Ryals, et al. (1992) SEB Symposium 49, 205-229.
Ryan, C.A. (1990) Ann. Rev. Phytopathol. 28, 425-449.
Santa Cruz, et al. (1993~ Molecular Plant-microbe Interactions 6, 707-714.
Scofield, S., et al. (1992) The Plant Cell 4:573-582.
Scott, A., et al. (1994) The Plant Cell 6, 1845-1857.
S t ein , J . C ., et al . ( 1 9 9 1 ) Pro c . Nat l . Acad . S c i . USA
88: 8816-8820 .
Swinburne, et al (1992) The Plant Cell 4, 583-592.
T~kAh~h;, et al (1989) Molecular & General Genetics 216, 188-194.
Thomas, C.M., et al. (1993) Molecular and General Genetics (In Pre8s) .
Thorsness, et al (1983) The Plant Cell 5, 253-261.
Triglia, T., et al. (1988) Nucleic Acids Res. 16:8186.
Uknes, et al. (1992) The Plant Cell 4, 645-656.
Valon, C., et al. (1993) Pl.Molec.Biol. 23:415-421.
van den Ackerveken, et al. (1993) Plant Physiol.
van der Beek, et al. (1992) Theor.App.Genet. 84:106-112 .
van den Elzen, et al. (1985) Plant. Mol. ~3iol. 5, 149-154 .
van Kan J.A.L., et al. (1991) MPMI 4 :52-59 .
van den Elzen, et al. (1985) Plant. Mol. Biol. 5, 299-302 .
Van Den Ackerveken, et al. (1992) Plant Journal 2, 359-~ ~O 95l~5~4 2 1 8 8 5 6 2 P~r/GB9S/01075 366 .
Walbot, V., et al. (1983) T. Kosuge et al, eds. (Plenum Press), pp. 431-430.
Walker, J.C., (1993) Plant Journal 3 :451-456.
Ward, et al (1991) The Plant Cell 3, 1085-1094.
Nei, et al. (1992) Science (Wash. ) . 257, 85-88.
Whitham, S., et al. (1994) Cell 78, 1101-1115.
Wolter, M., et al. (1983) Molecular & &eneral &enetics 23 9, 122 - 128 .

WO 95131564 2 1 8 8 5 6 2 PCrlGB95/0107~
~2 BEQ ID NO. 1:
CATAGTCTTT r,r r T~TTTRr ATTAAACAGG GGCATTATTG r r rr~ 7` r rTr TTAGATGTAT 6 0 GAaAATTTTG G~rr7~ Tr~ TTGACAACAC GAACATTTTT rr'`''rrD7`rT ATTAACTCAG 120 AATATTTTCC GTTGAATGAA Tr~ TrrrT rrTrrTrD7~T TTTTAGACCA AACTATGAAG lB0 AACATGCCAT GTCTGGACTC CTGCACTATC TTCCATCAAC AGGTCAATTC TCTCAACTCT . 240 ATTGGTGGAA r~rTrr~rr~T ACAaATTGAA TTATATTAD~A AGACAAGCTC rrrTrr~ rT 300 CACTGTTATA rADrDDrrrr r7~DrTrrrrT TCAGCCCCAA ArrrTrrTr~A CCCGAATCAT 360 CATTCTGATG CTTCGTACAA ATTTATTGAA TTTTcAacTT TAAAGGTTTA TGAACCAAAT 4 a o ATTACGCTTA rTrTrATrrr ~ L ' ' ' L GATTAATCAA ACTTATTGAA TTTTCAACTT 540 TADAGGTTTT "~L~7L'il ATrrrrDr7~r TA~Dr~.T TTAaATTATA TAGTCTTTGG 600 ATGGTGACCT ATTTGGATGG TAACATTATT C~ ~ rA7~rrT ATTGATAACG rr"~ ATTrT .660 Tr~ rr r Dr TGAGAAGGAC ATGTCTGGAC L~l ~l L~ ~ ~i L_ LLil_ L~ r,rrr~r~TrrDT 720 TCTTGTGGAD AATTAGCTCG rrr~T~r,rrrr CTATGTGAGG TDrrTr~rTrr TAaATTTTTC 780 TTTGCTTAAT LL~L~ L~TA TDTArrTrDT cTA~D~ATTATT GAATAGTCAC DrD-~'"DDD 840 CATTTCTTGA LLL~LL~L`'L ATCAACATAA CAAGTTTTGA l~I L .L~G TGCDGAA 897 ATG GAT TGT GTA AAD. CTT GTA TTC CTT ATG CTA TAT ACC TTT CTC TGT 945 Met Asp Cy5 Val Ly8 Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys Oln Leu Ala Leu Ser Ser Ser Leu Prr~ }~is Leu Cys Pro Glu Asp Gln _5 1 5 Ala Leu Ser Leu Leu Gln Phe Lys Asn Met Phe Thr Ile ABn Pro ABn Ala Ser Asp Tyr Cys Tyr Asp Ile Arg Thr Tyr Val Asp Ile Gln Ser TAT CCA AGA A~:T CTT TCT TGG AAC ADA AGC ACA AGT TGC TGC TCA TGG 1137 Tyr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys Cys Ser Trp GAT GGC GTT CAT TGT GAC GAG ACG ACA GGA CAA GTG aTT GCG CTT GAC 1185 Asp Gly Val E~i3 Cys Asp Glu Thr Thr Gly Gln Val Ile Ala Leu Asp CTC CGT TGC AGC CAA CTT C~ GGC AaG TTT CAT TCC AAT AGT AGC CTC 1233 Le~ Arg Cys Ser Gln Leu Gln Gly Lys Phe Elis Ser Asn Ser Ser Leu Phe Gln Leu Ser Asn Leu LYR Arg Leu Asp Leu Ser Phe Asn Asn Phe go 95 100 105 Thr Gly Ser Leu Ile Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr _ _ _ _ _ _ W0 95/31564 73 1 ~ 107S

Xis Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu Ile Pro Ser Glu ATC TGT CAC CTT TCT A~ CTA CAC GTT CTT CGT ATA TGT GAT CAA TAT
Ile Cys E~is Leu Ser Lys Leu l~is Val Leu Arg Ile Cys Asp Gln Tyr 1425 GGG CTT AGT CTT GTA CCT TAC AAT TTT GAA CTG CTC CTT A~G AAC TTG 47 Gly Leu Ser Leu Val Pro Tyr A5n Phe Glu Leu Leu LeU Lys Asn LeU 1 3 Thr Gln Leu Arg Glu Leu Asn Leu Glu Ser Val Asn Ile Ser Ser Thr 1~0 175 180 185 Ile Pro Ser Asn Phe Ser Ser E~is Leu Thr Thr Leu Gln Leu Ser Gly Thr Glu Leu E~is Gly Ile Leu Pro Glu Arg Val Phe EIis Leu Ser Asn TTA CAA TCC CTT CAT TTA TCA GTC AAT AC AGG

