WO2000011196A1

WO2000011196A1 - Polynucleotide sequences

Info

Publication number: WO2000011196A1
Application number: PCT/GB1999/002720
Authority: WO
Inventors: Ian Jeffrey Evans; John Anthony Ray
Original assignee: Syngenta Limited
Priority date: 1998-08-18
Filing date: 1999-08-17
Publication date: 2000-03-02
Also published as: CA2335652A1; AU5434499A; BR9913042A; EP1104480A1; CN1316007A; JP2002523052A; GB9818003D0; AU772284B2

Abstract

Nucleotide sequences and the expression products thereof are described for use in the production of transgenic plants. In particular polynucleotides comprising a sequence selected from those depicted in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 are provided.

Description

POLYNUCLEOTIDE SEQUENCES The present invention relates to recombinant DNA technology, and in particular to nucleotide sequences (and expression products thereof) which are used in the production of transgenic plants. The present invention provides, inter alia, nucleotide sequences useful in the production of plants which show improved resistance to infection by microorganisms such as bacteria and fungi.

According to the present invention there is provided a polynucleotide comprising a sequence selected from those depicted in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.

Also included within the invention is the translation product of the said polynucleotide sequences depicted in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.

The invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 53 to 385 in SEQ ID No. 1, nucleotides 11 to 334 in SEQ ID No. 2, nucleotides 24 to 317 in SEQ ID No. 3, nucleotides 20 to 343 in SEQ ID No. 4 or nucleotides 1 to 446 in SEQ ID No. 5. Also included within the invention is the translation product of the region comprised by nucleotides 53 to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3 or by nucleotides 20 to 343 in SEQ ID No. 4 or nucleotides 1 to 446 in SEQ ID No. 5 and protein having an amino acid sequence which is at least 85% similar to said product. The said translation product is a preproprotein comprising a signal sequence, protein encoding sequence and C-terminal propeptide which is naturally processed to yield mature biologically active protein. The invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 137 to 286 in SEQ ID No. 1, nucleotides 95 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No. 5. These polynucleotide sequences are especially preferred. Also included within the invention and especially preferred is the translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3 or by nucleotides 104 to 253 in SEQ ID No. 4 and protein having an amino acid sequence which is at least 95% similar to said product. The translation product is an antimicrobial protein. The related antimicrobial proteins DmAMPl and Dm-AMP2 have been described in Published International Patent Application No. WO 93/05153 and in Osborn et al (1995) FEBS Lett. 368 257-262.

The invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5. These nucleotides are particularly preferred according to the invention and encode protein sequences which may be used as cleavable linkers in the co-expression of multiple proteins as is described further herein. The invention further extends to the translation product of nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5 and protein having an amino acid sequence which is at least 85% similar to said product. The invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 53 to 136 in SEQ ID No. 1, nucleotides 11 to 94 in SEQ ID No.2, nucleotides 24 to 107 in SEQ ID No. 3, nucleotides 20 to 103 in SEQ ID No. 4 or nucleotides 1 to 176 in SEQ ID No. 5 excluding the sequence encoding the intron marked at positions 65 to 156. These nucleotide sequences are signal sequences which may be linked to homologous and heterologous protein encoding regions to transport proteins extracellular ly. The invention further extends to the use of said sequences as signal sequences. The invention further extends to the translation product of nucleotides 53 to 136 in SEQ ID No. 1, nucleotides 11 to 94 in SEQ ID No.2, nucleotides 24 to 107 in SEQ ID No. 3, nucleotides 20 to 103 in SEQ ID No. 4 or nucleotides 1 to 176 in SEQ ID No. 5 and protein having an amino acid sequence which is at least 85% similar to said product.

It is preferred that the degree of similarity is at least 90%, more preferred that the degree of similarity is at least 95% and still more preferred that the degree of similarity is at least 97%. In the context of the present invention, two amino acid sequences with at least 85% similarity to each other have at least 85% similar (identical or conservatively replaced) amino acid residues in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected. Likewise, two amino acid sequences with at least 90% similarity to each other have at least 90% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 3 gaps with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected.

For the purpose of the present invention, a conservative amino acid is defined as one which does not alter the activity/function of the protein when compared with the unmodified protein. In particular, conservative replacements may be made between amino acids within the following groups:

(i) Alanine, Serine, Glycine and Threonine

(ii) Glutamic acid and Aspartic acid

(iii) Arginine and Lysine

(iv) Isoleucine, Leucine, Valine and Methionine (v) Phenylalanine, Tyrosine and Tryptophan

Sequence alignments to measure sequence similarity may be produced using the Lasergene program MegAlign (supplied by DNASTAR Inc. 1228 S. Park St. Madison WI 53715, USA). The Clustal Method and the PAM250 residue weight table with the following parameters may be used: Multiple Alignment Parameters gap penalty 10 gap length penalty 10 Pairwise alignment parameters Ktuple 1 gap penalty 3 window 5 Diagonals saved 5

The invention also includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from those encoded by SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5, which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted in SEQ ID No 1, SEQ ID No 2, SEQ ID No3, SEQ ID No.4 or SEQ ID No.5 with the proviso that the sequence is not that described in SEQ ID No.6 or 7.

The polynucleotide sequence provided in SEQ ID Nos 6 and 7 is the predicted DNA sequence for Dm- AMP 1 and Dm-AMP2 as described in Figure 31A of Published International Patent Application No. WO 93/05153.

The invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 53 to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3, by nucleotides 20 to 343 in SEQ ID No. 4 or by nucleotides 1 to 446 in SEQ ID No.5 which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 53to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3, by nucleotides 20 to 343 in SEQ ID No. 4 or by nucleotides 1 to 446 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No. 6 or SEQ ID No. 7.

The invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3, by nucleotides 104 to 253 in SEQ ID No. 4 or by nucleotides 177 to 326 in SEQ ID No.5 which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3, by nucleotides 104 to 253 in SEQ ID No. 4 or by nucleotides 177 to 326 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No. 6 or SEQ ID No. 7.

The invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5. which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65 °C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No. 6 or SEQ ID No. 7. It may be desired to target the translation products of the polynucleotide to specific sub-cellular compartments within the plant cell, in which case the polynucleotide comprises sequences encoding chloroplast transit peptides, cell wall targeting sequences etc. immediately 5' of the regions encoding the said translation products.

Translational expression of the protein encoding sequences contained within the polynucleotide may be relatively enhanced by including known non translatable translational enhancing sequences 5' of the said protein encoding sequences. The skilled man is very familiar with such enhancing sequences, which include the TMV-derived sequences known as omega, and omega prime, as well as other sequences derivable, inter alia, from the regions 5' of other viral coat protein encoding sequences. In a particularly preferred embodiment of the invention, the polynucleotide is modified in that mRNA instability motifs and/or fortuitous splice regions are removed, or plant preferred codons are used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if the thus modified polynucleotide comprises plant preferred codons, the degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein is less than about 60%.

The invention also includes a plant transformation vector comprising a plant operable promoter, a polynucleotide sequence comprising all or part of the sequence selected from those depicted in SEQ ID'No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.5 under the transcriptional control thereof and encoding an antimicrobial protein, and a plant operable transcription terminator. The promoter may be constitutive or inducible. In particular, the promoter may be such that it induces transcription in response to application to the plant material containing it of a chemical. The invention further provides a plant transformation vector comprising a polynucleotide sequence selected from the group consisting of nucleotides 137 to 286 in SEQ ID No. 1, nucleotides 95 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No.5 under the transcriptional control of a plant operable promoter, and a plant operable transcriptional terminator.

The polynucleotide sequences provided in SEQ ID No 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.5 are related sequences with the translated products thereof showing a high degree of sequence similarity and it is believed that they may belong to a multi gene family. The invention still further includes plant tissue transformed with the said polynucleotide or vector, and material derived from the said transformed plant tissue, as well as morphologically normal fertile whole plants comprising the tissue or material. Such transformed plants include but are not limited to, field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, etc. Particularly preferred genetically modified plants are bananas.

The invention still further includes the progeny of the plants of the preceding paragraph, which progeny comprises a polynucleotide of the invention stably incorporated into its genome and heritable in a mendelian manner and the seeds of such plants and such progeny. The invention also provides a method of producing plants which are substantially tolerant or substantially resistant to antimicrobial infection, comprising the steps of:

(i) transforming plant material with a polynucleotide or vector of the invention;

(ii) selecting the thus transformed material; and

(iii) regenerating the thus selected material into morphologically normal fertile whole plants.

Plant transformation, selection and regeneration techniques, which may require routine modification in respect of a particular plant species, are well known to the skilled man. The invention also provides the use of a polynucleotide as described herein or a vector described herein in the production of plant tissues and/or morphologically normal fertile whole plants which are substantially tolerant or substantially resistant to microbial infection.

In a further aspect the invention provides a method of selectively controlling microorganisms at a locus comprising the plants, progeny and/or seeds described herein comprising applying to the locus a microorganism controlling amount of the translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1.; nucleotides 95 to 244 in SEQ ID No.2, nucleotides 108 to 257 in SEQ ID No. 3, or nucleotides 104 to 253 in SEQ ID No. 4. In a still further aspect the invention provides the use of a polynucleotide according to the invention described herein , or a vector as described herein in the production of an antimicrobial protein.

The invention will be further apparent from the following description taken in conjunction with the associated figures and sequence listings in which : Figure 1 shows the polynucleotide and corresponding amino acid sequences for A) Dml (SEQ ID No5) and B) Dm2.18 (SEQ ID Nol),

Figure 2 shows the polynucleotide and corresponding amino acid sequences for A) Dm2.1 (SEQ ID No2) and B) Dm2.3 (SEQ ID No3), Figure 3 shows the polynucleotide and corresponding amino acid sequence for Dm2.5 (SEQ

ID No. 4),

Figure 4 shows a diagrammatic map of plasmids pMJBl, pDmAMPD and pDmAMPE;

Figure 5 shows a diagrammatic map of plasmid pFAJ3106; Figure 6 shows a diagrammatic map of plasmid pFAJ3109

Figure 7 shows the nucleotide sequence between the Xhol and Sad sites of pFAJ3106;

Figure 8 shows the nucleotide sequence between the Xhol and Sad sites of pFAJ3109;

Figure 9 shows a diagrammatic map of plasmid pZPS38;

Figure 10 shows a diagrammatic map of plasmid pZPS34; Figure 11 shows a diagrammatic map of plasmid pZPS35;

Figure 12 shows a diagrammatic map of plasmid pZPS37.

Figure 13 shows a plan of the construction of the Dm- AMP gene

Figure 14 shows one predicted polynucleotide sequence for DmAMPl (SEQ ID No. 6) and

Dm-AMP2 (SEQ ID No. 7). Figure 15 shows a diagrammatic map of plasmid pAID-MR7

EXAMPLE 1

Dm Gene Isolation and Vector Construction Dahlia cDNA library construction

Near-dry seeds were collected from flowers of Dahlia merkii .

Total RNA was purified from the seeds using the method of Jepson et al . (Plant Molecular

Biology Reporter 9 131-138 (1991)).

Seeds were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Phenol/m-cresol (9:1) was added followed by RNA homogenisation buffer and the mixture ground until a fine paste was obtained. The mixture was spun, the aqueous phase collected and extracted twice with phenol/chloroform (1 :1). Lithium chloride (12 M) was added to the resulting aqueous layer to a final concentration of 2 M and incubated overnight at 4°C.

Precipitated RNA was collected by spinning at 13,000 rpm in an Eppendorf centrifuge and the RNA pellet re-suspended in 5 mM Tris-HCI, pH 7.5. A second overnight lithium chloride precipitation was carried out and the RNA collected and re-suspended in 5 mM Tris-HCI, pH

7.5.

0.6 mg of total RNA was obtained from 2 g of D. merkii seed.

PolyATract magnetic beads (Promega) were used to isolate approximately 2 μg poly-A⁺ RNA from 0.2 mg of total RNA.

