THURICIN 17 FOR PROMOTING PLANT GROWTH AND DISEASE RESISTANCE AND TRANSGENIC PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of and priority from United States Provisional Patent Application No. 60/938,309, filed May 16, 2007, the content of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] 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.

BACKGROUND OF THE INVENTION

[0003] 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.

[0004] Bacteria-produced compounds of various kinds are known to have plant growth promoting activity. For instance lipo-chitooligosaccharides (LCOs) or nodulation (NOD) factors, produced by certain rhizobia, have been demonstrated to increase plant germination.

[0005] However, compounds known to improve plant growth at low concentrations have not been proteins produced by other organisms. Until the instant invention, there have not been reports of bacteriocins that increase plant growth or disease resistance.

SUMMARY OF THE INVENTION

[0006] The inventors have discovered, surprisingly, that bacteriocins may be used to

promote plant growth and/or promote disease resistance in plants.

[0007] Accordingly, in one aspect, 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.

[0008] In another aspect, 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:

(a) a polypeptide comprising the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO: 1);

(b) a polypeptide 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; and

(c) a 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).

[0009] In another aspect, the invention provides a composition comprising a purified polypeptide as described above, and a carrier or diluent.

[0010] In another aspect, the invention provides an isolated polynucleotide encoding a polypeptide as described above, or the complement thereto.

[0011 ] hi another aspect, the invention provides a vector comprising a polynucleotide or host cell as described above.

[0012] hi another aspect, 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.

[0013] In another aspect, 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.

[0014] In another aspect, the invention provides 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.

[0015] In another aspect, the invention provides a plant seed treated with the plant growth and/or disease resistance promoting composition as described above.

[0016] In another aspect, the invention provides a kit comprising a plant growth and/or disease resistance promoting composition as described above and instructions for use.

[0017] In another aspect, 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:

(a) providing a polypeptide;

(b) determining whether said polypeptide promotes plant growth and/or disease resistance; and

(c) determining whether said polypeptide has bactericidal and/or bacteristatic properties.

[0018] In another aspect, 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:

(a) providing a bacteriocin; and

(b) determining whether said bacteriocin has plant growth and/or disease resistance promoting properties.

[0019] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the figures, which illustrate, by way of example only, embodiments of the present invention:

[0021] 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.

[0022] 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 μL (circles), 100 μL (closed squares), 300 μL (triangles), and 600 μL (open squares) of PPBP (0.066 μg μl ^"1 ).

[0023] FIG. 3 illustrates a SDS-PAGE analysis on PPBP and the CFS, as well as direct detection of PPBP and CFS. 20 μL of PPBP and CFS were loaded into wells, media exposed to the same conditions as for the PPBP and CFS served as controls. For direct detection of bacteriocin activity, 35 μL 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).

[0024] 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.

[0025] 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.

[0026] FIG. 6A-C illustrate a visual representation of inhibition of thuricin 17 as it relates to its production. Age of culture, via optical density, was initially determined via spectrophotometry, (A) 1.46; (B) 1.30 and (C) 1.13. Inhibition by thuricin 17 was conducted via the disk diffusion assay on the indicator strain Bacillus thuringiensis ssp.

[0027] 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.

[0028] 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".

[0029] Fig. 7B provides the 625 bp genomic DNA sequence encoding the thuricin 17 repeat sequences depicted in Fig. 7.1.

[0030] Fig. 7C depicts the predicted amino acid sequence of thuricin 17.

[0031 ] 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.

[0032] 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).

[0033] FIG. lOA-C illustrates the characterization of the plant biological activity of thuricin 17 on soybean (Glycine max L.) germination (%). The chromatogram was initially separated into 5 fractions (61-70, 71-80, 81-90, 91-100 and 101-110 minutes) (Figure 10A), then further subdivided (B) 81-82, 83-84, 85-86, 87-88 and 89-90 (Figure 10B). Material from B, thuricin 17 purified from fraction 86-87) was then assessed to determine the optimum concentration for increasing germination, (Figure 10C). Bars represent ±SE (n = 10).

[0034] 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).

[0035] FIG. 12 illustrates a schematic diagram of planting methodology for corn seeds supplied with varied concentrations of thuricin 17 solutions.

[0036] FIG. 13 illustrates corn seedling emergence (%) at 72h, 76h, 8Oh and 84h after eight treatments with one of three different solutions of thuricin 17 (10 ^"9 M, 10 ^"10 M, or 10 ^"u M) or a control treatment. Values are means ± SE of n= 4-5 replicates.

[0037] FIG. 14 illustrates tomato seedling emergence (%) at 72h, 12Oh, 144h and

168h after eight treatment with one of three different solutions of thuricin 17 (10 ^"9 M, 10 ^"10 M, or 10 ^"π M) or control treatments. Values are means ± SE of n= 4-5 replicates.

[0038] 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.

[0039] 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 μmol/L)) and thuricin 17 (1 x 10 ⁸ M). Control (open circles), chitin hexamer [(GlcNAc) _ό ] (circles), thuricin 17 (triangles), chitin hexamer and thuricin 17 (squares). Each point represents the mean ± SE (n=3).

