AP924A - Transcription regulatory elements for gene expression in plant tissue. - Google Patents

Abstract

Qualitative transcriptional regulatory sequences functional in plants, plant tissue and in plant cells for inducible gene expression and quantitative transcriptional regulatory sequences for increasing the transcriptional expression of downstream generic information in plants, plant tissue and plant cells are disclosed. Also disclosed are methods and recombinant DNA molecules for improving the disease resistance of transgenic plants, especially wherein an inducible promoter controls the expression of a protein capable of evoking the hypersensitive response in a plant.

Description

TRANSCRIPTIONAL CONTROL SEQUENCES AND METHODS

The Field of the Invention

The field of this invention is the area of plant 5 molecular biology, and it relates in particular to transcription regulatory elements: a qualitative regulatory sequence which positively regulates downstream gene expression in plant tissue in response to the stress of am invading microbial pathogen, an elicitor, or other inducing chemical signals and quamtitative regulatory sequences which increase the transcriptional expression of associated sequences.

The Background of the Invention In plants, disease resistance to fungal, is bacterial, and viral pathogens is associated with a plant response termed the hypersensitivity response (HR). In the HR, the site in the plaint where the potential phytopathogen invades undergoes localized cell death, amd it is postulated that this localized plamt cell death aspect of the HR contains the invading microorgamism or virus, thereby protecting the remainder of the plant. Other plant defenses include the production of phytoalexins (antibiotics) amd/or lytic enzymes capable of averting pathogen ingress and/or cell wall modifications which strengthen the plant cell wall against physical amd/or enzymatic attack.

The HR of plants, including tobacco, can include phytoalexin production as part of the response to invading microorganisms. One class of compounds made by tobacco (Nicotiana tabacum) in response to microbial invaders sure the antimicrobial sesquiterpenes.

Cell suspension cultures have provided useful information regarding the regulation of terpene synthesis. Isoprenoids are ubiquitous in nature, and the

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- 2 early portions of the biosynthetic pathway are shared with the biosynthetic pathway for other isoprenoid compounds such as sterols, carotenoids, dolichol, and ubiquinone and growth regulators (e.g., gibberellic acid), which are classified as primary metabolites.

Isoprenoid compounds classified as secondary metabolites are not essential for growth, and include mono-, sesqui-, and diterpenoids. These secondary metabolite isoprenoids are important mediators of interactions between the plant and its environment.

A variety of compositions can serve as elicitors of plant phytoalexin synthesis. These include, but are not limited to, one or more toxic ions, e.g., mercuric ions, other chemically defined compositions, metabolic is inhibitors, cell wall glycans, certain glycoproteins, certain enzymes, fungal spores, chitosans, certain fatty acids, and certain oligosaccharides derived from plant cell walls [See, e.g., Sequeira, L. (1983) Anna. Rev. Microbiol. 37:51-79 and references cited therein].

Epi-5-aristolochene synthase (EAS) activity in tobacco plants has been shown to be induced by cell wall fragments of certain Phytophthora species and by Trichoderma viride cellulase but not Aspergillus japonicum pectolyase [Chappell et al. (1991) Plant

Physiol. 97:693-698]. Attack by other plant pathogens or an avirulent related strain can also induce phytoalexin synthesis; for example. Pseudomonas lachrymans induces sesquiterpenoid synthesis in tobacco [Guedes et al.

(1982) Phytochemistry 21:2987-29881.

Elicitins are proteins which are produced by plant pathogens and potential plant pathogens, which proteins induce the HR in tobacco plants. Amino acid and nucleotide coding sequences for an elicitin of Phytophthora parasitica have been published [Kamoun et al. (1993) Mol. Plant-Microbe Interactions 6:573-581].

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- 3 Plant pathogenic viruses including, but not limited to, Tobacco Mosaic Virus (TMV) induce the HR in infected „ plants. Bacteria which infect plants also can induce HR and thereby disease resistance; representative bacteria eliciting HR include, but are not limited to, Agrobacterium species, Xanthomonas species and Pseudomonas syringae. Plant pathogenic fungi (and certain avirulent strains as well) also induce the HR response, where these include, but are not limited to, io Phytophthora parasitica and Peronospora tabaci.

When tobacco cell suspension cultures are treated with an elicitor, squalene synthetase is suppressed, thus stopping the flow of common biosynthetic precursors into sterols. The concomitant induction of sesquiterpene is cyclase gene expression causes the flow of precursors into sesquiterpenes. The first step in the pathway from famesyl diphosphate to the sesquiterpene phytoalexin capsidiol in elicitor-induced tobacco tissue is catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. The coding sequence and deduced amino acid sequence for one member of the EAS gene family of tobacco have been published [Facchini and Chappell (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092]. The transcriptional expression of one or more members of the

EAS gene family is induced in response to elicitors.

There is a long felt need in the art for methods of protecting plants, particularly crop plants, from infection by plant pathogens, including but not limited to, phytopathogenic viruses, fungi and/or bacteria.

Especially important from the standpoint of economics and environmental concerns are biological or natural methods rather than those which depend on the application of chemicals to crop plants. There is also a long felt need in the art for plant transcriptional regulatory

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- 4 sequences for use in controlling the expression of heterologous DNA sequences in transgenic plants.

Summary of the Invention

In general, the invention features a recombinant 5 nucleic acid molecule that includes an inducible plant disease-resistance regulatory element. Such a recombinant nucleic acid molecule is, in general, at least 80% identical to a naturally-occurring inducible plant disease-resistance regulatory element; that is, up to 20% of the base pairs of the reference DNA sequence can be replaced with an alternative basepair (e.g., G-c replaced with A-T, T-A, or C-G) , provided that the transcription-promoting activity of the altered sequence is the same or greater than the reference sequence. In preferred embodiments, the recombinant nucleic acid molecule according to the invention is obtained from a gene encoding a terpene cyclase (e.g., a sesquiterpene cyclase). For example, the recombinant regulatory element of the invention is obtained from an epi-520 aristolochene synthase (EAS) gene that includes the nucleotide sequence shown in Fig. 3A (SEQ ID NO: 14) or an inducible plant disease-resistance fragment thereof.

In preferred embodiments, the recombinant nucleic acid molecule according to the invention has the nucleotide sequence shown in Fig. 3A (SEQ ID NO: 14).

In other preferred embodiments, the nucleic acid molecules of the invention includes any of the following sequences: nucleotides 463-473 of SEQ ID NO: 2; nucleotides 406-486 of SEQ ID NO: 2; nucleotides 463-572 of SEQ ID NO: 2; nucleotides 371-463 of SEQ ID NO: 2; and nucleotides 411-457 of SEQ ID NO: 2.

In preferred embodiments, the recombinant nucleic acid molecule of the invention is obtained from a dicot {e.g. , a member of the Solanaceae such as Nicotiana) .

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- 5 In other preferred embodiments, the nucleic acid of the invention is obtained from a monocot; a gymnosperm; or a conifer.

In yet other preferred embodiments, the nucleic 5 acid molecule of the invention is genomic DNA;

chemically-synthesized DNA; or is a combination of genomic DNA and chemically-synthesized DNA; genomic DNA and cDNA; chemically-synthesized DNA and cDNA; or genomic DNA, cDNA, and chemically-synthesized DNA.

In preferred embodiments, induction of the nucleic acid molecule of the invention is mediated by a plant pathogen such as a fungus (e.g., Phytophthora), a bacterium (e.g., Pseudomonas), or a virus (e.g., tobacco mosaic virus) as described herein. In other preferred is embodiments, such induction is mediated by an elicitor (e.g., by fungal or bacterial elicitors).

In another aspect, the nucleic acid molecule of invention is operably linked to and functions to regulate inducible transcription of nucleotide sequences encoding a heterologous polypeptide. Preferably, the heterologous polypeptide is capable of conferring disease-resistance to a plant. For example, the heterologous polypeptide may be an elicitin (e.g., a fungal elicitin such as the ParAl polypeptide from Phytophthora, a bacterial elicitin such as harpin, or a pharmaceutical polypeptide such as tissue plasminogen activator). Expression of such a heterologous polypeptide is mediated by one or more external agents (e.g., ethylene or methyl jasmonate). In other embodiments, the nucleic acid molecule of invention is capable of expressing the heterologous polypeptide in a cell-specific manner.

In another aspect, the invention features a vector including the nucleic acid molecule of the invention, a method of directing expression of a polypeptide by

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- 6 introducing the vector into a cell (e.g., a transgenic plant cell), and a cell containing the vector.

In another aspect, the invention features a method of providing disease-resistance to a transgenic plant, the method including the steps of: (a) producing a transgenic plant cell including the nucleic acid of according to the invention integrated into the genome of the transgenic plant cell; and (b) growing the transgenic plant from the plant cell wherein the expression of the nucleic acid molecule according to the invention- confers disease-resistance to the transgenic plant.

In preferred embodiments, the transgenic plant according to the methods of the invention is a dicot (e.g., is a member of the Solanaceae such as Nicotiana);

a monocot; a gymnosperm; or a conifer.

In another aspect, the invention features a method of increasing the transcriptional expression of a downstream DNA sequence in a transgenic plant cell, the method including the steps of: (a) producing a transgenic plant cell including the nucleic acid of invention positioned for increasing transcription of a downstream DNA sequence and integrated into the genome of the transgenic plant cell; and (b) growing the transgenic plant from the plant cell.

In addition to the above features, the present invention provides qualitative transcriptional regulatory sequences which regulate downstream gene expression in plant tissue in response to one or more elicitors, other defined inducing compounds, or in response to the stress of an invading phytopathogen (the inducible transcription regulatory sequence) and quantitative transcription regulatory sequences which increase the transcription of downstream sequences (the transcription-enhancing sequence). As specifically exemplified herein, these transcriptional regulatory sequences are found in nature

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- 7 upstream and operatively linked to the epi-5-aristolochene synthase gene (EAS4) of tobacco; when operatively linked to a coding sequence (and in the presence of an operatively linked promoter element, from the EAS4 gene or from a heterologous plant-expressible gene) these sequences mediate the inducible transcriptional expression of that coding sequence when the plant or plant tissue is invaded by a potential phytopathogen (e.g., a virus, fungus or bacterium) or in io response to elicitors such as Trichoderma viride cellulase or plant or fungal cell wall fragments for plants, plant tissue and/or plant suspension culture cells. That potential plant pathogen can be a virus including, but not limited to, tobacco mosaic virus or tobacco vein mottle virus; a bacterium including, but not limited to, Pseudomonas syringae, Xanthomonas campestris or Agrobacterium tumefaciens; or a fungus including, but not limited to, a species of Phytophthora (e.g., P. parasitica) or Peronospora (e.g., P. tabaci). The EAS4 promoter including the inducible transcription regulatory element(s) and the transcription-enhancing sequence(s) are disclosed herein as SEQ ID NO:2. In SEQ ID NO:2, the CAAT-homologous sequence of the EAS4 promoter is located at nucleotides 513 to 516, and the TATA-sequence motif is located at nucleotides 540 to 543.

Examples of inducible transcriptional regulatory elements within the N. tabacum PAS4 upstream sequence are from nucleotide 458 to nucleotide 473 of SEQ ID NO:2; from nucleotide 454 to 473; and from nucleotide 413 to

473 Of SEQ ID NO:2.

Another aspect of the '□resent invention is the transcription-enhancing element derived from the EAS4 promoter and promoter-associated sequences. When operatively linked upstream of a initiation sequences and a heterologous DNA sequence to be expressed.

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- 8 Further aspects of the present invention are transgenic plant cells, plant tissues, and plants which have been genetically engineered to contain and express a nucleotide sequence of interest, preferably a coding sequence, an antisense sequence, or other sequence under the regulatory control of the inducible transcription regulatory element. It is an object of this invention to provide the nucleotide sequences which mediate the induction of the expression of a downstream coding io sequence in response to elicitor exposure, potential, phytopathogen invasion in a plant, or certain other exogenous inducing signals such as exposure to methyl jasmonate and ethylene. An exemplary elicitor inducible transcription regulatory element is that from the 5' flanking region of the EAS4 gene of Nicotiana tabacum; as specifically exemplified herein, this sequence is presented in SEQ ID KO:2 from nucleotide 410 to nucleotide 473. Equivalents of the exemplified nucleotide sequence are those nucleotide sequences which similarly direct the induction of the expression of downstream nucleotide sequences. Preferably, the inducible transcription regulatory element is associated with the EAS4 promoter and promoter-associated sequences (e.g., the combination having the nucleotide sequence as given in SEQ ID NO :2 from nucleotide 410 to nucleotide 573 of SEQ ID NO:2, preferably from nucleotide 361 to 573 of SEQ ID NO:2, and more preferably from nucleotide 1 to 573 Of SEQ ID N0:2).

By disease-resistance regulatory element is meant a DNA sequence capable of regulating the expression of a gene product associated with a plant defense response (e.g., a hypersensitive response) that, in a native plant, is used to a combat pathogenic organism. Also included in this term are regulatory elements (and, as defined below, fragments of such regulatory elements)

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- 9 that are sufficient to render gene expression inducible by disease-associated external signals or agents (e.g., pathogen- or elicitor-induced signals or agents as described herein). In general, disease-resistance regulatory elements are located in the 5' region of a gene, but are not so limited.

By inducible is meant that a regulatory element is capable of mediating increased gene expression (e.g., mRNA or polypeptide production) in response to an io interaction between a plant cell and either a pathogen or elicitor. Also included in the invention are diseaseresistance regulatory elements that direct inducible gene expression in a cell- or tissue-specific manner.

By obtained from a gene is meant that the nucleotide sequence of a regulatory element is based on sequence information included in a naturally-occurring plant gene (e.g., tobacco EAS4) . Once identified, the regulatory element according to the invention is obtained from a natural source or can be prepared according to any standard method (e.g., by recombinant methods or chemical synthesis). Such a recombinant nucleic acid molecule is, in general, at least 80% identical to a naturallyoccurring inducible plant disease-resistance regulatory element; that is, up to 20% of the base pairs of the reference DNA sequence can be replaced with an alternative basepair (e.g., G-C replaced with A-T, T-A, or C—G), provided that the transcription-promoting activity of the altered sequence is the same or greater than the reference sequence.

By an inducible plant disease-resistance fragment is meant any stretch of nucleotides regardless of length, which is sufficient to direct increased gene expression (e.g., mRNA or polypeptide production) in response to an interaction between a plant cell and either a pathogen or elicitor. The term fragment

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- 10 preferably means about 6-10 nucleotides in length. However, fragments of DNA regulatory elements according to the invention can range in size from 10-100 nucleotides, 100-300 nucleotides in length, or even greater than 300 nucleotides in length. Such fragments are prepared by routine methods (e.g., by appropriate restriction digestion or by chemical synthesis of the fragment). Identification of an inducible plant diseaseresistance DNA fragment in a gene is carried out using standard methods in the art (e.g., those methods described herein). In one particular example, identification of an inducible plant disease-resistance fragment may be carried out using standard promoter deletion analysis. A construct including a disease15 resistance promoter that confers pathogen-inducible transcription to a reporter gene to which it is operably linked may be progressively deleted by 5', 3', and/or nested deletions until the effect of a pathogen on the reporter gene is diminished or eliminated. To confirm the identification of the pathogen-inducible element, point mutations may then be introduced into the element. An akternate approach to guage changes in transcription is to link the presumed regulatory element from the gene to the reporter gene. To test for complete promoters, the DNA fragment is placed directly in front of the reporter gene lacking endogenous promoter activity.

Using such techniques, any fragment of an inducible plant disease resistance regulatory element may be identified.

By tissue-specific” is meant capable of preferentially increasing expression of a gene product (e.g. , an mRNA molecule or polypeptide) in one tissue (e.g., xylem tissue) as compared to another tissue (e.g., phloem).

By cell-specific is meant capable of preferentially increasing expression of a gene product

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- 11 (e.g., an mRNA molecule or polypeptide) in one cell (e.g., a parenchyma cell) as compared to another cell (e.g., an epidermal cell).

By epi-5-aristocholene synthase” or EAS is 5 meant an enzyme capable of catalyzing the cyclization of trans, trans-farnesyl diphosphate to the bicyclic intermediate epi-5-aristocholene.

By pathogen is meant an organism whose infection into the cells of viable plant tissue elicits a disease io response in the plant tissue.

By elicitor is meant any molecule that is capable of initiating a plant defense response. Examples of elictors include, without limitation, one or more toxic ions, e.g., mercuric ions, other chemically defined is compositions, metabolic inhibitors, cell wall glycans, certain glycoproteins, certain enzymes, fungal spores, chitosans, certain fatty acids, and certain oligosaccharides derived from plant cell walls, and elicitins (e.g., harpin, cryptogein, and pariscein).

By elicitin is meant a protein elicitor.

By operably linked is meant that a regulatory sequence(s) and a gene are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

By heterologous polypeptide is meant any polypeptide that is expressed in a transformed plant cell from a gene that is partly or entirely foreign (i.e., does not naturally occur in) to the transformed plant cell.

By polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

3y transformed plant cell is meant a cell into 35 which (or into an ancestor of which) a recombinant

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- 12 nucleotide sequence (e.g., the EAS4 promoter(-1147 to +67):GUS reporter gene or gEAS4_600(cyclase) promoter: parAZ mature elicitin gene) has been introduced by means of recombinant DNA techniques (e.g., those techniques described herein).

By plant cell is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

is By transgenic is meant any cell which includes a

DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants, and the DNA is inserted by artifice into the genome of the organism.

By substantially pure DNA is meant DNA that is free of the genes or ancillary nucleotides which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene or regulatory element. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding an additional polypeptide sequence or a regulatory element.

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- 13 Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Brief Description of the Drawings

Figure 1 presents data for transient expression experiments for GUS regulated by the EAS 3 and EAS4 promoter region sequences in comparison to a CaMV 35S-GUS construct (Cauliflower Mosaic Virus 35S promoter /5-glucuronidase reporter gene). These experiments were io carried out in tobacco protoplasts into which the DNA constructs had been electroporated. Uninduced levels of expression for the EAS-GUS constructs were relatively high because fungal enzymes which digest plant cell walls were used in the preparation of the protoplasts. The y-axis shows units of GUS activity and the 5' extents of the EAS upstream sequences are given below each bar on the graph. As above, the numbering is relative to the natural transcription start site of EAS4 (or the corresponding base of EAS3); in. EAS4 (SEQ ID NO:2) a start site is at nucleotide 573; in EAS3 (SEQ ID NO:1) a potential transcription start site is at nucleotide 489.

Figures 2A-B present information concerning the reporter gene expression directed by the instant inducible transcription regulatory element of the invention. Fig. 2A presents a schematic of experiments carried out with the EAS4 promoter region controlling the expression of the GUS reporter gene in stably transformed transgenic plants.

Fig. 2B presents the results of gain of function assays for the EAS4 promoter-associated sequences regulating expression of the GUS reporter gene via the CaMV 35S minimal promoter. In both Figs. 2A and 2B, numbering for the EAS4 upstream region reflects numbering relative to £ W I 0 / L 6 /d/dV

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- 14 the natural transcription start site of the EAS4 gene (see also SEQ ID NO:2 wherein the transcription start site corresponds to nucleotide 573). In both Figs. 2A and 2B, the GUS expression is measured in fluorescence units per milligram of protein.

Figures 3A-B are schematic illustrations showing the DNA sequence of the 5' upstream region of the EAS4 gene and the structure of the GUS reporter gene carrying the EAS4 promoter(-1148 to +67). Fig. 3A shows the io nucleotide sequence of the 5* upstream region of the EAS4 promoter (-1148 to +67) . The CAAT and TATA boxes are marked in bold face. The transcription initiation site is marked as +1. Fig. 3B is a schematic illustration showing the structure of the EAS4 promoter (-1148 to +67): GUS reporter gene. The 5' flanking sequence of the EAS4 promoter (1148 to +67) was fused in correct reading frame with the GUS reporter gene in binary vector pBHOl.l.

Figures 4A-B present data concerning elicitor20 induced GUS gene expression in leaves, stems, and roots of transgenic tobacco plants containing the EAS4(-1148 to +67):GUS reporter gene. Fig. 4A is a graph illustrating elicitor-induced GUS activity in tobacco leaves over an 18 hour time course. 06i, 08r, and 09p each represent an independently transformed line of transgenic tobacco containing the EAS4 promoter(-1148 to +67):GUS reporter gene construct. Water (as control) and 25 nM of cryptogein were infiltrated symmetrically and simultaneously into the leaf (abaxially), and the infiltrated zones of leaf tissue were then collected for analysis of GUS activity over the 18 hour time course.

The presented data are the averaged results of two separate experiments. Fig. 4B is a bar graph showing elicitor-induced GUS activity in the stems and roots of transgenic tobacco containing the 2AS4 promoter(-1148 to

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- 15 +67):GUS reporter gene. 03f and O9p represent two independently transformed lines of transgenic tobacco.

C-0 (open boxes) represents GUS activity present in segmented roots and stems without elicitor treatment.

C-18 (shaded boxes) and E-18 (solid boxes) represent GUS activity in segmented roots and stems 18 hours after an incubation in water or 100 nM of elicitor cryptogein, respectively. Five independent plants were evaluated per transgenic line.

io Figure 5 is a bar graph illustrating pathogeninduced GUS activity by race 0 and race 1 of Phytophthora parasitica var. Nicotianae in transgenic tobacco.

Detached leaves from one line of transgenic tobacco (09p) containing the EAS4 promoter(-1148 to +67):GUS reporter gene were inoculated with mycelial plugs of 2-day-old P. p. var. Nicotianae cultures and then incubated in a growth chamber at 27°C with constant fluorescent light for 24 hours. Control leaves were inoculated with sterile oatmeal agar plugs. Infected zones of tissue were then examined for GUS activity. Values indicated by each bar are the means and standard errors for three separate experiments (n = 5).

Figure 6 is a bar graph illustrating the comparison of pathogen- and elicitor-induced GUS activity in transgenic plants treated with Pseudomonas syringae pv. Syringae 61 and its hrpH mutant, and the purified bacterial elicitor protein harpin. Leaves of one line of transgenic tobacco (09-P) containing the EAS4 promoter(1148 to +67):GUS reporter gene were infiltrated with either 50 pg/mL of purified Erwinia amylovora harpin elicitor, cell suspensions of wild type P. syringae pv. Syringae 61 or its hrpH mutant (A₆₀₀ = 0.05). After a 12 hour incubation, infiltrated zones of tissue were subsequently analyzed for GUS activity. Control values represent GUS activity observed in leaf tissue samples

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- 16 which were infiltrated with water. Values indicated by each bar are the average of results obtained from five plants.

Figures 7A-B show a Western blot analysis of induction of 5-epi-aristolochene synthase (EAS) in tobacco plant tissues. Proteins were extracted from control- and elicitor-treated tobacco leaves, stems, and roots. Equal quantities of proteins (25 pg per lane for leaf and stem, 15 μg per lane for root) were separated by

SDS-PAGE and transferred to nitrocellulose membranes,.

The blots were then reacted with EAS antiserum and immunoreactive bands visualized using goat anti-mouse antibodies coupled to alkaline phosphatase. Fig. 7A shows the Western blot analysis of leaf and stem tissue samples. Fig. 7B shows the Western blot analysis of root tissue samples.

Figures 8A-L are color photographs illustrating the histochemical localization of elicitor-induced GUS activity in transgenic tobacco containing the EAS4 promoter(-1147 to +67):GUS reporter gene. Leaf tissues from one representative line of transgenic tobacco were infiltrated with either 25 or 50 nM of the cryptogein elicitor. After an approximately 8 hour incubation, tissue sections were stained using l mM x-gluc(5-bromo-425 chloro-3-indoyl 0-glucuronide) for analysis of GUS activity. Stem and root segments were incubated with water or 100 nM of the elicitor cryptogein for approximately 12 hours before staining.

Fig. 8A is a color photograph shoving elicitor-induced

GUS activity in a transgenic tobacco leaf containing the EAS4 promoter(-1147 to +67):GUS reporter gene after treatment with two elicitor preparations: cryptogein and parasicein, both at concentrations of 25 nM and 50 nM (designated in Fig. 8A as C₂₅, C_s0, ?₂₅, and P₅₀, respectively). Fig. 8B is a color photograph of a leaf

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- 17 cross-section showing elicitor-induced GUS activity after treatment with cryptogein (magnification, 75X). Fig. 8C is a color photograph showing elicitor-induced GUS activity in a cross-section of a stem segment treated with cryptogein (magnification, 7.5X). Fig. 8D-F are color photographs showing successively higher magnifications (15X, 60X, and 75X, respectively) of the GUS-stained stem cross-section shown in Fig. 8C.

Fig. 8G is a color photograph showing a root tip treated io with water and stained for GUS activity (magnification, 60X). Fig. 8H is a color photograph showing elicitorinduced GUS activity of a longitudinal section of a root tip treated with cryptogein (magnification, 60X). Fig.

is a color photograph of a longitudinal section of a is root after treatment with water and staining for GUS activity (magnification, 75X). Fig. 8J is a color photograph showing GUS activity of a longitudinal section of a root treated with cryptogein (magnification, 75X). Fig. 8K is a color photograph of a root cross-section treated with water and staining for GUS activity (magnification, 95X). Fig. 8L is a color photograph showing GUS activity of a cross section of a root treated with cryptogein (magnification, 95X). C, denotes cryptogein; P, denotes parasicein; c, denotes cortex; ca, denotes raaMn·; e, denotes epidermis; p, denotes palisade parenchyma; pe, denotes periderm; ph, denotes phloem; pi, denotes pith parenchyma; s, denotes spongy parenchyma; t, denotes trichome; x, denotes xylem; vc, denotes vascular cylinder.

Figure 9 is a schematic of the genetic manipulations leading to generation of disease-resistant plants. The coding sequence for the ParAl elicitin is isolated by PCR so as to have BamHI and Sstl ends. gEAS4₆₀₀-GUS-pBHOl, which directs the expression of the

GUS reporter gene under the regulatory control of the EAS £ W I 0 / z 6 /d/dV

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- 18 promoter, is digested with BamHI and Sstl to release the GUS reporter gene. Then the BamHI/Sstl-digested parAl amplimer is ligated to the large fragment produced after digestion of gEAS4₆₀₀-GUS-pBI101 to produce gEAS4 ₆₀₀-parAl-pBHO 1, from which the ParAl elicitin in plant cells or plant tissue is synthesized after induction with a suitable elicitor.

Figure 10 is a bar graph illustrating the disease ratings of independent lines of transgenic tobacco io (Nicotiana tabacum cv. KY160) containing either -the jparAl mature elicitin gene under the control of the gEAS4_600(cyclaae) promoter gene after inoculation of detached leaves with either race O or race 1 of Phytopbthora parasitica var. nicotianae. The y-axis shows the lesion score (number of lesions per leaf) after 72 hours. The x-axis shows control plants and independent lines of transgenic tobacco which were tested for disease resistance. KY160 denotes non-transformed N. tabacum cv. KY160; G denotes independent lines of transgenic Ν'. tabacum cv. KY160 which were transformed with a construct containing the gEAS4_600(cyclaae) promoter fused to GUS; M denotes independent lines of transgenic

N. tabacum cv. KY160 which were transformed with a construct containing the gEAS4_600(cyclaae) promoter fused to the mature parAl elictin coding sequence; S denotes independent lines of transgenic N. tabacum cv. KY160 which were transformed with a construct containing the gEAS4_600(cyclaaa) promoter fused to a sequence encoding a mature parAl elictin including a signal sequence. Shaded box, denotes inoculation of detached leaf with race 0 of P. p. var. nicotianae. Solid box denotes inoculation of detached leaf with race 1 of P. p. var. nicotianae.

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Detailed Description of the Invention

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- 19 5-epi-aristolochene synthase (EAS) is a key enzyme in the synthesis of sesquiterpenoid phytoalexins, for example, in solanaceous plants, including but not T imited to Nicotiana species (e.g., tabacum), Capsicum annum and

Syoscyamus muticus. EAS catalyzes the reaction of faraesyl diphosphate to (+) gemacrene A to eudesmane carbocation to 5-epi-aristolochene. other plants, such as the crucifers, also have sesquiterpene cyclase enzymes.

Treatments which induce host defensive responses in plant tissue or plant cells such as phytoalexin synthesis include cell wall fragments of Phytophthora species and Trichoderma viride cellulase. However, pectolyase from Aspergillus japonicum does not function as an elicitor in tobacco cell culture. Elicitors which induce sesquiterpenoid phytoalexin synthesis have been shown to function at the level of controlling transcription of key biosynthetic enzymes [Vogeli and Chappell (1990) Plant Physiol. 94:1860-1866]. Similar patterns have been observed in other plants, but no transcriptional control sequences which mediate gene induction in response to phytopathogen challenge have been described.

Tobacco (N. tabacum) contains an EAS gene family with some 12-15 members, the coding sequence of EAS4 has been published [Facchini and Chappell (1992) supra]. However, since that time, the present inventors have discovered that the EAS3 does not appear to be expressed in response to the elicitor treatment, and surprisingly, the nucleotide sequences upstream of EAS3 do not appear to mediate the induction of a reporter gene in a chimeric gene construct in elicitor-induced transgenic cell culture. It is noted that the translation start site was incorrectly identified in the 1992 Facchini and Chappell publication- The nucleotide sequence of a genomic clone

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- 20 of EAS4, as it appears in Facchini and Chappell (1992) supra is presented in SEQ ID NO:7, with the deduced amino acid sequence being given in SEQ ID NO:8.

Other aspects of a plant's defenses against invasion and infection by a phytopathogenic microorganism include the hypersensitive response, which is characterized by necrosis, i.e., programmed cell death in the localized area of attack by the plant pathogen. Elicitor treatments as described above can also induce io the necrotic response.

Elicitins are proteins produced by fungal plant pathogens, which proteins elicit a hypersensitive response in an infected plant. Generally, but not necessarily, localized cell death is the result of the elicitin-induced response in the infected (or challenged) plant tissue. These responses mediate full or partial resistance to destructive infection by the invading, potentially plant pathogenic microorganism. For the purposes of the present invention, a protein of a plant pathogen or potential plant pathogen which induces the hypersensitive response in plant tissue after invasion of that plant tissue or after expression of that coding sequence in the plant tissue is considered to fall within a broad definition of an elicitin.

A relatively well-known elicitin of a plant-pathogenic fungus is the ParAl protein of Phytophthora parasitica. The parAl locus is a member of a gene family [Ricci et al. (1989) Eur. J. Biochem. 183:555-5631. The coding and amino acid sequences for the parAl gene product are described in Kamoun et al.

(1993) Mol. Plant-Microbe Interact. 4:423-432 and in SEQ ID NOS:12 and 13 herein.

Other phytopathogen proteins with potential elicitin activity have been characterized as to amino acid sequences and other properties [See, e.g.,

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- 21 Nespoulous et al. (1992) Planta 186;551-557; Huet et al. (1992) Phytochemistry 31:1471-1476; Huet and Pernollet (1992) FEBS Lett. 257:302-306; Kamoun et al. (1993) Mol. Plant-Microbe Interact. 5.:22-33]. Several avirulent genes from pathogenic bacteria which correspond to elicitin activity have been characterized. For example, Keen, N.T. (1990) Annu. Rev. Phytopathol. 24:447-463 has described an avirulence gene of Fulva fulvia.

Hammond-Kosack et al. (1994) Proc. Natl. Acad. Sci. USA io 2J.:10445-10449 have described the avr gene of- ~

Cladosporium fulvum, which functions in the same way as the P. parasitica elicitin.

Certain bacterial plant pathogens also express proteins with similar effects on the hypersensitivity response as those of the P. parasitica ParAl elicitin.

For the purposes of the present invention, these proteins fall within the scope of the term elicitin. Multiple homologs of the avirulence gene avrBs3 of Xanthomonas campestris pv. vesicatoria have been detected in other X.

campestris pathovars [Bonas et al. (1989) Mol. Gen.

Genet. 218:127-136; Knoop et al. (1991) J. Bacteriol. 173:7142-71501 and in other species of Xanthomonas [De Feyter and Gabriel (1991) Mol. Plant-Microbe Interact.

£:423-432; Hopkins et al. (1992) Mol. Plant-Microbe

Interact. 5:451-459], The avrD gene of Pseudomonas syringae pv. tomato can confer avirulence; P. syringae pv. glycinea expresses an altered avrD gene product [Kobayashi et al. (1990) Mol. Plant-Microbe Interact. 2:103-111].

It is understood that to be useful in the present invention as it applies to creating transgenic plants with improved disease resistance traits using an elicitin coding sequence expressed under the regulatory control of a pathogen-response transcription regulatory element (and with a minimal promoter functional in those plants) that

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- 22 elicitin proteins must be capable of promoting expression of defense genes (including but not limited to those genes governing phytoalexin synthesis, the hypersensitive response and/or localized necrosis) in those plants.

Many functional combinations of plant and phytopathogen are known to the art, and the skilled artisan knows how to test the functioning of a particular elicitin in a particular plant tissue (or cells) in the turning on of programmed cell death or phytoalexin synthesis or the like. It is also understood that treatment of plant cells or tissue with compositions such as certain fungal cellulases or certain plant polysaccharide fragments can also induce the host defensive (i.e., hypersensitive) response. Such treatments are used as models for actual plant pathogen attack or invasion.

A non-naturally occurring recombinant nucleic acid molecule, e.g., a recombinant DNA molecule, is one which does not occur in nature; i.e., it is produced either by natural processes using methods known to the art, but is directed by man to produce a desired result or it has been artificially produced from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized molecules or portions thereof, and wherein those parts have been joined by ligation or other means known to the art.

