AU772284B2 - Polynucleotide sequences - Google Patents

Description

WO 00/11196 PCT/GB99/02720 -1- 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. 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. The invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting ofnucleotides 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 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 WO 00/11196 PCT/GB99/02720 -2- 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 DmAMP 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 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 extracellularly. 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 similarity to each other have at least 85% similar (identical or conservatively replaced) WO 00/11196 PCT/GB99/02720 -3amino 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: Alanine, Serine, Glycine and Threonine (ii) Glutamic acid and Aspartic acid (iii) Arginine and Lysine (iv) Isoleucine, Leucine, Valine and Methionine 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 gap length penalty Pairwise alignment parameters Ktuple 1 gap penalty 3 window Diagonals saved 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 0 C in a WO 00/11196 PCT/GB99/02720 -4solution 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-AMP1 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 0 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 0 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 WO 00/11196 PCT/GB99/02720 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 0 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 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 WO 00/11196 PCT/GB99/02720 -6identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein is less than about 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 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 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.

-7- The invention also provides a method of producing plants which are substantially tolerant or substantially resistant to antimicrobial infection, comprising the steps of: 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 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. nucleotides 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.

20 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.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art X:\TaniakUMndmenma634941 SyngenLt speci.doc 7abase or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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) Dm (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), 6 6 ft f" ft ft ftf ft fo ot t tf ft t o oot ft ft ft ,t f ot .t f f f X:\Tania\Amendmenas\634941 Syngenta spccic.doc WO 00/11196 PCT/GB99102720 -8- 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 pMJB1, 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 XhoI and SacI sites of pFAJ3106; Figure 8 shows the nucleotide sequence between the XhoI and SacI 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 ofplasmid 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 DmAMP1 (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 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 Lithium chloride (12 M) was added to the resulting aqueous layer to a final concentration of 2 M and incubated overnight at 4 0

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-HC1, pH 7.5. A second overnight lithium chloride WO 00/11196 PCT/GB99/02720 -9precipitation was carried out and the RNA collected and re-suspended in 5 mM Tris-HC1, pH 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 pg 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 pl of a solution containing 0.2M Tris-HCl pH 8.5, 0.25M NaC1, 0.025 M EDTA and 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 pl of aqueous extract was transferred to a fresh Eppendorf tube. Genomic DNA was precipitated by the addition of 300 pC 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 p1 H 2 0.

To amplify a 144 bp fragment of DNA encoding 48 amino acids of the mature Dm-AMP1 a PCR was carried out with Dahlia genomic DNA and oligonucleotides AFP-5 (based on Dm- AMPI 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 0 C, seconds, 48°C, 12 seconds and 72 0 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 MC 1022 cells. Transformation mixes were plated onto L-agar plates containing 100 pg/ml ampicillin and incubated at 37"C for 16 hours. Colonies were picked WO 00/11196 PCT/GB99/02720 and cells shaken for 16 hours in 3 ml L-broth containing 100 .tg/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-AMP1 related genes.

PCR clone 4 contained the DNA sequence

AAGACGTGGTCGGGAAACTGTGGCAATACGGG

ACATTGTGACAACCAATGTAAATCATGGGAGGGTGCGGCCCATGGAGCGTGTCA

TGTGCGTAATGGGAAACACATGTGTTTCTGCTACTTCAAC, encoding a portion of the observed mature Dm-AMP1 protein sequence (KTWSGNCGNTGHCD

NQCKSWEGAAHGACHVRNGKHMCFCYFN).

The 144 bp PCR product mixture labelled with oa 2 -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 0 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 0 C. Autoradiography was carried out at -70 0 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-AMP 1 peptide sequence.

Isolation of a Mature Dm-AMPI Gene Using the sequence of PCR clone 4 (above) and information from the NH 2 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-AMP 1.

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, WO 00/11196 PCT/GB99/02720 -11 12 seconds and 72 0 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 half of a Dm-AMP gene was identified.

A PCR was carried out with Dahlia genomic DNA and oligonucleotides MATAFP-3 (based on the mature Dm-AMP1 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-AMP1 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-AMP core and C-terminal extension.

Vector Oligonucleotide design Four oligonucleotides were designed based on the DNA sequence of the mature Dm-AMP I gene: DMVEC-1 top strand priming at the 5' end of the mature DmAMP-1 gene incorporating a Nco I site at the translation start of DmAMP-1 allowing cloning into pMJB1 (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-1 gene incorporating a Nco I site at the translation start of DmAMP-1 allowing cloning into pMJB 1 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 pMJB 1.

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, seconds for 45 cycles. A PCR product of approximately 450 bp spanning the mature Dm- WO 00/11196 PCT/GB99/02720 12- AMP1 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 pMJB Icut with Nco I and Sac I. The ligation mix was used to transform competent E. coli MC 1022 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 pMJB 1 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 WO 00/11196 PCT/GB99/02720 13 with Nco I. DNA sequencing confirmed that one transformant termed pDmAMPE, contained DNA encoding Din-AMP leader, core and C-terminal extension.

Both pDrnAMPD and pDmAMPE vector sequences contained PCR derived base substitutions with respect to Din-AMP 1 gene DNA sequence however the base changes were silent having no effect on the expected amino acid sequence.

