MXPA00004939A - A pathogen and stress-responsive promoter for gene expression - Google Patents

Abstract

Nucleic acid sequences and methods for their use are provided for obtaining plants with inducible phenotypes, including novel or enhanced resistance to infection and stress. Novel nucleic acid constructs are provided, which include a transcriptional initiation region froma pathogen inducible-rapid response grape stilbene synthase gene, a DNA sequence of interest and a transcriptional termination region. The constructs are used to prepare expression cassettes which may then be introduced into plants and portions thereof for modulation of expression of endogenous products as well as production of exogenous products in the plant.

Description

U N PROMOTER OF TENSION AND PATHOGEN RESPONSE FOR EXPRESSION OF GENES I NTRODUCTION Technical Field This invention relates to the inducible regulation of gene expression to provide a host plant with resistance to infection of pathogens, as well as abiotic stresses. The invention is exemplified by the use of a Stilbene synthase promoter from Vitls to give a gene expression that is inducible upon infection of pathogens.

Background Stilbene synthase (EC 2.3.1.95) is a polyketide synthase that catalyzes the synthesis of 3,4 ', 5-trihydroxystilbene (resveratrol) from p-coumaroyl-CoA and three molecules of malonyl-CoA. This cytosolic enzyme is confined to a few genera of plants. In intact and unchallenged cells, the enzyme is weakly expressed, but it is synthesized de novo on attack by pathogens. The extractors derived from pathogens ignite the synthesis of stilbene phytoalexins, which are part of the resistance process induced in these plants. In vines, resveratrol and other hydroxystilbenes with fungicidal potential are the dominant phenols produced under biotic stress. Several plants including grapevine synthesize resveratrol when they are attacked by pathogens. Comparative studies have revealed that the Response model against microbial attack depends mainly on the stage of development of the individual plant cell. In this regard, systems have been established in parsley, beans and potatoes and compared with each other; each individual plant species is unique with respect to the speed with which gene expression changes, the duration of the transient stage and the degree to which the enzymes in the path are produced in a coordinated manner. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanuts (Arachis hypogaea), vine. { Vitis vinifera), pine (Pinus silvestris) and orchids. A few plants, such as spruce and rhubarb, contain stilbenes but do not exhibit increased de novo synthesis of stilbenes in response to extractors. In grapes, stilbenes (resveratrol and its oligomeric derivative viniferin) have antimicrobial activity against Botrytis cinerea and Plasmopara viticola, the main pathogens of this species. Liswidowati et al. , (Planta (1991) 18: 307-314) demonstrated that stilbene synthase is rapidly and transiently synthesized in cell cultures of grapes after extraction with B. cinerea. A clone for grape stilbene synthase has been isolated and it has recently been shown that stilbene synthase, when expressed in transgenic tobacco plants, confers an increased resistance to the disease against B. cinerea. The temporal expression of stilbene synthesis in grapes has been studied when analyzing RNAs present in different extraction stages by the addition of Phytophthora camelivora cell wall to Vitis cultures. vinifera Stationary mRNA levels reached maximum values (approximately 1.5 times the baseline) 6 hours after the start of the extractor treatment. Therefore, it would be of interest to obtain the gene coding for this early start of stilbene synthase and isolate the regulatory elements responsible for the rapid and high level of expression of the coding sequence; the regulatory elements provide a means to control the expression of genes for plant stress and disease resistance. There is a substantial interest in modifying a plant with promoters that provide transcription and expression induced by a biotic or abiotic strain of a gene introduced into the plant. Of particular interest are promoters that provide locally inducible gene expression. Also of interest is the ability to intensify or modify the properties of other promoters.

Relevant literature Melchior and Kindl, Arch. Biochem. Biophys. (1991) 288: 552-557, report the induction patterns of stilbene synthase genes cloned with pSV25, pSV21 and pSV368 in grapes. Hain et al, Nature (1 993) 361: 153-1 56, reports the production of transgenic tobacco plants containing vine stilbene synthase genes with increased resistance to Botrytis cinerea infection. Sparvoli et al. , Plant Mol. Biol. (1 994) 24: 743-755, reports the cloning and characterization of structural genes required for anthocyanin and stilbene biosynthesis in grapes. Wiese et al. , Plant Mol. Biol. (1994) 26: 667-677, report the isolation and sequencing of stilbene synthase Vst1, Vst2 and Vst3 genes from grape (Vitis vinifera) and compare the expression patterns of ethylbenzene synthase genes in cells treated with extractors.

