ZA200103234B - Utilization of an antifungal protein from steptomyces tendae against plant pathogenic fungi. - Google Patents
Utilization of an antifungal protein from steptomyces tendae against plant pathogenic fungi. Download PDFInfo
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- ZA200103234B ZA200103234B ZA200103234A ZA200103234A ZA200103234B ZA 200103234 B ZA200103234 B ZA 200103234B ZA 200103234 A ZA200103234 A ZA 200103234A ZA 200103234 A ZA200103234 A ZA 200103234A ZA 200103234 B ZA200103234 B ZA 200103234B
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- South Africa
- Prior art keywords
- afp
- antifungal protein
- plant pathogenic
- pathogenic fungi
- plant
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- 101710083587 Antifungal protein Proteins 0.000 title claims abstract description 44
- 244000000004 fungal plant pathogen Species 0.000 title claims abstract description 12
- 241000187179 Streptomyces tendae Species 0.000 claims abstract description 12
- 241000235349 Ascomycota Species 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims description 29
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 241001465180 Botrytis Species 0.000 claims description 9
- 230000009261 transgenic effect Effects 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 235000006008 Brassica napus var napus Nutrition 0.000 claims description 4
- 244000061456 Solanum tuberosum Species 0.000 claims description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000013339 cereals Nutrition 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 235000021374 legumes Nutrition 0.000 claims description 3
- 235000009973 maize Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 235000013311 vegetables Nutrition 0.000 claims description 3
- 241000219194 Arabidopsis Species 0.000 claims description 2
- 235000007319 Avena orientalis Nutrition 0.000 claims description 2
- 244000075850 Avena orientalis Species 0.000 claims description 2
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 claims description 2
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 claims description 2
- 240000002791 Brassica napus Species 0.000 claims description 2
- 240000000385 Brassica napus var. napus Species 0.000 claims description 2
- 240000007124 Brassica oleracea Species 0.000 claims description 2
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 2
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims description 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 2
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 claims description 2
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- 229920000742 Cotton Polymers 0.000 claims description 2
- 244000241257 Cucumis melo Species 0.000 claims description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 2
- 240000009088 Fragaria x ananassa Species 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 240000008415 Lactuca sativa Species 0.000 claims description 2
- 235000003228 Lactuca sativa Nutrition 0.000 claims description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 2
- 240000008790 Musa x paradisiaca Species 0.000 claims description 2
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 2
- 244000061176 Nicotiana tabacum Species 0.000 claims description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- 240000004713 Pisum sativum Species 0.000 claims description 2
- 235000010582 Pisum sativum Nutrition 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims description 2
- 235000021536 Sugar beet Nutrition 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 244000098338 Triticum aestivum Species 0.000 claims description 2
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 2
- 239000011814 protection agent Substances 0.000 claims description 2
- 235000021012 strawberries Nutrition 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 5
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- 230000003115 biocidal effect Effects 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
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- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- LWTDZKXXJRRKDG-KXBFYZLASA-N (-)-phaseollin Chemical compound C1OC2=CC(O)=CC=C2[C@H]2[C@@H]1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-KXBFYZLASA-N 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- 108010022172 Chitinases Proteins 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 description 2
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 2
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- 108020004414 DNA Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 241000908213 Metarhizium carneum Species 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 241001134446 Niveas Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 241001149509 Penicillium vulpinum Species 0.000 description 1
- 101710163504 Phaseolin Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101000611441 Solanum lycopersicum Pathogenesis-related leaf protein 6 Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- RIOXQFHNBCKOKP-UHFFFAOYSA-N benomyl Chemical compound C1=CC=C2N(C(=O)NCCCC)C(NC(=O)OC)=NC2=C1 RIOXQFHNBCKOKP-UHFFFAOYSA-N 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- LWTDZKXXJRRKDG-UHFFFAOYSA-N phaseollin Natural products C1OC2=CC(O)=CC=C2C2C1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-UHFFFAOYSA-N 0.000 description 1
- 108010004568 plant pathogenesis-related proteins Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000001974 tryptic soy broth Substances 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/14—Plant cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Abstract
The invention relates to the use of an antifungal protein (AFP) having a molecular weight of approximately 10kDa from Streptomyces tendae against plant pathogenic fungi from the Ascomycetes family.
Description
Utilization of an antifungal protein from Streptomyces tendae against plant pathogenic fungi
The present invention relates to the use of an antifungal protein (AFP) having a molecular weight of approx. 10 kDa from Streptomyces tendae against plant pathogenic fungi from the Ascomycetes family.
