WO2001048186A2

WO2001048186A2 - Induction of defence genes in plants transformed with mybnt1

Info

Publication number: WO2001048186A2
Application number: PCT/GB2000/004863
Authority: WO
Inventors: David Twell; Mehdi Barghchi; Sasan Amirsadeghi
Original assignee: University Of Leicester
Priority date: 1999-12-24
Filing date: 2000-12-18
Publication date: 2001-07-05
Also published as: AU2201901A; WO2001048186A3

Abstract

The present invention concerns methods of inducing plant defence genes, genetic constructs for effecting same, plants modified to have 'defence' phenotypes and contain such constructs, seed obtained by growing said plants, methods of conferring a 'defence' phenotype upon plants, plants obtained by said methods, and seed obtained by growing same.

Description

Induction of Plant Defence Genes

The present invention concerns methods of inducing plant defence genes, genetic constructs for effecting same, plants modified to have "defence" phenotypes and contain such constructs, seed obtained by growing said plants, methods of conferring a "defence" phenotype upon plants, plants obtained by said methods, and seed obtained by growing same.

In horticulture, it is extremely desirable to protect plants from attack by pathogens such as fungi and bacteria. The present invention succeeds in isolating a novel member (named NtmybPS 1 , also referred to as mybntl) of the myb family of plant genes, NtmybPS 1 normally being predominantly expressed in pollen and anthers, but which when expressed or over-expressed in other plant organs induces expression of plant defence genes - the PR (pathogenesis-related) genes, resulting in a "defence" phenotype, the extent of protection being dependent upon the level and location of expression of NtmybPS 1 (protection i.e. defence is essentially cell-autonomous).

NtmybPS 1 is disclosed in PCT/GB98/02010 as causing induction of a dwarf phenotype. However, PCT/GB98/02010 does not disclose that a defence phenotype may be achieved, and requires that genes, recombinant DNA constructs and methods using NtmybPS 1 all cause a dwarf phenotype.

The myb genes are a family of pathway regulating genes distinguished by a conserved DNA binding domain (Martin, C. and Paz-Ares, J., 1997, TIG, 11(2): 67- 73). Modified expression of myb family genes has previously been shown to affect pigment synthesis, cell shape and thus colour due to diffraction, root hair shape, timing of flowering, response to hormones during seed development and germination, and lignin synthesis. In the case of affected lignin synthesis (Tamagnone, L. et al., 1998, The Plant Cell, JO: 135-154), dwarfing was observed but it also resulted in premature senescence resulting in necrotic areas in older leaves, and also chlorosis (i.e. reduced chlorophyll content).

The present invention provides the opportunity to produce transgenic plants which express NtmybPS 1 for example in a constitutive, inducible, temporal or organ- specific manner, to effect induction of plant defence genes to effect e.g. a general induction of plant defences or for example organ-specific protection, or a general or specific enhancement of plant defences.

The methods, constructs, plants and seeds of the present invention are particularly advantageous insofar as they are able to provide for the protection of plants (including seeds) from pathogen attack without requiring the use of e.g. fungicides or other such chemical compounds which may have an uncertain or damaging effect upon the environment, particularly as they are concentrated in the higher regions of the food chain.

Although the genetic constructs used to cause induction of plant defences are generally synthetic, the genes which are induced are the plant's own defence genes.

According to the present invention there is provided a method of inducing plant defence genes to provide a "defence" phenotype (i.e. a method of making a plant which produces a defence phenotype), comprising the steps of: a) transforming or transfecting cells of a plant with a recombinant DNA construct comprising operatively linked in the 5' to 3' direction: i) a promoter region that directs the transcription of a gene in plants; ii) a DNA coding sequence encoding the expression product having the sequence of SEQ ID NO: 2; and iii) a 3 non-translated region which contains a polyadenylation signal for plants; b) selecting plant cells that have been transformed or transfected; c) regenerating plant cells that have been transformed or transfected to produce differentiated plants; and d) selecting a transformed or transfected plant which expresses said construct and which has a defence phenotype.

The "defence" phenotype of the present invention may take many forms, for example a constitutive enhancement of the expression of plant defence genes, a temporal or organ-specific expression (for example seed-specific expression or expression in tubers, vascular system or fruit) of plant defence genes. Similarly, expression could be activated by pathogens. The defence phenotype may also be inducible by an external stimulus, and thus the method may comprise the additional step of stimulating (for example by the administration of a stimulant) the selected transformed or transfected plant in order to induce the defence phenotype.

In particular the methods of the present invention may be such that they do not induce a dwarf phenotype in the selected transformed or transfected plant. This limitation (that a dwarf phenotype is not induced) may apply to all aspects of the present invention.

