WO2008138129A1 - Thuricine 17 permettant de favoriser la croissance végétale et la résistance des plantes à une maladie et plantes transgéniques - Google Patents

Thuricine 17 permettant de favoriser la croissance végétale et la résistance des plantes à une maladie et plantes transgéniques Download PDF

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Publication number
WO2008138129A1
WO2008138129A1 PCT/CA2008/000921 CA2008000921W WO2008138129A1 WO 2008138129 A1 WO2008138129 A1 WO 2008138129A1 CA 2008000921 W CA2008000921 W CA 2008000921W WO 2008138129 A1 WO2008138129 A1 WO 2008138129A1
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Prior art keywords
polypeptide
plant
plant growth
bacteriocin
activity
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PCT/CA2008/000921
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English (en)
Inventor
Donald Smith
Kyung Dong Lee
Elizabeth Gray
Alfred Souleimanov
Xiaomin Zhou
Trevor Charles
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The Royal Institution For The Advancement Of Learning/Mcgill University
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Publication of WO2008138129A1 publication Critical patent/WO2008138129A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • 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
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Definitions

  • This invention relates to purified polypeptides that are bacteriocins and that possess plant growth and/or disease resistance promoting activity, and their use in e.g. promoting plant growth, promoting disease resistance in plants, and as bactericidal or bacteristatic agents.
  • the invention further relates to nucleic acid molecules encoding such polypeptides, and transgenic plants comprising such nucleic acid molecules.
  • Bacteriocins are proteins produced by prokaryotes that are bactericidal and/or bacteristatic against organisms related to the producer strain, but that do not act against the producer strain itself.
  • LCOs lipo-chitooligosaccharides
  • NOD nodulation
  • bacteriocins may be used to promote plant growth and/or promote disease resistance in plants.
  • the invention provides a method for promoting plant growth and/or disease resistance comprising applying a purified polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity to a plant or plant seed, or in the growing environment thereof.
  • the invention provides a purified polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity, said polypeptide being selected from the group consisting of:
  • polypeptide of (a) possessing the bacteriocin and plant growth and/or disease resistance promoting activities of the polypeptide of (a), and which possesses at least 70% sequence identity to SEQ ID NO: 1 over its entire length;
  • polypeptide which is a fragment of the polypeptide of (a) or (b), said fragment possessing the bacteriocin and plant growth and/or disease resistance promoting activities of the polypeptide of (a).
  • the invention provides a composition comprising a purified polypeptide as described above, and a carrier or diluent.
  • the invention provides an isolated polynucleotide encoding a polypeptide as described above, or the complement thereto.
  • the invention provides a vector comprising a polynucleotide or host cell as described above.
  • the invention provides a plant comprising a polynucleotide as described above.
  • the plant may express the polypeptide encoded by the polynucleotide, obviating the need to apply the polypeptide to the plant in order to obtain the benefits of the plant growth and/or disease resistance promoting activities of the polypeptide.
  • the invention provides a method for producing a polypeptide comprising culturing the host cell as described above under conditions sufficient for expression of the polypeptide encoded by said polynucleotide, and recovering said polypeptide.
  • the invention provides a plant growth and/or disease resistance promoting composition
  • a plant growth and/or disease resistance promoting composition comprising a purified polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity, and a carrier or diluent.
  • the invention provides a plant seed treated with the plant growth and/or disease resistance promoting composition as described above.
  • the invention provides a kit comprising a plant growth and/or disease resistance promoting composition as described above and instructions for use.
  • the invention provides a method for obtaining a polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity comprising:
  • the invention provides a method for obtaining a polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity, comprising:
  • FIG. IA-C illustrate HPLC analysis of the three samples: (A) PPBP, Partially Purified Bacterial Peptide, prepared by HPLC purification; (B) medium control, exposed to the exact same conditions as PPBP, including butanol extraction, HPLC purification; (C) CFS, Cell Free Supernatant, prepared by differential centrifugation of the bacterial culture.
  • A PPBP, Partially Purified Bacterial Peptide, prepared by HPLC purification
  • B medium control, exposed to the exact same conditions as PPBP, including butanol extraction, HPLC purification
  • C CFS, Cell Free Supernatant, prepared by differential centrifugation of the bacterial culture.
  • FIG. 2A-C illustrate the bactericidal and/or bacteristatic effects on Bacillus thuringiensis NEB 17 (A), Bacillus cereus ATCC 14579 (B) and Bacillus thuringiensis ssp thuringiensis BtI 627 (C) exposed to 0 ⁇ L (circles), 100 ⁇ L (closed squares), 300 ⁇ L (triangles), and 600 ⁇ L (open squares) of PPBP (0.066 ⁇ g ⁇ l "1 ).
  • FIG. 3 illustrates a SDS-PAGE analysis on PPBP and the CFS, as well as direct detection of PPBP and CFS.
  • 20 ⁇ L of PPBP and CFS were loaded into wells, media exposed to the same conditions as for the PPBP and CFS served as controls.
  • 35 ⁇ L of PPBP and CFS were loaded into wells, and the respective media control was also used.
  • the gel overlaid with a soft agar King's medium, was inoculated with the indicator strain, Bacillus thuringiensis ssp. thuringiensis Btl627.
  • Lane 1 low molecular weight marker (MKR); Lane 2: loading dye control (LD), Lane 3: CFS; Lane 4: PPBP; Lane 5: centrifuged media control (CM ctl); Lane 6: purified media control (PM ctl); Lane 7: PPBP for direct detection; Lane 8: CFS for direct detection; Lane 9: purified media control (PM ctl) and Lane 10: centrifuged media control (CM ctl).
  • MKR low molecular weight marker
  • Lane 2 loading dye control
  • Lane 3 CFS
  • Lane 4 PPBP
  • Lane 5 centrifuged media control
  • CM ctl purified media control
  • Lane 7 PPBP for direct detection
  • Lane 8 CFS for direct detection
  • Lane 9 purified media control
  • CM ctl centrifuged media control
  • FIG. 4 illustrates MALDI-QTOF (Matrix Assisted Laser Desorption Ionization - Quadrapole Time of Flight) mass spectrometry analysis of the PPBP, partially purified via reversed phase HPLC, and collected in 60% acetylnitrile.
  • MALDI-QTOF Microx Assisted Laser Desorption Ionization - Quadrapole Time of Flight
  • FIG. 5 illustrates MALDI-QTOF mass spectrometry of partially purified thuricin 17 (PPT 17).
  • Thuricin 17 was partially purified via reverse phase HPLC, and collected in 60% acetonitrile. Sequence analysis via Edman degradation was determined and the presence of cysteines was detected via ms/ms fragment analysis of the parent ion. Analysis was conducted on two separate biological replicates that were grown and extracted separately; similar results were obtained from each.
  • FIG. 6A-C illustrate a visual representation of inhibition of thuricin 17 as it relates to its production.
  • FIG. 7 illustrates thuricin 17 production by Bacillus thuringiensis NEB 17 over time. Sample aliquots were removed at hourly intervals and the O.D. 600 n m recorded. In parallel, aliquots were diluted to determine the viable cell count (CFU). Production of thuricin 17 was quantified into activity units (AU) by preparing a series of two-fold dilutions and testing against the indicator strain B. thuringiensis ssp. thuringiensis Bt 1627.
  • AU activity units
  • FIG. 7 A depicts the thuricin 17 genetic region. Three direct repeats, each encoding a copy of the thuricin 17 encoding gene, lie upstream of an albA homologue.
  • the thuricin 17 encoding repeat sequences are identified by numbers 1, 2 and 3.
  • the NEB-R primer binding sites are identified as "a”.
  • the NEB-F2 primer binding site primer binding sites are identified as "b”.
  • the thurl7dn primer binding site is identified as "c”.
  • the thurl7albAup primer binding site is identified as "d”.
  • Fig. 7B provides the 625 bp genomic DNA sequence encoding the thuricin 17 repeat sequences depicted in Fig. 7.1.
  • Fig. 7C depicts the predicted amino acid sequence of thuricin 17.
  • FIG. 8A-C illustrate HPLC analysis of (A) the crude extract from Bacillus thuringiensis NEB 17; (B) partially purified thuricin 17, and (C) King's Medium B without bacteria, as a control.
  • FIG. 9 illustrates the bacteriocin effects of thuricin 17.
  • Controls were the producer strain, Bacillus thuringiensis NEB 17 (A), as well as purified media without thuricin 17 tested on B. cereus ATCC 14579 (B). Strains showing inhibition are B. cereus ATCC 14579 (C), and Brevibacillus brevis ATCC 8246 (D).
  • FIG. lOA-C illustrates the characterization of the plant biological activity of thuricin 17 on soybean (Glycine max L.) germination (%).
  • FIG. 1 IA-D illustrates HPLC chromatograms of the entire extract of Bacillus thuringiensis NEB 17 before the purification (A), and compounds eluted with 35% acetonitrile (B), 43% acetonitrile (C) and 100% acetonitrile (D).
  • FIG. 12 illustrates a schematic diagram of planting methodology for corn seeds supplied with varied concentrations of thuricin 17 solutions.
  • FIG. 15A-B illustrate soybean leaf area ( Figure 15A) and shoot dry weights (Figure 15B) at 14 days after treatment with the bacteriocin extracted from Bacillus cereus UW85 (cerecin 85) at 10 "9 M, 10 "10 M, or 10 "11 M.
  • FIG. 16A-B illustrate changes in phenylalanine ammonia lyase (PAL) ( Figure 16A) and tyrosine ammonia lyase (TAL) ( Figure 16B) activities in soybean leaves after treatment with chitin hexamer (0.5 ml (100 ⁇ mol/L)) and thuricin 17 (1 x 10 8 M).
  • Control open circles
  • chitin hexamer [(GlcNAc) ⁇ ] circles
  • thuricin 17 triangles
  • chitin hexamer and thuricin 17 squares.