The poly-A⁺ RNA was used to construct a cDNA library using a ZAP-cDNA synthesis kit (Stratagene). Following first and second strand synthesis double stranded cDNA was size fractionated on a Sephacryl S-400 column. The three largest cDNA size fractions were pooled and ligated with vector DNA. After phage assembly using Gigapack Gold (Stratagene) packaging extracts, approximately 1 x 10⁵ pfu were obtained. Probing

Dahlia genomic DNA was prepared from 100 mg of developing Dahlia seeds and flower tissue. Tissue was homogenised in a 1.5 ml Eppendorf tube with a conical plastic pestle. 400 μl of a solution containing 0.2M Tris-HCI pH 8.5, 0.25M NaCl, 0.025 M EDTA and 0.5% SDS was added and the tube vortexed for 5 seconds. Cell debris was pelleted by spinning at 13,000 rpm for 1 minute in a MSE bench top micro centrifuge. 300 μl of aqueous extract was transferred to a fresh Eppendorf tube. Genomic DNA was precipitated by the addition of 300 μl isopropyl alcohol and incubation at room temperature for 2 minutes. Genomic DNA was pelleted by spinning at 13,000 rpm for 5 minutes. The ethanol/aqueous supernatant was removed from the tube by pipette and the genomic DNA pellet allowed to air dry. Genomic DNA was then resuspended in 30 μl H₂O.

To amplify a 144 bp fragment of DNA encoding 48 amino acids of the mature Dm- AMP 1 a PCR was carried out with Dahlia genomic DNA and oligonucleotides AFP-5 (based on Dm- AMP1 N-terminal amino acid sequence CEKASKTW) and AFP-3EX (based on Dm- AMP 1 C-terminal amino acid sequence MCFCYFNC). Using the following conditions 94°C, 60 seconds, 48°C, 12 seconds and 72°C, 60 seconds for 35 cycles. A PCR product of approximately 150 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation. The PCR product was cloned into pBluescript by ligating blunt Bluescript vector and gel isolated PCR product together using T4DNA ligase and transforming into competent E. coli MCI 022 cells. Transformation mixes were plated onto L-agar plates containing 100 μg/ml a picillin and incubated at 37°C for 16 hours. Colonies were picked and cells shaken for 16 hours in 3 ml L-broth containing 100 μg/ml ampicillin at 37"C. Plasmid DNA was prepared from colonies using a Promega Wizard mini-prep kit. The inserts of 10 transformants were sequenced using a Sequenase kit (United States Biochemical). The cloned PCR product sequences represented 3 Dm-AMPl related genes. PCR clone 4 contained the DNA sequence

AAGACGTGGTCGGGAAACTGTGGCAATACGGG

ACATTGTGACAACCAATGTAAATCATGGGAGGGTGCGGCCCATGGAGCGTGTCA TGTGCGTAATGGGAAACACATGTGTTTCTGCTACTTCAAC, encoding a portion of the observed mature Dm-AMPl protein sequence (KTWSGNCGNTGHCD NQCKSWEGAAHGACHVRNGKHMCFCYFN).

The 144 bp PCR product mixture labelled with α³²-P d-CTP was used to probe Hybond N (Amersham) filter lifts made from plates containing a total of 6 x 10⁴ pfu of the cDNA library. The filters were hybridised at 46°C for 18 hrs in 5 x SSC, 0.1% SDS, 0.25% skimmed milk powder. Filters were washed in 2 x SSC, 0.1% SDS at 60°C. Autoradiography was carried out at -70°C with intensifying screens. Thirty potentially positive signals were observed. 22 plaques were picked and taken through two further rounds of screening. After in vivo excision 13 clones were characterised by DNA sequencing. Four classes of Dm- AMP related peptide were encoded by the 13 cDNA clones and the sequences of these peptides are provided in SEQ ID Nos 1- 4 in the accompanying figures. Three versions of the Dm- AMP core region were represented in the four classes. One of the classes (Dm2.5 type) contained a core region which may correspond to Dm-AMP2. None of the cDNAs encoded a core region equivalent to the observed mature Dm-AMPl peptide sequence. Isolation of a Mature Dm-AMPl Gene Using the sequence of PCR clone 4 (above) and information from the NH₂ and COOH ends of the peptides described by cDNA sequences two pairs of oligonucleotides were designed for amplification of a gene encoding the observed mature Dm-AMPl. A PCR was carried out with Dahlia genomic DNA and oligonucleotides MATAFP-5P (based on the codons present in Dm2.1, Dm2.3, Dm2.18 and Dm2.5 encoding the N-terminal amino acid sequence M(AV)(KN)(NR)SVAF) and MATAFP-5 (based on the mature Dm- AMP 1 amino acid sequence NGKHMCF) using the following conditions; 94°C, 60 seconds, 53°C, 12 seconds and 72°C, 60 seconds for 40 cycles. A PCR product of approximately 220 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation. The PCR product was cloned into pBluescript and clones sequenced as described above. A clone containing the

5' half of a Dm-AMPl gene was identified. A PCR was carried out with Dahlia genomic DNA and oligonucleotides MATAFP-3 (based on the mature Dm-AMPl amino acid sequence GACHVRN) and DM25MAT-3 (based on the last two amino acids and the 3' untranslated region of Dm2.5) using the following conditions; 94°C, 60 seconds, 53°C, 12 seconds and 72°C, 60 seconds for 40 cycles. A PCR product of approximately 170 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation. The PCR product was cloned into pBluescript and clones were sequenced as described above. A clone containing the 3' half of a Dm- AMP 1 gene was identified.

The 5' and 3' sections of the mature gene were combined to assemble the sequence of the mature Dm-AMPl gene (see Figure 1 SEQ ID No.5) which is comprised of exon 1, 64 bp encoding part of the leader peptide, 92 bp intron and exon 2 encoding the end of the leader sequence, Dm-AMPl core and C-terminal extension.