SEQ ID NO: 1 (Entrez accession AF 198499; GenBank 11066264) encodes an expression product having the sequence of SEQ ID NO: 2 (accession number AAG28526; GenBank 1 1066265). However, it is also possible to encode the same expression product using modified coding sequence, for example by substituting nucleotides whilst still encoding the same codons. Similarly it is possible to add or delete nucleotides to modify the coding sequence expression product. Thus the DNA coding sequence of the recombinant DNA construct is not limited solely to SEQ ID NO: 1, appropriate variants being readily apparent to a person skilled in the art, the results of the modification upon plant defence being readily determinable. The coding region of SEQ ID NO: 1 is located at nucleotides 182-1591 and thus it may have the sequence of nucleotides 182-1591 of SEQ ID NO: 1.

Also provided according to the present invention is a recombinant DNA construct whose expression in plants induces a plant defence phenotype, and comprising operatively linked in the 5' to 3' direction: i) a promoter region that directs the transcription of a gene in plants: ii) a DNA coding sequence encoding an expression product according to the present invention; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

The expression of the construct (i.e. the production of the expression product) may be in plant tissues other than pollen and/or anthers.

The DNA coding sequence in such a construct may have the sequence of SEQ ID NO: 1, or nucleotides 182-1591 of SEQ ID NO: 1.

The promoter region may comprise the CaMV35S promoter. The 3' non- translated region may comprise the 3' polyadenylation signal from the CaMV35S transcript.

Promoters useful in the induction of the defence phenotype of the present invention preferably possess one or more of the following properties: constitutive expression of inserted sequences throughout the plant; intermediate rate of expression of inserted sequences; organ-specific expression in stems and/or petioles and/or leaf sheathes; cell-specific expression in vascular tissues. These properties are defined as to drive expression of the inserted sequence(s) at a level that results in NtmybPS 1 stimulation to produce dwarfing when the modified plants are grown in the field.

Examples of promoters useful in the present invention include, inter alia, the cauliflower mosaic virus 35S (CaMV 35S) promoter, the maize polyubiquitin (ubl) promoter (Christensen, A.H. et al., 1992, Plant Mol. Biol. 18: 675-689), the Asparagus officinalis pathogenesis-related vascular-tissue-specific (AoPRl) promoter (Warner, S.A.J. et al., 1994, Plant Journal, 6: 31-43), the rice ribulose-bisphosphate carboxylase (rbcs) promoter (Kyozuka, J. et al., Plant Physiology, 102: 991-1000), and the maize shrunken- 1 promoter (Maas, C. et al, Plant Mol. Biol., 16: 199-207).

However, constitutive expression of the coding sequence typically results in a dwarf phenotype in addition to the defence phenotype. Dwarfing may not be desirable and so as discussed above, non-constitutive promoters which cause a specifically limited expression of the coding sequence may be more preferable, i.e. the invention extends to the use of promoters which result in the induction of the defence phenotype but which do not result in a dwarf phenotype.

Particularly useful are promoters which provide for an inducible (for example by chemical stimulants or pathogens) expression, organ-specific (for example fruit, tuber, vascular system or leaf-specific) expression, or temporally specific (e.g. seedling-specific, possibly specific to the "damping off stage) expression, particularly so when a dwarf phenotype is not induced in the plant transformed or transfected with recombinant DNA constructs containing them.

For example, inducible systems which may be employed in the present invention are alcohol-inducible systems such as the "AlcR" system (Zeneca) in which expression of a coding sequence is induced in a plant by spraying it with alcohol. Similarly, steroid-inducible systems are well known and may be readily employed. Pathogen-responsive promoters such as the PR promoter may be used, and can provide for a positive-feedback loop triggered by a pathogen - the PR promoter driving expression of NtmybPS 1 induces the PAL promoter (see "Experimental" section below) of other genes, inturn causing induction of PR (pathogen responsive) genes, providing a positive- feedback (i.e. autocatalytic) system.

Example of organ-specific promoters are fruit-specific promoters (for example in tomatoes, vine fruits etc.), root-specific promoters in e.g. potatoes, or promoters specific to the plant vascular system.

Temporally specific expression can be used to protect plants at particularly vulnerable stages of their life-cycle or at times when the most important or valuable growth occurs. For example, seeds are particularly vulnerable to fungal infection at the "damping off' stage and so a seedling-specific promoter can be used to induce a defence phenotype in seeds, whilst being temporally limited such that subsequent seedling growth is not accompanied by induction of a dwarf phenotype.

Since the defence phenotype induced in the present invention is essentially cell-autonomous, it need not result in dwarfing or in a wasting of valuable plant resources - if fruit is vulnerable but roots are not then it is possible to protect only the fruit.