BRIEF DESCRIPTION OF THE INVENTION Methods and compositions are provided to obtain plants that have inducible resistance to infection and stress. Nucleic acid constructs are provided which provide regulated transcription, such as pathogen-inducible transcription, in a plant tissue or plant part of interest or in response to external agents, such as UV exposure. Particularly, the transcription regions of the grape stilbene synthase genes bind to DNA sequences of interest and are introduced into a host plant cell for integration into the genome, to provide pathogen-induced transcription or strain. It is of considerable interest that the expression of the DNA sequences of interest is locally induced at the site of stress, as opposed to being induced systemically. The constructs provide modulation of the expression of endogenous products as well as the production of exogenous products in the plant. Novel nucleic acid constructs are also provided using a grape stilbene synthase promoter, particularly a promoter from a gene that delivers a high and rapid expression level on infection or extraction, bound to a DNA sequence of interest and a region of transcription termination. A DNA construct is introduced into a host plant cell for integration in the genome and regulated transcription on infection or extraction. In this manner, high levels of RNA are reached and, as appropriate, polypeptides at the time of infection or extraction.

BRIEF DESCRIPTION OF THE DIAMETERS Figure 1 shows the nucleotide sequence (SEQ I D NO: 1) of the promoter region of a genomic clone of stilbene synthase of grapes, STS8. The STS8 promoter clone also contains a partial coding sequence for the stilbene synthase. The partial coding sequence begins at the ATG located at nucleotides 1 21 2-1 214. Figure 2 shows a schematic representation of the primary DNA vector pCGN81 32 for plant transformation.

DESCRIPTION OF THE PREFERRED MODALI DADES In accordance with the present invention, nucleic acid constructs are provided which allow for the regulated modification of the plant phenotype in response to stress, for example, UV exposure, fungal extractors, interactions with microorganisms and injuries The nucleic acid constructs comprise a regulated transcription initiation region derived from a local and rapidly inducible stilbene synthase gene, particularly one having the characteristics of a transcription initiation region derived from a stilbene synthase gene SV25 of Vitis vinifera. The constructs can be included in a transcription cartridge or an expression cartridge in which, downstream of the regulated transcription initiation region is an nucleotide sequence of interest, which provides for the regulated modification of a plant phenotype, by modulating the production of an endogenous product, such as for quantity, relative or similar distribution, or the production of an exogenous expression product to provide a new function or product. One or more introns may also be present. Depending on the manner of introduction of the nucleic acid construct into a host plant, other DNA sequences may be required, such as sufficient T-DNA from an Agrobacterium plasmid for transfer to a host plant. Host plants of particular interest are fruit plants, such as strawberry and tomato. The rapidity and induction force of the gene expression provided by the transcription activators of the present invention are particularly advantageous where it is desired to shoot or greatly intensify the resistance responses of the plant to pathogens and pests, or to increase the effectiveness of mechanisms that protect against abiotic stresses (for example, antioxidant enzymes, repair of injuries and repair of genetic damage from UV radiation). In this manner, the DNA sequences of interest are only expressed at the time of infection and in the area of infection, said specificity can avoid potential detrimental effects to the plant, which may be caused by the constitutive expression of such sequences. The desired effects on plant resistance can be achieved by regulating the production of phytoalexins (eg, hydroxystilbenes), the expression of the resistance genes disease, such as, R genes, defense induction genes, such as, avr genes, and genes for resistance to insects or nematodes, for example. Examples of genes that can be expressed under the regulatory control of the stilbene synthase promoter constructs of the present invention for enhanced resistance to disease are described in Hammond-Kosack, K.E. and Jones, J.G. D. ("Plant disease resistance genes", Annu, Rev. Biochem. (1990) 59: 873-907). The ability to achieve high level transgene inducible expression is also of considerable importance in "molecular farms", where plants are used to produce industrial or pharmaceutical polypeptides and other biopolymers that are foreign to plants. It is also expected that the transcription primers of the present invention are advantageous for use in reporter gene constructs to develop rapid and sensitive cell-based screens