Antifungal proteins have been described for some time, some of these proteins also being termed PR proteins (plant pathogenesis-related proteins) in the case of plants (Bol, J.F. & Linthorst, H.J.M. (1990), Annu.
Rev. Phytopathol., 28, 113). These proteins are formed by plants under stress conditions, for example in the case of viral or fungal infection, inducing an active defense mechanism of the plant which is termed “induced resistance” (Lindhorst, H.J.M., (1991) Cri. Rev. Plant
Sci., 10(2), 123). Some of these proteins have, for example, a chitinase activity or f-1,3-glucanase activity.
Proteins with an antifungal activity have already been detected from microorganisms too, some of these proteins also showing chitinase activity or f-1,3- glucanase activity. Other proteins, in contrast, act via an interaction of the fungal cell wall.
For example, an antifungal protein (AFP) having a size of approx. 10 kDa which is active against the fungal species Paecilomyces varriotii, Byssoclamis nivea,
Pencillium puberulum and Eupenicillium terrum has been isolated from Streptomyces tendae. However, the action was restricted to these species. Other species such as, for example, Paecilomyces carneus, Paecilomyces lilacenus, Penicillium chrysogenum or Penicillium claviforme are not inhibited. In addition, the use of
AFP is not justified commercially either since the fungal species mentioned are not plant pathogenic.
The object of the present invention was therefore to find an antifungal protein which 1s active against plant pathogenic fungi.
Surprisingly, it has now been found that AFP from
Streptomyces tendae is active against plant pathogenic fungi from the Ascomycetes family.
The subject-matter of present the invention is therefore the use of an antifungal protein (AFP) with a molecular weight of approx. 10 kDa from Streptomyces tendae against plant pathogenic fungi from the
Ascomycetes family, in particular against Botrytis cinera.
The antifungal protein mentioned contains a typical amino-terminal signal sequence, i.e. a hydrophilic
N-terminus is followed by a hydrophobic transmembrane region, which cause extracellular secretion. Moreover, two forms are known, namely a shorter form with a molecular weight of approx. 9 860 Da and a longer form with a molecular weight of approx. 10 300 Da.
Since AFP is secreted by Streptomyces tendae into the culture medium, it can be isolated for example directly from the Streptomyces tendae culture filtrate, following methods known to the skilled worker. As an alternative, AFP can be produced by genetic engineering, the gene isolated preferably being cloned into a so-called multicopy vector, for example plJ702 (Hopwood, D.A. et al. (1985) Genetic Manipulation of
Streptomyces. A Laboratory Manual. Norwich, U.K.) and using this construction to transform a suitable strain, for example the non-nikkomycin-producing strain
Streptomyces tendae NPY. An example of an AFP-encoding nucleic acid is shown in SEQ ID No. 1. When this preferred transformant was grown, for example, for 7 days in 200 ml of nutrient solution, approx. 6 mg of
AFP were obtained, which corresponds to 30 mg of AFP/1.
The production of AFP by genetic engineering via a preferably controlled fermentation, is therefore especially preferred.
For the use in accordance with the invention, AFP can be applied directly for example in the form of a formulation together with preferably at least one additional auxiliary. To this end, for example, plants attacked by fungi or endangered by fungal attack are sprayed with the formulation described. The AFP- concentration of the formula is generally from approx. 10 ug/ml up to approx. 500 mg/ml. Suitable auxiliaries are, for example, protease inhibitors, stabilizers such as glycerol, sucrose, salts, solvents or generally known substances from the field of crop protection.
So-called transgenic plants which are capable of producing AFP themselves are another possibility of providing protection against fungal infection.
Another embodiment is therefore the use according to the invention of AFP, the AFP being formed in a transgenic plant. The transgenic plant is, preferably, genetically engineered maize, cotton, potato, banana,
Arabidopsis, casava, tobacco, oilseed rape (canola), potato, sugar beet, cereals such as, for example, wheat, barley or oats, strawberries, vegetables such as, for example, cabbage, legumes such as, for example, peas or beans, tomato, lettuce or melon.