As is shown in the experimental results given below (see Figure 2), PAL (phenylalanine ammonium lyase) mRNA levels and therefore PAL expression is enhanced in NtmybPS 1-overexpressing lines. It is known that PAL is upstream in the synthetic pathway for salicylic acid. The present invention also provides for up- regulating PAL expression in order to further enhance the downstream production of salicylic acid and to therefore further enhance the induction of the defence phenotype. This is achievec by incorporating in a construct according to the present invention, operatively linked in the 5' to 3' direction: i) a PAL promoter region; ii) a PAL coding sequence; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

Alternatively cells of a plant may be transformed or transfected with a recombinant DNA construct comprising operatively linked in the 5' to 3' direction: i) a PAL promoter region; ii) a PAL coding sequence; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

A number of PAL genes are known, for example PALA and gPALl (Fukasawa-Akada T. et al., Plant Mol Biol. 1996 Feb;30(4):71 1-22; PMID: 8624404; Pellegrini L. et al, Plant Physiol. 1994 Nov; 106(3):877-86; PMID: 7824656) and their promoters, coding sequences and 3' non translated regions can be used in the constructs of the present invention. In particular, Nicotiana PAL coding sequences and/or promoters can be used, especially those of Nicotiana tabacum.

Such a "dual cassette" provides for self-enhanced production of salicylic acid and also makes the induction of salicylic acid levels and of the defence phenotype independent of the ability of the myb gene product to regulate a plant's endogenous PAL gene.

Also provided according to the present invention are plants transformed or transfected with said genetic construct. Methods of transformation and transfection of both monocotyledonous and dicotyledonous plants are well known. Methods for producing transgenic plants in a variety of different monocots are currently available, and these methods are equally applicable to the present invention. Successful transformation and plant regeneration have been achieved in asparagus {Asparagus officinalis; Bytebier et al, 1987, Proc. Natl. Acad. Sci. USA 84: 5345); barley (Hordeum vulgarae; Wan and Lemaux, 1994, Plant Physiol. 104: 37); maize (Zea mays; Rhodes et al., 1988, Science 240: 204; Gordon-Kam et al, 1990, Plant Cell 2: 603; Fromm et al, 1990, Bio/Technology 8: 833; Koziel et al., 1993, Bio/Technology 1 1 : 194); oats (Avena sativa; Somers et al., 1992, Bio/Technology 10: 1589); orchardgrass (Dactylis glomerata; Horn et al., 1988, Plant Cell Rep. 7: 469); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al, 1988, Bio/Technology 6: 10; Zhang et al, 1988, Plant Cell Rep. 7: 379; Luo and Wu, 1988, Plant Mol. Biol. Rep. 6: 165; Zhang and Wu, 1988, Theor. Appl. Genet. 76: 835; Christou et al, 1991, Bio/Technology 9: 957); rye (Secale cereale; De la Pena et al, 1987, Nature 325: 274); sorghum (Sorghum bicolor; Cassas et al, 1993, Proc. Natl. Acad. Sci. USA 90: 11212); sugar cane (Saccharum spp.; Bower and Birch, 1992, Plant J. 2: 409); tall fescue (Festuca arundinacea; Wang et al, 1992, Bio/Technology 10: 691 ); turfgrass (Agrostis palustris; Zhong et al, 1993. Plant Cell Rep. 13: 1 ); wheat (Triticum aestivum; Vasil et al, 1992, Bio/Technology 10: 667; Troy Weeks et al, 1993, Plant Physiol. 102: 1077; Becker et al, 1994, Plant J. 5 : 299).

Methods for transforming and transfecting a wide variety of different dicots and obtaining transgenic plants are well documented in the literature (see Gasser and Fraley, 1989, Science 244: 1 93; Fisk and Dandekar, 1993, Scientia Horticulturae, 55: 5-36; Christou, 1994, Agro Food Industry Hi Tech (March/April 1994) p.17, and the references cited therein) and include for example electroporation, particle bombardment, silica fibres and PEG (polyethylene glycol)-mediated transfection, and can also be applied in the present invention. A DNA encoding the expression product of the present invention can be introduced into any of these dicotyledonous plants in order to produce transgenic plants that display dwarf phenotypes in the field. Plants may be agronomic crop plants, horticultural crop plants or ornamental crop plants. Agronomic and horticultural crop plants include cereals, non- cereal seed crops, root crops, vegetable crops and fruit crops. Cereal crops include wheat, rye, barley, oats, maize, buckwheat, sorghum, and rice; non-cereal seed crops include peas, beans, soybeans, oil-seed rape, canola, linseed, sunflower, and flax; root crops include potato, sweet potato, sugar beet, carrot, swede, and turnip; vegetable crops include asparagus, mustard, lettuce, tobacco, and cauliflower; horticultural crops include tomato, egg plant, cucumber, celery, melon, and squash; fruit crops include strawberry, blackberry, blueberry, apple, apricot, peach, pear, plum, orange, cranberry, and lemon. Additional crop plants include cotton and sugarcane. Ornamental plants include petunia, chrysanthemum, carnation, poinsettia, begonia, tradescantia and snapdragon.

Also provided is a plant produced according to the method of the present invention.

Also provided according to the present invention is seed obtained by growing a plant according to the present invention.