(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(AG)CACAT

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

AAACACATGTGTTTCCCATT

MATAFP-3

AGCGTGTCATGTGCGTAT

Din25MAT-3 TAAAGAAACCGACCCmTCACGG DMVEC-1

ATGACAGTATGTGTGGTTCC

DMVEC-2

AACGCGGTAGAGTACTTGA

DMVEC-3 ATGCATCCATGGTGAATCGGTCGGTTGCGTTCTCCGCGTTCG.1JCTGATCC=J~C

GTGCTCGCCATCTCAGATATCGCATCCGTAGTGGAGACTATGCGAGAAA

DMVEC-4

AGAGTTCGACTACATAGA

EXAMPLE 2 Constructions of plant transformation vectors WO 00/11196 PCT/GB99/02720 -14- Expression cassettes containing a Dm-AMP1 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 HindIII 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 HindIII 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-AMP 1 product is monitored by standard Western blot or ELISA methods using antibody which had been raised against Dm-AMP1 protein. A range of expression levels are detected. The Dm-AMP1 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 polvprotein 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 XhoI and SacI sites of these plasmids, which encompass the regions encoding antimicrobial proteins, are presented in Figures 7 and 8. The regions comprised between the XhoI and SacI 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

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, WO 00/11196 PCT/GB99/02720 Plant Cell 7, 573-588) and as primers a mixture of the PCR product of the first PCR reaction, primer OWB 175 and primer OWB 172 SacI site underlined). The resulting PCR product was digested with XhoI and SacI and cloned into the expression cassette vector pMJB I (see above). The expression cassette in the resulting plasmid, called pFAJ3099, was digested with HindIII (flanking the 5' end of the 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 HindIII-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, C.R. 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 DmAMP1 (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 DmAMP1 in coating buffer. Primary antisera were used as 1000- and 2000-fold diluted solutions (DmAMP1 and RsAFP2, respectively) in 3 gelatin in PBS containing 0.05 (v/v) Tween 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 NaH 2

PO

4 15 mM Na 2

HPO

4 100 mM KC1, 1.5 M NaC1. The WO 00/11196 PCT/GB99/02720 16homogenate 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 trifluoroacetic acid (TFA). The column was eluted at 1 ml/min in a linear gradient in 35 min. from 15 to 50 acetonitrile in 0.1 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 NaH 2

PO

4 15 mM Na 2

HPO

4 100 mM KC1, 1.5 M NaC1).

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 (BIO101/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 oftransgenic 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 DmAMP 1. The polyprotein precursor regions of these constructs all featured a leader peptide region from the DmAMP 1 cDNA, the mature protein domain of DmAMP 1, an internal propeptide region, and the mature protein domain of RsAFP2. Construct 3106 has a propeptide consisting of a part of the DmAMP1 propeptide and a putative subtilisin-like protease processing site (IGKR) at its Cterminus.

The rationale behind construct 3106, is based on our observations that the C-terminal propeptides of DmAMP 1 are cleaved off at their N-terminus when expressed as DmAMP 1preproproteins in tobacco, respectively, while this processing event does not prevent the WO 00/11196 PCT/GB99/02720 -17mature 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 C.A. and Fuller 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 ofDmAMP 1 (construct 3109, figure 6).

Expression levels of DmAMP1 and RsAFP2 were analysed in leaves taken from a series of T1 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 DmAMP1 and RsAFP2. There was generally a good correlation between DmAMPI and RsAFP2 levels. However, the RsAFP2 levels were generally 2 to 5-fold lower than the DmAMP 1 levels. It is not known whether the apparent lower expression levels of RsAFP2 versus DmAMP 1 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 DmAMP 1 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 DmAMP or RsAFP2 using Elisa assays.

WO 00/11196 PCT/GB99/02720 -18- DmAMP cross-reacting compound eluted at a position identical or very close to that of authentic DmAMP 1 in the line transformed with construct 3106. Likewise, a RsAFP2 crossreacting 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-DmAMP1 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 DmAMP 1 C-terminal propeptide with subtilisin-like protease site as a linker peptide) are somehow processed to yield separate DmAMP 1-cross-reacting and RsAFP2-cross-reacting portions that appear to be identical or very closely related to DmAMP 1 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 DmAMP1 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), DmAMP 1 and RsAFP2 in the extracellular fluid (EF) and intracellular extract (IE) fractions obtained from transgenic Arabidopsis plants.

Construct Relative abundance' of GPD DmAMPI RsAFP2 EF IE EF IE EF IE pFAJ3106 17 83 94 6 60 WO 00/11196 PCT/GB99/02720 -19- '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 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 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.

WO 00/1119620 PCT/GB99/02720 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 Construct Promoter Signal No. of Plants Plants Name Peptide Tested expressing Brazzein pZPS34 UBQ None 29 18 UBQ Dahlia 25 23 AMP1 pZPS37 PG None 15 7 pZPS38 PG Dahlia 13 11 AMP1 SUBSTITUTE SHEET (RULE 26) WO 00/11196 -21 Table 2 (continued) PCT/GB99/02720 Construct Max Brazzein Min Brazzein Mean Brazzein in Name ng/g Fresh wt ng/g Fresh wt those plants expressing the gene pZPS34 25.57 Not Detected 6.85 226.53 Not Detected 43.89 pZPS37 12.77 Not Detected 3.32 pZPS38 51745.77 Not Detected 12867.34 Example 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 pAID-MR7.

pAID-MR7 was constructed using the commercially available cloning vector pNEB 193 (New England Biochemicals) itself a modified version of pUC19 (Yanisch-Perron Vieira J. and Messing J. "Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors." Gene; 33:103-19 (1985)). Gene components to facilitate protein expression were inserted within the multiple cloning region of pNEB193, 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 (I 1) from maize (Dennis Gerlach Pryor Bennetzen Inglis Llewellyn Sachs Ferl R.J. and Peacock W.J. "Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize." Nucleic Acids Research; 12:3983-4000(1984)).

