GB2374598A - Transactivation elements for enhanced plant gene expression - Google Patents

Abstract

A method of enhancing plant gene expression as described. The method comprises transforming a plant cell with a construct comprising a nucleic acid sequence which encodes a transactivation domain of Rel A (p65) subunit of the human cellular transcription factor NF- k B, or a transactivating fragment thereof, or a transactivating mutant of either of these, or a transactivating multimer of said fragments or mutants of fragments of these, said sequence being arranged such that it enhances expression of a target gene in said plant cell.

Description

. Novel Expression System The present invention relates to control

elements useful in recombinant DNA technology, and in particular to transactivation elements useful in expression of genes in 5 plant cells, to nucleic acids encoding these and to plant cells and plants containing them.

It is well known that the rate of transcription of eukaryotic genes is regulated by transcriptional activators which are proteins which interact with DNA sequences located distal to the site of transcription initiation. These are known to comprise two discrete functional domains, a specificity domain which directs the activator to the appropriate lo DNA promoter sequence, and a transactivation domain, which functions to enhance transcription of the gene which is controlled by that promoter.

Transactivation domains are often included in constructs used to transform recombinant organisms in order to ensure that desired gene and in particular transgenes are expressed efficiently and effectively in recombinant systems.

15 Well known transaetivation domains which are often included in recombinant expression systems include the activator derived from VP16 of Herpes simplex vikus (see for example S. J. Triezenberg et al. Genes Dev. (1988) 2:718-729). Another is the Rel A (p65) subunit of the human cellular transcription factor NF-KB (1:). W. Ballard et al., Proc. Natl. Acad. Sci USA, (1991) 89:1875-1879).

20 This latter transactivation domain is 134 amino acids in length but has been the subject of mutational analysis which identified a minimal acidic activation module of about 11 amino acids (W.S. Blair et al., Molecular and Cellular Biology, (1994) 14, 11, 7226-7234). In that study, it was found that in mammalian cells, monomers of the transactivation domain produced only low levels of transcription activation, whereas Is multimers and particularly dimers and trimers of this module were highly effective.

Furthermore mutated forms of the peptides in which phenylalanine residues were changed to alanine residues were produced, and it was found that this had a significant impact on the activity.

The applicants have found that particular peptides derived from the Rel A 30 transcription factor or mutant forms thereof are particularly active in the transactivation of genes in plants.

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According to the present invention, there is provided a method of enhancing plant gene expression, which method comprises transforming a plant cell with a construct comprising a nucleic acid sequence which encodes a transactivation domain of Rel A (p65) subunit of the human cellular transcription factor NF-KB, or a s transactivating fragment thereof, or a mutant of either of these, or a transactivating multimer of said fragments or mutants thereof, said sequence being arranged such that it enhances expression of a target gene in said plant cell.

The term "multimer" refers to repeat copies of the peptide fragments, attached either directly or by way of short 'linker' sequences for example of from 1 to 4 amino lo acids. Multimers suitably contain for example from 2 to 6 and preferably about 3 copies (a trimer) of the peptide sequence.

The target gene may be a heterologous or homologous gene of the plant cell, depending upon the desired target. If it is a homologous plant gene, the construct used to transform the plant cell should be designed to insert in the genome of the plant cell at an 15 appropriate position to allow the peptide to interact with the target gene. Where a homologous gene is being introduced, it may be convenient to incorporate this into the same construct as the transactivation domain, although cotransformation methods may also be employed.

Suitably, the sequence used is a transactivating fragment of Rel A (p65) subunit 20 of the human cellular transcription factor NF-KB, or a mutant thereof, and in particular it is a peptide which comprises eleven amino acids found between amino acids 535 and 545 in the sequence of the human cellular transcription factor NF-KB, or a mutant. The native sequence is of SEQ ID NO 19 as follows: 25 535 SIADMDFSALL545 (SEQIDNO 19)

Preferably the sequence used is a mutant form of this peptide or a multimer, and particularly a trimer, of these.

In one embodiment, the peptide may be a mutant monomeric peptide fragment of 30 the transactivation sequence of llel A (p65) subunit of the human cellular transcription factor NP-KB and in particular, an 11-mer peptide of SEQ ID NO 1 SFADMDFSALL (SEQ ID NO 1)

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In this mutant peptide, an isoleucine residue at position 536 in the full length sequence is changed to a phenylalanine group.