The invention will be further apparent from the following description, with reference to the accompanying Figures which shows, by way of example only, results obtained with primary transformants. Of the Figures:

Figure 1 shows SDS-PAGE analysis of total soluble leaf protein extracted from two transgenic lines (myb3 and myb7). WT = wild type tobacco extract. HET = heterozygote. HOM = homozygotes. L3 - L6 = leaf 3 to leaf 6. Far-left and far- right columns are molecular weight markers. Visible in the Figure is increased expression of an apparent 17 Kd protein, identified as being PRla. Also visible is the induction of expression of an apparent 27 Kd protein, identified as being Chitinase P; Figure 2 shows Northern blot analysis of total RNA extract from leaves of wild type (WT1, WT2), and two different NtmybPS 1 over-expressing lines (left and right). Hom = homozygous lines. Het = heterozygous lines. Hybridisation probes used were (from top to bottom) NtmybPS 1 , Chitinase P, PR- 1 a, and Phenylalanine ammonium lyase (PAL). Results show that chitinase P and PRla mRNAs are activated in Hom and Het plants and that expression level is positively correlated with the level of expression of NtmybPS 1. Homozygotes clearly show increased PAL mRNA expression; and

Figure 3 shows height data for primary transformants. Vertical axis is plant height (cm); horizontal axis is Transgenic myb lines, with "C" (far left column) indicating control.

Experimental

The following experiments detaii the isolation, purification and cloning of RNA encoded by gene NtmybPS 1 and the isolation and sequencing of NtmybPS 1 from a cDNA library. Transformed tobacco plants expressing NtmybPS 1 under the control of the CaMV35S promoter were found to express pathogen-responsive genes, namely PR-P (tobacco acidic chitinase P), PR- la (tobacco pathogenesis-related protein) and to have PAL (phenylalanine ammonium lyase) mRNA induced. Mybl is also found to be induced. NtmybPS 1 expression using a constitutive promoter (below) also resulted in dwarfing, which was used as a marker to isolate stable transformed plants. However, other embodiments of the invention need not cause induction of a dwarf phenotype.

Materials and Methods

Unless stated otherwise, all procedures were performed using standard protocols and following manufacturer's instructions where applicable. Standard protocols for various techniques including PCR, molecular cloning, manipulation and sequencing, are described in texts such as McPherson, M.J. et al (1991, PCR: A practical approach, Oxford University Press, Oxford), Sambrook, J. et al. (1989, Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory, New York), Huynh and Davies (1985, "DNA Cloning Vol is - A Practical Approach", IRL Press, Oxford, Ed. D.M. Glover), Sanger, F. et al. (1977, PNAS USA 74(12): 5463-5467), Harlow, E. and Lane, D. ("Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998), Jung, G. and Beck-Sickinger, A.G. (1992, Angew. Chem. Int. Ed. Eng., 31: 367-486), Harris, M.A. and Rae, I.F. ("General Techniques of Cell Culture", 1997, Cambridge University Press, ISBN 0521 573645).

Reagents and equipment useful in, amongst others, the methods detailed herein are available from the likes of Amersham (http://www.amersham.co.uk), Boehringer Mannheim (http ://www .boehringer- ingeltheim . com), C lontech (http://www.clontech.com). Genosys (http://www.genosys.com), Millipore (http://www.millipore.com), Novagen (http://www.novagen.com), Perkin Elmer (http://wvAv.perkinelmer.com), Pharmacia (http://www.pharmacia.com), Promega (http://www.promega.com), Qiagen (http://www.qiagen.com), Sigma (http://www.sigma- aldrich.com) and Stratagene (http://www.stratagene.com).

Induction of plant defence gene expression

1. Two proteins are highly expressed in NtmybPS 1 plants

SDS-PAGE (Sambrook. J. et al, 1989, Molecular cloning: A Laboratory Manual, 2nd Edn. Cold spring Harbor. NY, Cold Spring Harbor Laboratory Press) was used as a simple and rapid technique to identify changes in total protein profile of NtmybPS 1 over- expressing lines. Two major proteins, a ca. 17 and a ca. 27 Kd protein were identified to be highly expressed in NtmybPS 1 plants compared with wild type untransformed plants. The levels of induction of these proteins in the homozygote NtmybPS 1 plants were higher than heterozygotes (Figure 1).

2. The ca. 27 Kd induced protein is tobacco acidic chitinase P (PR-P) The complete sequence of a tryptic peptide from the ca.27 Kd protein in NtmybPS 1 plants was determined using Edman degradation and was found to consist of the following 13 amino acids GPIQLTNQNN YEK (SEQ ID NO: 3). This sequence was used to search for sequence similarities using the BlastP algorithm and showed an exact match with the amino acids 123-135 of the tobacco acidic chitinase (PR-P) with the trypsin cleavage sites at arginine (amino acid 122) and lysine (amino acid 135). This short motif corresponds to the nucleotides 403-441 of tobacco acidic chitinase P in the lambda cht28 clone (Payne. G. et al. 1990. PNAS USA, 87: 98- 102). PR-P and its acidic isoform. PR-Q proteins consists of 253 amino acid residues with a 24 amino acid signal peptide. The mature PR-P and PR-Q proteins consist of 229 amino acid with calculated masses of 24.859 and 25.033 Kd respectively. The estimated molecular weight of the induced protein (ca. 27 Kd) from the SDS gel was in good agreement with the molecular weight of the identified PR-P protein.