for environmental and agricultural monitoring. Furthermore, it is expected that the present invention can be used to increase the production of pharmaceutically important secondary metabolites in transgenic plants by over-expression of heterologous genes or suppression of endogenous genes in metabolic pathways of plants. The desired transcription initiation region is one that is activated at or shortly after pathogen infection or extraction by abiotic stresses, such as UV irradiation and injury. An example of a desired transcription initiation region is that referred to herein as STS8. The initiation region of STS8 transcript is activated within one hour after infection and remains active until at least 72 hours post-infection. Importantly, transcription of the STS8 promoter is induced locally at the site of extraction or infection. Local induction is preferred in the expression of plant genes in cases where it is desired to provide resistance to invading pathogens, particularly fungal or bacterial pathogens. The identification of transcription initiation regions having the desired characteristics can be achieved in a variety of ways. For example, cDNA or genomic libraries can be prepared from a known source of inducible stilbene synthase genes with pathogen and extractor, such as vine, peanut, pine and orchid plants. A library of a given species is classified with oligonucleotide probes designed to contain sequences that are complementary to coding regions of other known inducible stilbene synthase genes. Alternatively, oligonucleotide probes can be designed on the basis of the amino acid sequence information obtained from purified stilbene synthase proteins. Oligonucleotide probes based on amino acid sequences can be degenerate or can be influenced to favor the preferred codons of the source plant. Oligonucleotide primers can be designed to be used in polymerase chain reaction-reverse transcription (RT-PCR) to amplify PCR fragments for cloning and sequencing. The sequences of the PCR fragments can be compared with coding regions known of stilbene synthase genes desired to identify the corresponding clones. As an example, the cDNA of a stilbene synthase gene that responds to a highly inducible pathogen is obtained by RT-PCR, using the total RNA prepared from specific gene primers and plant tissue infected with pathogen. The cDNA is then used as a bait to isolate the promoter region using genomic restriction libraries of the plant tissue in consecutive PCR reactions with nested primers. The PCR amplification products are cloned and sequenced. The sequence products are then compared to the known coding sequence of the stilbene synthase gene. Additional sequences comprising approximately 200 bp to approximately 1500 bp upstream from the start codon are identified, and then the clones containing upstream sequences are evaluated by desired promoter activity. The transcription initiation region may be natural or homologous to the host or foreign or heterologous to the intended host. The term "foreign" is intended to mean that the transcription initiation region is not usually found in the host plant into which it is introduced. In addition to the use of the complete transcription initiation regions of the present invention, the use of regulatory elements within the promoters to enhance or modify the properties of other plant promoters is also considered herein. For example, the use of a region of approximately 130 bp from nucleotides -456 to -324 of the STS8 promoter (nucleotides 678-81 0 of Figure 1) can provide an induction of response to ozone. In addition, a fragment of approximately 1 30 bp located at nucleotides -324 to -193 of the STS8 promoter (nucleotides 81 0-941 of Figure 1) can be used to add pathogen induction characteristics to other promoter plants. A sequence of nucleotides of interest is inserted downstream of and under the regulation of the transcription initiation region. The nucleotide sequence of interest provides for the modification of plant phenotype, for example, by altering the production of an endogenous product, such as by amount, relative or similar distribution, or by coding a structurally or functionally novel gene product. The nucleotide sequence can have an open reading frame that encodes a peptide of interest, eg, an enzyme, or a sequence complementary to a genomic sequence, where the genomic sequence can be an open reading frame, an intron, a leader sequence of non-coding, or any other sequence where the complementary sequence inhibits transcription, messenger RNA processing, eg, splicing or translation. The nucleotide sequence of interest may be of synthetic or natural origin or combinations thereof. Depending on the nature of the nucleotide sequence of interest, it may be desirable to synthesize the sequence with preferred codons of plants. The preferred codons of plants can be determined from the highest frequency codons in the proteins expressed in the greatest amount in the plant species of particular interest.