CCC ACA ACC A~A TGG AAT AGC AGT GCA TC~ CTC.ATG ACG TTA TAC GTC 1713 Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Tyr Val 235 240 24s GAT AGT GTG AAT ATT GCT GAT AGG ATA CCT A~A TCA TTT AGC C~T CTA 1761 Asp ser Val Asn Ile Ala Asp Arg Ile Pro Lys Ser Phe Ser Elis Leu Thr Ser Leu llis Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro ATT CCT AaA CCT CTA TGG AAT CTC ACC APC ATA GTG T~T TTG CAC CTT 1857 Ile Pro Ly3 Pro Leu Trp A5n Leu Thr Asn Ile Val Phe Leu Elis Leu GGT GAT AaC CAT CTT GAA GGA CCA ATT TCC CAT TTC ~CG ATA TTT GAA 1905 Gly Asp Asn His Leu Glu Gly Pro Ile Ser Ili5 Phe Thr Ile Phe Glu AaG CTC AaG AGG TTA TCA CTT GTA AAT AAC AAC TTT GAT GGC GGA CTT 1953 Lys Leu Lys Arg Leu Ser Leu Val Asn Asn Asn Phe Asp Gly Gly Leu GAG TTC TTA TCC T~T AAC ACC CAA CTT GAA CGG CTA GAT TTA TCA TCC 2001 Glu Phe Leu Ser Phe Asn Thr Gln Leu Glu Arg Leu Asp Leu Ser Ser Asn Ser Leu Thr Gly Pro Ile Pro ser Asn Ile Ser Gly Leu Gln Asn 3so 355 360 Leu Glu Cys Leu Tyr Leu Ser Ser Asn E~is Leu Asn Gly Ser Ile Pro 365 370 37s Ser Trp Ile Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn 380 38s 390 WO9a/31564 21 8~62 l~vl .v/a AAC ACT TTC AGT GGA AAA ATT CAA GAG TTC AAG TCC Aa~ ACA TTA AGT 2193 Asn Thr Phe Ser Gly Lys Ile Gln Glu Phe Lys Ser Lys Thr Leu Ser 3gS 400 405 GCC GTT ACT CTA AaA CAA AAT AaG CTG AAA GGT CGT ATT CCG AAT TCA 2241 Ala Val Thr Leu Lys Gln Asn Lys Leu Lys Gly Arg Ile Pro Asn Ser Leu Leu Asn Gln Lys AGn Leu Gln Leu Leu Leu Leu Ser HiD Asn Asn Ile Ser Gly His Ile Ser Ser Ala Ile Cys Asn Leu Lys Thr Leu Ile Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr Ile Pro Gln Cys GTG GTT GAG AGG AaC GAA TAC CTT TCG CAT TTG GAT TTG AGC AaA AAC 2433 Val Val Glu Arg Asn Glu Tyr Leu Ser His Leu Asp Leu Ser Lys Asn Arg Leu Ser Gly Thr Ile Asn Thr Thr Phe Ser Val Gly Asn Ile Leu AGG GTC ATT AGC TTG CAC GGG AAT AaG CTA ACG GGG AaA GTC CCA CGA 2529 Arg Val Ile Ser Leu His Gly Asn Ly9 Leu Thr Gly Lys Val Pro Arg ~10 515 520 TCT ATG ATC AAT TGC AaG TAT TTG ACA CTA CTT GAT CTA GGT AaC Aa~T 2577 8er Met Ile Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gln Leu AAG ATT TTA AGC TTG AGA TCA AAT AAG TTG CAT GGT CCC ATC AaA TCT 2673 Lys Ile Leu Ser Leu Arg Ser Asn Lys Leu His Gly Pro Ile Lys Ser 5er Gly Asn Thr Asn Leu Phe Met Gly Leu Gln Ile Leu Asp Leu Ser TCT AAT GGA TTT AGT GGG AaT TTA CCC GAA AGA ATT TTG GGG AAT TTG 2769 Ser Asn Gly Phe Ser Gly Asn Leu Pro Glu Arg Ile Leu Gly Asn Leu CAA ACC ATG AaG GAA ATT GAT GAG AGT ACA GGA TTC CCA GAG TAT ATT 2817 Gln Thr Met Lys Glu Ile Asp Glu Ser Thr Gly Phe Pro Glu Tyr Ile Ser Asp Pro Tyr A3p Ile Tyr Tyr Asn Tyr Leu Thr Thr Ile Ser Thr Lys Gly Gln Asp Tyr Asp Ser Val Arg Ile Leu Asp Ser Asn Met Ile Ile Asn Leu Ser Lys Asn Arg Phe Glu Gly His Ile Pro Ser Ile Ile ~ ~0 95131564 2 1 8 8 5 6 2 P l ~ C . ,07~
GGA GAT CTT GTT GGA CTT CGT ACG TTG AAC TTG TCT CAC AaT GTC TTG 3009 Gly Asp Leu val Gly Leu Arg Thr Leu Asn Leu Ser His Asn Val Leu 670 67s 680 Glu Gly His Ile Pro Ala Ser Phe Gl n Asn Leu Ser Val Leu Glu Ser 685 690 69s TTG GAT CTC TCA TCT AAT AaA ATC AGC GGA GAA ATT CCG CAG CAG CTT 3105 Leu Asp Leu Ser Ser Asn Lys Ile Ser Gly Glu Ile Pro Gln Gln Leu Ala Ser Leu Thr Phe Leu Glu Val Leu Asn Leu Ser His Asn His Leu GTT GGA TGC ATC CCC AAA GGA AaA CAA TTT GAT TCG TTC GGG AAC ACT 3201 Val Gly Cys Ile Pro Lys Gly Lys Gln Phe Asp Ser Phe Gly Asn Thr TCG TAC CAA GGG AAT GAT GGG TTA CGC GGA TTT CCA CTC TCA AaA CTT 3249 Ser Tyr Glr. Gly Asn Asp Gly Leu Arg Gly Phe Pro Leu Ser Lys Leu Cys Gly Gly Glu Asp Gln Val Thr Thr Pro Ala Glu Leu Asp Gln Glu Glu Glu Glu Glu Asp Ser Pro Met Ile Ser Trp Gln Gly Val Leu Val Gly Tyr Gly Cy8 Gly Leu Val Ile Gly Leu Ser Val Ile Tyr Ile Met Trp S Thr Gln Tyr Pro Ala Trp Phe Ser Arg Met A~p Leu Lya Leu GAA CAC ATA ATT ACT ACG AAA ATG AAA AAG CAC AAG AaA AGA TAT TAGTGAGTAG 3496 Glu His Ile Ile Thr Thr Lys Met Lys Lys His Lys Lys Arg Tyr CTATACCTCC AGGTATTCCA ~LL~I~Ll ATCTTTCAGA AGATTAmT I1~1~L~ 3556 ATGA~ATTAT CGACCTCCTT CATCCTCAAA GCTCTTA~CT TTCACTCTTC ATmTGAAA 3616 ATTTCAGGAT TcAaAGATTT CCGAGTTCCC AGTTGCTTGG GATGCAGATA AaAGccmT 3676 TATCTTTCAT AGTTTCTTAT CCTATGAATA AaGATTTTAT TTTCATTTGT CTATGGCACG 3736 TAGATATGTT CCGTCACTAA AaAcATTGTA TTTCTCTCAA ~L~LLO~L~ ACATGATATC 3796 r~r~rT TGACTTCAAT TAAGTTACTG TAGTCTGCTA TTTTAATTTT TTcCATTGaA 3856 ACACAACTGA CGTATCTTGA (''~ "T ATGATCCCCC GGGCTGCAG 3905 8EQ ID N0 . 2:
, Met Asp Cys Val Lys Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys Gln Leu Ala Leu Ser Ser ser Leu Pro His Leu Cys Pro Glu Asp Gln _5 1 5 _ _ _ _ . .. .. . .. , . . _ .. _ .. _ . . _ . _ _ . . . . , . .. , .. _ _ . , . _ WO95/31564 2 1 8 8 5 6 2 ~ 5~. /a Ala Leu Ser Leu Leu Gln Phe Lys Asn Met Phe Thr Ile As Asn 10 lS 20 n Pro la Ser Asp Tyr Cys Tyr Asp Ile Arg Thr Tyr Val Asp Ile Gln Ser yr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys C s Ser T
45 50 y rp Asp Gly Val Elis Cys Asp Glu Thr Thr Gly Gln Val Ile Ala As 60 65 70 Leu p Leu Arg Cys Ser Gln Leu Gln Gly Lys Phe His Ser Asn Ser Ser Leu Phe Gln Leu Ser Asn Leu Lys Arg Leu Asp Leu Scr Phe Asn Asn Phe hr Gly Ser Leu Ile Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr 110 llS 120 li5 Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu Ile Pro Ser Glu Ile Cys Elis Leu Ser Lys I,eu Hi8 Val Leu Arg Ile Cys Asp Gln Tyr 140 145 lS0 Gly Leu Ser Leu Val Pro Tyr Asn Phe Glu Leu Leu Leu Lys Asn Leu Thr Gln Leu Arg Glu Leu Asn Leu Glu Ser Val Asn Ile 170 - 175 180 Ser Ser Thr le Pro Ser Asn Phe Ser ser Hi5 Leu Thr Thr Leu Gln Leu Ser Gly 19~ 195 200 hr Glu Leu Hi8 Gly Ile Leu Pro Glu Arg Val Phe His Leu As 205 210 Ser n Leu Gln S~r Leu l~is Leu Ser Val ~sn Pro Gln Leu Thr Val Arg Phe Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Val 235 240 245 Tyr Asp Ser Val Asn Ile Ala ASp Arg Ile Pro Lys Ser Phe Ser P~is Lcu hr Ser Leu ~}is Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro le Pro Lys Pro Leu Trp Asn Leu Thr Asn Ile Val PhOE Leu His Leu Gly Asp Asn ~Iis Leu Glu Gly Pro Ile Ser ~Tis Phe Thr Ile Phe Glu Lys Leu Lys Arg Leu Ser Lcu Val Asn Asn Asn Phe Asp Gly Gly Leu Glu Phe Leu Ser Phe Asn Thr Gln Leu Glu Arg Leu Asp Leu Ser Ser 330 335 340 34s Asn Ser Leu Thr Gly Pro Ile Pro Ser Asn Ile Ser Gly Leu Gln Asn WO95131564 2 1 8 8 5 6 2 F~ J075 eu Glu Cys I.eu Tyr Leu ser Ser Asn llis Leu Asn Gly Ser Ile Pro 365 370 37s Ser Trp Ile Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn 380 3b5 390 Asn Thr Phe Ser Gly Ly6 Ile Gln Glu Phe Lys Ser Lys Thr Leu Ser Ala Val Thr Leu Lys Gln Asn Lys Leu Lys Gly Arg Ile Pro Asn Ser eu Leu Asn Gln Lys Asn Leu Gln Leu Leu Leu Leu Ser Elis Asn Asn le Ser Gly ~is Ile Ser Ser Ala Ile Cys Asn Leu Lys Thr Leu Ile Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr Ile Pro Gln Cys Val Val Glu Arg Asn Glu Tyr Leu Ser ~lis Leu Asp Leu Ser Lys Asn Arg Leu Ser Gly Thr Ile Asn Thr Thr Phe Ser Val Gly Asn Ile Leu Arg Val Ile Ser Leu Lis Gly Asn Lys Leu Thr Gly Lys Val Pro Arg er Met Ile Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gln Leu Lys le I.eu Ser Leu Arg 5er Asn Lys Leu His Gly Pro Ile Lys Ser Ser Gly Asn Thr Asn Leu Phe Met Gly Leu Gln- Ile Leu Asp Leu Ser er Asn Gly Phe ser Gly Asn Leu Pro Glu Arg Ile Leu Gly Asn Leu ln Thr ~et Lys Glu Ile Asp Glu Ser Thr Gly Phe Pro Glu Tyr Ile Ser Asp Pro Tyr Asp Ile Tyr Tyr Asn Tyr Leu Thr Thr Ile Ser Thr Lys Gly Gln Asp Tyr Asp Ser Val Arg Ile Leu Asp Ser Asn Met Ile 63s - 640 645 Ile Asn Leu Ser Lys Asn Arg Phe Glu Gly Lis Ile Pro ser Ile Ile ly Asp Leu val Gly Leu Arg Thr Leu Asn Leu Ser 3~is Asn Val Leu lu Gly Elis Ile Pro Ala Ser Phe Gln Asn Leu Ser Val Leu Glu Ser eu Asp Leu Ser Ser Asn Lys Ile Ser Gly Glu Ile Pro Gln Gln Leu WO95/31564 2 1 ~ /. CiO75 Ala ~er Leu Thr Phe Leu Glu Val Leu Asn Leu Ser His Asn iIis Leu 715 72n ~ 725 Val Gly Cys Ile Pro Ly6 Gly Lys qln Phe Asp Ser Phe Gly Asn Thr er Tyr Gln Gly Asn Asp Gly Leu Arg Gly Phe Pro Leu Ser Lys Leu ys Gly Gly Glu Asp Gln Val Thr Thr Pro Ala Glu Leu Asp Gln Glu Glu Glu Glu Glu Asp Ser Pro Met Ile Ser Trp Gln Gly Val Leu Val Gly Tyr Gly Cys Gly Leu Val Ile Gly Leu Ser Val Ile Ile Met 795 800 805 Tyr Trp Ser Thr Gln Tyr Pro Ala Trp Phe Ser Arg Met Asp Leu LyD Leu Glu Elis Ile Ile Thr Thr Lys Met Lys Lys E~is Lys Lys Arg Tyr 5BQ ID N0. 3:
ALLL~LL~iA lLL`'lL~L~L DTr~ 'pTP7l CAAGTTTTGA L~L~L~LL~G TGCAqAaATG 60 GATTGTGTAA PprTTr~TpTT rrTTPTGrTP TATACCTTTC TCTGTCPaCT l~i~LLLAi~ 120 TCATCCTTGC ~L~ALLL~L~i rrrrrrn--rT CAAGCTCTTT CTCTTCTACA ATTCDAGAAC 180 ATqTTTACCA TTAATCCTAA TGCTTCTqAT TATTGTTACG PrPTDn--r7~r ATPrr-TP~n-- 240 ATTCPqTCPT ATCCAAqAAC L~LLL~ LL ii~ p7~ p CAAGTTGCTG CTCATGGGAT 300 qqCGTTCATT GTGACGAGAC ~7~--Pr~r-Dn GTGATTGCGC TTGACCTcCq TTrrPrrr7 ?. 360 CTTCDAGGCA AGTTTCATTC rr nTDr.TPr~r CTCTTTCPAC TCTCCDATCT CPAAAqGCTT 420 ~AILL~iL~LL TTAATAATTT CACTqGATCP CTCATTTCPC CaaDATTTGq TGAGTTTTCA 480 AaTTTGACGC liTCTCqATTT GTCGCATTCT AGTTTTAcAq GTCTAATTCC TTCTqAaaTC .540 TGTCACCTTT rTP7~n~'TPrA ~ -, ATATGTGATC ~nTATr7nr7rT TPr.TrTTr.T~ 600 CCTTACAATT TTqAACTGCT rrTT~n--rn" TTrArrr7'7`T TAArArAnrT C-AAccTTqAA 660 TCTGTAAACA TCTCTTCCAC ~ AaTTTCTCTT rTrDTTTP7`r AaCTCTACAA . 720 CTTTCAGGCA CAqAGTTA13A ~iW~ALL~i rrrrn~ n-- TTTTTCACCT TTccaAcTTA 780 C,AATCCCTTC ATTTATCAGT CAATCCCCAq CTCACGGTTA GGTTTCCCAC A7.~rA-~-.Tnn 840 AATAGCAqTG CATCACTCAT GACGTTATAC GTCqATAGTG TqAATATTGc Tr~TpnnpTA 900 CCTAAATCAT TTAGCCATCT AACTTCACTT CATqAGTTGT AACATGGGTCG TTGTA~TCTG 960 TCAwGCCTA TTCCTAAACC TCT~TGGAAT CTCaCCAACA TAGTGTTTTT GCACCTTGGT 1020 GATAACCATC TTqAAGGACC AATTTCCCAT TTCACGATAT TTGAaAAGCT CaaGAGGTTA 1080 TCACTTGTAA ATAACAACTT TGATGGCGGA CTTGAqTTCT TATCCTTTAA CACCCAACTT 114 0 rrn ~rrrT~n AL1LA~ e~.I. CAATTCCCTA ACTwTCCAA TTCCATCCAA rPT~ rnD 1200 ~ WO95/31564 21 88~62 r~

