WO2001020008A2

WO2001020008A2 - Vascular-specific promoters

Info

Publication number: WO2001020008A2
Application number: PCT/EP2000/006248
Authority: WO
Inventors: Enrico Scarpella; Anneke HOGE-MEPPELINK; Annemarie H. Meijer; J. Harry C. HOGE
Original assignee: Universiteit Leiden
Priority date: 1999-09-16
Filing date: 2000-07-03
Publication date: 2001-03-22
Also published as: AU5824400A; WO2001020008A3

Abstract

A DNA fragment upstream of the rice Oshox1 gene or portions or variants of the DNA fragment wherein the DNA fragment or portions or variants thereof are capable of promoting vascular expression of an associated DNA sequence upon reintroduction into a plant, typically wherein vascular expression of the associated DNA sequence enables production of an antipathogenic protein.

Description

VASCULAR-SPECIFIC PROMOTERS

Field of the Invention The present invention relates to promoters and more specifically to inducible promoters able to drive expression in vascular cells of plants.

Background

A promoter is located in the 5'upstream region of a gene and contains certain sequences necessary for transcription of that gene. Inducible promoters include any promoter which, in response to exposure to an inducer, can initiate or enhance expression of a given gene. In the absence of an inducer, the DNA sequence will either not be transcribed or will be transcribed at a reduced level relative to transcription levels in the presence of an inducer. In certain instances, a factor may bind specifically to an inducible promoter to activate transcription, said factor being present in an inactive form and convertible (either directly or indirectly) to an active form by the inducer. The inducer may be a chemical/biochemical agent, such as a protein, metabolite (sugar, alcohol, etc.) a growth regulator, herbicide, or a phenolic compound. Alternatively, the inducer may be a directly imposed physiological stress (for example, heat, salt, wounding, toxic elements etc.) or an indirectly imposed physiological stress (for example, the action of a pathogen or disease agent, such as a virus). A plant cell containing an inducible promoter may be exposed to an inducer by external application of the inducer to the cell such as by spraying, watering, heating, or similar methods. Examples of inducible promoters include the inducible 70 kD heat shock promoter of Drosophila melanogaster (Freeling, M. et al, Ann. Rev. Genet. 19, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R.T. et al, in: Miflin, BJ. (ed.) Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3., pp. 384-438, Oxford Univ. Press, 1986). Examples of promoters that are inducible by a simple chemical are described in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/37609. A disadvantage of some inducible promoters of the prior art is the relatively slow rate at which they are induced upon application of the relevant inducer.

A further disadvantage of inducible promoters of the prior art is that they are generally not expressed throughout the different developmental stages of the plant, namely, the various stages encompassing development from embryo through to the mature plant.

Another disadvantage is that expression of some inducible promoters of the prior art is limited to certain plant cells/tissues/organs. Examples of this may be found in certain promoters from plant genes, such as certain members of the HD-Zip gene family. The HD-Zip promoter, Athb-8, from Arabidopsis (Baima et al, 1995, Development 121,

4171-4182) is a wound inducible promoter which is only expressed in procambium.

Similarly, the tomatoVahoxl promoter (Tornero et al, 1996, Plant J. 639-648) although not shown to be an inducible promoter, has only been found to be expressed in phloem during secondary growth. It is known that in plants the spread of some pathogens, such as fungi and bacteria is via the vascular system. Other pathogens, such as some insects (sucking pests or the like) gain access to the vascular system of a plant by means of a proboscis or the like. It would therefore be advantageous to have a promoter able to drive vascular-specific expression of an antipathogenic element. It is therefore an aim of the present invention to provide an inducible promoter which alleviates some of the aforementioned problems.

It is a further aim of the present invention to provide a promoter able to drive vascular-specific expression in plants.

A further aim of the present invention is to provide an inducible promoter which can be expressed throughout the different developmental stages of a plant, namely, the various stages encompassing development from embryo through to the mature plant.

It is a further aim of the present invention to provide a promoter that can be induced more rapidly than some inducible promoters of the prior art.

It is a further aim of the present invention to provide fragments of DNA comprising the promoter according to the present invention. It is a further aim of the present invention to provide transgenic plants (or parts and/or seeds thereof) comprising the promoter according to the present invention, including plants (or parts of plants) and seeds derived from said transgenic plants.

The Oshoxl gene derived from rice has previously been characterised and sequenced (Meijer et al, The Plant Journal (1997)11(2), 263-276). Studies carried out suggest that Oshoxl has a role in developmental regulation. Furthermore, Oshoxl mRNA was detectable by RNA blot analysis in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings and leaves of mature plants.

Summary of the Invention

The present invention provides inducible promoters able to drive vascular- specific expression in plants. The promoter according to the invention is advantageously inducible upon wounding of the plant. Further factors which cause induction of the promoter include, for example, hormones and sucrose. The promoter according to the present invention is a HD-Zip (homeodomain leucine zipper) gene promoter from rice and has advantageously been shown to drive vascular-specific expression in rice and Arabidopsis, in all organs and at all developmental stages. The gene driven by this promoter has been shown to be involved in the formation of vascular tissue by inducing the cell fate commitment. Furthermore, the promoter according to the present invention has been shown to be active in guard cells, pollen and trichomes of the shoot and flower.

As known to persons skilled in the art, the spread of some pathogens, such as fungi and bacteria occurs via the plant vascular system. Furthermore, certain insects, when attacking a plant, penetrate the plant surface by means of a proboscis or the like giving the insect direct access to the plant vascular system. Therefore, the promoter according to the present invention may be used to express, in a plant, genes conferring resistance to various pathogens. Introduction of the promoter of the invention into plant systems confers several advantages, as detailed throughout the application. The promoter according to the present invention is inducible upon wounding (for example, wounding by penetration of the plant surface by the insect) and may be used to drive expression (in the vascular system of a plant) of an element toxic to the insect but non-toxic to the plant. Examples of such elements include insecticidal peptides selected from, for example, mushroom fruitbodies or from the genus Paecilomyces or the genus Beauveria. Further examples include insecticidal peptides such as, lectins, serpins and haemolycins. Furthermore, certain fungi, such as Fusarium oxysporum, Verticillium dahliae,

Verticillium albo-atrum, Ceratocystis are known to infect and spread through the vascular system of a plant, therefore, the promoter according to the present invention could be used to drive expression of an element toxic to the fungi. Examples of such elements include antifungal proteins selected from chitinases, glucanases, osmotins, magainins, lectins, saccharide oxidase like hexose oxidases, oxalate oxidase, oxalate decarboxylase, toxins from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and Impatiens and albumin-type proteins, such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase. The promoter according to the present invention may also be used to drive expression of various phytoalexins or saponins.