The termination region is one that is functional in a plant host cell. In addition to containing at least one terminator sequence, the terminator region can include a poly A signal. In view of the relative exchangeability of the terminator regions, the selection of a terminator region to be used in the expression construct is primarily based on in the convenience. The termination region and the transcription initiation region, or the region of termination and nucleotide sequence of interest can originate from the same or different sources. Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the termination regions of the octopine synthase gene and the nopaline synthase gene. Additional DNA sequences can be included in the transcription cartridge, for example, adapters or linkers to join the DNA fragments in the proper orientation and, as appropriate, in the appropriate reading frame. Other DNA sequences may be needed to transfer transcription constructs in organisms used to transform plant cells, for example, A tumefaciens. In this regard, the use of T-DNA from the Ti or Ri plasmids, as a flanking region in a transcription construct is described in EPO Application No. 1 16,71 8 and PCT Applications Nos. WO84 / 0291 3, 0291 9 and 02920. See also Herrera-Estrella, Nature (1 983) 303: 209-21 3; Fraley et al. , Proc. Natl. Acad. Sci, USA (1983) 80: 4803-4807; Horsch et al. , Science (1988) 223: 496-498; and DeBlock et al. , EMBO J. (1988) 3: 1 681-1689.

The expression constructs of this invention, which contain the 5'-unregulated regulated regions of locally inducible stilbene synthase genes, are transformed into plant cells to assess their ability to function with a structural gene different from the open reading frame that it is naturally associated with the 5'-region without transferring and assessing its induction characteristics in response to a particular stimulus, such as infection with Botrytis. In addition, the use of the STS8 transcription initiation region of the present invention for expression of stilbene synthase genes is considered herein to provide increased resistance to plant diseases, particularly fungal or bacterial diseases. Such constructs can express STS genes regulated in a natural way by the gene promoter STS8, but preferably in host plants other than grapes, or may express other stilbene synt genes under the control of unnatural transcription of the STS8 promoter. The stilbene synt genes in such constructs may contain regions of natural introns associated with the stilbene synt genes, or they may be designed to remove any intron. Of interest is the use of such constructs to increase plant resistance to diseases, such as those caused by fungi, including Fusarium and Verticillium, and bacterial diseases, such as, root decomposition by bacteria. A variety of techniques are available for the introduction of DNA into a plant host cell. These techniques include transformation using Ti plasmid DNA and A. tumefaciens or A. rhizogenes as the transforming agent, protoplast fusion, injection, electroporation and the like. The transcription construct is usually linked to a marker that allows the selection of transformed cells in the treated population, for example, resistance to antibiotics, such as kanamycin, G41 8, bleomycin, chloramphenicol, and others. Any variety of plants can be used as a host cell according to this invention. Of particular interest are agricultural fruit crops, such as strawberries and tomatoes, although the use of the STS8 transcription initiation region in other plants, including other plants bearing fruits, is also considered. Examples of plants in which the STS8 transcription constructs can find use include grain plants, such as wheat, barley, rye, oats and rice; oilseed crop plants, such as, soybeans, Brassica oilseed plants, including canola and varieties of high erucic acid, corn, sunflower, safflower, oil palm and peanut; forest plants, such as spruce, spruce, pines and oaks; fruit plants, such as apple, melon, citrus, grape, banana, coconut and pineapple; and other diverse crop plants, including cotton, cocoa, tobacco and potatoes. Host cells of transformed plants are used to regenerate plants. See, for example, McCormick et al. , Plant Cell Reports (1 986) 5: 81 -84. These plants are grown and pollinated either with the same transformed strain or with different strains, and the resulting hybrid having the desired phenotypic characteristic identified. They can be done grow two or more generations to ensure that the desired phenotypic characteristic is maintained and inherited in a stable manner, and then seeds are collected to ensure that the phenotype or other desired property has been achieved. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL PHASE Materials and methods Cloning vectors The cloning vector pCR2.1 TA of I nvitrogen was obtained. A binary vector for plant transformation, pCGN5928 was constructed using the kanamycin resistance gene neomicin phosphotransferase (npt1) driven by the transcription initiation region of noplain synthase (nos 5 ') and the transcription termination sequences ( nos 3 ') (Fraley et al., Proc. Natl. Acad. Sci (1 983) 80: 4803-4807 and Depicker et al., J. Molec. Appl. Genet. (1 982) 1: 562-573) . Both nos 5 'and nos 3' were amplified by PCR from the strain of Agrobacterium tumefaciens C58 and were linked together with the nptl l gene of pCGN783 (Houck, et al., Frontiers Appl Microbiol (1 988) 4) as a EcoR I fragment to form pCGN5908. The fragment nos 5'-nptl l-nos3 'was then cloned into pCGN 1541, containing ori322, Right border (0.5 Kb), lacZ, Left Border (0.58Kb), as an Xho I fragment to create the intermediate pCG N591 0. The origins of replication Col EI and pRi, as well as the gene of resistance to Gentamicin were acquired from a suppressed Not I derivative of pCGN 1 532 (McBride and Summerfeit, Plant Molecular Biology, (1 990), 14: 269-276) as a BamH I fragment to create pCGN5924. Finally, a linker containing unique restriction sites was synthesized and cloned into the Asp 718 / Hind l l l sites (within the lacZ sequence) of pCGN5924 to create the binary vector pCGN5928.