CTTCAAAACC TAGAATGTCT CTACTTGTCA TCABACCACT TrAATGGGAG TATACCTTCC 1 '60 ~llO; ll- ACTGGTTGAG TTAGACTTGA Gr7`-TPnADr TTTCAGTGGA 1320 AaAGGTCGTA TTCCGAATTC ACTCCTAaAC rr~ 7~rr TACAATTACT ~ ~ 1440 rDrD~T~TD TCAGTGGACA TATTTCTTCA r~rT~TrTr-rA ATCTGAABAC ATTGATATTG 1500 TTAGACTTGG r71r~TDrlT~ TTTGGAGGGA ACAATCCCAC AATGCGTGGT Tr~ 1560 GAATACCTTT CGCATTTGGA TTTGAGCABA AACAGACTTA GTGGGACAAT rD~ TDrDDrT 1620 TTTAGTGTTG GA~ACATTTT AAGGGTCATT AGCTTGCACG C~ T7~7~r~rT 7~r~ 1680 TTGAATGACA CATTTCCBBA ~1~ 7~i~7~ TACCTATTTC D7~TT"'`~'"'T TTTAAGCTTG 1800 AGATCAaATA AGTTGCATGG TCCCATCABA TCTTCBGGGA ATACABACTT 7111~ W71 1860 CTTCAAATTC ll~ ATCTAATGGA TTTAGTGGGA ATTTACCCGA AAGAATTTTG 1920 GGGAATTTGC ~7~7~rrDTr7~ GGAAATTGAT r~ TDrDn GATTCCCAGA ~ -~l 1980 a~TrrDTATr. A~..lll~ll~ CBATTATTTG ACGACAATTT r~TDr~Dr""" ACBAGATTAT 2040 GATTCTGTTC GA~DTTTTGGA TTCTAACATG ATTATCAATC TCTCAAAGAA cAr~ATTTGAA 2100 GGTCATATTC rAAGCATTAT TGGAGATCTT GTTGGACTTC GTACGTTGBA CTTGTCTCAC 2160 AATGTCTTGG AAGGTCATAT ACCGGCATCA TTTCAaAATT T~TrDrTDrT CGAATCTTTG 2220 .l.l~:~T rT~7~T~ T rDrr~ ATTCCGCAGC AGCTTGCATC CCTCACATTC 2280 CTTGAAGTCT TABATCTCTC TCACBATCAT CTTGTTGGAT GCATCCCCBA 7~ 7 r7 r 2340 TCAABACTTT ~lu~ d7l~A AGATCAAGTG ACAACTCCAG rTrDrrTDrD TCAAGAAGAG 2460 r~ 7r. ATTCACCAAT GATCAGTTGG CAGGGGGTTC 1~ .l~ 2520 7 GACTGTCCGT 7`7'TDTDrDTA ATGTGGTCAA rTr~ TDTrr AGCATGGTTT 2580 TCGAGGATGG ATTTAAAGTT rr~ DrDTA ATTACTACGA D"7`T"''7.~ rrDrD~ 2640 AGATATTAGT r'"TDnrTDT ACCTCCAGGA TTCAAAGATT TCCGAGTTCC CAGTTGCTTG Z700 GGATGCAGAT ABAAGCCTTT 1~ lll~ TAGTTTCTTA TCCTATGAAT AAAGATTTTA 2760 TTTTCATTTG TCTATGGCAC rTD--`TDTr~T TCCGTCACTA AAAACATTGT A111.1~ A 2820 ACTCTTTCGT CACATGATAT rD~ DrDr TTGACTTCAA TTAaGTTAAA D7~ D7~7~ 2880 8EQ ID ~o . 4:

Met Gly Phe Val Leu Phe Ser Gln Leu Pro Ser Phe Leu Leu Val Ser Thr Leu Leu Leu Phe Leu Val Ile Ser Hia Ser cy8 Arg Ala Tyr Cys _ .. . _ .. .. _, _ .. _ .. _ .. .. _ . . . _ _ _, _ . . _ _ _ , _ .. _ _ ,, . _ .. _ . ~

WO 95/31564 2 ~ 8 3 5 6 2 ~ 1075 AAC AGT TCT TGT ACA AGA GCT TTT GAC TGT ~CTT GGA CAA TGT GGA AGA 144 A~n Ser Ser Cys Thr Arg Ala Phe Asp Cys Leu Gly Gln Cys Gly Arg Cys Asp Phe His Lys Leu Gln Cys Val ~is CTCGAGTTCG r~rrTDD~7~ rrTDT7\~DDT ATTAZ~TA~AA ATTTTAAaAT
51 GGTATATCAA l l l . l~--.A'L 1 D D rrD 7~ rr. TCAMATCGC TGMACAACA
101 GCGATTTCCT TrDrrrr`'`n AAGCAAaATc GCTACTACTG CAGCGATTTT
151 GCAMATGTA ACTTTTTTT~ ~MAMATGC Ai ~ L~L l ATD~r~rTDTD
2 01 TATTTGAATT TrD D 7~ 7. D D ~ D TATTTGAAAA TCAATAMAT l l ~i l l l l l ~--251 TACGATTTTC TTTTTAaAAT L~llLL~i GAMATCCCT DrrTDr~r-rDr~
301 w~IlL.~T TTTTAATTTT TTTTADATAA l~rrrrDr~rr~D TTTTCGAMA
351 ~DATTTT AAAAAMATT GADAAAGTCG CTGCCTAGGT AGCGATTTGA
401 ATTTTTTTAA AaAATGTTAT ATTTTGCAZLA ATCGTTGCAG Tarr~DrrDT
4 51 l L l~i~ 1111 .L TTGGAGGA~A l ~i~ l v L 1~1 TCCAGCGATT l L ~ , L L l l 501 rr-TTDDTATD ADATTTTATA TAaCGTTTTG AaATTTTTGT TAATATTTTA
551 TAACTTTTAG GCTCCGGACT CAAGATTACT ~l~ i~L TDrTTTDTD~
601 TGCATAGTCT GAATTTTGAA r`rrrD lDTD GTTTAATTTT rr~rrDTD~T
651 TCAGACATGA AATCTTTADA AaAGTTTAAA TAAaATTTGT ATATGTTGAA
701 ~rTDr~r'`DD A~DGTATTATA ATTCACGATA ATTTATTCAC MGCCATCGT

801 l~i:~l~ll~i~ GAGGTTGAGA TATCTTAATC TATCTCCA~DT P~D```D``r 901 CAAGATACTT CATCATATAA AaADATAATC TCCGTGMGA AATTCTTTTA
951 TT~GGAMAT CGATTTTAGA GTCATTGCAA TTTAATTTTA TCA~aATATT
1001 TGAGCATGAA AaATTTGMA TGGAGGTGTC ATDDD~'~TDD AATACCCTTT
1051 ~ DrDrr:r TTTATTGAGT TGACGATAGT TCADGTAGGG ~DDTD7~DTD
1101 ACTTATTAAT Tr``TDT7-7\D ACTTGCAAGA A~MAGTGAT ATTCMATTT
1151 AATTCTGACC ATTATCTCTT GATATTCTTT ~ l TATTTGAATA
1201 TTCATTTTTC A~D~AGTTcr-A CGTcATAaGA CATCADATAT CMGTAGGTC
1251 rrDTD7 ~\D7\T ~DDDTArrrT TCTCAACATG Dr~nDrDD`-- ATTGAaMAT

1301 GACTADCATT TTCTCADAGA rDDDDDrDD7~ ACATGTGAGA GMGACATTA
1351 wAATCATCA TAATCTCTGA GACTGAGMT TGTTAGATAT GGTCCACTAC

WO 9S/3 1~i64 8 P ~, I /~ S _ A075 1401 TrTr~Tr~ AGADTTTTGA rrr~r~TrTA TTATACACTA AGAGTGGTCA

1501 GGCTAAATTA GACCTCTAAC PrrrrnrD7~T CCAD~ArTTG ACTTGAGAAT

1551 GACAA~ATTT I~LL~:1UA TArrrrrrr7` ATTAGCAAAT TTGGAAAAAA

1601 L~ L~i~ll GTTGATCTTT AATTAGTATA Ar~TTACGTAC AATATCCTAT

1651 TGAATTGGAA rrr~TD71rrT rDrrrTDTr~ TGATaGTTTC Trr`'`r`"TD

1751 TGCTTCGACT r~r~ rD7~ ~ LU--~L~LL r~TnrDDr GCAACTTTCA
1801 ~ rrr~ TAATTCCTTT TTGGCTTCAT GGATTCCAAG TTCTAATGCA

1851 TGCAAGGACT GGTATGGAGT ~LU~ LL1 rnTr7rTrr~r, TAAACACGTT

1901 GA~TATTACA AATGCTAGTG TCATTGGTAC ACTCTATGCT LLL.~LLL1 1951 CATCCCTCCC TTCTCTTGA~ AATCTTGATC TTDrrDr''`~ CAATATCTAT

2001 GGTACCATTC CACCTGAGAT TGGTAATCTC ACAAATCTTG L~L~I~LLUA

2051 CTTGAACAAC AATCAGATTT C~ \rr~IT DrrrrrDrr~ ATCGGTTTAC
2101 TArrr7`''rrT TCAGATCATC ~LLL~ ACAATCAATT AAATGr~ATTT

2151 ATTCCTAAAG AAATAGGTTA CCTAaGGTCT rTTDrT~ T~1~LLLU~

2201 TATCAACTTT CTTAGTGGTT CCATTCCTGC TTCAGTGGGG AATCTGAaCA

2251 ACTTGTCT~T LLLUL~I~L TACAATAATC AGCTTTCTGG .L~L~LLLL
2301 r``'""~7.TPD GTTACCTAAG ATCTCTTACT GAGCTAGATT TGAGTGATAA
2351 TGCTCTTAAT ~ LL~L~IL~ L LU~LL~LL rr~r~rTDTr, AACAACTTGT
24 01 ~L L L L L LUL L L~ L L L~L~;A AATCAGCTTT ~LU~7~ Li1~T TCCTGAA~AA

2501 TAATGGCTCT AiL~LU~LL CATTGGGGAA TTTGAACAAC LLUL~LLLLL

2551 1ULLL~LLL~ Tr rTrPr ~L LL~ LU;~, ~L~LLi~LU~ r~`7~TDrrT

2601 TPrrTrr~PT CTCTTAATGT CCTAGGTTTG AGTGAGAATG CTCTTAATGG

2651 ~L~L~LL~ 1 GCTTCATTGG GGAATCTGAA AAACTTGTCT AGGTTGAATC
2 7 01 TTGTTAATAA TCAGCTTTCT ~. L .L~I L . CTGCTTCATT GGGGAATCTG

2751 AACAACTTGT ~1~1UL1U1~ TCTTTACAAT AACCAGCTTT 1U~ L~T
2801 1~1U~1L A TTGGGGAATC TGAACAACTT ~ 1.1~1U11U TATCTTTACA

2851 ATAATCAGCT 11~1U~1~L A1L~LU~1L CATTG~3GGAA TCTGAACAAC

2901 TTGTCTAGGT TGTATCTCTA CAATAATCAG ~LL1~1U~1 ~L~L1~1UA

2951 Ar`7`rTpr~GT TACTTGAGTT CTCTTACTTA TCTAGATTTG ArTrrTPrrT

3 0 01 CCATTAATGG A1 L LAL L .. 1 ~i. 11~1 L L U GCAATATGAG CAACTTGGCT

3051 1L111U111~ TTTATGAAAA TCAGCTTGCT AGCTCTGTTC rT~rr~r1~T

WO9S/31564 2 1 8~562 F~,l,. _. 1075 3101 AGGTTACCTA AGGTCTCTTA A''~l~L1L~ TTTGAGTGAG ~Tr,rTrTT~
3151 l~ ~ TTCGGGAATT TGAACAaCTT GTCTAGGTTG
3201 AATCTTGTTA ATAaTcAGcT I l~,b~l~ L ATTCCTGAAG 7~-~7`T~rr~TTI~