Furthermore, the promoter according to the present invention may be used to drive vascular expression of an element toxic to various bacterial pathogens. Examples of such elements include antibacterial agents selected from T4 lysozyme, AX protein from sugar beet, thionines. Ace-AMPl, Allium cepa and magainins. Bacterial pathogens that attack the vascular system of plants include Clavibacter, Erwinia, Pseudomonas and Xanthomonas.

Furthermore, the promoter according to the present invention has been found to be active in guard cells and trichomes. The promoter according to the present invention may be used to drive expression, in guard cells and trichomes, of antipathogenic elements, thereby enhancing the natural defence role of the guard cells and trichomes.

Furthermore, the promoter according to the present invention may be used to drive expression of genes involved in the regulation of vascular formation and differentiation. Further advantageously, in the case of rice and other cereals, expression does not take place in the part of the plant that is typically consumed (GUS expression was not found in the endosperm). This is a factor that may help lead towards consumer acceptance of such engineered crops.

According to the present invention, there is provided a DNA fragment upstream of the rice Oshoxl gene or portions or variants of said DNA fragment wherein said DNA fragment or said portions or variants thereof are capable of promoting vascular expression of an associated DNA sequence on reintroduction into a plant.

The DNA fragment according to the present invention is further characterised in that it comprises the nucleotide sequence represented by SEQ ID NO:l.

SEQ ID NO:l

1 GAGCTCCGAA ATCAATGCTT TGTATATTTT GCATGTACTA TTAAATCTGA TATATATACC

61 ATAAATAAAT ACGCTTTTAT TTCGTAGATG GTAAGAATTT TCACTATTAA TTCTAAGTGA

121 GGTAATCTCT CCTTCAAAGA AAGAAAATAC TCATACCTAC TATCCATACA ACCAACCAAA

181 CAAAATCTCT TTCAAACCGT ATCTTTGGTG CAAGCAACCA AGCAGAATCT CACGCTAACC 241 TGGCTAAACC AATACAACCA ACCAAACGGT TGCATATGTA TCTACCTAGC CAGGCCAAAC

301 TCAGCCTTGA TGGAAGAGAT GGGCCAAGCT AAAACAATGC AAGCAACCAA ACACACCCTG

361 ATTCTGAGAA GCATTCCAGT TTTTGACTTC TAGTTTACAA CTACAGCTAT AGAATCTAGA

421 AAAAACTTAG ACTGTTTGAG GAGCTTCTGA TTTCTGAGAG AAGCTGCAAC AGTTAAAAAC

481 TCCTCTAAAC TGGTCCATAG TTTTCAGTGG TCAAAAGTGA GATTATACAT GAGGTTAAAC 541 AAATAAT TA CTATTCATTA ATACTTGGAG AGAACAAGGA GAGGAGAAAA CTAGGGGAAT

601 GAGAGAGTAT TATTAGTTCG ATGGTTAAAA AAATAAATAA AAAGATGAAA TAGTAAATAG

661 TATTAAGGAA GAAAAATATT TTTTGTGAGC TAAAATTTTA AACTATAATT AGATTTTCTT

721 TTGGACAAGG AGATTAGAGA TTAATGTTTG GGAAAGACTC TGCTGGTATC TCTCATTT C

781 GGAGGAATTG AGGGTGATTG GATAAGATAA AAAGAAAGAT AATCAAATAG ACAAATAATA 841 CTACTCTCTA TTTAAGAT T AATAGTACAT AGAAAAATAG GGTTAGGGGA GTATAAAAAA

901 ATCTTAACAT TTGTGAATAA ACATAACACA CACTAGGAAA AATGTTTCAC TATATTAGTA

961 GACAAAGTGG ATAAGCCTCA GGAGATATAC TTGATAGTCC AATGATAAAA AGACGTGTGG

1021 TTTCAACCTT AAGAATCCTC AACACGATTA TAATTTTTTC TTAAAAAATA TTTGGAGGGG

1081 CGTTTCTCTC TATATATTTT TTTATTTTTT TAGCGTAAAG CCGTAAAACC TCAGGCTGCG 1141 AGCTATGGGC AGGCTCAACT GAAGGCATGG CCAGGAACCA GGGTCAAAGG AGCAACAAGT

1201 CATGTCCGGT GGGCCCCACC CGGCCCCAAC CCCGGGGGAC AAGTCAATGA GTGCCCCCGC

1261 TCCCTCCGAA CAGGAGAAGC TGCTCATCAT TTGCGCTGCG ACGCTGACGC GTGCGCCCAT 1321 TCCACGCAAA AGAATACTAG CAGCCCATGC ATGCACAATA CTACTACTAC TAGTATTTTC

1381 TTTGACTTTT CCTGCACCCC ACAAAATATC GCTTTTGATC TAATCTCCTC CTTAATTACC 1441 CCCTCTAATC AAGCTCCCCT GTCTCCCCCA CGCTTTAAAA GCTTCCTCCT CCTCCTCGCC

1501 AACCACCACC CTCAGTCCCT CACCTCCATT TCCATCAAGC CCAAGAACGA ACTCACCACG 1561 CTAGTGTTGA GAAGTGGAGA GAGTTGAGAG GAATC According to the present invention, a portion of said DNA fragment comprises a contiguous block of about 10% or more of SEQ ID NO:l. said portion being capable of promoting vascular expression of an associated DNA sequence when reintroduced into a plant. Furthermore, a variant of said DNA fragment encompasses a sequence showing homology to SEQ ID NO:l, said variant being capable of promoting vascular expression of an associated DNA sequence when reintroduced into a plant and said variant being able to hybridize to SEQ ID NO: 1 or a portion thereof under stringent conditions. In this case, stringent conditions are typically reactions at a temperature of between 60°C and 65°C in 0.3 strength citrate buffer saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffer saline containing 0.1 % SDS.

According to a preferred feature of the present invention, the portion of said DNA fragment comprises SEQ ID NO:2. SEQ ID NO:2 below consists of nucleotides 1214- 1233 of SEQ ID NO:l. This portion is thought to be responsible for the binding of Myb proteins, thereby producing the vascular specificity of the promoter according to the present invention.