SUBSTITUTE SHEET (RULE 26) WO 00/11196 PCT/GB99/02720 -22- 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 (Pietrak et al Nucleic Acid Research 14 5857-5868 (1986), Franck Guilley Jonard 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 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 RI 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 MC 1022 cells. Tranformation mixes were plated onto L-agar plates containing 100 .tg/ml ampicillin and incubated at 37 0 C for 16 hours. Colonies were picked and cells shaken for 16 hours in 3 ml L-broth containing 100 pg/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.

WO 00/11196 PCT/GB99/02720 -23- Example Vector DNA was prepared as in Example Plasmid DNA of a cDNA clone containing a Dm2.3 ORF was digested with Eco RI and Sca 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 MC 1022 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 Vector DNA was prepared as in Example Plasmid DNA of a cDNA clone containing a Dm2.5 ORF was digested with Eco RI and Dra I and the ends filled in with Klenow DNA polymerase. Insert DNA containing the 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 MC1022 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 cellulase RS (Yakult Honsha Co., Ltd), 0.2% pectolyase Y23 (Yakult Honsha Co., Ltd), 0.5M mannitol, 5mM CaCl 2 2H20, 0.5% MES, pH5.6, -660mmol/kg) using WO 00/11196 PCT/GB99/02720 -24cells, incubating at 25 0 C, rotating gently for 2 hours. The digestion mixture is sieved through 250gm and 38um sieves, and the filtrate centrifuged at 700rpm for 3.5 minutes. Protoplasts are resuspended in wash buffer (0.358M KC1, 1.0mM NH 4

NO

3 5.0mM CaCl 2 2H20,

KH

2

PO

4 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, Crawley, UK). Protoplasts are resuspended at 2 x 10 6 /ml in MaMg medium (0.4M mannitol, 15mM MgCl 2 0.1% MES, pH5.6, -450mmol/kg) aliquotting 0.5ml treatment 1x106 protoplasts/treatment). Samples are heat shocked at 45°C for 5 minutes then cooled to room temperature. Each transformation is carried out with 1Ogg of pAID-MR7 alone or 10 Og 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 0 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.

EDITORIAL NOTE APPLICATION NUMBER 54344/99 The following Sequence Listing pages 1 to 23 are part of the description. The claims pages follow on pages "25" to "28".

WO 00/11196 WO 0011196PCT/GB99/02720 1 SEQUENCE LISTING <110> ZENECA Limited Evans, Ian J Ray, John A <120> Polynucleotide sequences <130> PPD 50355 <140> <141> <150> <151> GB 9818003.7 1998-08-18 <160> 37 <170> Patentln Ver. 2.1 <210> 1 <211> 399 <212> DNA <213> Dahlia merckii <220> <221> CDS <222> (53)..(388) <400> 1 gtgccccggq tcacgaagtt cggcacatct tagcgttatg cataagtcaa aa atg gcc 58 Met Ala 1 Lys Asn Ser Val Ala Phe Phe Ala Leu Cys Leu Leu Leu Phe Ile Leu 10 WO 00/11196 WO 0011196PCT/GB99/02720 gct Ala agc Ser tca gaa atc Ser Giu Ile aca tgg tct Thr Trp Ser aga tcg gtg aag ggg gaa Arg Ser Val Lys Gly Giu gga aat tgt ggc aat aca Gly Asn Cys Giy Asn Thr 40 45 gag ggt gca gcc cat gga Giu Gly Ala Ala His Gly tta tgt gag aag gca Leu Cys Giu Lys Ala aga cac tgt gat gac Arg His Cys Asp Asp qct tgt cac gtg cgc Ala Cys His Val Arg tgt ccc aaa gcc cag Cys Pro Lys Ala Gin 154 202 250 298 cag tgc aag tct Gin Cys Lys Ser ggt ggg aaa cac Gly Gly Lys His atg tgc ttc tgc tac ttc aac Met Cys Phe Cys Tyr Phe Asn 75 aag ttg gct Lys Leu Ala gag gat aaa ctc Glu Asp Lys Leu aga Arg gca gca gag cta Ala Ala Glu Leu aag gag aag Lys Glu Lys 346 aat aat Asn Asn 100 att gga gct gaa aag Ile Gly Ala Glu Lys 105 gtg cct tca gcc aca cct tga Val Pro Ser Ala Thr Pro 110 388 gtactaacaa a <210> <211> <212> <213> <220> <221> <222> 2 523

DNA

Dahlia merckii

CDS

(337) WO 00/11196 WO 0011196PCT/GB99/02720 <400> 2 ggcacgagta atg gcc aaa aat tca gtt gct ttc tta gca ttt ctt ctg Met Ala Lys Asn Ser Val Ala Phe Leu Ala Phe Leu Leu ctt ctt ttc Leu Leu Phe is gtt ctt gct atc Val Leu Ala Ile 20 tca gaa atc gga Ser Glu Ile Gly tcg gtg aag ggg gaa Ser Val Lys Gly Giu aat tgt ggc aat aca Asn Cys Gly Asn Thr tta Leu tgt gag aag gca.