A transgenic plant is one which has been genetically modified to contain and express heterologous DNA sequences, either as regulatory RNA molecules or as proteins. As specifically exemplified herein, a transgenic plant is genetically modified to contain and express a heterologous DNA sequence operably linked to and under the regulatory control of transcriptional control sequences by which it is not normally regulated,

i.e., under the regulatory control of the inducible transcriptional control sequences of the EAS4 gene of

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- 23 Nicotians tabacvun. ks used herein, a transgenic plant also refers to those progeny of the initial transgenic plant which carry and are capable of expressing the heterologous coding sequence under the regulatory control of the qualitative and/or quantitative transcription control sequences described herein. Seeds containing transgenic embryos are encompassed within this definition.

When production of a heterologous gene or coding io sequence of interest is desired under conditions of.

potential pathogen invasion or inducer (e.g., elicitor) treatment, that coding sequence is operably linked in the sense orientation to a suitable promoter and under the regulatory control of the inducible regulatory sequences, in the same orientation as the promoter, so that a sense (i.e., functional for translational expression) mRNA is produced. A transcription termination signal functional in a plant cell can be placed downstream of the coding sequence, and a selectable marker which can be expressed in a plant, can be covalently linked to the inducible expression unit so that after this DNA molecule is introduced into a plant cell or tissue, its presence can be selected and plant cells or tissue not so transformed will be killed or prevented from growing. Similarly, a heterologous coding sequence can be expressed under the regulatory control of the inducible transcription regulatory element or the transcription-enhancing element in transgenic plant cell suspension culture, with induction occurring in response to the addition of an elicitor to the cell culture medium.

Where inhibition of gene expression is desired in a plant being invaded by a microbial pathogen, such as a phytopathogenic fungus, then either a portion or all of that coding sequence or cDNA sequence can be operably linked to a promoter functional in plant cells, but with

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- 24 the orientation of the coding sequence opposite to that of the promoter (i.e., in the antisense orientation) so that the transcribed RNA is complementary in sequence to the mRNA, and so that the expression of the antisense molecule is induced in response to pathogen invasion. In addition, there may be a transcriptional termination signal downstream of the nucleotides directing synthesis of the antisense RNA.

The present inventors have isolated a DNA sequence which mediates the inducible expression of a downstream gene in plant cells in response to invasion by a potential plant pathogen and/or treatment with an elicitor or other chemical signals. For example, a combination of ethylene and methyl jasmonate may serve to induce downstream gene expression via the qualitative transcription regulatory sequence. It is understood that there may be a multiplicity of sequence motifs within that regulatory sequence, where individual motifs each respond to one or more distinct environmental signal. As specifically exemplified, this transcription-regulating sequence is derived from the EAS4 locus of N. tabacun, and it is given in SEQ ID NO:7. The deduced amino acid sequence for the EAS protein is given in SEQ ID NO: 8.

The open reading frame of the EAS4 gene, which is interrupted by six introns, is provided in SEQ ID NO:7.

A computer search of Genbank for nucleotide sequences homologous to the SEQ ID NO:2 sequence revealed no known nucleotide sequences with significant homology.

Organization of the EAS genes in the N. tabacum genome was described in Facchini and Chappell (1992) supra using an EAS probe and Southern hybridization experiments. Under conditions of high stringency, multiple fragments hybridized with analysis indicating that there is a gene family with some 12-16 members in the tobacco genome. In these experiments, however, the

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- 25 probe included the EAS coding sequence rather than the promoter and promoter-associated regulatory sequences.

EAS homologous genes can be identified and isolated from plant species other than N. tabacum based s on significant degrees of nucleotide sequence homology;

i.e., DNA:DNA hybridization under conditions of moderate to high stringency with a tobacco EAS coding sequence probe allows the identification of the corresponding gene from other plant species. A discussion of hybridization io conditions can be found for example, in Hames and Higgins (1985) Nucleic Acid Hybridization, IRL Press, Oxford,

U.K. Generally sequences which have at least about 70% nucleotide sequence homology can be identified by hybridization under conditions of moderate stringency.

Under such conditions, it is generally preferred that a probe of at least 100 bases be used. Host preferably, in the present case, the probe will be derived from the coding portion of the EAS4 coding sequence. Labels for hybridization probes can include, but are not limited to, radioactive groups, fluorescent groups, and ligands such as biotin to which specific binding partners (which are in turn labeled) bind. It is the label which allows detection of the hybridization probe to the target nucleic acid molecule. Alternatively, well-known and widely accessible polymerase chain reaction (PCR) technology is advantageously used to amplify sequences with significant nucleotide sequence homology to a target sequence.

It is understood that nucleic acid sequences other than the EAS coding sequence disclosed in SEQ ID NO :7 will function as coding sequences synonymous with the exemplified EAS4 coding sequence. Nucleic acid sequences are synonymous if the amino acid sequences encoded by those nucleic acid sequences are the same. The degeneracy of the genetic code is well-known to the art;

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- 26 i.e., for many amino acids, there is more than one nucleotide triplet which serves as the codon for the amino acid. It is also well-known in the biological «arts that certain amino acid substitutions can be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function, sim-i lar amino acids can be those that are similar in size and/or io charge properties, for example, aspartate and glutamate and isoleucine and valine are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas o£ Protein Sequence and

Structure, Vol. 5, Suppl. 3, pp. 345-352, which is incorporated by reference herein, provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources.

Terpene cyclase genes can be found in solanaceous plants, including N. tabacum and Hyoscyastus muticus, as disclosed herein, and in members of the mint family (Labitaceae) and the Euphorbiaceae, including but not )imited to those which have been demonstrated to contain sequences of significant homology, and in substantially all plants. Preferably, EAS4 homologs will be selected from the Solanaceae. Such sequences can be identified by nucleic acid hybridization experiments or when cloned in expression vectors, by cross reaction to tobacco EAS-specific antibody, or any other means known to the art, including the use of PCR technology carried out using oligonucleotides corresponding to portions of SEQ

ID NO:7, preferably in the region encoding EAS. Antibody

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- 27 can be prepared after immunizing an experimental animal with EAS purified as described in Vogeli et al. (1990) Plant Physiology 93:182-187 or using a peptide conjugate, where the amino acid sequence of the peptide is taken from a hydrophilic portion of the EAS amino acid sequence (SEQ ID NO:8). Monoclonal and polyclonal antibody production techniques are readily accessible to the art (See, e.g., Campbell (1994) Monoclonal Antibody Technology. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon and Knippenberg, eds, Elsevier, Amsterdam? Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Alternately, a cDNA library (in an expression vector) can be screened with EAS-specific antibody, or EAS peptide-specific antibody can be prepared using peptide sequence(s) from hydrophilic regions of the EAS protein (SEQ ID NO:8) and technology well-known in the art.

An inducible transcription regulatory sequence can be operably linked to any promoter sequence functional in plants as understood by the skilled artisan; where a regulatory element is to be coupled to a promoter, generally a truncated (or minimal) promoter is used, for example, the truncated 35S promoter of Cauliflower Mosaic Virus, CaMV). Truncated versions of other constitutive promoters can also be used to provide CAAT and TATAhomologous regions; such promoter sequences can be derived from those of A. tmaefaciens T—DNA genes such as nos, ocs, and mas and plant virus genes such as the CaMV 19S gene. It will be understood that the goals of a skilled artisan will determine the choice, of particular promoters used with the inducible transcription regulatory sequences. It is further understood that when a protein capable of generating a cell death response is

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- 28 to be expressed, then there is preferably no basal transcriptional (and translational) expression in the absence of inducer. The minimization of basal expression is less critical in applications for inducible gene expression where the gene product has no significant toxicity to the plant cells producing it.

A minimal promoter contains the DNA sequence signals necessary for RNA polymerase binding and initiation of transcription. For RNA polymerase II promoters, the promoter is identified by a TATA-homologous sequence motif about 20 to 50 bp upstream of the transcription start site and a CAAT-homologous sequence motif about 50 to 120 bp upstream of the transcription start site. By convention, the skilled artisan often numbers the nucleotides upstream of the transcription start with increasingly large numbers extending upstream of (in the 5' direction) from the start site. Generally, transcription directed by a minimal promoter is low and does not respond either positively or negatively to environmental or developmental signals in plant tissue. An exemplary minimal promoter suitable for use in plants is the truncated CaMV 35S promoter, which contains the regions from -90 to +8 of the 35S gene.

The inducible transcription regulatory sequence (qualitative transcription regulatory sequence) is localized to the region between -167 and -100 relative to the EAS4 transcription initiation site (nucleotides 406 to 473, initiation at nucleotide 573 in SEQ ID NO:2). It is understood that there may be a plurality of sequence motifs which respond to particular stimuli. Operably linking this sequence directly upstream of a minimal promoter functional in a plant cell confers inducible expression of a coding sequence operably fused just downstream of the promoter, e.g., a heterologous coding

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- 29 sequence, and the skilled artisan understands spacing requirements and other requirements for translational expression of the coding sequence. The heterologous coding sequence is preferably for an elicitin-like protein of a plant pathogenic microorganism (e.g., a virus, bacterium or fungus), for example, the sequence encoding the parAl gene product (elicitin) of Phytophthora parasitica where disease resistance via the hypersensitivity response to an invading potential plant pathogen is desirable. Harpin proteins of certain . phytopathogenic bacteria also can serve as inducers of expression mediated by the EAS4-derived inducible transcriptional regulatory sequences. Inclusion of additional 5' flanking sequence from the EAS4 gene allows for increased levels of downstream gene expression.

Preferred is the use of a sequence including the -266 to +1 region of EAS4 (nucleotides 307 to 573 of SEQ ID NO:2), and more preferred is the sequence including -567 to +1 (nucleotides 1 to 573 of SEQ ID NO:2).

An alternative to the use of the fusion of the

EAS4 transcription regulatory sequence fused to a heterologous minimal promoter is the use of the promoter region of EAS4 in conjunction with the upstream promoter-associated regulatory elements. In such an application the use of nucleotides 307 to 463, or more preferably for greater levels of downstream expression, nucleotides 371 to 463, 311 to 462, and 10 to 573 of SEQ ID NO:2.

In a plant such as N. tabacua, the instant inducible transcription regulatory element directs the induction of downstream gene expression in response to invading plant pathogens and certain compositions such as some fungal cellulases and certain plant and fungal cell wall fragments. Plant pathogens which can trigger this expression include, but are not limited to, Xanthoaonas,

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- 30 Pseudomonas syringae, Phytophthora species including parasitica, and Peronospora species (e.g., tabaci).

Coding sequences suitable for expression in a plant are operably linked downstream of the regulated promoter construct. Transgenic plants can be constructed using the chimeric gene consisting essentially of the regulated promoter, any additional transcription-enhancing sequences, and the desired coding sequence including the necessary sequence signals for its translation. Where disease resistance is to be advantageously induced in response to invasion of a transgenic plant tissue by a potential plant pathogen or in response to treatment with an elicitor or other chemical signal which induces EAS4 gene expression, the coding sequence is preferably for an elicitin of a plant pathogenic microorganism, e.g., the parAl gene product of Phytophthora parasitica (as described in Kamoun et al. (1993) supra). Other elicitin-like proteins have been described in the readily available scientific literature, and include those from Phytophthora species, Peronospora species, and Xanthomonas species, among others.

Alternative coding sequences which can be expressed under the regulatory control of the present inducible transcription regulatory element for improvement of the resistance of a (transgenic) plant or plant tissue exposed to a viral, bacterial or fungal plant pathogen include, but are not limited to, chitinase, TMV coat protein or other plant virus coat protein, NIa virus gene and others.

Additionally, or alternatively, induction of the regulated construct can be induced, for example, by treating the transgenic plant or tissue with an elicitor or with a bacterium, virus or fungus (preferably not pathogenic for the host plant) capable of inducing expression via the inducible transcription regulatory

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- 31 element of a coding sequence not capable of turning on the HR, or disease resistance directly could be achieved. Coding sequences which may be advantageously expressed include an insecticidal protein, such as one of the

Bacillus thuringiensis crystal proteins, which when expressed would protect the plant from insect pests.

Phytoalexin synthesis from the native EAS4 gene, or induction of gene expression mediated by the present regulated EAS4 promoter or the inducible transcription io regulatory element in combination with at least a heterologous minimal promoter, can be induced by treating the plant tissue or cells with a wide variety of defined chemicals, crude fungal culture filtrates, fungal cell wall extracts, and oligosaccharides from plant or fungal cell walls [Albersheim and Valent (1978) J. Cell. Biol.

22.:627-643]. Other compounds capable of inducing the HR include certain cellulases, for example, Trichodenoa viride cellulases, and certain plant or fungal cell wall fragments, among others.

A transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens-mediated DNA transfer, preferably with a disarmed T-DNA vector, electroporation, direct DNA transfer, and particle bombardment (See Davey et al. (1989) Plant Mol. Biol. 12:275; Walden and Schell (1990) Eur. J. Biochem. 192:563: Joersbo and Burnstedt (1991) Physiol. Plant. 81:256: Potrykus (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. £2.:205; Gasser and Fraley (1989) Sci. 244:1293: Leemans (1993) Bio/Technology.

H:522; Beck et al. (1993) Bio/Technology. 11:1524;

Koziel et al. (1993) Bio/Technology. 11:194; and Vasil et al. (1993) Bio/Technology. 11:1533.). Techniques are well-known to the art for the introduction of DNA into aonocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those

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- 32 tissues. Monocots which have been successfully transformed and regenerated include wheat, corn, rye, rice and asparagus. For example, U.S. Patent No. 5,350,689 (1994, Shillito et al.) describes transgenic

Zea mays plants regenerated from protoplasts and protoplast-derived cells. For efficient production of transgenic plants, it is desired that the plant tissue used for transformation possess a high capacity for regeneration. Transgenic aspen tissue has been prepared io and transgenic plants have been regenerated [Devellard et al. (1992) C.R. Acad. Sci. Ser. VIE 314:291-298K; Nilsson et al. (1992) Transgenic Res. 1:209-220; Tsai et al.

(1994) Plant Cell Rep. 14:94-97]. Poplars have also been transformed [Wilde et al. (1992) Plant Physiol.

98:114-1201. Technology is also available for the manipulation, transformation and regeneration of Gymnosperm plants in the laboratory. For example, U.S. Patent No. 5,122,466 (1992, Stomp et al.) describes the bio-ballistic transformation of conifers, with preferred target tissue being meristematic and cotyledon and hypocotyl tissues. U.S. Patent No. 5,041,382 (1991,

Gupta et al.) describes enrichment of conifer embryonal cells.

Techniques and agents for introducing and selecting for the presence of heterologous DNA in plant cells and/or tissue are well-known. Genetic markers allowing for the selection of heterologous DNA in plant cells are well-known, e.g., genes carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. The marker allows for selection of successfully transformed plant cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene.

Other techniques for genetically engineering plant cells and/or tissue with an expression cassette

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- 33 comprising an inducible promoter or chimeric promoter fused to a heterologous coding- sequence and a transcription termination sequence are to be introduced into the plant cell or tissue by Agrobacterium-mediated transformation, electroporation, microinjection, particle bombardment, or other techniques known to the art. The expression cassette advantageously further contains a marker allowing selection of the heterologous DNA in the plant cell, e.g., a gene carrying resistance to an ic antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin.

The transcription regulatory sequences, particularly the inducible transcription regulatory element (or the EAS4 promoter with the inducible and preferably the transcription-enhancing element) is useful in controlling gene expression in transgenic plant cells in suspension cell culture as an alternative to expression in transgenic plants. For example, the EAS4 promoter including the transcription initiation signals, the inducible transcription regulatory element and the transcription-enhancing element, can be used to mediate the inducible expression of one or more heterologous coding sequence(s) in transgenic plant cells in suspension cell culture. When desired, expression of the coding sequence of interest is induced by the addition of an elicitor or other inducing chemical signal to the culture. Suspension culture cells respond to elicitors readily in comparison to intact plants. The heterologous coding sequence(s) can encode proteins which mediate synthesis of pharmaceutical compounds, poly-0-hydroxybutyrate synthesis or other secondary metabolites, cellulose, starch, sugars, oils, or the heterologous sequences can encode pharmaceutical proteins, insecticidal toxin proteins, antifungal proteins, antiviral proteins (such as coat proteins to

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- 34 mediate resistance to virus infection), the Nla protein, chitinases, glucanases, male sterility proteins or sequences, proteins to improve nutritional quality or content, or developmental and/or tissue-specific p-rogr-ams or patterns. It is understood that transgenic plants can be similarly used to express heterologous coding sequences as can transgenic plant cells.

Where transgenic plants are to be induced for phytoalexin synthesis or for the expression of a heterologous coding sequence under the regulatory control of the EAS4 promoter or the inducible transcription regulatory element derived therefrom and/or the transcription-enhancing sequence derived from the EAS4 promoter as well, the elicitor must penetrate the cuticle of the plant to have an inductive effect. Alternatively, the plant tissue can be wounded to facilitate or allow the uptake of the elicitor into the plant tissue. A wide variety of inducing compositions, including elicitors and other chemical signals, such as the combination of ethylene and methyl jasmonate, can be effectively introduced into the transgenic plant suspension cell cultures, where there is significantly less of a barrier to the uptake and/or sensing of the elicitors. Where ethylene and methyl jasmonate serve to induce gene expression, the ethylene is used at a concentration between about 1 and about 50 ppm and the methyl jasmonate is used at a concentration between about 0.1 mM and about 1 mM.

