MXPA00009576A - Pathogen-inducible promoter - Google Patents

Abstract

This invention describes pathogen-inducible promoters which normally drive expression of plant hexose oxidases, especially those which can be isolated from Helianthus annuus and Lactuca sativa, more specifically those promoters which naturally are the regulatory regions driving expression of the hexose oxidase MS59 and WL64, respectively. Also claimed are chimeric constructs where these pathogen-inducible promoters drive expression of antipathogenic proteins or of proteins which can elicit a hypersensitive response.

Description

INDOCTBLE PROMOTER FOR PATHÓ-ENOS FIELD OF THE INVENTION This invention relates to the field of pathogen-inducible promoters and the chimeric DNA sequences comprising said promoters, especially in the area of plant biotechnology.

BACKGROUND OF THE INVENTION Inducible promoters include any promoter capable of increasing the size of the products of a given gene in response to an inducer. In the absence of the inducer, the DNA sequence is not transcribed. Typically, the factor that specifically binds with an inducible promoter to activate transcription is present in an inactive form that is then directly or indirectly converted into an active form by the inducer. Said inducer may be a chemical agent such as a protein, a metabolite (sugar, alcohol, etc.), a growth regulator, a herbicide, a phenolic compound or a stress indicator.

Ref.:123342 physiological imposed directly by heat, salt, wounds, toxic elements, etcetera, or indirectly through the action of a pathogen or a disease agent such as a virus. A plant cell containing an inducible promoter can be exposed to an inducer by externally applying the inducer to the cell by means of spraying, watering, heating or the like. Those skilled in the art know inducible promoters and there are several that can be used to drive the expression of genes of interest. Examples of inducible promoters include the 70kD heat shock inducible promoter from Drosophi la mel anogas ter (Freeling, M. et al, Ann.Rev.Genet.l9, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, RT et al, in Milflin, B. J, (ed.) Oxford Surveys or Plant Molecular and Cell Biology, Vol. 3, pp. 384-438, Oxford, Univ. Press, 1986). Examples of which are inducible by a simple chemical are the promoters described in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/3769.

An important subclass of inducible promoters are the promoters that are induced in plants upon infection by a pathogen. Examples of pathogen-inducible promoters are the PRP1 promoter (also known as the gastl promoter) that can be obtained from potatoes (Martini, N. Et al (1993), Mol.Gen. Genet. 263, 179-186), the Fisl promoter. (WO96 / 34949J, the Bet v 1 promoter (S oboda, 1. et al, Plant CelL and Env. 1_8, 865-874, 1995), the Vatl promoter (Fischer, R, Dissertation, Univ. Fr Hohenhei, 1994 Schubert, R. et al, Plant Mol. Biol. 3_4, 417-426, 1997), the promoter of sesquiterpene cyclase (Yin, S. Et al, Plant Physiol. 115, 437-451, 1997) and the promoter gstAl (Mauch, F. and Dudler, R, Plant Physiol 102 1193-1201, 1993) among which may be mentioned.A disadvantage of some of these promoters is that they are also constitutively active or do not react to certain types of pathogens. , it would be advantageous to have promoters that regulate the expression rapidly after infection by a pathogen, that is, with an induction time as small as possible.

Then, there is still a need for promoters that are inducible by pathogen that overcome the disadvantages of the prior art.

BRIEF DESCRIPTION OF THE INVENTION DNA fragments upstream of the regulatory regions of plant genes coding for hexose oxidase have recently been found capable of promoting pathogen-inducible transcription of an associated DNA sequence when they are reintroduced into a plant. Preferably, such is a fragment obtainable from Hel i anus a nn uus said DNA fragment is specifically the regulatory region upstream of the gene encoding hexose oxidase, termed MS59, more specifically characterized in that it comprises the nucleotide sequence of 1 to 1889 illustrated in SEQ ID NO: 15.

Also part of the invention is a DNA fragment obtainable from La ct uca sa ti va capable of promoting the pathogen-inducible transcription of an associated DNA sequence when it is reintroduced into a plant, specifically the DNA fragment which is the current regulatory region. above the gene encoding hexose oxidase, designated WL64 (SEQ ID NO: 18).

Also included in the invention is a portion or variant of a DNA fragment according to that described above capable of promoting pathogen-inducible transcription of an associated DNA sequence when it is reintroduced into a plant.

Embodiments of the invention are chimeric DNA sequences comprising a DNA fragment according to any of the DNA fragments described above in the transcription direction and a DNA sequence which is expressed under its transcriptional control and which are not naturally occurring under the control of said DNA fragment. A preferred embodiment is one in which the chimeric DNA sequence in which the DNA sequence will be expressed causes the production of an antipathogenic protein that is preferably selected from the group consisting of chitinases, glucanases, osmotins, megainins, lectins, saccharide oxidases , oxalate oxidases, Bacillus thuringiensis toxins, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia: Cnicus, Lathyrus, Clitoria, seeds of Alli? M, Aralia and Impatiens and albuminoid proteins such as thionin, napina, trypsin inhibitor of barley, cereal gliadin and wheat alpha amylase.

Another embodiment of the chimeric DNA sequences of the invention is a chimeric DNA sequence in which the DNA sequence to be expressed causes the production of a protein that can induce a hypersensitive response, preferably selected from the group consisting of Cf proteins. and PtO of the tomato, the avr proteins of Cl adospori um ful vum and the inducing proteins of Ps and udomona so Xan th omona s.

Another part of the invention are replicons comprising the chimeric DNA sequences mentioned above, preferably containing a recognition site for a restriction endonuclease for the insertion of a sequence of DNA to be expressed under the control of said DNA fragment. Also included in the invention are microorganisms that contain said replicon, plant cells having a chimeric DNA sequence incorporated in their genome according to those described above and plants consisting essentially of said cells. Such plants are preferably dicotyledonous. Also part of the invention are parts of said plants which may be seeds, flowers, tubers, roots, leaves, fruits, pollen and wood.

Yet another embodiment of the invention is the use of a DNA fragment as described above to identify homologs capable of promoting pathogen-induced transcription in a plant.

Another use of a chimeric DNA sequence according to the invention is the transformation of plants and the use of a portion or variant of the DNA fragments according to the invention to create hybrid regulatory DNA sequences.

Another object of the invention is the use of a chimeric DNA sequence as described above to confer pathogen resistance to a plant.

DETAILED DESCRIPTION OF THE FIGURE Figure 1. Schematic drawing of the MS59 gene (genomic) and its promoter region.

DETAILED DESCRIPTION OF THE INVENTION The main aspect of the invention is the regulatory sequences that naturally occur in plants and drive the expression of the genes encoding hexose oxidase. Specifically, the regions that appear in Heli anus annu us and / or Lac t uca sa ti va, especially in the 5 'region (upstream) of the MS59 gene (SEQ ID NO: 15) or of the WL64 gene (SEQ ID NO. NO: 17), respectively. These genes encode a hexose oxidase that is toxic to fungal pathogens that are described in WO 98/13478, incorporated herein by reference. It has been discovered that, in the face of infection by pathogens, these genes are expressed to a large extent, indicating inductibility by pathogen. Pathogen-inducible promoters are of great value in biotechnological resistance engineering.

Although the invention is especially exemplified with respect to the promoter that drives the expression of hexose oxidase in sunflower, designated MS59, it is believed that all promoters that conduct homologous genes to this MS59 will have properties more or less identical to those of the MS59 promoter, that is, expression activity that is induced by pathogenic infection, preferably by fungal infection. It is commonly known that promoters of homologous genes have similar properties, especially those related to induction by pathogens. Examples of these are the promoters that drive the expression of the osmotin protein related to pathogenesis, of which inducibility has been established by pathogen in potato (Zhu et al, Plant Physiol 108 929-937, 1995), tobacco (Liu et al, Plant Mol. Biol. 29, 1015-1026, 1995) and tomato (Rui z-Medrano et al., Plant Mol. Bol. 20_, 1199-1202, 1992); and the promoters that drive the expression of PR-10 group genes, of which inductibility has been demonstrated in the pSTH-2 promoter in potato (Matton, D. P: and Brisson, N. Mol. Plant - Microbe lnterac. , 325-331, 1989), the AoPRl promoter in Aspara gus offi ci nal is (Warner et al, The Plant J. 3 ^ 191-201, 1993) and the Bet v 1 promoter in Be t ul a verrucosa (Swoboda et al. al, Plant Cell, Environm. 1_8 885-874, 1995).

In this description the terms "regulatory sequence" and "promoter" will be used interchangeably The present invention provides, inter alia, chimeric DNA sequences comprising the regulatory sequences according to the invention. The term chimeric DNA sequence should be interpreted as any DNA sequence comprising sequences not normally found in nature. Then, chimeric DNA should encompass DNA sequences comprising regulatory regions inducible by pathogen at a non-natural site of the plant genome without forgetting the fact that the genome of said plant normally contains a copy of said regulatory region at its normal chromosomal site . Similarly, said regulatory region can be incorporated into the genome of the plant in places where it can not be found naturally, or in a replication vector where it is not found in nature, such as in a bacterial plasmid or in a viral vector. Chimeric DNA should not be limited to DNA molecules that can replicate in a host, but should also encompass DNA capable of being ligated into the open reading structure of a replicon according to this invention, for example by specific adapter sequences. The open reading structure may or may not be linked to its regulation elements in the 3 'direction.

The open reading structure of the gene whose expression is driven by a pathogen-inducible promoter can be derived from a gene library. In this case, it can contain one or more introns separating the exons that constitute the open reading structure that codes for the protein according to the invention. The open reading structure may also be encoded by a single exon or by a mRNA of the mRNA coding for the protein according to the invention. Open reading structures according to the invention can also be those in which introns have been added or artificially removed. Each of these variants is encompassed by this invention.

