WO2000055337A1 - Transgenic plants that are resistant to a broad spectrum of pathogens - Google Patents
Transgenic plants that are resistant to a broad spectrum of pathogens Download PDFInfo
- Publication number
- WO2000055337A1 WO2000055337A1 PCT/CA2000/000288 CA0000288W WO0055337A1 WO 2000055337 A1 WO2000055337 A1 WO 2000055337A1 CA 0000288 W CA0000288 W CA 0000288W WO 0055337 A1 WO0055337 A1 WO 0055337A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- amino acid
- dermaseptin
- temporin
- peptides
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates to plants that are genetically engineered to express one or more peptides belonging to the temporin and/or dermaseptin families.
- Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. These pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops. The most widely practiced methods of reducing the damage caused by such pathogens involve the use of various chemical agents. Unfortunately, many pathogens develop resistance to such chemicals, and some pathogens (especially viruses) are not susceptible to control by chemical means. In addition, many of the chemical agents used are broad-spectrum toxins, and may cause serious environmental damage, as well as toxicity in humans. Plant breeding and, more recently, genetic engineering techniques have also been employed to combat plant pathogens.
- Transgenic plants provided by the invention may be used in conventional agricultural applications, such as food crops. Alternatively, the plants may be harvested and processed to extract the expressed temporin and/or dermaseptin peptides, which may then be purified for use in medical and other applications.
- the invention thus encompasses transgenic plants that express at least one dermaseptin or temporin peptide, and methods of making such plants.
- Parts of such plants including seeds, fruit, stems, leaves and roots, may be utilized in conventional ways as food sources, or as a source of the dermaseptin or temporin peptides. Because all plant types are susceptible to one or more plant pathogens, the present invention may be usefully applied to produce broad-spectrum resistance in any plant type.
- the invention may be applied to both monocotyledonous, dicotyledenous and gymnosperm plants, including, but not limited to maize, wheat, rice, barley, soybean, cotton, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts; and flowers such as orchides, carnations and roses.
- the invention provides transgenic plants that express one or more dermaseptin and/or temporin peptides.
- dermaseptin and temporin peptide families are well known in the art.
- Examples of dermaseptins that may be used in the invention include, but are not limited to, the dermaseptins described by Mor. et al., Biochemistry, 30:8824-8830, 1991, Strahilevitz, Biochemistry, 33: 10951- 10960, 1994 and Kirberger, Biochim. Biophys. Ada 1388: 279-283, 1998.
- temporins that may be used include, but are not limited to, the temporins described by Simmaco et al., Eur. J.
- both dermaseptin and temporin peptides are produced as precursor forms that are subsequently processed by proteolytic cleavage to form mature proteins.
- the mature forms of dermaseptins are typically about 27-34 amino acids in length, while the mature forms of temporins are typically about 10-13 amino acids in length.
- the invention contemplates the use of both the naturally occurring full-length (unprocessed) forms of these peptides, as well as the mature (processed) forms of the peptides and intermediate forms.
- synthetic forms of the peptides may also be employed.
- Synthetic forms of the peptides include any form that is not naturally occurring, and encompasses peptides that differ in amino acid sequence from the naturally occurring peptides, but which still retain dermaseptin or temporin biological activity. Such sequence variants will typically retain at least 40% amino acid sequence identity with at least one naturally occurring dermaseptin or temporin peptide.
- dermaseptins and temporins that may be employed include forms having N-terminal peptide extensions.
- Such peptide extensions may comprise portions of the precursor forms of dermaseptins or temporins that are usually removed during protein processing, or may be synthetic sequences.
- These N-terminal peptide extensions may serve to provide enhanced resistance to proteolytic cleavage, and may also enhance the antimicrobial activity of the peptides.
- these N-terminal extensions are of between 2 and 25 amino acids in length, although longer extensions may also be employed.
- Examples of N-terminal extension sequences that are utilized in certain embodiments include the peptide sequences MAMWK and MASRH.
- the AMWK sequence is a naturally-occurring peptide extension; it is part of the full-length dermaseptin-b peptide sequence that is normally cleaved during processing.
- the ASRH is a synthetic extension sequence. In each case, the N-terminal methionine is added to the extension peptide to ensure proper expression of the peptide.
- the fundamental aspect of the invention is based on the expression of temporin and dermaseptin peptides in transgenic plants, other amino acid sequences may be joined to the peptides in order to produce fusion peptides.
- Expression of such fusion peptides in transgenic plants may provide even more effective broad-spectrum pathogen resistance than expression of temporin or dermaseptin peptides alone, or may enhance stability of the expressed dermaseptin/temporin molecule to provide higher expression levels, and thereby facilitate purification of the peptide from plant tissues.
- the invention provides transgenic plants that express a fusion peptide comprising:
- the second peptide sequence is typically, but not necessarily, linked to the amino
- the second peptide sequence comprises an anionic
- pro-region peptide sequence Such pro-region peptides serve to neutralize the cationic nature of the dermaseptin or temporin and may thus provide enhanced stability in the cellular environment.
- these pro-regions generally include a number of negatively charged amino acids, such as glutamate (Glu or E) and aspartate (Asp or D).
- Suitable pro-regions include those that are found in naturally occurring unprocessed (full-length) dermaseptin and temporin peptides, as well as anionic pro- regions from other peptides, including those of mammalian origin, such as the pro-region from sheep cathelin proteins. Fusion peptides that include such pro-regions may be represented as P-D or P-T, wherein P is the pro-region peptide, T is a temporin peptide and D is a dermaseptin peptide.
- pro-region peptides may be directly joined to the N-terminus of the dermaseptin or temporin peptide, it may be beneficial to join the two peptides using a spacer peptide.
- spacer peptides to join two peptide domains is well known in the art; such spacer peptides are typically of between 2 and 25 amino acids in length, and provide a flexible hinge connecting the first peptide sequence to the second peptide.
- Spacer sequences that have been used to provide flexible hinges connecting two peptide sequences include the glycine(4) serine spacer (GGGGS x3) described by Chaudhary et al., Nature 339: 394-397, 1989.
- an N-terminal peptide extension as described above may serve to provide the spacer peptide function.
- Fusion peptides that comprise a pro-region peptide, a spacer peptide and a dermaseptin or temporin peptide may be represented as P-S-D or P-S-T, wherein S represents the spacer peptide.
- Spacer sequences may also include a cleavage site, such as a peptide sequence recognized and cleaved by a protease. Such sites facilitate removal of the pro-region from the dermaseptin or temporin peptide following purification from plant tissues.
- nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand
- SEQ ID 1 shows the dermaseptin b cDNA sequence
- SEQ ID 2 shows the amino acid sequence of the precursor (unprocessed) dermaseptin b peptide
- SEQ ID 3 shows the 27 amino acid sequence of the mature dermaseptin b peptide
- SEQ ID 4 shows the 31 amino acid sequence of the mature dermaseptin B peptide
- SEQ IDs 5-14 show the amino acid sequences of various mature (processed) dermaseptin peptides
- SEQ ID 15 shows a cDNA sequence encoding temporin G
- SEQ ID 16 shows the amino acid sequence of the precursor (unprocessed) form of temporin G
- SEQ ID 17 shows the 13 amino acid sequence of the mature temporin G peptide
- SEQ IDs 18-26 show the amino acid sequences of various mature (processed) temporin peptides
- SEQ ID 27 shows the nucleic acid sequence encoding MSRA 2
- SEQ ID 28 shows the amino acid sequence of MSRA
- SEQ IDs 29-32 show the oligos used to generate the nucleic acid sequence encoding MSRA 2
- SEQ IDs 33 shows the nucleic acid sequence encoding MSRA 3
- SEQ ID 34 shows the amino acid sequence of MSRA 3
- SEQ IDs 35-38 show the oligos used to generate the nucleic acid sequence encoding
- SEQ IDs. 39-41 show the amino acid sequences of various N-terminal extension sequences Brief Description of the Figures
- Figure 1 is a graph that shows the results from assays that tested the resistance of transgenic potato tubers to soft rot Discs prepared from tubers of Desiree control and transgenic plants expressing Demaseptin B (sample Nos Dl, D2, D6, D10) or Temporin A (sample Nos Tl, T2, T3) were infected with E. carotovora After 6 days at room temperature, rotted tissue was gently removed from the discs and the sensitivity/resistance to E. carotovora was expressed as the loss of weight of tuber tissue
- Figure 2 is a graph that shows the bactericidal effect of the peptides MSRA 2 (Dermaseptin B) and MSRA 3 (Temporin A) on E. coh
- MSRA 2 Demaseptin B
- MSRA 3 Temporin A
- Dermaseptin B (DSB, 7 ⁇ g/ml, 30 ⁇ g/ml, and 75 ⁇ g/ml), Temporin A (TA, 75 ⁇ g/ml, 133 ⁇ g/ml, 200 ⁇ g/ml) and a combination of Temporin A and Dermaseptin B (133 ⁇ g/ml Temporin A and 30 ⁇ g/ml Dermaseptin B) for 4 hours, diluted and plated on LB plates After overnight incubation at 37°C, the colonies were counted and the survival of bacteria was scored
- Figure 3 is a graph that shows the bactericidal effect of the peptides MSRA 2 (Dermaseptin B) and MSRA 3 (Temporin A) on E. carotovora.
- the cell cultures were incubated at room temperature in the presence of indicated concentration of Dermaseptin B (DSB, 23 ⁇ g/ml, 45 ⁇ g/ml) or Temporin A (TA, 67 ⁇ g/ml, 133 ⁇ g/ml) for 4 hours, diluted and plated on LB plates After overnight incubation at 28°C, the colonies were counted and the survival of bacteria was scored
- Dermaseptin refers to any member of the family of naturally occurring cationic peptides termed dermaseptins
- Dermaseptins were first identified in skin extracts from the South American arboreal frog Phyllomedusa sauvagn (Mor et al , J. Biol. Chem., 269 31635-31641, 1994) They are broad-spectrum microbiocidal peptides that inhibit growth of filamentous fungi as well as bacteria, yeast, and protozoa (Strahilevitz, Biochemistry, 33 10951-10960, 1994) Since the first dermaseptin, dermaseptin S, was identified, a number of other members of this peptide family have been characterized and cloned, including dermaseptin-b, isolated from the skin of Phyllomedusa bicolor (Mor et al , J. Biol.
- Dermaseptin peptides are typically expressed as precursor forms of around 60- 80 amino acids in length, and are subsequently processed to mature forms of around 27-34 amino acids in length
- dermaseptin-b SEQ ID 1, Amiche et al , J. Biol. Chem 269: 1747-1852, 1994, Chapentier et al , Biol
- This precursor form of dermaseptin-b is processed to produce two mature forms, termed dermaseptin b and dermaseptin B (Strahilevitz, Biochemistry, 33 10951-10960, 1994)
- Dermaseptin b (SEQ ID 3) is 27 amino acids in length and comprises amino acid residues 49-75 of the precursor form Dermaseptin B is an alternative cleavage product of 31 amino acids in length, and includes an N terminal extension of 4 amino acids (AMWK) (SEQ ED: 4).
- Dermaseptin B comprises amino acid residue 45-75 of the precursor form.
- SEQ IDs: 1 and 2 which show the full-length precursor form of dermaseptin b
- the dermaseptin peptides shown in the Sequence Listing represent the processed, mature forms of the peptides.
- the N-terminal V-helical amphipathic segment of the mature dermaseptin peptides has been identified as important for antimicrobial activity (Mor et al., J. Biol. Chem., 269:31635-31641, 1994; Mor and Nicolas, Journal Biochemical Chemistry, 269:1934-39, 1994) and this fragment may be used in place of the full- length mature dermaseptin.
- the term "dermaseptin” also encompasses variant dermaseptin peptides, as well as fragments of the naturally occurring peptides, that share a specified level of sequence identity with a naturally occurring dermaseptin peptide, or that differ from a naturally occurring dermaseptin peptide by one or more conservative amino acid substitutions.
- variant peptides and fragments retain dermaseptin biological activity, which may be assayed by the methods described below.
- a variant dermaseptin will typically share at least 40% amino acid sequence identity with a naturally occurring dermaseptin peptide (such as the one shown in SEQ ID: 3) as determined by the methods described below.
- Dermaseptin biological activity the ability of a dermaseptin peptide to inhibit bacterial growth and/or fungal growth. Dermaseptin biological activity can readily be ascertained by using the protocols given below.