  • FIG. 17 illustrates changes of total phenolics in soybean leaves after treatment with chitin hexamer and thuricin 17.
  • TO control; Tl: chitin hexamer [(GIcNAc) 6 ], T2: T17; T3: chitin hexamer and thuricin 17.
  • FIG. 18A-B illustrate changes of peroxidase ( Figure 18A) and superoxide dismutase (Figure 18B) activities in soybean leaves after treatment with chitin hexamer and thuricin 17.
  • TO control; Tl: chitin hexamer [(GIcNAc) 6 ]; T2: thuricin 17; T3: chitin hexamer and thuricin 17.
  • FIG. 19A-C illustrate active staining of peroxidase (POD) ( Figure 19A), catalase (CAT) ( Figure 19B) and superoxide dismutase (SOD) ( Figure 19C) in soybean leaves after treatment with chitin hexamer and thuricn 17 ((a) PAGE; (b) inactivated by H 2 O 2 ; and (c) inactivated by KCN).
  • TO control
  • Tl chitin hexamer [(GIcNAc) 6 ]
  • T2 thuricin 17
  • T3 chitin hexamer and thuricin 17.
  • the invention provides a method for promoting plant growth and/or disease resistance comprising applying a purified polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity to a plant or plant seed, or in the growing environment thereof.
  • the polypeptides used in the methods of the invention exhibit at least one plant growth and/or disease resistance promoting property and also have at least one property of a bacteriocin.
  • the polypeptides demonstrate at least one bactericidal or bacteristatic activity against a related or unrelated bacterial strain, preferably a related strain.
  • polypeptide encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100 or more amino acids) or post-translational modification (e.g., glycosylation or phosphorylation) or the presence of e.g. one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptoids, peptidomimetics, etc.
  • amino acids either D- or L-amino acids
  • post-translational modification e.g., glycosylation or phosphorylation
  • non-amino acyl groups for example, sugar, lipid, etc.
  • bacteriocin means a protein or peptide produced by a prokaryote (typically a Gram-negative or Gram-positive bacterium) and that is bactericidal and/or bacteristatic against organisms related to the producer strain, but that does not act against the producer strain itself. Many, but not all, bacteriocins are of low- molecular weight, in the range of about 100 to about 10,000 Daltons. Bacteriocins are known to inhibit growth of closely related microorganisms thereby eliminating or significantly reducing competition for available nutrients (Jack et al. Microbiol. Rev., 59:171-200, 1995). Bacteriocins have also been implicated as playing a role as antibiotics against pathogenic bacteria and as natural food preservatives.
  • plant growth promoting activity encompasses a wide range of improved plant properties, including, without limitation, improved nodulation (e.g. increased number of nodules), nitrogen fixation (e.g. increased nitrogen concentration as measured by mg g "1 dry weight of plant material), increased leaf area, increased seed germination, increased leaf greenness (e.g. as measured by SPAD), increased photosynthesis ( ⁇ mol cm “2 s "1 ), or an increase in accumulated dry-weight of the plant.
  • improved nodulation e.g. increased number of nodules
  • nitrogen fixation e.g. increased nitrogen concentration as measured by mg g "1 dry weight of plant material
  • increased leaf area e.g. as measured by SPAD
  • increased leaf greenness e.g. as measured by SPAD
  • increased photosynthesis ⁇ mol cm “2 s "1 )
  • plant disease resistance promoting activity encompasses, without limitation, increased resistance to pathogen attack or increased production of one or more secondary metabolites that function to improve the resistance of a plant to pathogen attack, as discussed herein.
  • Polypeptides useful in practicing the methods of the invention can be obtained in a number of ways.
  • any polypeptide of interest may be screened, either sequentially in either order, or simultaneously, for a plant growth and/or disease resistance promoting activity and for activity as a bacteriocin.
  • the polypeptide will be produced by a bacterial strain known to be a plant growth promoting strain such as a PGPR.
  • the polypeptide is obtained from a bacterial strain and known to be a producer of bacteriocin.
  • a zone of inhibition assay such as an agar disc diffusion assay may be used to test the polypeptides of interest or bactericidal or bacteristatic activity against various indicator strains.
  • a polypeptide of interest may be applied by leaf spray or root irrigation to test plants, such as soybean plants. Plants may then be grown under controlled environment conditions (growth chamber or greenhouse) for e.g. about 40 days. At harvest, data may be collected concerning e.g. plant height, leaf greenness, leaf area, nodule number, nodule dry weight, shoot and dry root weight or length, nitrogen content and photosynthesis and compared to controls.
  • Assessment of plant disease resistance promoting activity of polypeptides may also be accomplished by known methods, such as by detecting or measuring a reduction in pathogen infestation of a plant, or indirectly by detecting or measuring increased production of one or more secondary metabolites that function to improve the resistance of a plant to pathogen attack.
  • Exemplary secondary metabolites include lignification- related enzymes such as phenylalanine ammonia lyase (PAL), and tyrosine ammonia lyase (TAL), antioxidative enzymes such as peroxidase (POD), catalase (CAT), and superoxidase dismutase (SOD), and total phenolic compounds.
  • PAL phenylalanine ammonia lyase
  • TAL tyrosine ammonia lyase
  • POD peroxidase
  • CAT catalase
  • SOD superoxidase dismutase
  • total phenolic compounds e.g. PAL, TAL, POD
  • An increase or improvement in plant growth or disease resistance means a statistically significant increase or improvement in the measured criterion of plant growth or disease resistance in a plant treated with a polypeptide according to the invention relative to an untreated control plant.
  • Bacteria that are known to produce bacteriocins include, but are not limited to, Bacillus, Pseudomonas, Rhizobium, Braydyrhizobium and Lactoccus species.
  • bacteriocins Depending on their structure, mode of action and chemical properties, four distinct classes of bacteriocins are recognized (Klaenhammer 1993). Current classifications of bacteriocins include Class I- type A lantibiotics, Class I-type B lantibiotics, Class Ha, Class lib, Class Hc and Class III (Eijsink et al. 2002; Chen and Hoover 2003). Nisin, for example, is a widely characterized bacteriocin produced from the lactic acid bacterium, Lactococcus lactis, and has been accepted by the World Health Organization (WHO) as a food biopreservative (Hansen 1994). Current applications of bacteriocins are as food preservatives while less research has been conducted on the agricultural applications of bacteriocins.
  • WHO World Health Organization
  • B. thuringiensis HD2 synthesizes thuricin HD2, 950 kDa (Favret and Yousten 1989). Thuricin 7, 11.6 kDa, is produced by a soil isolate, B. thuringiensis BMGl.7 (Cherif et al. 2001).
  • B. thuringiensis ssp. tochigiensis HD868 produces tochicin, 10.5 kDa, effective against over 20 B. thuringiensis members (Paik et al. 1997).
  • thuringiensis B439 produces two antibiotic peptides, thuricin 439A and 439B (Ahern et al. 2003), both ⁇ 3 kDa, differing by 100 Da. Torkar and Matijasic (2003) report several bacteriocins, l-8kDa, from B. cereus milk isolates and B. cereus ATCC 14579 produces a BLIS (bacteriocin like inhibitory substance) with a molecular weight of 3.4kDa (Risoen et al. 2004). B. cereus BC7 produces cerein 7, 3.94 kDa (Oscariz et al.
  • Bacteriocins such as those described above may be tested for plant growth and/or disease resistance promoting activity as described herein.
  • the polypeptides of the invention may also be obtained from bacterial species that are known to have plant growth promoting activity or to produce compounds that promote plant growth, but that are not necessarily known to produce bacteriocins. These include, for example, plant growth promoting rhizobacteria (PGPR). PGPR increase plant growth and include bacteria in the soil near plant roots, on the surface of plant root systems, in spaces between root cells or inside specialized cells of root nodules (Kloepper et al., 1978).
  • PGPR plant growth promoting rhizobacteria
  • PGPRs are known to produce bacteriocins, and bacteriocin production by PGPR members is illustrated by Pseudomonas ssp. (Parret and De Mot 2002) and bacteriocins denoted as "rhizobiocins" from rhizobia (Schwinghamer 1975).
  • Rhizobium leguminosarium bv. viciae strain 306 produces the bacteriocin, pRle306c, with a type I secretion system required for export (Venter et al. 2000).
  • Wilson et al. (1998) found a R. leguminosarum isolate that produces a virulent bacteriocin lethal to 68% of soil isolate strains. The bacteriocin may have facilitated its persistence in the soil (Wilson et al. 1998).
  • PGPR can be classified as extracellular PGPR (ePGPR) or intracellular PGPR (iPGPR) based on their degree of association with plants (Gray and Smith, 2005).
  • iPGPR are the nodulating rhizobia housed within the cells of anatomically sophisticated nodules and provide reduced nitrogen to plants.
  • ePGPR are those that reside in the soil, on the surface of plants or in the extracellular spaces in plant root tissue.
  • ePGPR increase plant growth through a broad range of mechanisms, for instance by producing phytohormones (Bastian et al., 1998; Jameson, 2000) or metal chelating siderophores (Carson et al.,
  • ePGPR ePGPR
  • iPGPR rhizobia (species in the genera, for example, Rhizobium, Sinorhizobium, and Bradyrhizobium species such as Bradyrhizobium japonicum), or species of Frankia.
  • proteins from other sources may be tested for bactericidal and/or bacteristatic activity as well as plant growth and/or disease resistance-promoting activity.
  • the polypeptide is obtained from or obtainable from Bacillus (e.g. B. thuringiensis or B. cereus), Pseudomonas, Rhizobium, or Bradyrh izobium .
  • Bacillus e.g. B. thuringiensis or B. cereus
  • Pseudomonas e.g. Rhizobium, or Bradyrh izobium .