SEQ ID NO:2

CCCCACC CGGCCCCAAC CCC 2 0

SEQ ID NO:3 shown below is a fragment of the Oshoxl gene which comprises the promoter according to the present invention.

SEQ ID NO:3 1 GAGCTCCGAA ATCAATGCTT TGTATATTTT GCATGTACTA TTAAATCTGA TATATATACC

61 ATAAATAAAT ACGCTTTTAT TTCGTAGATG GTAAGAATTT TCACTATTAA TTCTAAGTGA

121 GGTAATCTCT CCTTCAAAGA AAGAAAATAC TCATACCTAC TATCCATACA ACCAACCAAA

181 CAAAATCTCT TTCAAACCGT ATCTTTGGTG CAAGCAACCA AGCAGAATCT CACGCTAACC

241 TGGCTAAACC AATACAACCA ACCAAACGGT TGCATATGTA TCTACCTAGC CAGGCCAAAC 301 TCAGCCTTGA TGGAAGAGAT GGGCCAAGCT AAAACAATGC AAGCAACCAA ACACACCCTG

361 ATTCTGAGAA GCATTCCAGT TTTTGACTTC TAGTTTACAA CTACAGCTAT AGAATCTAGA

421 AAAAACTTAG ACTGTTTGAG GAGCTTCTGA TTTCTGAGAG AAGCTGCAAC AGTTAAAAAC 481 TCCTCTAAAC TGGTCCATAG TTTTCAGTGG TCAAAAGTGA GATTATACAT GAGGTTAAAC

541 AAATAATATA CTATTCATTA ATACTTGGAG AGAACAAGGA GAGGAGAAAA CTAGGGGAAT

601 GAGAGAGTAT TATTAGTTCG ATGGTTAAAA AAATAAATAA AAAGATGAAA TAGTAAATAG

661 TATTAAGGAA GAAAAATATT TTTTGTGAGC TAAAATTTTA AACTATAATT AGATTTTCTT 721 TTGGACAAGG AGATTAGAGA TTAATGTTTG GGAAAGACTC TGCTGGTATC TCTCATTTAC

781 GGAGGAATTG AGGGTGATTG GATAAGATAA AAAGAAAGAT AATCAAATAG ACAAATAATA

841 CTACTCTCTA TTTAAGA AT AATAGTACAT AGAAAAATAG GGTTAGGGGA GTATAAAAAA

901 ATCTTAACAT TTGTGAATAA ACATAACACA CACTAGGAAA AATGTTTCAC TATATTAGTA

961 GACAAAGTGG ATAAGCCTCA GGAGATATAC TTGATAGTCC AATGATAAAA AGACGTGTGG 1021 TTTCAACCTT AAGAATCCTC AACACGATTA TAATTTTTTC TTAAAAAATA TTTGGAGGGG

1081 CGTTTCTCTC TATATATTTT TTTATTTTTT TAGCGTAAAG CCGTAAAACC TCAGGCTGCG

1141 AGCTATGGGC AGGCTCAACT GAAGGCATGG CCAGGAACCA GGGTCAAAGG AGCAACAAGT

1201 CATGTCCGGT GGGCCCCACC CGGCCCCAAC CCCGGGGGAC AAGTCAATGA GTGCCCCCGC

1381 TTTGACTTTT CCTGCACCCC ACAAAATATC GCTTTTGATC TAATCTCCTC CTTAATTACC

1441 CCCTCTAATC AAGCTCCCCT GTCTCCCCCA CGCTTTAAAA GCTTCCTCCT CCTCCTCGCC

1501 AACCACCACC CTCAGTCCCT CACCTCCATT TCCATCAAGC CCAAGAACGA ACTCACCACG

1561 CTAGTGTTGA GAAGTGGAGA GAGTTGAGAG GAATCAATGG AGATGATGGT TCATGGGAGG 1621 AGAGACGAGC AGTATGGCGG GCTCGGGCTC GGGCTTGGGC TTGGGCTCAG CCTCGGCGTC

1681 GCCGGTGGTG CAGCCGACGA CGAGCAGCCG CCGCCGCGCC GTGGTGCCGC CCCGCCGCCG

1741 CAGCAGCAGC TGTGCGGCTG GAACGGCGGC GGTCTCTTCT CCTCGTCTTC CTCCGGTGAG

1801 TATAGATGGA CGGACTTACG TCGAAGTCGT GCGTGCATGC ATGGATGCAT GGATCGATCT

1861 TACGAAGGTT AGTTTGCGTG CAGATCATCG GGGGAGGTCG GCGATGATGG CGTGCCACGA 1921 CGTCATCGAG ATGCCGTTCC TACGGGGGAT CGACGTGAAC CGTGCGCCGG CGGCAGAGAC

1981 GACCACGACG ACGGCGAGGG GGCCCAGCTG CAGCGAGGAA GACGAGGAGC CCGGCGCGTC

2041 CTCCCCCAAC AGCACGCTCT CCAGCCTCAG CGGCAAGCGC GGCGCACCAT CTGCCGCCAC

2101 CGCCGCCGCC GCCGCCGCCA GCGACGACGA GGACTCCGGC GGCGGATCC

Nucleotides 1-1596 comprise the promoter and leader sequence as represented by

SEQ ID NO: l ; nucleotides 1597-1784 comprise an Oshoxl coding sequence; nucleotides

1785-1872 comprise the first intron; and nucleotides 1873-2149 comprise an Oshoxl coding sequence (part of second exon). Furthermore, nucleotides 1525-2149 (except for the intron from 1785-1872) correspond to the 5' end of the Oshoxl mRNA.

The present invention further includes a chimeric DNA sequence comprising, in the direction of transcription, at least one DNA fragment (or portion or variant thereof) as hereinbefore described and at least one DNA sequence to be expressed under the transcriptional control of said DNA fragment, wherein the DNA sequence to be expressed is not naturally under the transcriptional control of the DNA fragment.