Cys Glu Lys Ala agc Ser 35 aag aca tgg tct gga Lys Thr Trp Ser Gly 40 aga cac tgt gat Arg His Cys Asp gct tgt cac gtg Ala Cys His Val cag tgc aag tct tgg Gin Cys Lys Ser Trp gag ggc gca gcc cat gga Giu Gly Ala Ala His Gly tgc ttt tgc tac ttc aac Cys Phe Cys Tyr Phe Asn cgc ggt ggg aaa cac Arg Giy Gly Lys His 70 tgt Cys tcc Ser aaa gcc cag aag ctg Lys Ala Gin Lys Leu aag gag aag agt gaa Lys Giu Lys Ser Glu 100 cag gat aaa ctc aaa Gin Asp Lys Leu Lys gcc gac aag Ala Asp Lys Ctc gc Leu Ala gcc gaa aag gtg cca gct aca cct Ala Giu Lys Val Pro Ala Thr Pro 105 tga 337 gtactaacaa gtgttgtatg attatgaata aagagaaaat gctttctagt taccatattt 397 agcattctct aatgtgtaat gtttgttgct tttggaacta attgcttaac tatgattcca 457 gctaataatg ttttaagtat ataatataag ttatcttatt ttgaagcctg taaaaaaaaa 517 aaaaaa WO 00/11196 WO 0011196PCT/GB99/02720 <210> 3 <211> 385 <212> DNA <213> Dahlia merckii <220> <221> CDS <222> (320) <400> 3 cggcacgagg cacaatctca aaa atg gcc aaa aat tcq gtt gct ttc ttt gca 53 Met Ala Lys Asn Ser Val Ala Phe Phe Ala ttt gtc ctg ctt ctt ttc gtt ctt gct atc Phe Val Leu Leu Leu Phe Val Leu Ala Ile 20 tca gaa att gga tcg gtg Ser Glu Ile Gly Ser Val 101 aag gga gaa tta Lys Gly Glu Leu ggc atc aca tca Gly Ile Thr Ser tgt gag aag gca agc Cys Glu Lys Ala Ser 35 aag aca tgg tct Lys Thr Trp Ser gga aat tgt Gly Asn Cys gag ggt gca Glu Gly Ala cac tgt gac His Cys Asp cag tgc cgg tcg Gin Cys Arg Ser tgg Trp at c Ile cat His gga gct tgt cac gtg Gly Ala Cys His Val 65 cgc ggt ggg aaa cac atg tgc ttc tgc Arg Gly Gly Lys His Met Cys Phe Cys tac Tyr ttc aac tgt tcc aaa Phe Asn Cys Ser Lys gcc gat gag ctc Ala Asp Glu Leu aag gag aag att Lys Giu Lys Ile gcc gaa aag atg cca Ala Glu Lys Met Pro gcc aca cct Ala Thr Pro tga gtactaacaa atgctatatg 340 WO 00/11196 WO 0011196PCT/GB99/02720 attataaata aagagaaaat gctttctaaa aaaaaaaaaa aaaaa <210> 4 <211> 577 <212> DNA <213> Dahlia merckii <220> <221> <222>