  • the polypeptide is a class IE) bacteriocin.
  • the polypeptide is a polypeptide that is obtained from or obtainable from Bacillus thuringiensis, especially Bacillus thuringiensis strain NEB 17, originally isolated from soybean root nodules (Bai et al. 2003), and which was deposited at the International Depositary Authority of Canada (K)AC) on March 27, 2003 under Accession No. 270303-02.
  • Thuricinl7 discussed below, and Bacthuricin F4 are two novel bacteriocins having plant growth and/or disease resistance promoting activity isolated by the inventors from B. thuringiensis strain NEB 17 and their uses are contemplated herein.
  • the polypeptide is a bacteriocin (designated BF4) which is obtainable from or obtained from B. thuringiensis strain BUPM4.
  • the polypeptide is a bacteriocin (designated C85) which is obtainable from or obtained from B. cereus strain UW85.
  • BF4 strain BUPM4
  • C85 strain UW85
  • Tl 7 strain NEB 17
  • T17, F4 and C85 have HPLC elution times that, while not identical, are very similar. While the total amino acid composition indicates differences between Tl 7 and BF4, the first 17 amino acids from the amino end are the same.
  • UW85 has been deposited in the American Type Culture Collection under accession number ATCC 53522.
  • BUPM4 is in the collection of the Medical Faculty of Sfax, in Tunisia.
  • polypeptide is equivalent (i.e. has the same amino acid sequence) to one expressed by the mentioned bacterial strain but is not limited to the polypeptide only when produced by that strain.
  • the polypeptide could be produced recombinantly in a host cell or organism or synthesized chemically.
  • polypeptide may possess one or more of the following properties:
  • the polypeptide may maintain bactericidal and/or bacteristatic activity after exposure to a temperature of 100°C for at least 15 minutes;
  • the polypeptide may maintain bactericidal and/or bacteristatic activity after treatment with ⁇ -amylase or catalase, but exhibit loss of activity after treatment with proteinase K or protease;
  • the polypeptide may have molecular weight in the range of about 3100 to 3200 Da.
  • the polypeptide is a novel polypeptide denoted thuricin 17 (T 17) identified by the inventors.
  • Tl 7 comprises the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO: 1).
  • such a polypeptide is obtained from or obtainable from Bacillus thuringiensis strain NEB 17 (IDAC 270303-02).
  • the polypeptide is a polypeptide that retains at least some of the bacteriocin and plant growth and/or disease resistance promoting activity of Tl 7 but differs in sequence from Tl 7 by one or more amino acid insertions, deletions, or substitutions, particularly conservative amino acid substitutions.
  • conservative amino acid substitutions refers to the substitution of one amino acid for another at a given location in the polypeptide, where the substitution can be made without substantial loss of the relevant function, hi making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • such a polypeptide may possess at least one activity of a bacteriocin and plant growth promoting activity and possess at least 70, 80, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, when optimally aligned.
  • identity refers to sequence similarity between two polypeptide or polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid or nucleic acid sequences is a function of the number of identical or matching amino acids or nucleic acids at positions shared by the sequences, for example, over a specified region.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at http://clustalw.genome.ad.jp, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoI. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.).
  • Sequence identity may also be determined using the BLAST algorithm (e.g. BLASTn and BLASTp), described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/ ' ).
  • An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions.
  • Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • Naturally occurring variant sequences may be more likely to retain bacteriocin and plant growth and/or disease resistance promoting activities, such as homologs produced by closely related bacterial species.
  • the polypeptides are preferably in purified form.
  • purified is meant that the polypeptide is substantially separated or isolated from the components such as other polypeptides, proteins, or lipids, carbohydrates, etc. that accompany the polypeptide in its natural environment.
  • a polypeptide that is chemically synthesised or produced by recombinant technology will generally be substantially free from its naturally associated components and be considered to be purified.
  • the purified polypeptide will constitute at least 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% by weight, of the total material in a sample (i.e.
  • a sample of the purified polypeptide will contain less than 40%, 30%, 20%, 10%, 5%, 2% or 1% by weight of components such as other polypeptides, proteins, lipids, carbohydrates, etc. that accompany the polypeptide in its natural environment).
  • a substantially purified polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant polynucleotide encoding the polypeptide compound or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
  • polypeptides may be obtained from bacterial species that express the polypeptides.
  • the bacterial strain may be cultured under conditions sufficient for expression of the polypeptide and the polypeptide recovered from the culture medium.
  • the polypeptide may be purified by e.g. by chromatography (e.g. high-performance liquid chromatography), gel electrophoresis, filtration, dialysis, precipitation, centrifugation, etc. or combinations thereof.
  • the polypeptide is purified by solid phase extraction, e.g. using a Cl 8 solid phase extraction column such as a PREPSEP Cl 8 column (Fisher Scientific, Pittsburgh PA, USA).
  • the polypeptides may be expressed recombinantly, by culturing a host cell transformed or transfected with nucleic acid encoding the polypeptide.
  • the host cell may be a prokaryotic host cell, for example a bacterial cell, or a eukaryotic cell, such as a yeast, plant, or animal cell.
  • polypeptides may be synthesized chemically via known procedures.
  • Polypeptides may be applied either before, during or after planting and may be applied to, for example, plant leaves, stems, roots, or seeds.
  • plant includes without limitation whole plants, plant parts, organs, leaves, stems and roots.
  • plant seeds are discussed separately in the claims as it is envisaged that the plant growth and/or disease resistance promoting compositions may be applied to the seeds well e.g. in advance of planting.
  • the polypeptide may additionally or alternatively be applied to the growing environment of the plant or seed rather than directly to the plant or seed.
  • growing environment is meant the area sufficiently proximal to the plant or seed (such as to the soil adjacent to the plant or seed) that the polypeptide can effect a growth- or disease resistance- promoting effect on the plant. If the polypeptide is applied to the soil, it may be applied before, during or after planting. [0077]
  • the polypeptide may be applied by any suitable means, either in solid (e.g. as a free-flowing powder) or liquid form (such as in an aqueous carrier). Leaf spray and root irrigation are two preferred techniques.
  • the polypeptide may also be applied to various portions of the plant or seed in slow-release formulations, such as beads or gels.
  • the polypeptide is applied in an aqueous carrier at a concentration of about 10 "9 , 10 "10 or 10 "n M, equivalent to a total of 15.8, 1.58 and 0.158 ng pot "1 (where each pot contained ten plants), respectively.
  • the polypeptides may be used alone or in the form of a plant growth and/or disease resistance promoting composition.
  • Such compositions may contain diluents, adjuvants, excipients, carriers, etc. suitable for inclusion in a plant growth and/or disease resistance promoting compositions as are known in the art.
  • the compositions may be in, for example, solid (such as powdered) or liquid form.
  • the plant growth and/or disease resistance promoting composition may be provided in the form of a kit containing the composition and e.g. instructions for use of the composition for promoting plant growth.
  • the composition may take the form of plant seeds pre-treated with the plant growth and/or disease resistance promoting composition.
  • Plants are able to synthesize a broad range of secondary metabolites capable of improving their resistance to pathogen attack, hi many cases these are only synthesized when the plants are exposed to compounds that indicate the presence of the pathogen (Somssich et al., 1986) - elicitors such as oligosaccharides.
  • the major molecular events of plant-pathogen interactions can be divided into three steps: i) generation and recognition of signal compounds, ii) inter-and intracellular signal conversion and transduction, and iii) activation of signal-specific responses in target cells (Ebel and Cosio, 1994).
  • Elicitor molecules produced by microorganisms are extremely diverse in nature.
  • Four major classes of elicitor-active oligosaccharides have been identified as oligoglucan, oligochitin, oligochitosan from fungi and oligogalacturonide of plants (Cote and Hahn, 1994).
  • Chitin is an elicitor molecule produced by fungal cell walls; it is a polysaccharide and is composed of /3-1-4-linked N-acetylglucosamine units.
  • Glucans which have the ability to stimulate the production of phytoalexins, newly synthesized antimicrobial compounds of low molecular weight, were initially detected in culture filtrates of the oomycete Phytophthora sojae, a pathogen of soybean (Ayers et al., 1976). Glucans similar to those active as elicitors in soybean occur as extracellular polysaccharides in the symbiotic partner of soybean, Bradyrhizobium japonicum (Rolin et al., 1992). Cyclic ⁇ -l,3-l,6-glucans of the microsymbiont of soybean, Bradyrhizobium japonicum USDA 110 have been shown to have elicitor activity (Miller et al., 1990).
  • elicitors of plant pathogen defence mechanisms may be used in conjunction with the methods and compositions of the invention.
  • Such elicitors may be applied to plants, seeds, or the growing environment of the plant together with or separately from the polypeptide possessing plant growth and/or disease resistance promoting activity.
  • Plant growth and/or disease resistance promoting compositions of the invention may contain such elicitors, or be packaged together with the elicitor.
  • Preferred elicitors include oligosaccharides, such as oligoglucans, oligochitins, oligochitosans, (preferably from fungi) and oligogalacturonides.
  • Plants planted, germinated or grown in the presence of the plant growth and/or disease resistance promoting polypeptides of the invention may exhibit an increase in plant growth, such as an increase in one or more of nodulation, nitrogen fixation, height, increased seedling emergence, leaf area, seed germination, leaf greenness, photosynthesis, or shoot, root, or total dry weight, relative to a plant that has not been treated with the plant growth and/or disease resistance promoting composition.
  • plants planted, germinated or grown in the presence of the plant growth and/or disease resistance promoting polypeptides of the invention may exhibit one or more characteristics of improved disease resistance, such as, for example, reduced or inhibited pathogen infestation, increased activity of a lignif ⁇ cation-related enzyme such as PAL or TAL or an antioxidative enzyme such as POD, CAT or SOD.