Materials Tth DNA polymerase and the GenomaWalker® set were obtained (formerly known as the PromoterFinder set) from Clontech. The sources of enzymes and additional reagents are as follows. Restriction enzymes: Dra I, EcoR V and Sac I were provided with the GenomeWalker set; Pvu I I and Stu I were obtained from Boehringer Mannheim Enzymes: reverse transcriptase was from Boehringer Mannheim; ligase was provided in the GenomeWalker set. Chemicals: the deoxynucleotides were from the Advantage® set Genomic PCR from Clontech and the general chemicals were from Sigma Chemicals Example 1 Cloning of the STS8 promoter 1 A. Preparation of genomic restriction libraries of grapes To isolate genomic DNA from grapes, fresh tissue was milled to a fine powder in liquid nitrogen using a cooled mortar and pestle. The powder was added to a DNA extraction buffer (200 mM CHES (2- (N-cyclohexylamino) ethanesulfonic acid) / NaOH pH 9.1, 200 mM NaCl, 1 00 mM EDTA pH 9.0, 2% SDS, 0.5% sodium deoxycholate, 2% Nonidet NP-40, 20 mM ß- mercaptoethanol) and the sample was incubated at 65 ° C for 10 minutes. Potassium acetate was added at a concentration of 1.1 M, followed by incubation on ice for 30 minutes. The DNA was centrifuged at 20,000 x g for 20 min at 4 ° C. The supernatant was filtered through two layers of Miracloth® (Calbiochem, La Jolla, CA). The DNA was centrifuged again as before. DNA was precipitated by adding 15 ml of isopropanol and centrifuging for 25 minutes at 25,000 x g. The pelleted DNA was resuspended in TE (10 mM Tris, 1 mM EDTA). Optical grade cesium chloride (0.97 g / ml) and 3 mg of ethidium bromide were added to the DNA solution. The DNA was then ultracentrifuged at 65,000 rpm for 1 8 h at 1 5 ° C. The DNA was extracted with 1-butanol saturated with water until clear. The DNA was then precipitated by adding two volumes of 1 00% ethanol and centrifuging for 25 minutes at 8,000 x g. The DNA pellet was resuspended in 200 ul of TE. The DNA restriction libraries of grapes were constructed from grape genomic DNA as instructed in the GenomeWalker set. Genomic DNA was digested individually with 5 different restriction enzymes blunt end; Dra \, EcoR V, Pvu I I, Be I and Stu I. After digestion, the GenomeWalker Adapters (48 bases) were ligated to both ends of the restriction fragments to create the restriction libraries. 1 B. Isolation of STS cDNA Leaves of grapes infected with Botrytis were prepared to be used as a source of RNA as follows. A spore suspension of Botrytis cinérea CaM strain of 1 06 was used to atomize separate grape leaves. The inoculated grape leaves were incubated at 20 ° C, with light periods of 12 hours for 3 days in humidified trays. The leaves were collected 3 days post inoculation and were used for RNA extractions. Grape RNA was extracted using an adaptation of the method described by Loulakakis, K.A. , Roubelakis-Angelakis, K.A. , and Kanellis, A. K., American Journal of Enology and Viticulture (1 996) 47: 1 81 -1 85. The cesium chloride gradient step described in this reference was omitted. The cDNA of a stilbene synthase gene was obtained by the reverse transcription polymerase chain reaction (RT-PCR) technique, using the total RNA prepared from grape leaves infected with Botrytis and the specific primers of the stilbene synthase gene. : RES 1 5 'TAGGATCCATGGCTTCAGTTGAGGAAT 3' (SEQ ID NO: 2) Res2 5 'GCGAATTCCTATTTGATACATTACGCCATTG 3' (SEQ ID NO: 3) The nucleotide sequences of the primers were designed according to the published sequence of a highly inducible STS gene by pathogen, psV25 [Melchior and Kindl, Arch. Biochem. Biophys. (1 991) 288: 552-557]. RES1 is the forward primer that contains the sequence encoding the STS gene from the 5 'end of the cDNA, including the ATG start codon (underlined above) and the restriction cloning sites. RES2 is the reverse initiator containing the sequence complementary to bases 1208 to 1232 in the 3 'untranslated region (numbering according to Melchior and Kindl) and restriction cloning sites. Following PCR using the primers RES 1 and RES 2 and the RNA prepared as described above, the resulting STS gene fragment of approximately 1280 base pairs was digested with restriction enzymes EcoR I and Bam H ly was cloned into pBluescript II SK- (Stratagene) to create pCGN81 05. The nucleotide sequence of the cloned STS cDNA (designated STS8) was determined by automated sequencing using the forward and backward M1 3 primers. No product was obtained when the total RNA of healthy grape leaves was used, suggesting that the cloned STS gene is expressed only during infection.

C. C. Cloning of Stilbene Synthase Gene Promoter For cloning of the STS promoter, the cDNA sequence obtained as described in the previous section was used as a template to isolate the promoter region using the Clonetech GenomeWalker set. The GenomeWalker set is a set based on PCR to scan genome in genomic DNA without cloning. Genomic grape restriction libraries prepared as described above were used in consecutive PCR reactions with nested primers. The primary reaction was performed using a specific primer for the linked GenomeWalker adapter, AP 1, (5'-GTAATACGACTCACTATAGGGC-3 ') and the specific STS gene primer, RES2 (described above). In addition to the oligonucleotide primers (0.2 μM each), the PCR reaction mixture contained 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.0% glycerol, 0.2 mM Tris-HCl (pH 8.3) , 4.6 mM KCl, 1.5 mM EDTA, 1 5 μM dithiothreitol, 7.3 μg / ml BASA, 1. 1 mM KOAc and 0. 1 unit Tth DNA polymerase. The mixtures were amplified using the following conditions: 7 cycles of 94 ° C for 2 seconds, 72 ° C for 3 minutes, 32 cycles of 94 ° C for 2 seconds, 67 ° C for 3 minutes and 1 cycle of 67 ° C for 4 minutes in a Perkin-Elmer 9800 thermocycler. Secondary PCR reaction was performed with internal primers to the primary PCR primers. The specific primer of GenomeWalker was AP2: 5'-ACTATAGGGCACGCGTGGT-3 '(SEQ ID NO: 4) and the stilbene synthase gene-specific primer was RES-Í1: 5'-TAGAGCTCTGCAGTTCAATGCTGCATCCCTACCAAGTCTA-3' (SEQ ID NO: 5) . RES-11 is the reverse primer containing the sequence complementary to nucleotides 309 to 335 of STS8 stilbene synthase cDNA and restriction cloning sites. The second PCR reaction was run using the same buffering conditions as in the primary reaction and under the amplification conditions of 5 cycles of 94 ° C for 2 seconds, 72 ° C for 3 minutes, 20 cycles of 94 ° C for 2 hours. seconds, 67 ° C for 3 minutes and 1 cycle of 67 ° C for 4 minutes. The main products of the PCR reaction were cloned into the vector pCR2, 1 TA (I nvitrogen) and sequenced. The sequence products showed identity in the coding region for the STS gene, and included an additional sequence upstream of 200 to 1 200 bp from the start codon, depending on the size of the template fragment of the restriction library. A clone containing an upstream sequence of 121 1 bp was designated as the STS8 promoter. This clone, pCGN81 23, also included approximately 700 bp of sequence coding for stilbene synthase, including an intron of 359bp.