CDS

(346) <400> 4 ggcacgagcc tattaaaaa atg gtg aat cga tcg gtt gct ttc tcc gtg ttc Met Val Asn Arg Ser Val Ala Phe Ser Val Phe gtt ctg atc ctt Val Leu Ile Leu ttc gtg ctc gcc atc tca gat atc aca agt gtg aga Phe Val Leu Ala Ile Ser Asp Ile Thr Ser Val Arg 20 gga gaa gta GJly Giu Val tgc gag aaa gct agc Cys Giu Lys Ala Ser 35 aag aca tgg tca Lys Thr Trp Ser gga Gly aac tgt ggc Asn Cys Gly aac acg Asn Thr gga cac tgt gac aac Gly His Cys Asp Asn 50 caa tgt aaa tac Gin Cys Lys Tyr tgg Trp, gag ggg gcg gcc Giu Gly Ala Ala cat His ggg gcg tgc cac gtg cgt gga ggg aaa Gly Ala Cys His Val Arg Gly Gly Lys 65 cac atg tgt ttc tgc tac His Met Cys Phe Cys Tyr 70 caa gac aaa gtt aat gcc Gin Asp Lys Val Asn Ala ttc aag tgt ccc aaa gcc Phe Lys Cys Pro Lys Ala gaa aag ctt gct Giu Lys Leu Ala 85 WO 00/11196 WO 0011196PCT/GB99/02720 6 caa gag ctt gac cgt gat gcc aag aaa gtg att. ccg aac gtt gaa cat 340 Gin Glu Leu Asp Arg Asp Ala Lys Lys Val Ilie Pro Asn Vai Giu His 100 105 ccg tga aagggtcggt ttctttaaat agaaLagtctt agattacgaa tgcgaataac 396 Pro tatagaaaat gtttgctaaa tgtcacatta taattagaac tttatgattg ttgtcaatag 456 ggcattttct tgttagtgat atgtgtaata aggtgatgct tttatgcttt tcgtgcgtaa 516 gagttttcga ctatgtgtaa taaagaaagg gtcttttttt tttaaaaaaa aaaaaaaaaa 576 a 577 <210> <211> 446 <212> DNA <213> Dahlia merckii <220> <221> CDS <222> (64) <220> <221> CDS <222> (446) <400> atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt ctg atc ctt ttc 48 Met Val Asn Arg Ser Val. Ala Phe Ser Ala Phe Vai Leu Ile Leu Phe 1 5 10 1s gtg ctc gcc atc tca g gttatcaaat ctttagttca tttattgaat atgatagtat 104 Val Leu Ala Ile Ser WO 00/11196 WO 0011196PCT/GB99/02720 7 ttatattctt ttatggtttt atgtgttctg acaagttgca aatattgagt ag at atc Asp Ile gca tcc Ala Ser gtt agt gga gaa cta Val Ser Gly Giu Leu 30 tgc gag aaa gct Cys Giu Lys Ala agc Ser aag aca tgg tcg Lys Thr Trp Ser gga Gly aac tgt ggc aat Asn Cys Gly Asn acg Thr 45 gga cat tgt gac Gly His Cys Asp aac caa tgt aaa tca tgg Asn Gin Cys Lys Ser Trp 50 cgt aac ggg aaa cac atg Arg Asn Gly Lys His Met 209 257 305 353 gag ggt gcg gcc Glu Gly Ala Ala tgt ttc tgt tac Cys Phe Cys Tyr cat gga gcg tgt cat His Gly Ala Cys His ttc aat tgt aaa aaa Phe Asn Cys Lys Lys 80 gtg Val1 65 gcc gaa aag ctt Ala Giu Lys Leu gct Ala caa gac Gin Asp aaa ctt aaa Lys Leu Lys gcc gaa caa ctc Ala Giu Gin Leu gct caa Ala Gin 95 gac aaa ctt aat Asp Lys Leu Asn 100 gcc caa aag Ala Gin Lys ctt gac Leu Asp 105 cgt gat gcc aag aaa Arg Asp Ala Lys Lys 110 gtg gtt cca aac gtt Val Val Pro Asn Val 115 gaa cat ccg Giu His Pro <210> 6 <211> 150 <212> DNA <213> Dahlia merckii <400> 6 gagctttgcg agaaggcttc taagacttgg tctggaaact gcggaaacac tggacattgc gataaccaat gcaagtcttg ggagggagct gctcatggag cttgccatgt tagaaacgga 120 aagcatatgt gcttctgcta cttcaactgc. 150 WO 00/11196 WO 0011196PCT/GB99/02720 8 <210> 7 <211> <212> DNA <213> Dahlia merckii <400> 7 gaggtttgcg agaaggcttc taagacttgg tctggaaact gcggaaacac tggacattgc <210> 8 <211> 111 <212> PRT <213> Dahlia merckii <400> 8 Met Ala Lys Asn Ser 1 5 Val Ala Phe Phe Ala 10 Leu Cys Leu Leu Leu Phe Ile Leu Ala Lys Ala Ser Ile Ser Glu Ile Arg Val Lys Gly Glu Leu Cys Glu Arg His Cys Lys Thr Trp Ser Gly 40 Asn Cys Gly Asn Thr Asp Asp Gin Cys Lys Ser Trp 55 Glu Gly Ala Ala His Gly Ala Cys His Val Arg Gly Gly Lys Met Cys Phe Cys Phe Asn Cys Pro Lys Ala Gln Lys Leu Ala Glu Asp Lys Leu Arg 90 Ala Ala Glu Leu Ala Lys Glu Lys Asn Asn Ile Gly Ala Glu Lys Val Pro Ser Ala Thr Pro WO 00/11196 WOOO/1196PCT/GB99/02720 <210> 9 <211> 108 <212>. PRT <213> Dahlia rnerckii <400> 9 Met Ala 1 Lys Asn Ser Val Ala Phe Leu Ala Phe Leu Leu Leu Leu Phe Val Leu Ala Lys Ala Ser Ile Ser Glu Ile Gly Ser 25 Val Lys Gly Glu Leu Cys Glu Arg His Cys Lys Thr Trp Ser Asn Cys Gly Asn Thr Asp Asp Gin Cys Lys Ser Glu Gly Ala Ala His Gly Ala Cys His Val Arg Gly Gly Lys His 70 Met Cys Phe Cys Phe Asn Cys Ser Ly s Ala Gin Lys Leu Glu Lys Ser Glu 100 Gin Asp Lys Leu Lys Ala Asp Lys Leu Ala Lys Ala Glu Lys Val Ala Thr Pro <210> <211> 98 <212> PRT <213> Dahlia merckii <400> Met Ala Lys Asn Ser Val Ala Phe Phe Ala Phe Val Leu Leu Leu Phe 1 5 10 WO 00/11196 WO 0011196PCT/GB99/02720 Val Leu Ala Lys Ala Ser Ile Ser Glu Ile Gly Ser 25 Val Lys Gly Giu Leu Cys Glu Ser His Cys Lys Thr Trp Ser Asn Cys Giy Ile Thr 4S Asp Asn Gin Cys Arg Ser Trp 55 Glu Gly Ala Ilie His Gly Ala Cys His Arg Gly Gly Lys His Met Cys Phe Cys Phe Asn Cys Ser Ala Asp Giu Leu Lys Glu Lys Ile Glu Ala Giu Lys Met Pro Ala Thr Pro <210> 11 <211> 108 <212> PRT <213> Dahlia rnerckii <400> 11 Met Val Asn Arg Ser Val Ala Phe Ser Val Phe Val Leu Ile Leu Phe Val Leu Ala Lys Ala Ser Ile Ser Asp Ile Thr Val Arg Gly Giu Val Cys Glu Gly His Cys Lys Thr Trp Ser Gly 40 Asn Cys Gly Asn Thr Asp Asn so Gin Cys Lys Tyr Giu Gly Ala Ala Gly Ala Cys His Val Arg Gly Gly Lys His 70 Met Cys Phe Cys Ty r 75 Phe Lys Cys Pro Lys WO 00/11196 WO 0011196PCT/GB99/02720 Ala Glu Lys Asp Ala Lys Leu Ala Gin Asp Lys Lys Val Ile Pro Asn 100 Val Asn Ala Gin Glu Leu Asp Arq 90 Giu His Pro <210> 12 <211> 118 <212> PRT <213> Dahlia merckii <400> 12 Met Val Asn Arg Ser Val Ala Phe Ser Phe Vai Leu Ile Leu Phe Cys Giu Val Leu Ala Ser Asp Ile Ala Ser Val Ser Gly Giu Leu Lys Ala Ser Lys Thr Trp Ser Asn Cys Gly Asn Thr Gly His Cys Asp Asn Gln Cys Lys Ser Glu Gly Ala Ala His Gly Ala Cys His Val Arg Asn Gly Lys His Met Cys Phe Cys Phe Asn Cys Lys Ala Giu Lys Leu Ala Gln Asp Lys Leu Ala Giu Gin Leu Ala Gin Asp Lys Leu Asn 100 Ala Gin Lys Leu Asp Arg 105 Asp Ala Lys Lys Val Val 110 Pro Asn Val 115 Giu His Pro WO 00/11196 WO 0011196PCT/GB99/02720 <210> 13 <211> 8 <212> PRT <213> Dahlia merckii <400> 13 Cys Glu Lys Ala Ser Lys Thr Trp 1 <210> 14 <211> 8 <212> PRT <213> Dahlia rnerckii <400> 14 Met Cys Phe 1 <210> <211> 126 <212> DNA <213> Dahli Cys Tyr Phe Asn Cys a merckii <400> aagacgtggt cgggaaactg tqgcaatacg ggacattgtg acaaccaatg taaatcatgg gagggtgcgg cccatggagc gtqtcatgtg cgtaatggga aacacatgtg tttctgctac 120 ttcaac 126 <210> <211> <212> <213> 16 42