According to a preferred embodiment of the present invention, there is provided a chimeric DNA sequence wherein said DNA sequence to be expressed causes the production of an antipathogenic element including antifungal proteins selected from chitinases, glucanases, osmotins, magainins, lectins, saccharide oxidase like hexose oxidases, oxalate oxidase, oxalate decarboxylase, toxins from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and Impatiens and albumin-type proteins, such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase and/or insecticidal peptides from, for example, mushroom fruitbodies or from the genus Paecilomyces or the genus Beauveria or insecticidal peptides such as, lectins, serpins and haemolycins and/or an antibacterial agent selected from T4 lysozyme, AX protein from sugar beet, thionines, Ace-AMPl. Allium cepa and magainins.

The present invention further provides replicons comprising the abovementioned chimeric DNA sequences.

Also included in the present invention are microorganisms containing such a replicon, plant cells having incorporated into their genome, a chimeric DNA sequence as described above and plants essentially consisting of said cells. Such a plant may be a dicotyledonous plant or a monocotyledonous plant. Also parts of said plants selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood, form part of the invention. According to a further aspect of the present invention, there is provided use of a chimeric DNA sequence in the transformation of plants and use of a portion or variant of the DNA fragments according to the invention for making hybrid regulatory DNA sequences.

A further aspect of the present invention provides use of a chimeric DNA sequence as described above for conferring pathogen resistance to a plant. According to a further aspect of the present invention, there is provided a method for making a plant resistant to insects and/or fungi and/or bacteria, which method comprises introducing into said plant a DNA fragment capable of promoting vascular expression of an element toxic to any or all of insects, bacteria and fungi.

Description of the Figures

The present invention will now be described with reference to the following Figures which are by way of example.

Figure 1 is a schematic representation of a 35S-GUS reporter gene construct. pCambia-derived binary vectors harbouring the reporter gene constructs were used for Agrobacterium -mediated transformation of rice and Arabidopsis. The promoter and the leader sequences of the CaMV 35S promoter were fused to the ATG start codons (within Ncol site: CCATGG) of the GUS reporter gene.

Figure 2 is a schematic representation of a pOshoxl-GUS reporter. The 35S promoter represented in Figure 1 was replaced by Osho l promoter. For this, the l .όkb region upstream of the ATG start codon of the Oshoxl start codon was used. The Oshoxl start codon was modified to an Ncol site to fuse it to the GUS reporter gene.

Figure 3 is an illustration of vascular expression of the promoter according to the invention in various parts of the rice plant, namely, the root of the plant (Figure 3a), leaf (Figure 3b), stomatal guard cells (Figure 3c), pollen (Figure 3d), floral organs (Figure 3e), spikelet (Figure 30, leaf sheath (Figure 3g), trichomes in a leaf (Figure 3h), anther (Figure 3i), auricle (Figure 3j) and embryo (Figure 3k). Intense GUS staining can be seen in vascular regions of the plant. Figures 3c, 3d and 3h illustrate expression of the promoter in non-vascular areas, namely, guard cells, pollen and trichomes. Figure 4 is an illustration of vascular expression of the promoter according to the invention in various parts of the Arabidopsis plant. Figure 4a is an illustration of vascular expression in embryo, Figure 4b in flower, Figure 4c in leaf and Figure 4d is an illustration of expression in a root of Arabidopsis. Again, intense GUS staining can be seen in vascular regions in the various parts of the Arabidopsis plant, indicating vascular specific expression. Figure 5 illustrates expression of the Oshoxl promoter upon wounding of the plant. The mesophyll cells between the two vascular bundles shown were wounded using a needle; expression was observed after about 30 minutes following wounding. Intense

GUS staining is shown in vascular cells and also in mesophyll cells surrounding the wounded area.

Figure 6 is a comparative illustration of expression driven by the 35S promoter and the Oshoxl promoter. Vascular specific expression can be seen for the Oshoxl promoter. Figure 7 is a dendrogram representing the evolutionary relationships between members of the HD-Zip family. The pairwise alignments of sequences and clusters of sequences that together generate the final alignment of HD-Zip promoter family members is shown. The distance along the horizontal axis is proportional to the differences between the sequences. The distance along the vertical axis has no significance.

Detailed Description of the Invention The present invention primarily concerns promoters or regulatory sequences naturally occurring in rice. It has been found that upon wounding, genes under the regulatory control of these promoter or regulatory sequences are newly expressed or show enhanced expression, indicating wound inducibility.

The terms "regulatory sequence" or "regulatory region" and "promoter" are used interchangeably herein.

The present invention further provides chimeric DNA sequences comprising the DNA fragments of the present invention. The expression chimeric DNA sequence, as used herein, shall encompass any DNA sequence comprising DNA sequences not naturally found. For instance, chimeric DNA, as used herein, shall encompass DNA comprising the regulatory region which is inducible in a non-natural location of the plant genome, notwithstanding the fact that said plant genome normally contains a copy of said regulatory region in its natural chromosomal location. Similarly, said regulatory region may be incorporated into a part of the plant genome where it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector. The term "chimeric DNA", as used herein, shall not be limited to DNA molecules which are replicable in a host, but shall also encompass DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor sequences, physically linked to the regulatory region according to the invention. The regulatory region may or may not be linked to its natural downstream open reading frame. The open reading frame of the gene whose expression is driven by the wound- inducible regulatory regions of the invention may be derived from a genomic library. In this situation, it may contain one or more introns separating the exons making up the open reading frame that encodes a protein according to the invention. The open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention. Chimeric DNA sequences according to the invention also comprise those in which one or more introns have been artificially removed or added. Each of these variants is embraced by the present invention.

In order to be capable of being expressed in a host cell, a regulatory region according to the invention will usually be provided with a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ribosome recognition and attachment. In eukaryotic cells, an expression cassette usually also comprises a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur. Also, it is often the case that a signal sequence may be encoded, which is responsible for the targeting of the gene expression product to subcellular compartments. The principles governing the expression of a chimeric DNA construct, in a chosen host cell, are commonly understood by those of ordinary skill in the art. Furthermore, the construction of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic.

In order for the chimeric DNA sequence to be maintained in a host cell, it will usually be provided in the form of a replicon comprising said chimeric DNA sequence (according to the invention) linked to DNA which is recognised and replicated by the chosen host cell. Accordingly, the selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary person skilled in the art.