The following examples use many techniques well-known and accessible to those skilled in the arts of molecular biology, in the manipulation of recombinant DNA in plant tissue and in the culture and regeneration of transgenic plants. Enzymes are obtained from commercial sources and are used according to the vendors' recommendations or other variations known to the art.

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- 35 Reagents, buffers and culture conditions are also known to the art. References providing standard molecular biological procedures include Sambrook et al. (1989) Molecular Cloning, second edition. Cold Spring Harbor Laboratory, Plainview; NY; R. Wu (ed.) (1993) Methods in

Enzymology 218; Wu et al. (eds.) Methods in Enzymology 100 and 101; Glover (ed.) (1985) DNA Cloning, Vols. I and

II, IRL Press, Oxford, UK; and Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, TRL Press, Oxford, UK. References related to the manipulation and transformation of plant tissue include R. A. Dixon (ed.) (1985) Plant Cell Culture: A Practical Approach, IRL Press, Oxford,

UK; Schuler and Zielinski (1989) Methods in Plant Molecular Biology, Academic Press, San Diego, CA; Weissbach and Weissbach (eds.) (1988) Methods for Plant Molecular Biology, Academic Press, San Diego, CA; I. Potrykus (1991) Ann. Rev. Plant Physiol. Plant Mol. Biol. £2.:205; Weising et al. (1988) Annu. Rev. Genet. 22:421; van Wordragen et al. (1992) Plant Mol. Biol. Rep. 19:12; Davey et al. (1989) Plant Mol. Biol. 13:273; Walden and Schell (1990) Eur. J. Biochem. 192:563; Joersbo and Brunstedt (1991) Physiol. Plant. 22.:256 and other work cited in the foregoing references. Abbreviations and nomenclature, where employed, are deemed standard in the field and are commonly used in professional journals such as those cited herein.

All references cited in the present application are expressly incorporated by reference herein.

The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified compositions and methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

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- 36 EXAMPLES , Example 1. EAS-specific Antibodies

Monoclonal and polyclonal antibodies specific for tobacco EAS were prepared as described by Vogeli et al.

(1990) Plant Physiology 93:182-187. Additional antibody preparations could be made as polyclonal antibodies using purified EAS as antigen or using a peptide sequence conjugated to a carrier protein using well-known techniques. The amino acid sequence of a peptide for io antibody production is selected from a particularly hydrophilic region of the protein (For antibody production techniques, see, for example, Campbell (1994) Monoclonal Antibody Technology. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon and

Rnippenberg, eds, Elsevier, Amsterdam; Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, cold Spring Harbor, NY).

Example 2. DNA and Protein Sequence Determination

Sequence determinations of single-stranded and double stranded DNAs were carried out by the dideoxynucleotide chain termination procedure [Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:8073-8077], with a Sequenase kit from United States Biochemical Corp., Cleveland, OH) or an automated fluorescence based system (Applied Biosystems, Foster City, CA).

Example 3. Construction of a Full-length EAS Clone

Nicotiana tabacum L. cv. KY14 cell suspension cultures were treated with Trichoderma viride cellulase (Type RS, Onozuka) at a final concentration 0.5 /xg/ml during rapid growth phase to induce the expression of EAS. Parallel suspension cell cultures which did not receive cellulase served as controls. Cells were collected by gentle vacuum filtration 4 hours after the

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- 37 addition of the cellulase elicitor to the induced culture.

A cDNA library was prepared in pcDNAII (Invitrogen, San Diego, CA) from polyA⁺ RNA extracted from the N. tabacua cells treated for 4 hours with elicitor. The library was screened by differential hybridization using polyA+ RNA prepared from the induced and control cultures. Clones appearing to be positive were further screened by hybrid selection-in vitro translation-immunoprecipitation analysis as described by Alwine et al. (1979) Methods Enzymol. 68:220-242.

A putative positive EAS cDNA clone was used as a hybridization probe for the isolation of additional cDNA and genomic clones. The genomic library thus screened was one constructed in 1EMBL3 using Mbol partially digested DNA prepared from N. tabacun L. cv. NK326 hypocotyl DNA {Clontech, Palo Alto, CA). This screening yielded 8 independent clones, each of which appeared to represent a different chromosomal locus. EAS4 and EAS3 genomic clones were described in Facchini and Chappell (1992) supra, but are now known to have been incomplete.

Facchini and Chappell (1992) supra had misidentified the translation start sites of the EAS3 and EAS4 coding sequences in the genomic clone described therein. The correct translation start site for the EAS3 and EAS4 coding sequences have been determined to be methionine codons 165 bp upstream of the ATG codons previously identified as start sites. The corrected start site for EAS4 was mapped using a combination of primer extension assays to identify the transcription start site and additional N-terminal amino acid sequencing data of purified enzyme as noted hereinabove.

An amplimer of 110 bp was prepared by a polymerase chain reaction to provide a DNA sequence corresponding to amino acids 56-92 of the EAS4 protein (see SEQ ID NO: 12)

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- 38 and Facchini and Chappell (1992) supra. This amplimer was used as a hybridization probe to screen a cDNA library in pcdNAII (Invitrogen, San Diego, CA) prepared from polyA+ RNA from tobacco cell culture cells 4 hours after elicitor treatment (Trichoderma viride cellulase). This amplimer was made using a sense primer (ATGCTGTTAGCAACCGGAAGG; SEQ ID NO:3) and a reverse primer (ATCCAAAATCTCATCAATTTC; SEQ ID NO:4), and the genomic EAS4 template in a standard PCR reaction [Saiki et al.

(1988) Science 239:487-4911. The 110 bp amplimer was isolated after polyacrylamide gel electrophoresis using DE—81 paper (Whatman International, Inc., Clifton, NJ). The isolated fragment was then radiolabeled with [a-³²P]-dCTP using a random priming kit from Stratagene (La Jolla, CA) for use as a hybridization probe in colony lifts of the cDNA library as previously described [Hanahan and Meselson (1980) Gene 10:63-671. The longest clone obtained in these experiments appeared to lack 80 bp of 5' coding sequence.

To obtain a full-length clone, a RT/PCR approach was used. First strand cDNA was prepared from polyA+ RNA prepared from tobacco cells after induction with elicitor as described [Facchini and Chappell (1992) supra] using reverse primer having the sequence ATGAGTCCTTACATGTGA (SEQ ID NO:5). This sequence corresponds to nucleotides 459-477 downstream of the translation start site. The reverse transcriptase reaction was carried out in a 10 μ1 reaction (1 pg polyA+ RNA, 25 pmol reverse primer, 10 mM DTT, 2.5 mM each dATP, dGTP, dCTP, dTTP, 8 units RNase

Block I (Stratagene, La Jolla, CA), first strand synthesis buffer used according to the manufacturer's instructions (Stratagene) for l hr at 37°C. This reaction was terminated by treating at 99°C for 5 min. Then 40 μΐ of master PCR mix was added to the first strand reaction; PCR master mix contains 10 mM Tris-HCl

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- 39 pH 8.3, 50 πΜ KC1, 1.5 mM MgCl₂, 0.01% Tween-20, 0.01% (w/v) gelatin, 0.01% NP-40, 2.5 mM each deoxynucleotide triphosphate, 1 unit of TaqI polymerase, and 25 pmol forward primer (GGGAGCTCGAATTCCATGGCCTCAGCAGCAGCAGTTGCAAACTAT_f SEQ ID

NO:6, EcoRI and Ncol recognition sites underlined and ATG translation start site in bold). PCR was carried out under standard conditions [Back et al. (1994) Arch. Biochem. Biophys. 315:523-5321

The 492 bp reaction product was digested with

EcoRI and Hindlll and subcloned into similarly cut pBluescript SK (Stratagene). A Hindlll/Xhol fragment from another partial cDNA clone was subsequently cloned into the corresponding sites of the 5'-terminal sequence clone to generate a full-length cDNA clone named pBSK-TEAS. pBSK-TEAS DNA was transformed into Escherichia coli TB1 using a CaCl₂ protocol [Sambrook et al. (1989) supra]. Determination of the DNA sequence of the insert confirmed that this plasmid had the expected and desired structure (dideoxynucleotide chain termination procedure, United States Biochemical Corp., Cleveland, OH).

Example 4. Identification of EAR Homologous Sequences

Tobacco leaf genomic DNA was isolated as described in Murray and Thompson (1980) Nucleic Acids Research £:4321-4325. After digestion of aliquots with desired restriction enzymes, the digested DNA samples were electrophoresed on 0.8% agarose gels and the size-separated DNAs were transferred to nylon membranes.

DNA blots were hybridized with random primer radiolabeled cEASl, which is truncated at the 5' end of the coding region, (prepared as in Sambrook et al. (1989) supra) at 60C in 0.25 M sodium phosphate buffer, pH 8.0, 0.7% SDS, i% bovine serum albumin, 1 mM EDTA. The blot was then

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- 40 washed twice at 45°C with 2X SSC, 0.1% SDS and twice with

0.2X SSC, 0.1% SDS (IX SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Relative hybridization levels were estimated from autoradiograms using a video densitometer (MilliGen/Biosearch, Ann Arbor, MI).

Facchini and Chappell (1992) supra reported that Southern hybridization results indicated that there were 12-16 copies of EAS-homologs in the N. tabacvun genome.

To address the presence of significantly homologous sequences to tobacco EAS and apparent number of copies per genome of those sequences, Southern hybridization experiments were carried out using DNA isolated from other plant species.

Restriction endonuclease-digested genomic DNAs are separated by agarose gel electrophoresis (0.8% agarose), and then transferred to a Hybond-N⁺ membrane (Amersham Corp., Arlington Heights, IL). Radiolabeled probe comprising coding sequences of EAS, and hybridizations are carried out essentially as described in Sambrook et al. (1989) supra. Moderate stringency conditions are used (hybridization in 4X SSC, at 65°C) last wash in IX SSC, at 65®C).

Alternatively, PCR can be carried out using target DNA as template and primers derived from the EAS4 coding sequence in highly conserved regions (see SEQ ID NO:7) using well-known techniques.

Example 5. Detection of EAS Protein

The enzymatic activity of an expression product can be confirmed using the techniques described in Facchini and Chappell (1992) supra and in Back et al. (1994) Arch. Biochen. Biophys. 315:527-532.

For detecting the presence of EAS cross-reacting protein material, total protein fractions are prepared from 100 sil aliquots of bacterial culture harvested and £71 I· 0 / Z 6 /d/dV

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- 41 concentrated by centrifugation for 2 minutes in a microfuge. After discarding the culture supernatant, cell pellets are resuspended in 100 pi 50 mM Tris-HCl, pH 6.8, 10 mM dithiothreitol, 2% sodium dodecyl sulfate,

0.01% bromophenol blue, 10% glycerol. For immunological detection 15 pi aliquots are electrophoresed over 11.5% SDS-polyacrylamide gels; for Coomassie blue-staining of the proteins, 35 pi aliquots are similarly electrophoresed. For soluble protein samples, the cells io are processed as in the procedure for determination of enzymatic activity (see Back et al. (1995) supra or Facchini and Chappell (1992) supra). For immunological detection 10 pi aliquots are electrophoresed as above; for Coomassie blue-staining, 10-50 pi aliquots were electrophoresed.

After electrophoresis the proteins are stained with Coomassie blue, or the proteins are transferred to nitrocellulose membranes as described [Towbin and Gordon (1984) Journal of Immunological Methods 72.:313-340] for imnunodeteetion. After incubating for 30 minutes in 5% low-fat milk in IX TBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl), the nitrocellulose blots were incubated overnight in the same solution containing monoclonal antibody specific for tobacco EAS (1:1000 dilution; Vogeli et al.

(1990) Plant Physiology 93:182-187). Goat anti-mouse antibodies linked to alkaline phosphatase and the specific chromogenic dye were then incubated to visualize the binding of the EAS-specific antibody to the proteins immobilized on the nitrocellulose membranes [Leary et al.

(1983) Proc. Natl . Acad. Sci . USA 80:4045-40491.

Example 6. Genomic EAS4 Clone

The 5'—truncated cDNA clone cEASl described in Facchini and Chappell (1992) supra was used as a hybridization probe for screening a N. tabacum cv. NK326

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- 42 genomic library in the 1EMBL3 vector (Clontech, Palo Alto, CA). DNA sequences were determined using routine subcloning and DNA sequencing protocols.

The DNA and deduced amino acid sequences of the

EAS4 genomic clone are presented in SEQ ID NO: 7-8.

Example 7. Generation of Transgenic Plants

For studies of the function of portions of the upstream untranslated region of the EAS4 gene, Hindlll/BamHI-ended fragments of this upstream DNA were io cloned into pBHOl (Clontech, Palo Alto, CA) so that expression of the β-glucuronidase (GUS) reporter gene could be monitored in transformed plant cells. The 5'-flanking sequence of the EAS3 gene is given in SEQ ID NO:1 and the 5'-flanking sequence of the EAS4 gene is given in SEQ ID NO:2. In each of these sequences, the translation start site (ATG) is the last three nucleotides. By primer extension techniques, the EAS4 transcription start site was estimated at nucleotide 573 in SEQ ID NO:2. CAAT and TATA box motifs are identified at nucleotides 429 to 432 and at nucleotides 456 to 459 in SEQ ID NO:1 (EAS3) and at nucleotides 513 to 516 and at nucleotides 540 to 543 in SEQ ID NO:2 (EAS4).

The transformed plant cell lines were produced using a modified Agrobacterium tumefaciens transformation protocol. The recombinant plasmids containing the sequences to be introduced into plant tissue were transferred into A. tumefaciens strain GV3850, by triparental mating with Σ. coli TB1 (pRK2013). N. tabacum leaves at a variety of stages of growth were cut into 1 cm² pieces, and dipped in a suspension of agrobacterial cells (about 10⁴ to 10⁵ cells/ml). After 3 to 10 minutes, the leaf segments were then washed in sterile water to remove excess bacterial cells and to reduce problems with excess bacterial growth on the “reared leaf segments.

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- 43 After a short drying time (30 to 60 seconds), the treated leaf segments are placed on the surface of Plant Tissue Culture Medium without antibiotics to promote tissue infection and DNA transfer from the bacteria to the plant tissue. Plant Tissue Culture Medium contains per liter: 4.31 g of Murashige and Skoog Basal Salts Mixture (Sigma Chemical Company, St. Louis, MO), 2.5 mg of benzylaminopurine (dissolved in 1 N NaOH), 10 ml of o.l mg/ml indoleacetic acid solution, 30 g sucrose, 2 ml of io Gamborg's Vitamin Solution (Sigma Chemical Co., St.

Louis, MO) and 8 g of agar. The pH is adjusted between pH 5.5 and 5.9 with NaOH. After 2 days, the leaf segments were transferred to Plant Tissue Culture Medium containing 300 Mg/ml of kanamycin, 500 Mg/ml of mefoxin (Merck, Rahway, NJ) . The kanamycin selects for transformed plant tissue, and the mefoxin selects against the agrobacterial cells.

It is necessary to minimize the exposure of the explant tissue to agrobacterial cells during the transformation procedure in order to limit the possible induction of the regulated parAl coding sequence during the production of the transgenic plant cells, which would cause a cell death response. Accordingly, the bioballistic technique for the introduction of heterologous

DNA containing cell suicide genes under the regulatory control of the inducible transcriptional regulatory element is a useful alternative transformation technique because it does not entail the use of agrobacterial cells or fungal cell wall digestive enzymes (as necessary for the generation of protoplasts for electroporation), both of which lead to induction of the coding sequences under the control of that regulatory element.

Transgenic plants were regenerated essentially as described by Horsch et al. (1985) Science 227:1229-1231.

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- 44 The resulting transgenic plants were tested for the expression of the β -glucuronidase (GUS) reporter gene using 5-bromo-4-chloro-3-indolyl-/3-D-glucuronic acid as described by Jefferson et al. (1987) EMBO Journal 6:3901-3907, using untreated (control) conditions and inducing conditions. An inducing condition is the intercellular application of T. viride cellulase to tobacco tissue in the transgenic plants (using a mechanical pipetter to apply 50-100 μΐ or 10-100 nm protein (e.g., cryptogein) inducing composition to interstitial tissue); controls were mock-applied but not treated with cellulase elicitor. Tobacco tissue was wounded with a scalpel in some experiments to facilitate exposure to the inducing compounds.

Example 8. Deletion Analysis of Promoter and Promoter-Associated Region

In separate reactions, the EAS4-derived DNA sequence encompassed by -567 to +67 relative to the transcription start site (nucleotides 6 to 642, SEQ ID NO:2, EAS4) was substituted for the Cauliflower Mosaic Virus (CaMV) 35S promoter [Benfey et al. (1990) EMBO Journal 2.:1677-1684] in the GUS-reporter vector pBI221 (Clontech, Palo Alto, CA). Deletion mutants in the EAS4 upstream regions were then isolated after restriction endonuclease. Analysis of the gEAS promoter-GUS constructs was carried out in electroporated tobacco cell protoplasts (Fig. 1) and in stably transformed tobacco lines (Fig. 2A, Table 1) . Preliminary data for the transient expression demonstrated that SEQ ID NO :1 did not function for elicitor inducibility and SEQ ID NO :2 functioned in regulating gene expression.