To generate a transgenic plant, a nucleic acid encoding
AFP, for example in the form of naked DNA, viral DNA or
RNA, or in the form of plasmid DNA, is introduced into a plant cell. An example of an AFP-encoding nucleic acid is shown in SEQ ID No. 1. However, the present invention also covers those nucleic acids which differ from the nucleic acid sequence of SEQ ID No. 1 owing to the degeneracy of the genetic code, but which encodes the same AFP amino acid sequence. Moreover, the invention covers those mutants or variants of the nucleic acid of SEQ ID No. 1 which encode an AFP protein which is active against plant pathogenic fungi of the Ascomycetes family, in particular against
Botrytis cinera. These include, for example, fusion proteins of the AFP protein with other foreign proteins or an AFP protein with an N-terminally deleted methionine. Other examples of variants are nucleic acids which hybridize on the stringent conditions with the nucleic acid of SEQ ID No. 1. The stringent hybridization conditions can be determined for example by Sambrook, J. et al. in Molecular Cloning, A
Laboratory Manual, 2nd Edition, Cold Spring Habour
Laboratory Press, 1989. Transgenic plants are subsequently regenerated from the transformed plant cells. Preferably, an AFP-encoding nucleic acid can be introduced into the plant cell by means of recombinant agrobacteria, by electroporation, by bombardment with microparticles and/or by means of polyethylene glycol.
The infection with recombinant Agrobacterium tumefaciens bacteria in plant cells of dicots is described, for example, by Klee, H. et al. (19897) Annu.
Rev. Plant Physiol. 38, 467 or EP-B2-0122791). The infection with recombinant Agrobacterium tumefaciens bacteria in plant cells of monocots is described, for example, by Ishida, Y. et al. (1996) Nature Biotech. 14, 745 with reference to maize (Zea mays L.). The agrobacteria used for this purpose have a T-DNA into which the AFP-expressing gene and, 1f appropriate, a suitable promoter, for example a plant phaseolin promoter (see, for example, EP-B2-0122791) or a viral 355 promoter (see, for example, Ishida, Y. et al.
(1996), above) had been inserted. The T-DNA can be transferred into plant cells for example by coculturing the recombinant agrobacteria together with immature embryos of the plant in question (see, for example,
Ishida, Y. et al. (1996), above). To select the transformed plant cells, it is preferred to use T-DNA constructs which are capable of expressing, in the plant cell, a resistance gene from the AFP gene to be expressed. A suitable resistance gene is, for example, the gene encoding phosphinothricin acetyltransferase (see, for example, Ishida, Y. et al. (1996), above).
Thus, successfully transformed plant cells can be selected for example by means of the corresponding antibiotic phosphinothricin. The regeneration of a plant from transformed plant cells is generally known (see, for example, Sagi et al. (1995), Nature Biotech, 13, 481-485, Ishida, Y. et al. (19%¢), above, or
Schopke et al. (1996), Nature Biotech, 14, 731-735.
In addition to the use of Agrobacterium tumefaciens as transformation means, other transformation methods are known and suitable, such as, for example, electro- poration, the bombardment with microparticles or the use of polyethylene glycol (see, for example, Potrykos, (1991) Annu. Rev. Plant Physiol. Mol. Biol., 42, 205;
Estruch, J.J. et al. (1997), Nature Biotech., 15, 137- 141 or Dingermann, T. (1995) BIOforum, 18, 252). For example, casava (Schopke et al. (1996), above) or banana (Sagi, et al. (1995), above) have already been transformed by bombarding plant cells with DNA-coated particles. In this method too, the AFP gene to be expressed 1s cloned into a suitable vector which preferably has a resistance gene allowing the subsequent selection of the transformed plant cells.
The recombinant vector is preferably applied to tungsten particles with which, for example, suspensions of embryogenic cells are bombarded (see, for example,
Sagi, et al. (1995), above). The «cell suspensions treated thus are subsequently selected for transformed plant cells using a suitable antibiotic, for example phosphinothricin, and a transgenic plant is regenerated therefrom, for example as already described above.
A considerable advantage of the present invention is that AFP is active against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis
Cinera, which have already developed a resistance in particular to one or more crop protection agents.
The examples which follow and the figure are intended to describe the invention in greater detail without imposing any limitation:
SEQ ID No. 1 shows a nucleic acid sequence encoding an antifungal (223) protein (AFP) from
Streptomyces tendae.