A special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame to the plant cell. Replicons with such capability are herein referred to as vectors. An example of such vector is a Ti-plasmid vector which, when present in a suitable host, such as Agrobacterium tumefaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell. Different types of Ti-plasmid vectors (vide: EP 0 116 718 Bl ) are now routinely being used to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes. A particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 Bl and US 4,940,838). Other suitable vectors, which may be used to introduce DNA according to the invention into a plant host, may be selected from the viral vectors, for example, non-integrative plant viral vectors, such as derivable from the double stranded plant viruses (for example, CaMV) and single stranded viruses, gemini viruses and the like. The use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines.

The expression "host cells incorporating a chimeric DNA sequence according to the invention in their genome" shall encompass cells and multicellular organisms comprising or essentially consisting of such cells which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis. According to a preferred embodiment of the invention, plants are provided which essentially consist of cells which incorporate one or more copies of said chimeric DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion. By virtue of the transcription and translation of the chimeric DNA of the invention in some or all of the plant's cells, those cells that comprise said regulatory region will respond to wounding and thus produce the protein encoded by the open reading frame which is under control of the regulatory region. In specific embodiments of the invention, this protein will be an antipathogenic protein capable of conferring resistance to pathogen infections.

As is well known to those skilled in the art, regulatory regions of plant genes consist of disctinct subregions with interesting properties in terms of gene expression. Examples of such subregions include enhancers and silencers of transcription. These elements may work in a general (constitutive) way, or in a tissue-specific manner. Deletions may be made in the regulatory DNA sequences according to the invention, and the subfragments may be tested for expression patterns of the associated DNA. Various subfragments so obtained, or even combinations thereof, may be useful in methods of engineering pathogen resistance, or other applications involving the expression of heterologous DNA in plants. The use of DNA sequences according to the invention to identify functional subregions, and the subsequent use thereof to promote or suppress gene expression in plants is also encompassed by the present invention.

Furthermore, it is generally believed that use of a transcriptional terminator region enhances the reliability as well as the efficiency of transcription in plant cells. Use of such a region is therefore preferred in the context of the present invention.

Examples of proteins that may be used in combination with the regulatory region according to the invention include, but are not limited to, β-l,3-glucanases and chitinases which are obtainable from barley (Swegle M. et al, Plant Mol. Biol. 12, 403-412, 1989; Balance G.M. et al. Can. J. Plant Sci. 56, 459-466, 1976 ; Hoj P.B. et al, FEBS Lett. 230, 67-71. 1988; Hoj P.B. et al. Plant Mol. Biol. 13, 31-42, 1989). bean (Boiler T. et al, Planta 157, 22-31, 1983; Broglie K.E. et al, Proc. Natl. Acad. Sci. USA 83, 6820-6824, 1986: Vδgeli U. et al, Planta 174, 364-372, 1988); Mauch F. & Staehelin L.A., Plant Cell 1, 447-457, 1989); cucumber (Metraux J.P. & Boiler T., Physiol. Mol. Plant Pathol. 28, 161-169, 1986); leek (Spanu P. et al, Planta 177, 447-455, 1989): maize (Nasser W. et al, Plant Mol. Biol. 11, 529-538, 1988), oat (Fink W. et al, Plant Physiol. 88, 270-275, 1988), pea (Mauch F. et al, Plant Physiol. 76, 607-611, 1984; Mauch F. et al, Plant Physiol. 87, 325-333, 1988), poplar (Parsons, TJ. et al, Proc. Natl. Acad. Sci. USA 86. 7895-7899. 1989), potato (Gaynor J.J., Nucl. Acids Res. 16, 5210, 1988; Kombrink E. et al, Proc. Natl. Acad. Sci. USA 85, 782-786, 1988; Laflamme D. and Roxby R., Plant Mol. Biol. 13, 249-250, 1989), tobacco (e.g. Legrand M. et al, Proc. Natl. Acad. Sci. USA 84, 6750-6754, 1987; Shinshi H. et al. Proc. Natl. Acad. Sci. USA 84, 89-93, 1987), tomato (Joosten M.H.A. & De Wit P.J.G.M., Plant Physiol. 89, 945-951, 1989), wheat (Molano J. et al, J. Biol. Chem. 254, 4901-4907, 1979), magainins, lectins, toxins isolated from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa (EP 0 576 483) and Amaranthus (EP 0 593 501 and US 5,514,779), albumin-type proteins (such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase, EP 0 602 098), proteins isolated from Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus and Clitoria (EP 0 603 216), oxalate oxidase (EP 0 636 181 and EP 0 673 416), saccharide oxidase (PCT/EP 97/04923), antimicrobial proteins isolated from Allium seeds and proteins from Aralia and Impatiens (WO 98/13478) and the like.

Application of the present invention is advantageously not limited to certain plant species. Any plant species may be transformed with chimeric DNA sequences according to the invention, allowing the regulatory region to be induced upon wounding, thereby triggering, for example, production of antipathogenic proteins to be produced in some or all of the plant's cells. The promoter according to the invention, although isolated from a monocotyledonous species (rice), has been demonstrated herein to also be functional in a dicotyledonous species (Arabidopsis).

Although some of the embodiments of the invention may not be practicable at present, for example, because some plant species are as yet recalcitrant to genetic transformation, the practising of the invention in such plant species is merely a matter of time and not a matter of principle, because the amenability to genetic transformation as such is of no relevance to the underlying embodiment of the invention.

Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyledoneae as well as the Monocotyledoneae. In principle, any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell, as long as the cells are capable of being regenerated into whole plants. Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al, Nature 296, 72-74, 1982; Negrutiu I. et /„ Plant Mol. Biol. 8, 363-373, 1987), electroporation of protoplasts (Shillito R.D. et al., Bio/Technol. 3, 1099-1102, 1985), microinjection into plant material (Crossway A. et al, Mol. Gen. Genet. 202, 179-185, 1986), DNA (or RNA-coated) particle bombardment of various plant material (Klein T.M. et al, Nature 327, 70. 1987), infection with (non-integrative) viruses and the like. A preferred method according to the invention comprises Agrobacteriwn- ediated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,940,838. A further preferred method for transformation is the floral dip method essentially as described by Clough and Bent (1998) Plant J. 16: 735-743.

Tomato transformation is preferably essentially as described by Van Roekel et al (Plant Cell Rep. 12, 644-647, 1993). Potato transformation is preferably essentially as described by Hoekema et al. (Hoekema, A. et al, Bio/Technology 7, 273-278, 1989).

Generally, after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant expressible genes co- transferred with the nucleic acid sequence encoding the protein according to the invention, after which the transformed material is regenerated into a whole plant.

Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material. Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or electroporation

(Shimamoto et al., Nature 338, 274-276, 1989). Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar-gene, which encodes phosphinothricin acetyltransf erase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm, Plant Cell, 2, 603-618, 1990). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, Plant Mol. Biol. 3, 21-30, 1989). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil, Bio/Technol. 8, 429-434, 1990). The combination with transformation systems for these crops enables the application of the present invention to monocots.

Monocotyledonous plants, including commercially important crops, such as rice and corn are also amenable to DNA transfer by Agrobacterium strains (vide WO 94/00977; EP 0 159 418 Bl; Gould J, et al, Plant. Physiol. 95, 426-434, 1991).

Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis to monitor the presence of the chimeric

DNA according to the invention, copy number and/or genomic organization. Additionally or alternatively, expression levels of the newly introduced DNA may be undertaken, using Northern and/or Western analysis, techniques well known to persons having ordinary skill in the art. After the initial analysis, which is optional, transformed plants showing the desired copy number and expression level of the newly introduced chimeric DNA according to the invention may be tested for resistance levels against pathogens.

Alternatively, the selected plants may be subjected to another round of transformation, for instance to introduce further genes, in order to enhance resistance levels, or broaden the resistance.

Other evaluations may include the testing of pathogen resistance under field conditions, checking fertility, yield, and other characteristics. Such testing is now routinely performed by persons having ordinary skill in the art. Following such evaluations, the transformed plants may be grown directly, but usually they may be used as parental lines in the breeding of new varieties or in the creation of hybrids and the like.

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

B. Cross-pollination of transgenic plants each already capable of expressing one or more chimeric genes, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coupled to another selectable marker. The seed, obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the chimeric genes themselves. The plants obtained from the selected seeds can then be used for further crossing. In principle, the chimeric genes are not on a single locus and the genes may therefore segregate as independent loci.

C. The use of a number of a plurality of chimeric DNA molecules, for example, plasmids, each having one or more chimeric genes and a selectable marker. If the frequency of co-transformation is high, then selection on the basis of only one marker is sufficient. In other cases, the selection on the basis of more than one marker is preferred.

D. Consecutive transformation of transgenic plants already containing a first, second etc., chimeric gene with new chimeric DNA, optionally comprising a selectable marker gene. As in method B, the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci.

E. Combinations of the above mentioned strategies.

The actual strategy may depend on several easily determined considerations, such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids). The actual strategy is not critical with respect to the described invention.

In this context it should be emphasised that plants already containing chimeric DNA may form a suitable genetic background for introducing further chimeric DNAs according to the invention, for instance in order to enhance the production antipathogenic substances, thereby enhancing resistance levels. The cloning of other genes corresponding to proteins that can suitably be used in combination with the regulatory DNA fragments, and the obtention of transgenic plants, capable of relatively over- expressing same, as well as the assessment of their effect on pathogen resistance in planta, is now within the scope of the ordinary skilled person in the art.

Plants with improved resistance against pathogens may be grown in the field, in the greenhouse, or at home or elsewhere. Plants or edible parts thereof may be used for animal feed or human consumption, or may be processed for food, feed or other purposes in any form of agriculture or industry. Agriculture shall encompass horticulture, arboriculture, flower culture, and the like. Industries which may benefit from plant material according to the invention include but are not limited to the pharmaceutical industry, the paper and pulp manufacturing industry, sugar manufacturing industry, feed and food industry, enzyme manufacturers and the like.

The present invention advantageously reduces the need for biocide treatment, thus lowering costs of material, labour, and environmental pollution. Further advantageously, shelf-life of products (for example, fruit, seed, and the like) may be extended. Plants for the purpose of this invention shall encompass multicellular organisms capable of photosynthesis, and subject to some form of pathogen attack. They shall at least include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants.

Another use of the promoter of the present invention is to influence the formation of vascular tissue in plants. As is shown, the promoter has been found to be highly active during provascular development, and when expressing the Oshoxl gene product it is even capable of causing anticipation of vascular differentiation and altering the timing of provascular cell fate commitment. This has been shown by placing the Oshoxl gene under control of the 35S promoter, which caused changes in the root apical region of the Oshoxl overexpressing plants and a clear cytological evidence of vascular differentiation in the precursor of the central large late metaxylem element at a distance of 0.5 mm from the root tip could be detected. This finding indicates that expressing Oshoxl ectopically in procambial cells that do not yet express the gene is sufficient to anticipate their entrance into a vascular differentiation pathway. This specific ability of the promoter can be used in applications to increase the amount of vascular tissue in plants. This can be done by either transforming the plants with a construct comprising both the Oshoxl promoter driving expression of the Oshoxl gene or by transforming plants with a chimeric construct in which the Oshoxl promoter is used to drive genes which are involved in signalling, cell cycling, cell elongation and/or cell division. Genes which can be used for this purpose are, for instance, genes involved in brassinosteroid biosynthesis and perception, such as det-2 (Fujioka S., et al., Plant Cell 9, 1951-1962, 1997) and BRI (Li, J. and Chory, J., Cell 90, 929-938, 1997); genes involved in sugar signalling, such as trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) (WO 97/42326), invertase, hexokinase and hexokinase inhibitors, sucrose synthase (SuSy); and genes involved in hormonal signalling, such as aba-insensitive genes, gibberellic acid insensitive genes and genes coding for enzymes in hormonal biosynthesis pathways.

Examples 1. Isolation and sequencing of the Oshoxl promoter region

Oryza sativa Indica IR58 total DNA, partially digested with Sau3Al, was cloned in λGEMl 1 (Promega) BamHl arms. Plaques of the amplified genomic library were lifted with nylon filters and hybridized with a 0.55 kb DNA fragment containing the 5' end of the Oshoxl cDNA up to the BamHl site (Meijer et al. 1997, Plant J. 11, 263-276). Hybridization conditions were as described in Memelink et al. (1994, Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, FL 1-23). Positive plaques were purified through additional rounds of plaque lifting and hybridization. DNA from purified phages was isolated with a lambda DNA purification kit (Qiagen). λ DNA was digested with SacV BamHl and subjected to Southern blot analysis with hybridization conditions as during library screening. From one of the positive lambda clones a 2149 bp fragment (SEQ ID NO:3) hybridizing to the Oshoxl cDNA probe was isolated and subcloned in pBluescript Et SK+ (Stratagene). The insert of resulting construct (pSK- OshoxlSB) was sequenced by the Double strand sequencing service of Eurogentec. This revealed that the fragment contained 1596 bp (SEQ ID NO: l) of promoter and leader sequence upstream of the Oshoxl ATG start codon.