Example 2 Sequence analysis of the promoter region The sequence of the STS8 promoter element was aligned with the stilbene synthase promoter sequence vstl of one. The alignment of the STS8 promoter sequence from -460 to the start codon (ATG), including 76 bp from the untranslated leader sequence, shows 51% identity to -a sequence from -430 to ATG (including 73 bp from the leader). untranslated) of transcript vst 1. Two regions, -340 to -280 and -280 to -140, have been identified in the vstl promoter as responsible for the inducibility of ozone and pathogen respectively (Scubert, et al., (1997 ), Plant Molecular Biology, 34: 417-426, and references used therein). Alignment of the sequence from -430 to -280 upstream of the start of transcription of vstl with the STS8 promoter revealed 61% identity with the sequence of -456 to -324 of the STS8 promoter (nucleotides 678-81 of the Figure 1 ). The sequence of -280 to -140 upstream of the start of vstl transcription has been identified as important for the response to pathogens. A comparison of this sequence with STS8 showed that the sequence between -324 and -193 of STS8 (nucleotides 81 0-941 of Figure 1) is 76% identical to the sequence responding to vstl pathogen. The alignment of the region -193 to -1 of the STS8 promoter (nucleotides 941-1 135 of Figure 1) with the -40 to -1 region of the vstl promoter region indicates that the STS8 promoter region contains 2 small insertions (approximately 15 and 30 nucleotides), which are not present in the corresponding region of the vst1 promoter.

Example 3 Construction of an expression construct 3A. Preparation of a promoter-reporter gene construct A fusion construct of promoter DNA was made with a reporter gene encoding β-glucuronidase (GUS) as follows. The STS8 promoter from pCGN8123 was ligated to the 5 'end of the NOS 3'-gene G' N terminator fusion fragment as a Sal 1 -? / Co 1 fragment, producing the plasmid gone pCGN81 31. The GUS-NOS fragment was prepared as follows. A 1.8 kb fragment encoding β-glucuronidase (GUS) from Escherichia coli (Jefferson et al., Proc. Natl. Acad. Sci. (1 986) 83: 8447-8451) was linked to the 3 'region without transferring Nopaline synthase (US 3 '0.3 Kb) of T-DNA from Agrobacterium tumefaciens (Fraley et al., Proc. Natl. Acad. Sci (1983) 80: 4803-4807 and Depicker et al., J. Molec. Appl. Genet. (1982) 1: 562-573) to generate the GUS-NOS terminator fusion. The GUS gene included an intron acquired from the second intron (IV2) of the ST-LS 1 gene from potato (Vancanneyt et al., Mol.Gen.Genet. (1 990) 220: 245-250). 3B. Preparation of the expression vector The STS8-GUS-NOS 3 'cartridge from pCGN81 31 was cloned into the binary vector pCGN5928 as a Not 1 fragment, producing the expression vector pCGN81 32 shown in Figure 1. 3C. pCGN81 32 was transformed into the strain of Agrobacterium tumefaciens LBA4404 by the method of Holsters et al. , Molecular and General Genetics (1 979) 1 63: 1 81 -1 87.