In order to be expressed in a host cell, the regulatory sequences according to the present invention will usually be accompanied by transcription initiation sequences which will be suitably derived from any gene capable of being expressed in the chosen host cell, as well as from initiation regions. of translation for recognition and adhesion of ribosomes. In eukaryotic cells, an expression cassette usually comprises a transcription termination region located downstream of the open reading fr allowing transcription to terminate and polyadenylation of the primary transcript to occur. further, the use of codons can be adapted to the chosen host. In addition, a signal sequence which is responsible for the orientation of the gene expression product towards subcellular compartments can commonly be encoded. The principles governing the expression of a chimeric DNA construct in a given host cell are commonly understood by those skilled in the art and the creation of expressible constructs of chimeric DNA is now routine for any host cell, be it prokaryotic or eukaryotic.

For the chimeric DNA sequence to be maintained in a host cell it must usually be placed in the form of a replicon comprising said chimeric DNA sequence according to the invention linked to DNA that is recognized and replicated in the chosen host cell. Accordingly, the selection of the replicon is largely determined by the host cell chosen. Such principles governing the choice of suitable replicons for a certain type of host cell are well known to any person skilled in the art.

A special type of replicon is one capable of transferring, in whole or in part, to another host cell, such as a plant cell, thus co-transferring the reading structure opened according to the invention to said plant cell. Replicons with this capacity are referred to here as vectors. An example of such vectors is the vector plasmid Ti which, when present in a suitable host, such as Agroba c t eri um t umefa ci ens, is capable of transferring part of itself, the so-called T region, to a plant cell. Currently, numerous types of Ti vector plasmids (see: EP O 116 716 Bl) are commonly used to transfer chimeric DNA sequences to plant cells, or protoplasts, from which new plants can be generated with said incorporated chimeric DNA sequences. Stably in your genome. A particularly preferred form of plasmid vectors Ti is that of the so-called binary vectors as described in (EP or 120 Bl and US 4940838). Other suitable vectors that can be used to introduce DNA according to the invention into a host plant can be selected from viral vectors, for example, non-integrative viral vectors, such as those that can be derived from double-stranded plant viruses (e.g. , CaMV), virus with simple chain, gemini virus and the like. The use of such vectors can be advantageous, particularly when it is difficult to transform the host plant stably. This may be the case of woody species, especially trees and vines.

The term "host cells incorporating a chimeric DNA sequence according to the invention to its genome" must comprise all cells and multicellular organisms comprising them or consisting of them that stably incorporate said chimeric DNA into their genome. , maintaining is the chemetic DNA and preferably transmitting a copy of it to cells of its progeny, either by mitosis or by meiosis. According to a preferred embodiment of the invention, plants are provided which essentially consist of cells which incorporate one or more copies of said chimeric DNA in their genome and which are capable of transmitting one or several copies to their progeny, preferably in a Mendelian manner. Thanks to the transcription and translation of the chimeric DNA according to the invention in some or all of the cells of the host plant, those cells comprising said regulatory region will respond to the attack of the pathogen and then produce the protein encoded by the open reading structure that is under the control of the regulatory region. In specific embodiments of the invention, this protein will be an antipathogenic protein capable of conferring resistance to pathogenic infections.

As is well known to those trained in the art, the regulatory regions of plant genes consist of different subregions with interesting properties in terms of gene expression. Examples of such sub-regions as indicated herein are the enhancers, also transcription silencers. These elements can work in a general (constitutive) way or in a specific tissue way. Deletions may be made in the regulatory regions of the DNA according to the invention, the subfragments being evaluated for patterns of expression of the associated DNA. The various fragments obtained, as well as combinations thereof, may be useful in pathogen resistance engineering methods or in other applications involving the expression of heterologous DNA in plants. The use of the DNA sequences according to the invention to identify functional sub-regions and the subsequent use thereof to promote or suppress the expression of genes in plants are also included in the present invention.

Considering the need for a transcription termination region, it is generally believed that such a region improves both the confidence and efficiency of transcription in plant cells. The use thereof is then markedly preferable in the context of the present invention.

Examples of proteins that can be used in combination with the ICS regulatory region according to the invention include, but are not limited to: avble β-1,3-glucanases and chitinases from barley (S egle, M. et al, Plant Mo Biol 12, 403-421, 1989; Balance, GM et al., Can. J. Plant Sci. 56, 459-466, 1976; Hoj PB et al, FEBS Lett. 230, 67-71, 1988; Hoj PB. et al., Plant Mo. Biol. 13, 31-42, 1989), bean (Boller T et al., Planta 157, 22-31, 1983; Broglie KE et al., Proc. Nati. Acad. Sci. USA 83, 6820-6824, 1986; Vógeli U. et al., Plant 174, 364-372, 1988); Mauch F. & Staehelin L.A., Plant Cell 1, 447-457, 1989); cucumber (Metraux J.P. &Boller T., Physiol, Mol Plant Pathol, 28,161-159, 1986); leek (Spanu P. et al, Planta 177, 447-455, 1989); corn (Nasser W. et al, Plant Mo. Biol. 11, 529-538,1988), oats (Fink W. et al, Plant Physiol. 270-275, 1988); Pea (Mauch F. et al, Plant Physiol 76, 607-611, 1984, Mauch F. et al, Plant Physiol 87, 325-333, 1988); poplar (Parsons, T.J. et al, Proc. Nati Acad. Sci. USA 86, 7895 -. 7895-7899, 1989); Papa (Gaynor JJ, Nucí Acids Res. 16, 5210, 1988; Kombrink E. et al., Proc, Nati., Ac .. Sci. USA 85, 782-786, 1988; Laflamme D. and Roxby R., Plant Mol. Biol. 13, 249-250, 1989); tobacco (for example, Legrand M. et al Proc. Nati, Acad. Sel. USA 84, 6750-6754, 1987; Shinshi H. et al, Proc. Nati. Acad. Sci. USA 84, 89 -. 89-93, 1987); tomato (Jooste M.H.A. & De Wit P.J.G.M., Plant Physiol. 89, 945-951, 1989); wheat (Molano J. et al, J. Biol. Chem 254, 4901-4907, 1979); magainins, lectins, toxins isolated from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa (EP O 576 483) and Amaranthus (EP O 593 501 and US 5,514,779), albuminoid proteins (such as thionin, napina, barley trypsin inhibitor , cereal gliadin and wheat high amylase, EP O 602 098), isolated proteins from Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus and Clitoria (EP or 603 216), protein isolated from Capsicum, Briza, Delphinium, Catapodium, Baptista and Microsensis, oxalate oxidase (EP or 636 181 and EP O 673 416), saccharide oxidase (PCT / EP 97/04923), antimicrobial proteins isolated from Allium seeds and proteins from Aralia and Impatiens (WO 95/24485), Heuchera and Aesculus proteins (PCT / GB94 / 02766), mutant peptides of the above-mentioned proteins (PCT / GB96 / 03065 and PCT / GB96 / 03068), and the like.

Another use of the inducible promoter is the conduction of proteins that play a role in the interaction between genes for resistance (for example, as described in WO 91/15585). Such proteins are, for example, plant proteins as described in Karrer, EE et al (Plant Mol. Biol 36 681-690, 1998) activated ndrl, activated edsl, activated Xa21, Cf proteins, BS3 protein and tomato Pto proteins, the Rpml and Rps2 proteins of Arabidopsis thaliana, the N gene of tobacco, the avr inducing proteins of Cladosporium fulvum, the avrBs3 protein of Xanthomonas, the harpins of Erwinia and the avrPto protein of Pseudomonas.

The actual applicability of the invention is not limited to some plant species. Any species of plant that is subject in some way to some kind of attack by pathogens can be transformed with genes according to the invention, allowing the regulatory region to be induced by a pathogenic infection, thus triggering the production of antipathogenic proteins that they will be produced in several or all of the cells of the plant.

Although some embodiments of the invention may not be practicable in the present, for example, because some plant species are still resistant to genetic transformation, the practice of the invention in such plant species is merely a matter of time and not of principle. , because resistance to genetic transformation as such is not relevant to the underlying realization of the invention.

The transformation of plant species is now routine for a now impressive number of plant species, including mono and dicotyledons. In principle any transformation method can be used to introduce chimeric DNA according to the invention into a suitable ancestral cell, as long as the cells are capable of regenerating an entire plant. The methods can be chosen from the calcium / polyethylene glycol method for protoplasts (Krens, FA Et al, Nature 296 72-74, 1982, Negrutiu, I. Et al, Plant Mol Biol, 363-373, 1987), electroporation of protoplasts. (Shillito, RD Et al, Bio / Technol 3, 1099-1102, 1985), microinjection in plant material (Crossaway, A. Et al, Mol.Gen.Genet 202 179-185, 1986), bombardment of particles covered with DNA or RNA on varied plant material (Klein, TM et al, Nature 327, 70, 1987), virus infection (non-integrative) and the like. A preferred method according to the invention comprises the transfer of DNA mediated by Agroba ct eri um. The use of so-called binary vector technology as described in EP A 120 516 and US Patent 4940838 is especially preferred.

The tomato transformation is preferably done as described in Van Rokel et al (Plant Cell Rep. 1_2 644-647, 1993). The transformation of para is preferably done as described in Hoekma et al (Hoekma, A. et al, Bio / Technology 1, 273-278, 1989). Generally, after transformation, cells or groups of plant cells are selected based on the presence of one or more markers that are encoded by the expressible genes of the plant cotransferred with the nucleic acid sequence coding for the protein according to the invention. invention. Then, the transformed material is regenerated in a complete plant.

Although considered more resistant to genetic transformation, monocotyledonous plants are susceptible to transformation, and fertile transgenic plants can be regenerated from cells, embryos or other transformed plant material. Currently preferred methods for the transformation of monocotyledons are the bombardment with microprojectiles of embryos, explants or cell suspensions, and the direct taking of DNA or electroporation (Shimamoto et al, Nature 338, 274-276, 1989). Transgenic maize plants have been obtained by introducing the bar gene Streptomyces hygroscopi cus, which codes for phosphinothricin acetyltransferase (an enzyme that inactivates the herbicide phosphinothricin) in embryogenic cells of a suspension of a corn crop by bombardment with microprojectiles (Gordon-Kamm , Plant Cell, 2 603-618, 1990). The introduction of genetic material into aleurone protoplasts or other monocotyledonous crops such as wheat and barley has also been reported (Lee, Plant Mol. Biol 1_3 21-30, 1989). Wheat plants have been regenerated from embryogenic suspension cultures by selecting only the oldest compact and nodular embryogenic callus tissues for the establishment of briogenic suspension cultures (Vasil, Bio / Technol 8_ 429-434, 1990). The combination with transformation systems for these crops allows the application of the present invention in monocots.