Example 1:
To prepare test plates, 50 pl of a spore suspension of
Botrytis cinerae were added to 100 ml of test agar (20 g/1 malt extract, 10 g/1 glucose, 2 g/l yeast extract, 0.5 ammonium sulfate, 15 g/l agar, pH 6.0). 25 ml of this were poured into Petri dishes. The final spore concentration was 10° spores/ml. Sterile anti- biotic test disks (Schleicher und Schuell, Dassel, Frg) 0.5 cm in diameter were subsequently applied to a test plate. Then, 5, 10, 15 and 20 pul of the AFP solution (100 mg/ml lyophilized culture filtrate of S. tendae- transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.7 cm for 5 ul of AFP solution, 1 cm diameter for 10 ul, 1.3 cm for 15 nl of solution and 1.9 cm for 20 ul.
Example 2:
To prepare test plates, 50 pl of a spore suspension of
Botrytis cinerae ZF 3629 (resistant to BCM (Benlat,
Benomyl™, Hoechst Schering AgrEvo GmbH, Frankfurt, carbendazem) were added to 100 ml of test agar (20 g/1 malt extract, 10 g/l glucose, 2 g/l yeast extract, 0.5 ammonium sulfate, 15 g/1 agar, pH 6.0). 25 ml of this were poured into Petri dishes. The final spore concentration was 10° spores/ml. Sterile antibiotic test disks (Schleicher und Schuell, Dassel, Frg) 0.5 cm in diameter were subsequently applied to a test plate.
Then, 5, 10, 15 and 20 ml of the AFP solution (100 mg/ml lyophilized culture filtrate of S. tendae- transformants) were subsequently pipetted onto the test disks and the test plate was incubated for 4 days at 30°C. The diameter of the inhibitory zone was 0.9 cm for 5 ul of AFP solution, 1.3 cm diameter for 10 ul, 1.9 cm for 15 nl of solution and 2.2 cm for 20 ul.
Example 3 - Improved production by fermentation:
In a flask equipped with a hose, 100 ml of the preculture medium (103 g/l1 sucrose, 20 g Tryptic Soy
Broth (Oxoid, Wesel), 10 g/1 MgCl, and 10 g/l yeast extract) were inoculated with an agar section of strain
S. tendae. The culture was grown for 5 days at 28°C at 200 rpm on an orbital shaker. 10 1 of the production medium (103 g/l sucrose, 20 g
Tryptic Soy Broth (Oxoid, Wesel), 10 g/1 MgCl,, 10 g/1 yeast extract, 100 pl desmophen, pH 7.5) were inoculated with 100 ml of the above-described preculture.
The fermenter employed was a Biostat E (Braun,
Melsungen). Fermentation was carried out for 4 days at 28°C, 700 rpm and 0.5 vvm of air. Thereupon, fermentation was stopped, and the culture supernatant was harvested by centrifugation at 3000 rpm and 4°C.
The batch was subsequently dried by lyophilization. It was possible to employ the powder directly. If 15 pul of the 1lyophilizate (100 mg/ml) were employed against
Botrytis cinerae in an inhibitory-zone test, an inhibitory zone of 1.7 cm and, against Botrytis cinerae
ZF 3629, an inhibitory zone of 2.3 cm were detected.
Claims (10)
1. The use of an antifungal protein (AFP) having a molecular weight of approx. 10 kDA from Streptomyces tendae against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis cinera.
2. The use as claimed in claim 1, wherein AFP is used in the form of a formulation together with preferably at least one auxiliary.
3. The use as claimed in claim 1, wherein AFP is formed in a transgenic plant.
4, The use as claimed in claim 3, wherein the transgenic plant 1s selected from among maize, cotton, potato, banana, Arabidopsis, casava, tobacco, oilseed rape (canola), potato, sugar beet, cereals, strawberries, vegetables, legumes, tomato, lettuce or melon.
5. The use as claimed in claim 4, wherein cereals are selected from amongst wheat, barley or oats, the vegetable used is cabbage, or the legumes are selected from amongst peas or beans.
6. The use as claimed in any of claims 3 - 5, wherein the transgenic plant comprises a nucleic acid as claimed in SEQ ID No. 1, SEQ ID No. 1 being part of the claim.
7. The use as claimed in any of claims 1 - 6, wherein the plant pathogenic fungus stated is resistant to one or more crop protection agents.
-9/A
8. The use as claimed in any one of the preceding claims, substantially as herein described and exemplified.
9. An antifungal protein (AFP) having a molecular weight of approx. 10 kDA from Streptomyces tendae, for use against plant pathogenic fungi from the Ascomycetes family, in particular against Botrytis cinera.