Other particular mutants of the native sequence of SEQ ID NO 19 have one or 5 more of the following mutations which are preferably additional to the 1536P mutation: 1. An additional two S residues at the end of the sequence (which act as linkers in the case of a multimer) such as SEQ ID NO 2 10 SFADMDFSALLSS (SEQ U) NO 2)

2. A sequence in which at least one acidic residue has been placed adjacent a phenylalanine residue such as SEQ ID NO 3, where a serine residue is replaced with an aspartic acid residue 5 DFADMDFDAIL i (SEQ ID NO 3) 3. A sequence in which number of acidic residues has been maintained but that these have been rearranged such as SEQ ID NO 4 20 SFDAMDFSALL (SEQ ID NO 4)

Preferably mutants of the invention will have a combination of the mutations listed above, in particular a combination of 1 and 2 (such as SEQ ID NO 5) DFADMDFDAlLSS (SEQ I1:) NO 5) or 1 and 3 (such as SEQ ID NO 6) 30 SFDAMDFSAILSS (SEQ ID NO 6)

pen 50610 4 or 2 and 3 such as SEQ ID NO 20, where in addition, the position alanine and aspartic acid residues are reversed so that a phenylalanine residue is flanked by two aspartic acid residues DFDAMDFDALL (SEQ ID NO 20).

A preferred sequence includes all 3 mutations such as SEQ ID NO 21 DFDAMDPDALLSS (SEQ ID NO 21).

lo Most preferably, multimeric forms of these peptides are used in the method of the invention, in particular trimeric forms or variants thereof such as peptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ R) NO 10.

SFADMDFSALLSSSFADMDFSALLSRPSFADMDFSALLSS (SEQ ID NO 7)

15 SFADMDFSALLSSSFADMDFSAILSSSFADMDFSALLSS (SEQ 11) NO 8)

DFADMDFDALLSSDFADMDFDALLSSDFAD FDALLSS (SEW ID NO 9)

DFDAMDFDAlLSSDFDAMDFDALLSSDFDAMOFDAIlSS (SEQ ID NO 10) SEQ ID NOs 2, 4, 5, 6, 8, 9, 10, 20 and 21 are novel sequences and as such, form 20 a further aspect of the invention.

The applicants have found that in plant cells, trimenc peptides have an activity level which is better than that of the full length Rel A (p65) subunit of the human cellular transcription factor NF-KB. IJse of a small activation peptide of this size has significant advantages in terms of the size of the constructs used in the transformation 2s process can be reduced, making them easier to produce and use, and reducing the genetic burden on the resultant recombinant plant.

Furthermore such short peptide sequences are advantageous over the full length transcription factor (which may be 100-200 amino acids in length) since any deleterious sequence within the domain would have been dispensed with and therefore, the peptides 30 behave more robustly, irrespective of their protein surroundings.

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In a further aspect, the invention provides a nucleic acid which encodes a novel peptide as described above. Suitably the nucleic acid is one in which codons have been optimised for expression in the target plant species.

Plant cells transformed using the constructs of the invention and plants derived 5 therefrom, form yet further aspects of the invention.

Any transformation method suitable for the target plant or plant cells may be employed, including infection by Aerobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplast and microprojectile transformation and pollen tube transformation. The transformed cells may then in lo suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.

Suitable plant cells are cells from crop including field crops, cereals, fruit and

vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, 15 barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.

The target gene which is controlled by the transactivation peptide in accordance with the invention may be any desirable gene or transgene, such as those involved in conferring pesticide resistance such as herbicide or insecticide resistance, to control 20 fertility (in particular in the production of hybrid plants) or in improving the nutritional or aesthetic value of a crop plant. In particular, however, the method of the invention may be used to drive expression of genes involved in "gene switches" such as for example those described in WO 96/37609 and WO 93/21334.

Expression of genes and particularly trahsgenes is controlled by regulatory 25 elements and when transforming an organism, it is important to ensure that suitable regulatory elements are included and arranged so that the target gene is expressed in the desired manner. For example, it may be required that the gene is expressed only for a limited period, in order to achieve the desired modification to the organism.