3. Chitinase P (PR-P) mRNA is induced in NtmybPS 1 plants

To determine whether PR-P is activated in mybPS l plants, a 0.79 kb PR- P probe was prepared by reverse transcription and PCR amplification of PR-P RNA using two oligonucleotide primers with the following sequences: 5'-GAGAGGAAAA TGGAGTTTTC TGGATCACCA C -3' (SEQ ID NO: 4) and 5'-CATAACATGA TCTAACGAAT CCTAGCCTTG GG -3' (SEQ ID NO: 5) corresponding to the nucleotides 28-58 (coding strand) and 788-819 (complementary strand) of the PR-P in lambda cht28 clone (Payne et al, 1990, supra).

A Northern blot was prepared according to standard methods (Sambrook et al, 1989, supra) from RNA isolated from leaves of wild type, homozygote and heterozygote NtmybPS 1 plants. This blot was probed with the 0.79 kb PR-P cDNA. The results showed that PR-P mRNA is induced in a gene dosage dose dependent manner according to the level of over-expression of NtmybPS 1 (Figure 2). Homozygote NtmybPS 1 plants (lanes 3 and 7; Figure 2; left and right) showed a greater level of expression of PR-P than heterozygotes (Figure 2). The level of expression of PR-P was positively correlated with the level of expression of NtmybPS 1 (Figure 2).

4. The ca. 17 Kd protein is the tobacco pathogenesis related protein, PR- la

An incomplete sequence of a tryptic peptide from the ca. 17 Kd induced protein in NtmybPS 1 plants consisted of 14 amino acid residues was determined as ADVGVEPLTW DDQV (SEQ ID NO: 6). This 14 amino acid sequence showed an exact match to amino acids 46-59 of tobacco pathogenesis-related protein la (PRla). The PR-la, PR-lb and PR-lc are highly homologous with a protein weight of 15 Kd (Pfitzner, U.M. and Goodman, H.M., 1987, Nucleic Acid Research, 15: 4449-4465) . The estimated size of the induced protein (ca. 17 Kd) from SDS gel is in good agreement with the size of PR- la. PR- la is induced during systemic acquired resistance (SAR) in tobacco and implicated in pathogen resistance (Bol, J.F. et al, 1990, Annual Review of Phytopathology 28: 113-138; Linthorst and Cornelissen, 1990; Ryals, J.A. et al, 1996, Plant Cell, 8: 1809-1819; Rauscher, M. et al, 1999, Plant J., 19: 625-633). The expression of PR- la is under complex control and is induced by tobacco mosaic virus and salicylic acid (Cornelissen, B.J.C. et al, 1987, Nucleic Acids Research, 15: 6799-6811).

5. PR- la mRNA is induced in NtmybPS 1 plants

To determine whether PR- la is also activated in NtmybPS 1 plants, a 0.56 kb PR- la probe was prepared by reverse transcription and PCR amplification of PR- la RNA using two oligonucleotide primers with the following sequences: 5'-ATACAACATT TCTCCTATAG TCATGGG -3' (SEQ ID NO: 7) and 5' -ATTAACGTGA AATGGACGTA GGTCG -3' (SEQ ID NO: 8) corresponding to nucleotides 1486-1512 (coding strand) and 2021-2045 (complementary strand) of the PR- la in clone 1 (Cornelissen et al, 1987, supra). The same RNA blot used for analysis of expression of PR-P (Figure 2) was probed with the 0.56 kb PR- la cDNA. Analysis of gene expression showed that the PR la like PR-P is also activated in a gene dosage dependent manner. Homozygote NtmybPS 1 plants showed a greater level of expression of PR- la than heterozygotes (Figure 2). This pattern of expression of PR- la correlated well with the pattern of expression of NtmybPS 1 (Figure 2).

6. Phenylalanine ammonia lyase (PAL) mRNA is induced NtmybPS 1 plants RT-PCR was performed using the ABgene Reverse-i TTM One Step system RT-PCR Kit (AB-0845) according to the manufacturer's instructions. Two oligonucleotide primers: 5'-GCTGAATCCT TAAGAGGGAG TCATTTGG-3' (nt. 85-1 12) (SEQ ID NO: 9) and 5'- CAAGCCATTG TGGAGATGTT CGGAG-3' (1054-1078) (SEQ ID NO: 10) were used to amplify a 994 bp (nt. 85-105 ) fragment of gPALl cDNA (Fukasawa-Akada, T. et al, 1996, Plant Molecular Biology 30: 711-722). The 994 bp tobacco gPALl fragment was used to probe a northern blot as described in Figure 2. The results showed that PAL mRNA was induced above wild type levels in homozygote NtmybPS 1 plants (Figure 2)

PAL is a key enzyme involved in phenylpropanoid synthesis and is proposed to provide cinnamate to enzymes involved in salicylic acid (SA) synthesis (Yalpani, N. et al, 1993, Plant Physiology, 103: 315-321). It has been found that the induction of the defence genes in NtmybPS 1 plants occurs via the increased production of SA.