- the antibacterial activity of a given dermaseptin peptide is assessed by determining its ability to inhibit the growth of a pectino lyric bacterial strain such as Erwinia carotovora or Escherichia coli DH5 V
- the activity of a given peptide is determined by serially diluting the peptide in LB and aliquoting 100 :1 to wells in a 96 well microtiter plate A fresh bacterial culture ( ⁇ 0 3 A550) is then grown on Luria- Bertani medium (LB) (1% w/v tryptone and 0 5% w/v yeast extract) and diluted to 10 " 2 in LB to represent approximately 10 4 - 10 5 colony forming units (CFU) ml "1 10 :1 of the bacterial culture is then
- the dermaseptin peptide is determined to have biological activity if, under the conditions of this assay, it is capable of inhibiting bacterial growth by at least 10% at a concentration of 7 :g per ml (i e , at this concentration, the number of bacterial colonies is no more than 90% that of the control plate)
- the antifungal activity of a given dermaseptin peptide is assessed by utilizing the fungal strains Phytophthora cactorum and/or Fusarmm solam
- the selected fungal strain is grown on Five Cereal Agar (FCA containing 20 gL "1 five cereal baby food instant flakes, and 8 gL "1 agar 3 (Terras et al , The Plant Cell 7 573-588, 1995)
- FCA Five Cereal Agar
- FCA Five Cereal Agar
- the dermaseptin peptide is determined to have biological activity if, under the conditions of this assay, it is capable of inhibiting fungal growth at a concentration of 5 :g per ml (i e , there is a discernible zone of inhibition around a well containing this concentration of peptide)
- Temporin refers to any member of the family of naturally occurring cationic peptides termed temporins (Simmaco et al , Eur. J. Biochem., 242 788-92 1996) as well as fragments and variants of these naturally occurring peptides that display temporin biological activity as defined below
- Temporins are small cationic peptides with anti-microbial activity that were initially identified from a cDNA library prepared from the skin of the frog, Rana temporaria These peptides show some sequence similarity to hemolytic peptides isolated from Vespa venom, however, the temporin peptides are not hemolytic (Simmaco et al , Eur. J. Biochem., 242 788-92 1996)
- temporins A, B, C, D, E, F, G, H, K, and L have been described by Simmaco et al , Eur. J. Biochem., 242 788-92 1996
- temporins are typically expressed in a precursor form and subsequently processed to produce a mature form
- the cDNA molecule that encodes temporin G (shown in SEQ ID 15, and located in the GenBank nucleotide database under accession number Y09395) encodes a 61 amino acid precursor form of temporin G (shown in SEQ ID 16)
- Amino acids 1-22 comprise a signal sequence
- amino acids 23-46 comprise a pro-region
- amino acids 47-59 comprise the processed, mature temporin G peptide (the mature form is shown in SEQ ID 17)
- the predicted mature temporin peptides are between 10 and 13 amino acids long, and some have been found to be amidated at the C-terminus (Sim
- temporin also encompasses variant temporin peptides, as well as fragments of the naturally occurring peptides, that share a specified level of sequence identity with a naturally occurring temporin peptide, or that differ from a naturally occurring temporin peptide by one or more conservative amino acid substitutions
- variant peptides and fragments retain temporin biological activity, which may be assayed by the methods described below
- a variant temporin will typically share at least 40% amino acid sequence identity with a naturally occurring temporin peptide (such as the one shown in SEQ ID 17) as determined by the methods described below
- Temporin biological activity the ability of a temporin peptide to inhibit bacterial growth
- the antibacterial activity of a given temporin peptide is assessed by determining its ability to inhibit the growth of a pectinolytic bacterial strain such as Erwinia carotovora or Escherichia coli DH5V
- the activity of a given peptide is determined by serially diluting the peptide in LB and aliquoting 100 :1 to wells in a 96 well microtiter plate
- a fresh bacterial culture ( ⁇ 0 3 A550) is then grown on Luria- Bertani medium (LB) (1% w/v tryptone and 0 5% w/v yeast extract) and diluted to 10 " 2 in LB to represent approximately 10 4 - 10 5 colony forming units (CFU) ml "1 10 :1 of the bacterial culture is then inoculated into the wells containing the peptide and the samples are incubated at 37°C for 4 hours The
- the temporin peptide is determined to have biological activity if, under the conditions of this assay, it is capable of inhibiting bacterial growth by at least 10% at a concentration of 100 :g per ml (i e , at this concentration, the number of bacterial colonies is no more than 90% that of the control plate)
- the antifungal activity of a given temporin peptide is assessed by utilizing the fungal strains Phytophthora cactorum and/or Fusarium solani
- the selected fungal strain is grown on Five Cereal Agar (FCA containing 20 gL "1 five cereal baby food instant flakes, and 8 gL "1 agar 3 (Terras et al , The Plant Cell, 7 573-588, 1995) After 5 days growth at room temperature a mycelial plug is removed and placed upside down in the center of a fresh FCA plate A sterile solution (10 :1) of the test peptide is then introduced into a well 3 cm from the edge of the plate and a control well
- the temporin peptide is determined to have biological activity if, under the conditions of this assay, it is capable of inhibiting fungal growth at a concentration of 5 :g per ml (i e , there is a discernible zone of inhibition around a well containing this concentration of peptide)
- Transgenic plant As used herein, this term refers to a plant that contains recombinant genetic material not normally found in a wild-type plant of this species
- a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually)
- Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences Sequence identity is typically expressed in terms of percentage identity, the higher the percentage, the more similar the two sequences are Methods of alignment of sequences for comparison are well known in the art
- NCBI Basic Local Alignment Search Tool (Altschul et al., J. Mol. Biol 215 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx It can be accessed at http//www ncbi nlm nih gov/BLAST/ A description of how to determine sequence identity using this program is available at http //www ncbi nlm nih gov/BLAST/blast_help html
- Variants of naturally occurring dermaseptin and temporin peptides useful in the present invention are typically characterized by possession of at least 40% sequence identity counted over the full-length alignment with the amino acid sequence of a naturally occurring temporin or dermaseptin peptide when aligned using the NCBI Blast 2 0 1 (described in Altschul et al , Nucleic Acids Res 25:3389- 3402, 1997)
- the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1)
- the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties) Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 70%, at least 80%, at
- a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g , by genetic engineering techniques
- Oligonucleotide (oligo) A linear polynucleotide sequence of up to about 100 nucleotide bases in length
- Probes and primers may readily be prepared based on the amino acid sequence provided by this invention
- a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule
- Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989 and Ausubel et al , Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences, 1987
- Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme Primer pairs can be used for amplification of
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0 5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA)
- Primer Very Well Chemical Research, Cambridge, MA
- probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides
- Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
- Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
- a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
- a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
- a vector may also include one or more selectable marker genes and other genetic elements known in the art.
- a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Two peptide sequences may be operably linked through a normal peptide bond, or by other covalent linkage.