The transient expression data obtained with the N. tabacum protoplasts into which various EAS3 and EAS4 promoter-GUS constructs were introduced are given in Fig.

1. Progressive deletions from the 5’ end of the EAS4

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- 45 promoter regions reduce the levels of expression, but inducibility is maintained for the -262, -202 and -110 constructs (relative to the transcription start site at nucleotide 573 of SEQ ID NO:2). These data indicated that only very low levels of GUS are expressed via either EAS3 promoter region construct. Similarly, the CaMV 35S promoter alone is not induced by the elicitor treatment.

The data in Figs. 2A-2B indicate that deletion of genetic material between -567 and -160 reduce the level io of downstream gene expression but does not destroy the inducibility of expression. Therefore, the DNA sequences between -567 and -160 appear to contain transcription-enhancing activity. Most of the transcription-increasing activity appears to reside between -567 and -212, but additional enhancement appears to be mediated by sequence information between -212 and -160 relative to the transcription start site (transcription start site is nucleotide 573; -567 is nucleotide 1, -212 is nucleotide 361, and -160 is nucleotide 413, all in SEQ ID NO:2).

In the Gain of Function assay data in Fig. 2B, the

DNA sequence information necessary to mediate induction in response to elicitor treatment is located between -160 and -87 relative to the EAS4 transcription start site (i.e., between nucleotides 413 and 486 of SEQ ID NO:2).

In these experiments the EAS-derived sequences were placed in front of a truncated CaMV 35S promoter [Benfey et al. (1990) EMBO J. 1677-1684]. This figure also demonstrates that the EAS4-derived transcription regulatory region functions when fused to a heterologous minimal promoter.

In a more extensive analysis of independent transformants, either the entire -567 to +67 EAS4 upstream region or 5' deletions thereof were inserted upstream of the GUS (0-glucuronidase) reporter gene in

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- 46 vector ρΒΙΙΟΙ (Clontech, Palo Alto, CA), and expression levels of the GUS reporter were assayed under inducing and noninducing conditions. 160 bp upstream of the transcription start site of EAS4 were sufficient to direct the regulated expression of the GUS reporter gene, although the presence of additional upstream sequences mediated increased expression. Constructs containing a minimum of 167 bp upstream of the EAS4 transcription start site gave transient gene expression in io electroporated protoplasts and confer elicitor-inducibility of GUS reporter gene expression (minimum of 2.5-fold increase in gene expression). By contrast, the EAS3 upstream region (SEQ ID N0:l) does not appear to support high levels of reporter gene expression in the transient expression system, nor does it appear to confer elicitor-inducibility to the downstream reporter gene.

In part, the elicitor-inducible GUS reporter gene expression was expected in the protoplast system because those protoplasts were generated using fungal cell wall digestive enzymes, and those enzymes have been shown to elicit phytoalexin production and sesquiterpene cyclase gene expression in plants [Chappell et al. (1991) Plant Physiology 27:693-698]. A possible explanation as to why the protoplasts respond to a second elicitor treatment is that the cells are allowed to recover for 6-8 hours before the second treatment. This recovery phase allows the cells to return to an elicitor-responsive state.

pBHOl is commercially available from Clontech (Palo Alto, CA). It contains the CaMV 35S promoter upstream of the GUS reporter gene in a pUC19 vector; thus it serves as a vector for transient expression experiments where the recombinant vector is introduced into plant protoplasts. The presence of this plasmid and its derivatives is selected by growth on kanamycin. A

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- 47 promoter-less” GUS cassette in the Agrobacterium binary plasmid vector pBINl9 (Bevan, M. (1984) Nucl. Acids Res.

12.:8711) similarly carries a plant-expressible kanamycin resistance determinant.

r Example 9. Identification of Inducible Transcription Regulatory Element

The 5' flanking domains of genomic EAS3 and EAS4 clones were mapped by SI nuclease protection and primer extension experiments [Sambrook et al. (1989) supra].

Subclones, comprising up to 1 kb 5 · to the translation start site were sequenced and fused to the /3-glucuronidase (GUS) reporter gene in pBHOl for studies in transgenic plant tissue. The resulting recombinant plasmids were then electroporated into tobacco protoplasts. GUS activity was measured in transient expression assays, and stable transformed tobacco cell lines were also isolated for studies of GUS induction and expression.

Constructs were prepared containing a minimum of about 200 bp of nucleotide sequence upstream of the EAS4 transcription start site in the modified pBHOl vector, and a ^-glucuronidase (GUS) reporter gene were made and analyzed for ability to drive regulated GUS expression. 200 bp of flanking sequence appeared sufficient to drive transient gene expression in electroporated protoplasts and confers elicitor inducibility to GUS expression (minimum of 2.5 fold induction). Similar experiments with the EAS3 flanking sequence indicated that 200 bp from the EAS3 locus did not support either high levels of

GUS expression or elicitor responsiveness in transformed plant cells. Cellulase and elicitins from Phytophthora [Ricci et al. (1989) Sur. J. Biochem. 1^3:555-563] serve to induce gene expression mediated by the EAS4-derived regulatory sequences.

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AP000924

- 48 Further studies related to the identification of sequences important in mediating induced gene expression in response to pathogen invasion, as modeled using cellulase or elicitins, were carried out after oligonucleotide site-directed mutagenesis [Kunkel (1985) Proc. Natl. Acad. Sci. USA £2.:488-492] of the putative regulatory region of EAS4. Substitution of GT for the wild-type CA at -233 and -234 relative to the EAS4 transcription start site (nucleotides 334-335 of SEQ ID io NO:2) did not appear to alter the expression of the GUS reporter gene as measured after incubation in the presence of elicitor (cellulase) for 20 hours.

Preliminary methylation interference and gel retardation studies carried out essentially as described [Sambrook et al. (1989) supra] indicated that an octameric sequence centered around -233 relative to the translation start site (centered around 334 of SEQ ID NO:2) binds proteins from plant cell nuclei. Methylation interference data suggested that the G at position -233 was preferentially protected against methylation by DMS (dimethyl sulfate) if first allowed to interact with nuclear extracts. The results of gel retardation studies were consistent with those obtained in the methylation protection experiments. When DNA fragments containing the -343 to -140 region (relative to the translation start site) (nucleotides 230 to 433 of SEQ ID NO:2) were examined after reaction to nuclear extracts, mobility in native acrylamide gel electrophoresis appeared retarded. Protein binding was abolished by the GT to CA substitution at positions -234 and -233. Similar results were observed in control and elicitor-induced cell extracts, and reporter gene expression was not changed by this 2 bp mutation. Thus, it is concluded that the region around -233 is not directly involved in the

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- 49 induction of gene expression in response to pathogen invasion or elicitor treatment.

Preliminary experiments indicate that EAS4 DNA sequences between -253 and -48 relative to the EAS4 transcription start site (between nucleotides 320 and 525 of SEQ ID NO:2) have qualitative and quantitative effects on downstream reporter gene expression. Sequences between -110 and -1 of EAS4 relative to the transcription start site of EAS4 (nucleotides 463 to 572 of SEQ ID

NO:2) to mediate the inducible response, while sequences between -202 and -110 relative to the EAS4 transcription start site (nucleotides 371 to 463 of SEQ ID NO:2) enhance the levels of both induced and uninduced reporter gene expression.

Example 10. Construction of a Chimeric

EAS4 Promoter(-1148 to +68):GUS Reporter Gene

In another series of experiments, we examined the activation of the EAS4 promoter (-1148 to +67) in transgenic tobacco plants. To obtain the 5' flanking sequence of the tobacco EAS4 gene promoter (-1148 to +67), an approximately 1.9 kb Hindlll-Hindlll fragment containing a portion of the EAS4 5' sequence was isolated from the gEAS4 genomic clone described above. The isolated fragment was then cloned into the polylinker of the pBluescript KS(+) plasmid vector (Stratagene). Using standard PCR methodology, the resulting pKS(+) plasmid containing the EAS4 Hindlll-Hindlll fragment was used as a DNA template to generate an approximately 1.2 kb EAS4 promoter subfragment (-1148 to +67) having sequences 1148 bp upstream and 67 bp downstream of the EAS4 transcription start site. The nucleotide sequence of this fragment is shown in Fig. 3A. The 1215 bp Hindlll-BamHI fragment containing rhe EAS4 promoter (1148 to +67) was then ligated in correct reading frame with the coding region of GUS in the binary vector

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- 50 pBHOl.l. Fig. 3B shows a schematic illustration of the structure of the resulting EAS4 promoter(-1148 to +67):GUS reporter gene fusion.

Example 11. Elicitor- and Pathogen-inducible Expression of a Chimeric EAS4 Promoter(-1148 to +67,:GDS

Gene in Transgenic Tobacco

To assess the function of the tobacco EAS4 promoter (-1148 to +67), gene expression of the GUS reporter construct shown in Fig. 3B in transgenic tobacco io plants treated with either an elicitor or pathogen was monitored as follows.

The EAS4 promoter(-1148 to +67):GUS reporter gene shown in Fig. 3B was transferred to the disarmed Agrobacterium tumefaciens strain GV3850 using the triparental mating procedure described by Schardl et al. (Gene £1: 1-11, 1987). Tobacco plants (Nicotiana tabacum cv Xanthi) were then transformed with the disarmed Agrobacterium heir boring the reporter construct using the leaf disc transformation method described by Horsch et al. (Science 227: 1229-1231, 1985). As control plants, lines of transgenic tobacco expressing the GUS reporter gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter were also regenerated. Seeds from regenerated transgenic tobacco plants were germinated on medium containing 100 mg/L kanamycin. The resulting kanamycin-resistant plants were subsequently transferred into soil and grown in a greenhouse. These plants were then tested for the expression of the GUS reporter gene using the assay described above, using untreated (water control) conditions and inducing (elicitor- and pathogentreated ) conditions.

To determine the elicitor inducibility of GUS gene expression driven by the EAS4 promoter(-1148 to +67), GUS activity was monitored in the leaves of transgenic tobacco plants treated with the fungal elicitor protein

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- 51 cryptogein or water as follows. Half of a fully expanded leaf (8th leaf from bottom of the plant) from a two-month old transgenic tobacco plant was infiltrated from the abaxial side using a pipette with approximately 50 pi of

25 nM cryptogein [prepared according to the method of Ricci et al., Eur. J. Biochem. 183: 555-563, 1989]; the other half of the leaf was infiltrated with approximately 50 pi of water in a similar fashion. At various times after infiltration, the cryptogein and water infiltrated zones of the leaf were collected from intact plants and analyzed for GUS activity.

As shown in Fig. 4A, leaves from three independent lines of transgenic tobacco which were infiltrated with 25 nM of cryptogein showed that GUS gene expression was induced «4 hours after elicitor treatment. In contrast, the 35S CaMV promoter:GUS reporter gene was not induced by elicitor treatment (data not shown). In addition, GUS activity was not induced in leaves of transgenic plants containing the EAS4 promoter(-1148 to +67):GUS reporter gene when treated with water (Fig. 4A) . At the microscopic level, GUS activity was found to be restricted exclusively to areas of leaf tissue challenged with elicitor, indicating that the EAS4 promoter(-1148 to +67) was not induced by a secondary signal (Fig. 8A).

To characterize the organ-specificity of GUS gene expression driven by the EAS4 promoter (-1148 to +67), roots and stems of transgenic tobacco plants were treated with cryptogein elicitor and analyzed for GUS activity as follows. Roots were obtained from transgenic tobacco plants and maintained on sterile Murashige and Skoog woH-iπ-m until tested. Stems (removed from one to one and half inches under the apex) were obtained from two-month old greenhouse-grown transgenic tobacco plants. Prior to elicitor treatment, roots and stems were cut into ~ 15-30 uM segments. Segmented roots and stems were then

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- 52 incubated on filter paper moistened with 100 nM of cryptogein or water for 18 hours at room temperature.

GUS activity in root and stem segments was subsequently analyzed using the assay described above.

Fig. 4B shows that GUS activity was induced in both the roots and stems of two independent lines of transgenic tobacco after an 18 hour treatment with cryptogein. A 15-fold increase in GUS activity relative to samples treated with water (control) was observed in both stems and roots, respectively. Roots, showed higher GUS activity them stems. These results show that the EAS4 promoter (-1148 to +67) is inducible in root and stem tissue in response to elicitor treatment.

To determine if the EAS4 promoter(-1148 to +67):GUS reporter gene was induced by fungal pathogens, GUS activity in the leaves of transgenic tobacco treated with two different races of Phytophthora parasitica var. Nicotianae was analyzed as follows. Young apical leaves were detached from about 45-day old transgenic tobacco plants and inoculated with a mycelial plug (»1 cm in diameter) of 2-day-old race 0 or race 1 of Phytophthora parasitica var. Nicotianae cultures grown on oatmeal agar as described by Tedford et al. (Plant Disease 74:

313-316, 1990). Inoculated leaves were then incubated on filter paper moistened with distilled water in a growth chamber at 25°C with constant fluorescent light for 24 hours. Control leaves were inoculated with blank oatmeal agar plugs. Inoculated tissues were subsequently collected and analyzed for GUS activity as described above.

As shown in Fig. 5, GUS activity was induced in the leaves of transgenic tobacco when treated with either race 0 or race 1 of Phytophthora parasitica var. Nicotianae. Although race 0 and race 1 of P. p. var.

Nicotianae typically induce different disease symptoms,

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- 53 no significant difference in dise;.„se symptoms caused by these two races was observed on the transgenic leaves of

N. tabacum cv. Xanthi. Furthermore, we observed that both race 0 and race 1 of P. parasitica induced the expression of the GUS reporter gene equally well.

To determine if GUS gene expression was induced by bacterial pathogens or elicitors, GUS activity in the leaves of transgenic tobacco treated with Pseudomonas syringae pv. Syringae 61 and its hrpH mutant, and Erwinia io amylovora harpin elicitor was analyzed as follows.Young apical leaves were detached from an approximately 45 day old transgenic tobacco plant and infiltrated with a cell suspension (As₀₀=0.05) of Pseudomonas syringae pv.

Syringae 61 or its hrpH mutant, or harpin elicitor (50 gg/ml) using a pipette according to the methods described above. Control leaves were infiltrated with water.

After approximately 12 hours, tissue was collected and analyzed for GUS activity as described above.

As shown in Fig. 6, GUS activity was induced in the leaves of transgenic tobacco after 12 hours when treated with either Pseudomonas, hrpH mutant, or harpin elicitor. In contrast, low GUS activity was detected in transgenic leaves treated with water. Furthermore, a hypersensitive response was observed to develop within the tissue zones infiltrated with either wild type P.

syringae Pv. Syringae 61 or purified harpin protein about 12-15 hours after treatment. These results indicate that the EAS4 promoter (-1148 to 67) is activated in response to either bacterial pathogens or a bacterial-derived elicitor.

Example 12. xamunoblot Analysis

The expression of EAS in different tissues of transgenic plants in response to cryptogein treatment was analyzed by standard Western blotting methods as follows.

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- 54 Proteins were extracted from control and cryptogein elicitor-treated tobacco tissues by homogenization with 80 mM potassium phosphate buffer (pH 7.0) containing 20% (w/v) glycerol, 10 mM sodium metabisulfite, 10 mM sodium ascorbate, 15 mM MgCl₂, and 5 mM /J-mercapthoethanol as described by Vogeli and Chappell, (supra). Protein concentrations were determined by the Bio-Rad assay. Equal quantities of protein were then separated by SDSPAGE, transferred to nitrocellulose membranes, and immunodetected as described by Voegeli and Chappell (gypsa)·

As shown in Figs. 7A-B, EAS protein was not detected in the leaves of tobacco plants by Western blotting experiments. In contrast, EAS protein was induced in the leaves of transgenic plants after treatment with elicitor. In segmented stems and roots, EAS protein was detected in the absence of elicitor treatment, indicating that one or more members of the EAS multigene family, but not EAS4 as determined by histochemical localization data described above, sire activated upon wounding.

Example 13. Cell-specific Expression Pattern of the EAS4 Promoter(-1148 to +67,:GUS Reporter Gene in Transgenic Tobacco

Figures 8A-K show the results obtained when tissues from different transgenic lines of tobacco were stained for GUS activity after treatment with elicitins. The data presented in Figures 8A-K are representative of a series of observations made at different times with several independent lines of transgenic tobacco expressing the EAS4 promoter(-1148 to +67):GUS reporter gene shown in Fig. 3B. GUS activity was localized in transgenic tissues by staining sections with 1 mM X-gluc (5-bromo-4-chloro-3-indolyl 0-glucuronide) staining solution containing 50 mM NaPO₄, pH 7, 0.05% Triton X—

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- 55 100, and 0.1% β-mercaptoethanol for 12-16 hours at 37^eC, and then fixed in 50% ethanol, 5% glacial acetic acid, and 10% formaldehyde for 2 hours. Chlorophyll was removed from appropriate tissues by incubating the sections in 70% ethanol. Sections of stained plant tissue were observed using a Zeiss IIIRS microscope and photographed using an MC63 photomicrographic camera.