Monocotyledonous plants, including commercially important crops such as rice or corn are also susceptible to DNA transfer by Agroba ct erium strains (see WO 94/00977, EP 0 159 418 Bl, Gould, J. et al, Plant Physiol. , 426-434, 1991).

Upon completion of DNA transfer and regeneration, the putatively transformed plants can be evaluated using, for example, Southern analysis, for the presence of the chimeric DNA according to the invention, number of copies and / or genomic organization. In addition, or alternatively, the expression levels of the new introduced DNA can be measured using Northem or Western analysis, techniques well known to those skilled in the art. After the initial analysis, which is optional, transformed plants with the number of copies and the desired level of expression of the new introduced chimeric DNA can be evaluated with respect to their levels of resistance against pathogens. Alternatively, the selected plants may be subjected to another transformation session, for example, to introduce other genes, so as to improve the levels of resistance or increase it.

Other evaluations may include the evaluation of pathogenic resistance in the open field, verifying fertility, yield and other characteristics. Such evaluation is now carried out routinely by persons with ordinary knowledge in the art.

After such evaluations, the transformed plants can be cultivated directly, but commonly they can be used as parental lines in the creation of new varieties or in the creation of hybrids and the like.

To obtain transgenic plants capable of constitutively expressing more than one chimeric gene, there are several alternatives, including the following: A. The use of DNA, for example, T-DNA in a binary plasmid, with a number of physically modified genes attached to the gene of the selective marker. The advantage of this method is that the chimeric genes are physically linked and thus migrate as a single Mendelian locus.

B. Cross-pollination of transgenic plants capable of expressing one or more chimeric genes, preferably linked to a selective marker gene, with pollen from a trapezic plant that contains one or more chimeric genes bound to another selective marker. Then, the seed obtained by this crossing can be selected based on the presence of both markers, or based on the presence of the chimeric genes themselves. The plants obtained from the selected seeds can be used for additional crosses. In principle, the chimeric genes are not in the same locus and can then segregate as independent loci.

C. The use of a number or a plurality of chimeric DNA molecules, for example, plasmids, each with one or more chimeric genes and a selective marker. If the cotransformation frequency is high, then, the selection based on a single marker is sufficient. In other cases, the selection based on more than one marker is preferred.

D. The consecutive transformation of transgenic plants already containing a first, a second, etc., chimeric gene with the new chimeric DNA, optionally comprising a selective marker gene. As in method B, the chimeric genes are not in principle in the same locus, and can be segregated as independent loci.

E. The combination of the strategies mentioned above.

The actual strategy may depend on various considerations such as can easily be determined according to the purpose of the parent lines (direct cultivation, use in a breeding program, employment to produce hybrids) but is not critical to the described invention.

In this context it should be emphasized that plants already containing the chimeric DNA can form a suitable genetic framework for introducing chimeric DNA according to the invention, for example, to improve the production of antipathogenic substances, thereby improving the induction capacity, thus improving the resistance levels. The cloning of other genes corresponding to proteins that can be used appropriately in combination with DNA regulatory fragments and the obtaining of transgenic plants able to overexpress relatively same, as well as the evaluation of their effect on the resistance to pathogens in pl an ta , is now within the knowledge of anyone trained in the art.

Can be grown in the field, the greenhouse, in the home or other places plants or parts of them with improved resistance against pathogens. The complete plants or part of them can be used for human or animal consumption the plants or the edible parts thereof, and they can also be processed as food, food and other purposes in any form of agriculture or industry. Agriculture also involves horticulture, arboriculture, floriculture and the like. Industries that would benefit from plant material according to the invention include but are not limited to the pharmaceutical industry, the pulp and paper manufacturing industry, the sugar industry, the food and feed industry, the enzyme manufacturing industry and It was before.

The advantages of the plants or parts thereof according to the invention are the lower need for biocides, decreasing the cost of the material, work and environmental pollution, also prolonging the shelf life of the products (for example, fruits, seeds and similar) of such plants. Plants for the purpose of this invention should encompass any multicellular organism capable of photosynthesis and subject to some form of pathogenic attack. Angiosperms should be included, as well as gymnosperms, mono and dicotyledonous plants.

EXAMPLE 1 INDUCTION OF MESSENGERS MS59 IN SUNFLOWER PLANTS Leaves of 7 to 8 weeks of sunflower plants (Hel i anus annus cv Zebulon) were induced by rolling them 5 times with 5 mM salicylic acid (SA), spraying once with 1 mM SA, one with jasmonic acid (JA ) 0.1 mM, one with ACC (1-aminocyclopropane-1-carboxylic acid, a precursor of the plant hormone ethylene) or generating a wound. The plant samples were harvested from induced leaves after 24 hours (SA l M, 0.1 mM JA, 1 mM ACC and wounds) and after 5 days (5 mM SA). Control samples were taken 24 hours after induction in non-induced plants EXAMPLE 2 EXTRACTION OF SUNFLOWER LEAF RNA AND SYNTHESIS OF cDNA Total RNA was extracted from 10 g of leaf material using a hot phenol method and purified using the Quiagen RNA buffer kit and tip-100 columns (Quiagen GmbH, Germany). The contaminating DNA was degraded using DNase 1 treatment ( Gibco BRL).

The cDNA was prepared using 1 μg of total RNA, I μl of primers oligo (dT) i2-i8 (500 μg / ml, Gibco BRL) and 200 units of reverse transcriptase Superscript II RT RNAse H + (Gibco BRL) as described by the manufacturer.

EXAMPLE 3 CONSTRUCTION OF MS59 BY MIMIC AND ANALYSIS OF THE SAMPLES BY PCR - COMPARATIVE RT MS59 levels were determined using comparative RT-RT technology. In this technique, the competition between the cDNA target and an artificial PCR MIMIC carries out the quantification of possible transcript levels (Paul D. Siebert and James W. Larrick (1992), Nature 359, 557-558).

For the construction of a MIMIC PCR the following primers were constructed: FR-pUC-208 (SEQ ID NO: 1) 5 'GTT CCG GAG GTT GTG ACC GTG GGA TGT GCT GCA AGG CG3', FR-pUC-209 (SEQ ID NO: 2) 5 ', CTG GGG AAG CCC GTG TAG TAA AGC CCC CGC GCG TTG GCC GAT TC3', Fr-MS59-47 (SEQ ID NO: 3) 5 ', CTG GGG AAG CCC GTG TAG TAA AGC3 * and FR -MS59-77 (SEQ ID NO: 4) 5 'GTT CCG GAG GTT GTG ACC GTG3 The primers FR-pUC-208 and FR-pUC-209 were used to amplify a 387 bp fragment of the pUC18 plasmid (Yanisch-Perron, C, Vieira, J and Messing, J. (1985) Gene 33, 103-119) by PCR (10 cycles of 1 'at 95 ° C, 1' at 55 ° C, 2 'at 72 ° C). From this PCR product 1 μl was amplified using the primers FR-MS59-47 and FR-MS59-77 by PCR to produce a large amount of MIMIC PCR (30 cycles of 1 * at 95 ° C, 1 'to 55 ° C, 2 'to 72 ° C). The primers FR-MS59-47 and FR-MS59-77 will amplify a 312 bp band of the MS59 cDNA so that it can be easily distinguished from the 387 bp MIMIC when separated on a 2% agarose gel. The dilutions of MIMIC PCR were made in a range of 100 ng / μl to 0.01 ag / μl in water with 0.2 μg / μl of glycogen as transporter.

TABLE 1: induction levels of the MS59 messenger in sunflower leaves after different stress treatments compared to the control.

Induction Method Induction parts Control Ia 5 mM salicylic acid 1000 1 mM acid 1 mM salicylic acid ACC 10 0.1 mM jasmonic acid 10 Wounds 10 Notes: 3 could not be detected, arbitrarily set to 1.

The cDNA samples were analyzed in a competitive RT-PCR. Then, 2 μl of the samples were combined in a 0.5 ml tube with 1 μl of diluted MIMIC (amounts 0.1 pg, 10, fg, 1 fg and 0.1 fg). DNA and MIMIC amplification was performed using 10 μM of the primers FR-MS59-47 and FR-MS59-77, 0.5 μL of 20 M dNTP, lx PCR buffer, MgCl2 and 2.5 units of Taq DNA polymerase (Gibco BRL) , letting it proceed for 35 cycles, 1 '95 ° C, 1' 55 ° C, 2 '72 ° C. The PCR products were separated on a 2% agarose gel and visualized by dyeing with ethidium bromide using a UV illuminator.

EXAMPLE 4 TESTS AND INFECTION IN SUNFLOWER PLANTS WITH DIFFERENT FUNGI Infections with fungi developed in plants from 7 to 8 weeks. The leaves were inoculated by placing small drops (15 to 20 μl) of a spore suspension of Bo tryti s neerea, a suspension of hyphae fragments from Di aporth eh el ani thi] (PH9905) or a suspension of hypha fragments. Scl ero ti ni scl ero ti orum in small cuts made on the leaves to allow the fungi to penetrate. The infections were allowed to proceed at 18 ° C and at high relative humidity (+ 90%). The infection of the fungus was allowed to proceed 18 ° C at a high relative humidity (+ _90%). Discs were collected from the leaves (13 mm in diameter) that contained the site of the infection 4 days after inoculation. Leaf disks of non-infected leaf tissue were collected as control.

EXAMPLE 5 EXTRACTION OF RNA-POLY + SUNFLOWER LEAF AND SYNTHESIS OF cDNA Poly-A + 100 mg of leaf tissue was collected using the Quickprep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Upsala, Sweden). The relative amount of mRNA was determined using nucleic acid visualization by staining 10 μl of the samples with 4 μl of 1 μg / ml ethidium bromide in a UV illuminator.