PRT

Dahlia merckii WO 00/11196 WO 0011196PCT/GB99/02720 <400> 16 Lys Thr Trp Ser Gly Asn Cys Gly Asn Thr Gly His Cys Asp Asn Gin 1 5 10 Cys Lys Ser Gly Lys His Glu Gly Ala Ala His Gly Ala Cys His Val Arg Asn Met Cys Phe Cys Tyr Phe Asn <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 17 8

PRT

Dahlia merckii

SITE

(2) Xaa=Ala or Val

SITE

(3) Xaa=Lys or Asn

SITE

(4) Xaa=Asn or Arg <400> 17 Met Xaa Xaa Xaa Ser Val Ala Phe 1 WO 00/11196 WO 0011196PCT/GB99/02720 <210> 18 <211> 7 <212> PRT <213> Dahlia merckii <400> 18 Asn Gly Lys 1 <210> 19 <211> 7 <212> PRT <213> Dahli His Met Cys Phe a merckii <400> 19 Gly Ala Cys His Val Arg Asn 1 <210> <211> <212> <213> <220> <221> <222> <223> <220> <223> 24

DNA

Artificial Sequence misc feature 9, 12, 15, 21) n is any residue Description of Artificial Sequence: Oligonucleotide <400> tgyganaang cnwsnaarac ntgg WO 00/11196 PCT/GB99/02720 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <222> 12, <223> n is any residue <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 21 carttraant ancanaaarc acat 24 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <222> 12, 21) <223> n is any residue <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 22 atggcsaanm rntcrgttgc ntt 23 WO 00/11196 PCT/GB99/02720 16 <210> 23 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 23 aaacacatgt gtttcccatt <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 24 agcgtgtcat gtgcgtaat 19 <210> <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> taaagaaacc gaccctttca cgg 23 WO 00/11196 PCT/GB99/02720 17 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 26 atcgtagcca tggtgaatcg gtcggttgcg ttctccgcg 39 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 27 aaaccgaccg agctcacgga tgttcaacgt ttggaac 37 <210> 28 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 28 atgcatccat ggtgaatcgg tcggttgcgt tctccgcgtt cgttctgatc cttttcgtgc tcgccatctc agatatcgca tccgttagtg gagaactatg cgagaaa 107 WO 00/11196 PCT/GB99/02720 18 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 29 agcaagcttt tcgggagctc aacaattgaa gtaa 34 <210> <211> <212> DNA <213>.Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> aggaagttca tttcatttgg <210> 31 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 31 gcctttggca caacttctgc ctctttccga tgagttgttc ggctttaagt ttgtc WO 00/11196 PCT/GB99/02720 19 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 32 ttagagctcc tattaacaag gaaagtagc <210> <211> <212> <213> <220> <223> 33 4

PRT

Artificial Sequence Description of Artificial Sequence: Synthetic sequence <400> 33 Ile Gly Lys Arg 1 <210> <211> <212> <213> <220> <221> <222> 34 534

DNA

Artificial Sequence

CDS

(76)..(525) WO 00/11196 WO 0011196PCT/GB99/02720 <220> <223> Description of Artificial Sequence: Synthetic sequec <400> 34 cicgagtatt titacaacaa iiaccaacaa caacaaacaa caaacaacat tacaattact atttacaatt acacc atg gtg aat cgg tcg gtt gcg tic tcc gcg tic gtt 11 Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val cig Leu aic ctt Ile Leu ttc gig ctc gcc atc Phe Val Leu Ala Ile 20 ica gat atc gca icc git agi gga Ser Asp Ile Ala Ser Val Ser Gly gaa cia Giu Leu tgc gag aaa gct agc Cys Giu Lys Ala Ser 35 aag acg igg icg ggc Lys Thr Trp Ser Gly aac igt ggc aac Asn Cys Gly Asn acg Thr gga cat tgi gac aac Gly His Cys Asp Asn so caa tgi aaa tca Gin Cys Lys Ser t gg Trp 55 gag ggt gcg gcc Glu Gly Ala Ala cat His 207 255 303 351 gga gcg tgt cat gig Gly Ala Cys His Val aat tgt aaa aaa gcc Asn Cys Lys Lys Ala cgt aac ggg aaa cac Arg Asn Gly Lys His 70 gaa aag cti gct caa Giu Lys Leu Ala Gin 85 atg tgt tic tgt tac tic Met Cys Phe Cys Tyr Phe gac aaa cit aaa gcc gaa Asp Lys Leu Lys Ala Giu caa cic aic gga aag agg cag Gin Leu Ile Gly Lys Arg Gin tig igc caa agg cca agt ggg aca Leu Cys Gin Arg Pro Ser Gly Thr 105 tgg ica gga Trp Ser Gly 110 gic igi gga aac aai aac Val Cys Gly Asn Asn Asn 115 gca igC aag Ala Cys Lys 120 aai cag tgc ati Asn Gin Cys Ile WO 00/11196 WO 0011196PCT/GB99/02720 aga ctt gag Arg Leu Glu 125 cac aag tgt His Lys Cys aaa gca cga cat Lys Ala Arg His 130 gga tct tqc Gly Ser Cys aac tat gtc ttc cca gct Asn Tyr Val Phe Pro Ala 135 140 atc tgc tac ttt cct tgt Ile Cys Tyr Phe Pro Cys 145 taa taggagctc 150 <210> <211> <212> <213> <223> 149