3301 ~ i~ TTCATTCGGG AATTTGA/~CA ACTTGTCTAG GTTGAATCTT

3351 GTTA~AATC ~r~rTTTrTan ~ ~1~ r~7~r7~ T~r~ GTTACCTAAG
3401 ATCTCTTAAT GACCTAGGTT TGAGTGAGAA TGCTCTTAAT ~i~.~l.l~l~

3451 CTGCTTCATT r~--r~ TrTr. ~7~r7~rTTrT ~ ~ Ll~l~ TCTTTACAAT
3501 AATCAGCTTT ~l~,w_l~fLI TCCTGAaGAA ATAGGTTACT TGAGTTCTCT

3551 TACTTATCTA ~ ATAACTCTCT TAATGGACTT }.I ~
3601 CATTTGGCAA TATGAGAAAT CTGCAAGCTC TGATTCTCAA Tr~T7~-~r~-~T

3651 CTCATTGGGG AaATTCCTTC Al.l~ i~ AATTTGACAT rz-rT--~-~rT
3701 ~ , rrr~ r~ ATTTGAaGGG AZU~GTTCCG CAATGTTTGG

3751 GTAATATCAG T7~7~rrTTrz~r- ~Lll~l~W~ TrTr~TrT~7` TAGTTTCAGT
3801 rA~r~rrTrr ~l~ ~I TTCCAATTTA ACATCACTAC AAATACTTGA

3851 TTTTGGCAGA AACAATCTGG Drrrrrr7-7T Z~rr1~r7`'`TGT TTTGGCA7~TA

3901 TTAGTAGCCT CGAGGTTTTT GATATGCAGA ~rr~\r~ rT TTCTGGGACT

3951 rTTrr~7 r7~7~ ATTTTAGCAT TGGATGTTCA CTGATAAGTC TCAACTTGCA
4001 TGGCAATGAA rT~r~ Tr AAATCCCTCG GTCTTTGGAC A1 TTGCAAAA

4051 AGCTGCAAGT I.1.L~1 ~- nr-''r''r~TC ~7`rTr~`rr-~ CACATTTCCC

4101 ~ GAACTTTGCC ~r'`--rTr7'r~ GTTTTAaGGT Tr7~r2~Trr7~7~

4151 Tr~ TT~r~r1~T rr''rrTATal~ r~Tr1~Tr~7~r GGCrGAAATC ~I~511L' ~1~7 4201 ATCTTCGAaT CATAGATCTC TCTCGCAATG CATTCTCGCA AGACTTACCA

4251 ACGAGTCTAT TTGAaCATTT r~ r~ Tr~ AGGAcAGTTG DT~ r2~7,T
4301 GC-~C--'`"rrA l~rTT~Tr''l\7\ arTI~TTDra~ TGACTCGGTG r-T~r-TTaTa~

4351 C~AAGGGATT GGAGCTTGAA ATTGTGAGAA '.L.l~=.l~.L~ GTACACAGTT

4401 ATCGATCm r7\7~rrr71r~7~ ATTTr''~'----'` ~.A'l~,Il.~ll ~l-~l~:~l~i 4501 r,rT~T21Tl~rC ATCATCACTT GGAAGTTTAT CTATACTGGA ATCACTArAc 4551 ~l~ \rr~ rTTTc zlr_7~ _7.T~ rr~r~
4601 TACGTTTCTT GAATTCTTAA 1~ ~ rr-TT~TrTr r~ Trr~

4701 AATGATGGAT TDrr,Tral~TA TCCAGTTTCA AaAGGTTGTG GCAAAGATCC

4751 TGTGTCAGAG ~ 7.7lrT~T;~ CAGTGTCTGC GrT~r7~ T r~

~ ~O 9S131564 2 1 8 8 5 6 2 ~ ~ ' ~ ~ 1 A J j 4801 ATTCTGAATT TTTCAATGAT TTTTGGADAG CAGCTCTGAT (il .i., 113.L~

4901 ADATCTAAGA TGGCTTGCAA GAATCATTGA D~ rTC~~D CACAAAATTA
4951 TCATGCAAAG r'-~ - CAGCGAGGTC ADAGA~DATTA rD-~ T~~
S001 AATAATCACT TCTAGACAAG TTACCAATAC AGA~DAGATTT GATTTCAGAA
SOSl CTTCAGGTAT TCACGCTAaG CTCTAACACT TA;~ AGTTTATTCT
5101 A~rDD~-T~7~T ATATGGTTTT TTTTTATCAA r7\D~TDrTTD TTAAGGCTTG
5151 ATACAAATTG CTATAATCAC TTGGAAGCTG TrDT~TDT~D r~ ---rT7~D
5201 A~DATTTATAG TTGTGTGACT CACTTTCTTA TTTTTCAGAT TTTCAGGAGC
5251 CAaGAATTAG AAGACGCTGG TGTADAGGAT 1 l ~ L1~ ~ L~
5301 GCTTATGATT GTTGGATTTG A~ l TTATAAGGTT TTCTTCAGTT
5351 rrrrDD~TrT AATATTTTGA ATTTTGATGA TDTDT7`DT7~D Al~
5 4 01 TTGAATGATG l ~ ~I . L ~ L ~:bW~l ~ ATAATACTCA CCTCAaaGAA
5451 TCTAD~GAG TTAGCGCACG DT7~ r~TD r~D~'DTDrP~I D--~D~ ~DTDr 5501 ATTACAACCT l~ ll~l TATCTTACAC CCCAAAGCTT 41L~' l~
5551 Dr~--`D7`--- CAAGTTTTAT TTTTAGATAT GGGGAGCCTT lil l'ljlli~'l`;'i 5651 DTDrrD--''-r TGTGTTTADA ~lL'~:L~ 7 TATTAGTTTG TAATATTTGr, 5701 AGGTCTTAAA TTGAACAGAT GCACATCTGT TCGTGAD~GA r,rDTr~ -TDT
5751 TCTTATAAGT CAACTCTCAA GTTCTATAD~A TDTD~---r--T rrTDDD''TDr 5801 rATD~ DDD AACTGCAGTA TDrT~D''----a TTGTTGGATC rTrr~ D
5851 TTGCTGGTAA Cr~rrrT~D~r AaCATAcGTT AL~LL~j~L~; rrrrT~`--7'`--5901 r.TDrrrDrTr ADATAATCTA GGTTTGCATA ~ 7 r7~ \rD7~rDD
5951 TTATTADACA AAATCCACAC Pr7-rTDrirDr DTr-~-r-TJ~D ADAATTTAAT
6001 GACGAGATGA AAGAAACTCA rrrr~7-DTG GACTTTATCA P71r~7`"7`7`DT
6051 ACATTGTTTG TPrrTTTTr~ ACAACCATTT ATCACTCAAA GAAGATCAAG
6101 ~ ~ ~ TTACATCGTT CTTGGAACAA AATTATGTAC ATA~AACTTA
6151 CAGGAATCAT ~.5 ' 111~7 ' ~71~7 Trr,T7~-~ D7\~ T rrDTr 7~ TAGTCCADriA

6251 GATCCCCGAA CTGCCAGCAC r7`D7`----Dr7~D r~D~`pDD~Tr TDrpTrr----r, 6301 AGTTACTGAG ATCAAAGAGC ATr~7`DDDD'' GCACTTCATA CTAATATGAT
6351 AACTTCATAC TAATATGATA CAATTATTTA r~\r~rD-~D-~ r-.`D~'.`DTDr 6401 r7~Drrr~7~r rr,rD~rPTPr TTTATCTATT PD''r~""PrT GCACTCAaGA
64S1 TAACTAGTAT lll~ A G

WO95/3l564 ~ 1 88~6~ PCTIGP95/0l075 SEQ ID N0. 6:

ohKVV~i SLQFFTLFYL FTVAFASTEE PTPT.T.TrwT~PT FKNQNw'SFLA

51 SWIPSSNACK IJwY~VV~rw~i RVwTLNITNA SVIGTLYAFP FSSLPSLEwL
101 nT.' ~,1 IPPEIGNLTN LVYLDLN~WNQ l~-ill~Ul~i T.T.~T~T.QTTT~T

151 F_NQLNGFIP T~T Tr.VT.PqT.T KLSLGINFLS GSIPASVGNL w'NLSFLYLYw' 201 NQLSGSIPEE TCYT.PqT.TT.'T. nT.qn~ T~T.~ TPPqT.I LSFLFLYGNQ

251 LSGSIPEELC YLRSLTYLDL qF~.TT.T,~..Tp PqT "~TT. .C FLFLYGNQLS
301 ~.~lVh~ l. RSLNELGLSE rTT~T,~rTp::~q Tr.T~T.~T.qTiT. NLVwNQLSGS

351 TP~CT.aNT LqMT.YT, _ LSGSIPASLG NLNNLSMLYL YNrTQT.qnqTP

401 2~qTr.NT. .q T~T.YT, _ .q ~ibl~l~ Yh SSLTYLDLSN NSINGFIPAS

501 NT. .qT T.rTT. ~ _ .qr.CTP Rr'Tr.YT.PqT.rT VLDLSENALN GSIPASFGNL

551 Nw'LSRLNLT~N NQLSGSIPEE TaYT.PqT.`--lT. r.T.q~rT~T.~ rPI~qTr.NT.
601 T.qr~T.YT. _ LSGSIPEEIG YLSSLTYLSL .~-.rP PqT~ Q

651 PT,TT, .T ~ V Wh TSLEVLYMPR Nw'LKGKVPQC LGNISNLQTtL
701 ' .~ i LPSSISNLTS LQILDFGRrwN LEGAIPQCFG NISSLEVFDM
751 1 .qr7TT.P TNFSIGCSLI qT.T~T.~-''.r! DEIPRSLDNC KRLQVLDLGD

801 NQL`wDTFPMW LGTLPELRVL T~T. .TTaP TT~qCPZ-T~rMF PnT.rTTnT.Cr~

851 NAFSQDLPTS LFEELKGMRT V~ ..,Y ~YYL~ IVVV VTKGLELEIV

901 RILSLYTVID L! rr.~rnl PSVhGDLIAI RILNVSENAL QGYIPSSLGS

951 T..qTT.T.'.CT.nT.C FNQLSGEIPQ QLASLTFLEF T.NT.CT~YT.Qr. I.:l~y~5~yrh~

1001 1~ GLRGYPVSKG ~iKu~v~irw YTVSALEDQE ~l~rrl~w 1051 K~ahMGYGSG T.rTnTCMTYT LISTGNhRWL PT~TTT~'T~T.E~TR ~ v 1101 ~ _ EF~

8EQ ID N0. 7:

KKVV~i SLQFFTLFYL FTVAFASTEE PTI~T T-T~WlrPT FRNQNNSFLA

51 SWIPSSNACK ~WI~iVV~l'W~ RVWTLNITNA SVIGTLYAFP FSSLPSLENL

101 nT. ~l IPPEIGNLTN LVY'LDLN.NNQ l~ll~yl~i T.T.7-~TQTTT~T
151 FENQLNGFIP T~T~TrYT.T qT.T KLSLGINFLS GSIPASVGNL NwLSFLYLYN
201 NQLSGSIPEE ISYLRSLTEL nT qn~TrT.~q IPASLGNMNN LSFLFLYGNQ
251 LSGSIPEEIC YLRSLTYLDL SEN~LNGSIP PqTnNT. .C FLFLYGNQLS