2. Reporter gene constructs

In all constructs, the promoter and leader sequences of either the Oshoxl promoter (pOshoxl) or the CaMV 35S promoter were fused to the ATG start codons (within Ncol site: CCATGG) of the GUS or GFP reporter genes. There was no occurrence of polylinker sequences between the promoter and the reporter gene sequences which might have influenced expression pattern. Therefore, the promoter strength/patterns of pOshoxl and 35S are directly comparable. The comparison between the 35S and Oshoxl was made only in rice (see Figure 6). In Arabidopsis, only the pOshoxl constructs were examined, since 35S expression is well described, (i) 35S-GUS Reporter pCambia-derived binary vectors, pCAMBIA1301 (Roberts et al 1997, Rockefeller

Foundation Meeting of the International Program on Rice Biotechnology, 15-19 Sept. 1997, Malacca, Malaysia) harbouring the reporter gene constructs were used for

Agrobacterium-mediated transformation of rice and Arabidopsis. A diagrammatic representation of the 35S-GUS reporter is shown in Figurel.

(ii) pOshoxl-GUS Reporter

The 35S promoter in pCambial301 (as described above) was replaced by the Oshoxl promoter. In this case, the 1.6kb region upstream of the ATG start codon of the

Oshoxl start codon was used. To allow fusion of the Oshoxl promoter/leader region to reporter genes the sequence around the start codon (CAATGG) was modified to an Ncol site (CCATGG). To introduce this mutation a subfragment from pSK-OshoxlSB was amplified by PCR. The PCR product was digested with HindlWNcol and the resulting 116 bp fragment (containing the Oshoxl upstream region from the Hindlll site at position

1479 in the OshoxlSB sequence to the introduced Ncol site) was inserted in cloning vector pUC21 (Vieira and Messing, 1991 Gene 100, 189-194) resulting in construct pUC21-OshoxlHN. After verification (using sequence analysis) of the sequence of the cloned PCR product, the Oshoxl promoter/leader region was reconstructed by first cloning the 1479 bp 5' pOshoxl region as Sαcl/Hmdlll fragment from pSK-OshoxlSB in the corresponding sites of cloning vector pIC19R (Marsh et al, 1984, Gene 32, 481-485).

The fragment was then excised as HindTH/Bglll fragment and cloned between the

Hmdiπ/ββrøΗI sites of pUC21-OshoxlΗN, resulting in construct pUC21 -pOsho l.

From this construct, the reconstructed Oshoxl promoter/leader was cloned as SaWNcol fragment (Sail site is in vector polylinker) in binary vector pCambial391z (Roberts et al, Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, 15- 19 Sept. 1997, Malacca, Malaysia) to obtain the pOshoxl-GUS reporter construct. A diagrammatic representation of the pOshoxl-GUS reporter is shown in Figure 2. (iii) 35S-GFP Reporter A pCambia-based binary vector was constructed which was similar to pCambia

1301 except that it contained a 35S-GFP reporter gene in place of the 35S-GUS reporter gene. The 35S promoter in front of the GFP reporter gene was identical to that in front of the GUS reporter gene in pCambial301. The 35S promoter was excised as Sall/Ncol fragment from pCAMBIA1301 (Roberts et al, 1997, Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, 15-19 Sept. 1997, Malacca, Malaysia) and cloned into the corresponding sites of pCAMBIA1390-GFP. The GFP version used had a S65T mutation, a plant codon and contained a ST-LS1 intron (from vector pMON30063, Plant Phys, 1996, 112: 893-900). The fusion to the 35S promoter was made at the Ncol site containing the ATG start codon of the GFP gene. (iv) pOshoxl-GFP Reporter

This reporter was analogous to the 35S-GFP reporter except that the GFP gene is driven by the l.όkb Oshox promoter sequence which is the same as in the pCambia- pOshoxl-GUS construct. A NcoI/BamHI fragment containing the GFP coding region and nos terminator was excised from plasmid pMON30063 (Pang et al., Plant Physiol. 1996, 112: 893-900) and cloned in the Ncol/Bglll sites of pCAMBIA1390 (Roberts et al, Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, 15- 19 Sept. 1997, Malacca, Malaysia) resulting in construct pCAMBIA1390-GFP. The Oshoxl promoter/leader was then introduced as Sall/Ncol fragment from plasmid pUC21-pOshoxl (see (ii) above) into the corresponding sites of pCAMBIA1390-GFP.

3. Expression Pattern of the Oshoxl Promoter in Rice

Expression was monitored in 10 independent pOshoxl -GUS rice lines and in a similar number of pOshoxl-GFP lines (first generation transformants). The relevant developmental stages of all plant organs, throughout the whole life cycle of the plant, were examined. As detailed below, reporter gene activity was observed at all developmental stages and in parts of all plant organs. Reporter gene activity was specifically observed in the vascular system and was always apparent during the stage of vascular element differentiation from procambium derivatives. Three exceptions to the vascular-specificity were noted, namely, the guard cells of stomata, trichomes of the shoot and flower and pollen. Expression patterns observed with the GUS or GFP reporter gene constructs were identical. For a number of lines, second generation plants were analysed and shown to have the same expression characteristics as the first generation transform ants.

A. Expression at the organ level (i) Roots

Expression was found in the central stele (including pericycle) starting from the transition zone throughout the whole root (see Figure 3a). (ii) Leaves and stem

Expression was found in vascular bundles of leaf sheath, auricle, ligule, leaf blade and stem. Expression was also found in stomatal guard cells and in trichomes (see Figures 3b, 3c, 3g, 3h and 3j). (iii) Flowers

Expression was found in vascular bundles of sterile lemmas, palea, lemma, lodicules, ovary, style and stamen filament. Expression was also found in stomatal guard cells and trichomes of palea and lemma and in pollen (see Figures 3d, 3e, 3i, and 3k).

(iv) Embryos

Expression was found in vascular bundles of the scutellum and embryonic axis (see Figure 3k).