[0040] 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. Each bar represents the mean ± SE (n=3).

[0041] 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. Each bar represents the mean ± SE (n=3).

[0042] 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.

DETAILED DESCRIPTION

[0043] 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. Specifically, the polypeptides demonstrate at least one bactericidal or bacteristatic activity against a related or unrelated bacterial strain, preferably a related strain.

[0044] As used herein, the term "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.

[0045] As used herein, the term "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.

[0046] As used herein, the term "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 (μmol cm ^"2 s ^"1 ), or an increase in accumulated dry-weight of the plant.

[0047] As used herein, the term "plant disease resistance promoting activity" or the

like, 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.

[0048] Polypeptides useful in practicing the methods of the invention can be obtained in a number of ways. For example, 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. In one embodiment, the polypeptide will be produced by a bacterial strain known to be a plant growth promoting strain such as a PGPR. In another embodiment, the polypeptide is obtained from a bacterial strain and known to be a producer of bacteriocin.

[0049] Methods for testing compounds for bactericidal or bacteristatic properties are known in the art. For example, 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.

[0050] Assessment of the plant growth promoting activity of polypeptides may be accomplished by known methods. For instance, 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.

[0051] 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. Various methods for

detecting or measuring increases in enzyme activity levels in plants (e.g. PAL, TAL, POD, CAD and SOD) are known in the art and exemplary techniques are described in the examples herein. Similarly, techniques for determining concentrations or levels of total phenolic compounds are known and exemplary methods are described in the examples herein.

[0052] 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.

[0053] Bacteria that are known to produce bacteriocins include, but are not limited to, Bacillus, Pseudomonas, Rhizobium, Braydyrhizobium and Lactoccus species.

[0054] 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.

[0055] Most known bacteriocin producing Bacillus species are from either soil or food isolates. 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). B. 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.

1999) and B. cereus strain 8 A, from the soils of Brazil, produces cerein 8 A (Bizani and Brandelli 2002).

[0056] Bacteriocins such as those described above may be tested for plant growth and/or disease resistance promoting activity as described herein.

[0057] 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).

[0058] Some 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).

[0059] 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.,

2000) and by suppressing disease through antibiosis (Maurhofer et al., 1992). Both ePGPR and iPGPR may be used in the practice of the invention. Illustrative examples of

ePGPR include Pseudomonas, Lactobacillus and Bacillus species, while illustrative examples of iPGPR include the rhizobia (species in the genera, for example, Rhizobium, Sinorhizobium, and Bradyrhizobium species such as Bradyrhizobium japonicum), or species of Frankia.

[0060] The foregoing is not limiting and proteins from other sources (for example fungi, protists or cyanobacteria) may be tested for bactericidal and/or bacteristatic activity as well as plant growth and/or disease resistance-promoting activity.

[0061] In an embodiment, the polypeptide is obtained from or obtainable from Bacillus (e.g. B. thuringiensis or B. cereus), Pseudomonas, Rhizobium, or Bradyrh izobium .

[0062] In an embodiment, the polypeptide is a class IE) bacteriocin.

[0063] hi one embodiment 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.

[0064] hi one embodiment, the polypeptide is a bacteriocin (designated BF4) which is obtainable from or obtained from B. thuringiensis strain BUPM4. hi another embodiment, the polypeptide is a bacteriocin (designated C85) which is obtainable from or obtained from B. cereus strain UW85. BF4 (strain BUPM4) and C85 (strain UW85) bear strong likenesses to Tl 7 (strain NEB 17). Each of these bacteriocins does not kill the strains that produce the other two, indicating the same mechanism of action (and the same mechanism for protecting against T17, BF4 and C85). 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.

[0065] As used herein, by "obtainable" it is meant that the 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. For instance, the polypeptide could be produced recombinantly in a host cell or organism or synthesized chemically.

[0066] In one embodiment the polypeptide may possess one or more of the following properties:

(a) the polypeptide may maintain bactericidal and/or bacteristatic activity after exposure to a temperature of 100°C for at least 15 minutes;

(b) the polypeptide may maintain bactericidal and/or bacteristatic activity after treatment with α-amylase or catalase, but exhibit loss of activity after treatment with proteinase K or protease; and

(c) the polypeptide may have molecular weight in the range of about 3100 to 3200 Da.

[0067] In one embodiment, 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). In one embodiment such a polypeptide is obtained from or obtainable from Bacillus thuringiensis strain NEB 17 (IDAC 270303-02).

[0068] In one embodiment, 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. As used herein, the terms "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.

[0069] Accordingly, 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. The term "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/ ^' ).

[0070] 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). Alternatively, 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). Generally, 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.

[0071 ] 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.

[0072] The polypeptides are preferably in purified form. By "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. Thus, for example, 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. Typically, 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.

[0073] hi one embodiment, polypeptides may be obtained from bacterial species that express the polypeptides. For instance, 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. In a preferred embodiment, 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).

[0074] 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.

[0075] Alternatively, the polypeptides may be synthesized chemically via known procedures.