Equal amounts of poly-A + RNA- (+ _100 ng) were used to synthesize cDNA using 200 units of Superscript II RT RNAse H-reverse transcriptase (Gibco BRL) and 1 μl oligo (dT) primers 12 to 18 (500 μg / ml, Gibco, BRL) as described by the manufacturer.

EXAMPLE 6 ANALYSIS OF SAMPLES BY PCR - COMPETITIVE RT The various cDNA samples were analyzed as described in Example 3.

TABLE 2: levels of induction of the messenger after infection with different fungi.

Notes: a = arbitrarily set at 1 b = 13 mm leaf disc around the area of fungal infection c = 13 to 25 mm leaf ring around the site of fungal infection.

EXAMPLE 7 CONSTRUCTION OF A MIMIC PCR gapC AND ANALYSIS OF THE SAMPLES BY COMPETITIVE PCR As an internal control of RNA quality and cDNA preparation, a PCR-RT was included in the gapC maintenance gene using the same samples described in Examples 4, 5 and 6.

For the construction of a PCR MIMIC, the following primers were developed: FR-pUC-224 (SEQ ID NO: 5) 5 'CCA TGG GCT CAA ACT GGA GCC GGC CGG GAG CAG ACA AGC CCG 3', FR-pUC-225 ( SEQ ID NO: 6) 5 'CGA GAC GTC AAC AGT CGG GAC CCA CTC ATT AGG CAC CCC AGG C3', FR-gapC-211 (SEQ ID NO: 7) 5 'CCA TGG GCT CAA ACT GGA GCC G 3' and FR-gapc-212 (SEQ ID NO: 8) 5 'CGA GAC GTC AAC AGT CGG GAC C3'. The primers FR-pUC-224 and FR-pUC-225 were used to amplify a 527 bp fragment of the pUC18 plasmid (Yanisch-Perron, C, Vieira, J and Messing, J. (1985) Gene 3_3 103-119) by PCR (10 cycles of 1 'at 95 ° C, 1' at 55 ° C, 2 'at 72 ° C). From this PCR product 1 μl was amplified using the primers FR-gapC-211 and FR-gaPC-212 by PCR to produce a large amount of MIMIC PCR (30 cycles of 1 'at 95 ° C, 1' to 55 ° C, 2 'to 72 ° C).

The FR-gapC-211 and FR-gapC-212 primers will amplify a 470 bp band of the MS59 cDNA so that it can be easily distinguished from the 527 bp MIMIC when separated on a 2% agarose gel. The dilutions of MIMIC PCR were made in a range of 100 ng / μl to 0.01 ag / μl in water with 0.2 μg / μl of glycogen as transporter.

The cDNA samples were analyzed in a competitive PCR. Then, 2 μl of each sample was combined in a 0.5 ml tube with 1 μl of diluted MIMIC (quantities 0.1 pg, 10, fg, 1 fg and 0.1 fg) or without MIMIC. Amplification of cDNA and MIMIC was performed using 10 μM of the FR-gapC-211 and FR-gapC-212 primers, 0.5 μM of 20 mM dNTP, lx PCR buffer, MgCl2 and 2.5 units of Taq DNA polymerase (Gibco BRL) , leaving it to proceed for 35 cycles, l1 95 ° C, 1 '55 ° C, 2' 72 ° C. The PCR products were separated by staining with ethidium bromide and using a UV illuminator.

TABLE 3: induction levels of the control gapC messenger in sunflower leaves after infection with different fungi.

Notes: a = arbitrarily set at 1 The results show that RNA quality and cDNA preparation are not affected by these fungal infections. The induction of gapC messengers by plant pathogens and environmental stress factors was previously described in Laxalt et al (1996) Plant Mol. Biol. 20: 961-972.

EXAMPLE 8 ISOLATION OF THE MS59 PROMOTER OF THE SUNFLOWER GENOME For the isolation of the MS59 promoter, genomic DNA was isolated from sunflower leaves using a CTAB extraction method. About 10 μg of genomic DNA were digested with enzymes, BspHI, EcoRV, NlalV, Hphl, Rsal and SsplV for 16 hours at 37 ° C. These restriction sites are all located in the first part of the Ms59 cDNA. The digestion mixtures were extracted with phenol / chloroform / isoamyl alcohol and precipitated 0.1 volumes of NaAc (pH = 5.2) 3 M and 2.5 volumes of 96% ethanol. The DNA precipitate was dissolved in 50 μl of distilled water and washed with 70% ethanol, using 25 μl of each sample to separate them in a 0.7% agarose gel for 16 hours. The DNA was transferred to a nylon membrane (Hybond-N +, Amersham Life Science) using Southern Blotting with 0.4 M NaOH. The imprint was hybridized (16 hours at 65 ° C) using a 320 bp fragment (from the initial ATG condom until BspHI site) labeled with 32P-dCTP as a probe. Then the imprint was washed with a stringency of 0.2x SSC at 65 ° C. The results of the Southern Blot are indicated in Table 4.

The remaining 25 μl of the digestion mixture were ligated in such a way that the circularization of the DNA fragments was stimulated. This was accomplished by ligating the DNA in a (large) volume of 300 μl in Ix T4 binding buffer and 5 weiss units of T4 DNA ligase (Gibco BRL) for 1 hour at 16 ° C. Again the mixture was extracted with phenol / chloroform / isoamyl alcohol and precipitated with ethanol, the precipitate of DNA being dissolved in 50 μl of water.

The primers were designed in the first part of the cDNA (between the codon ATG and the first restriction site used, BspHI) and directed forward. The primers FR-MS59-11 (SEQ ID NO: 9) 5 ', CAG GCA GCT GTT GTG TGT GGC 3', and FR-MS59-49 (SEQ ID NO: 10) 5 ', CGG GAA GCA GAA TGG were used. GTT G 3 'in a PCR reaction in 1 μl of the binding mixture using the Klentaq polymerase mixture (Clontech Laboratories, Inc., Palo Alto, CA, USA), 200 μM dNTP and 10 μM of each primer. The polymerase mixture was activated by 1 'at 94 ° C followed by 35 cycles of 30"at 94 ° C 1' at 55 ° C and 3 'at 68 ° C. The PCRT products were analyzed on an agarose gel 1% without detecting any specific band, then a native PCR was developed as described above but now with the native primers FR-MS59-34 (SEQ ID NO: 11) 5 'ACG TAG ATA TGC AAC AAG AAA CGC 3' and FR-MS59 (SEQ ID NO: 12) 5 ', GAG CAA GAG AAG AAG GAG AC 3' using 1 μl of the PCR product from the first round After the analysis of the PCR products in an agarose gel 1 Very specific bands were detected, and the results of the inverse PCR are shown in Table 4.

TABLE 4: Results of the inverse PCR and Southern Blot expressed in band sizes (nd = not determined) The 1.9 kb band of HindIII of the iPCR was isolated from the gel, the sequence of the ends being determined by using the primers FR-MS59-34 and FR-MS59-50 in an automatic DNA sequencer (Applied Biosystems).

New PCR primers were developed on the basis of the DNA sequence of the MS59 promoter of the sunflower genome. The primer FR-MS59-226 (SEQ ID NO: 13) 5 'GCA AGC TTT ATA GTT TAC GAT CC 3' is located downstream, in the upstream part of the promoter region MS59, superimposed with the restriction site of HindIII. The primer FR-Ms59-227 (SEQ ID NO: 14) 5 ', TTG CCA TGG TGC ATG GTT TAG CG 3' can be annealed to the most downstream part of promoter / leader MS59 overlaying with the start of the ATG translation and introducing a Ncol restriction site spanning the initial ATG codon. The DNA sequence of the complete promoter fragment from the Hind III to Neo I site upstream (SEQ ID NO: 15, nucleotides 1-1889) was determined using automated DNA sequence analysis (Applied Biosystems).

Using Pfu DNA polymerase (Stratagene) and both primers, the MS59 promoter region of the sunflower genomic DNA was amplified. The 1.9 kb PCR product was digested with HindIII and Ncol and ligated to a multiple donor vector also digested with HindIII and Ncol. The promoter was fused with the reporter gene of the GUS intron (Jefferson et al (1957) EMBO J. 6: 3901-3907) followed by the 3 'untranslated region of the potato proteinase inhibitor II gene (Thornburg et al. 1987, Proc Nati, Acad. Sci. USA, 8_4_, 744-748) that contains the necessary sequence for polyadenylation (An et al, 1989, Plant Cell 1_, 115-122) using the restriction sites Ncol and EcoRI, obtaining pMOG1367 The entire chimeric gene flanked by the restriction sites EcoRI and HindIII was then transformed into pMOGdOO (for the description of this plasmid see, for example, WO 97/42326) digested with EcoRi and HindIII.

The resulting binary vector pMOG1368 was introduced into the strain of Agrobacterium tumefaciens EHA105 for the transformation of potato and tomato white cultures, into the strain MOG101 for the transformation of tobacco and Arabidopsis thaliana and into the strain MOG301 for the transformation of Brassica napus.