10. An antifungal protein (AFP) as claimed in claim 9, substantially as herein described and exemplified. AMENDED SHEET
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19848517A DE19848517A1 (en) | 1998-10-21 | 1998-10-21 | Controlling phytopathogenic fungi, useful particularly against Botrytis cinerea resistant to known fungicides, by treatment with peptide from Streptomyces tendae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200103234B true ZA200103234B (en) | 2002-11-07 |
Family
ID=7885179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200103234A ZA200103234B (en) | 1998-10-21 | 2001-04-20 | Utilization of an antifungal protein from steptomyces tendae against plant pathogenic fungi. |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP1123004B1 (en) |
| JP (1) | JP2002527456A (en) |
| KR (1) | KR20010083915A (en) |
| CN (1) | CN1324214A (en) |
| AT (1) | ATE217757T1 (en) |
| AU (1) | AU748455B2 (en) |
| CA (1) | CA2347706A1 (en) |
| DE (2) | DE19848517A1 (en) |
| ID (1) | ID29012A (en) |
| IL (1) | IL142597A0 (en) |
| MX (1) | MXPA01004057A (en) |
| NZ (1) | NZ511213A (en) |
| WO (1) | WO2000022932A1 (en) |
| ZA (1) | ZA200103234B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100883190B1 (en) * | 2007-08-09 | 2009-02-12 | 경상대학교산학협력단 | Antibiotics preparation containing heat-stable protein having antimicrobial activity |
| CN103708960B (en) * | 2013-12-24 | 2016-06-01 | 安徽师范大学 | A kind of liquid composite microbic bacterial fertilizer and production method and purposes |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2537028A1 (en) * | 1975-08-20 | 1977-03-10 | Bayer Ag | ANTIBIOTICUM, A METHOD FOR ITS MANUFACTURING AND ITS USE AS A PLANT PROTECTION PRODUCT |
| US4158608A (en) * | 1975-08-20 | 1979-06-19 | Bayer Aktiengesellschaft | Fungicidally active antibiotic from Streptomyces tendae Ettlinger |
-
1998
- 1998-10-21 DE DE19848517A patent/DE19848517A1/en not_active Withdrawn
-
1999
- 1999-10-05 AU AU64679/99A patent/AU748455B2/en not_active Ceased
- 1999-10-05 JP JP2000576718A patent/JP2002527456A/en active Pending
- 1999-10-05 ID IDW20010845A patent/ID29012A/en unknown
- 1999-10-05 DE DE59901521T patent/DE59901521D1/en not_active Expired - Fee Related
- 1999-10-05 KR KR1020017004884A patent/KR20010083915A/en not_active Application Discontinuation
- 1999-10-05 EP EP99952494A patent/EP1123004B1/en not_active Expired - Lifetime
- 1999-10-05 MX MXPA01004057A patent/MXPA01004057A/en not_active Application Discontinuation
- 1999-10-05 CN CN99812433A patent/CN1324214A/en active Pending
- 1999-10-05 WO PCT/EP1999/007364 patent/WO2000022932A1/en not_active Application Discontinuation
- 1999-10-05 CA CA002347706A patent/CA2347706A1/en not_active Abandoned
- 1999-10-05 AT AT99952494T patent/ATE217757T1/en not_active IP Right Cessation
- 1999-10-05 NZ NZ511213A patent/NZ511213A/en unknown
- 1999-10-05 IL IL14259799A patent/IL142597A0/en unknown
-
2001
- 2001-04-20 ZA ZA200103234A patent/ZA200103234B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ATE217757T1 (en) | 2002-06-15 |
| DE19848517A1 (en) | 2000-04-27 |
| MXPA01004057A (en) | 2003-03-10 |
| CA2347706A1 (en) | 2000-04-27 |
| AU6467999A (en) | 2000-05-08 |
| JP2002527456A (en) | 2002-08-27 |
| IL142597A0 (en) | 2002-03-10 |
| KR20010083915A (en) | 2001-09-03 |
| EP1123004A1 (en) | 2001-08-16 |
| DE59901521D1 (en) | 2002-06-27 |
| AU748455B2 (en) | 2002-06-06 |
| WO2000022932A1 (en) | 2000-04-27 |
| CN1324214A (en) | 2001-11-28 |
| NZ511213A (en) | 2003-03-28 |
| ID29012A (en) | 2001-07-26 |
| EP1123004B1 (en) | 2002-05-22 |
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