Vector Oligonucleotide design

Four oligonucleotides were designed based on the DNA sequence of the mature Dm-AMPl gene: DMVEC-1 top strand priming at the 5' end of the mature DmAMP-l gene incorporating a

Nco I site at the translation start of DmAMP-l allowing cloning into pMJBl (see Figure 4).

DMVEC-2 bottom strand priming in the 3' end of the C-terminal extension and a Sac I site for cloning in pMJB 1.

DMVEC-3 top strand priming at the 5' end of the mature DmAMP-l gene incorporating a Nco I site at the translation start of DmAMP-l allowing cloning into pMJBl also encoding complete signal peptide (minus intron).

DMVEC-4 bottom strand priming in the 3' end of the core region and a Sac I site for cloning in pMJBl.

A PCR was carried out with Dahlia genomic DNA and oligonucleotides DMVEC-1 and DMVEC-2 using the following conditions; 94°C, 60 seconds, 60°C, 12 seconds and 72°C, 60 seconds for 45 cycles. A PCR product of approximately 450 bp spanning the mature Dm- AMP 1 gene was obtained, this PCR product was isolated from an agarose gel and used as a template for PCRs described in vector construction below.

Vector Construction pDmAMPD

A PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-1 and DMVEC-4 using the following conditions, 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles. The PCR product was cut with Nco I and Sac I the 60 bp Nco I/Sac I fragment was isolated and ligated with pMJBlcut with Nco I and Sac I. The ligation mix was used to transform competent E. coli MCI 022 cells and plasmid DNA of ampicillin resistant transformants was obtained as described above.

The identity of the fragment in one of the resulting transformants was confirmed by sequencing, the clone was termed pDmAMPA. A PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-3 and DMVEC-4 using the following conditions; 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles. The PCR product was cut with Nco I, the resulting 150 bp Nco I fragment isolated and cloned into pDmAMPA cut with Nco I. DNA sequencing confirmed that one transformant termed pDmAMPD, contained DNA encoding Dm-AMP leader and core region. pDmAMPE

The PCR product obtained with DMVEC-1 and DMVEC-2 was cut with Nco I and Sac I the 180 bp Nco I/Sac I fragment was isolated and cloned into pMJBl cut with Nco I and Sac I as described above. The identity of the fragment in one of the resulting transformants was confirmed by sequencing, the clone was termed pDmAMPB.

A PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-3 and DMVEC-4 using the following conditions; 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles. The PCR product was cut with Nco I and the resulting 150 bp Nco I fragment isolated and cloned into pDmAMPB cut with Nco I. DNA sequencing confirmed that one transformant termed pDmAMPE, contained

DNA encoding Dm-AMP leader, core and C-terminal extension.

Both pDmAMPD and pDmAMPE vector sequences contained PCR derived base substitutions with respect to Dm-AMPl gene DNA sequence however the base changes were silent having no effect on the expected amino acid sequence.

AFP-5 (to CEKASKTW)

TG(T,C)GANAANGCN(A,T)(G,C)NAA(A,G)ACNTGG

AFP-3EX (to MCFCYFNC)

CA(A,G)TT(A,G)AANTANCANAAA(A,G)CACAT MATAFP-5P

ATGGC(C,G)AAN(A,C)(A,G)NTC(A,G)GTTGCNTT

MATAFP-5

AAACACATGTGTTTCCCATT

MATAFP-3 AGCGTGTCATGTGCGTAAT

Dm25MAT-3

TAAAGAAACCGACCCTTTCACGG

DMVEC-1

ATCGTAGCCATGGTGAATCGGTCGGTTGCGTTCTCCGCG DMVEC-2

AAACCGACCGAGCTCACGGATGTTCAACGTTTGGAAC

DMVEC-3

ATGCATCCATGGTGAATCGGTCGGTTGCGTTCTCCGCGTTCGTTCTGATCCTTTTC

GTGCTCGCCATCTCAGATATCGCATCCGTTAGTGGAGAACTATGCGAGAAA DMVEC-4

AGCAAGCTTTTCGGGAGCTCAACAATTGAAGTAA

EXAMPLE 2

Constructions of plant transformation vectors Expression cassettes containing a Dm-AMPl open reading frame functionally linked to an enhanced 35S promoter, a TMV omega translational enhancer and a Nos 3' region are isolated as restriction fragments. pDmAMPD and pDmAMPE are both digested with the restriction endonucleases Hindlll and EcoRI and the appropriate restriction fragment isolated and purified. Each fragment is ligated into a binary vector (a pBIN19 derivative named pBinl9i) which has also been digested with Hindlll and EcoRI. The resulting constructs, named pDmAMPLC and pDmAMPLCC, incorporate the expression cassettes from pDmAMPD and pDmAMPE respectively. pDmAMPLC and pDmAMPLCC are subsequently introduced into Agrobacterium tumefaciens strain LBA4404 and introduced into tobacco and oil seed rape using standard plant transformation methodology.

Plants are regenerated from callus tissue resistant to the selective agent kanamycin and expression of the Dm-AMPl product is monitored by standard Western blot or ELISA methods using antibody which had been raised against Dm-AMPl protein. A range of expression levels are detected. The Dm-AMPl expressed in selected transgenic is further characterised following extraction and partial purification from leaves of such lines. The product is of the predicted mass, as indicted by mass spectrometry. It is also demonstrated to retain biological activity after extraction as demonstrated by retention of antifungal activity in in-vitro (micro-titre plate) assays. EXAMPLE 3