  • Increases of enzyme activity of more than 10, 20, 30, 40, 50, 60, or 70% maybe obtained by the methods of the invention.
  • Increases in concentration of total phenolics of more than 1, 5, 10, 15 or 20% may be obtained by the methods of the invention.
  • compositions of the invention may be used and the methods of the invention practiced wherever plants are grown, such as in greenhouse, field, or laboratory conditions.
  • the compositions may be used with plants that are grown at temperatures above 30°C, at which temperatures nitrogen fixing rhizobacteria are generally most active, or also at low temperatures, such as at an average daily root zone temperature below 28, 26, 24, 22, 20, 18, 16, 14, 12, or 1O 0 C.
  • the methods of the invention are not limited to use with any particular plant or plant-type.
  • Exemplary plants with which the methods of the invention may be practiced include, without limitation: legumes, such as soybean, peanut, pulses (e.g. pea and lentil), bean, forage crops (e.g. alfalfa and clover), plants of lesser agricultural importance (e.g lupine, sainfoin, trefoil, and even some small tree species); tomato; corn; horticultural tree species (e.g. peach, apple, plum, pear, mango), forestry tree species (e.g. spruce, pine, fir, maple, oak, poplar), and small grain cereals such as wheat, barley, oat, and canola.
  • legumes such as soybean, peanut, pulses (e.g. pea and lentil), bean, forage crops (e.g. alfalfa and clover), plants of lesser agricultural importance (e.g lupine, sainfoin, trefo
  • polypeptides of the invention may also be used as bacteriostatic and/or bactericidal agents in any application in which it would be desirable or advantageous to prevent or inhibit growth of bacteria.
  • the polypeptides of the invention may be used to treat or prevent bacterial infection in a subject, such as a mammalian subject, especially a human subject.
  • the polypeptide may be formulated as a pharmaceutical composition comprising a polypeptide of the invention together with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Such compositions may include additional bactericidal and/or bacteriostatic agents as are known in the art.
  • Pharmaceutical compositions may be formulated for administration, for example, topically, intravenously, orally, rectally, parenterally, etc. Suitable dosages and routes of administration can be determined by the skilled person.
  • polypeptides of the invention may also be employed to inhibit or prevent growth of bacteria in other applications, such as on a surface, in a liquid, in a nutrient medium, in a food product, etc., and the polypeptide may be formulated into a bactericidal and/or bacteristatic composition comprising the polypeptide together with one or more suitable carriers, excipients and diluents, and optionally one or more additional bactericidal and/or bacteristatic agents.
  • the invention provides polynucleotides encoding the polypeptides of the invention.
  • polynucleotide refers to a polymeric form of nucleotides of any length and may also be referred to in the art as a "nucleic acid” or “nucleic acid molecule".
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide.
  • the term includes single and double stranded forms of DNA or RNA.
  • DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the polynucleotides of the invention include full-length genes and cDNA molecules as well as a combination of fragments thereof.
  • the polynucleotides of the invention are preferably "isolated" polynucleotides by which it is meant that they are not present in their naturally occurring form associated with the 5' and/or 3' sequences with which they are normally found.
  • the polynucleotides are separated from at least one or both of the 5 ' or 3' sequences with which they are normally associated.
  • a nucleic acid molecule of the invention inserted into a vector or linked to a foreign promoter, is in "isolated" form.
  • the polynucleotide encodes a polypeptide that is a bacteriocin and that possesses plant growth and/or disease resistance promoting activity, said polypeptide being selected from the group consisting of:
  • polypeptide of (a) possessing the bacteriocin and plant growth and/or disease resistance promoting activities of the polypeptide of (a), and which possesses at least 70% sequence identity to SEQ ID NO: 1 over its entire length;
  • polypeptide which is a fragment of the polypeptide of (a) or (b), said fragment possessing the bacteriocin and plant growth and/or disease resistance promoting activities of the polypeptide of (a).
  • the isolated nucleic acid molecule comprises the entire sequence set forth in SEQ ID NO: 9 or the complement thereto, or a sequence comprising SEQ ID NO: 9 from position 21 to position 143, from position 250 to postion 372, or from position 478 to position 600, or the complement thereto.
  • the invention also encompasses isolated nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule that comprises the entire sequence set forth in SEQ ID NO: 9 or the complement thereto, or a sequence comprising SEQ ID NO: 9 from position 21 to position 143, from position 250 to postion 372, or from position 478 to position 600, or the complement thereto.
  • Fragments of the isolated nucleic molecules of the invention having lengths of at least about 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 nucleotides are encompassed by the invention and are useful as e.g. probes in hybridization reactions to identify polypeptides related to thuricin 17 that have bacteriocin and plant growth and/or disease resistance promoting activity or as PCR primers for amplifying such sequences.
  • the invention also provides vectors, such as plasmid vectors, viral vectors, expression vectors, etc. comprising the polynucleotides of the invention, as well as host cells transformed or transfected with polynucleotides of the invention.
  • the host cells may be host cells as described above.
  • vector refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • the recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art.
  • the vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells, including plant cells.
  • the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for selectable markers and reporter genes are well known to persons skilled in the art.
  • a recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector.
  • the living cell may include both a cultured cell and a cell within a living organism, preferably a plant. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention.
  • host cell and "recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Vector DNA can be introduced into cells via conventional transformation or transfection techniques.
  • transformation and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. Transformation methods particularly suited to plant transformation are discussed further below.
  • Vectors of the invention may contain one or more transcriptional regulatory sequences or elements.
  • Transcriptional regulatory sequence or element is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably linked.
  • 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 sequences.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably- linked but not contiguous.
  • a transcriptional regulatory sequence or element may be a promoter that directs tissue-selective expression of a coding sequence, e.g. seed-selective expression in a plant, or that directs expression of the coding sequence at a desired time in the life cycle of the host organism.
  • a promoter may be constitutive or inducible. Inducible promoters which initiate expression in response to an external stimulus, such as exogenous application of a chemical agent may be used in the practice of the invention, e.g. to direct expression of the polypeptide of interest in a plant at a preferred time in the life cycle of the plant.
  • a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid is considered “transformed”, “transfected", or “transgenic”.
  • a transgenic or transformed cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing a transgenic organism as a parent and exhibiting an altered phenotype resulting from the presence of a recombinant nucleic acid construct.
  • a transgenic organism is therefore an organism that has been transformed with a heterologous nucleic acid, or the progeny of such an organism that includes the transgene.
  • the introduced DNA may be integrated into chromosomal DNA of the cell's genome, or alternatively may be maintained episomally (e.g. on a plasmid).
  • selectable marker is used broadly to refer to markers which confer an identifiable trait to the host cell.
  • selectable markers include markers affecting viability, metabolism, proliferation, morphology and the like.
  • Preferred selectable markers include those that confer resistance to drugs, such as antibiotics.
  • Nucleic acids encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the peptide compound or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the invention provides a plant that is transformed with a nucleic acid molecule encoding a polypeptide of the invention or otherwise comprises such a nucleic acid molecule such that the plant can express the polypeptide.
  • exemplary plants with which the methods of the invention may be practiced include, without limitation, those described above i.e.: legumes, such as soybean, peanut, pulses (e.g. pea and lentil), bean, forage crops (e.g. alfalfa and clover), plants of lesser agricultural importance (e.g lupine, sainfoin, trefoil, and even some small tree species); tomato; corn; horticultural tree species (e.g. peach, apple, plum, pear, mango), forestry tree species (e.g. spruce, pine, fir, maple, oak, poplar), and small grain cereals or canola.
  • legumes such as soybean, peanut, pulses (e.g. pea and lentil), bean, forage crops (e.g. alf
  • Transgenic plants stably transformed with a nucleic acid molecule encoding a polypeptide of the invention sequence may be prepared by any of a variety of methods such as, without limitation Agrobacterium-mediated transformation, particle bombardment or electroporation.
  • a Ti or Ri plasmid is disarmed by deletion of tumor inducing genes.
  • the nucleic acid to be introduced into the plant is cloned into the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed.
  • Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. Transformation with Agrob ⁇ cterium is typically achieved in one of two ways. In the first, protoplasts or leaf-discs can be incubated with the Agrob ⁇ cterium and whole plants regenerated using plant tissue culture. Alternatively, some plant species may be transformed simply by dipping flowers into a suspension of Agrob ⁇ cterium and then planting the seeds in a selective medium.
  • particles typically of gold or tungsten
  • the transformation efficiency is lower than that of Agrob ⁇ cterium-mediated transformation, a broader range of plant species may be transformed by particle bombardment. Transformation of corn embryogenic cultures by bombardment with gold particles coated with plasmid DNA comprising a thuricin-17 coding sequence is described in the examples herein.
  • an electrical field is used to create pores in the plant cell wall, to permit entry of the nucleic acid of interest.
  • viral vectors may be used to transform plant cells.
  • Bacillus thuringiensis NEB17 (BtNEB17) was cultured in King's Medium B consisting of proteose peptone #3 (20 g L “1 ), K 2 HPO 4 (0.66 g L '1 ), MgSO 4 (0.09 g L '1 ) and glycerol (0.06 mL L “1 ) (Atlas 1995). The initial broth inoculum was taken from plated material and grown in 250 mL flasks, containing 50 mL of medium.
  • the bacterium was cultured at 28 ⁇ 2 0 C on an orbital shaker (Model 5430 Table Top Orbital Shaker, Forma Scientific Inc., Mariolta, Ohio, USA) for 48 h, rotating at 150 rev min "1 .
  • a 5 mL sample of subculture was added to 2 L of broth and cultures were grown in 4 L flasks under the same conditions as for the initial culture.
  • Bacterial populations were determined spectrophotometrically using an Ultrospec 4050 Pro UV/ Visible Spectrophotometer LKB (Cambridge, England) at 600 nm (Dashti et al. 1997) 96 h after culture preparation.