EXAMPLE 9 TRANSFORMATION OF pMOG1368 IN PAPA CV KARDAL PMOG1368 was transformed into potato essentially as described in Hoekma, et al (Hoekma, A. et al, Bio / Technology 7, 273-278, 1989): Briefly transformed potatoes (Solanum tuberosum cv. Kardal) with the Agrobacterium strain EHA 105 pMOG1368. The basic culture medium MS30R3 consisted of MS salts (Murashigue and Skoog (1962) Physiol. Plant 1_4 473), vitamins R3 (Ooms et al (1987) Theor. Appl. Genet. 13_, 744), 30 g / 1 of sucrose, 0.5 g / 1 of MES with final pH of 5.8 (adjusted with KOH) solidified when necessary with 8 g / 1 of Daichin agar. Tubers of Sol an um t uberos um cv. Kardal, sterilizing its surface by burning 96% ethanol for 5 seconds. The flames were extinguished in sterile agar, then pieces of approximately 2 mm thick were cut. Disks containing a piece of vascular tissue were cut and incubated for 20 minutes in MS30R3 medium with 1 to 5 x 108 bacteria / ml Agroba ct erium EHA 105 containing the binary vector. The tuber discs were washed with MS30R3 medium and transferred to solidified post-culture medium (PM). The PM consisted of MS30R3 medium supplemented with 3.5 mg / ml of zeatin riboside and 0.03 mg / ml of indoleacetic acid (IAA). After two days, the discs were transferred to fresh PM medium with 200 mg / l of cefotaxin and 100 mg / l of vancomycin. Three days later, the tuber discs were transferred to shoot induction medium (SIM) consisting of PM medium with 250 mg / l of carbenicillin and 100 mg / l of kanamycin. 4 to 8 weeks later, the shoots that emerged from the discs were separated and placed in medium for root development (MSS30R3 medium with 100 mg / l of cefotaxime, 50 mg / l of vancomycin and 50 mg / l of kanamycin). 4 to 8 weeks later, the shoots that emerged from the discs were separated and placed in medium for root development (MS30R3 medium with 100 mg / l of cefotaxime, 50 mg / l of vancomycin and 50 mg / l of kanamycin). The shoots were propagated axially by cuts in the meristems.

EXAMPLE 10 EVALUATION OF THE ROLE OF THE PROMOTER IN PLANTS OF TRANSGENIC POTATO Transgenic potato plants containing the promoter construct pMOG1368 MS59-GUS were grown in in vitro tubes and analyzed for their expression of the GUS gene. For this purpose, samples of leaves, stems and roots were taken and stained (results in Table 5). GUS expression levels were visually determined on a scale of 0 to 5, where 0 is an undetectable expression and 5 is the highest level of GUS observed in the leaves of the transgenic plant, of the rare transgenic line of tobacco 35S- GUS (line 96306). Samples of the leaves of this plant were included in all experiments as an internal reference.

TABLE 5: Expression of the GUS gene driven by the MS59 promoter in leaves, stems and roots of small seedlings i n vi tro Seedlings in the same age were infected with the fungus that caused late potato aphid, Phyt oph th ora i nfes t ans. Small droplets of water containing a high concentration of spores were applied on the surface of the leaf. The infection was allowed to proceed at room temperature for 96 hours. The leaves that showed symptoms of the disease of the seedlings were removed and stained for the expression of the GUS gene by GUS histochemical analysis (Godjin et al.

The Plant Journal (1993) 4 (5), 863-873). The expression in the lesion resulting from the infection of the fungus was monitored in the primary zone (the area around the site of infection) and in the uninfected part of the leaf (background).

TABLE 6: Expression of the GUS gene driven by the MS59 promoter in leaves of potato seedlings infected with P. infes tans.

Number of Before Injury Bottom line Plant primary infection 1 1336688--11 0 0 0 0 1368-3 0 0 0 0 1368-4 0 0 1 0 1368-5 0 0 1 0 1368-6 0 0 0 or 1368- 7 0 0 0 or 1368-8 0 0 0 or 1368-9 0 0 1 or The performance of the promoter was also evaluated in potato leaves grown before and after infection with P. Inf s t ans. Before inoculation, the leaves were removed and stained for GUS expression. The plants were then sprayed with a spore suspension at 5x105 spores / ml, allowing the infection to develop for 4 days (96 hours). Again, the leaves were removed and stained for GUS expression. The results are shown in Table 7. GUS expression levels were measured in the lesion, in the primary zone and in the uninfected part of the leaf.

TABLE 7: Expression of the GUS gene driven by the MS59 promoter in leaves of transgenic potato plants before and after infection with P. Infes t ans.

The results indicate that the MS59 promoter responds to fungal infection. The level of induced expression is quite low and may fall below detection levels in some cases. This would explain the low frequency of inducible GUS expression detected.