Example 4 Evaluation of the function of the STS8 promoter in transformed plants 4A. Transformation of tomato plants Vector pCGN81 32 was introduced into tomato plants by Agrobacterium-mediated transformation according to Fillatti et al. ,. { Bio / Technology, (1987) 5: 726-730). Positive transformants were identified as plants that were resistant to 150 μg / ml kanamycin. Positive transformants were tested for the inducible promoter function by spotting detached leaf tissue challenged with Botrytis with 5-bromo-4-chloro-3-indo! -β-D-glucuronide (X-Gluc) at 24-fold spots. hours. The detached transformed tomato leaves were inoculated with a single drop, 1 00 μl of spore suspension of the Botrytis cinerea Cal i strain of 1 06 conidiospores / μl along the middle vein. The inoculated leaves were incubated at 20 ° C with a light period of 1 2 hours in humidified plastic trays. At 24-hour points, the leaves were collected and infiltrated with GUS buffer (50 mM potassium phosphate (pH 7), 1 mg / ml X-Gluc and 0.1% Triton X-100), were allowed to stain overnight at 37 ° C. The next day, the leaves were stained with 1 hour washes using 70% ethanol and 4 to 6 hours using 100% ethanol. The inducible expression was characterized by GUS staining increased over time, which was limited to tissue in an area encompassed by a circumference of 22 mm surrounding the site of infection, as well as the site of infection itself, which was approximately of 5 mm in diameter. This expression pattern provides evidence of a local induction pattern, which is opposed to the systemic one. Twenty tomato lines resistant to kanamycin were analyzed for induction of GUS activity. Eight lines showed a weak induction of GUS expression 48 hours post-inoculation, five lines showed a mild induction, two lines showed a strong induction and five lines showed no induction. The strength of the induction was determined by visual inspection of the relative color intensity after spotting with GUS. The weak and soft expression by STS8 showed no staining with GUS at 0 hours after inoculation, while the two highest expression lines had a low basal level of GUS staining at the tips of the leaves. In order to determine the speed and duration of the induction at the mRNA transcript level, three lines were used for further analysis using Northern hybridizations, two medium levels and one high level of expressor. Twenty leaves detached from each line were inoculated by spraying with a suspension of spores of strain Botrytis cinerea CaM of 106 conidiospores / ml. The inoculated leaves were incubated in a humidified tray at 20 ° C. Two leaves of each line were collected at times 0, 1, 2, 4, 8, 12, 24, 48 and 72 hours post inoculation and were frozen at -80 ° C. Total RNA was isolated using TRIzol reagent (Gibco-BRL Life Technologies) following the manufacturers protocol. The total RNA, 30 μg, was separated on a denaturing agarose gel and transferred to a nylon membrane (Sambrook et al., 1989). Hybridizations with G US fragments of labeled random primer (using the Primer-lt II Random Primer set from Stratagene) were performed in 35% formamide, 5x SSC, 0.5x Denhardts, 1% SDS and 300 μg of yeast tRNA at 42 ° C. In the three lines examined, GUS mRNA was expressed within one hour after inoculation and the signal was sustained for approximately 48 hours after inoculation. Maximum transcript levels were reached approximately 2 hours after infection. 4B. Transformation of leaves of Nicotinana benthamiana Nicotinana benthamiana leaves were transiently transformed with Agrobacterium containing the fusion construct pSTS8-GUS. The Agrobacterium cells harboring pCGN81 32 were grown at 30 ° C to the density of 0.8 OD600- The cells were washed with sterile water and resuspended in 10 mM MgSO 4. The leaves of Nicotinana benthamiana were infiltrated with the Agrobacterium cell suspension using a sterile syringe without a needle. It was allowed that the infiltrated leaves will grow for two days before promoter activation as described above for transformed tomato leaves. The leaves were detached from the plant and placed in a humidified plastic tray. The upper surface of the leaves was inoculated by a 50 μl drop of conidiospore suspension of Botrytis cinerea CaM at 1 07 spores / ml. The activity of the STS8 promoter was determined by examining the GUS activity at 24 and 48 hours after the infection of Nicotinana leaves with Botrytis cinerea using the histochemical staining procedure as described by Stomp, in: GUS Protocols: using the GUS gene as a repórter of gene expression (GUS Protocols: using the GUS gene as a reporter of gene expression), ed. Gallgdher, S. R., Academic Press, San Diego, USA, 1 992, pp. 1 03-1 14 and a published fluorometric assay (Jefferson, Plant Mol. Biol. Repórter (1987) 5: 387-405). Histochemical staining at the site of fungal infection was observed indicating that the STS8 promoter was locally inducible by Botrytis cinerea infection. An intense spotting was observed at the infection site of Nicotinana leaves. Fluorometric assays showed that the GUS activity in tissues of infected leaves increased significantly when compared with healthy controls without infecting.

Example 5 Expression driven by STS8 promoter of stilbene synthase cDNA in tomato A STS8 promoter DNA fusion construct was prepared with a clone of stilbene synthase grape cDNA. A 1.2 Kb STS8 protor fragment of pCGN8123 was cloned as a 1-Nco1 salt fragment to the 5 'end of the stilbene synthase with a transcription terminator sequence Nos 3'. The STS8-stilbene synthase-Nos 3 'fragment was cloned into pCGN5928 as a Not 1 fragment to create pCGN81 26. The vector pCGN81 26 was transformed into the Agrobacterium tumefaciens strain LBA4404 by the method of Holster et al. , Molecular and General Genetics (1979) 1 63: 1 81 -1 87. The vector pCGN8126 was introduced into tomato plants by means of Agrobacterium-mediated transformation according to Fillatti et al. (Bio / Technlogy, (1987) 5: 726-730). Positive transformants were identified as plants that were resistant to 1 50 μg / ml kanamycin. Forty-five positive lines for kanamycin resistance were generated with pCGN81 26 and were classified by PCR to confirm the presence of the transgenic stilbene synthase gene. The positive transformants are analyzed by STS8 stilbene synthase gene expression and resveratrol accumulation. The selected lines are self-sufficient to obtain homozygous tansformants for use in field trials to test the effects of stilbene synthase gene expression on resistance to plant diseases.