There is some published evidence linking myb genes with the SA pathway of defence gene expression in that a tobacco myb gene mybl has been shown to be induced by exogenous application of SA and by tobacco mosaic virus (TMV) infection (Yang, Y.O. and Klessig, D.F., 1996, PNAS USA, 93: 14972-14977).

The myb gene class including NtmybPS 1 therefore may be of use in manipulating the plant defence response and may be of further use in manipulating phenylpropanoid synthesis and flux through these pathways.

This may provide a valuable way in which to provide increased protection against fungal, bacterial and viral infection and disease.

Isolation and expression of NtmybPS 1

1. Gene isolation

A member of the myb gene family was isolated from a cDNA library prepared from polyA+ RNA isolated from mature pollen of tobacco (Nicotiana tabacum cv. Samsun NN). cDNA library was prepared in the vector lambda ZAP using the techniques of Sambrook, J., Frisch, E.F., and Maniatis, T. ("Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989). The cDNA library was screened using degenerate oligonucleotide probes based on the consensus myb DNA binding domain (Jackson, D. et al, 1991, Plant Cell, 3: 115-125)

A full-length clone (pmybntl) was isolated and fully sequenced (SEQ ID NO: 1).

2. Starting with the NtmybPS 1 gene, this was modified by the addition of the CaMV35S 5' and 3' terminal sequences (i.e. the promoter and terminator) (Topfer, R. et al, 1987, Nucleic Acids Res., 15: 5890). CaMV35S promoters and terminators and cloning vectors are commercially available, and include promoters having duplicate enhancer regions at approximately -90 to -343. The CaMV35S5'-NtmybPSl-CaMV35S3' was then modified by the addition of terminal nucleotides to give the fragment SalI-CaMV35S5'-myb- CaMV35S3'-SacI, which was then inserted into plasmid pBIN19 (Bevan, M. et al., 1984, Nucleic Acids Res., 12: 871 1-8721) at restriction sites between nos3' and LB to give plasmid pBINl 9. This was then introduced into the disarmed Agrobacterium tumefaciens strain LBA4404(pAL4404) (Bevan, M. et al, 1984, supra). This Agrobacterium strain is referred to herein as agrobacterium dwarf (ADW)

3. The Agrobacterium strain ADW was used to infect leaf discs of tobacco (Nicotiana tabacum cv SRI) and kanamycin resistant, stably transformed plants regenerated. Primary transformants showed a reduction in mature plant height (see Figure 3) referred to as a dwarf phenotype.

4. Plants showing the dwarfing phenotype were allowed to set seed and shown to segregate approximately 3: 1 for kanamycin resistance:sensitivity, consistent with the presence of the T-DNA at a single genetic locus. All kanamycin resistant progeny showed the dwarf phenotype and two classes of dwarf plants were identified, one being severely dwarfed and the other showing intermediate plant height relative to the wild type. Seed collected from the severely dwarfed plants gave rise to 100% kanamycin- resistant progeny and therefore were homozygous for the T-DNA. Plants with intermediate level of dwarfing segregated 3: 1 for kanamycin resistance and were therefore heterozygous for the T-DNA. These data demonstrate that the introduced T-DNA carrying the 35S-NtmybPSl gene fusion can cause a dominant or gene-dosage dependent dwarfing phenotype.

5. The dwarfing phenotype was shown to be genetically transmissible and the severity of dwarfing was positively correlated with the level of expression of the introduced NtmybPS 1 gene in the transgenic plants.

6. Transcript levels of NtmybPS 1 in transgenic plants containing the 35S-NtmybPSl construct were determined using Northern blot analysis. Northern blotting was performed using standard methodology. Total RNA was isolated from wild-type, myb hemizygous and corresponding homozygous plants and analysed using Northern blot analysis, the Northern blot being probed by incubating it with a radiolabelled NtmybPS 1 coding sequence and then autoradiographing, RNA transcripts having the NtmybPS 1 sequence appearing as dark bands on the autoradiograph.

The results of Northern blotting showed that dwarf plants homozygous for the transgene show higher (approx. 2 fold) levels of expression than heterozygous plants (strictly speaking hemizygous). Plants without the transgene or those which did not show dwarfing did not show detectable expression of NtmybPS 1.

It was concluded that the NtmybPS 1 over-expression level is positively correlated with the degree of dwarfing. This was confirmed in several independent transgenic lines.