Constructions of plant transformation vectors for polyprotein expression Schematic representations of the plant transformation vectors used in this work, pFAJ3106 and pFAJ3109, are shown in figures 5 and 6, respectively. The nucleotide sequences comprised between the Xhol and Sad sites of these plasmids, which encompass the regions encoding antimicrobial proteins, are presented in Figures 7 and 8. The regions comprised between the Xhol and Sad sites of plasmid pFAJ3106 (shown in Figure 7) was constructed following the two-step recombinant PCR protocol of Pont-Kindom G.A.D. (1994, Biotechniques 16, 1010- 1011). Primers OWB175 (5'AGGAAGTTCATTTCATTTGG) and OWB279 (5'- GCCTTTGGCACAACTTCTGCCTCTTTCCGATGAGTTGTTCGGCTTTAAGTTTGTC); were used in a first PCR reaction with plasmid pDMAMPE (see above) as a template. The second PCR reaction was done using as a template plasmid pFRG4 (Terras F.R.G. et al., 1995, Plant Cell 7, 573-588) and as primers a mixture of the PCR product of the first PCR reaction, primer OWB175 and primer OWB172 (5 TAGAGCTCCTATTAACAAGGAAAGTAGC, Sad site underlined). The resulting PCR product was digested with Xhol and Sad and cloned into the expression cassette vector pMJBl (see above). The expression cassette in the resulting plasmid, called pFAJ3099, was digested with Hindlll (flanking the 5' end of the CaMV35S promoter) and EcoRI (flanking the 3' end of the nopaline synthase terminator) and cloned in the corresponding sites of the plant transformation vector pGPTVbar (Becker D. et al., 1992, Plant Mol. Biol. 20, 1195-1197) to yield plasmid pFAJ3106. Plasmid pFAJ3109 was constructed by cloning the Hindlll-EcoRI fragment of plasmid pDMAMPD (see above) into the corresponding sites of plant transformation vector pGPTVbar (see above). Plant transformation

Arabidopsis thaliana ecotype Columbia-O was transformed using recombinant Agrobacterium tumefaciens by the inflorescence infiltration method of Bechtold N. et al. (1993, CR. Acad. Sci. 316, 1194-1199). Transformants were selected on a sand/perlite mixture subirrigated with water containing the herbicide Basta (Agrevo) at a final concentration of 5 mg/1 for the active ingredient phosphinothricin. Elisa assays and protein assays Antisera were raised in rabbits injected with either RsAFP2 (purified as described in Terras F.R.G. et al., 1992, J. Biol. Chem. 267, 15301-15309) or DmAMPl (purified as in Osborn R.W. et al., 1995, FEBS Lett. 368, 257-262). ELISA assays were set up as competitive type assays essentially as described by Penninckx I.A.M.A. et al. (1996, Plant Cell 8, 2309-2323). Coating of the ELISA microtiter plates was done with 50 ng/ml RsAFP2 or DmAMPl in coating buffer. Primary antisera were used as 1000- and 2000-fold diluted solutions (DmAMPl and RsAFP2, respectively) in 3 % (w/v) gelatin in PBS containing 0.05 % (v/v) Tween 20.

Total protein content was determined according to Bradford (1976, Anal. Biochem. 72, 248- 254) using bovine serum albumin as a standard. Purification and characterisation of expressed proteins Arabidopsis leaves were homogenized under liquid nitrogen and extracted with a buffer consisting of 10 mM NaH₂PO₄, 15 mM Na^PO^ 100 mM KCl, 1.5 M NaCl. The homogenate was heated for 10 min at 85°C and cooled down on ice. The heat-treated extract was centrifuged for 15 min at 15 000 x g and was injected on a reserved phase high pressure liquid chromatography column (RP-HPLC) consisting of C8 silica (0,46 cm x 25 cm; Rainin) equilibrated with 0.1 % (v/v) trifluoroacetic acid (TFA). The column was eluted at 1 ml/min in a linear gradient in 35 min. from 15 % to 50 % (v/v) acetonitrile in 0.1 % (v/v) TFA. The eluate was monitored for absorbance at 214 nm, collected as 1 ml fractions, evaporated and finally redissolved in water. The fractions were tested by ELISA assays. Preparation of extracellular fluid and intracellular extract Intercellular fluid was collected from Arabidopsis leaves by immersing the leaves in a beaker containing extraction buffer (10 mM NaH₂PO₄, 15 mM Na₂HPO₄, 100 mM KCl, 1.5 M NaCl). The beaker with the leaves was placed in a vacuum chamber and subjected to six consecutive rounds of vacuum for 2 min followed by abrupt release of vacuum. The infiltrated leaves were gently placed in a centrifuge tube on a grid separated from the tube bottom. The intercellular fluid was collected from the bottom after centrifugation of the tubes for 15 min at 1800 x g. The leaves were resubjected to a second round of vacuum infiltration and centrifugation and the resulting (extracellular) fluid was combined with that obtained after the first vacuum infiltration. After this step the leaves were extracted in a Phastprep (BlOlOl/Savant) reciprocal shaker and the extract clarified by centrifugation (10 min at 10,000 x g) and the resulting supernatant considered as the intracellular extract. Characterization of transgenic plants and expression analysis

To explore the possibility of expressing polyprotein precursor genes in plants, three different plant transformation vectors were made with the aim to co-express two different cysteine-rich plant defensins with antifungal properties, namely RsAFP2 and DmAMPl. The polyprotein precursor regions of these constructs all featured a leader peptide region from the DmAMPl cDNA, the mature protein domain of DmAMPl, an internal propeptide region, and the mature protein domain of RsAFP2. Construct 3106 has a propeptide consisting of a part of the DmAMP 1 propeptide and a putative subtilisin-like protease processing site (IGKR) at its C- terminus. The rationale behind construct 3106, is based on our observations that the C-terminal propeptides of DmAMPl are cleaved off at their N-terminus when expressed as DmAMP 1- preproproteins in tobacco, respectively, while this processing event does not prevent the mature proteins from being sorted to the apoplast (De Bolle et al., 1996, Plant Mol. Biol. 31, 993-1008; R.W. Osborn and S. Attenborough, personal communication). This infers that the processing enzymes are either in the secretory pathway or in the apoplast. On the other hand, C-terminal cleavage of the internal propeptide in these constructs should be executed by a subtilisin-like protease, a member of which in yeast (Kex2) is known to occur in the Golgi apparatus (Wilcox CA. and Fuller R.S., 1991, J. Cell. Biol. 115, 297- ), while a member in tomato occurs in the apoplast (Tornero P. et al., 1997, J. Biol. Chem. 272, 14412-14419). Proteins deposited in the apoplast, the preferred deposition site for antimicrobial proteins engineered in transgenic plants (Jongedijk E. et al., 1995, Euphytica 85, 173-180; De Bolle et al., 1996, Plant Mol. Biol. 31, 993-1008) are normally synthesized via the secretory pathway, encompassing the Golgi apparatus.