L IS S S E CUENC IA < 110 > MOGEH in erxu-tioaal nv < 120 > New pathogen-inducible promoter < 141 > < 150 > BF 98201024.1 < 151 > 1998-04-01 < 170 »paeßatia See» 2.1 < 210 > i < 211 > 38 < 212 > BRA < 313 > Artificial Sequence < 220 > < 323 > Description of Artificial Sequence: primer «400 > l gttceggagg ttgtga? cgt gggatgtgct gcaaggcg 38 «310» 2 < 211 > 44 < 212 > DNA < 2ia > Artificial Sequence < 220 > 223 > Artificial Sequence Description: Primer < 4Q0 > 2 ctggggaagc ccgtgeagta nagcccccgc gcgtggccg atte 44 < 210 > 3 «211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 3 ctggggangc ccgtgtagta «age 24« 310 »4 < -tll > 31 < 312 > DKA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer < 4DD > 4 getccggagg tcgtgaccgt g 21 < 210 > s H 43 < 312 > DA < 213 > Artificial Sequence < 330 > «223 Description of Artificial Sequence: primer < 400 > 5 ccaegggctc aaastggagc cggccgggag cagacaagcc eg 42 < 210 > 6 < 31 43 < 212 > DBA < 213 > Artificial Sequence < 220 > < 333 »Description of Artificial Sequence: starter < 400 > 6 cgagaeg ea acagtcggga cccactcatt aggcaeccca ggc 43 < 210 > 7 < 211 > 22 < 213 > DMA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 »7 ceatgggote aaactggagc cg 22 «2io > ß < 2U > 33 < 313 > DK? «313 > Artificial Sequence < 330 > 323 > Description of Artificial Sequence: primer < 400 > 8 cgagacgtca acagtcggga ce 22 < 210 > 9 < 211 > 21 < 212 > DMA < 3i3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer «400» »caggcagctg tggtttgtgg e 21 < 210 »10 < 211 > 2! < 312 > DBA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer «400» 10 cgggaagttg cagaagattg ggttg 5 < 210 > II < 211 > 25 < 212 > OKA < 213 > Artificial sequence < 220- > < 223 »Description of Artificial Sequence: primer« 400 > XX aegtagaat cgaacaagaa accgc 25 < 210 > 12 < 2il > 30 < 312- > DNA < 2i3 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 12 gageugaga agaaggagac 2o < 210 > 13 < 211 > 23 < 312 > DMA < 2i3 > Artificial Sequence < 320 »223» Description of Artificial Sequence: primer «400 > 13 gcaagcttta ta, gttt «cga tcc 23 < 210 > 14 < 211 > 23 < 212 > DMA < 213 > Artificial Sequence < 3 »Description of Artificial Sequence r starter < 40t »14 ttgccatgge gcaeggfctta gg 2 < 210 > 15 < 211 > 3ßB0 < 212 > DNA < 213 > Hßliaathuß apnuna «22» < 22i > promoter < 222 > (1) .. (1889) < 220 > < 221 > QD8 < 222 > . { 1890.}. .. (3503) «400 > 15 aagcttttata gtttacgatc eaaggttoga tg gttagtt oßaacaacgt tggag CGAA, gttcccttta ßo ggagcttctt tttttttttc aaactagtgt tggtagaa & a to g lao atttaet aaagtggtgt t ctcaaßaa to atteacca aaataatgtc tttgtstßct ttctcßtctt 180 aaetaaatta aatgaatata caattctat tattcatetc tgttaaeaet t atttacta 240 fctaettttta gaaacccaab aaaactaaat TSS taatttt attatsttact ataetgacac 300 tttatettct ttttgaaact cagacttgag gttctceatt atgtcaccta atattatctt 360 atcta & taat ttaiacatga ttaacataae ttactoaatt tacaattata tetaaattea 420, ccttaacata tatttegtta tttttagttg tucu to agtgttaagt tagttagcta 480 taaaaaagge cagaxagtaa aatagtttag to gtttgtt aacataagat tacaaaaaag 540 taaaaaaagc aagccataaa aataaaattt gggagttcgfc ttectatgao ttgacaaeae 500 tteaaagtag ettßatcgat caaaaataea tgatat & tta tteateeagt aaaaaataaa "60 a &taaaaeaa tattagcgta gataágagtg ataaataatt tttttattaa ataattgaaa 720 etttfcaaaaa agatcatttt etaaaaatco gtagcgagta aagttatga gttcgtfitaa 780 stttettatg tttc taßtt eatactgtt * aatatataaa aagataagga gttggt aaaa 840 agagttggtt caaaatataa aaaggtaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaae 900 tacaatggafc tgattggagc aaaacaaaat attttattga aaatataaca ttgaataaaa 960 aaaacaeaat gaataagaag taetacacta aattggcttt aaaaaataat eaagtattaa 1020 caatgaatta tagaaataaa ttetaetcaa ctaaactagc atagagaaaa tgagataaga 1080 ttagtaaata aaaeaceatt ctcttgaaaa acaecatttg atgaatattt ttttteaeca 1140 tcactactct aggaaaaaaa aaaaaaaccc ttctttttag tetaatattg agcttttaaa 1200 atacttatca tacttattag aeaaaaeaat eattttagag taaccttccc ctatacccta 1360 aagtacatat agacgattct ccatataata aattaaggta cagtttaaaa tgatttcatt 1320 aattatccaa aacttttact tctgtttaat tctttttggt tcgtatgtac ttgtataaet 1380 gtggatttaa eaaagtctcc attactttga atcaaaaata tgtttgaaat agctgaeatg 1440 tgtatttttt caaattgcae ataaatatae gcaaaetega aattgaaaag ttgaaagaaa 1500 ttttttgaaa acgtgtctca agtaggeett atgcaacttg ctattttcte gaetatccae 1560 gtttatctcc agatttetaa ttcatttgtg tttetttata tctttaacaa gttacaagea 1620 catggaatta tgeacc atg caetcteaet taaaatttat gtcttgttag gagaaattat 16 80 tttaaaatea atgttetaat atataaaaat eeaaattaag eaatgacaet taaaacatat 1740 gaecaatgac acgatacgat gagtacgtae eeaatatgaa tttteaactt tgattagcta 1800 tatttggtat gtcatttttg aaggteaaac attacgactt tcaatcgcct ataaattgca 1860 tgcatcaagc aaacgctaaa ccatgcaaa atg gea aat ate here tet tet tte 1913 Met Ala Aan He Thr Ser be Pue l 5 AAE atg ca act tec att ect act etc ctt ctt etc ttg etc tea aee 1961 Aßn Met C n Thr Ser lie Leu Thr Leu Leu Leu Leu Leu Leu Ser Thr 10 13 30 ca tet ect gea act tce c gt tec att a g e t ct t att t tt 3009 Gln Ser Ser Ala Thr Ser Arg Ser lie Thr Aap Arg Phe lie ain Cys 25 30 35 40 tea falls gac cgg gee gac cet tea ttt ceg ata acc gga gag gtt tac 3057 Leu His Asp Arg Ala Aap Pro Ser phe Pro lie Thr Gly ßlu Val Tyr 45 SO 55 act ecc gga aae tea tet ttt ect acc gtc ttg caa aac tac ate cga 2105 Thr Pro Gly Aan be Ser Phe Pro Thr val Leu Gln Asn Tyr Zl * Arg 60 65 70 aac ctt cgg tte aat gaa act ac ce aaa ecc ttt tta ate ate 2153 Aan Leu Arg Phe Asn Glu Thr Thr Thr Pro Lyß Pro Phe Leu lie Zle 75 80 B5 here gee gaa cat gtt tce falls act cag gea gct gctg gct tgt ggc aaa 2201 Thr Ala Clu Kis Val Ser His He Gln Ala Ala Val Val Cys Gly Lys 90 9S 100 caá aac cgg ttg cta ceg aaa acc aga age ggt ggt cat? ae tat gaa 2249 Gln Asn Arg Leu Leu Leu Lys Thr Arg Ser Gly Gly Kis Asp Tyr ßlu 105 110 115 120 ggt etc cec tac ctt here aac ac aac c a c a c a t a c a c a t a c a t a c a t a c a t a c a t a c a t a c a t a c a t a t He Val Asp 12S 130 135 aegte aat ate agg tec ata aae gea gat ate gaa ca gaa acc gea 234S Mee Phe Asn Leu Arg Ser Xle Asn Val Asp He Glu Gln Glu Thr Wing 140 145 150 tgg gtc ca g ge ggt gcg act ctt ggt gaa gcg falls tac cga ata gcg 2333 Trp Val Gln Wing Gly Wing Thr Leu Oly Glu Val Tyr Tyr Arg He Wing 155 160 165 gag aaa agt aac aag cat ggt Ctt ceg gea ggg gte tgt cea aeg gct 2441 Glu Lys Ser Aen Lys Mis Gly Phe Pro Wing Gly Val Cys Pro Thr Val 170 175 180 ggc gtc ggt ggg drops tet agg ggt ggt ggg tat ggt aat teg atg aga 2489 Gly Val Gly Gly His Phe Ser Gly Gly Gly Tyr Gly Aan Leu Met Arg 185 190 195 200 aaa falls gge eeg ecg gee gat aat att gee gat gct caa aea ata gae 2537 Lys Tyr Gly Leu Ser Val Aap Asa He Val Asp Ala Gln He He Asp 205 210 215 geg aac ggc aag etc ttg gat cga aag age atg ggt gag gae eeg eet 2585 Val Asn Gly Lys Leu Leu Asp Arg Lys Ser Ket Gly Glu Asp Leu Phe 220 225 230 tgg gcg ate acc ggc gge gge gege agt eee ge ge gee eea gee 2633 Trp Ala lie Thr Gly Gly Gly Gly Val Ser Ph * Gly Val Val Leu Ala 235 240 245 eac aaa aec aaa cea gtt cgt gtt ceg gag gtt gtg aee geg ect acc 2681 Tyr Lyß He Lys Leu Val Arg Val Pro Glu Val Val Thr Val Phe Thr 250 25S 260 act gaga aga gaga gaa caac aac ccc age acc acc gcg gaga cga egg 2729 He Glu Arg Arg Glu Glu Gln Asn Leu Ser Thr Z e Wing Glu Arg Trp 265 270 275 280 gta ca gtc gee gac aag cta gat aga gae cce eee cec cga aeg aec 2777 Val Gln Val Wing Asp Lys Leu? Sp? Rg Asp Leu Phe Leu Arg Met Thr 285 290 295 Ctt age gee here aac gat acag ggc gga aag gtc cge gct cct ate 2B25 Phe Ser Val He Aan Asp Thr Asn Gly Gly Lys Thr Val Arg Ala He 300 305 310 ecc cea aeg ctg tac etc gga aac ceg agg aac ce e gec here cet ceg 2873 Phe Pro Thr Leu Tyr Leu Gly Asn Ser Arg Asn Leu Val Thr Leu Leu 315 320 325 aac aaa ecc cec gag cea ggg ctg caa gaa ceg gat tgt act gaa 2921 Asn Lys Asp Phe Pro Glu Leu Gly Leu G n Glu Be Asp Cys Thr Glu 330 335 340 aeg age cgg gtt gag ect gcg ect tac tac aeg g? C tec ccc age ggt 2969 Mee Ser Trp val Glu Ser Val Leu Tyr Tyr Thr Gly Phe Pro Ser Gly 345 350 355 360 acc cea ace aeg gcg etc eea age cgc acc ect ca aga cte aae cea 3017 Thr Pro Thr Thr Ala Leu Leu Ser Arg Thr Pro Gln Arg Leu Asn Pro 365 370 37S etc aag acc aaa ccc gac tat geg ca aac ect att tec aaa ega cag 3065 Phe Lys He Lys Ser Asp Tyr al Gln Asn Pro He Ser Lys Arg Gln 380 385 390 eee gag etc ace tte gaa agg etg aaa gaa ctt gaa aac caa aeg teg 3113 Phe Glu P e He Phe Glu Arg Leu Lys Glu Leu Glu Asn Gln Met Leu 395 400 405 gct tte aac cea tat ggt ggt aga atg agt gaa ata tec gaa tte gea 3161 Wing Phe Asn Pro Tyr Gly Gly Arg Met Ser Glu He Ser Glu Phe Wing 410 415 420 aag ect ecc cea cat aga eeg ggt aac here gcg aaa act ac eac gaa 3209 Lys Pro Phe Pro His Arg Ser Gly Asn He Wing Lys Xle Gln Tyr Glu 425 430 435 440 gea aac tgg gag gae cet age gac gaa gee gaa aae cgt tac eeg aat 3257 Val Asn Trp Glu? Sp Leu Ser Asp Glu Wing Glu Asn Arg Tyr Leu Asn 445 450 455 tec here agg ceg aeg falls gat tae atg ace cea ttt gtg teg aaa aac 3305 Phe Thr Arg Leu Met Tyr Asp Tyr Met Thr Pro Phe Val Ser Lys Asn 460 465 470 eet aga aaa gea ect etg aac tat agg gat ttg gat ate gge ate aac 3353 Pro Axg Lys Wing Phe Leu Asn Tyr Arg Asp Leu Aßp He Gly He? An 475 480 485 age cat ggc agg aat gct tat aec gaa gga atg gtc eae ggg falls aag 3401 Ser His Gly? rg Asa Wing Tyr Thr Glu Gly Mee Val Tyr Gly Kis Lyß 490 495 SO0 falls etc aaa gag here aat tac aag agg cta gea agt gcg aag ace aaa 3449 Tyr Phe Lys Glu Thr Ase Tyr Lyß Arg Leu Val Ser Val Lyß Thr Lys 505 510 515 S20 gee gat ect gae aac tte ttt agg aat gag ca age tie cea acc eeg 3497 to Asp Pro Asp Asn Phe Phe Arg Asn Glu G n Ser I Pro Thr Leu 525,530,535 tea tet tgaagaaege acacacataa ataaataect ttgcgcaegg tattttcagg 3553 Ser Ser gtgttaaagt gatatteaga tatetatgat agaattcega eecgtacttt acacaatcaa 3613 aactgtatgg eectccgaac eeeecccete aacecegaaa aaeacaCaet agtattgtca 3673 aaaaaaa 3680 < 210 > 16 < 211 > S3S < 212 > PRT < 213 > Heliacchus annuus < 400 > 16 Met Wing Asn He Thr Ser Being Ph * Asn Met Gln Thr Ser He Leu Thr 1 5 10 15 Leu Leu Leu Leu Leu Leu Ser Thr Glo Ser Ser Ala Thr Ser Arg Ser 30 25 30 He Thr Asp Arg Phe He Gla Cys Leu His Asp Arg Wing Asp Pro Ser 35 40 45 Phe Pro He Thr Gly Glu Val Tyr Thr Pro Gly Asn Ser Ser Phe Pro 50 55 60 Thr Val Leu Gln Asn Tyr He Arg? Sn Leu? rg Phe? sn Glu Thr Thr 65 70 75 80 Thr Pro Lys Pro Phe Leu He He Thr Wing Glu His Val Ser His He 85 90 95 Gln Ala Ala Val Val Cys Gly Lys Gln Asn? Rg Leu Leu Leu Lys Thr 100 105 110 Arg be Gly Gly His Asp Tyr Glu Gly Leu Be Tyr Leu Thr Asn Thr 115 120 125 Asn Gln