  • BtNEB 17 cells were cultured as described above. Two liters of bacterial culture was phase partitioned against 0.8 L butanol for 12 h. The upper butanol layer was collected and evaporated using the rotary evaporator (Yamota RE500, Yamato, USA) at 50 0 C under vacuum. After evaporation, the resulting light brown viscose extract was resuspended in 25 mL of 18% acetonitrile (AcN:H 2 0, v/v).
  • Conditions of the fractionation chromatography were as follows: 45 minutes at 18% acetonitrile, 45 to 110 minutes of gradient elution with 18 to 60.4% of acetonitrile, 110 to 115 minutes at 60.7 to 100% of acetonitrile and 115 to 120 minutes at 100 to 18% of acetonitrile.
  • the HPLC elutions were collected at 1 minute intervals (Bai et al. 2002b). Preparative HPLC samples were separated into 120 minute fractions and were analyzed for peaks with retention times between 80 and 82 minutes, as this is when the peptide elutes.
  • the peptide elutes in approximately 60% acetonitrile, and is denoted as partially purified bacterial peptide (PPBP).
  • PPBP partially purified bacterial peptide
  • the BtNEB 17 compound was initially assessed for protein content via the
  • Antimicrobial activity of the BtNEB 17 peptide was assessed via agar disk diffusion assay (Kimura et a 1998) on all indicator strains listed in Table 1 below.
  • a host of Bacillus members and non-Bacillus members were tested for their inhibition by the BtNEB 17 peptide (Table 1).
  • the peptide was inhibitory to other Bacillus strains, including 16/19 B. thuringiensis strains, 4/4 B. cereus strains, 2/2 B. megatarium strains, 2/3 B. licheniformis strains and 1/2 B. pumilus strains (Table 1).
  • Bradyrhizobium japonicum 532C USDA 2 Bradyrhizobium japonicum 532C USDA 2 —
  • Bradyrhizobium japonicum USDA 3 t USDA 2 Bradyrhizobium japonicum USDA 3 t USDA 2 —
  • Bradyrhizobium japonicum USDA 110 USDA 2 Bradyrhizobium japonicum USDA 110 USDA 2 —
  • Paenibacillus dendritiformis C 168 ⁇ BGSC 3 9
  • KTCC Korean Type Culture Collection
  • NEB Non-Bradyrhizobium endophytic bacterium. * Strains cultured in King's Medium (Atlas 1995), ⁇ Strains cultured in Yeast Extract Mannitol (Vincent 1970), £ Strains cultured on MacConkey Agar (Difco), ⁇ Strains cultured on Tryptose Blood agar (Oxoid). Source*: SLC 1 I Dr.
  • BGSC 3 Bacillus Genetic Stock Center, University of Ohio, Department of Biochemistry, Cleveland, Ohio, USA
  • ARSCC 4 Agricultural Research Service Culture Collection, Peoria, Illinois, USA
  • ATCC 5 American Type Culture Collection
  • KU 6 Kuwait University, Department of Biology, Kuwait, Kuwait.
  • Indicator strains were cultured and tested for purity prior to running the assay and were then streaked onto agar plates. Due to the large volumes of material required, two replicates of the CFS were tested, instead of the PPBP. 15 ⁇ L of sample was spotted onto sterilized disks (6 mm) and allowed to dry. Petri dishes were maintained for at least 48 h at 27 0 C after which the zone of inhibition was measured (mm).
  • tryptose Blood Agar prepared according to manufacturers instructions (Difco, USA): tryptose blood agar base (10 g L “1 ), NaCl (4.8 g L “1 ), agar (12 g L '1 ) and sterile defribinated sheeps' blood (72 rnL L “1 ).
  • the activity of the BtNEB17 peptide was quantified by using a series of two fold dilutions (modified from Mayr-Harting et al. 1972) and was conducted on separate replicates. Briefly, 15 ⁇ L of two-fold dilution factors were spotted onto sterilized disks (6 mm) and allowed to dry; duplicates were conducted for each sample. The specific activity of samples was calculated as the reciprocal of the highest dilution that gave a clearly visible inhibition zone. This was expressed in activity units (AU) and determined using the indicator strain B. cereus ATCC 14579. By weighing lyopholized peptide an estimate of peptide concentration ( ⁇ g L "1 ) was determined and compared with the AU.
  • AU activity units
  • thuringiensis NEB 17 was cultured in the same manner and exposed to the same treatments as a negative control.
  • Cell density O.D. 60Onm was then read using an UltrospecTM 4050 Pro UV/ Visible Spectrophotometer LKB (Cambridge, England). Results were confirmed by the number of viable colony forming units (CFU) log mL "1 . Briefly, subsamples of cell cultures were taken each hour and diluted in 0.9% NaCl solution, 50 ⁇ L of diluted bacterial culture was inoculated onto agar plates, and viable cell count determined. Values are expressed on a log scale. The entire experiment was also repeated with CFS (0.071 ⁇ g ⁇ L '1 ).
  • SDS-PAGE indicated that the peptide present in the PPBP and CFS weighed 2500 - 3000 Da ( Figure 3, lanes 3 and 4). Results show it is also responsible for directly inhibiting bacterial growth. Due to the high percentage of acrylamide in the gel, it took many attempts to grow the indicator strain and colonies appear as an uneven lawn. Despite this, the inhibitory effects of the peptide were observed and it is inferred that the BtNEB 17 peptide is responsible for direct inhibition of bacterial growth. SDS-PAGE provided an estimate of the peptide's molecular weight and MALDI mass spectrometry data confirmed these results. A strong mass peak from MALDI analysis is observed at 3162.3 Da (See Figure 4 below). Additional testing, using FAB mass spectrometry, yielded similar results (data not shown).
  • Proteinase K from Tritirachium album, Sigma No. P-2308,, Protease (from Streptomyces griseus, Sigma No. P-6911), ⁇ -amylase (from barley malt VIII-A, Sigma No. A-2771) and catalase (from Corynebacterium glutamicum, Sigma No. 02071).
  • protease K from Tritirachium album, Sigma No. P-23008
  • Protease from Streptomyces griseus
  • ⁇ -amylase from barley malt VIII-A, Sigma No. A-2771
  • catalase from Corynebacterium glutamicum
  • protease and ⁇ -amylase enzymes were added to final concentrations of either 1 mg mL “1 or 2 mg mL "1 .
  • Catalase was added at either 40,000 U mL “1 or 60,000 U mL “1 .
  • Samples were incubated for 120 min at 37 °C, then heated at 100 0 C for 2 min for enzyme inactivation. Controls were as follows: PPBP plus the corresponding enzyme buffer, CFS plus the corresponding enzyme buffer, enzymes in corresponding buffer, purified medium and centrifuged medium.
  • the pH levels were determined using an Accumet Dual Channel pH/ Ion Conductivity Meter model AR50 (Fisher Scientific, Montreal). Inhibitory activity was conducted at 21 0 C and assessed on the indictor strain Bacillus thuringiensis ssp thuringiensis Bt 1627 and/or B. cereus ATCC 14579 (Table 2).
  • NEB17 Bacillus thuringiensis NEB17 (NEB 17) was cultured in King's Medium B:
  • Proteose peptone #3 (20 g L “1 ), K 2 HPO 4 (0.66 g L “1 ), MgSO 4 (0.09 g L “1 ) and glycerol (0.06 mL L “1 ) (Atlas 1995).
  • the bacterial cultures were grown in 4 L flasks containing 2 L of liquid media for at least 72 h at 28 ⁇ 2 0 C on an orbital shaker (Model 5430 Table Top Orbital Shaker, Forma Scientific Inc., Mariolta, Ohio, USA). Cultures were grown until an O.D.
  • Tl 7 partial purification was conducted by phase partitioning 2 L of bacterial with 0.8 L butanol for 12 h. The aqueous layer was removed and the organic layer concentrated at 50 0 C under vacuum by rotary evaporation (Yamota RE500, Yamato, USA). The remaining material was then resuspended in 25 mL of 18% acetonitrile (AcN:H 2 0, v/v). Prior to purification, all material was stored in a sterilized, sealed vial at 4 0 C. Purified media alone, without added bacteria, was subjected to the same extraction protocol, and this material acted as a control.
  • the molecular weight of the ion for sequencing was slightly less than the initially determined molecular weight of 3162 Da (Gray et al. 2006a). It was difficult to fragment the ion for sequencing and in fragmenting the intact peptide, partial amino acid residues were lost at the site of a putative site of post- translational modification (PTM). Nonetheless, we are still able to obtain partial sequence data which does coincide with amino acid analysis.
  • PTM post- translational modification
  • a signal drop-off (difficulty in sequencing past a specific amino acid residue) has also been reported for other bacteriocins. Ahern et al. (2003) found a signal drop off at the 20 th cycle when trying to sequence both thuricin 439A and 439B.
  • the peptide was then treated with carboxypepsidase Y and trypsin to generate peptide ladders for mass spectrometry based C-terminal sequencing. However, the peptide was resistant to further digestion (data not shown). Again, this is not uncommon.
  • a BLIS from B. cereus ATCC 14579 is resistant to trypsin, RNAse and lysozyme, but not to proteinase K and pronase E (Risoen et al. 2004).
  • Coagulin (Hyronimus et al. 1998) is resistant to degradation by trypsin. Exposure of thuricin 17 to carboxypepsidase Y and W yielded sufficient fragments for C-terminus analysis. A C-terminus sequence of CAS - C-terminus was then determined.
  • no exact match was found via BLAST searches, and in comparison with existing sequence information on currently published bacteriocins, confirming that Tl 7 is a novel compound.