Constructs were also made for expression of DmAMPl (construct 3109, figure 6). Expression levels of DmAMPl and RsAFP2 were analysed in leaves taken from a series of Tl transgenic Arabidopsis plants resulting from transformation with the constructs described above. Most of the tested lines transformed with the polyprotein constructs 3106 clearly expressed both DmAMPl and RsAFP2. There was generally a good correlation between DmAMPl and RsAFP2 levels. However, the RsAFP2 levels were generally 2 to 5 -fold lower than the DmAMPl levels. It is not known whether the apparent lower expression levels of RsAFP2 versus DmAMPl are real or whether they result from a bias in the extraction procedure or the assays. The expression levels in the lines transformed with the polyprotein constructs 3106 were generally much higher compared to those in lines transformed with the single protein construct 3109. Hence, the use of polyprotein constructs appears to result in enhanced expression, which is an unexpected finding. Analysis of the proteins expressed for polyprotein constructs A transgenic line was selected among each of the populations transformed with construct 3106 and the selected lines were further bred to obtain plants homozygous for the transgenes. In order to analyse whether DmAMPl and RsAFP2 were correctly processed in these lines, extracts from the plants were prepared as described in Materials and Methods and separated by RP-HPLC on a C8-silica column. Fractions were collected and assessed for presence of compounds cross-reacting with antibodies raised against either DmAMPl or RsAFP2 using Elisa assays. DmAMP 1 cross-reacting compound eluted at a position identical or very close to that of authentic DmAMPl in the line transformed with construct 3106. Likewise, a RsAFP2 cross- reacting compound was detected in the 3106 lines at an elution position identical or very close to that of authentic RsAFP2. None of the fractions reacted with both the anti-DmAMPl and anti-RsAFP2 antibodies, indicating that an uncleaved fusion protein was not present in the extracts. No cross-reacting compounds were observed in a non-transformed line. It is concluded that the primary translation products of the transcription units of construct 3106 (partial DmAMPl C-terminal propeptide with subtilisin-like protease site as a linker peptide) are somehow processed to yield separate DmAMPl -cross-reacting and RsAFP2-cross-reacting portions that appear to be identical or very closely related to DmAMPl and RsAFP2, respectively, based on their chromatographic behavior. Analysis of the subcellular location of coexpressed plant defensins In order to determine whether the coexpressed plant defensins are either secreted extracellularly or deposited intracellularly, extracellular fluid and intracellular extract fractions were obtained from leaves of homozygous transgenic Arabidopsis lines transformed with constructs 3106. The cytosolic enzyme glucose-6-phosphate dehydrogenase was used as a marker to detect contamination of the extracellular fluid fraction with intracellular components. As shown in Table 1, glucose-6-phosphate dehydrogenase was partitioned in a ratio of about 80/20 between intracellular extract fractions and extracellular fluid fractions. In contrast, the majority of DmAMPl and RsAFP2 content in all transgenic plants tested was found in the extracellular fluid fractions. These results indicate that both plant defensins released from the polyprotein precursors are deposited primarily in the apoplast. Hence, all processing steps that result in cleavage of the polyprotein structure must occur either in the apoplast or along the secretory pathway. Table 1 : Relative abundance of glucose-6-phosphate dehydrogenase activity (GPD),

DmAMPl and RsAFP2 in the extracellular fluid (EF) and intracellular extract (IE) fractions obtained from transgenic Arabidopsis plants. Construct Relative abundance¹ (%) of

GPD DmAMPl RsAFP2

EF IE EF IE EF IE pFAJ3106 Ϊ7 83 94 6 60 40 ¹ Relative abundance is expressed as % of the sum of the contents in the EF and IE fractions.

EXAMPLE 4 Expression of the sweet tasting protein Brazzein in tomato

Production of transgenic tomato plants with increased accumulation of sweet tasting protein Brazzein.

Constructs were prepared containing the Dahlia (Dahlia merckii) antimicrobial protein signal peptide fused with the Brazzein gene under the transcriptional control of the Arabidopsis polyubiquitin extension protein promoter (UBQ) or the Polygalacturonase promoter (PG). Constructs were also prepared which encoded Brazzein without a signal peptide but with an N-terminal methionine by the insertion of ATG nucleotides upstream of the Brazzein gene under the expressional control of either the UBQ promoter or the PG promoter. These were prepared as follows:

Construction of the transformation vector for expression in tomato with Dahlia signal peptide fused to Brazzein under the expressional control of either the UBQ promoter or the PG promoter:

A synthetic DNA was produced which coded for the Dahlia signal peptide fused to Brazzein. The codons were optimised for expression in tomato. Using appropriate restriction sites the coding sequence was cloned into a plasmid vector. The coding region was excised from the plasmid and cloned between the promoter in question and the terminator in the correct orientation for expression.

Generation and analysis of plants transformed with the transformation vector. The vector was transferred to Agrobacterium tumefaciens LBA4404 (a microorganism widely available to plant biotechnologists) and used to transform tomato plants. Transformation of tomato stem segments followed standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Up to 30 individual plants were regenerated with each construct and grown to maturity. The presence of the construct in all of the plants was confirmed by polymerase chain reaction analysis. DNA Southern blot analysis on all plants indicated that the insert copy number was between 1 and 10. Northern blot analysis on fruit from one plant indicated that the Brazzein gene was expressed. Brazzein production in the fruit of all plants was measured by ELISA (enzyme linked imunoabsorption assay) using a polyclonal and a monoclonal antibody raised against native Brazzein protein isolated from the fruit of the plant Pentadiplandra brazzeana Baillon Two fruit were collected from each transgenic plant at 7 days post breaker (the term breaker is used to indicate when the tomato fruit first show signs of the orange colouration characteristic of most mature tomato fruit). Total fruit protein was extracted from a sample of the pericarp of each of the fruit. The amount of Brazzein protein in the total protein extract was measured by ELISA and calculated as the amount of Brazzein per gram fresh weight of the fruit. For each plant the average Brazzein content of the two fruits was calculated. In some plants Brazzein could not be detected in the fruit using the ELISA technique. Western blot analysis of the total protein extract from some of the fruit revealed a 6.5kD protein band, which matches the predicted size of the mature Brazzein protein. This confirmed that the fruit contained Brazzein and that the signal peptide had been cleaved as if the signal peptide had not been cleaved, one would expect the protein to be larger. The Brazzein in fruit from plants which had been transformed with a construct lacking a signal peptide was not detected by Western blot. This is because the Brazzein content in these fruit is below the level of detection by western blot. ELISA is a more sensitive technique than western blot and protein was detected in these fruit by this method. The results are summarised in Table 2 below. TABLE 2