Gene switches provide a very useful addition to the "armouri' of the 30 biotechnologist. The expression "gene switch" used herein refers to a control sequence and regulator of such sequence which are responsive to an applied exogenous chemical

PPo 5061Q 5 inducer, enabling expression of a gene controlled by said control sequence and regulator to be managed externally, by applying or withholding the chemical inducer from the organism. An example of a gene switch which has been found to be particularly useful in s the manipulation of plants is derived from the fungal organism Aspergillus nidulans.

This organism expresses the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alc A only when it is grown in the presence of various alcohols and ketones. The induction is relayed through a regulator protein encoded by the ale R gene which is constitutively expressed. In the presence of inducer (alcohol or ketone), the regulator 10 protein activates the expression of the inducer. This means that high levels of the ADH 1 enzyme are produced under inducing conditions (i.e. when alcohol or ketone are present). Conversely, the ale A gene and its product, ADH 1, are not expressed in the absence of inducer. Expression of ale A and production of the enzyme is also repressed in the presence of glucose.

5 Thus, the alcA gene promoter is an inducible promoter, activated by the ale R regulator protein in the presence of inducer (i.e. by the protein/ alcohol or protein/ ketone combination). The ale R and ale A genes of Aspergillus nidulans (including the respective promoters) have been cloned and sequenced (Lockington RA et al., 1985, Gene, 33: 137-149; Felenbok B et al., 1988, Gene, 73: 385-396; Gwynne et al., 1987, 20 Gene, 51: 205-216?.

Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region of adh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. No equivalent to the alcR regulator 25 protein has, however, been found in any plant. Hence the ale A/alc R type of gene regulator system is not known in plants. This means that constitutive expression of ate R in plant cells does not result in the activation of any endogenous adh activity. It is therefore a particularly useful gene switch for plant use, since it can be used to control a transgene, without interfering with or interrupting any other plant cell function.

30 WO 93/21334 describes the production of transgenic plants which include such a system as a gene switch. This document specifically describes a chemically inducible

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plant gene expression cassette comprising a first promoter operatively linked to a regulator sequence which encodes a regulator protein and in particular the ale R protein of Aspergillus nidulans, and an inducible promoter such as the Aspergillus nidulans ale A promoter or a chimeric promoter including elements of this promoter, operatively s linked to target gene. The inducible promoter is activated by the regulator protein in the presence of an effective exogenous inducer, whereby application of the inducer causes expression of the target gene. Exogenous chemical inducers which can be applied include those described by Creaser et al, (1984) such as butan-2-one (ethyl methyl ketone), cyclohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol and ethanol lo and esters as described in WO00/44917.

Thus the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer (i.e. by the protein/alcohol or protein/ketone combination). These switches are particularly useful in allowing controlled gene expression by 15 switching on activity by application of an exogenous inducer and in particular an exogenous chemical inducer. Such controlled expression is useful in many aspects of agriculture, for example in allowing the production of plants which are reversibly male sterile, which are used in hybrid production, as described in WO 90/08830.

It is important in constructs used in gene switches that the regulatory components 20 such as the regulator sequence, is effectively and efficiently expressed at the point at which the it is required in the system. Thus placing such sequences under the control of good transactivation sequence such as those of the present invention, is particularly preferred. The invention will now be particularly described only by way of example with 2s reference to the accompanying diagrammatic drawings in which: Figure 1 illustrates a cloning Strategy for Trimers of Mutant RelA Acidic Activation Modules (AAM3).

Figure 2 is a schematic drawing of the expression cassettes containing lacAElis fusions to Gal4, VP16, RelA and SEQ ID NO 7 activation domains. The reporter 30 plasmid pXBKGUS used to assess the activation potency of the activators is also shown.

Figure 3 is a graph showing reporter gene activity of pLhG4, pLhVP16, pLhRelA and pLh(SEQ 1D NO 7) activators in Arabidopsis mesophyll protoplasts.

PPo 50610 8 Figure 4 is a schematic representation of expression and reporter constructs used to assess the trimerised activation domains of SEQ ID NO 8, SEQ ID NO 9 and SEQ NO 10.