As shown in Figs. 8B-F, GUS activity was present in elicitor-infiltrated zones of transgenic leaves, io indicating that the EAS4 promoter (-1148 to +67). was activated in response to cryptogein or parasicein infiltration throughout the leaf tissue, except in the epidermal cell layers where minimal GUS activity was observed. A similar weak staining pattern was observed in root and stem epidermal cells (Figs. 8G and 8L).

While minimal GUS activity was found in the epidermal cells of the transgenic leaves, GUS activity was observed in trichomes of both the leaf and root of transgenic plants (Figs. 8B and 8L).

In addition, as shown in Figs. 8C-F and Fig. 8L, elicitor-inducible GUS activity was observed in a distinct subepidermal layer of both stem and root sections (Figs. 8C-F and 8L). Fig. 8F shows that GUS activity in stem tissue was also present in the cambial, phloem, and primary xylem tissue layers; minimal GUS activity was observed in cortical regions of the stem. Furthermore, like stem tissue, GUS activity was localized within the vascular system of the roots (Fig. 8L).

No GUS activity was observed in flower petals, including pigmented and non-pigmented tissue, either before and after treatment with cryptogein.

Example 14. 5' Deletion Analysis of the EAS4 Promoter (-1147 to 4*57) in Transgenic Tobacco Plants

In another series of experiments, several 5' deletion constructs of the EAS4 promoter (-1147 to +67)

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- 56 were generated using standard PCR methodology. These promoter deletions were then fused to the GUS reporter gene in either pBIlOl.l and pBI221 vectors and subsequently transformed into tobacco as described above.

TABLE 1

5' Deletions	GUS Activity (MU/mg protein/min)	Fold Induction	# of independent transgenic lines tested
Control (water- infiltrated)	Elicitor- treated
-1148	173	1889	10.9	11
-567	120	921	7.7	12
-212	23	187	8.1	11
1 -160	2	34	17.0	9
1 -115	3	3	1	12
1 ~63	0	0	0	13

As shown in Table 1, deleting the EAS4 promoter from -1148 to -567 decreased inducible GUS activity to 50% of the level of that found in plants with a full-length EAS4 promoter (-1148 to +67). Further deletion of the promoter to -212 decreased inducible GUS activity by an average of 90%, and deleting to -160 decreased GUS activity to 2% of that exhibited by plants having a full-length EAS4 promoter (—1148 to +67) . Deletion to -63, a promoter which still contains putative CAAT and TATA boxes, completely abolished expression of the ribi-me-ric gene in control and elicitor-treated leaves.

These data indicated that multiple positive regulatory elements which control the quantitative expression levels of EAS4 in tobacco are contained within the -1148 to -160 region.

£ * I I· 0 / L 6 /d/dV

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- 57 Comparison of the results obtained with deletion to -567 and to -212 also indicated that the sequence within

-567 to -212 contributed to the levels of expression, because deletion from -567 to -212 resulted in a 4-fold decrease in expression levels with a loss of large amounts of GUS activity. Although a deletion to -160 largely removed inducible GUS activity, the sequence downstream of -160 was found to be sufficient to direct io inducible gene expression in leaf tissue. As shown in Fig. 9B, a 17-fold induction was observed in leaf tissues treated with elicitor. These data demonstrated that a qualitative element controlling the elicitor inducibility is located between -160 to -115.

Example 15. Disease-Resistant Transgenic Plants

The parAl coding sequence was isolated from

Phytophthora parasitica race 0 as follows: Genomic DNA was isolated and used as template in PCR with a 5IG forward primer f CGTTGGATCCCCACCTCATCCGAAATGAAC: SEQ ID

NO:9; BamHI site underlined); nucleotides 25-27 correspond to the translation start site and reverse primer (GGCTGAGCTCCTGGACGFCAGAGATCAAACC; SEQ ID NO: 10; Sstl site underlined) to amplify the coding region of the ParAl elicitin including the signal peptide coding sequence. To isolate an amplimer corresponding to the coding sequence of the ParAl elicitin, the MAT forward primer f GCCGGATCCTTATGACTAGTTGCACCACCACGCAGCAAACTG. SEQ ID NO:11; BamHI site, GGATCC, and Spel site, ACTAGT, underlined; translation start site at nucleotides 12-14) and the reverse primer as before (SEQ ID NO: 10) were used with genomic DNA as template.

For subcloning into pBluescript (Stratagene, La Jolla, CA) or into pEAS4 constructs, the amplimer DNA was digested with BamHI and Sstl. Where the mature protein's

AP/P/ 9 7/01143

APO 00 92 4

- 58 coding sequence is used, the mature elicitin/pEAS4 construct can be digested with Spel to insert a plant signal sequence at the 5' end of the open reading frame. The pEAS4—GUS vector is digested with BamHI and Sstl, with the large fragment of DNA being purified after agarose gel electrophoresis.

Figure 9 illustrates the molecular manipulations leading to the generation of disease-resistant plants.

The coding sequence for the ParAl elicitin is isolated by io PCR so as to have BamHI and Sstl ends. ~ gEAS4₆₀₀j_cycla8ej-GUS-pBI101, which directs the expression of the GUS reporter gene under the regulatory control of the EAS promoter, is digested with BamHI and Sstl to release the GUS reporter gene. Then the

BamHI/Sstl-digested parAl amplimer is ligated to the large fragment produced after digestion of gEAS4_600(cycla8e)-GUS-pBHOl to produce gEAS4_600(cycla8e)-parAl-pBI101, from which the ParAl elicitin is synthesized following induction with a suitable elicitor once plant cells or tissue have been transformed.

To assess resistance to plant pathogens, transgenic tobacco plants (Nicotiana tabacua cv. KY160) containing either the parAl mature elicitin gene (amino acids 21-118 of SEQ ID NO: 12) or parAl elicitin-withsignal sequence gene (amino acids 1-118 of SEQ ID NO: 12) under the control of the gEAS4_600(cyclaaej promoter were regenerated using the Agrobacterium-mediated gene transfer technique as described above. The resulting transgenic plants were then tested for disease resistance against either race 0 or race l isolates of Phythopthora parasitica var. Nicotianae using the standard detached leaf assay described by Tedford et al. (suura).

As shown in Fig. 10, several independent lines of transgenic tobacco containing the parAl mature elicitin

AP/P/ 9 7/01143

AP000924

- 59 gene under the control of the gEAS4_600(cyclaBe) promoter showed enhanced disease resistance to both race 0 and race 1 of P. p. var. Nicotianae relative to control plants (that is, relative to non-transformed N. tabacua cv. KY160 or transgenic N. tabacua cv. KY160 containing the EAS4_600(cyclaee) promoter:GUS reporter gene).

While various embodiments of the present invention have been described in detail, it is apparent that 'modifications, extensions, adaptations and optimizations io may occur to those skilled in the art. It is to-be expressly understood that such modifications and adaptations and so on are within the spirit and scope of the present invention, as set forth in the claims.

AP/P/ 9 7/01143

ΑΡ υΟ Ο 9 2 4

- 60 SEQUENCE LISTING (1) GENERAL INFORMATIONχ (i) APPLICANT: Board of Trustees of the University of Kentucky (ii) TITLE OF INVENTION: Transcriptional Control Sequences and Methods (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson, P.C.

(B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: US (F) ZIP: 02110-2804 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NDMBER: US 08/577,483 (B) FILING DATE: 22 December 1995 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NDMBER: US 08/471,983 (B) FILING DATE: 06 June 1995 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NDMBER: US 08/443,639 (B) FILING DATE: 18 May 1995 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul T. Clark (B) REGISTRATION NUMBER: 32,164 (C) REFERENCE/DOCKET NUMBER: 07678/003BR1 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070 (B) TELEFAX: (617) 542-8906 (C) TELEX: 200154

AP/P/ 9 7/01 143

ΑΡ ϋ ϋ ϋ 9 2 4 - 61 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 512 base pairs (Β) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CCGCGATTGG	AGGATGTTGT	ACGTCGAGCT	ACGCGGCACC	GCGCTTAATT	TTACTCGGTC	60
AAGAAGGAAC	GGGGATGGTG	GTCAACGAAA	CACGACGGGC	CCGACATCAT	GCCTGACAAC	120
CCGCCGTGGG	TGAAGAAGTC	GACGTTGGAA	AAGAGCTACA	GCCTGCTCCA	CGCGGATGCG	180
GGGATGGCCG	CTGACTACAG	AAAGTGCGTT	TCCCGCCACC	CGGGGCGAGC	CCGGGTTTTG	240
AAGATCAATG	CTGACCGAAC	CAGACGGCGG	TACGTCATCC	GCTTGAGGGT	AGAGACGGAT	300
CAGTTCTTGT	TGTCGTGTGT	CGAACTCGGG	ACGTTTGTCA	CATGGCTGGA	CGGGTTATTC	360
GCCGCCATCA	ACGTGTCGCC	GCCAATCGAC	GAGCGCGACT	TTCCCAGAGA	CTTTAGCGTG	420
CCACGGATCA	ATTACATTAA	CTAGTCTCTC	ACCACTATAT	ATACTTGTCC	CTTCTCTTCC	480
ATTTAAGTAG	AGTTCCTTTC	TTTCTTCCTT	AA			512

AP/P/ 9 7/01 143 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 642 base pairs (B) TYPE: nucleic acid <C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TAGGTGAATG	TCAGGGCTTA	TGCTCCACGA	TACTTATGCC	CTGCCAGTAC	ACCTCGCAGT	60
GGGACTCGCT	GAAAAAACGT	CTTTGTTGTG	AGAAATTGCA	ATTTTGAACC	TCTACAATTT	120
CGACAAAACC	TTGGTTCGTG	AAAACTGTTT	GATTAACTTT	TAGACCATCC	AGTCAATTTA	180

APO 00 9 2 4

- 62 ACTCTAAACT GACCTAAATA AATACTACGT ACACTAGTCT TTAAGTTCAT CAAAGTGGAC 240 TCTGCATTAA TAATTGAAAT TTATGCCGCA ACAATGACAT TAGGTTTTAT AAATAAAGTA 300 ATAGGAATTT GATAGTTCCA GGAAACAACT CTACAGTACT CCCTTATTTT GTGCCTTTTT 360 AAATAATATT ATTCAGTTGA CGAAACAAAT AAATAAAATA TTTGGGAAAC TGGATCAATA 420 GACCCCAGAC GCCAACAATG AATCAAAAGG CTGCTAGCTA GTGTAAAGTC TAGTAAGGCA 480 ACTGGGAAAT TAAATGATTA GGTGCTTTTG ATCAATTACA TTAACTAGTC TCTCACCACT 540 ATATATACTT GTCCCTTCTC TTCCATTTAA GTAGAGTTCC TTTCTTTCTT CCTTAAAACT 600 TAAAAGAACA AGTAAAAATA CACTCATCTT TAATTAGCAA TG 642 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc Oligonucleotide primer for

PCR (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATGCTGTTAG CAACCGGAAG G (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc « Oligonucleotide primer for

PCR (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ATCCAAAATC TCATCAATTT C

AP/P/ 9 7/01143

APUUU924

- 63 (2) INFORMATION FOR SEQ ID NOi5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (Β) ΤΏΈ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc « Oligonucleotide primer for

PCR.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATGAGTCCTT ACATGTGA 18 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc “ Oligonucleotide primer for

PCR.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGGAGCTCGA ATTCCATGGC CTCAGCAGCA GCAGTTGCAA ACTAT 45

AP/P/ 9 7/01143 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4253 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

ΑΡ θ ο ο 9 2 4

- 64 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1216..1327 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1454..1718 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1805..2182 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 2259..2477 (ix) FEATURE:

(A) NAME/KEY: CDS ' ' (B) LOCATION: 2609..2747 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 2902..3148 (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 3261..3555 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AAGCTTTACG AATTAGATGT AAAAAGACGC AAACTACTTA TATATATTAC CAAAGTAACT 60

TGAAAGTTTA AAATTTCAAT TAGAACTATA GTAGGGTAAA ACTGTCTATT TAAAATCAGT 120

ATTTAAAAAG GCATGAGCGA AAGATGAGGC GTTTTATCTA ACACGAAGCG AGGTGTAAGC 180

CCCATGOTGT TTTATTTTTA TATTTTATAA ATTTATAAAA TCATTATATA AATCAGAftAA 240

ATACACTAAA ATTGTGAAAA GTTAAAGAAA ATTATAGAAT TAATATATAT ATATATATAT 300

ATATATATAT ATATATATAT ATATATATAT ATATAXATAA ATGTATGTGT GTGTGTGTGT 360

GTATCGGATG CGCGCGACCA TGCAACTTTT TTTTCTTGAA AAAATAAAAG GCGTAAAGAT 420

ACAXTATACC TATGTCATCA AAACAATATA AAAACTAGAG CGATACCAAA GGAAATTTTA 480

AATTCAAAAA CTAACTTGAA ATTAATATAT TTAAAATTTC ATTTTTTTTT GTGTGGAGAA 540

AACAAAGCAT AACACTTTGC TTTGTAACAC TTTGCCTAGG TGAATGTCAG GGCTTATGCT 600

CCACGATACT TATGCCCTGC CAGTACACCT CGCAGTGGGA CTCGCTGAAA AAACGTCTTT 660

GTTGTGAGAA ATTGCAATTT TGAACCTCTA CAATTTCGAC AAAACCTTGG TTCGTGAAAA 720

CTCTTTGATT AACTTTTAGA CCATCCAGTC AATTTAACTC TAAACTGACC TAAATAAATA 780

CTACGTACAC TAGTCTTTAA GTTCATCAAA GTGGACTCTG CATTAATAAT TGAAATTTAT 840

GCCGCAACAA TGACATTAGG TTTTATAAAT AAAGTAATAG GAATTTGATA GTTCCAGGAA 900 acaactctac AGTACTCCCT TATTTTGTGC CTTTTTAAAT AATATTATTC AGTTGACGAA 960

ACftAATAAAT AAAATATTTG GGAAACTGGA TCAATAGACC CCAGACGCCA ACAATGAATC 1020

AP/P/ 9 7/01 143

APO 0 0 9 2 4

- 65 AAAAGGCTGC TAGCTAGTGT AAAGTCTAGT AAGGCAACTG GGAAATTAAA TGATTAGGTG 1080

CTTTTGATCA ATTACATTAA CTAGTCTCTC ACCACTATAT ATACTTGTCC CTTCTCTTCC 1140

ATTTAAGTAG AGTTCCTTTC TTTCTTCCTT AAAACTTAAA AGAACAAGTA AAAATACACT 1200

CATCTTTAAT TAGCA ATG* GCC TCA GCA GCA GTT GCA AAC TAT GAA GAA GAG 1251

Met Ala Ser Ala Ala Val Ala Asn Tyr Glu Glu Glu

1 5	10
ATT GTT CGC CCC GTC GCC GAC TTC TCC CCT AGT lie Val Arg Pro Val Ala Asp Phe Ser Pro Ser 15 20	CTC TGG GGT GAT CAG Leu Trp Gly Asp Gin 25	1299
TTC CTT TCA TTC TCC ATT GAT AAT CAG G TAATTTAACT AATACTAGTA Phe Leu Ser Phe Ser lie Asp Asn Gin 30 35	1347
TTCTTTATTT ATATTTATAG TTTGTTCTCC ATTGATAATC	AGGTAGTTTA TTTATGTTGA	1407
ACAACATTAA TTTTGCTAAT TTCAGTTTAA TGTACATTAC	ATATAG GTT GCG GAA Val Ala Glu 1	1462
AAG TAT ATA TAT GCT CAA GAG ATT GAA GCA TTG Lys Tyr He Tyr Ala Gin Glu Xie Glu Ala Leu 5 10	AAG GAA CAA ACG AGG Lys Glu Gin Thr Arg 15	1510
AGT ATG CTG TTA GCA ACC GGA AGG AAA TTG GCC Ser Met Leu Leu Ala Thr Gly Arg Lys Leu Ala 20 25 30	GAT ACA TTG AAT TTG Asp Thr Leu Asn Leu 35	1558
ATT GAC ATT ATT GAA CGC CTT GGT ATA TCC TAC Xie Asp lie lie Glu Arg Leu Gly Xie Ser Tyr 40 45	CAC TTT GAG AAA GAA His Phe Glu Lys Glu 50	1606
ATT GAT GAG ATT TTG GAT CAG ATT TAC AAC CAA Xie Asp Glu lie Leu Asp Gin Xie Tyr Asn Gin 55 60	AAC TCA AAC TGC AAT Asn Ser Asn Cys Asn 65	1654
GAT TTG TGC ACC TCT GCA CTT CAA TTT CGA TTG Asp Leu Cys Thr Ser Ala Leu Gin Phe Arg Leu 70 75	CTC AGG CAA CAC GGT Leu Arg Gin His Gly 80	1702
TTC AAC ATC TCT CCT G GTAAGTTCAT CATGAAGTTG Phe Asn Xie Ser Pro 85	TTAAAATTAT	1748
TATCCATTTA TTGGAAGAAG GCTAATTCAT CTTGAGTTTT	CTTTCTTGAA ATACCA	1804
GAA ATT TTC AGC AAA TTC CAA GAT GAA AAT GGC Glu Xie Phe Ser Lys Phe Gin Asp Glu Asn Gly 15 10	AAA TTC AAG GAG TCT Lye Phe Lys Glu Ser 15	1852
CTT GCT AGT GAT GTC TTA GGA TTA TTA AAC TTG Leu Ala Ser Asp Val Leu Gly Leu Leu Asn Leu 20 25	TAT GAA GCT TCA CAT Tyr Glu Ala Ser His 30	1900
GTA AGG ACT CAT GCT GAC GAT ATC TTA GAA GAC Val Arg Thr His Ala Asp Asp Xie Leu Glu Asp	GCA CTT GCT TTC TCC Ala Leu Ala Phe Ser	1948