- dermaseptin polypeptides A listing of exemplary dermaseptin peptides is provided above. Nucleic acid molecules encoding these dermaseptin polypeptides may either be derived by simple application of the genetic code to the peptide sequence, or the naturally occurring cDNA or gene sequence may be employed. For example, a cDNA sequence encoding dermaseptin-b is provided in SEQ ID: 1 (and disclosed in Amiche et al., J. Biol. Chem. 269:1747-852, 1994). Typically, the mature form of the dermaseptin peptide will be selected for expression.
- any fragment of a full-length dermaseptin peptide may be selected, contingent upon that fragment having dermaseptin biological activity if it is to be used to produce pathogen-resisting plants.
- the various dermaseptin peptides have varying degrees of anti-microbial activity, with some working more effectively against certain pathogens then others. Therefore, when selecting peptides for producing transgenic plants with enhanced pathogen resistance, the selection of a particular dermaseptin will be depend upon, among other factors, the type of plant in which the peptide is to be expressed, and the types of pathogens that commonly infect that plant type
- a nucleic acid molecule encoding the peptide may be produced by standard molecular biology techniques Because the dermaseptin peptides are relatively short, the simplest way to synthesize the nucleic acid molecule will likely be via synthesis of overlapping oligonucleotides on a commercially available oligonucleotide synthesizer The oligonucleotides can then be assembled into a full-length coding region in vitro This approach also permits the selection of codons encoding particular amino acid residues that reflect the codon usage bias of the plant into which the nucleic acid molecule is to be introduced, thereby enhancing the expression efficiency Detailed examples of the production of coding sequences using this approach are provided in the examples below b.
- Temporin Peptides A listing of exemplary temporin peptides is provided above Nucleic acid molecules encoding these temporin peptides may either be derived by simple application of the genetic code to the peptide sequence, or the naturally occurring cDNA or gene sequence may be employed For example, the cDNA sequence encoding temporin G is provided in SEQ ID 15 Typically, the mature form of the temporin peptide will be selected for expression However, any fragment of a full- length temporin peptide may be selected, contingent upon that fragment having dermaseptin biological activity if it is to be used to produce pathogen-resisting plants As with the selection of dermaseptin peptides, one of ordinary skill in the art will appreciate that the various temporin peptides have varying degrees of anti- microbial activity, with some working more effectively against certain pathogens then others Therefore, when selecting peptides for producing transgenic plants with enhanced pathogen resistance, the selection of a particular temporin will be depend upon, among other factors, the type
- the temporin and dermaseptin proteins may be also expressed in transgenic plants in the form of fusion proteins
- any desired peptide may be fused to the selected dermaseptin or temporin protein for expression in plants
- the expression of fusion proteins comprising an anionic pro-region peptide operably linked to the amino terminus of the dermaseptin or temporin is expected to be particularly beneficial
- Any anionic pro-region peptide may be employed for this purpose, including the anionic pro-regions that are found in naturally occurring full-length (i e , unprocessed) dermaseptin and temporin peptides
- the pro-region comprising amino acids 23-46 of temporin G shown in SEQ ID 16
- Such pro-region peptides serve to neutralize the cationic nature of the dermaseptin or temporin and may thus provide enhanced stability in the cellular environment
- these pro-regions generally include a number of negatively charged amino acids, such as glutamate (Glu or E) and aspart
- pro-region peptides examples include pro-region peptides of the following proteins the human neutrophil defensin protein (Daher et al , Proc. Natl. Acad. Sci USA, 85:7327-7331, 1988), the bovine antimicrobial cathelicidin protein BMAP28 (Skerlavaj et al , J. Biol. Chem 271: 28375-381, 1996), the sheep antimicrobial cathelin family of proteins (Mahoney et al , FEBS Lett. 377:519-522, 1995), bovine indolicidin (Del Sal et al , Biochem. Biophys. Res.
- human neutrophil defensin protein Disher et al , Proc. Natl. Acad. Sci USA, 85:7327-7331, 1988
- bovine antimicrobial cathelicidin protein BMAP28 Skerlavaj et al , J. Biol. Chem 271: 28375-381,
- an alternative embodiment involves linking the pro-region peptide to the dermaseptin or temporin peptide using a spacer peptide sequence.
- spacer peptides to join two peptide domains
- spacer peptides are typically of between 2 and 25 amino acids in length, and provide a flexible hinge connecting the first peptide sequence to the second peptide
- Spacer sequences that have been used to provide flexible hinges connecting two peptide sequences include the glycine(4)-serine spacer (GGGGS x3) described by Chaudhary et al , Nature 339: 394-397, 1989
- an N-terminal peptide extension as described below may serve to provide the spacer peptide function
- Spacer sequence peptides may also include a cleavage site, such as a peptide sequence recognized and cleaved by a protease, such as Factor Xa Such sites facilitate removal of the pro-region from the dermaseptin or temporin peptide following purification from plant tissues
- anionic pro- region peptides and spacer peptides to express certain cationic peptides
- an N-terminal extension peptide sequence may be added to the dermaseptin or temporin peptide
- Such peptide extensions may comprise portions of the precursor forms of dermaseptins or temporins that are usually removed during protein processing, or may be synthetic sequences
- These N-terminal peptide extensions may serve to provide enhanced resistance to proteolytic cleavage, enhance transcription levels, or enhance the antimicrobial activity of the peptides
- these N-terminal extensions are of between 2 and 25 amino acids in length, although longer extensions may also be employed
- Examples of N-terminal extension sequences that are utilized in certain embodiments include the peptide sequences AMWK, ASRH, and ALWK
- the AMWK (SEQ ID 39) sequence is a naturally-occurring peptide extension, it is part of the full-length dermaseptin-b peptide sequence that is normally cleaved during processing The addition of this sequence to the N-terminus of dermaseptin b (to produce dermaseptin B) has been
- variant Dermaseptin and Temporin Peptides As described above, a number of naturally occurring temporin and dermaseptin peptides are known, exemplified by those shown in the Sequence Listing Variants on these naturally occurring peptides may be selected by introducing amino acid substitutions, additional amino acid residues, or by deleting amino acid residues These variant peptides may either be produced by chemical synthesis (for example, in order to confirm that the variant peptide retains functional activity), or may be produced in a biological expression system. In the latter instance, the nucleic acid sequence encoding the corresponding naturally occurring peptide can be manipulated so that it encodes the variant peptide.