Figure 5 shows a comparison of Reporter gene activity in Arabidopsis mesophyll 5 protoplasts transformed with expression vectors expressing the monomer and trimerised activation domain fusions to lacAhis.

Figure 6 shows the activity of LhG4 compared to transformants.

In these Figures "SEQ ID NO" has been abbreviated to "NO".

10 Example 1: Des an of Synthetic Acidic Activation Modules The RelA Acidic Activation Module (AAM) m (residues 535-545) was modified to increase its efficiency of transcriptional activation in plant cells. The native sequence (535SIADMDFSALL545 (SEQ ID NO 19) was altered at key residues to increase the efficiency of activation as follows: 15 SEQ ID NO 1 mutant sequence: SFADMDFSALL (modification from native sequence: I53GF).

SEQ ID NO 3: DFADMDFDAIL (modifications from native sequence: S535D, I536F, S542D)

SEQ ID NO 20: DIl)AMDFDALL (modifications from native sequence: S535D, I536F, 20 A537D; D538A; S542D).

The idealised sequence of the RelA AAM is a bulky hydrophobic residue (Phenylalanine, Methionine or Isoleucine) flanked by one or more acidic residues (preferentially Aspartic Acid).

The I536F mutation (SEQ ID NO 1), tested previously as a monomer by Blair et 2s al (1994) in yeast (Sacccharom;yces cerevisiae) was trimerised in the form of SEQ ID NO 7. The I536F mutation is assumed to increase activity over the native sequence by providing an additional phenyalanine residue, vital to RelA Acidic Activation Module activity. Further to this the mutant sequence of SEQ ID NO 3 was designed to increase the 30 efficiency of the I536F mutation by flanking F536 and P541 with an increased number of aspartic acid residues. This sequence, previously tested in monomer form in yeast (Blair et al, 1994) had shown an improved activity over the I536F mutation.

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The nucleotide sequence encoding SEQ ID NO 3 was optimised for Arabidopsis codon usage (SEQ ID NO 5) and tested in protoplast transient expression. A trimer of this sequence (SEQ ID NO 9) shows a considerable increase in activity compared to LhG4. 5 The mutant sequence of SEQ ID NO 20 was designed to idealise the two phenylalanine residues of the I536F sequence by flanking both phenylalanine residues directly with two aspartic acid residues each. The S535D, A537D and S542D mutations achieved this, while the D538A mutation retained the 1 lea length of the sequence to allow direct comparison with the other AAMs.

10 SEQ ID NO 20 is a novel sequence and has not been tested previously in any system. The nucleotide sequence for SEQ ID NO 20 was idealised for Arabidopsis codon usage (SEQ ID NO 21) and tested in Arabidopsis protoplast transient expression. A trimer of this sequence (SEQ ID NO 10) also shows greater activity than LhG4, as set out below.

15 The SEQ ID NO 8 mutant sequence was a trier of the I536F mutation optimised for Arabidopsis codon usage. SEQ I1) NO 8 was designed to provide an equivalent to the SEQ ID NO 7 trimer but with uniform linkers between the three AAMs. SEQ ID NO 7 trimer sequence, contained arginine and praline residues in the linker between the second and third copies of the activation module. In the linker 20 between the first and second AAM, as well as between all AAMs of SEQ ID NO 9 and 10, the linker between monomer units is composed of two Serine residues. In SEQ 1D NO 8, the arginine and proline residues of SEQ ID NO 7 are replaced with serine, thus creating uniform linkers of two serine residues between the AAM monomers for all the AAMs tested.

Examule 2: Svnthesis and Clonine Strateav The sequences were synthesised as monomers for initial cloning.

Oligonucleotides were synthesised by MWG Biotech (Low Salt, HPSF pure) in pairs of overlapping, complementary sequences, as follows (AAM regions in capitals):

PPI) 5061Q 1C

Oli onuclotide Sequences encoding SEQ ID NO 1: (5'-3'):

aattcgctagcTCCTTCGCGGACATGGACTTCTCAGCCTTGCTGtCtagactcgagactagttg (SEQ ID NO 11)

5 (3'-5'):

aaKcaactagtctcgagtctagacAGcAAGGcTGAGAAGTccATGTccGcGAAGGAgctagcg (SEQ ID NO 12)