~ WO95/31564 21 88562 I~l. 5.'01075 301 ~ lr~:r;l-i~h RSLNVLGLSE ~T~T''r'lTV~q T.r.NT.T~NT.CTT. NLVNNQLSGS
351 Tpl~cT~r~NT LSMLYLYNNQ T..CrCTP~qT.r. NhNNLSMLYL YNNQLSGSIP
401 ~CTrNT c ~TYTYNNQTC ~ Lrrr;1~.5Yh SSLTYLDLSN NSINGFIPAS
451 FGNMSNLAFL FLYENQLASS VPEEIGYLKS LNv~DLSENA LNGSIPASFG
501 NT. .CTT.~T. v _ cacTP RRTr.~T.VcT.N VLDLSENALN GSIPASFGNL
551 NNLSRLNLVN NQT.Cr.CIPRR TrYT.T~CT.~"lT. r~T~cRNi~T~r-c Tp~cT~r~T~
601 T.CMT.YT. _ LSGSIPEEIG YLSSLTYLSL ! ,~TrT,Tp ~ 'Q
651 Z~T,TT, .T ~Ll~ V~ h TSLEVLYMPR NNLRGRvPQC LGNISNLQVL
701 '. 2~ri LPSSISNLTS LQILDFGRNN LEGAIPQCFG NISSLEVFDM
751 _ ~Cr:TT~p TNFSIGCSLI cT.NT.~Tr~TT.~T.R DEIPRSLDNC RRLQVLDLGD
801 NQLNDTFPMW LGTLPELRVL T~T.T~'7,~Tr.p TRCCT~T TMF pnT~T~TTnT~qT~
851 NAFSQDLPTS LFEHLKGMRT VL~ Y ~ YLILJ:.VVV VTKGLELEIV
901 RILSLYTvID T' r~ll PSVLGDLIAI RILNVSHNAL QGYIPSSLGS
951 TCTTWCTnTC FNQLSGEIPQ QLASLTFLEF LNLSI~YLQG l~Q~ yrKl 1001 rLs~.,Yr.~) GLRGYPVSRG ~ }'V~LSJ'.N YTVSALEDQE ~ rrl~rW
1051 RAA~MGYGSG T.rTaTCTTYT LISTGNLRWL ~vTTIPET.T.~nr llrlyKK~yK
1101 ~ _ _F~

8~eQ ~D ~O. 8:

GGTTTCTAGA AAAGTAGTCT CTTrACTTCA GTTTTTCACT CTTTTCTACC
51 TCTTTACAGT ~ l TCGACTGAGG AGGCAACTGC CCTCTTGAAA
101 TGGAAAGCAA CTTTCAAGAA rr~r~T~DT ~ " T
151 TCCAAGTTCT AATGCATGCA ~rr~rTr,GT~ TGGAGTTGTA TGCTTTAaTG
201 r.TDnr~nT~ CACGTTGAAT ATTACAAATG CTAGTGTCAT TGGTACACTC
251 TATGCTT~TC CATTTTCATC ~ ~l CTTGAA~ATC TTGATCTTAG
301 r~-~r~1~r~T ~1~1J~1~ CCATTCCACC TGAGATTGGT AATCTCACAA
351 ~~ .l.lA TCTTGACTTG AACAACAATC AGATTTCAGG 71Tr~TDrr~
401 CCACAAATCG GTTTACTAGC CAAGCTTCAG A1~ TATTTCACAA
451 TCAP,TTAAAT GGATTTATTC rT~ r~ T AGGTTACCTA AGGTCTCTTA
5~1 CTAAGCTATC ~ il~l-- AACTTTCTTA GTGGTTCCAT l~l~i~llr:~

551 GTGGGGAATC TGAACAACTT ~l~l ~ TATCTTTACA ATAATCAGCT
601 1 l~l~ ATTCCTGAAG AAATAAGTTA CCTAAGATCT CTTACTGAGC
651 TAGATTTGAG TGATAATGCT CTTAATGGCT ~ 8ll~ -- TTCATTGGGG
701 AATATGAPCA ACTTGTCTTT ~ lll~ll TATGGAAATC AGCTTTCTGG

WO95/3l564 7 1 ~ a 751 ~L~L~1L~1 r`~r`7'7`T~T r.TT~rrT~-~r ATCTCTTAC T TrrrTrrrTT
801 TGAGTGAGAA TGCTCTTAAT iJI ~ , LL~ L1L rrrr`rTrTr Bsi AACAACTTGT ~LL~L-L~L 1~LLL~L~ AATCAGCTTT,,, ~ T
901 Tr,rTr~r~r AT~rrTTDrr TAAGATCTCT TAATGTCCTA GGTTTGAr~TG
951 AGAATGCTCT TA~TGGCTCT A', ~l .. rr~TTGr-r-r~rr TCTGAAAAAC
1001 TTGTCTAGGT TGAATCTTGT TAATAATCAG ~,, ~ ~l I T l ~1 1051 L ~ W~7 rrTrTr`~rr~ ACTTGTCTAT GTTGTATCTT TDrrrTrrrr 1101 AGCTTTCTGG ~L~_L~L~1 ~rTTrr~TTriri GGAATCTGAA rrrrTTr.TrT
1151 ATGTTGTATC TTTACaATAA TCAGCTTTCT ~ ~L~l~ILi ~L`~ ~I.
1201 GGGGAATCTG AacAAcTTGT CTAGGTTGTA TCTCTACAAT AaTCAGCTTT
1251 ~ -L~L--T T~rrTr`rr`r ATAGGTTACT TGAGTTCTCT TACTTATCTA
1301 GATTTGAGTA ATAaCTCCAT TAATGGATTT A1L~L~ L C~TTTwCAA
1351 Tr~Tr~--rrrr '1L~j~LLLLL 1 7~LL~ LLL~ TGAAAATCAG CTTGCTAGCT
1401 ~L~1L~--L~ AGAAATAGGT TrrrT~rrT CTCTTAATGT ~L~ L L~j 1451 i~GTGAGAATG CTCTTAATGG ~L~LL ~1 ~ BIL~L; GGAATTTGAA
1501 r7~r rTTr~TrT Anr.TTGAATC TTGTTAATAa TCaGCTTTCT ~ L~
1551 rTr~r~ T AwTTACCTA rr-r~TrTrTTA LT~ I ~ TTTGAGTGAG
1601 AATGCTCTTA l TI ~ _T L~ ~ L~ IL TTCr~rr` rTT TGAACAACTT
1651 GTCTAGGTTG liATCTTGTTA DTrrTrArrT ~L~L~j~ L~ ATTCCTGAAG
1701 rrrTAnrTTA CCTAAGATCT CTTAATGACC TAGGTTTGAG TGAGAATGCT
1751 rTTrrTrnrT ~ L~LL~ AATCTGAACA ACTTGTCTAT
1801 J1L~ ~L~ TACAi~AATC rnrTTTrTrr ~:~L~i~L r'~rr'~ ~TAn 1851 GTTACTTGAG TTCTCTTACT TA~ ~ ~ TGGwTaATAA rTrTrTTrl`T
1901 wACTTATTC ~L~i~L~ TGwcaATATG rr~ TrTrr AAGCTCTGAT
1951 TcTcaATG~T AACa~TCTCA TTGGGGAAAT L~1 l ~i~l GTGTGCAATT
2001 TGACATCACT rr~_TrTTn ~L~ A GAAAcaATTT r7lDrrr~7 7 ~
2051 GTTCCGCAAT r.TTTGr,r,TA" TATrDr.Tr"r CTTCaGwTTT TGTCGATGTC
2101 ATCTAATAGT TTCAGTGGAG AGCTCCCTTC ~I,.~.I,~ AATTTAACAT
2151 r~rT~rrrrT ACTTGATTTT rr.rrr~- rrr ATCTGGAGw Ar.r~TArrA
2201 CaATGTTTTG GcaATATTAG TAGCCTCGAG GTTTTTGATA Tr'rA--~r~r~7~
2251 CAAACTTTCT GwACTCTTC CAACAAATTT TAr.rrTTrnA TGTTCACTGA
2301 TAAGTCTCaA CTTGCATGGC 7 rTr"rrTrn AGGATGAAAT ~l~
2351 TTwACaATT r7rA7,r7~-rT GCAAGTTCTT GATTTAwAG ACAATCAACT
2401 r~rr`~ArA ~ l GGTTwGAAC TTTGccaGAG CTGAGAGTTT

~ WO95131564 ~ 1 8 D 5 62 2451 TAAGGTTGAC ATCGAATAAA TTGCATGGAC CTATAAGATC DTrDDrr~rrT
2501 GADATCATGT L~ L'CI TCGAATCATA ~ GCAATGCATT
2551 rTrrr-.rr~r TTACCAACGA ~'C'~ ~ ACATTTGAAA ,rRr.DTr~r--~
2601 CAGTTGAIAA ~Drr7.Trr~ GAACCAAGTT ATGAAAGCTA TTDrC`T~r`r 2651 ,~.. ~:.,,~R TTGTGACaAA GGGATTGGAG CTTGAAATTG TGAGAATTTT
2701 GTCTTTGTAC ~CAGTTATCG ATCTTTCAAG CAACAhATTT r~rr~--DTD
2751 l~ l rrTRGrrr`T CTCATTGCGA TCCGTATACT TAATGTATCT
2801 rhT!\DTr~rAT TerD1`rrrTA TDTDrrDTrD TCACTTGGAA GmATCTAT
2851 ACTGGAATCA CTAGACCTTT CGTTTAACCA ACTTTCAGGA c~r~TDrrAr 2901 AAr--A-AcTTGc TTCTCTTACG mCTTGAAT TCTTAAATCT rTrr,rDrDDT
2951 TATCTCCAAG GATGCATCCC l'rADrr`rrT ChATTCCGTA CCTTTGAGAG
3001 CAATTCATAT GAAGGTAATG ATGGATTACG TGGATATCCA GTTTC~AAAG
3051 GTTGTGGCAA AGATCCTGTG Tr7~r~r~D~D DrTATDrDRT ~~
3101 GAaGATCAAG AAAGCAATTC TGAATTTTTC AATGATTTTT Cr~D~r-Dnr 3151 l.~ihI~ TATRr~rTr~ c~rTGTr~TAT TGGCATATCC AT7`DTDTDTD
3201 TCTTGATCTC r~rTr.r:~D7~T rT7~r~TrRr TTGCAAGAAT CATTGAaGAA
3251 CTGGAACACA AAATTATCAT r~rDD~r--`r` DDr`~'r--DRr, GAGGTcaAAG
3301 AAATTACAGA Ar~r~D7~TD AiCb~l~.~ GACAAGTTAC rD-~TDrrr~D
3351 AGATTTGATT TCAGAACTTC AGACTTTCaG c~rrr7\Dr7~D TD`r``r`rr 3401 CTGGTGTAAA W/~ 1 TGCAGCTTAT ~ ii,hT
3451 TAGAmTTA GTTTTATAAG l~:I.L.Ll~.L~:A GTTGGGAaAA Tr.TD7`TDTTD
3501 TGAATTTGAT GDTDTDrDDT AAATGTTGTG TTTATTGAAA ~D~DDD~ D
3551 DDDDDDDD7~ D~"DD~`D7'D AAA

gBQ ID N0 . 9:

1 tAtAtAtrtt ~I~La.lLyL~ AttrJ~t~-rA aagtgattaa At~t~t~r 51 y~y~y~y~Ly aaatcaggta y~yLLLLyLy ttgttgtttc 101 ~y~yL~ yy t~rttr-_~Jr ~dY~Lyy~yLL ~ -~tr~ ~ J ~ J-- y~
151 Arnr~ ~y~yc~yLLL ctagacgcaa ttrr~rrJ~r~r gcttcttgaa 201 ~L~,yLLyJti~ y~LyLLLy~L ~iyLL~a~ _y ~:.N' :1 Y tr~Jrr~ rJA
251 ~rJrAtJ~rtrtr aagcacgagt trttrtAtrr agtacatgaa arr,rtt~r~
301 Arr~--tJrt rrttr----~Jr . ~~ - ~ Jl --trrJrArJcr tacagttgtt 351 Jrrtr---Jrr~r t:..rJr~-~--- tttr~-~rt:.~ ttatacaatt ctt-------t 401 aaaagagtaa L~ - tagagagtta AttttrArtt ~ A
451 gagagttaat tt~ttt~r _~-t--ttA tAttttrArt ttagtataca 501 attcttagtg LL~aLLLclyL attttr--tt atattatttg --tt~.--.tr 551 rtrAt-~tr~ ~tAtArttAt trtrr-tr~tc catgtgcatg L~LyLe~LLyy 601 r~ ~ tttrJ~tAttA -~ ~-t, --l r. agtacattct tA,rrJ~t-...
651 tgtcttgtac ~ rt rJ~-~rrrr-A aaatatgtgt gtttcaaaat 701 atctgtgtag ily~ y - - LyL~dyL~LC tgtctaattg rrt,r,.rtt 751 r~-'tAttAt ttctgtcttg tArp~-~rt ~gArtt~tr ataattaagt 801 rJ~r~rrArA r---ttr-~t rtrt~---t atctttgtat gtagtgtaaa WO 9~1~1564 2 ~ 8 8 5 ~ 2 PCTIG1395101075 851 aaagctttcg aggaaagtaa gacgaagttt ctrrtrtrtt trtr.~^ArtA
901 LyL.LLy.Ly atttacttct rti~ ir ttcgtctctt ctctgagttc 951 r~rtrtA~rAt ~rtrrrDTnrr ~L~1L~L~ L T,, 11 1.~ r~rrr~r~Tr.
1001 CGACGTTm CCa~AGCTTCA ~ W~ ~ TGTTCGCaAG ACGTTCC~CA
1051 GCCA~CTTCT C~AGGCTCTC r~r~rr-GrrnDT rDPTrrDTrr ~TTrr~TnaT
1101 CATwAATCG rrPrr~ rr rrrr-PTrrrr CCTGAGCTTA TA'~ ,L~
1151 Trrar`DrrT AGGATCTCAA TrrTrr~TrTT CTCTAAGAAC TA~,L~ L
1"01 rPrrr~TGr~Tr~ CTTAaATGAA TTGGTTGAGA TrrrrrPrTQ CTTTAATGAT
1~51 TTAGGTCAAA TGGTGATTCC AGTTTTCTAC GACGTTGATC rTTrrr~a--T
1~01 Tr~~D~arr-r- r-rrr,Qrr~DT TTGGAAAGGT rTTTraDD~r ACATGCGAGG
1~51 TcaGcAAGGA rrPPrrDrrr- GwGATCAGA rprr-a~~~TG GGTGCAAGCT
1401 cTca - rAGATA Tr~Qrr DnTrT ,Drrrrr~~~r GATCTTCTGA ACGGgtacgt 1451 LyLL.It!tc.LL rr--tAtAtr Ly~LLy~yLL ttcaattgtc tr,~--~t,t 1501 ~ I l L Ll ~. ~l ~y~LL~:yyL tcttctttta ggggtgcttc tt~--ttJr----A
1551 -''tt~'rtt LLyLL.lLLd~ GCCTAATGAA GCGCATATGG TTGAAMGAT

1651 ACTTCGTCGG AATTGAAGCT CATATTGAGG. cAaTAAAATc AGTATTGTGC
1701 TTGGAATCCA rrr~rrTPQ AATGGTCGGG ATTTGGGGAC arTrDrrr.DT
1751 TGGTADGAGT ACCATCGGAA GAGCTCTTTT Cr~ GTCAACTC TCTAGCCAGT
1801 TCCACCATCG L~L1LL~L~ DrTTDTDP`` GrDrrDQTr~r~ TDQTr.r~r~.Tr 1851 TCTGGCATGA AGTTGAGTTG QrrDnD`r`r LL I ~ ~ i ~ ~ AAATCTr GG
1901 TCAAD~AGGAC rTD~Dr`TDQ AGCATTTTGG Tr~TQQTrr~~ CaAAGG~ AA

2001 rTTr~r~~rT TGGTGGGA~A DQrT~~nTrr. TTTGGATCTG r~rrrDr, IDT
2051 AATTGTGATC ACTCAAGATA GGCAACTTCT CAAGGCTCAT GAGATTG~CC
2101 TTGTATATGA GGTGAAGCTG rrDTrTrPPr ~Pl LL~ LL~1 TrPr~TQDTD
2151 TCCCAA~TG CTTTTGGGAD AGACTCTCCA CCTGATGATT TTr~ D~T
2Z01 AGCATTTGAA GTTGCCGAGC TTrTrQrTDQ 'L~LL~L1 1 ~} QQTrTrDrTQ
2251 ~~ bLLL~ ATCTTTAaAA rr~Prrr~rD A,DGATGAGTG rQTr~
2301 ATGCCTAGGC TTCGAAATGA TTCaGATGAT A~AATTGAGG DDDrDrTD~~
2351 AGTCGGCTAC GATAGGTTAA DTP`DDPP-D TDr'~`~TTD TTTAAGTGCA
2401 TTGCATCTTT TTTCAA~GGT TTT~AAGTCA GTDDrr.TrPP Dr`PTTACTT
2451 r``r`TI rTQ L'~iWL L~C AATGTTGGCT GAGA~GTCCC TrDTDrrTDT
2501 TprDrrrr~rT Cr`TDT~TDQ DrDTrrDrDD TTTGCTAGAG AAATTGGGTA
2551 GAGAAA'`TGA TCGTGCAAAG Trr~'`r~~TD DTCCTr~GaAA ACGTCAATTT
2601 CTGACGAATT TTGAGG~TAT TCG~GAAGTA TTr`rrr`r` AaAcTgtaag 2651 1 1 1 ~ I ~ . J, ~ I , ~,, ~ I . _ .rl yLL L,l~- Ly~: At~rtttAt Atr''tAt'' 2701 tr~t.~~tt~ r~tt,-tA --~ tPO~rA ,.l L~ -J. catgcgtaat 2751 taaaacgtag ~LLLy~LyLy tr~ --t aaaaagggtt y~y~LLyLLtl 2801 P~-ttAtAtt agttttcttc Jy- I Ll ~ tr-~r~rr~rr GAAACTCTTC
2851 TTGGAATACG TTTGCCACDC LLL~ ~I~ TTACGACAAG ~L~iL~l L~L
2901 ATAGATGAaA AATCATTCAA DQrrDTr,~rrT r~TrTrrr7~T DTrTD~~--.T
2951 1~jLLL~'LL~j~j TCAGATGGGG TTCTACCTCA GAGCCTCGTT L~L~L~L~L~
3001 GTAaAcTcAA AaGGcTATGG TGGGATAATT GTCCATTGAA ~il l_. I 1~71 ~ .
3051 TCTAATTTTA DQQrTr`~TD TrTrQT~r`' CTCaGAATGG Tr~'TDrTD-3101 rrTTr~r~r ~ G~CTCAGGT Art~-ttttt ttagtgatca 3151 Attt,rt~ r Ar~ ~tA ~__t~ tgtttaaaat gttcattaac 3201 gtgtgtgctc trttttrrrr L,~LLLL.~LLL tcagCCCCTT GGAAGTCTCA
3251 AGAAGATGGA TTTGTATAAT TrrTDrD7~7.T Tr`'`r'7~DT TCCDGATCTT
3301 TCTTTAGCCA TAaAccTcGA GGAATTADAT CTTGMAGAAT GCGAATCTTT
3351 GGAGACACTT ~ TTrrr~Trc CATTAAACTG AGGGAGT~rAA
3401 I~LLI~LL~; ~ ~L~1L~ ATAGATTTAA D~Trr~TTArr~ Drr,rATrTrT
3451 AATCTCGAAT ATCTATCAGT TCCTAGTTGG Trr~rTpr-r~r7 AATGCACTCA
3501 W~L/ 1~LL LJLILL~L~LL GTAAACTCAA AAGTGTATTG TGGACTAATT
3551 GTCCATTGaA ~--hLLL~LLL TCTAATTTTA Drr.rTr~rTr, TrTrrTTr.-3601 CTCATAATGG Dr.TrrDQTQA GCTTGAGAAG CTGTGGGATG GTACTCAGgt 3651 Artl~-ttrtA ttagtgataa taaatatgtt æ~'-'-~rtA ---,tr--_ 3701 tgtttaa;~at gttcattaac gtgtgtgctc trttttrrrr tattttgtta 3751 tcagTCACTT GGAAGTCTCA AGGAGATGAA TTTGAGGTAT TccAac~DrATT
3801 T~ 'r'-7~T TCCAGATCTT TrTTTrQrrD TAA~ACCTCGA GGAATTAGAT
3851 LL_LLL~jj~L GCGTATCTTT GGTGACAC--T ~LL~L~h~ TTrD"~DTrr 3901 rDrTD~rTr ATCTATTTAG DTDTrDQTrD rTQrr~D~7~T AGAGAGTT
3951 TTCCAACCGT TTTCrACT~G AAATCTCTCG AGTACCTCGA TCTCAGGA
4001 TGCCCGAATT .TGAGAaa~TT rrrDrrD-,--r r~-TrQQDT rTGrrTrr.~
4051 TAGATTATCT rQDDrD'--'T I~LLL~1L-~ Drrrar~ T GAGATCGTGG
4101 TAGAAGATTG TTTGGAAC AAGAATCTCC CTGCTGGDCT r~QrTTDTrTr 4151 GACTGCCTTA TGAGATGTAT GCCTTGTGAA TTTrQrTrDQ r-rr-rTrDr ... .. , . .. ... .. . . . . .... . .. ...... . . . : . . _ . _ . _ . _ .. .

WO95131564 2 1 ~3~562 r , ~
4201 TTTTCTCAAT GTGAGCGGCT GCAAGCTTGA GAAGCTAT~G GAAGGCATCC
4251 AGgtacattg tt~tr,rtAt r,rt~-ttttt gtttaccttc tgttatataa 4301 ctaattaagt AtArrr7~-t ttgtttttat yy~.LLyLyyL rJr ~trrArX
4351 ttatgtctta r~t=rAtArA taataatgtt tP-ttAt~At tttA -~-r51tA
4401 tataggtata --tt~ t ~r?ttAtrAtr gataatgatt ~_ ~J ~
4451 .I~LyLLLLLL tcagTCGCTT GGAAGTCTCG AAGAGATGGA TCTGTCaGAA

4551 ~ 71 CTCAGCGGGT GCAA~AGTTT GGTGACACTT CCTTCTACAA
4601 TTGGGAATCT TCAAAATTTG AGACGTTTGT ACATGAACAG DTGrDrDrnr.
4651 CTGGAGGTTC TTCCGACCGA TGTCAACTTG TCATCTCTCG AaACCCTCGA
4~01 TCTCAGTGGT TGCTCAAGTT TGAGAACTTT TCCTCTGATT TCAACTAATA
4751 ~ CTATCTGGAA 7`rrDrrr-rrD TTGAAGAAAT TCCAGATCTT
4801 TCA~AGGCCA CCAAGCTCGA GTCTTTGATA CTCAACAACT GCAD~GTTT
4851 GGTGACACTT CCTTCTACAA TTGGGAATCT TCAAAATTTG AGACGTT~GT
4901 DrDTr`7~rDr ATGCACAGGG CTGGAGCTTC TTCCGACCGA TGTCAACTTG
4951 TCATCTCTCG AaACCCTCGA TCTCA~TGGT TGCTCAAGTT TGAGAACTTT
5001 TCCTCTGATT TCAACTAGAA TCGAATGTCT CTATCTAGAA D~rDrrr7rrD
5051 TTGAAGAAGT l~ ATTGAGGATT TcAcrArGcT rDrTrTDrTl~
5101 ~ il~il GTTGCCAGAG GTTGAAD~AC ATCTCCCCDA ArATTTTCAG
5151 ACTGACTAGT CTTACGCTCG CCGACTTTAC AGAC''GTAGA (~