Pro Phe He Val Aßp Met Ph * Asn Leu? Rg Ser He Asn 130 135 140 Val Asp He Glu Gis Glu Thr Ala Trp Val Gln Ala Gly Ala Thr Leu 145 150 155 160 Gly Glu Val Tyr Tyr Arg He Wing Glu Lys Ser Aßn Lys His Cly Phe 165 170 175 Pro Wing Gly Val Cys Pro Thr Val ßly Val Gly Gly His Phe Ser Gly 180 185 190 Gly Gly Tyr Gly Asn Leu Met Arg Lys Tyr Gly Leu Ser Val Asp Asn 195 200 205 He Val Asp Ala Gln He He? ßp Val Asn Gly Lyß Leu Leu Asp Arg 210 215 220 Lys Ser Met ßly Glu Asp Leu Phe Trp Wing I * Thr Gly Gly Gly Gly 225 230 235 240 Val Ser Phe Gly Val Val Leu Ala Tyr Lys He Lyß Leu Val Arg Val 245 250 255 Pro ßlu Val Val Tnr Val Phe Thr He Glu Arg Arg ßlu Glu Gln Aan 260 265 270 Leu Ser Thr He Wing Glu Arg Trp Val ßln to Wing? Sp Lys Leu? ßp 275 280 285 Arg Asp Leu Phe Leu Arg Met Thr Phe Ser Val He Asn Asp Tbr Asn 290 295 300 Gly Gly Lys Thr Val Arg Wing He Phe Pro Thr Leu Tyr Leu Gly Asn 305 310 315 320 would be Leu Val Thr Leu Lau? ßn Lyß? Sp Phß Pro Olu Leu 325 325 335 Leu Gln Siu Ser? ßp Cyß Thr Glu Met Ser Trp Val Glu Ser Val Leu 340 345 350 Tyr Tyr Thr Gly Phe Pro Ser Oly Thr Pro Thr Thr? Leu Leu Ser 355 360 36S? Rg Tbr pro sn? Rg Leu? ßn p? O Ph? Lys Xle Ly? Ser? ßp Tyr Val 5 370 375 380 Oln? ßn Pro? L? Ser Lys? Rg Oln Ph? Olu Ph * Zl * Ph? Alu? Rg L * u 38S 390 355 400 Ly * Olu L * ßlu? ßa ßln Met Leu? The Phe? An Pxo? r Oly Oly? xg 403 410 415 Met be Ola Xle Ser ßlu Phe? the Lys Pro Ph? Pro Ria? rg ßer Gly 420 425 430 Aon xle Ala Lys l e Gln Tyr Siu Val? ßa Trp ßl? ? ßp Leu ser Aap 10 435 440 445 Olu Wing Olu? ßn? xg Tyx Leu? ßn Phe Thr? rg LeuZM? t Tyr? sp Tyx- 450 455 460 Ket Thr Pro Phe Val Ser Lyß? ßn Pro? rg Lyß? the Phe Leu ? ßn Tyr 465 470 47S 480? rg? ap Leu Aßp He Gly Xle? ßn Ser Hiß Oly? rg ßn? the Tyr Thr 485 490 495 -, c ß u ßly Mee Val Tyr Oly Hiß Lyß Tyr Phe Lys slu Thx? an Tyr Lys 500 505 S10? rg Leu Val Ser val Lyß Thr Lys Val Asp Pro? ßp? sn Pb * Phe? rg 515 520 525 Aan Olu ßl »be xle Pro Tbr Leu Ser Ser S30 535 538 < 210 > 17 < 211 »1981 20 < 212 > DHA «21 > Lactuca ßaeiva < 220 »< 231 > CDS < 222 > (7) .. (1626) < 220 > < 22i > unsafe < 252 > (372) «223 > replace (372, "") 25 < 220 > "321" insecure < 222 »(379) < 223 »replace (379," e ") < 220 > «231» insecure < 222 > (766) «223 > replace t7Bd '"*"' < 220 > «221» insecure < 222 > (1105) .. (1106) < 323 > replace («05..H06," ga "or" gg "or" aa ") < 400 > 17 acaaaa atg gea ate eee eee eee etc aae eee aaa ect eat att tte 48 Mat? La Xle Thr Tyr Ser Phe? ßn Phe Lys be Tyr Xle Phe i S 10 ect etc ete ett gee tea act 98 Pro Leu Leu Leu Val Leu Leu Ser Thr Bis Ser Ser? Thr Ser Thr 15 30 25 30 tee ate atae ee ee ee ee ee eta eta aac eae gee gae eet 144 Ser Xle Xle? sp? rg Phe Thr Gla Cys Leu Asn Asn? Xg? The Asp Pro 35 40 45 tet tte ceg etc age gga caa ctt tae act oac tc t tc tot ttt 193 Ser Phe Pro Leu Ser Oly ßln Lea Tyr Thr Pro? Ap? ßn ser Ser Phe SO $ 3 60 eca tee gtc teg caa gee tae eee cgg aae ete tte aat gaa tee 240 Pro ser val Leu Oln? La Tyr He? Rg? An Leu? Rg Phe? ßn slu Ser 65 70 75 • cg act ccc ••• eco «te tfc» abe ate mac gee tta eae cet tet aae 3TB Thr Thr Pro Lys Pro Xla Leu X *? le Thr? la Leu Hia Pro Ser Ble SO B5 90 aet eaa gea gct gtt gtg tgc gee aaa here falls cgc ctg eta atg aaa 336 Xl * Wave? The? Val Val Cyß? The Lyß Thr Hi? Rg Leu Leu Mee Ly? S 55 100 105 110 aee aga age gga gge eae gat tat gag ggg ett tec tat gtg aee aat 384 Thr? Rg Ser ßly ßly Hiß? sp Tyr Olu ßly Leu Ser Tyr Val Thr? ßn 115 120 123 tsg uß oa * ccß ttt ttt gtt gtt gae aeg tee aac tea ogo toe aea 432 Ser? ßa ßla Pro Pha Pha Val Val? ßp Het Phe? an Leu? rg be Xle 130 135 140 aac geg agt ate ga * gae aet gae tgg gtc eaa get ggt gog aet 4ßo? sn to the Ser Xle Olu? ßp Thr? wave the Trp Val ain? la Gly? the Thr 35 ISO iss ett age gaa gee eae eae ega ega eag gea gag aaa age aae agt eae get S2S Leu oly alu val Tyr Tyr? rg Xle? la ßlu Ly? ser? ßn ser Hi? ala 180 165 170 ttt ceg gcc ggc gtt tgc cet acc gtc gga gct ggt ggc cat tet age 376 Phß Pro Wing Gly Val Cyß Pro Thr Val Gly Val Gly Gly Hiß Phe Ser 175 180 185 190 ggt ggt tat ggt aac ttg aeg gga aaa tac g? e ctt tet gcc gac 624 Gly Gly Gly Tyr ßly Aßn Leu Met Gly Lys Tyr Gly Leu Ser Val Asp 195 200 205 aac att gcc gae get c ag tta acc gac gtg aac ggt aaa ctt ctg aac 672 Asn He Val Asp Ala Gln Leu He Asp al? an Gly Lys Leu Leu Aßn 210 215 220 cgg aaa tea atg ggt gaa gat ctt tet tgg gcc ate here ggt ggt ggt 720 Arg Lys Ser Mee Gly Glu? Sp Leu Phe Trp Wing He Thr Gly Gly Gly 225 230 235 10 ggt gcc age tet ggt gcg gtt gta gcg tac aag acc aaa ctg gtt cgt 768 Gly Val Ser Pha Gly Val Val Val Ala Tyr Lys He Lys Leu Val Arg 240 245 250 gtt cec acc acc gtg acc gee ecc aac gt * caa aga acce cce gag cag 816 Val Pro Thr Thr Val Thr Val Phe Asn val Gln? Rg Thr Ser Glu Gln 255 360 265 270 aae eca age acc ata gec falls ega egg here ca gee geg gac aag ccc 864 Asn Leu Ser Thr Zl «Ala His Arg Trp He ßln Val Ala Aßp Lys Leu 275 280 285 15 gat aat gac cee ecc etc cga atg acc ttt aac gtg here aac aac aac 912 Asp? Asp Leu Phe Leu Arg Mee Thr Phe Asn Val He? Sn Asn Thr 290 295 300 aat ggc gaa aag aeg ata ege ggt ttg ttt cea aea etg tac cte gga 960 Asn aiy Glu Lyß Thr He Arg Gly Leu Phß Pro Thr Leu Tyr Leu Gly 305 310 315 aae cet acc get etc gtt gec etc ctg aac aag gat tee ccc gaa tea 1008 Asn Ser Thr Ala Leu Val Ala Leu Leu? Sn Lys Asp Phe Pro Glu Leu 320 325 330 ggt gta gaa ate eea gae cgc att gaa atg agt tgg ate gag ecc gec 1056 20 Gly val Glu He Ser Asp Cys He ßlu Mee Ser Trp He Glu Ser Val 335 340 345 350 cce cee Caa acae ece ece ece aec ggc acc ccg acc accc ccg cet cea 1104 Leu Phe Tyx Thr Asn Phe Pro He Gly Thr Pro Thr Thr Wing Leu Leu 3SS 360 365 age ege here ccc ca ca aga cta aac cea t a t aaa aaa Cce gat eac 1152 Ser? Rg Thr Pro Qln? Rg Leu? An Pro Phe Lys He Ly? Ser Asp Tyr 370 375 380 gta aaa aac -act ate tec aaa cag gga tte gaa tec ata ttt gaa agg 1200 val Lys Aßn Thr He Ser Lyß Gln Gly Phe Glu Ser He Phe Glu Arg 25 385 390 395 atg aaa gáa eee gaa aac caá atg cea gcc tte aac ect tat ggt gga 1248 Met Lys Glu Leu Glu Asn Gln Met Leu? the Phe? sn Pro Tyr Gly Gly 400 405 410 aga aeg age gaa ate te gaa eec gea aag cce eee ccc cae cga tea 1296 Arg Met S er ßlu He Ser Glu Phe Ala Lye Pro "Phe Pro His Arg Ser 41S 420 425 430 ggg aat ata gcg aag ate caa tac gaa gea aac cgg gac gaa cee gge 1344 Gly Asn He? The Lys He Gla Tyr Glu Val A? N Trp Asp alu Leu Gly 435 440 445 gcc gaa gea gcc aat cgg tac ttg aac tte ac agg gtg atg tat gat 1392 Val Glu? the wing Asn Arg Tyr Leu Asn Phe Thr Arg val Met Tyr Aßp 450 455 460 tat aeg act ccg ttt gtt tec aag aac ccc agg gaa gea ctt ctg aac 1440 Tyr Mee Thr Pro Phe Val Ser Lyß Aßn Pro Arg Glu? The Phe Leu? ßn 465 470 475 tac agg gae eea gac aee gge gec aac age falls ggc aag aae gee cae 1488 Tyr? Rg? ßp Leu Asp Zle Gly Val? Sn Ser Hi? Gly Lys? sn the Tyr 480 485 490 ggt gaa gga atg gtt tat ggg falls aag tae eec aaa gag aeg aae eae 1536 Gly ßlu Gly Mee val Tyr ßly His Lys Tyr Ph * Lyß ßlu Thr? sn Tyr 495 500 505 510 aag agg cea aeg aeg geg aag aeg agg gee gae eee age aae cee eec 1584 Lys? rg Leu Thr Mee val Lys Thr Arg Val? sp Pro Ser? sn Phe Phe 515 520 525 agg aae gag caa age aec cea ace ecg tea cet cea egg aag 1626 ? rg Aan Glu Gln Ser He Pro Thr Leu Ser Ser Ser Trp Lys 530,535,540 taaattceaa acteacctgt gaaattgaac aaaagtacgg ctttttcaag gtcatggtat 1686 acgatattga ecagatteag eaeaaeeetg acttgeaeee aeacaaacaa aaeeacacta 1746 eaeeeeeeeg aateeagaec eeeeaeeeec eacgaaeaee eggaaaaaca gaegecgaca 1806 ceeecaagaa ecacagaece tgaacattgt gaacaatgaa taaaeegagg acteeececg 1866 ggtteetttt ataageaege aaeagcaege ctttaatcaa gataacegat cattggatgc 1926 aatttattat caeaaacece attcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1981 < 2 0 > 18 < 2U > 540 < 212 > PH < 213 > Laceuca ßaciva < 400 > 18 Mee Wing II * Thr Tyr Being Phe? An Phe Lyß Being Tyr He Phe Pro Leu 1 5 10 15 Leu Leu Val Leu Leu Being Thr His Being Being Wing Thr Ser Thr Being He 20 25 30 He Asp? Rg Phe Thr Gln Cys Leu? ßn? Sn? Rg Wing Asp Pro Ser Phe 35 40 45 Pro Leu be oly Gln Leu Tyr Thr Pro Asp? Sn Ser Ser Phe Pro Ser 50 55 60 val Leu Gln? The Tyr He Arg? Sn Leu? Rg Phe? sn Glu Ser Thr Thr 65 70 75 80 Pro Lys Pro He Leu He He Thr? La Leu His Pro Ser His He Glp 85 90 95 Ala? The Val Val Cys? The Lys Thr His? Rg Leu Leu Mee Lys Thr? Rg 100 105 110 Ser Gly Gly His? Sp Tyr Glu Gly Leu Ser Tyr Val Thr? ßn Ser? Sn 115 120 125 Gln Pro Phe Phe Val Val Asp Mee Phe Asn Leu Arg Ser He Asn Val 130 135 140 Ser He ßlu Asp ßlu Thr Ala Trp Val ßln? la ßly Ala Thr Leu Gly 145 150 155 160 Glu Val Tyr Tyr? Rg He? The Glu Lys Ser Asn Ser His? The Phe Pro 165 170 175? The Gly Val Cy? Pro Thr Val Gly Val Gly Gly His Phe Ser Gly Gly 180 185 190 Gly Tyr Gly Asn Leu Mee Oly Lys Tyr Gly Leu Ser Val? ßp Asn He 195 200 205 Val Aßp Ala Gln Leu He? Sp Val? ßn Gly Lys Leu Le? ßn? Rg Lys 210 215 220 Ser Mee Gly Glu? Sp Leu Phe Trp? The Zle Thr Gly Gly Gly Gly Val 225 230 235 240 Be Phe Gly Val Val Val Ala Tyr Lys He Lys Leu Val? Rg Val Pro 245 250 255 Thr Thr Val Thr Val Phe Asn Val G n? Rg Thr Ser Glu ßln? Sn Leu 260 265 270 Ser Thr Xle? His? Rg Trp He Gln Val Ala? Sp Lys Leu? Sp Asn 275 380 385 Asp Leu Phe Leu Arg Mee Thr he Aßn Val XI * Aßn Aßn Thr? ßn ßly 290 295 300 Glu Lyß Thr Xle? Rg Gly Leu Phe Pro Thr Leu Tyr Leu Gly Aßn Ser 305 310 315 320 Thr? La Leu Val Ala Leu Leu? ßn Lys Aßp Phe Pro Glu Leu Gly Val 32S 330 335 Glu He Ser Aßp Cys He Glu Mee Ser Trp H * Olu be Val Leu Phe 340 34S 350 Tyr Thr Aßn Pbe Pro He a and Thr Pro Thr Thu the Leu Leu Ser? Rg 355 360 365 Thr Pro Glp? Rg Leu? Sn Pro Phe Lys He Lys Ser? ßp Tyr Val Lys 370 375 380? Sn Thr Xle Ser Lys Gln Gly Phe Glu Ser Xle Phe Glu Arg Met Lys 385 390 395 400 Glu Leu Glu? Sn Gln Met Leu? The Phe? Sn Pro Tyr Gly Gly? Rg Mee 405 410 415 Ser Glu Xle Ser Glu Pbe Wing Lys Pro Phe Pro His? Rg Ser Gly? Sn 420 425 430 Xle? The Lys Xle Gln Tyr Glu Val? Sn Trp Asp Glu Leu Gly Val slu 435 440 445? The? La? Sn? Rg Tyr Leu Aan Phe Thr Arg Val Met Tyr Asp Tyr Met 450 455 460 Thr Pro Phe Val Ser Lyß Asn Pro Arg Glu? The Phe Leu? Sn Tyr? Rg 465 470 475 480? Sp Leu? Sp xle Gly Val? Sn Ser His Gly Lyß? Sn? The Tyr Gly Glu 485 490 495 Gly Met Val Tyr Oly Hia Lya Tyr Phe Lys slu Thr? An Tyr Lys? Rg 500 505 510 Leu Thr Met Val Lyß Thr? Rg Val? Sp Pro Ser? Sn Phe Phe? Rg? An 515 520 525 Glu Gln Ser Xle Pro Thr Leu Being Being Trp Lys 530 535 540 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following