B. Expression at a cellular level

Reporter gene activity was observed in all the cell types in the vascular bundle: outer bundle sheath cells, inner bundle sheath cells, proto- and metaphloem sieve elements and companion cells, parenchymic cells of the phloem, protoxylem tracheary elements and lacuna, early and late metaxylem tracheary elements, thin and thick walled parenchymatic cells of the xylem. Although some of these cell types (sieve elements and xylem tracheary elements) are dead at maturity, GUS-staining was still observed in these cell types, which is explained by the stability of the GUS protein. C. Wound inducibility

Increased reporter gene activity was observed upon wounding. Mesophyll cells between vascular bundles were wounded using a needle and expression was observed approximately 30 minutes following wounding. Increased GUS activity was observed in the vascular cells near the wounded area. GUS activity was also found in mesophyll cells (expression of the promoter not normally being observed in these cells).

Expression Pattern of the Oshoxl Promoter in Arabidopsis

For both the pOshoxl-GUS and the pOshoxl-GFP construct, 10-20 independent

Arabidopsis lines (second generation transformants) were analysed. In all organs (roots, leaves and stem, flowers, siliques and embryos) the same expression pattern was observed as described for rice, except for petals in which the vascular bundles did not show GUS or

GFP expression (see Figures 4a, b, c and d).

4. Oshoxl expression at procambium development

Analysis of Osho l -GUS plants (as produced in the previous examples) revealed that the provascular and vascular-specific expression was absent from the most distal part of the root apex, although a prostele was there already clearly identifiable. This remarkable feature of Oshoxl expression would suggest that a hidden change in the developmental fate of procambial cells is taking place in such area of the central cylinder. Therefore, a selection of organs was examined to ascertain whether oshoxl expression could be associated with a specific cytological event during vascular differentiation.

In a series of root transverse sections, the first signs of Oshoxl expression was detected approximately 0.25 mm from root tip in provascular cells of the mitotic region. Expression started in the precursor of the central late metaxylem element, which is the first cell to undergo vascular differentiation. Subsequently, expression spread basipetally and radially to the rest of the prostelar elements, pericycle included, following the order in which they are known to commence vascular differentiation, but clearly before any cytological sign of this process could be recognised.

During lateral root formation Oshoxl was found to be down-regulated in pericycle cells which, upon dedifferentiation, resume their meristematic activity to give rise to lateral primordium. Expression reappeared as soon as a provascular strand was recognisable in lateral root primodium.

Also, analysis or provascular tissue in other rice organs revealed that Oshoxl expression invariably starts in procambial cells that do not yet show any cytological progression towards vascular differentiation.

Claims

CLAIMS:

1. A DNA fragment upstream of the rice Oshoxl gene or portions or variants of said DNA fragment wherein said DNA fragment or portions or variants thereof are capable of promoting vascular expression of an associated DNA sequence on reintroduction into a plant.

2. A DNA fragment according to claim 1 inducible upon wounding of said plant.

3. A DNA fragment according to claims 1 or 2, characterised in that said fragment comprises the nucleotide sequence represented by SEQ ID NO: l .

4. A portion of the DNA fragment according to any of claims 1 to 3. wherein said portion comprises the nucleotide sequence represented by SEQ ID NO:2.

5. A chimeric DNA sequence comprising in the direction of transcription: (i) a DNA fragment according to any of claims 1 to 4; and

(ii) a DNA sequence to be expressed under transcriptional control of said DNA fragment, which DNA sequence is not naturally under transcriptional control of said DNA fragment.

6. A chimeric DNA sequence according to claim 5. wherein said DNA sequence to be expressed causes production of an antipathogenic protein.

7. A chimeric DNA sequence according to claim 6, wherein said antipathogenic protein is an antifungal protein selected from chitinases, glucanases, osmotins, magainins, lectins, saccharide oxidase, oxalate oxidase, oxalate decarboxylase, toxins from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and Impatiens and albumin-type proteins, such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase and/or wherein said antipathogenic protein is an insecticidal protein selected from mushroom fruitbodies or from the genus Paecilomyces or the genus Beauveria or includes insecticidal peptides such as, lectins, serpins and haemolycins and/or wherein said antipathogenic protein is an antibacterial agent selected from T4 lysozyme, AX protein from sugar beet, thionines, Ace-AMPl, Allium cepa and magainins.

8. A chimeric DNA sequence according to claim 5, characterised in that the DNA sequence to be expressed codes for a protein involved in signalling, cell cycling, cell division and/or cell elongation.

9. A chimeric DNA sequence according to claim 8, characterised in that the DNA sequence to be expressed codes for a protein selected from the group essentially consisting of det-2, BRI-1, TPS, TPP. ABI-4, GAI, HXK and SuSy.

10. A replicon comprising a chimeric DNA sequence according to any of claims 5 to 9.

11. A microorganism containing a replicon according to claim 10.

12. A plant cell comprising a chimeric DNA sequence according to any of claims 5 to 9.

13. A plant comprising cells according to claim 12.

14. A part of a plant selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood, obtained from a plant according to claim 13.

15. Use of a DNA fragment according to any of claims 1 to 4 in identifying homologues capable of promoting wound-induced transcription and/or vascular expression in a plant.

16. Method of transforming plants using a chimeric DNA sequence according to any of claims 5 to 9.

17. Method of producing hybrid regulatory DNA sequences using a portion or variant of said DNA fragment according to any of claims 1 to 4.

18. Use of a chimeric DNA sequence according to any of claims 5 to 7 for conferring pathogen resistance to a plant.

19. Method for making a plant resistant to insects comprising introducing into said plant a DNA fragment capable of promoting vascular expression of an element toxic to insects.

20. Method for making a plant resistant to fungi comprising introducing into said plant a DNA fragment capable of promoting vascular expression of an element toxic to fungi.

21. Method for making a plant resistant to bacteria comprising introducing into said plant a DNA fragment capable of promoting vascular expression of an element toxic to bacteria.

22. A method according to any of claims 19 to 21, wherein said DNA fragment comprises the DNA fragment according to any of claims 1 to 4.

23. A method to induce vascular tissue formation in plants by transforming them with a gene coding for the Oshoxl protein.

24. A method according to claim 23, characterised in that the gene comprises the sequence as depicted in SEQ ID NO:3, basepairs 1597-1784 and 1873-2149.

25. A method according to claim 23 or 24, characterised in that the protein is under regulatory control of the DNA fragment according to any of claims 1-4.