5301 l~il.:i71~711~L GATTATTACT CTGATGACTT TGAGGTAAAT rrrr~rrrDr 5351 TTAGATTGTC DDrr`TrDrT GTCAACGATG TGGAGTTTAA 7llll~ill~
5401 TCCATTACGA TCAAAGAATG rr~r-Tr~T7~rrD ~lC,~ , TCTATCDJ~QA
5451 ~rrDrr--rDr 7~DrrDrrDrr rTDrrrr~r-- rDrrrrrrrr DTr,rrr~:TDD
5 5 01 ~ D t ~ ~ t t ~ r ~ t--~ _ ~r~ r~ ~ t r l 5551 atttgtttta t~rJ~tr~-~ at~rr~t~gr r~--~rJ---tAr I ~.J_I ~J `
5601 cgatcgtttg AtArAt~-t~ r~r~tr~- - At r,rrJ~rJ---~tr ~.J ~ .J_~
5651 Atr-~~tt~~, . I -I IJ . - ~dLLyL~y~ rJ~r~r~rJ~-r ~ J .~
5701 ~-tAr~rJ ctttcgttag ~J~ J -~_ ~rJrArrAtrA tr-~~Atrtr 5751 t-r-~rJJrrJrJ- a~,_LLL ~LyL ~LLy.-L~ L ---AtrJ~t~-~ tr-~
5801 rJ~trJ~---r-~ t~rrtAtrtt gtatcCtgtt tatggtaact 5851 ts~trAttrt~ ttttr~ctrtt ~l I I _,JJ~I _ ArttrJJrJrAtr J_, _l I _l 5901 At~--tttAt ~-t,J_I ~ r~t LLc ~L~ yLt ~
5g51 ~rAr--grrA ttttttrt~c ~g~tAt~r-~ gatgatatgt ,r,~r~trAtt 81!:Q ID N0 10:

~'`rq.CCCaVT~ uv~r~r~i VDVRKTFLS. TTRDTnrrCT c~lrlu~

51 SRTIAPELIS AIREARISIV IFSKNYAS5T WCLNEL-vEI~. KCFNDLGQMV
101 l~VI'YJVU~S ~k:v~Kyl~ yi KVrr.~ V~ ~UI~U~LUl~Y RWVQALTDIA
151 NTDrFnT.T~7r ~ Kl SNDVSNKLIT ~rUUL~V~i TT~roTFDT~Tc 201 vT rT T~CTr;~Dv ~ L GKSTIGRALF SQLSSQ~R AFLTYKSTSG

251 SDVSGMKLSW QRFTT..CFTTr. yKUlKl~:~r~ VvEQRLNERK VLILLDDVDN

301 LEFL-KTLVGK AEWFGSGSRI IVITQDRQLL KA~3EIDLVYE VRLPSQGLAL

351 KMISQYAFGR Da~uurK ~ AF_VAELVGS LPLGLSVLGS ,CT.Rr-r)RnFw 401 VRMMPRLRND ~nnRrFFTT.T~ vGyDRLr~KRN RELFRCIACF ~n~ Y ~K
451 ELLEDDVGLT MT-DFRCT~TT~T l~l~Yl~ ~ T.T.FRT~rVFTn RARSKGNPGK

501 RQFLTNFEDI REVLT_KTGT FTTTrTVT.P PGYLTTRSFL LUDi~ }~

601 LRMVNSKLEK LWDGTQPLGS LKKMDLYNSY KLREIPDLSL DTNT.FFT.NT.T.~

651 EOESLETLPS CTQr DTT~T T~T.` T.~-rWrrT.T.Tn T RCT FrMr~T EYLSVPSWSS

701 ~ ilVY~' PRRLKSVLwT NCPLKRLPSN FRAEYLVELI ~TYcr.~T.RRT.W

751 Dr-TQcT~r~qT~R , . KEIPDLSLAI NLEELDLFGC VSLVTLPSSI
801 ~T~TTYTn MSEOENLESF PTVFNLKSLE YLDLTGCPNL RNFPbI~MGC
851 pWT17T,CT~Tr~T, 1"~i7 ~N~lVV ~IJ~rl .P AGLDYLDCLM RCMPCEFRSE
901 QLTFLNVSGC ~T.TCT T.W~rTQ CT.r~CT. . CT.~CT.~NT.RFT,P nT.CRDTNT.RT.
951 LCLSGC~SLV TLPSTIGNLQ NT.rJT T. ~ TGLEVLPTDV l~rr.qCT,T.'TT,nT, 1001 SGCSSLRTFP LISTNIVCLY T.T.~NTDTr~T TP nT.cRDTRT.r c r.TT: , 1051 TLPSTIGNLQ NT.T TiT, - TGLELLPTDV NT.C.qT.T'TT.nT. SGCSSLRTFP
1101 LISTRIECLY LENTAIEEVP CCIEDFTRLT VLRMYCCQRL KNl:jJ'Nlr~

1151 TSLTLDDFTD CRGVIKDLSD ATVVATMEDH VSCVPLSENI l~ iK~WL~A
lZ01 ~OIJYI~ VNRNPIRLST rll~ V~;rK~ ~l'l'lK~ li VRLLYvYQET

1251 _~llr~JK KRMRVSLLP

82Q ID No. 11:

GACCADACTG GACTCCTGCT ~l.~ A TCbGCAGGTC 7~TTrTrr~Tr~

51 GbD~ATTAGC Trr~--rTrrr r~rDrTDTrTr. Prr.TDrrTDr. TDrT~DTr.T

101 1l~lll~ ,. bATTTGTGCT DT~TPTDrrT rDTrTPDDTT DTTr~DT~r~
151 rDrDrr~ rr A~DACATCTCT TbATTbGTTT TGATCATTTT TDr.Tr,r~r--~

201 bI~ T7~7~D~rTTr~T GTTTTTCATG rTDTDTr.TrT Ll~' ll '~251 ACTTGTTTCC ~ T~ ~ I ~ACCTrDTTT r.Trrrr,rr~7l GATCAAGCTC

301 ll~ AGAATTCDAG AACATGTTTA CCGTTAATCC TAPTGCTTCT
351 l l...,., .~., . Drr~rDr~Dr ADCTCTTTCT Trr`~r~7`D GCACAAGTTG

401 ~l~=~.l~l~ GATGGCGTTC DTTr.Tr`"nD 7~-~rr~rDr.nD CA~GTGATTG

501 AGCCTCTTTC AACTCTCCAA ~CTCDAAAGG ~ ., ..-..., ... rTTDT7~~~TaD
551 TTTCACTGGA TCGCCCATTT CACCTADATT TGGTGAGTTT TrDnDTT~nD
601 CGCATCTCGA TTTGTCGCAT TCAAGTTTTA a-r~a-r~Ta-TDnT CCCTTCTGAA

651 ATCTCTCA~C TTTCTADACT ATACGTTCTT CGTATTAGTC TADATGAGCT

701 TACTTTTGGT CCTCACA~TT TTGAATTGCT TCTTAAGAAC TTGACCCAAT
751 TA~DAGTGCT rr~rrTTC~ TCTATCAACA TCTCTTCCAC Ll~

801 AATTTCTCTT CTCATTTADC AAATCTATGG CTTCCATacA CAGAGTTACG
851 l~ Al~ 7 rrrr~D~r~ TTTTCCACCT TTCCGACTTA GAATTTCTCG

901 ATTTATCAAG CAATCCCC~G CTCACGGTTA GGTTTCCCAC ~DrrP~rTr~r~
951 PDTpnrDnTn CATCACTCAT GAAGTTATAT CTCTATAATG TGAATATTGA

WO 95/31564 1~~ . .075 2~ 88562 1001 TGATAGGATA CCTGAATCAT TTAGCCATCT AacTTcAcTT CATAAGTTGT
1051 ACATGAGTCG TTCTAATCTG TCAGGGCCTA TTCCTAAACC TrTATrA~7.T
1101 CTCACCAACA TAGTGTTTTT GGACCTTAaT AATAACCATC TTr7~7~rA~Ar 1151 AATTCCATCC Prrr~TA~rrr, ~ ATAr~ATr7~ rrT~rr~TA
1201 CATCAAACAA CTTAAATGCG ArTATDrrPT CCTGGATATT ~
1251 TCACTGATAG GGTTAGACTT ~A''~r7`rTr~r ACTTTCAGTG GAAAAATTCA
1301 AGAGTTCAAG TCCAAAACAT TA~r.TPrrnT TACTCTAAAA rA~ .TA-~rr 1351 TAAAAGGTCC TATTCCGAAT TCACTCCTAa Arrrr~"r~'` CCTACAATTC
1401 L~ .lll. rArPrr~Tr'~ TATCAGTGGA CATATTTCTT CAGCTATCTC, 1451 CAATCTGAAA ACATTGATAT TGTTAGACTT Arr.Aar.T~ T AATTTGGAGG
1501 A---`rA7'TAr~A GCAATGCGTG GTTGAGAGGA 7\rr.~.~TrrrT TTCGCATTTG
1551 GATTTGAGCA ArA7~rrA~rT TPr.TrrA~Ap ATCAATACAA CTTTTAGT~T
1601 TGGAAACATT TTAAGGGTCA TTAGCTTGCA rcrr~7~Trrr rTArrr.rrr.P
1651 AaGTCCCACG ~I~ AL~: P71TTrrr'"'T A~TTGACACT ACTTGATCTA
1701 Gr~Tr'`rDrT~ TGTTGAATGA QCATTTCCA AACTGGTTGG r~TPrrTATT
1751 TCAATTGAAG ATTTTAaGCT TGAGATcAaA TAAGTTGCAT ~ ~L~_A
1801 AATCTTCAGG r~ TrrA~7lr ~ ."i~ . GTCTTCAaAT ..~ AI-~1851 TCATCTAATG GATTTAGTGG GAATTTACCC GAAAGAATTT TGGGGAAm 1901 GrA7~ArrpTr~ AAGGAAATTG DTr~r~--Trr AGGATTCCCA GAGTATATTT
1951 rTr7~T~rATP l~I~1.l~I Trr~TTATT TGACGACAAT TTCTACAAAG
2001 GGACAAGATT ~uAl~ l TCGAATTTTG GATTCTAACA TGATTATCAA
2051 TCTCTCAAAG AACAGAmG AAGGTCATAT TCCAAGCATT ATTGGAGATC

2151 ATPrrr~:rPT CATTTCAaAA TTTATCAGTA CTCGAATCAT l~
2201 ATCTAATAaA ATCAGCGGAG AaATTCCGCA GCAGCTTGCA TCCCTCACAT
2251 TCCTTGAAGT CTTAAATCTC TCTCACAATC A~ A~
Z301 ~ 'r'`2`'~rr AATTTGATTC GTTCGGGAAC ACTTCGTACC AAGGGAATGA
2351 TGGGTTACGC GGATTTCCAC TCTCAAAACT ll~~ GAAGATCAAG
2401 TGACAACTCC Pr~rTr~rrT~ GATCAAGAAG 7~ r~7~r~ AGATTCACCA
2451 ATGATCAGTT nrrAr~r~r-T l., ,~,~, TACGGTTGTG GACTTGTTAT
2501 TGGACTGTCC rTDATAThrA TAATGTGGTC AACTCAATAT CCaGCATGGT

2551 TTTCGAGGAT GGATTTAAAG TTGGAACACA TAATTACTAC GAaAATGAAA
2601 r~rrPr~r~A AAAGATATTA GTGAGTAGCT ATACCTCCAG GTATTCCACT
2651 TGATCATTAT cTTTcAGAaG ATTATTTTTT GTATATCGAT GAAATTATCG

WO 95/31564 21 ~ ~ ~ 6 2 PCT/GB9 2701 ACCTCCTTCA TCCTCA~AGC TCTTAACTTT CACTCTTCAT TTTTGAI~AAT
2751 TTCAGGATTC AaAGATTTCC GAGTTCCCAG ~ .~ TGCAGATAAA
2801 AGCCTTTTTA TCTTTCATAG ~ TATr'~`T~ GATTTTATTT
2851 ~ TrGrDraTA GATATGTTCC GTCACTAAAA ACATTGTATT