- the coding region for a variant peptide can simply be synthesized de novo and introduced into a suitable expression vector
- substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
- substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
- a hydrophilic residue e.g
- variant peptides having one or more of these more substantial changes may also be employed in the invention, provided that temporin or dermaseptin biological activity is retained. More extensive amino acid changes may also be engineered into variant dermaseptin or temporin peptides. As noted above however, these variant peptides will typically be characterized by possession of at least 40% sequence identity counted over the full-length alignment with the amino acid sequence of their respective naturally occurring sequences using the alignment programs described above In addition, these variant peptides will retain biological activity
- a dermaseptin or temporin peptide has biological activity may be achieved using the assay systems described above Following confirmation that the peptide has the desired activity, a nucleic acid molecule encoding the peptide may be readily produced using standard molecular biology techniques Where appropriate, the selection of the open reading frame will take into account codon usage bias of the plant species in which the peptide is to be expressed
- Selection of progeny plants containing the introduced transgene may be made based upon the detection of an altered phenotype Such a phenotype may result directly from the disease resistance conferred by the introduced sequence or may be manifested as enhanced resistance to a chemical agent (such as an antibiotic) as a result of the inclusion of a dominant selectable marker gene incorporated into the transformation vector
- dermaseptins and/or temporin peptides may be introduced into plant species including, but not limited to, maize, wheat, rice, barley, soybean, cotton, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, tobacco, flax, peanut, clover, cowpea, grapes, vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers, tree fruits such as citrus, apples, pears, peaches, apricots, walnuts, fur trees such as Douglas fir and loblolly pine, and flowers such as carnations and roses b.
- plant transformation vectors include one or more cloned sequences under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker
- plant transformation vectors typically also contain a promoter regulatory region (e g , a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal Examples of constitutive plant promoters that may be useful for expressing
- tissue specific (root, leaf, flower, and seed for example) promoters (Carpenter et al., The Plant Cell 4 557-571, 1992, Denis et al., Plant Physiol 101 1295-1304, 1993, Opperman et al , Science 263 221-223, 1993, Stockhause et al , The Plant Cell 9 479-489, 1997, Roshal et al , The EMBO J 6 1155, 1987, Schernthaner et al , E 5O J. 7 1249, 1988, Yamamoto et al , Plant Cell 3 371-382, 1990, and Bustos et al , Plant Cell 1 839, 1989) can be fused to the coding sequence to obtain particular expression in respective organs
- Plant transformation vectors may also include RNA processing signals, for example, mtrons, which may be positioned upstream or downstream of the ORF sequence in the transgene
- the expression vectors may also include additional regulatory sequences from the 3 '-untranslated region of plant genes, e g , a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase (NOS) 3' terminator regions
- plant transformation vectors may also include dominant selectable marker genes to allow for the ready selection of transformants Such genes include those encoding antibiotic resistance genes (e g , resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (e g , phosphinothricin acetyltransferase) c. Transformation and Regeneration Techniques
- Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the appropriate transformation technique will be determined by the practitioner
- Suitable methods may include, but are not limited to electroporation of plant protoplasts, liposome-mediated transformation, polyethylene glycol (PEG) mediated transformation, transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens (AT) mediated transformation.
- Typical procedures for transforming and regenerating plants are described in the patent documents listed at the beginning of this section. d. Selection of Transformed Plants
- transformed plants are usually selected using a dominant selectable marker incorporated into the transformation vector.
- a dominant selectable marker will confer antibiotic resistance on the seedlings of transformed plants, and selection of transformants can be accomplished by exposing the seedlings to appropriate concentrations of the antibiotic.
- Selection can also be accomplished by exploiting the pathogen resistance that is conferred to the plant via the transgene. As described in the Examples below, such screening may be accomplished either after the transgenic plants have been regenerated, or (depending on the transformation method used) may be performed on green transgenic callus prior to plant regeneration.
- the level of resistance that is conferred by a single copy of a transgene encoding a dermaseptin or a temporin peptide may be enhanced by introducing multiple copies of a single cationic peptide gene, or several genes encoding different cationic peptides.
- such vectors comprise two or more dermaseptin and/or temporin open reading frames each operably linked to its own 5' and 3' regulatory sequences.
- such vectors can result in the expression of multiple varieties of cationic peptides.
- transgene encoding a first cationic peptide can be introduced into a first plant and a second transgene encoding a second cationic peptide can be introduced into a second plant.
- the resulting transgenic plants can then be crossed to produce progeny that carry both transgenes.
- compositions and methods described above may be used not only to produce plants having enhanced, broad spectrum pathogen resistance, but may also be used for the large scale production of dermaseptins and temporins for a wide range of other applications
- temporins and dermaseptins produced in large quantities in plants may be purified and used in medical applications
- the production of biologically active peptides in plants is now widely practiced, and bulk expression and purification methods are well known Examples of constructs that facilitate the production of biologically active proteins in plants can be found in U S Patent 4,956,282 to Goodman et al These constructs generally contain a promoter region and an additional nucleic acid sequence that encodes an amino acid sequence that is later utilized in the purification process The amino acid sequence that is used to facilitate the isolation of the dermaseptin and/or temporin peptides can be subsequently cleaved and discarded
- a nucleic acid molecule was designed to encode the mature 27 amino acid form of dermaseptin b (SEQ ID 3) with a 5 amino acid N-terminal extension sequence, MAMWK
- This nucleic acid construct was designated MSRA and was synthesized using four overlapping oligonucleotides in a single PCR reaction
- the oligonucleotides used are shown in Table 2
- the first two oligonucleotides (oligo #1 and oligo #2) contained the nucleic acid sequences encoding the N-terminal and the C-terminal portions of the peptide, respectively These oligonucleotides were used in the PCR reaction at a 20 nM concentration
- the second two oligonucleotides contained sequences recognized by various restriction enzymes Specifically, oligo #3 contained restriction sites for Xbal, Kpnl and Ncol, and oligo #4 contained restriction site for Sstl, Pstl and Hindlll These oligonucleotides were
- oligo #1 is identical to underlined portion of oligo #3, thus allowing oligo #3 to bind to the PCR product from the initial elongation of oligos #1 and #2
- the underlined portions of oligo #4 are identical to the underlined portion of oligo #2 This allows oligo #4 to bind to the PCR product created by elongation of oligos #1 and #2