The above results demonstrate that the SSTS8 promoter can be used to provide an inducible expression of the DNA sequences of interest in plants, where the response to induction is rapid, the expression is locally induced and the expression reaches a high level. Such an expression pattern is particularly desirable for the expression of genes related to resistance to plant diseases, such as, the stilbene synthase gene, and can provide for the production of improved plants that have enhanced resistance to plant pathogens. All publications and patent applications mentioned in this specification are indicative of the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same degree as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (9)

1. REIVI NDI CATIONS
2. 1 . A DNA construct comprising: in the 5 'to 3' direction of transcription, a transcription initiation region derived from a wine stilbene synthase gene, a DNA sequence of interest and a functional transcription termination region in plants, wherein said initiation region promotes the rapid and locally induced initiation of gene transcription in response to pathogen infection and abiotic stresses. The DNA construct according to claim 1, wherein said transcription initiation region provides induction of transcription of said DNA sequence of interest within one hour of said pathogen infection or abiotic stress.
3. 3. The DNA construct according to claim 2, wherein said transcription initiation region is derived from the STS8 stilbene synthase gene of Vitis vinifera.
4. 4. The DNA construct according to claim 2, wherein said transcription initiation region comprises the DNA sequence shown in Figure 1.
5. 5. The DNA construct according to claim 1, wherein said DNA sequence of interest is an open reading frame that encodes an amino acid sequence.
6. 6. The DNA construct according to claim 1, wherein said DNA sequence of interest is complementary to an endogenous mRNA for a plant cell.
7. 7. The DNA construct according to claim 1, which comprises the transcription initiation region STS8 in the plasmid PCGN8131. The construct according to claim 1, which comprises the transcription initiation region STS8 in the expression vector pCGN8132. 9. A transcription cartridge comprising: in the 5'-3 'direction of transcription, a transient initiation region of a grape stilbene synthase gene, rapid response, locally inducible, to a pathogen, operably linked to a sequence of nucleotides of interest and a region of transcription initiation. 1 0. The transcription cartridge according to claim 9, wherein said transcription initiation region is derived from the STS8 stilbene synthase gene of Vitis vinifera. eleven . The transcription cartridge according to claim 9, wherein said transcription initiation region has the DNA sequence shown in Figure 1. 12. A DNA construct comprising: in the 5'-3 'direction of transcription, an adjustable transcription initiation region of a grape stilbene synthase gene, fast response, locally inductible, to a pathogen, a linker or polylinker which has a site or plurality of restriction sites for the insertion of a gene to be expressed under transcription control of
8. said transcription initiation region and a functional transcription termination region in plants. The DNA construct according to claim 1, wherein said transcription initiation region is derived from the STS8 stilbene synthase gene of Vitis vinifera. The DNA construct according to claim 1, wherein said transcription initiation region has the DNA sequence shown in Figure 1. 15. The DNA construct according to claim 1, wherein a DNA sequence of heterologous interest to the stilbene synthase gene is inserted into at least one of said restriction sites. 1 6. A DNA construct comprising a functional transcription initiation region in a plant cell, wherein said transcription initiation region comprises nucleotides 678-81 0 of the sequence shown in Figure 1. 17. A DNA construct comprising a functional transcription initiation region in a plant cell, wherein said transcription initiation region comprises nucleotides 81 0-941 of the sequence shown in Figure 1. 1 8. A DNA construct comprising a transcription initiation region in a plant cell, wherein said transcription initiation region comprises nucleotides 941 -1 1 35 of the sequence shown in Figure 1.
9. 9. A plant or portion thereof comprising a DNA construct according to any one of claims 1, 3, 12, 14 or 1 6-1 8. 20. A method for modulating resistance to disease in a plant, wherein said method comprises growing a plant that contains in its genome a construct, which provides the expression of a gene useful for providing resistance to a plant pathogen under the regulatory control of the stilbene synthase STS8 grape promoter. twenty-one . The method of claim 20, wherein said gene is stilbene synthase.

   SUMMARY

   Nucleic acid sequences and methods for their use are provided to obtain plants with inducible phenotypes, including novel or enhanced resistance to infection and stress. Novel nucleic acid constructs are provided, which include a transcription initiation region of a grape stilbene synthase gene, rapid response, inducible, pathogen, a DNA sequence of interest and a transcription termination region. The constructs are used to prepare expression cartridges, which can then be introduced into plants and portions thereof for the modulation of the expression of endogenous products, as well as the production of exogenous products in the plant.