PRT

Artificial Sequence Description of Artificial Sequence: Synthetic sequence <400> Met Val Asn Arg Ser 1 5 Val Ala Phe Ser Ala Phe Val Leu Ile Val Leu Ala Lys Ala Ser Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu Gly His Cys Lys Thr Trp Ser Gly Asn Cys Gly Asn Asp Asn Gin Cys Lys Ser Trp 55 Giu Gly Ala Ala His Gly Ala Cys His Val Arg Asn Gly Lys His 70 Met Cys Phe Cys Phe Asn Cys Lys Lys Ala Glu Lys Leu Ala Gin Asp Lys Leu Lys 90 Ala Glu Gin Leu Ile Gly Lys Arg Gln Lys 100 Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val 110 WO 00/11196 WO 0011196PCT/GB99/02720 Cys Gly Asn 115 Asn Asn Ala Cys Lys Asn 120 Gln Cys Ile Arg Leu Glu Lys 125 Phe Pro Ala His Lys Cys Ile 140 Ala Arg 130 His Gly Ser Cys Asn Tyr Val 135 Cys Tyr Phe Pro Cys 145 <210> 36 <211> 316 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (312) <220> <223> Description of Artificial Sequence: Synthetic sequence <400> 36 ctcgagtatt tttacaacaa ttaccaacaa. caacaaacaa caaacaacat tacaattact atttacaatt acacc atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val ill ctg atc ctt ttc gtg ctc gcc atc tca gat atc qca tcc gtt agt gga Leu Ile Leu Phe Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly 20 gaa cta tgc gag aaa gct agc aag acg tgg tcg ggc aac tgt ggc aac Glu Leu Cys Glu Lys Ala Ser Lys Thr Trp Ser Gly Asn Cys Gly Asn 35 159 207 WO 00/11196 WO 0011196PCT/GB99/02720 acg Thr gga cat tgt gac Gly His Cys Asp aac Asn 50 caa tgt aaa tca Gin Cys Lys Ser tgg Trp gag qgt gcg gcc Glu Gly Ala Ala gga gcg tgt cat gtg Gly Ala Cys His Val cgt aat ggg aaa cac Arg Asn Gly Lys His 70 atg tgt ttc tgt tac Met Cys Phe Cys Tyr ttc 303 Phe aat tgt tga gctc Asn Cys <210> 37 <211> 78 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic sequence <400> 37 Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe Val Leu Ala Lys Ala Ser Asp Asn Gin Ile Ser Asp Ile Ala Val Ser Gly Giu Leu Cys Glu Gly His Cys Lys Thr Trp Ser Asn Cys Gly Asn Thr Cys Lys Ser Trp 55 Glu Gly Ala Ala His Gly Ala Cys His Val Arg Asn Gly Lys His Met Cys Phe Cys Tyr Phe Asn Cys 70

Claims (19)