- a nucleic acid molecule was designed to encode the mature 13 amino acid form of temporin A (SEQ ID 33) with a 6 amino acid N-terminal extension sequence, MASRHM
- This nucleic acid construct was designated MSRA 3 and was synthesized using four overlapping oligonucleotides in a single PCR reaction
- the oligonucleotides used are shown in Table 3
- the first two oligonucleotides (oligo #1 and oligo #2) contained the nucleic acid sequences encoding the prototype peptide
- these oligonucleotides were fully complementary, thus eliminating the need for an initial elongation cycle prior to the binding of oligos #3 and #4 Oligos #1 and #2 were used in the PCR reaction at a 20 nM concentration
- the second two oligonucleotides contained sequences recognized by various restriction enzymes Specifically, oligo #3 contained restriction sites for Xbal, Kpnl and N
- nucleotides in bold represent the regions of complementanty between oligo #1 and oligo #2 As depicted in the diagram these sequences are fully complementary
- the underlined portions oligo #2 are complementary to the underlined portions of oligo #3 and the underlined portions of oligo #1 are complementary to the underlined portions of oligo #4
- the nucleic acid sequences encoding MRSA 2 and MRSA 3 were assembled into various plant transformation vectors, thereby placing them under the transc ⁇ ptional control of a variety of different promoters
- Cloning MRSA 2 and MRSA 3 sequences into one such vector placed the respective sequences under the control of a promoter that contained two copies of the CaMV 35S promoter and an AMV RNA4 translation enhancing element (Kay et al , Science 236 1299-1302, 1987)
- the clones that resulted from hgation into this vector were given the prefix pD as a designation Therefore, pDMSRA designates a vector which contains the MSRA 2 construct under the control of the double CaMV 35S promoter and the AMV RNA4 translational enhancer
- pDMSRA 3 designates a vector that contains the MSRA 3 construct under the control of the double CaMV 35S promoter with the AMV RNA4 translational enhancer
- Potato cultivars, Russert Burbank and Desiree, as well as tobacco were used as representative plant species for transformation
- the transformation of the plants was performed according to DeBlock, Theoret Appl Genet 76 161 -11 A, 1988, with some modifications Briefly, transformations of tobacco and potato were carried out by isolating leaves (5 mm) and stems from 4-week-old shoots These leaves and shoots were cut and further wounded by scratching with glass pipette tips Wounded leaves and stems were then floated upside down on 15 ml of S2 medium in a petri dish. This medium contained the components listed in Murashige and Skoog, Physiol. Plant.
- the plant tissue samples were placed at room temperature (RT) at 3000 lux to allow for callus formation After two weeks, many small calli formed at the wounded edges of the leaves and stems The small calli were removed and transferred to fresh S6 medium (S4 without NAA) After 2-3 weeks, the calli were transferred to S8 medium (S6 supplemented with 0 1 mg/L GA 3 ) to allow for shoot formation
- RT room temperature
- S8 medium S6 supplemented with 0 1 mg/L GA 3
- SI medium contained the components described in Gamborg et al , Exp. Cell Res 50 151-158, 1968, with the addition of 20 g/L sucrose, 150 mg/L CaC12, 0 4% agarose, pH 5 8, 1 g/L carbenicillin and 50 ⁇ g/ml kanamycin
- the SI medium and the shoots were placed in Magenta jars to allow for root formation After one week the shoots had rooted In order to avoid selecting identical shoots, transfer of shoots from the same or closely linked calli was avoided
- the regenerated putative transgenic plants were transferred to MS medium containing 1 g/L carbenicillin and 50 ⁇ g/ml kanamycin for further analysis
- DNA was isolated from the transgenic potato and tobacco plants using the methods described below In some instances purification involved a more rigorous protocol and in others a simple crude extract procedure was performed The more rigorous extract procedure started by obtaining ten grams of fresh leaf tissue, and immediately freezing the sample in liquid nitrogen The frozen tissue was then ground into a fine power and extracted with 20 ml extraction buffer (50 mM Tris-HCl buffer, 5 mM EDTA, 0 35 M sorbitol, 0 1% BSA, 0 1% mercaptoethanol, 10% PEG 4000) The homogenate was filtered through several layers of cheesecloth and one layer of miracloth The final purification steps were then performed in accordance with Wagner et al , Proc. Natl. Acad. Sci U.S.A. 84 2097-5100, 1987
- the crude extract procedure was used mainly when isolation was being done for the purposes of PCR analysis
- about 200 mg of fresh leaves were collected and ground in liquid nitrogen into a powder 100 ⁇ l of 0 5 N NaOH was added to the powder and mixed (vortexed) for 30 seconds
- the suspension was centrifuged for 5 minutes and 5 ⁇ l of the supernatant was added into 45 ⁇ l of 100 mM of Tris buffer (pH 8 0)
- This crude genomic DNA extract was used as the template for PCR amplification
- Detection of the presence or absence of the MSRA 2 construct was achieved by performing a PCR reaction with the extracted genomic DNA and oligos # 3 and # 4 from Table 2 above.
- the expected size of the product from the reaction was 129 bp. This method allowed for the identification of transgenic tobacco and potato plants transformed with the pDMSRA 2 or pRSHMSRA constructs
- Detection of the MSRA 3 construct was achieved by performing PCR reactions either with oligo #3 and oligo # 4 from Table 3 above or with a primer specific for the 2 x 35S CaMV promoter in combination oligo #4 from Table 3 above Transgenic tobacco plants containing the pDMSRA 3 construct were identified
- a control plant was also engineered to contain a GUS gene under the control of a super promoter containing the mas (mannopine synthase) promoter/activator region preceded by a trimer of the ocs (octopine synthase) upstream activating sequence (Ni et al , The Plant J.
- RNA substrate for these experiments was isolated and purified from the transgenic tobacco and potato plants The protocol used for this isolation was performed in accordance with Verwoerd et al , Nucl. Acids Res 17 2362, 1989
- Transgenic potato plants containing pDMSRA 2 and transgenic tobacco plants containing either pDMSRA 2 or pRSHMSRA 2 were tested as described and showed resistance to the pathogen After one week of growth in the presence of the bacterial culture the transgenic plants were uninfected (as determined by visual inspection) and continued to grow In sharp contrast, a control plant challenged with bacterial culture was severely infected after one week of incubation, growth was inhibited and the plant died after 2-3 weeks of exposure to fungal pathogens
- Mature plants were tested for their resistance to various fungi using the following protocol One cm 2 x 0 5 cm of Fusarium or Phytophthora sp. -containing media slices were cut and put in the center of fresh plates of V8 agar media (250 ml/L V8 juice, 7 grams/L agar) in a 9 cm petri dish and grown for about one month at room temperature, or until the fungal mycelia completely covered the petri dish Shoots of transgenic plants (-10 cm) were cut and transferred into MS medium for further growth According to different treatments, plants were allowed to grow for 3 days or 2 weeks until the shoots rooted Two 1 cm 2 x 0 5 cm slices of the fungal agar were then applied to both sides of the plant shoots without wounding the plant The resulting degree of infection was then determined visually In a representative experiment, a transgenic potato plant transformed with pDMSRA 2 , tobacco plants transformed with either pRSHMRS A 2 or pDMSRA 2 , and control potato and tobacco plants were exposed to
- the experiment described above was also used to test the transgenic plant's resistance to Phytophthora infestans. The results from these assays showed that the transgenic plants were also resistant to Phytophthora infestans.