7. Additional height data: The height of plants homozyg DUS and hemizygous for the NtmybPS 1 transgene construct was examined. Results show that for several independent transgenic lines that the homozygous plants are significantly more dwarfed than the heterozygotes.

From the above data it was conclude that gene dosage of the NtmybPS 1 construct in plants is positively correlated with the degree of dwarfing.

8. Additional SA (salicylic acid) expression data

Extraction and partitioning:

Weighed leaf tissue was ground in liquid nitrogen using a mortar and pestle to produce a fine power which was extracted with methanol containing 20 mM sodium diethyldithiocarbamate (3 ml.g^"1 fresh weight). After 15 minutes the ground tissue and methanol were transferred to an Eppendorf tube and centrifuged at 5000 g for 1 minute. The supernatant was decanted and the pellet re-extracted with methanol. The two methanolic supernatants were combined and 100,000 dpm [7-^l4C]benzoic acid (40 mCi mM^'1) added as an internal standard prior to reduction to dryness in vacuo. Each extract was redissolved in 2 ml 0.1 M, pH 8.0 phosphate buffer and partitioned three times against 1 ml dichloromethane after which the aqueous phase was slurried with insoluble polyvinylpolypyrrolidone (100 mg.g ') which was removed by centrifugation. The aqueous supernatant was adjusted to pH 2.5 with H₂S0₄ and partitioned three times against 1 ml volumes of ethyl acetate which were combined, dried and reduced to dryness. Samples were redissolved in 1 ml methanol and aliquots taken for measurement of radioactivity and analysis of salicylic acid by reversed phase HPLC.

HPLC

Samples were analysed on Shimadzu liquid chromatograph comprising a controller, two LC-10ATVP pumps, a SIL-10ADVP autoinjector with a sample cooler, a CTO-10AVP oven, a SPD10A UV-vis absorbance monitor and a RF-10AXL fluorescence detector. Both detectors were linked to a Reeve Analytical (Glasgow, UK) 2700 data handling system. Reversed phase separations were carried out at 40 °C on a 250 x 4.6 mm i.d. 4 μm Synergy RP-MAX 80A column fitted with a MAX-RP guard column (Phenomenex, Macclesfield, UK). The column was eluted at 1 ml. min^"1 with a 15 min gradient of 25-40% acetonitrile in 1% aqueous formic acid. Column eluent was directed first to the aborbance monitor operating at 270 nm then to the fluorometer at excitation 305 nm and emission 420 nm. For analysis, 55 μl aliquots of the acidic, ethyl acetate fraction in 1 ml methanol were mixed with 165 μl of 1% aqueous formic acid and maintained at 4 °C in the sample cooler of the autoinjector prior to injection of x μl volumes onto the HPLC column. Samples were analysed in triplicate.

Results are given in the table below, which shows levels of salicylic acid (SA) determined in leaf samples of wild type (WT) and plants overexpressing NtmybPS 1. Hom = homozygous, Het = heterozygous for C aMV35 S -NtmybPS 1 construct. Myb7 and Myb29 are two independent transgenic lines. Means, standard deviation (SD) and standard errors (SE) are derived from the analysis of samples in triplicate. SA concentration is shown as ng SA per g fresh weight of leaf tissue.

This data shows that SA levels are elevated approx 17 fold in heterozygous plants and 30-100 fold in homozygous plants. This data proves that induction of PAL leads to increased SA.

Taken together with the results of Northern blotting, these results establish that NtmybPS 1 over-expression using the 35S promoter (and in this example in tobacco) results in dwarfing that is dependent upon gene dosage acting through expression level.

Therefore tissue-targeted and inducible control of plant and organ growth can be expected to be possible using such an expression-dependent strategy.

As well as the basic constructs containing the NtmybPS 1 coding sequence under the control of the CaMV35S promoter ("Isolation and expression of NtmybPS 1 " section above) additional constructs are made, so called "dual cassettes" containing (i) the NtmybPS 1 coding sequence under the control of promoters such as the CaMV35S promoter; and (ii) a PAL coding sequence under the control of a PAL promoter. In particular PAL promoters from Nicotiana, especially Nicotiana tabacum, are used, and can be used with the PAL coding sequence specific for the plant in which it is to be expressed.

Using standard cloning techniques (Sambrook, J., Frisch, E.F., and Maniatis, T., "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989) the genomic sequence (including the regulatory sequences upstream of the coding sequence) of PALI is isolated and purified. PCR primers for this are produced on the basis of the PALI sequence identified by Pellegrini L. et al, 1994 (supra). This is then ligated to the 35S-NtmybPSl construct to give a dual construct 35S-NtmybPS l-PALl , which is then introduced into the disarmed Agrobacterium tumefaciens strain LBA4404(pAL4404) (Bevan, M. et al, 1984, supra). This is then used to infect leaf discs of tobacco and stably transformed plants regenerated as detailed above for ADW.