1. 2. JAN. 2004 17:29 CLAIMS: 1. A subs selecte, SEQ ID PHILLIPS ORMONDE 96141867 NO, 2637 P. 6 2. A substanti selected froi nucleotides nucleotides:
2. 3. A substanti; selected fror nucleotides nucleotides No.
3. 4. A substantii selected fror nucleotides: 3, nucleotide lly isolated or purified polynucleotide comprising a sequence m those depicted in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, 4 and SEQ ID No. illy isolated or purified polynucleotide comprising a sequence n the group consisting of nucleotides 53 to 385 in SEQ ID No. 1, I1 to 334 in SEQ ID No. 2, nucleotides 24 to 317 in SEQ ID No. 3, !0 to 343 in SEQ ID No. 4 or nucleotides 1 to 446 in SEQ ID No. illy isolated or purified polynucleotide comprising a sequence n the group consisting of nucleotides 137 to 286 in SEQ ID No. 1, )5 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID illy isolated or purified polynucleotide comprising a sequence i the group consisting of nucleotides 287 to 385 in SEQ ID NO. 1, !45 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. s 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID Ily isolated or purified polynucleotide encoding an antimicrobial g substantially similar antimicrobial activity to that encoded by any No. 1, No 2, No. 3, No. 4 or No. 5 which polynucleotide is iry to one which hybridises to any of SEQ ID No. 1, No. 2, No. 3, 5 when incubated at a temperature of between 55 and 65°C in a aining 6 X SSC (saline sodium citrate buffer) containing 0.1% SDS powdered skimmed milk followed by washing at the same with 0.1, 0.5 or 2x SSC containing 0.1% SDS with the proviso that e is not that described in SEQ ID No. 6 or 7 or SEQ ID No. 34 of ly isolated or purified polynucleotide according to any preceding r comprising a region encoding a peptide which Is capable of Stranslation products of the sequence to plastids such as mitochondria, other organelles or plant cell walls. A substantie protein havir of SEQ ID complement. No. 4 or No. solution cont and 0.25% temperature the sequenc WO 94/1607
4. 6. A substantia claim, furthe targeting th chloroplasts, W.BmG fi0wursfl3441 a y MIy a 09 COMS ID No: SMBI-00567006 Received by IP Australia: Time 18:44 Date 2004-01-12 2. JAN. 2004 17:29
5. 7. A polyr enhancil by the p
6. 8. A polyn, mRNAi express similar F express protein i
7. 9. A polyni plant p polynucl unmodif modifiec
8. 10. A plant polynucl transcri; PHIL LIPS ORMONDE 96141867 NO. 2637 P. 7 26 tide according to any preceding claim, wherein translational ,quences are inserted 5' of the protein encoding regions comprised cleotide. tide according to any preceding claim, which is modified in that ility motifs andlor fortuitous splice regions are removed such that f the thus modified polynucleotide in a plant yields substantially n having a substantially similar activity/function to that obtained by f the unmodified polynucleotide in the organism in which the ing regions of the unmodified polynucleotide are endogenous. tide according to any one of claims 1 to 7 which is modified in that red codons are used, and wherein when said modified e is introduced into and expressed in a plant in which the olynucleotides is endogenous, the degree of identity between the nucleotide and the endogenous polynucleotide is less than isformation vector comprising a plant operable promoter, a e according to any of the preceding claims under the I control thereof and a plant transcription terminator. transformed with the polynucleotide of any one of claims 1 to 9 or f claim 10 and material derived from the said transformed plant in said material comprises said polynucleotide or said vector. ally normal fertile whole plants comprising the tissue or material of g claim. of the plants of the preceding claim, which progeny comprises the le of any one of claims 1 to 9 stably incorporated into its genome e in a mendelian manner, the seeds of such plants and such Sproducing plants which are substantially tolerant or substantially picrobial infection, comprising the steps of: forming plant material with the polynucleotide of any one of claims vector of claim oooo oooo
9. 11. Plant tissue the vector c tissue where
10. 12. Morphologic the precedin
11. 13. The progen) polynucleoti and heritabl progeny.
12. 14. A method o resistant to r trans 1 to 9 or the W.seramensmn&48'w, Svuiwarad* COMS ID No: SMBI-00567006 Received by IP Australia: Time 18:44 Date 2004-01-12 JAN. 2004 17:29 (ii) s (iii) r fertile wl
13. 15. Use of t in the p plants v infectior
14. 16. The sut by nucli sequent PHILLIPS ORMONDE 96141867 NO. 2637 P. 8
15. 17. The substar by nucleotic sequence w
16. 18. The substar by nucleotid sequence w
17. 19. The substar by nucleotid sequence w The substar by nucleotid No. 2, nude ID No. 4 or I acid sequen
18. 21. A method c plants, prog to the locus region comr: 244 in SEQ 104 to 253 i 27 ing the thus transformed material; and erating the thus selected material Into morphologically normal l1ants. 3lynucleotide of any one of claims 1 to 9. or the vector of claim .tion of plant tissues and/or morphologically normal fertile whole are substantially tolerant or substantially resistant to microbial ially isolated or purified translation product of the region comprised s 137 to 286 in SEQ ID No. 1, or protein having an amino acid fich is at least 97% identical to said product. :ially isolated or purified translation product of the region comprised 3s 95 to 244 in SEQ ID No.2 or protein having an amino acid iich is at least 97% identical to said product. :ially isolated or purified translation product of the region comprised -s 108 to 257 in SEQ ID No. 3, or protein having an amino acid iich is at least 95% identical to said product. tally isolated or purified translation product of the region comprised 3s 104 to 253 in SEQ ID No. 4 or protein having an amino acid lich is at least 95% identical to said product. :ially isolated or purified translation product of the region comprised is 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID )tides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 In SEQ ucleotides 327 to 446 in SEQ ID No.5 and protein having an amino ;e which is at least 85% identical to said product. selectively controlling microorganisms at a locus comprising the ny and/or seeds of either of claims 12 or 13, comprising applying t microorganism controlling amount of the translation product of the ised by nucleotides 137 to 286 in SEQ ID No. nucleotides 95 to ID No.2, nucleotides 108 to 257 in SEQ ID No. 3, or nucleotides i SEQ ID No. 4. oo ooe o •gig o o o gOOD o*O go ooooo 9 W.W2rSanl0AV~m0Wse\ Syiigwtu4 COMS ID No: SMBI-00567006 Received by IP Australia: Time 18:44 Date 2004-01-12
19. 412. JAN. 2004 17:29 22. Use of tl in the pn PHIL 23. A plant or according to 24. A polynucle hereinbefore Examples. A plant trc hereinbefore Examples. 26. Plant tissue described, 27. Plants accor described, LIPS ORMONDE 96141867 NO. 2637 P. 9 28 )lynucleotide of any one of claims 1 to 9, or the vector of claim tion of an antimicrobial protein. ;lant tissue as described herein when produced by a process claim 14 or claim ,tide according to any one of claims 1 to 9 substantially as described, with reference to any of the Figures, Tables and/or insformation vector according to claim 10 substantially as described, with reference to any of the Figures, Tables and/or according to claims 11 or 23 substantially as hereinbefore ith reference to any of the Figures, Tables and/or Examples. ling to any one of claims 12, 13 or 23 substantially as hereinbefore ith reference to any of the Figures, Tables and/or Examples. cording to claims 14 or 21 substantially as hereinbefore described, e to any of the Figures, Tables and/or Examples. ling to claims 15 or 22 substantially as hereinbefore described, with any of the Figures, Tables and/or Examples. Ily isolated or purified translation product according to any one of 20 substantially as hereinbefore described, with reference to any of Tables and/or Examples. nuary, 2004 )E FITZPATRICK 5ENTA LIMITED 28. A method ai with refereni 29. A use accor reference to A substantik claims 16 to the Figures, DATED: 12 J; PHILLIPS ORMONI Attorneys for: SYN C* eeeC e C ooo o ooo o weBewneindomtMales 1 synema ae COMS ID No: SMBI-00567006 Received by IP Australia: Time 18:44 Date 2004-01-12