40 45

AP/P/ 9 7/01143

ΑΡΟ ϋΟ 924

- 66 ACT ATC CAT CTT GAA TCT GCA GCT CCA CAT TTG AAA TCT CCA CTT AGG 1996

Thr lie Hie Leu Glu Ser Ala Ala Pro His Leu Lys Ser Pro Leu Arg

55 60

GAG CAA GTG ACA CAT GCC CTT GAG CAA TGT TTG CAC AAG GGT GTT CCT 2044

Glu Gin Val Thr His Ala Leu Glu Gin Cys Leu His Lys Gly Val Pro

70 75 80

AGA GTC GAG ACC CGA TTC TTC ATC TCA TCA ATC TAT GAC AAG GAA CAA 2092

Arg Val Glu Thr Arg Phe Phe He Ser Ser lie Tyr Asp Lys Glu Gin

90 95

TCG AAG AAT AAT GTG TTA CTT CGA TTT GCC AAA TTG GAT TTC AAC TTG 2140

Ser Lys Asn Asn Val Leu Leu Arg Phe Ala Lys Leu Asp Phe Asn Leu

100 105 110

CTC CAG ATG TTG CAC AAA CAA GAA CTT GCT CAA GTA TCA AGG 2182

Leu Gin Met Leu His Lys Gin Glu Leu Ala Gin Val Ser Arg ~ . 115 120 125

TGCGATATAT AAAAACGATG AACCCTTTTT GATTCATCAT ATCTCAAGTA CTCATGTTAA 2242

TTTCTTATGC TGCAGG TGG TGG AAA GAT TTG GAT TTT GTA ACA ACA CTT 2291

Trp Trp Lys Asp Leu Asp Phe Val Thr Thr Leu

10

CCA TAT GCT AGA GAT CGA GTA GTT GAA TGC TAC TTT TGG GCA TTA GGA 2339

Pro Tyr Ala Arg Asp Arg Val Val Glu Cys Tyr Phe Trp Ala Leu Gly

20 25

GTT TAT TTT GAG CCT CAA TAC TCT CAA GCT CGC GTC ATG CTC GTT AAG 2387

Val Tyr Phe Glu Pro Gin Tyr Ser Gin Ala Arg Val Met Leu Val Lys

35 40

ACC ATA TCA ATG ATT TCG ATT GTC GAT GAC ACC TTT GAT GCT TAC GGT 2435

Thr He Ser Met He Ser He Val Asp Asp Thr Phe Asp Ala Tyr Gly

50 55

ACA GTT AAA GAA CTT GAG GCA TAC ACA GAT GCC ATA CAA AGG 2477

Thr Val Lye Glu Leu Glu Ala Tyr Thr Asp Ala He Gin Arg

65 70

TATGAACTTC ATCAATTCAC TTATTCCTTG ATAGTGAATG TCGTCGTGAA AAGATTAAGA 2537

CGAATTTCTA CTCATTATAG TTGTGCTCTT TCAAAATGCA TGAATTCACC TTAATTTTGT 2597

CATCCTGCAG A TGG GAT ATC AAC GAA ATT GAT CGG CTT CCT GAT TAC ATG 2647

Trp Asp He Asn Glu He Asp Arg Leu Pro Asp Tyr Met

10

AAA ATC AGT TAT AAA GCT ATT CTA GAT CTC TAC AAG GAT TAT GAA AAG 2695

Lys He Ser Tyr Lys Ala lie Leu Asp Leu Tyr Lys Asp Tyr Glu Lys

20 25

GAA TTG TCT AGT GCC GGA AGA TCT CAT ATT GTC TGC CAT GCA ATA GAA 2743

Glu Leu Ser Ser Ala Gly Arg Ser His lie Val Cys His Ala lie Glu

35 40 45

AGA G TATGTTCGAG CAACTTAACT ATCGAAATAC ATTTTTTCCT TAATCCATTT 2797

Arg

CTCACTTTGG TTTACCTTGT GTTCGTCTTT TAGTGATTAG AAACTTGATA CAGTTCAATC 2857

AP/P/ 9 7/01143

ΑΡϋθϋ924

- 67 AATATTTTCT AACACTTGAA CACATATATG TTTTGTATTC ACAG ATG AAA GAA era 2913 Met Lys Glu Val

GTA AGA AAT TAT AAT GTC GAG TCA ACA TGG TTT ATT GAA GGA TAT ATG 2961

Val Arg Asn Tyr Asn Val Glu Ser Thr Trp Phe Xie Glu Gly Tyr Met

10 15 20

CCA CCT GTT TCT GAA TAC CTA AGC AAT GCA CTA GCA ACT ACC ACA TAT 3009

Pro Pro Val Ser Glu Tyr Leu Ser Asn Ala Leu Ala Thr Thr Thr Tyr

30 35

TAC TAC CTC GCG ACA ACA TCG TAT TTG GGC ATG AAG TCT GCC ACG GAG 3057

Tyr Tyr Leu Ala Thr Thr Ser Tyr Leu Gly Met Lys Ser Ala Thr Glu

45 50

CAA GAT TTT GAG TGG TTG TCA AAG AAT CCA AAA ATT CTT GAA GCT AGT 3105

Gin Asp Phe Glu Trp Leu Ser Lys Αβη Pro Lys lie Leu Glu Ala ser

60 65

GTA ATT ATA TGT CGA GTT ATC GAT GAC ACA GCC ACG TAC GAG G 3148

Val Xie lie Cys Arg Val lie Asp Aep Thr Ala Thr Tyr Glu

75 80

TATGATTTGC ATCTCAAGAA ATTATATCAT TATATGGGAT TTGGACAAAC AAAGTGTTGC 3208

GACGACAATT AAGGCAATAT AAAAGCTAAC CTTTAATTTA TCTGCTTTCT AG GTT 3263

Val

GAG AAA AGC AGG GGA CAA ATT GCA ACT GGA ATT GAG TGC TGC ATG AGA 3311

Glu Lys Ser Arg Gly Gin Xie Ala Thr Gly lie Glu Cys Cys Met Arg

10 15

GAT TAT GGT ATA TCA ACA AAA GAG GCA ATG GCT AAA TTT CAA AAT ATG 3359

Asp Tyr Gly Xie Ser Thr Lye Glu Ala Met Ala Lys Phe Gin Asn Met

25 30

GCT GAG ACA GCA TGG AAA GAT ATT AAT GAA GGA CTT CTT AGG CCC ACT 3407

Ala Glu Thr Ala Trp Lys Asp Xie Asn Glu Gly Leu Leu Arg Pro Thr

40 45

CCC GTC TCT ACA GAA TTT TTA ACT CCT ATT CTC AAT CTT GCT CGT ATT 3455

Pro Val Ser Thr Glu Phe Leu Thr Pro Xie ILeu Asn Leu Ala Arg Xie

55 60 65

GTT GAG GTT ACA TAT ATA CAC AAT CTA GAT GGA TAC ACT CAT CCG GAG 3503

Val Glu Val Thr Tyr Xie His Asn X^u Asp Gly Tyr Thr His Pro Glu

75 80

AAA GTC TTA AAA CCT CAC ATT ATT AAC CTA CTT GTG GAC TCC ATC AAA 3551

Lys Val Leu Lye Pro His Xie Xie Asn Leu Leu Val Asp Ser lie Lys

90 95

ATT T GAGCTGCCAT TTGTTGCTCA TCTCAAGGAA ACTTCATTCT TCTTTGTGCA 3605

Xie

GTTGTGCAGT AGACTTCCTA ACTAGGAGCT TCTTAAGATC CTTGTAAGAA ATAATCTTCA 3665

AGTGTTATGA ATCCGCATTG TGGAGAAATC TTTTTATATG ACAATAAGTT ATGTTATGAA 3725

GAATGTTATG GGGGTCTCTT ATGACCTATT TGTCAGTGTA TGAAGTAATC TGAGCCTGTC 3785

AP/P/ 9 7/01143

AP O 0 0 9 2 4

GAAAAAAAAG	GTAATCTGAG	CCTTTTGCTC	GTCCTTCCTT	TAGTATTTCT	TTTTATCATA	3845
CTTGGTCTCA	CAAAAATTAG	TTTTTGGCAC	CTTTGTTTTT	CCTTGTGGCG	CATGTGTATA	3905
TACATCTGAA	ACATATACTT	AAAGGTTAAG	AGGACATTGA	CATATTGAAT	CAACACTAGT	3965
GTTATTGGCA	TACAGGAGAG	AATCTATGTG	TAAAGGACGG	GGTGGAACCC	CACCCACAAG	4025
ACTTGGTCGA	GACTATTGTT	TATCGAAAAA	ACGGTACAGT	TGAATTTATA	CGTGGTTTAT	4085
AGACAAGTGA	ATTAATTTGA	TCCTAAAATA	ATAGGCGAAT	TAGATAAAAA	TGTAATTCTT	4145
AGCCTTGAGT	TGGAGACGAA	ATAGCAGAAA	TAGTGATTCC	AGGAGAAAGG	CTTTCGGGTA	4205
CCACAACAAT	GATATCAAAA	AATAAAAAGA	TAAAATTGTA	TTAAGCTT		4253

(2) INFORMATION FOR SEQ ID NO:8:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 550 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ro *r (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met 1

Ala

Ser

Ala

Ala 5

Val

Ala

Asn

Tyr

Glu 10

Glu

Glu

lie

Val

Arg 15

Pro

Val

Ala

Asp

Phe 20

Ser

Pro

Ser

Leu

Trp 25

Gly Asp

Gin

Phe

Leu 30

Ser

Phe

Ser

lie

Asp 35

Asn

Gin

Val

Ala

Glu 40

Lys

Tyr

lie

Tyr

Ala 45

Gin

Glu

lie

Glu

Ala 50

Leu

Lys

Glu

Gin

Thr 55

Arg

Ser

Met

Leu

Leu 60

Ala

Thr

Gly

Arg

Lys 65

Leu

Ala

Asp

Thr

Leu 70

Asn

Leu

He

Asp

lie 75

lie

Glu

Arg

Leu

Gly 80

lie

Ser

Tyr

His

Phe 85

Glu

Lys

Glu

lie

Asp 90

Glu

He

Leu

Asp

Gin 95

lie

Tyr

Asn

Gin

Asn 100

Ser

Asn

Cys

Asn

Asp 105

Leu

Cys

Thr

Ser

Ala 110

Leu

Gin

Phe

Arg

Leu 115

Leu

Arg

Gin

His

Gly 120

Phe

Asn

He

Ser

Pro 125

Glu

He

Phe

Ser

Lys 130

Phe

Gin

Asp

Glu

Asn 135

Gly

Lye

Phe

Lys

Glu 140

Ser

Leu

Ala

Ser

Asp 145

Val

Leu

Gly

Leu

Leu 150

Asn

Leu

Tyr

Glu

Ala 155

Ser

Hie

Val

Arg

Thr 160

Bis

Ala

Asp

Asp

lie 165

Leu

Glu

Asp

Ala

Leu 170

Ala

Phe

Ser

Thr

lie 175

His

cn c£ £

Ap v 0 0 9 2 4

Leu Glu

Ser

Ala 180

Ala

Pro

His

Leu

Lys 185

Ser

Pro

Leu

Arg

Glu 190

Gin

Val

Thr His

Ala 195

Leu

Glu

Gin

Cye

Leu 200

His

Lys

Gly

Val

Pro 205

Arg

Val

Glu

Thr Arg 210

Phe

Phe

He

Ser

Ser 215

He

Tyr

Asp

Lys

Glu 220

Gin

Ser

Lys

Asn

Asn Val 225

Leu

Leu

Arg

Phe 230

Ala

Lys

Leu

Asp

Phe 235

Asn

Leu

Leu

Gin

Met 240

Leu His

Lys

Gin

Glu 245

Leu

Ala

Gin

Val

Ser 250

Arg

Trp

Trp

Lys

Asp 255

Leu

Asp Phe

Val

Thr 260

Thr

Leu

Pro

Tyr

Ala 265

Arg

Asp

Arg

Val

Val 270

Glu

Cys

Tyr Phe

Trp 275

Ala

Leu

Gly

Val

Tyr 280

Phe

Glu

Pro

Gin

Tyr 285

Ser

Gin

Ala

Arg Val 290

Met

Leu

Val

Lys

Thr 295

lie

Ser

Met

lie

Ser 300

He

Val

Asp

Asp

Thr Phe 305

Asp

Ala

Tyr

Gly 310

Thr

Val

Lys

Glu

Leu 315

Glu

Ala

Tyr

Thr

Asp 320

Ala lie

Gin

Arg

Trp 325

Asp

lie

Asn

Glu

He 330

Asp

Arg

Leu

Pro

Asp 335

Tyr

Met Lys

lie

Ser 340

Tyr

Lys

Ala

lie

Leu 345

Asp

Leu

Tyr

Lys

Asp 350

Tyr

Glu

Lys Glu

Leu 355

Ser

Ser

Ala

Gly

Arg 360

Ser

His

He

Val

Cys 365

His

Ala

He

Glu Arg 370

Met

Lys

Glu

Val

Val 375

Arg

Asn

Tyr

Aen

Val 380

Glu

Ser

Thr

Trp

Phe lie 385

Glu

Gly

Tyr

Met 390

Pro

Pro

Val

Ser

Glu 395

Tyr

Leu

Ser

Asn

Ala 400

Leu Ala

Thr

Thr

Thr 405

Tyr

Tyr

Tyr

Leu

Ala 410

Thr

Thr

Ser

Tyr

Leu 415

Gly

Met Lys

Ser

Ala 420

Thr

Glu

Gin

Asp

Phe 425

Glu

Trp

Leu

Ser

Lys 430

Asn

Pro

Lys lie

Leu 435

Glu

Ala

Ser

Val

lie 440

lie

Cys

Arg

Val

He 445

Asp

Asp

Thr

Ala Thr 450

Tyr

Glu

Val

Glu

Lys 455

Ser

Arg

Gly

Gin

He 460

Ala

Thr

Gly

He

Glu Cys 465

Cys

Met

Arg

Asp 470

Tyr

Gly

lie

Ser

Thr 475

Lye

Glu

Ala

Met

Ala 480

Lys Phe

Gin

Asn

Met 485

Ala

Glu

Thr

Ala

Trp 490

Lys

Asp

lie

Asn

Glu 495

Gly

AP/P/ 9 7/01 143

Leu Leu Arg Fro Thr Pro Val Ser Thr Glu Phe Leu Thr Pro He Leu

500 505 510

APVυυ 9 2 4

- 70 Asn Leu Ala Arg lie Val Glu Val Thr Tyr lie Hie Asn Leu Asp Gly 515 520 525

Tyr Thr His Pro Glu Lys Val Leu Lys Pro His lie lie Asn Leu Leu 530 535 540

Val Asp Ser lie Lys lie

545 550 (2) INFORMATION TOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = Oligonucleotide primer for

PCR.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CGTTGGATCC CCACCTCATC CGAAATGAAC 30 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc « Oligonucleotide primer for

PCR.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GGCTGAGCTC CTGGACGCAG AGATCAAACC 30

AP/P/ 9 7/01143 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: eingle (D) TOPOLOGY: linear

ΑΡ ο ϋ 0 9 2 4

- 71 (11) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: /desc = Oligonucleotide primer for

PCR (reverse).