Table 2 (continued)

Example 5

Construction of Dm- AMP Transient Expression Vectors

To produce proteins for assessment of the relative activity of the variants of Dm- AMP, three vectors were constructed for transformation of black Mexican sweet (BMS) maize cell suspensions to give transient expression of Dm-AMPs.

The vector chosen for these experiments was pALD-MR7. pALD-MR7 was constructed using the commercially available cloning vector pNEB193

(New England Biochemicals) itself a modified version of pUC19 (Yanisch-Perron C, Vieira J. and Messing J. "Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and UC19 vectors." Gene; 33:103-19 (1985)). Gene components to facilitate protein expression were inserted within the multiple cloning region of pNEB 193, namely:

1) A plant promoter to drive transcription, a 1.9Kb Xba I fragment of the MR7 promoter (MR7 prom.) from maize (as described in US Patent No. 5837848)

2) A sequence known to enhance gene transcription, the alcohol dehydrogenase intron 1 (1 1) from maize (Dennis E.S., Gerlach W.L., Pryor A.J., Bennetzen J.L., Inglis A., Llewellyn D., Sachs M.M., Ferl R. J. and Peacock W.J. "Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize." Nucleic Acids Research; 12:3983-4000(1984)). 3) A multiple cloning region for insertion of open reading frames containing the site for restriction endonuclease Xba I.

4) A 3' region for mRNA transcript termination and polyadenylation from the cauliflower mosaic virus 35S RNA transcript (3') (Pietrak et al Nucleic Acid Research 14 5857-5868 (1986), Franck A., Guilley H., Jonard G., Richards K. and Hirth L. "Nucleotide sequence of cauliflower mosaic virus DNA." Cell; 21: 285-94 (1980)).

To produce proteins for assessment of the relative activity of the variants of Dm- AMP, three vectors were constructed for use in a transient expression system.

All plasmid DNA described in the following examples was prepared using Promega Wizard mini-prep or Promega Wizard midi-prep kits using the manufacturer's suggested protocols.

DNA sequencing was carried out using USB Sequenase kits and the manufacturer's suggested protocols.

Example 5a Vector DNA was prepared by digesting plasmid DNA of pAIDMR7 with Xba I and the ends filled in with Klenow DNA polymerase. The linear vector was isolated from an agarose gel by electroelution and ethanol precipitation. The DNA pellet was air dried and taken up in a small volume of water.

Plasmid DNA of a cDNA clone containing a Dm2.1 ORF was digested with Eco Rl and Sea I and the ends filled in with Klenow DNA polymerase. Insert DNA containing the Dm2.1 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.

The DNA pellet was air dried and taken up in a small volume of water.

Vector and insert DNA were ligated together using T4 DNA ligase and transformed into competent E. coli MCI 022 cells. Tranformation mixes were plated onto L-agar plates containing 100 μg/ml ampicillin and incubated at 37°C for 16 hours. Colonies were picked and cells shaken for 16 hours in 3 ml L-broth containing 100 μg/ml ampicillin at 37°C.

Plasmid DNA was prepared from several colonies and used in DNA sequencing reactions to identify transformants containing the Dm2.1 coding region in the appropriate orientation with respect to the MR7 promoter. One such clone was identified and named pAIDMR721. Example 5b

Vector DNA was prepared as in Example 5a.

Plasmid DNA of a cDNA clone containing a Dm2.3 ORF was digested with Eco Rl and Sea

I and the ends filled in with Klenow DNA polymerase. Insert DNA containing the Dm2.3 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.

The DNA pellet was air dried and taken up in a small volume of water.

Vector and insert DNA were ligated together, transformed into E. coli MCI 022 and colonies characterised by DNA sequencing as described in Example 5a. A clone was identified containing the Dm2.3 ORF in the desired orientation and named pAIDMR723.

Example 5c

Vector DNA was prepared as in Example 5a.

Plasmid DNA of a cDNA clone containing a Dm2.5 ORF was digested with Eco Rl and Dra

I and the ends filled in with Klenow DNA polymerase. Insert DNA containing the Dm2.5 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.

The DNA pellet was air dried and taken up in a small volume of water.

Vector and insert DNA were ligated together, transformed into E. coli MCI 022 and colonies characterised by DNA sequencing as described in Example 5a. A clone was identified containing the Dm2.5 ORF in the desired orientation and named pAIDMR725.