- Another experiment was performed challenging a pDMSRA 2 transgenic potato plant with Fusarium solani. After 6 days, Fusarium grew all over the surface of MS medium, the damage to the roots in the control plants was severe, and the base of stem was penetrated by Fusarium and the stems were softened and veins of infected leaves showed clear browning and necrosis. After several days, the control plant collapsed and died. However, the transgenic plant continued to grow even under the extreme fungal infestation by Fusarium solani.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00910462A EP1163352A1 (en) | 1999-03-17 | 2000-03-16 | Transgenic plants that are resistant to a broad spectrum of pathogens |
AU32679/00A AU772964C (en) | 1999-03-17 | 2000-03-16 | Transgenic plants that are resistant to a broad spectrum of pathogens |
JP2000605754A JP2002538828A (en) | 1999-03-17 | 2000-03-16 | Transgenic plants resistant to broad-spectrum pathogens |
US09/936,885 US6835868B1 (en) | 1999-03-17 | 2000-03-16 | Transgenic plants expressing dermaseptin peptides providing broad spectrum resistance to pathogens |
CA2365099A CA2365099C (en) | 1999-03-17 | 2000-03-16 | Transgenic plants that are resistant to a broad spectrum of pathogens |
US10/719,623 US7081568B2 (en) | 1999-03-17 | 2003-11-20 | Transgenic plants expressing temporin peptides |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12507299P | 1999-03-17 | 1999-03-17 | |
US60/125,072 | 1999-03-17 |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09936885 A-371-Of-International | 2000-03-16 | ||
US09/936,885 A-371-Of-International US6835868B1 (en) | 1999-03-17 | 2000-03-16 | Transgenic plants expressing dermaseptin peptides providing broad spectrum resistance to pathogens |
US10/719,623 Division US7081568B2 (en) | 1999-03-17 | 2003-11-20 | Transgenic plants expressing temporin peptides |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000055337A1 true WO2000055337A1 (en) | 2000-09-21 |
Family
ID=22418071
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2000/000288 WO2000055337A1 (en) | 1999-03-17 | 2000-03-16 | Transgenic plants that are resistant to a broad spectrum of pathogens |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1163352A1 (en) |
JP (1) | JP2002538828A (en) |
CN (1) | CN1351667A (en) |
AU (1) | AU772964C (en) |
CA (1) | CA2365099C (en) |
WO (1) | WO2000055337A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2853538A1 (en) | 2013-09-27 | 2015-04-01 | Université Pierre et Marie Curie (Paris 6) | Analogues of temporin-SHa and uses thereof |
WO2018115798A1 (en) | 2016-12-22 | 2018-06-28 | Sorbonne Universite | Antimicrobial peptides and uses thereof |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3201223A4 (en) * | 2014-10-01 | 2018-06-27 | Plant Health Care, Inc. | Elicitor peptides having disrupted hypersensitive response box and use thereof |
CN106397568B (en) * | 2016-12-02 | 2019-10-01 | 河南牧翔动物药业有限公司 | A kind of cecropin D ermaseptin-M and its application |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0497366A2 (en) * | 1991-02-01 | 1992-08-05 | ENICHEM S.p.A. | Antimicrobial peptides and their use against plant pathogens |
EP0552559A2 (en) * | 1991-12-23 | 1993-07-28 | Unilever Plc | Transgenic plants resistant to microbian infection |
WO1995018855A2 (en) * | 1994-01-07 | 1995-07-13 | Pioneer Hi-Bred International, Inc. | Synthetic antimicrobial peptides |
WO1996028559A1 (en) * | 1995-03-13 | 1996-09-19 | University Of British Columbia | Method for the production of cationic peptides |
EP0798381A2 (en) * | 1996-03-25 | 1997-10-01 | National Institute Of Agrobiological Resources, Ministry Of Agriculture, Forestry And Fisheries | Pathogen-resistant plants and method for production thereof |
WO1998006860A1 (en) * | 1996-08-14 | 1998-02-19 | Novartis Ag | Peptide with inhibitory activity towards plant pathogenic fungi |
WO1998025961A1 (en) * | 1996-12-13 | 1998-06-18 | Sbl Vaccin Ab | Antimicrobially active polypeptides |
WO1998040401A2 (en) * | 1997-03-10 | 1998-09-17 | Micrologix Biotech Inc. | Compositions and methods for treating infections using cationic peptides alone or in combination with antibiotics |
WO1998050543A1 (en) * | 1997-05-02 | 1998-11-12 | Meristem Therapeutics S.A. | Recombinant lactoferrin, methods of production from plants and uses |
WO1999006564A1 (en) * | 1997-07-31 | 1999-02-11 | Sanford Scientific, Inc. | Expression of antimicrobial peptide genes in plants, and their use in creating resistance to multiple plant pathogens |
WO2000026344A1 (en) * | 1998-10-30 | 2000-05-11 | Interlink Biotechnologies Llc | Peptides with enhanced stability to protease degradation |
WO2000031279A2 (en) * | 1998-11-20 | 2000-06-02 | Micrologix Biotech Inc. | Producing antimicrobial cationic peptides as fusion proteins |
-
2000
- 2000-03-16 WO PCT/CA2000/000288 patent/WO2000055337A1/en not_active Application Discontinuation
- 2000-03-16 CN CN 00805132 patent/CN1351667A/en active Pending
- 2000-03-16 CA CA2365099A patent/CA2365099C/en not_active Expired - Fee Related
- 2000-03-16 JP JP2000605754A patent/JP2002538828A/en active Pending
- 2000-03-16 EP EP00910462A patent/EP1163352A1/en not_active Withdrawn
- 2000-03-16 AU AU32679/00A patent/AU772964C/en not_active Ceased
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0497366A2 (en) * | 1991-02-01 | 1992-08-05 | ENICHEM S.p.A. | Antimicrobial peptides and their use against plant pathogens |
EP0552559A2 (en) * | 1991-12-23 | 1993-07-28 | Unilever Plc | Transgenic plants resistant to microbian infection |
WO1995018855A2 (en) * | 1994-01-07 | 1995-07-13 | Pioneer Hi-Bred International, Inc. | Synthetic antimicrobial peptides |
WO1996028559A1 (en) * | 1995-03-13 | 1996-09-19 | University Of British Columbia | Method for the production of cationic peptides |
EP0798381A2 (en) * | 1996-03-25 | 1997-10-01 | National Institute Of Agrobiological Resources, Ministry Of Agriculture, Forestry And Fisheries | Pathogen-resistant plants and method for production thereof |