Olizonuclotide Sequences encoding SEQ ID NO 3: lo (5 -3): aattcgCtagcGATTTCGCGGACATGGACTTCGACGCGTTGCTGtctagactcgagactagttg (SEQ ID NO 13)

(3 -st) aattcaactagtctcgagtctagaCAGCAACGCGTCGAAGTCCATGTCCGCGAAATCgctagcg 15 (SEQ ID NO 14)

SEQ ID NO 4 Oli onuclotide Sequences: (5 -3):

aattcgctagcGATTTCGATGCCATGGAC'l l'l'GATGCCTTGCTGtctagactcgagactagttg 20 (SEQ ID NO 15)

(3 -5'):

aattcaactagtctcgagtctagaCAGCAAGGCATCAAAGTCCATGGCATCGAAATCgctaBcg (SEQ 1D NO 16)

Once annealed, the AAM fragments possess open EcoR1 cut sites at the 5' and 3' 25 ends, allowing for cloning directly into EcoR;I sites within the activator region of - pKI102.LhlO7. Within each fragment is a NheI site upstream of the AAM, and XbaI, XhoI and SpeI sites, consecutively, followed by the STOP codon downstream of the AAM (Figure l(a)).

Each pair of complementary oligonucleotides was mixed to a final concentration 30 of lmM in water, boiled for 10, then allowed to cool naturally to room temperature to permit annealing of the complementary sequences. 5 pMol of the annealed oligonucleotide mix was used directly to ligate into EcoRI-cut plJCAP (figure l(b)) for

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verification by sequencing and for amplification of the plasmid in E. coli. An NheYXbaI excised fragment from this pUCAP.AAM plasmid (Figure l(c)) was used to multimerise the AAM sequence to form trimers for comparison to pLhlO7. Dimerisation of the AAM was performed by cutting pUCAP.AAM with XbaI and ligating the NheIIXbaI s fragment from pUCAP.AAM into this cut site (Figure l(d)). This re-constitutes the XbaI site at the 3' end of the second AAM, and creates a non-functional NheYXbaI site between the two AAMs (Figure l(e), hatched box). This process was repeated using the same fragment inserted into XbaI-cut pUCAP.AAM2 to produce a trimer of the AAMs (Figure 1), pUCAP.AAM2, which possesses a non-functional NheYXbaI site between 10 each monomer (encoding two Serine residues), an in-tact NheI site at the 5' end and XbaI, XhoI and SpeI enzyme sites between the 3' end of the trimer and the Stop codon (Figure l(g)). Finally, an XbaIlSpeI digest followed by self-ligation (Figure 1(h) ) allowed for the removal of these 3' sites (leaving a non-functional XbaYSpeI site immediately downstream of the Trimer, in frame (encoding two Serine residues), enabling the 15 function of the STOP codon (Figure l(i)). This plasmid (pUCAP AAM3 3 was cut EcoRJ and the AAM3A trimer fragment was ligated into pK1102-Lh107 cut with EcoRI (Figure l(i)) to permit testing of the AAM tnmer in protoplast transient expression using pK I!.h AAM3A (;igure l(j)) as the activator construct, and pXBKGUS as reporter.

The monomer of each AAM was also created by taking the initial pUCAP.AAM 20 construct, deleting the XbaI, XhoI and SpeI enzyme sites as described above (Figure l(i)), and cloning as described into pKI102.LhlO7.

Examnle 3. Protonlast preparation from Arabidopsis BY2 cell culture for PEG mediated transformation to test relA transcription activator potency 25 Protoplasts were prepared from 25ml Arabidopsis BY2 cell cultures maintained at a 1/10 dilution in 1xMS Salts (SIGMA), 3% Sucrose, 0.5mgA NAA, 0.51lgA Kinetin.