Example 6

Transient Expression of Dm-AMPs

Plasmid DNA of clones pAIDMR721 pAIDMR723, pAIDMR725 and pAIDMR7 is used to transform cultured maize BMS cells using the PEG method. Protoplast preparation and transformation

Protoplasts are isolated from a maize suspension of Black Mexican Sweet Corn suspension culture (BMS) [Green, Hort. Sci., 12 (1977) 131 ; Smith et al., Plant Sci. Lett., 36 (1984) 67] subcultured in BMS medium (MS medium supplemented with 2% sucrose, 2 mg/1 2,4-D, pH5.8). Cells from suspensions two days post subculture are digested in enzyme mixture (2.0% cellulase RS (Yakult Honsha Co., Ltd), 0.2% pectolyase Y23 (Yakult Honsha Co., Ltd), 0.5M mannitol, 5mM CaCl₂2H₂O, 0.5% MES, pH5.6, ~660mmol/kg) using ~10ml/g cells, incubating at 25°C, rotating gently for 2 hours. The digestion mixture is sieved through 250μm and 38μm sieves, and the filtrate centrifuged at 700rpm for 3.5 minutes. Protoplasts are resuspended in wash buffer (0.358M KCl, l .OmM NH₄NO₃, 5.0mM CaCl₂2H₂O, 0.5mM KH₂PO₄, pH4.8, ~670mmol/kg) and pelleted twice prior to resuspending in wash buffer and counting. Transformation is achieved using PEG (PEG 3350, Sigma Co) mediated uptake (Negrutiu et al., 1987) employing plasmid DNA prepared using Qiagen midi plasmid preparation kit (Qiagen Ltd, Crawl ey, UK). Protoplasts are resuspended at 2 x lOVml in MaMg medium (0.4M mannitol, 15mM MgCl₂, 0.1% MES, pH5.6, ~450mmol/kg) aliquotting 0.5ml / treatment (i.e. lxlO⁶ protoplasts/treatment). Samples are heat shocked at 45°C for 5 minutes then cooled to room temperature. Each transformation is carried out with lOμg of pAID-MR7 alone or lOμg of each construct pAIDMR721, pAIDMR723 or pAIDMR725. Each protoplast treatment is resuspended in 1.5ml culture medium (MS medium, 2% sucrose, 2mg/l 2,4-D, 9% mannitol, pH5.6, ~700mmol/kg). Samples are incubated in 3cm dishes at 25°C, in the dark, for 48 hours prior to harvesting.

After 48 hours incubation cells are separated from media by centrifugation.

Cells are osmotically lysed by the addition of water. Cell debris is pelleted by centrifugation and the proteins remaining in solution are freeze dried. The freeze dried proteins are taken up in a small volume of water and the concentration of protein determined using Bradford reagent.

Culture media removed from cells is freeze dried and taken up in a small volume of water, the concentration of protein is determined as above.

Protein samples isolated from all BMS transformations are assayed for spore germination inhibition in a bioassay against Fusarium culmorum spores as described in Published International Patent Application No. WO 93/05153.

Claims

1. A polynucleotide comprising a sequence selected from those depicted in

SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.

2. A polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 53 to 385 in SEQ ID No. 1, nucleotides 11 to 334 in SEQ ID No. 2, nucleotides 24 to 317 in SEQ ID No. 3, nucleotides 20 to 343 in SEQ ID No. 4 or nucleotides 1 to 446 in SEQ ID No. 5.

3. A polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 137 to 286 in SEQ ID No. 1, nucleotides 95 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No. 5.

4. A polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5.

5. A polynucleotide encoding a protein having a substantially similar activity to that encoded by any of SEQ ID No. 1, No 2, No. 3, No,4 or No. 5 which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65┬░C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted in SEQ ID No 1, SEQ ID No 2, SEQ ID No3, SEQ ID No.4 or SEQ ID No.5 with the proviso that the sequence is not that described in SEQ ID No.6 or 7.

. A polynucleotide encoding a protein having a substantially similar activity to that encoded by nucleotides 137 to 286 in SEQ ID No. 1, nucleotides 95 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No. 5., which polynucleotide is complementary to one which when incubated at a temperature of between 55 and

65┬░C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3, by nucleotides 104 to 253 in SEQ ID No. 4 or by nucleotides 177 to 326 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No.

6 or SEQ ID No. 7.

7. A polynucleotide according to any preceding claim, further comprising a region encoding a peptide which is capable of targeting the translation products of the sequence to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls.

8. A polynucleotide according to any preceding claim, wherein translational enhancing sequences are inserted 5' of the protein encoding regions comprised by the polynucleotide.

9. A polynucleotide according to any preceding claim, which is modified in that mRNA instability motifs and/or fortuitous splice regions are removed, or plant preferred codons are used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if the thus modified polynucleotide comprises plant preferred codons, the degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein is less than about 60%.

10. A plant transformation vector comprising a plant operable promoter, a polynucleotide sequence according to any of the preceding claims under the transcriptional control thereof and a plant transcription terminator.

11. Plant tissue transformed with the polynucleotide of any one of claims 1 to 9 or the vector of claim 10 and material derived from the said transformed plant tissue.

12. Morphologically normal fertile whole plants comprising the tissue or material of the preceding claim.

13. The progeny of the plants of the preceding claim, which progeny comprises the polynucleotide of any one of claims 1 to 9 stably inco╧åorated into its genome and heritable in a mendelian manner, the seeds of such plants and such progeny.

14. A method of producing plants which are substantially tolerant or substantially resistant to microbial infection, comprising the steps of: (i) transforming plant material with the polynucleotide of any one of claims 1 to

9 or the vector of claim 10

(ii) selecting the thus transformed material; and

15. Use of the polynucleotide of any one of claims 1 to 9, or the vector of claim 10 in the production of plant tissues and/or morphologically normal fertile whole plants which are substantially tolerant or substantially resistant to microbial infection.

16. The translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1.; nucleotides 95 to 244 in SEQ ID No.2, nucleotides 108 to 257 in SEQ ID No. 3, or nucleotides 104 to 253 in SEQ ID No. 4 and protein having an amino acid sequence which is at least 95% similar to said product.

17. The translation product of the region comprised by nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID

No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5 and protein having an amino acid sequence which is at least 85% similar to said product.

18. A method of selectively controlling microorganisms at a locus comprising the plants, progeny and/or seeds of either of claims 12 or 13, comprising applying to the locus a microorganism controlling amount of the translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1.; nucleotides 95 to 244 in SEQ ID No.2, nucleotides 108 to 257 in SEQ ID No. 3, or nucleotides 104 to 253 in SEQ ID No. 4.

19. Use of the polynucleotide of any one of claims 1 to 9, or the vector of claim 10 in the production of an antimicrobial protein.