  • Production of the material by B. thuringiensis NEB 17 was determined by preparing subcultures of cells taken from Petri plates and culturing for at least 12 h. One mL of this material was then added to 250 mL of King's medium. Subsamples were taken every hour and the O.D. 6 oo nm (Optical Density) and log CFU (Colony Forming Units) mL "1 were determined ( Figure 7). The O.D. was determined spectrophotometrically with an UltrospecTM 4300 Pro UV/Visible Spectrophotometer. The CFU was determined by diluting subsamples, taken each hour, in 0.9% NaCl solution.
  • Tl 7 Fifty ⁇ L of diluted bacterial culture was then inoculated onto agar plates, and viable cell count determined.
  • the activity of Tl 7 was quantified as specific activity units (AU) using the indicator strain, B. thuringiensis ssp. thuringiensis Bt 1627. This was done by preparing a CFS (Cell Free Supernatant), extracting material every hour, preparing a series of two fold dilutions. For detection of inhibition, the disk diffusion assay was used; 15 ⁇ L of diluted T17 was spotted onto sterilized filter paper disks (6 mm diameter). Production of T17 begins at the mid-exponential growth phase and continues well into the stationary phase ( Figure 7), which coincides with the results for thuricin, B.
  • AU specific activity units
  • DNA samples were manipulated using standard techniques. Sequencing primers used in this work were NEB-F2, 5'-GTATGTGCAGCATGTTCTGTAG-S' (SEQ ID NO: 5); NEB-R, 5'-AACAAGACCAACATGTCC-S' (SEQ ID NO: 6); NEB17albAup, 5'-GTGGCGGTTTTATTTATCG-S' (SEQ ID NO: 7); and thurl7dn 5'-TAAAACATAGGGAGTTATACTTAG-S' (SEQ ID NO: 8). All sequencing was performed at Mobix Lab (McMaster University, Hamilton, Ontario, Canada) using whole genomic DNA as a template.
  • Genomic DNA was isolated using a DNA spooling method (Meade et al, 1982; modified by Oresnik et al, 1994). Sequence data were analyzed using ApE (M. Wayne Davis) and database searches were performed using BLAST (Altschul et al. 1997).
  • subtilis which encodes a bacteriocin of 448 aa (Kunst et al., GenBank accession number CAB 15764).
  • a region of this sequence data which agreed with the published albA coding sequence was used to design a reverse primer, NEB17albAup, which revealed the presence of three copies of a gene with homology to a sequenced peptide, thuricin-S of Bacillus thuringiensis sv. entomocidus (Chehimi et al., GenBank accession number P84763).
  • Ambiguities in the sequence obtained from NEB17albAup were resolved by designing and sequencing from a parallel sequencing primer, thurl7dn.
  • FIG. 7A The genomic DNA region encoding this region, 625 bp in length, is shown in Figure 7.B.
  • Each copy of the thuricin-17-encoding gene is 123 nt in length and predicts a peptide of 39 aa matching that of the thuricin-17 peptide sequence, as well as an N- terminal extension which we believe represents an export signal sequence that would be cleaved from the mature peptide.
  • the predicted mature peptides agree with the N-terminal sequences of both the thuricin-17 peptide and Thuricin-S; these predicted peptides also match the predicted molecular weight of thuricin-17 derived from the sequenced peptide ( Figure 7.C).
  • the presence of three copies of the gene, all coding for the same protein may suggest that there are constraints on the evolution of the gene, perhaps related to more than one function for the protein.
  • a stock culture of bacteria was grown in 250 mL flasks, containing 50 mL of broth. Bacteria were cultured at 28 ⁇ 2 0 C on an orbital shaker (Model 5430 Table Top Orbital Shaker, Forma Scientific Inc., USA) for 32 h, rotating at 150 rpm. Culture populations were determined at 600 nm using an Ultrospec 4300 Pro UV/Visible Spectrophotometer (Biochem Ltd., England), then adjusted with broth to a 1% inoculation ratio (final volume) in 4.0 L flasks containing 1.0 L of the broth culture medium. The resulting subculture was grown for 48 h.
  • the resulting viscose extract was resuspended in 18% acetonitrile (AcN:H 2 O, v/v) and further purified through HPLC (Waters 510 system, Waters, USA).
  • HPLC Waters 510 system, Waters, USA.
  • the HPLC was equipped a C 18 reverse-phase column (Vydac218TP54, 300 nm, 5 ⁇ m, 4.6 x 250 mm), model 441 absorbance detector at 214 nm and column temperature at 2O 0 C.
  • the elution was performed as follows: 0-45 min with isocratic 18% AcN and 45-110 min with a gradient from 18 to 60.7% AcN.
  • HPLC eluates were collected as 110 fractions, 1 min of elution time per fraction, and maintained at 4 0 C until use. Culture medium, without bacteria, was put through the same extraction and purification procedure, and the resulting material was used as a negative control.
  • the 110 collected fractionations were initially assayed to assess their plant biological activity.
  • fractions 61 to 110 were aggregated into 5 groups (61- 70, 71-80, 81-90, 91-100 and 101-110 minute fractions; Figure 10A), pooled and tested for their ability to enhance seed germination of soybean cultivar OAC Bayf ⁇ eld.
  • the active fractions selected in the first step were further divided into five groups (81-82, 83-84, 85-86, 87-88 and 89-90 minute fractions; Figure 10B) and retested.
  • Soybean seeds were surface-sterilized in 2% sodium hypochlorite for 3 min and then rinsed 5 times with distilled water (Bhuvaneswari et al., 1980). Ten soybean seeds were placed on two layers of sterilized filter paper wetted with 7 mL of treatment solution, in Petri dishes. Treatment application marked the beginning of the assay. Petri dishes were maintained in an incubator (Conviron El 5 Growth Chamber, Controlled Environments Ltd., Winnipeg, Canada) at 25 ⁇ I 0 C and 70-80% humidity. Germination was determined to have occurred when the root tip had clearly penetrated the seed coat. The number of germinated seeds was recorded periodically for 30 h and germination was expressed as a percentage (%) of the total number of seeds in the dish.
  • thuricin 17 concentration causing the greatest increase in germination was determined.
  • Thuricin 17 solutions were prepared by lyophilizing purified material at -60 0 C, under vacuum pressure using a Savant Modulyo Freeze-dryer fitted with a Savant Model VPOF oil pump and Savant Model VPL200 air pump. The dried fraction was then resuspended in sterilized, distilled water.
  • thuricin 17 was investigated for its ability to enhance soybean nodulation, photosynthesis and growth under greenhouse conditions. Soybean seeds of OAC Oxford (an early maturing cultivar) and Korada (a late maturing cultivar) were surface-sterilized in 2% sodium hypochlorite for 3 min, and rinsed 5 times with distilled water (Bhuvaneswari et al., 1980). These two cultivars were selected as they have been widely grown in eastern Canada. Seeds were placed in sterilized vermiculite to germinate.
  • BJ 532C was cultured in yeast extract mannitol culture medium (YEM) (Vincent, 1970). Broth was inoculated with slant material and cultured on an orbital shaker at 150 rpm for 7 days at 28 0 C. A subculture was prepared by inoculating new broth medium with the initial culture such that the added inoculant material constituted 1% of the volume of the subculture. After 5 days the subculture was centrifuged at 2,800 x g for 20 min at 4 0 C.
  • yeast extract mannitol culture medium YEM
  • Thuricin 17 was applied to soybean plants by either leaf spray or root irrigation, hi both types of application thuricin 17 was applied at 0, 5 x 10 "11 (T 17-1), 5 x 10 '10 (T17-2) and 5 x 10 "9 M (T17-3). Treatments were applied three times to each plant, when soybean plants were at the Vl, V2 and V3 stages (Fehr et al., 1971). For leaf sprays, Tween 20 (0.01%) was added into treatment solutions and also the control. The top surfaces of the pots were covered with vinyl plastic to ensure the treatment solutions did not drip onto the soil. Treatment solutions were sprayed, with an atomizer, onto leaves until wet.
  • treatment solutions including the control, did not contain Tween 20.
  • Treatment solution 1 mL, was diluted with distilled water to become 20 mL and poured on the rooting medium surface at the base of the plant stem. Plants were grown for 40 days following the initial application of treatment solutions.
  • the pot experiment was structured as a randomized complete block design (RCBD) with four replications. Data were analyzed via analysis of variance (ANOVA) using CoStat software (CoStat Software, Monterey, USA). Since there was no interaction between cultivar and application method, cultivar and concentration, or cultivar, application method and concentration, but there was an application method by concentration interaction, data are presented as application method by thuricin 17 concentration interaction means. Means comparisons were conducted using an ANOVA protected the least significant difference (LSD) (P ⁇ 0.05) test.
  • LSD least significant difference
  • thuricin 17 did not inhibit the growth of B. thuringiensis NEB 17 ( Figure 9A), the thuricin producer, or B. japonicum 532C.
  • T 17-2 increased leaf photosynthetic rates about 6% over the control (from 13.75 to 14.55 ⁇ mol cm '2 s "1 ) (Table 3).
  • Leaf greenness (SPAD reading) was similarly affected and the average value for Tl 7-2 was 29.2, as compared with the control at 27.3 (Table 3).
  • Increases in leaf area were also observed for all three treatments, with T 17-2 causing the greatest increase.
  • Tl 7-2 increased plant dry weight by 15% from 1.137 for the control to 1.304 g plant "1 in the T17-2 treatment (Table 3).
  • Table 3 Effects of thuricin 17 on soybean (cultivars OAC Oxford and Korada) photosynthesis, leaf greenness, leaf area, plant height and plant dry weight at harvesting time. The application method (leaf spray and root irrigation) and concentration interaction means are shown. Treatment Photo-synthetic Leaf color Leaf area Plant Dry weight rate (SPAD) height
  • T17-l, T17-2 and T17-3 represent thuricin 17 concentrations of 5 x 10 "n , 5 x 10 "10 and 5 x 10 "9 M, respectively.