2951 AGTTACTGTA GTCTGCTATT TTAATTTCTT CCATT.GAAAC ACAACTGACG
3001 TATCTTGAGA ~r`"`"T7~T GATCTCAGAA ATGGGAATCT CCCAATCCAA
S2Q ID No. 12:
MGCvRLVFFM LYVFLFQLVS SSSLPHLCPE DQALALLEFK l....~lvl.. ~c 51 DYCYDRRTLS ~ ~bW CI~iVSl~:lJr l l~i QVIELDLRCI QLQGKFHSNS
101 SLFQLSNLKR LDLSYNDFTG bflb~l~rl~r;l~ CnT.TUT.nT.C~T 5br~1ivl~:,r 151 Tc~TT.cRT.Y~7T. RISLNELTFG PHNFELLL~N LTQLKvLDLE slNl5b~
201 NFSSHLTNLW LPYTELRGIL PERVFHLSDL EFLDLSSNPQ LT'vRFPTTKw 2Sl Naa`CT.MRT.Y LYNVNIDDRI PESFSHLTSL HKLYMSRSNL SGP}PXPLw`N
301 LTNIVFLDLN NNHLEGPIPS NVSGLRNLQI T.WT. ~ SIPSwIFSLP
351 CT.TaT,nT, lr~ lyrir~ SKTLSTVTLK QNKLKGPIPN CT.T~TQR7~T~T7 401 T.T.T. ~TTgC~TrWT.R TT.TT.T.TlT.~`' NLEGTIPQCV VERNEYLSHL
451 nT~c ~CaT ll llrbV~ l LRvIsL~u~GNK LTGKVPRSMI NrR~7T,TT,T,nT, 501 GN~MLNDTFP NWLGYLFQLK TT.CT17~` 'TU ~ LFMGLQILDL
551 SSNGFSGNLP ERILGNLQTM KrilLIri7,~-ir~ riYl::lJrlLllY YNYLTTISTK
601 ~iy~ vr~ll, DSNMIINLSK ~rrr~ bl IGDLVGLRTL NLS~vLEGU
651 IPASFQNLSV T.T.'.CT.nT.! ISGEIPQQLA SLTFLEVLNL SHNHLVGCIP
701 r~yrJ~r-,r TCYQ''`~r.T.T~ GFPLSKLCGG EDQVTTPAEL L~ .v 751 MISWQGVLVG YGCGLVIGLS v ~ PAWFSRMDLK LEHIITTKMX
~01 =~

WO 95131564 2 1 8 8 5 6 2 ~ ~ i /a ~Trr-~G~r~ _~.,___~.____~CAATTGCC'I"'`CI~LL_,___~L ,.... ,., ArAr~r~rCT

T~rrr~TlD~ r~a~ Ar~TGT~rAArr~AAGTAAAr~AAr~AArAr~AcATc-TGA~GA

CTIllAT~l~'rT~r~T~AT~ rrrAl L~LL~ AArrrrrrAAAArTrAA~--rATArAA
cAA~ rr.~r,~ TAArr,n~r G Arrr.rA~ ~7L L L ~ ~ 120 L F L V I S }~ S C R A 3C A. P K T Q P Y N
rrr-q~rr-~-~rrrA-r-AAr.TrATrrArArrA-rTr~rATrr,r,TCCCAACGA~VL-~
a~ ac =A~_~7L L~7L 1~ ,~A-GTA~.vlv~7L r~ Ar~ArrrAr.{~rjTT~r~A~rAr,~_.,~
P C R P Q E V I D ~r K C ~ G } 1~ D C L Y
r~rrr~Arrrr,~-Ar.~PTr.~r7.Arr~r~r~Lr.Tr~r.T~rrnrTrr~--r~AAGTTrr,rAA
lal I I I I I 1 240 ~L-iL~ ~ACATGTTGGAi~ I.,~ Ar-Ar~pcc~rr~ Tr~rlrTcA~ccGTT
P N P D S C T T Y I Q C V P L D E V G N
rr~ LAAGccATGTrrAAAAr~`~Ttjr~r-Tr~`rArc~ rc~TcGcA}~
~CTTcrr-ArArrAAATTrcr-~ rA~r~l LL .~ rrr--- - . L~-l . _, .~.I'AArrnTT
~ R P V V ~ P C P R G L Q W N D ~ V G R
r~n~nn~nrr~ lrr~7.rrT~ ,AAAnArrrrnrA~r~r~~"r 301 1 . I I I 1 360.
cTTr~rr~rr~rTGATAGGT~TGGAcTrArrr~rAr~Ar-r~rr~ ' L ~ L~ 7 X W C D Y P ~ L S T C P V ~C '[! P Q P R P
r~lr~rr--~ r-Ar~ ;Ar~ ArTCG~j 36~ 1 1 1 1 1 1 420 Ll~_L~ ACAGb:~rl-~LL~ nrr~rrll~r~lpAr~r~rr~ r~r~nrr K R G G V G G R ~ A S V G L P G Y
ArAAr~ r~ Allr~r-TTrlrG~r~rr~r~ , v~ . rrc~
421 1 1 1 ~ ~ 1 4~0 L~7L.L._LL'I I I .'~rrr~-~TTGTr-~r~-rr~Trnll~rTrn~ rr~rr~,.'l 1'1 ,_rnrA
C~ AC
481 ~
GCTG

Claims (54)

CLAIMS:

1. A method of providing increased pathogen resistance in a plant, or a part or propagule of a plant, by induction of variegation in which a gene is expressed or suppressed in cells resulting in the activation of a plant defence response, which comprises:
(i) inactivating a nucleotide sequence which contributes to a plant defence response or inactivating one or more nucleotide sequences forming a part of a combination of nucleotide sequences which contributes to a plant defence response;
(ii) introducing said nucleotide sequence or sequences into the genome of a plant; and (iii) restoring said nucleotide sequence or sequences to a functional form in cells of the plant or a descendant thereof, or a part or propagule of the plant or descendant, to result in increased pathogen resistance.

2. A method of providing increased pathogen resistance in a plant, or a part or propagule thereof, by induction of variegation in which a gene is expressed or suppressed resulting in necrosis, which comprises:
(i) inactivating a nucleotide sequence which contributes to necrosis or inactivating one or more nucleotide sequences forming part of a combination of nucleotide sequences which contributes to necrosis;
(ii) introducing said nucleotide sequence or sequences into the genome of a plant; and (iii) restoring said inactivated nucleotide sequence or sequences to a functional form in cells of the plant or a descendant thereof, or a part or propagule of the plant or descendant, to result in necrosis.

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

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

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

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

7. A method according to claim 6 wherein said nucleotide sequence comprises a plant pathogen resistance gene (R) or a mutant, variant or derivative thereof 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 (E), or both (i) 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 (E).

8. A method according to claim 7 wherein said plant pathogen resistance gene (R) is a tomato Cf-9 gene or a mutant, variant 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 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 (R) 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 (R) 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, 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-4 elictor; or wherein said plant pathogen resistance gene (R) 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 (R) 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.

10. A method according to claim 5 wherein said nucleotide sequence encodes a Cauliflower Mosaic Virus gene VI protein, a bacterial harpin gene protein, an Arabidopsis RPP5 gene protein, a ubiquitin conjugating enzyme, an RNase such as Barnase, a mutant, variant or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or a 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.

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

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

13. A method according to claim 5 in which the plant defence response or necrosis is dependent on the expression of a mutant allele of a gene from a nucleotide sequence which is capable of activating the plant defence response and the expression of an enfeebled negative regulator of the defence response, leading to the plant defence response and/or necrosis.

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

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

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

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

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

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

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

21. A transgenic plant, or descendant thereof, or part or propagule of the plant or descendant, obtainable using a method of any of the preceding claims with increased pathogen resistance compared with wild-type.

22. A plant, or a descendant thereof, or a part or propagule of the plant or descendant, or a derivative of any of these, which is phenotypically variegated, comprising a cell or clone expressing a first phenotype and other cells expressing a second phenotype comprising increased pathogen resistance compared with wild-type, 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.

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 (R) 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 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:
(i) a tomato pathogen resistance gene;
(ii) a tobacco pathogen resistance gene;
(iii) a potato pathogen resistance gene;
(iv) a Arabidopsis pathogen resistance gene;
(v) 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;
(x) 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 (i) to (x);

28. A plant, descendant, derivative, part or 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 N'; 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:
(i) a Cladosporium fulvum avirulence gene or another elicitor of a resistance gene for a Cladosporium fulvum avirulence gene;
(ii) a suitable TMV coat protein or another N' elicitor;
(iii) a suitable PVX coat protein or another Rx elicitor; or (iv) a mutant, variant or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or homologue of any of (i) to (iii).

30. A plant, descendant, derivative, part or propagule according to claim 29 wherein said Cladosporium fulvum avirulence gene is Avr2, Avr4, Avr5 or Avr9.

31. A cell containing (i) nucleic acid encoding one or more than one nucleotide sequence which causes or contributes to the plant defence response and/or cell necrosis, at least one said nucleotide sequence being 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 (R) 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.

35. 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 derivative thereof by way of insertion, addition, deletion or substitution of one or more nucleotides or homologue thereof, or encodes another Cf-9 elicitor.

36. A cell according to claim 35 wherein said R gene is:
(i) a tomato pathogen resistance gene;
(ii) a tobacco pathogen resistance gene;
(iii) a potato pathogen resistance gene;
(iv) a Arabidopsis pathogen resistance gene;
(v) 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;

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

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 N'; 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:
(i) a Cladosporium fulvum avirulence gene or another elicitor of a resistance gene for a Cladosporium fulvum avirulence gene;
(ii) a suitable TMV coat protein or another N' elicitor;
(iii) a suitable PVX coat protein or another Rx elicitor; or (iv) a mutant, variant or derivative by way of insertion, addition, deletion or substitution of one or more nucleotides or a homologue of any of (i) to (iii).

39. A cell according to claim 38 wherein said Cladosporium fulvum avirulence gene is Avr2, Avr4, Avr5 or Avr9.

40. 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.

44. A method of producing a cell according to any one of claims 31 to 43 comprising introduction of nucleic acid (i) and/or (ii) into the cell or an ancestor thereof.

45. A composition comprising any of the following combinations of nucleotide sequences:
(i) a nucleotide sequence comprising R, a nucleotide sequence comprising I and a nucleotide sequence comprising A;
(ii) a nucleotide sequence comprising R, and a nucleotide sequence comprising I and A;

(iii) a nucleotide sequence comprising I, and a nucleotide sequence comprising A and R;
(iv) a nucleotide sequence comprising A, and a nucleotide sequence comprising R and I;
(v) a nucleotide sequence comprising R, I and A;
wherein R encodes a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and A encodes a substance able to reactivate R inactivated by I.

46. A composition comprising any of the following combinations of nucleotide sequences:
(i) a nucleotide sequence comprising R, a nucleotide sequence comprising L, a nucleotide sequence comprising I, and a nucleotide sequence comprising A;
(ii) a nucleotide sequence comprising R, a nucleotide sequence comprising L and I, and a nucleotide sequence comprising (A);
(iii) a nucleotide sequence comprising R, a nucleotide sequence comprising L and A, and a nucleotide sequence comprising I;
(iv) a nucleotide sequence comprising R, a nucleotide sequence comprising I and A, and a nucleotide sequence comprising L;
(v) a nucleotide sequence comprising L, a nucleotide sequence comprising I and R, and a nucleotide sequence comprising A;

(vi) a nucleotide sequence comprising L, a nucleotide sequence comprising A and R, and a nucleotide sequence comprising I;
(vii) a nucleotide sequence comprising I, a nucleotide sequence comprising L and R, and a nucleotide sequence comprising A;
(viii) a nucleotide sequence comprising R, and a nucleotide sequence comprising L, I and A;
(ix) a nucleotide sequence comprising L, and a nucleotide sequence comprising I, A and R;
(x) a nucleotide sequence comprising I, and a nucleotide sequence comprising A, R and L;
(xi) a nucleotide sequence comprising A and a nucleotide sequence comprising A, R and I;
(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 45 to 47 wherein a cell contains any of said combinations of nucleotide sequences.

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

50. 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.