A 5 day 1/10 or 6 day 1/20 subculture of cells was used to make protoplasts fresh for each transformation. All manipulations were carried out under sterile conditions in a laminar flow hood. 25rnl of cell culture was transferred to a 50ml sterile centrifuge tube 30 and the cells pelleted in a Beckman Coulter Allegra 21R bench-top centrufuge at 45g (minimum acceleration and deceleration speeds) at 18 C for 10 minutes. The pellet was resuspended by gentle rocking in 30ml Plasmolysis Solution (0.4M D(-)Mannitol, 3%

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(w/v) Sucrose, 8rnM CaCl'; pH 5.6-5.8, autoclaved 12 minutes) and the tube of cells left on its side for 20 minutes. The tube was then centrifuged as above and the pellet resuspended by gentle rocking in 40ml Enzyme solution (1% (w/v) Cellulase "Onozuka" R10, 0.25% (w/v) Macerozyme R10 (Yakult Honsha Co., Tokyo, Japan) dissolved in 5 Plasmolysis solution in the dark at 4 C, pH 5.6-5.8 with KOH, filter sterilized, stored for up to 48 hours in the dark at 4 C) and divided between two 50ml centrifuge tubes. The re-suspended cells were left at room temperature in the dark for 1 hour, followed by 30 mins gentle rocking, then by 1 hour stationary. A sample of cells was then checked under an inverted microscope to verify that cell walls had been digested to reveal 10 protoplasts. Clumps of cells could be dispersed by filtration through a 100pm sterile mesh. The protoplasts were pelletted by the addition of 30ml Mannitol WS (0.4M D(-) Mannitol, lmM D()Glucose, 30.8mM NaCl, 25rnM CaC12, lmM KCl, 0.3mM 2- (N-

Morpholino) ethane sulphonic acid monohydrate (IS) (BDH Chemicals); pH 5. 6-5.8 5 with KOH, autoclave 12 minutes) to each tube, gentle but thorough mixing and centrifugation at 29g (minimum acceleration and deceleration speeds) at 18 C for 10 minutes. The pellets were re-suspended by gentle rocking in 15ml each of Mannitol Mg solution (0.4M D(-)Mannitol, 0.1% (w/v) MES, 15mM MgC12, pH 5.6-5.8, autoclave 12 minutes), recombined into a single tube and centrifuged at 29g (minimum acceleration 20 and deceleration speeds) at 18 C for 10 minutes. The pellet was washed again by resuspending gently in 30ml of Mannitol Mg solution and centrifugation at 29g (minimum acceleration and deceleration speeds) at 18 C for 10 minutes. Finally the pellet was resuspended gently in 5ml Mannitol Mg and placed on ice for up to 30 minutes during preparations for the transformation.

PEG-mediated transformation of Arabidonsis Protoplasts Each individual transformation was performed in a sterile disposable petri dish under sterile conditions. Activator and Reporter DNA was purified using Qiagen Midi and Maxi Plasmid purification kits (Qiagen), followed by PhenoVchloroforrn extraction 30 and two ethanol precipitations (with 3M NaOAc). Heering Sperm DNA (SIGMA) was added as carrier DNA, after phenoVchloroform extraction and two ethanol precipitations and sonication to a mean fragment size of lkb. Activator and Reporter DNA was mixed

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in a microfuge tube with 250ilg sonicated Herring Sperm DNA in a final volume of 50,ul.

25 1 Chloroform was added to the tube and the mixture vortexed to sterilise the DNA solution. The chloroform phase was settled out by centrifugation in a microfuge before the DNA was placed in the petri dish for transformation. The sterile DNA was spotted 5 in a ring of small droplets on the petri dish, and 350,u1 of PEG-CMS solution (0.4M Mannitol, 100mM Ca2NO4, 40% PEG 4000 (BDH Chemicals), filter sterilised) was placed adjacent to this. 300,u1 of protoplast suspension was added to the DNA using a sterile filter-tip lml gilson pipette (with the tip removed to a wider aperture). The Protoplasts and DNA were mixed gently for 20 seconds using the pipette tip. 1 minute 10 after the addition of protoplasts to the DNA, the PEG-CMS droplet was then taken up into the pipette and expelled slowly into the protoplast/DNA mix while moving the pipette tip through the protoplast/DNA droplet in a swirling motion. One transformation event could therefore be performed every 2 minutes.

30 minutes after the addition of the protoplasts to the DNA, the 15 protoplastlDNA/PEG-CMS droplet was washed with 600 1 Mannitol W5 placed gently, drop-wise, into the periphery of the protoplast droplet. A further 15 minutes later lml of Mannitol W5 was added in an identical manner. After a further 15 minutes the pellet was washed with 2ml Mannitol W5, dropwise as above, and the droplet was guided so that it touched the rim of the petri-dish. After a further 15 minutes a final wash of 4ml 20 Mannitol W5 was added to the edge of the droplet furthest from the rim of the petri-dish.