WO1998006860A1 (en) * | 1996-08-14 | 1998-02-19 | Novartis Ag | Peptide with inhibitory activity towards plant pathogenic fungi |
WO1998025961A1 (en) * | 1996-12-13 | 1998-06-18 | Sbl Vaccin Ab | Antimicrobially active polypeptides |
WO1998040401A2 (en) * | 1997-03-10 | 1998-09-17 | Micrologix Biotech Inc. | Compositions and methods for treating infections using cationic peptides alone or in combination with antibiotics |
WO1998050543A1 (en) * | 1997-05-02 | 1998-11-12 | Meristem Therapeutics S.A. | Recombinant lactoferrin, methods of production from plants and uses |
WO1999006564A1 (en) * | 1997-07-31 | 1999-02-11 | Sanford Scientific, Inc. | Expression of antimicrobial peptide genes in plants, and their use in creating resistance to multiple plant pathogens |
WO2000026344A1 (en) * | 1998-10-30 | 2000-05-11 | Interlink Biotechnologies Llc | Peptides with enhanced stability to protease degradation |
WO2000031279A2 (en) * | 1998-11-20 | 2000-06-02 | Micrologix Biotech Inc. | Producing antimicrobial cationic peptides as fusion proteins |
Non-Patent Citations (7)
Title |
---|
BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1388, no. 1, 14 October 1998 (1998-10-14), pages 279 - 283, ISSN: 0006-3002 * |
CHARPENTIER STEPHANE ET AL: "Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 24, 12 June 1998 (1998-06-12), pages 14690 - 14697, XP002142518, ISSN: 0021-9258 * |
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 14 October 1998 (1998-10-14), WECHSELBERGER CHRISTIAN: "Cloning of cDNAs encoding new peptides of the dermaseptin-family.", XP002142520, Database accession no. PREV199800505321 * |
MOR AMRAM ET AL: "The NH-2-terminal alpha-helical domain 1-18 of dermaseptin is responsible for antimicrobial activity.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 3, 1994, pages 1934 - 1939, XP002142516, ISSN: 0021-9258 * |
MOR AMRAM ET AL: "The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 50, 1994, pages 31635 - 31641, XP002142519, ISSN: 0021-9258 * |
SIMMACO M ET AL: "TEMPORINS, ANTIMICROBIAL PEPTIDES FROM THE EUROPEAN RED FROG RANA TEMPORARIA", EUROPEAN JOURNAL OF BIOCHEMISTRY,DE,BERLIN, vol. 242, no. 242, December 1996 (1996-12-01), pages 788 - 792-792, XP000856339, ISSN: 0014-2956 * |
STRAHILEVITZ JACOB ET AL: "Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes.", BIOCHEMISTRY, vol. 33, no. 36, 1994, pages 10951 - 10960, XP002142517, ISSN: 0006-2960 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2853538A1 (en) | 2013-09-27 | 2015-04-01 | Université Pierre et Marie Curie (Paris 6) | Analogues of temporin-SHa and uses thereof |
WO2018115798A1 (en) | 2016-12-22 | 2018-06-28 | Sorbonne Universite | Antimicrobial peptides and uses thereof |
FR3061178A1 (en) * | 2016-12-22 | 2018-06-29 | Centre National De La Recherche Scientifique | ANTIMICROBIAL PEPTIDES AND USES THEREOF |
Also Published As
Publication number | Publication date |
---|---|
JP2002538828A (en) | 2002-11-19 |
CN1351667A (en) | 2002-05-29 |
CA2365099A1 (en) | 2000-09-21 |
EP1163352A1 (en) | 2001-12-19 |
AU772964B2 (en) | 2004-05-13 |
AU3267900A (en) | 2000-10-04 |
CA2365099C (en) | 2012-05-29 |
AU772964C (en) | 2004-10-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CZ311998A3 (en) | Antifungal polypeptide and control methods of plant pathogenic mould fungi | |
MX2014009113A (en) | Pathogen resistant citrus compositions, organisms, systems, and methods. | |
WO2001007635A1 (en) | Bs2 resistance gene | |
US6835868B1 (en) | Transgenic plants expressing dermaseptin peptides providing broad spectrum resistance to pathogens | |
Colova-Tsolova et al. | Genetically engineered grape for disease and stress tolerance | |
US20070067876A1 (en) | Production of Transgenic Poinsettia | |
US5446127A (en) | Antipathogenic peptides and compositions containing the same | |
WO1998004687A1 (en) | Antimicrobial peptides | |
CA2253946A1 (en) | Method for regulating cell death | |
AU772964C (en) | Transgenic plants that are resistant to a broad spectrum of pathogens | |
US6784337B1 (en) | Method of improving nematode resistance in plants via transformation with a DNA encoding a proteinase inhibitor fusion protein | |
AU2001284482B9 (en) | Disease-resistant plants and method of constructing the same | |
AU739960B2 (en) | Expression of antimicrobial peptide genes in plants, and their use in creating resistance to multiple plant pathogens | |
CA2391128C (en) | A method of making transgenic plants expressing a cecropin-mellitin hybrid cationic peptide imparting broad-spectrum pathogen resistance | |
US6025544A (en) | Processes for modifying plant flowering behavior | |
Norelli et al. | Genetic engineering of apple for increased resistance to fireblight | |
US6121511A (en) | Production of transgenic impatiens | |
MISRA et al. | Sommaire du brevet 2365099 | |
MISRA et al. | Patent 2365099 Summary | |
JP2000512503A (en) | Ozone-induced gene expression in plants | |
CN114230649B (en) | Tn1 protein related to rice tillering force, related biological material and application thereof | |
Stirn et al. | Genetically modified plants | |
KR100718549B1 (en) | Vector comprising hrp gene from erwinia pyrifoliae transgenic agrobacterium tumefaciens and pathogenic resistant transgenic plant using the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 00805132.1 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2365099 Country of ref document: CA Ref document number: 2365099 Country of ref document: CA Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2000 605754 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09936885 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 32679/00 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000910462 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2000910462 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWG | Wipo information: grant in national office |
Ref document number: 32679/00 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000910462 Country of ref document: EP |