Claims (22)

1. A DNA sequence that naturally drives the expression of a plant gene encoding hexose oxidase, characterized in that it is capable of promoting a pathogen-inducible transcription of a DNA sequence when it is reintroduced into a plant.

2. A DNA sequence according to claim 1, characterized in that it can be obtained from Helianthus annuus.

3. A DNA sequence according to claim 1, characterized in that it can be obtained from Lactuca sativa.

4. A DNA sequence according to claim 2, characterized in that it is the regulatory region upstream of the gene encoding hexose oxidase as shown in SEQ ID NO. NO: 15

5. A DNA sequence according to claim 3, characterized in that it is the regulatory region upstream of the gene encoding hexose oxidase as shown in SEQ ID NO: 18.

6. A DNA sequence according to claim 4, characterized in that it comprises the nucleotide sequence from 1 to 1889 illustrated in SEQ ID NO: 15.

7. A portion or variant of a DNA sequence according to any of claims 4 to 6 characterized in that it is capable of promoting pathogen-inducible transcription of an associated DNA sequence when it is reintroduced into a plant.

8. A chimeric DNA sequence characterized in that it comprises in the transcription direction (i) a DNA sequence according to any of claims 1 to 7 and (ii) a DNA sequence to be expressed under its transcriptional control, not being naturally under the transcriptional control of said DNA fragment.

9. A chimeric DNA sequence according to claim 8, characterized in that the DNA sequence to be expressed causes the expression of an antipathogenic protein.

10. A chimeric DNA sequence according to claim 9, characterized in that said antipathogenic protein selected from the group consisting of chitinases, glucanases, osmotins, magainins, lectins, saccharide oxidases, oxalate oxidases, Bacillus thuringiensis toxins, isolated antifungal proteins of Mirabilis jalapa , Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and lmpatiens and albuminoid proteins such as thionin, napina, trypsin inhibitor of barley, cereal gliadin and wheat alpha amylase.

11. A chimeric DNA sequence according to claim 8, characterized in that the DNA sequence to be expressed causes the production of a protein capable of inducing a hypersensitive response, preferably selected from the group consisting of the Cf, Bs3 and PtO proteins of the tomato , Rpml and Rps2 of Arabi dopsi s th ali, N protein of tobacco, avr proteins of Cl to dospori um ful vum, harpins of Erwi ni and the inducing proteins (avrBs3, avrRpml, avrRpt2) of Pse udomona so Xa n th omona s.

12. A replicon characterized in that it comprises a chimeric DNA sequence according to any of claims 8 to 11.

13. A replicon characterized in that it comprises in the transcription direction a DNA sequence according to any of claims 1 to 7 and contains at least one recognition site for a transcription endonuclease for the insertion of a DNA sequence to be expressed under the control of said DNA sequence.

14. A microorganism characterized in that it contains a replicon according to any of claims 12 to 13.

15. A plant cell characterized in that it has incorporated in its genome, a chimeric DNA sequence according to any of claims 8 to 11.

16. A plant characterized in that it essentially consists of cells according to claim 15.

17. A plant according to claim 16 characterized in that it is a dicotyledonous plant.

18. A part of a plant that has inserted in its genome, at least one additional copy of a chimeric DNA sequence, said part of the plant is selected among seeds, flowers, tubers, roots, leaves, fruits, pollen and wood, characterized because it is obtained from a plant according to claims 16 and 17.

19. Use of a DNA sequence according to any of claims 1 to 7, which serves to identify homologs capable of promoting pathogen-induced transcription in a plant.

20. Use of a chimeric DNA sequence according to any of claims 8 to 11 for transforming plants.

21. Use of a portion or variant according to claim 7 to create hybrid regulatory DNA sequences.

22. A use of a chimeric DNA sequence according to any of claims 9 to 11, for conferring pathogen resistance to a plant.