  • Table 4 Effects of thuricin 17 on soybean (cultivars OAC Oxford and Korada) nodulation and nitrogen fixation (at final harvest). The application method (leaf spray and root irrigation) and concentration interaction means are shown.
  • T17-l, T17-2 and T17-3 represents a thuricin concentration of 5 x 10 " ", 5 x 10 "10 and 5 x 10 "9 M, respectively.
  • the Bacillus thuringiensis strain NEB 17 was cultured in King's liquid medium at 25 °C on an orbital shaker for 48 h, rotating at 150 rev min "1 .
  • the composition of this medium was as follows: protein peptone #3 -20 g; K 2 HPO 4 -1.5 g; MgSO 4 -0.75 g; glycerol -15 mL; distilled water -1000 mL.
  • the entire culture was extracted by adding 0.4 volume of n-butanol.
  • the butanol-water mixture had been shaken for 30 min and kept overnight at 4 0 C.
  • the separated butanol phase was collected and evaporated at 450 0 C using the rotary evaporator.
  • the dried extract was resuspended in 20% acetonitrile and used for the purification of Tl 7.
  • Butanol-so ruble compounds in 20% acetonitrile, were loaded on Cl 8 solid phase cartridges and fractionated using 35 % (acetonitrile:water, v/v) , 43% and 100% acetonitrile. These fractions were collected. Aliquots of 0.2 mL were taken from them and used for the HPLC analyses to quantify T17 in fractions.
  • Thuricin 17 was not detected in the fraction with 35% acetonitrile ( Figure 1 IB) but was abundant (134.1 mg) in the fraction with 43% acetonitrile ( Figure HC). Only 1.4 mg of Thuricinl7 was present in the fraction with 100% acetonitrile ( Figure 1 ID). Multiple repetitions (10 times) of the procedure for purification of T17 showed that only 1.0 ⁇ 0.4% and 0.3 ⁇ 0.2% of the total loaded bacteriocin were eluted with 35% and 100% acetonitrile, respectively. The maximum of 98.7 ⁇ 0.3% was detected in fraction with 43% acetonitrile.
  • the pots were placed in a growth chamber under these conditions: 25/22 °C (day/night), 16 h photo period, and with a light intensity of 340 ⁇ moles m "2 s "1 .
  • the study consisted of eight treatments of Tl 7 concentrations of 10 "9 , 10 "10 , 10 "u M dissolved in either dH 2 O or Hoagland's solution (HS, H strength) and two controls (dH 2 O and HS only).
  • Tomato plants were watered daily (50 mL) with their respective Tl 7 solution or HS. Tomato seedlings began to emerge after 4 days. Emergence for tomato was considered when seedlings were 2 or 3 mm above the medium ( Figure 13). Plants were harvested after 23 days of growth. Data were collected on plant height and leaf area. Tomato plants were separated into shoot and roots before oven drying at 60 0 C for a minimum of 72 h, then measured for dry weight.
  • Tomato plants showed a similar pattern to that of corn when supplied with Thuricin 17 solutions of 10 "9 , 10 "10 and 10 'n M.
  • Tomato seeds treated with Tl 7 solution of 10 "9 M had higher emergence rates from 96 to 144 h after seeding than the control plants, which were only given distilled water ( Figure 12).
  • tomato plants treated with T17 10 "9 , 10 "10 and 10 "11 M solutions had higher shoot and total plant dry weights than the control plants (Table 5).
  • the seeds were watered with 100 mL of the respective BF4 solution or dH 2 O for the control and then covered with 200 mL of vermiculite. The seeds were given another 80 mL of the respective BF4 solution or dH 2 O.
  • the pots were placed in a growth chamber under these conditions: 25/22 °C (day/night), 16 h photoperiod, and with a light intensity of 340 ⁇ moles m '2 s "1 . In total, there were 20 pots with 5 pots per treatment. Soybean plants were watered daily (50 mL) with their respective BF4 solution or dH 2 O for the control. Plants were harvested after 15 days of growth. Data were collected on plant height and leaf area. Soybean plants were separated into shoot and roots before oven drying at 80 0 C for a minimum of 72 h, then measured for dry weight.
  • Seeds of soybean ⁇ Glycine max L. Merr. cv. OAC Bayfield were surface sterilized with 400 mL L "1 commercial bleach solution for 2-3 minutes and rinsed several times with distilled water (dH 2 O). The seeds were then imbibed in the respective C85 (10 "9 , 10 "10 , 10 “11 M) or control (dH 2 O) solutions for 30 minutes prior to transfer into individual Petri plates. Ten seeds of soybean were placed in previously surface sterilized 400 mL pots containing a Whatman filter paper (A4) and 200 mL of fine vermiculite. The seeds were watered with 100 mL of the respective C85 solution or dH 2 O for the control and then covered with 200 mL of vermiculite.
  • A4 Whatman filter paper
  • the seeds were given another 80 mL of the respective UW85 solution or dH 2 O.
  • the pots were placed in a growth chamber under these conditions: 25/22 °C (day/night), 16 h photoperiod, and with a light intensity of 340 ⁇ moles m "2 s "1 .
  • Soybean plants were watered daily (50 mL) with their respective C85 solution or dH 2 O for the control. Plants were harvested after 14 days of growth, and leaf area and shoot dry weight were measured. Soybean plants treated with the bacteriocin produced by Bacillus cereus UW85 at 10 "9 , 10 "10 and 10 "11 M had higher leaf area and shoot dry weights than the control plants ( Figure 14).
  • Soybean ⁇ Glycine max L. Merr. cv. OAC Bayfield seeds were surface sterilized in 10% bleach, rinsed several times with distilled water and then germinated and grown in VermiculiteTM (Holiday, Montreal) in a growth chamber under a 16h/8h (day/night) regime (natural light supplemented with high pressure sodium lamps to reach the appropriate daylight), at 25 ⁇ 1°C, until they reached vegetative cotyledon (VC) stage (Fehr and Caviness, 1977).
  • VermiculiteTM Holiday, Montreal
  • 16h/8h day/night regime
  • the plants were excised at the base of the stem with a sharp scalpel and promptly placed in 2 mL EppendorffTM tubes containing 0.5 mL of 100 ⁇ mol L "1 chitin hexamer [(GIcNAc) 6 ], 0.5 mL of 1 x 10 8 mol L "1 thuricin 17, and chitin hexamer + thuricin 17 mixed (1:1) solution in phosphate buffer (15 mM sodium phosphate, pH 6.5).
  • the control plants were treated with phosphate buffer solution alone. Once all the solution was taken up by the plants (4-6 h), they were immediately transferred to glass test tubes containing 20 mL distilled water.
  • the plants were kept under constant white light (85 ⁇ mol-m "2 -s "1 ). Leaves were collected at 12, 24, 36, 48, 60 and 72 h after elicitor treatment, weighed, placed in plastic bags and stored immediately at -80 °C.
  • PAL phenylalanine ammonia lyase
  • TAL tyrosine ammonia lyase
  • the reaction mixture at a final volume of 3 mL, consisted of 1.9 mL of 50 mM Tris-HCl buffer (pH 8.0), 100 ⁇ L of enzyme preparation and either 1.0 mL of 15 mM L-phenylalanine for PAL or 1.0 mL of 15 mM L-tyrosine for TAL.
  • the assay was started by the addition of enzyme extract after an initial incubation for 60 min at 40°C.
  • the reactions were stopped by the addition of 200 ⁇ L of 6 N HCl.
  • the amounts of trans- cinnamic and p-coumaric acids formed were determined by measuring absorbance at 290 and 330 inn, respectively, against an identical mixture in which D-phenylalanine was substituted for L-phenylalanine and D-tyrosine for L-tyrosine.
  • the enzyme activity was expressed in nmoles (cinnamic or coumaric acid) mg protein "1 min "1 , where 1 unit is defined as 1 nmoles (cinnamic or coumaric acid) mg protein "1 min "1 .
  • the assay mixture contained 50 ⁇ L of sample with 0.475 mL of 0.25 N Folin-Ciocalteu reagent (Sigma Chemical Co.). After 3 min, 0.475 mL of 1 mol L '1 Na 2 CO 3 was added and after 1 h absorbance was measured. The phenolic contents were estimated using a standard curve prepared with gallic acid. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg g "1 fresh weight (FW).
  • GAE gallic acid equivalents
  • the activity of peroxidase was assessed using the method of Chance and Maehly (1955).
  • the reaction mixture consisted with 50 ⁇ L of 20 mM guaiacol, 2.8 mL of 50 mM Tris-HCl buffer (pH 8.0) and 0.1 mL extract.
  • the reaction was started with addition of 20 ⁇ L of 40 mM H 2 O 2 and the change in the absorbance at 470 nm was recorded for 1 min.
  • the activity of peroxidase was calculated using an extinction coefficient for the tetraguaiacol of 26.6 mM "1 cm "1 at 470 nm.
  • One unit of enzymatic activity was defined as the amount of enzyme required for the formation of 1 ⁇ mol of tetraguaiacol per minute.
  • SOD superoxide dismutase
  • NBT nitroblue tetrazolium
  • the reaction mixture (3.0 mL) consisted of 63 ⁇ M NBT (nitroblue tetrazolium), 1.3 ⁇ M riboflavin, 13 mM methionine, 0.1 mM EDTA, 50 mM Tris-HCl (pH 8.0), and 50 ⁇ L extract.
  • the mixture was held in a test tube and placed for 20 min under light at 78 ⁇ mol photons s "1 m "2 . Absorbance was recorded at 560 nm.
  • a non-illuminated reaction mixture that did not develop color served as the control, and its absorbance was subtracted from the A 560 of the reaction solution.
  • One unit of enzyme activity was defined as the amount of enzyme required to inhibit 50% of the NBT photoreduction, in comparison with tubes lacking the plant extract.
  • SOD activity staining after 12.5 % PAGE was performed to determine any change in the activity of SOD isozymes.