An alternative PAL construct is made by isolating and purifying the PALI promoter region located from the PALI transcriptional start codon and sequence lkb upstream thereof. This is then ligated to the PALA coding sequence (Fukasawa-Akada T. et al. , \996, supra). This is then ligated to the 35S-NtmybPS l construct to give a dual construct 35S-NtmybPS l-PALl-PALA, which is then introduced into the disarmed Agrobacterium tumefaciens strain LBA4404(pAL4404) (Bevan, M. et al, 1984, supra). This is then used to infect leaf discs of tobacco and stably transformed plants regenerated as detailed above for ADW.

In order to test the efficacy of the induced defence phenotype in protecting against disease, transgenic plants or cell cultures are exposed to infection by pathogenic organisms and the effect of the pathogen observed. In particular, TMV (tobacco mosaic virus) particles can be rubbed onto leaves of the transgenic plant to determine their resistance against it. Similarly, plants can be exposed to RNA or whole viral particles from other virii and their status monitored. Plants can also be inoculated with fungi and infected with fungi or bacteria-like pseudomonas to determine their disease resistance. This testing of disease resistance can of course be combined with the use (or absence) of conditions which induce the production of NtmybPS 1 according to the specific promoter or promoter complex used in the constructs of the present invention.

The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety.

Where "PMID:" reference numbers are given for publications, these are the PubMed identification numbers allocated to them by the US National Library of Medicine, from which full bibliographic information and abstract for each publication is available at http://www.ncbi.nlm.nih.gov. This can also provide direct access to electronic copies of the complete publications, particularly in the case of e.g. PNAS, JBC and MBC publications.

Claims

1. A method of making a plant which produces a "defence" phenotype, comprising the steps of: a) transforming or transfecting cells of said plant with a recombinant DNA construct comprising operatively linked in the 5' to 3' direction: i) a promoter region that directs the transcription of a gene in plants; ii) a DNA coding sequence encoding the expression product having the sequence of SEQ ID NO: 2; and iii) a 3' non-translated region which contains a polyadenylation signal for plants; b) selecting plant cells that have been transformed or transfected; c) regenerating plant cells that have been transformed or transfected to produce differentiated plants; and d) selecting a transformed or transfected plant which expresses said construct and which has a defence phenotype.

2. A method according to claim 1 , said recombinant DNA construct not inducing a dwarf phenotype in said selected transformed or transfected plant.

3. A recombinant DNA construct whose expression in plants induces a plant defence phenotype, and comprising operatively linked in the 5' to 3' direction: i) a promoter region that directs the transcription of a gene in plants; ii) a DNA coding sequence encoding an expression product having the sequence of SEQ ID NO: 2; and iii) a 3' non-translated region which contains a polyadenylation signal for plants; said recombinant DNA construct not inducing a dwarf phenotype in plants transformed or transfected with it.

4. A method or recombinant DNA construct according to any one of the preceding claims, the production of the expression product being in plant tissues other than those selected from the group consisting of pollen and anthers.

5. A method or recombinant DNA construct according to any one of the preceding claims, the DNA coding sequence having a sequence selected from the group consisting of SEQ ID NO: 1 and nucleotides 182-1591 of SEQ ID NO: 1.

6. A method or recombinant DNA construct according to any one of the preceding claims, said promoter region having at least one characteristic selected from the group consisting inducible expression, organ-specific expression, temporally specific expression, and pathogen-responsive expression.

7. A method or recombinant DNA construct according to claims 7, said promoter region comprising any one of the group consisting the CaMV35S promoter, the maize polyubiquitin (ubl) promoter, the Asparagus officinalis pathogenesis-related vascular-tissue-specific (AoPRl) promoter, the rice ribulose-bisphosphate carboxylase (rbcs) promoter, the maize shrunken- 1 promoter, and the pathogen responsive (PR) promoter..

8. A method or recombinant DNA construct according to any one of the preceding claims, said 3' non-translated region comprising the 3' polyadenylation signal from the CaMV35S transcript.

9. A method according to any one of claims 1, 2 and 4-8, said recombinant DNA construct additionally comprising operatively linked in the 5' to 3' direction: i) a PAL promoter region; ii) a PAL coding sequence; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

10. A method according to any one of claims 1, 2 and 4-8, said cells of said plant additionally being transformed or transfected with a recombinant DNA construct comprising operatively linked in the 5' to 3' direction: i) a PAL promoter region; ii) a PAL coding sequence; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

11. A recombinant DNA construct according to any one of claims 3-8, said recombinant DNA construct additionally comprising operatively linked in the 5' to 3' direction: i) a PAL promoter region; ii) a PAL coding sequence; and iii) a 3' non-translated region which contains a polyadenylation signal for plants.

12. A plant transformed or transfected with a recombinant DNA construct according to any one of claims 3-8 and 1 1.

13. A plant produced according to the method of any one of claims 1 , 2 and 4- 10.

14. Seed obtained by growing a plant according to either one of claims 12 or 13.