The droplet was then left at a very slight incline for a further 15 minutes before being transferred gently by sterile 10ml disposable pipette into a l5ml falcon tube. All transformations were left in 15ml faclon tubes on ice for at least 30minutes until the protoplasts sank naturally to the base of the tube. The supernatant was removed and 25 replaced with 2ml Protoplast Culture Medium (0.4M Sucrose, 250mg/L Xylose, lxMS Salts, pH 5.6-5.8 with KOH, autoclave 12 minutes) and the protoplasts left in their Falcon tubes at a slight incline (approximately 15 ) in the dark at 20 C. Maximal expression was observed at 48 hours.

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Harvesting of protoPIasts and fluorometric MUG assay for GUS actinty The protoplasts were harvested and total protein extracted to assayer for GUS activity. Each protoplast culture was mixed gently but thoroughly with Sml Mannitol W5 and pelleted by centrifugation at 3645g for lo minutes at room temperature. The 5 pellet was resuspended in 400 1 GUS Extraction Buffer (50mM Sodium Phosphate Buffer, pH7.0; 10mM,B Mercaptoethanol; 10mM Na2EDTA, 0.1% Sarcosyl, 0.1% Triton X-100, REED, placed in a 1,5ml microfuge tube and sonicated for 10 seconds.

The cell debris was pelleted in a rnicrofuge (13,000 rpm at 4 C for 10 minutes). Protein extracts were kept on ice or frozen quickly in liquid N2 and stored at -80 C. Total 10 protein concentration for each proteinextract was measured using the BIORAD Bradford Assay. 50'Lg of protein extract from each transformation was assayed for GUS activity at 37 C, with lmM Methylumbelifferone glucuronide (Melford Laboratories) dissolved in GUS Extraction Buffer) as a substrate. The reaction was performed in a total volume of 280 1 GUS Extraction Buffer in a 96-well microtitre plate. 40!11 of each assay was is removed at time intervals between 10 and 50 minutes and mixed with 200,u1 0.2M Na2CO3 in a separate multi-well microtitre plate (kept in the dark). Fluorescence of the Methylumbelliferone (MU) product was measured using a BMG POLARstar microtitre plate reader (BMG Laboratory Technologies GmbH, Offenburg, Germany) with excitation at 365nm, emission at 455nm. Fluorescence readings were compared to MIJ 20 standards (100 to 400 pmoles MU) and activity expressed as pmoles MU/ lg protein/minute. RelA activator is active in plant cells In a first set of experiments comparison of the Gal4, VP1G and RelA 2s transactivation domains was carried out. Figure 2, shows the expression cassettes of the vectors used in this experiment while Figure 3 shows the results of 4 experiments, carried out as described above. The data shows that at low activator vector DNA input activity of the RelA activator is best. The data demonstrates that relA is capable of activating gene expression in plant cells and the activity is comparable if not higher to 30 other activators known to be functional in plants.

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Example 4. RelA derived Pentide is a transactivator of gene expression in plant cells. In order to try to understand and.further reduce the size of the fragment needed as an activator of gene expression, a small acidic peptide from the RelA sequence was s introduced into the activator vector for testing in plant protoplasts using a method similar to that described in Example 3. Figure 4 shows that the single peptide sequence is responsible for 30% of the activity seen with the whole RelA transactivation domain.

Example 5. Mutagenesis and trimerisation provide a novel Potent transcriptional 10 activator in Plant cells.

A question that remained was whether multiple copies of the peptide in question would produce a transactivator which was as at least as active as the RelA full length sequence. Transformation of activator plasmids containing the expression cassettes Shown in Figure 5 were carried out in conjunction with the reporter plasmid, pBXGUS.

15 The data generated shows that the activator plasmids containing the trimer sequences SEQ ID NO 9 and SEQ ID NO 10 were more active the the transactivator proteins containing either the monomer of RelA, mutated monomers of ReLA, full sequence of RelA, Gal4 and VP16. The data generated indicates that the trimers generated are preferred transactivators for use in plant cells with improved potency.