  • the gel was soaked in 50 mM Tris-HCl (pH 8.0) containing 2.5 mM NBT for 25 min at room temperature. Cu/Zn-SODs were inhibited with KCN and H 2 O 2 and Fe-SODs were inhibited with H 2 O 2 ; Mn-SODs are resistant to both inhibitors (Fridovich, 1989).
  • the gel was rinsed in distilled water and then incubated in the same buffer, containing 28 mM TEMED and 28 ⁇ M riboflavin, for 30 min. The gel was placed under an illuminator for 30 min to develop the purple color, except for the areas where SOD was localized in gel.
  • chitosan oligomers Fully deacetylated chitooligosaccharides (chitosan oligomers) induce, depending on their degree of polymerization and concentration, PAL activation in Arabidopsis thaliana cell suspensions whereas reacetylation of the chitosan oligomer elicitors did not affect the activation of PAL (Cabrera, 2006).
  • TAL activity in T17 treated leaves increased until 48 h after treatment and thereafter slightly decreased (Figure 16B).
  • TAL activity in chitin hexamer treated leaves increased continuously throughout experiment period, while TAL levels in chitin hexamer and Tl 7 treated leaves were unaffected by treatment and remained low.
  • TAL activity was increased by 57.0% in Tl 7 treated leaves but by only 18.8% in chitin hexamer treated leaves, as compared with the control treatment.
  • TAL activity was increased by 5.0% in T17 and 23.8% in leaves of chitin hexamer treated plants, respectively, compared with the control.
  • This example describes transformation of corn plants with a thuricin-17 coding sequence by particle bombardment.
  • Embryo genie cultures of corn are produced and bombarded with gold particles coated with plasmic DNA containing a gene fragment of thuricin-17 and a selectable marker gene following the methods described by Ramputh et al. (2002).
  • Immature corn embryos (1.5-2.0 mm long) are isolated and cultured on medium consisting of N6 salts and vitamins, 2% sucrose, 1 mg L "1 2,4-D, 25 mM proline, 100 mg L "1 vitamin- free case amino acids, 10 ⁇ M silver nitrate and 0.6% Phytagar (GibcoBRL).
  • the embryogenic cultures are maintained at 25 °C in the dark and transferred to fresh medium every 2 weeks.
  • the thuricin-17 gene fragment is isolated from genomic DNA, and is then blunt-ended and ligated into a small site of a plasmid vector using a selectable marker. The constructed vector is then used for particle bombardment.
  • the cultures are transferred to medium containing 0.5 mg L "1 2,4-D and 10 mg/1 benzyl adenine and incubated for 1 week in the dark.
  • the cultures are then transferred to 0.5 x salts, hormone-free, 1% sucrose medium and cultured at 25 0 C in a 16 h photoperiod (50 ⁇ E ⁇ T 2 s "1 ) for plant development.
  • Rooted plants are established in soil and grown to maturity in greenhouses.
  • Transgenic lines are confirmed by Southern analysis and transgene activity by Northern analysis and by in-planta thuricin 17 production activity.
  • Agrawal AA Overcompensation of plants in response to herbivory and the by-product benefits of mutualism, Trends in Plant Science, 5:309-313, 2000.
  • Bai Y. et al Enhanced soybean plant growth resulting from co-inoculation of Bacillus strains with Bradyrhizobium japonicum, Crop Science, 43:1774-1781, 2003.
  • Bottini R. et al, Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase, Applied Microbiology and Biotechnology, 65:497-503, 2004.
  • Degrassi G. et al, Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacterial sensors, Current Microbiology, 45:250-254, 2002.
  • Driscoll, B.T. et al A novel bacteriocin, thuricin 17, produced by PGPR strain Bacillus thuringiensis NEB 17: isolation and classification, Journal of Applied Microbiology, 100:545-554, 2006.
  • Fridovich Superoxide dismutase. an adaptation to a paramagnetic gas, Journal of Biological Chemistry, 264:7761-7764, 1989.
  • Gutierrez Manero, FJ. et al The influence of native rhizobacteria on European alder (Alnus glutinosa L. Gaertn.) growth. II. Characterization and biological assays of metabolites from growth promoting and growth inhibiting bacteria, Plant and Soil, 182:67- 74, 1996. Gutierrez Manero, FJ. et al, The plant-growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins, Physiologia Plantarum, 111 :206-211 , 2001.
  • Gyobu Y. et al, Proposal to transfer Actinomadura carminata to a new subspecies of the genus Nonomuraea as Nonomueraea roseoviolaceae subsp. Carminata comb, nov, InternationalJournal of Systematic Evolution and Microbiology, 51:881-889, 2001.
  • Hyronimus, B. et al Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I 4 , Journal of Applied Microbiology, 85:42-50, 1998. Jack W.R. et al, Bacteriocins of Gram-positive bacteria, Microbiology Reviews, 59:171-200, 1995.
  • Lian, B. et al Evidence for the production of chemical compounds analogous to Nod factor by the silicate bacterium Bacillus circulans GY92, Microbial Research, 156:289-292, 2001. Lithgow, J.K. et al, The regulatory locus cinRl in Rhizobium leguminosarum controls a network of quorum-sensing loci, Molecular Microbiology, 37:81-97, 2000.
  • lichenin bacteriocin-like compound
  • Probanza A. et ah, Pinus pinea L. seedling growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus (B. licheniformis CET 5106 and B. pumilus CECT 5105), Applied Soil Ecology, 20:75-84, 2002.
  • Quadri L.E. et al, Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B, Journal of Biology and Chemistry, 16:12204-12211, 1994.
  • Bacteriocin small o ⁇ Rhizobium leguminosarum belongs to the class of N-acyl-Lhomoserine lactone molecules, known as autoinducers and as quorum-sensing co-transcription factors, Journal of Bacteriology, 178:366-371, 1996.
  • Torkar, K.G. et al. Partial characterization of bacteriocins produced by Bacillus cereus isolates from milk and milk products, Food Technology and Biotechnology, 41:121-129, 2003.
  • Venter A.R. et al. Analysis of the genetic region encoding a novel bacteriocin from Rhizobium leguminosarum viciae strain 306, Canadian Journal of Microbiology, 47:495-502, 2001.
  • Wilson R.A. et al Bacteriocin production and resistance in a field population of Rhizobium leguminosarum biovar viciae, Soil Biology and Biochemistry, 30:413-417, 1998.

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Abstract

L'invention porte sur un procédé permettant de favoriser la croissance végétale et/ou la résistance des plantes à une maladie, comprenant l'application sur une plante ou une graine de plante ou à l'environnement de croissance de celles-ci, d'un polypeptide purifié qui est une bactériocine et qui possède une activité favorisant la croissance végétale et/ou la résistance des plantes à une maladie. L'invention porte également sur des polynucléotides codant le polypeptide et sur des plantes comprenant de tels polynucléotides.
PCT/CA2008/000921 2007-05-16 2008-05-15 Thuricine 17 permettant de favoriser la croissance végétale et la résistance des plantes à une maladie et plantes transgéniques WO2008138129A1 (fr)

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CN103992971A (zh) * 2014-05-15 2014-08-20 郝之奎 一种Bacillus cereus MBRH3菌株及其筛选方法和应用
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CN109769615A (zh) * 2019-01-25 2019-05-21 广西壮族自治区农业科学院 一种促进花生根瘤固氮的方法
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CN102321554B (zh) * 2011-08-30 2012-12-05 新疆农业科学院微生物应用研究所 一种蜡状芽孢杆菌及其作为植物根际促生菌的应用
CN102321554A (zh) * 2011-08-30 2012-01-18 新疆农业科学院微生物应用研究所 一种蜡状芽孢杆菌及其作为植物根际促生菌的应用
CN103992971A (zh) * 2014-05-15 2014-08-20 郝之奎 一种Bacillus cereus MBRH3菌株及其筛选方法和应用
WO2019185129A1 (fr) * 2018-03-27 2019-10-03 The University Court Of The University Of Glasgow Résistance dérivée d'un pathogène bactérien chez les plantes
CN108588174A (zh) * 2018-04-20 2018-09-28 塔里木大学 一种检测杨树感染腐烂病的方法
CN108606003A (zh) * 2018-04-28 2018-10-02 金华市景和科技有限公司 一种提高植物抗辐射性能的调节剂的制备方法
CN109769615A (zh) * 2019-01-25 2019-05-21 广西壮族自治区农业科学院 一种促进花生根瘤固氮的方法
CN111557308A (zh) * 2020-04-24 2020-08-21 山东省农业科学院生物技术研究中心 一种花生专用微生物组合物及其制备方法和应用
CN114516905B (zh) * 2020-11-19 2024-04-09 武汉大学 植物光合调控基因tl7及其蛋白与应用
CN114516905A (zh) * 2020-11-19 2022-05-20 武汉大学 植物光合调控基因tl7及其蛋白与应用
CN114375989A (zh) * 2021-12-29 2022-04-22 淮阴工学院 杰米拉类芽孢杆菌w51在草莓和桃采后病害防治中的应用及应用方法
CN114375989B (zh) * 2021-12-29 2024-03-26 淮阴工学院 杰米拉类芽孢杆菌w51在草莓和桃采后病害防治中的应用及应用方法
CN115073575B (zh) * 2022-06-29 2023-07-21 北京林业大学 一种促进木本植物扦插成活的多功能肽及促进扦插生根的方法
CN115073575A (zh) * 2022-06-29 2022-09-20 北京林业大学 一种促进木本植物扦插成活的多功能肽及促进扦插生根的方法
CN116478238A (zh) * 2023-03-15 2023-07-25 北京衍微科技有限公司 用于促进根瘤生长的脂肽组合物
CN116478238B (zh) * 2023-03-15 2024-02-06 北京衍微科技有限公司 用于促进根瘤生长的脂肽组合物

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