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GCCGGATCCT TATGACTAGT TGCACCACCA CGCAGCAAAC TG 42 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 593 base pairs ' (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Phytophthora parasitica (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 207..563 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GTCGACGAAA GCCGAAGTGC GTGGCAGATC TTGCCGTTCG AATGCTACGC GCCACGGCAA 60

AACCTACACG GTACAACAGC TTCAAATAAA CCTGCAAGCG AGCCGCCAGC CCAACTCCAG 120

CTAGTCAAGC CTAGTTTGCC TCCAACTGCC ATTGTGCAAT TTGCTCTCAT CCACACCCAC 180

AP/P/ 9 7/01 143

CCCACTTCTC CCCCACCTCA TCCGAA ATG AAC TTC CGC GCT CTG TTC GCC GCC 233

Met Asn Phe Arg Ala Leu Phe Ala Ala

555 560

ACC GTC GCC GCC CTC GTC GGC TCC ACC TCC GCC ACC ACG TGC ACC ACC

281

Thr Val Ala Ala Leu Val Gly Ser Thr Ser

Ala

Thr

Thr

Cys

Thr 575

Thr

565

570

ACG

CAG

CAA

ACT

GCG

GCG

TAC

GTG

GCG

CTC

GTA

AGC

ATC

CTC

TCG

GAC

329

Thr

Gin

Gin

Thr

Ala

Ala

Tyr

Val

Ala

Leu

Val

Ser

lie

Leu

Ser

Asp

580

585

590

ACG

TCG

TTC

AAC

CAG

TGC

TCG

ACG

GAC

TCT

GGC

TAC

TCA

ATG

CTG

ACG

377

Thr

Ser

Phe

Asn

Gin

Cys

Ser

Thr

Asp

Ser

Gly

Tyr

Ser

Met

Leu

Thr

595

600

605

GCC

ACC

TCG

TTG

CCC

ACG

ACG

GAG

CAG

TAC

AAG

CTC

ATG

TGC

GCG

TCG

425

Ala

Thr

Ser

Leu

Pro

Thr

Thr

Glu

Gin

Tyr

Lys

Leu

Met

Cvb

Ala

Ser

610

515

620

AP0 00 92 4

ACC

GCG

TGC

AAG

ACG

ATG

ATC

AAC

AAG

ATC

GTG

ACG

CTG

AAC

CCG

CCC

473

Thr

Ala

Cys

Lys

Thr

Met

lie

Asn

Lys

He

Val

Thr

Leu

Asn

Pro

Pro

625

630

635

640

GAC

TGC

GAG

TTG

ACG

GTG

CCT

ACG

AGC

GGC

CTG

GTA

CTC

AAC

GTG

TTC

521

Asp

Cys

Glu

Leu

Thr

Val

Pro

Thr

Ser

Gly

Leu

Val

Leu

Asn

Val

Phe

645

650

655

ACG

TAC

GCG

AAC

GGG

TTC

TCG

TCT

ACG

TGC

GCG

TCA

CTG

TAA

563

Thr

Tyr

Ala

Asn

Gly

Phe

Ser

Ser

Thr

Cys

Ala

Ser

Leu

660

665

GCGGGTTTGA TCTCTGCGTC CAGAATCGAT 593 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TIPS: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

AP/P/ 9 7/01 143 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Met 1

Asn

Phe

Arg

Ala 5

Leu

Phe

Ala

Ala

Thr 10

Val

Ala

Ala

Leu

Val 15

Gly

Ser

Thr

Ser

Ala 20

Thr

Thr

Cys

Thr

Thr 25

Thr

Gin

Gin

Thr

Ala 30

Ala

Tyr

Val

Ala

Leu 35

Val

Ser

lie

Leu

Ser 40

Asp

Thr

Ser

Phe

Asn 45

Gin

Cys

Ser

Thr

Asp 50

Ser

Gly

Tyr

Ser

Met 55

Leu

Thr

Ala

Thr

Ser 60

Leu

Pro

Thr

Thr

Glu 65

Gin

Tyr

Lys

Leu

Met 70

Cys

Ala

Ser

Thr

Ala 75

Cys

Lys

Thr

Met

He 80

Asn

Lys

lie

Val

Thr 85

Leu

Asn

Pro

Pro

Asp 90

Cys

Glu

Leu

Thr

Val 95

Pro

Thr

Ser

Gly

Leu 100

Val

Leu

Asn

Val

Phe 105

Thr

Tyr

Ala

Asn

Gly 110

Phe

Ser

Ser

Thr

Cys

Ala

Ser

Leu

115 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1368 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: CDNA

APO00924

- 73 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

AAGCTTTACG AATTAGATGT AAAAAGACAC AAACTACTTA TATATATTAC CAAAGTAACT 60

TGAAAGTTTA AAATTTCAAT TAGAACTATA GTAGGGTAAA ACTGTCTATT TAAAATCAGT 120 ATTTAAAAAG GCATGAGCGA AAGATGAGGC GTTTTATCTA ACACGAAGCG AGGTGTAAGC 180 CCCATGGTGT TTTATTTTTA TATTTTATAA ATTTATAAAA TCATTATATA AATCAGAAAA 240

ATACACTAAA ATTGTGAAAA GTTAAAGAAA ATTATAGAAT TAATATATAT ATATATATAT 300

ATATATATAT ATATATATAT ATATATATAT ATATATATAA ATGTATGTGT GTGTGTGTGT 360 GTATCGCATG CGCGCGACCA TGCAACTTTT TTTTCTTGAA AAAATAAAAG GCGTAAAGAT 420

ACATTATACC TATGTCATCA AAACAATATA ATATATATAT ATATATATAT ATATATATAT 480

ATATATATAA ATGTATGTGT GTGTGTGTGT GTATCGCATG CGCGCGACCA TGCAACTTTT 540

TTTTCTTGAA AAAATAAAAG GCGTAAAGAT ACATTATACC TATGTCATCA AAACAATATA 600

AAAACTAGAG CGATACCAAA GGAAATTTTA AATTCAAAAA CTAACTTGAA ATTAATATAT 660

TTAAAATTTC ATTTTTTTTT GTGTGGAGAA AACAAAGCAT AACACTTTGC TTTGTAACAC 720

TTTGCCTAGG TGAATGTCAG GGCTTATGCT CCACGATACT TATGCCCTGC CAGTACACCT 780

CGCAGTGGGA CTCGCTGAAA AAACGTCTTT GTTGTGAGAA ATTGCAATTT TGAACCTCTA 840

CAATTTCGAC AAAACCTTGG TTCGTGAAAA CTGTTTGATT AACTTTTAGA CCATCCAGTC 900

AATTTAACTC TAAACTGACC TAAATAAATA CTACGTACAC TAGTCTTTAA GTTCATCAAA 960

GTGGACTCTG CATTAATAAT TGAAATTTAT GCCGCAACAA TGACATTAGG TTTTATAAAT 1020

AAAGTAATAG GAATTTGATA GTTCCAGGAA ACAACTCTAC AGTACTCCCT TATTTTGTGC 1080

CTTTTTAAAT AATATTATTC AGTTGACGAA ACAAATAAAT AAAATATTTG GGAAACTGGA 1140

TCAATAGACC CCAGACGCCA ACAATGAATC AAAAGGCTGC TAGCTAGTGT AAAGTCTAGT 1200

AAGGCAACTG GGAAATTAAA TGATTAGGTG CTTTTGATCA ATTACATTAA CTAGTCTCTC 1260

ACCACTATAT ATACTTGTCC CTTCTCTTCC ATTTAAGTAG AGTTCCTTTC TTTCTTCCTT 1320

AP/?/ 9 7/01143

AAAACTTAAA AGAACAAGTA AAAATACACT CATCTTTAAT TAGCAATG

Claims (51)

1. A method of using a DNA molecule comprising a nucleotide sequence encoding an elicitin protein operably linked to and expressed under the regulatory control of an inducible transcription regulatory element to produce a transgenic plant with improved disease resistance, said method comprising the steps of:

(a) providing a nucleotide sequence encoding a heterologous coding sequence of an elicitin protein operably linked to an inducible transcription regulatory element and a promoter functional in a plant cell to produce an inducible expression construct;

(b) cloning the inducible expression construct of step (a) into a plasmid vector adapted for use in a plant cell to produce an inducible expression vector;

(c) introducing said inducible expression vector of step (b) into a plant tissue to produce transgenic plant tissue; and (d) regenerating said transgenic plant tissue to produce a transgenic plant, whereby said elicitin is expressed in response to potential plant pathogen invasion or elicitor treatment of said transgenic plant, thereby inducing the hypersensitive response, increasing the resistance of said transgenic plant to a plant pathogen.

2. The method of claim 1, wherein said inducible transcription regulatory element comprises a nucleotide sequence as given in SEQ ED NO:2 from nucleotide 455 to nucleotide 473; a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 406 to nucleotide 473; or a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 473.

3. The method of claim 1. wherein said transgenic plant is a dicot, monocot, gymnosperm. or conifer.

4. The method of claim 3, wherein said transgenic plant is a member c-f the Solanaceae.

P/F/ 9 7/01143

5. The method of claim 4, wherein said elicitin protein is a fungal or bacterial elicitin.

6. i ne method of claim 5, wherein said fungal elicitin is from Pkytophthora.

7. The method of claim 5, wherein said elicitin protein comprises a FarAl, cryptogein, pariscein, or harpin protein.

ΑΡΟ 0 0 9 2 4

8. The method of claim 1, wherein said elicitin coding sequence has at least 70% nucleotide sequence homology to sparA 1 coding sequence as given in SEQ ID NO: 12 from nucleotide 207 to nucleotide 563 and encodes a functional elicitin protein; encodes a ParAl protein having an amino acid sequence as given in SEQ ID NO: 13; or is aperAI coding sequence as given in SEQ ID NO; 12 from nucleotide 207 to nucleotide 563.

9. A transgenic plant (a seed or a cell thereof) comprising a sequence encoding an elicitin, said coding sequence being operably linked to an inducible transcriptional regulatory element and a promoter functional in a plant, wherein said plant expresses said elicitin coding sequence in response to invasion of said plant by a potential plant pathogen or in response to treatment with an elicitor or other inducing chemical signal.

10. The transgenic plant of claim 9, wherein said inducible transcription Tegulatory element comprises a nucleotide sequence as given in SEQ ID NO;2 from nucleotide 458 to nucleotide 473; a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 406 to nucleotide 473; or a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 473.

11. The transgenic plant of claim 9, wherein said transgenic plant is a dicot. monocot, conifer, or gymnopserm.

12. The transgenic plant of claim 11, wherein said transgenic plant is a member of the Solanaceae.

13. The transgenic plant of claim 12, wherein said transgenic plant is M'ccrfcnc tabacum.

14. The transgenic plant of claim 9, wherein said elicitin is a fungal or bacterial elicitin.

15. Tne transgenic plant of claim 14, wherein said fungal elicitin is from

Phytophthora.

16. The transgenic plant of claim 14, w’herein said elicitin comprises a ParAl, cryptogein, pariscein, or harpin protein.

AP/P/ 9 7/01143 £p u ο Ο 9 2 4

17. The transgenic plant of claim 1, wherein said elicitin coding sequence has at least 70% nucleotide sequence homology to apcrAl coding sequence as given in SEQ H) NO: 12 from nucleotide 207 to nucleotide 563 and encodes a functional elicitin protein; encodes a ParAl protein having an amino acid sequence as given in SEQ ID NO: 13; or is zparA.1 coding sequence as given in SEQ ID NO: 12 from nucleotide 207 to nucleotide 563,

18. A method of producing a transgenic plant having greater resistance to disease than a corresponding parental plant, said method comprising the steps of:

(a) introducing a nucleotide sequence encoding a heterologous coding sequence cf an elicitin protein operably linked to an inducible transcription regulatory element and a promoter functional in a plant cell into a plant cell or plant tissue;

(b) regenerating a transgenic plant from the transgenic plant cell or tissue of step (a); and ' (c) growing the transgenic plant under conditions which allow the expression of said nucleotide sequence when said transgenic plant is invaded by a potential plant pathogen capable of infecting a corresponding parent plant whereby the expression of said elicitin is induced with the result that a resistance response is induced in response to an invading potential plant pathogen.

19. The method of claim 18, wherein said inducible transcription regulatory element comprises a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 458 to nucleotide 473; a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 406 to nucleotide 473; or a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 473.

20. The method of claim 18, wherein said transgenic plant is a dicot, monocot. gymnosperm, or conifer.

21. The method of claim 20, wherein said transgenic plant is a member cf the Solanaceae.

22. The method of claim 21, wherein said transgenic plant is Nicotiana tabacum.

AP/P/ 9 7/01143

23. The method of claim 18, wherein said elicitin is a fungal or bacterial elicitin

24. The method of claim 23, wherein said fungal elicitin is from Paytophtbora.

ΑΡϋ00924

25. The method of claim 23, wherein said elicitin protein comprises a ParAl, ctyptogein, pariscein, or harpin protein.

26. The method of claim 18, wherein said elicitin coding sequence has at least 70% nucleotide sequence homology to aparAl coding sequence as given in SEQ ID NO:12 from nucleotide 207 to nucleotide 563 and encodes a functional elicitin protein; encodes a ParAl protein having an amino acid sequence as given in SEQ ID NO; 13; or is zparA.1 coding sequence as given in SEQ ID NO: 12 from nucleotide .207 to nucleotide 563.

27. A transgenic plant (or a seed or cell thereof) genetically engineered to contain a chimeric gene comprising an inducible transcription regulatory element, a promoter functional in a plant and a coding sequence not naturally associated with said inducible transcription regulatory element, said coding sequence being expressed under the regulatory control of said inducible transcription regulatory element.

28. The transgenic plant of claim 27, wherein said inducible transcription regulatory element is comprises a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 458 to nucleotide 473; a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 406 to nucleotide 473; or a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 473.

1 43

29. lhe transgenic plant claim 27, wherein said promoter functional in a plant cell is the Cauliflower Mosaic Virus 35S gene minimal promoter.

30. The transgenic plant of claim 27, wherein said transgenic plant is a dicor, monocot, gymnosperm, or conifer.

21. Tne transgenic plant of claim 30, wherein said transgenic plant is a member of the Solanaceae.

σ>

32. 1 ne transgenic plant of claim 31, wherein said transgenic plant is Niccticna tabacvm.

33. A recombinant nucleic acid molecule comprising an inducible plant diseaseresistance regulatory element.

ΑΡϋΰϋ924

34. The nucleic acid molecule of claim 33, wherein said regulatory element Is obtained from a gene encoding a terpene or a sesquiterpene cyclase.

35. The nucleic acid molecule of claim 33, wherein said regulatory element directs expression of an epi-5-aristolochene synthase (EAS).

36. The nucleic acid molecule of claim 33, said nucleic acid molecule comprising the nucleotide sequence shown in Fig. 3A (SEQ ID NO:T4) dr an inducible plant disease-resistance fragment thereof; the nucleotide sequence shown in Fig. 3 A (SEQ ID NO: 14); the nucleotides 463-473 of SEQ ID NO: 2; nucleotides 406-486 of SEQ ID NO: 2; nucleotides 463-572 of SEQ ID NO: 2; the nucleotides 371-463 of SEQ ID NO: 2; or the nucleotides 411-457 of SEQ ID NO: 2.

37. The nucleic acid molecule of claim 33, wherein a heterologous codins sequence is operably linked downstream of and under the regulatory control of, said ' inducible transcription regulatory element.

•

38. The nucleic acid molecule of claim 37, wherein said heterologous coding sequence encodes an elicitin protein.

39. The nucleic acid molecule of claim 38, wherein said heterologous coding sequence encodes an elicitin of Phytophthoraparasitica.

40. The nucleic acid molecule of claim 39, wherein said elicitin of Pkylophlhora parasitica is the ParAl elicitin protein.

41. The nucleic acid molecule of claim 33, wherein said nucleic acid molecule is obtained from a dicot, monocot, gymnosperm. or conifer.

42. The nucleic acid molecule of claim 41, wherein said dicot is a member of the Solanaceae.

AP/P/ 9 7/01143

43. The nucleic acid molecule of claim 42, wherein said Solanaceous plant is a member of the genus Nicotiana.

44. A vector comprising the DNA of claim 33.

45. The vector of claim 44, said vector being capable of inducibly regulating the expression of a nucleotide sequence in a vector-containing cell.

AP Ο Ο Ο 9 2 4

46. Tr.e vector of claim 45, said nucleotide sequence coding for a heterologons polypeptide.

47. The vector of claim 46, wherein said heterologous peptide is. a pharmaceutical protein.

. 4S. A transgenic plant (or a seed or cell thereof) that contains a nucleic acid molecule of claim 33 integrated into the genome of said plant.

49. A non-natumlly occurring DNA. molecule comprising a transcription-enhancing element having a nucleotide sequence as given in-SEQJD NO:2 from nucleotide 371 to nucleotide 463.

50. A non-narurally occurring DN'A molecule comprising a transcription-enhancing sequence hawing a nucleotide sequence as given in SEQ ID NQ:2 from nucleotide 1 to nucleotide 463.

• ·

51. A. method of increasing the transcriptional expression of a downstream DN’A. sequence in a transgenic plant cell, said method comprising the steps of:

(a) operably linking a transcription-enhancing element having a nucleotide sequence substantially as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 463 to a promoter functional in a plant cell, said promoter being operably linked to a downstream DNA sequence for which transcriptional expression is to be directed to produce an enhanced expression cassette; and (b) introducing the enhanced expression cassette of step (a) into a plant cell to produce a transgenic plant cell.

52. The method of claim 51, wherein said tzanscriotion enhancing sequence further comprises a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 1 to 370 operably linked immediately upstream of the transcription-enhancing sequence of step (a).

53. The method of claim 51, wherein said promoter functional in a plant cell is a minimal promoter of Cauliflower Mosaic Virus.