20 Other modifications to the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

Claims (1)

1. PPD5061Q À6

   Claims

   1. A method of enhancing plant gene expression, which method comprises transforming a plant cell with a construct comprising a nucleic acid sequence which 5 encodes a transactivation domain of Rel A (p65) subunit of the human cellular transcription factor NF-kB, or a transactivating fragment thereof, or a transactivating mutant of either of these, or a transactivating multimer of said fragments or mutants of fragments of these, said sequence being arranged such that it enhances expression of a target gene in said plant cell.

   2. A method according to claim 1 wherein the nucleic acid sequence encodes a transactivating fragment of Rel A (p65) subunit of the human cellular transcription factor NF-K, or a mutant thereof.

   15 3. A method according to claim 2 which peptide comprises the eleven amino acids found between amino acids 535 and 545 in the sequence of-the human cellular transcription factor NF-KB, or a mutant thereof 4. A method according to claim 1 wherein the peptide is an 11-mer peptide of SEQ 20 ID NO 1

   SFAD FSAlL (SEQ ID NO 1) 5. A method according to claim 1 wherein the peptide comprises a mutant form of SEQ ID NO 1 as defined in claim 4 or a multimeric form thereof.

   6. A method according to claim 5 wherein said peptide has one or more of the following mutations: 1. an- additional two S residues at the end of the sequence; 2. a sequence in which at least one acidic residue has been added in place of a 30 serine residue adjacent a phenylalanine residue; or 3. a sequence in which number of acidic residues has been maintained but that these have been rearranged.

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   7. A method according to claim 6 wherein said peptide comprises SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO, SEQ ID NO 6, SEQ ID NO 20, or SEQ ID NO 21:

   5 SFADMDFSALLSS (SEQ ID NO 2)

   DFADMDFDALL (SEQ ID NO 3)

   SFDAMDFSAIL (SEQ ID NO 4)

   DFADMDFDALLSS (SEQ ID NO 5)

   SFDAMDFSALLSS (SEQ ID NO 6)

   10 DFDAMDFDALL (SEQ ID NO 20)

   DFDAMDFDALLSS (SEQ ID NO 21)

   8. A method according to any one of claims 2 to 7 wherein the peptide is a multimer. 9. A method according to claim 8 wherein the multimeric peptide is of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10.

   SFADMDFSALLSSSFADMDFSAILSRPSFADMDFSALLSS (SEQ ID NO 7)

   20 SFADMDFSALLSSSFADMDFSALLSSSFADMDFSALLSS (SEQ ID NO 8)

   DFADMDFDAI1 SSDFADMDFDALLSSDFADMDFDALLSS (SEQ ID NO 9)

   DFDAMDFDALLSSDFDAMDFDALLSSDFDAMDFDALLSS (SEQ ID NO 10)

   10. A peptide sequence selected from SEQ ID NOs 2, 20, 4, 5, 6, 21, 8, 9 and 10 as 25 defined hereinbefore.

   11. A nucleic acid which encodes a peptide as defined in claim 10.

   12. A nucleic acid which encodes a transactivation domain of Rel A (p65) subunit of 30 the human cellular transcription factor NF-KB, or a transactivating fragment thereof, or a transactivating mutant of either of these, or a transactivating multimer of said fragments

   - PPD 50610

   or mutants of fragments of these, wherein codons have been optimised for expression in the plant cell.

   13. A plant cell which has been transformed with a construct comprising a nucleic 5 acid sequence which encodes a transactivation domain of Rel A (p65) subunit of the human cellular transcription factor NP-KB, or a transactivating fragment thereof, or a transactivating mutant of either of these, or a transactivating multimer of said fragments or mutants of fragments of these, said sequence being arranged such that it enhances expression of a target gene in said plant cell.

   14. A plant cell according to claim 13 wherein said the target gene is a transgene.

   IS. A plant cell according to claim 14 wherein said transgene encodes a regulator protein effective in a gene switch.

   16. A plant or seed comprising a plant cell according to any of claims 13 to 15 and progeny thereof.

   17. A method, a peptide sequence, nucleic acid or plant cell substantially as 20 hereinbefore described with reference to the Figures.