[0076] Polypeptides may be applied either before, during or after planting and may be applied to, for example, plant leaves, stems, roots, or seeds. As used herein and in the claims, the term "plant" includes without limitation whole plants, plant parts, organs, leaves, stems and roots. For greater certainty, 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. By "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 skilled person can readily determine suitable application regimes for the polypeptide. In one embodiment, 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.

[0078] In practicing the methods of the invention, 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.

[0079] The composition may take the form of plant seeds pre-treated with the plant growth and/or disease resistance promoting composition.

[0080] 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.

[0081] 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). Various elements of the multi-component plant defense mechanism induced by elicitors include the hypersensitive reaction (HR) (Artat et al., 1994), the production of activated oxygen species (oxidative burst)

(Apostol et al., 1989), the modification of plant cell walls by deposition of callose (Conrath et al., 1989), and the synthesis and accumulation of antimicrobial phytoalexins (Dixon et al., 1983). In addition to these localized defenses, systemic acquired resistance (SAR), which increases the plant's resistance to subsequent pathogen attack, is activated in many plants; it can also be induced by specific elicitor compounds (Somssich et al., 1986).

[0082] 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 β-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).

[0083] Accordingly, 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.

[0084] 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.

[0085] Similarly 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 lignifϊcation-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.

[0086] The 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.

[0087] 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.

[0088] The 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.

[0089] For example, 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. In this embodiment, 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.

[0090] The 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.

[0091] hi another embodiment, the invention provides polynucleotides encoding the polypeptides of the invention. The term "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.

[0092] 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. For example, a nucleic acid molecule of the invention, inserted into

a vector or linked to a foreign promoter, is in "isolated" form.

[0093] In an embodiment, 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:

(a) a polypeptide comprising the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO: 1);

(b) a polypeptide 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; and

(c) a 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).

[0094] In an embodiment, 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] The term "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".

[0099] 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. In addition, 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.

[00100] 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. The terms "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.

[00101 ] Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms "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.

[00102] 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. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example 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.

[00103] In an embodiment of the invention, 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.

[00104] A cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid (e.g. exogenous or heterologous DNA [e.g. a DNA construct]), 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).

[00105] For stable transformation of plant cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (such as resistance to antibiotics) may be introduced into the host cells along with the gene of interest. As used herein, the term "selectable marker" is used broadly to refer to markers which confer an identifiable trait to the host cell. Non- limiting example of 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).

[00106] In one embodiment, 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. This avoids the requirement of applying the polypeptide to the plant independently. 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.

[00107] 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.

[00108] hi Agrobαcterium-mediated transformation, 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 Agrobαcterium is typically achieved in one of two ways. In the first, protoplasts or leaf-discs can be incubated with the Agrobαcterium and whole plants regenerated using plant tissue culture. Alternatively, some plant species may be transformed simply by dipping flowers into a suspension of Agrobαcterium and then planting the seeds in a selective medium.

[00109] hi the particle bombardment or biolistic method, particles, typically of gold or tungsten, are coated with the nucleic acid of interest and propelled into plant cells at high velocity. Although the transformation efficiency is lower than that of Agrobαcterium-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.

[00110] hi the electroporation method, an electrical field is used to create pores in the plant cell wall, to permit entry of the nucleic acid of interest.

[00111 ] Alternatively, viral vectors may be used to transform plant cells.

[00112] These and other transformation methodologies suitable for preparing transgenic plants stably transformed with a thuricin 17 coding sequence are well known in the art as reviewed in Rakoczy-Trojanowska M. (2002) and Newell, CA. (2000).

[00113] The invention is further exemplified by the following non- limiting examples.

EXAMPLES

Example 1

a) Bacterial Strains and Culture Preparations

[00114] 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. A cell free supernatant (CFS), containing the BtNEB 17 compound, was prepared by centrifuging the bacterial culture at 13,000g for 10 min on a Sorvall Biofuge Pico (Mandel Scientific). The supernatant was collected and the bacterial compound was detected via analytical-HPLC on a Vydac C: 18 reversed-phase column

(0.46 x 25 cm; 5 μM) (Vydac, CA, USA; catalogue # 218TP54). The HPLC was fitted with Waters 1525 Binary HPLC pump and a Waters 2487 Dual λ Absorbance detector set at 214 nm. All other bacterial strains, sources and culture media are given in Table 1 below.

b) Extraction and Partial Purification of the Bacterial Peptide

[00115] For partial purification of the bacterial peptide, 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). Prior to HPLC analysis, samples were centrifuged on a Sorvall Biofuge Pico (Mandel Scientific) at 13,000 g for 13 min, and the supernatant collected for chromatography. HPLC analysis (Waters, MA, USA) was conducted on a Vydac C: 18 reversed-phase column as described above.

[00116] 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). As a control, purified culture media, without btNEB17, was subjected to the same purification procedures.

[00117] As shown in Figure 1, PPBP and CFS shows a distinctive peak when analyzed via HPLC and in both cases the peak retention times were 80 -82 min (Figure IA and Figure 1C, respectively). In purified culture media without Bacillus thuringiensis NEB 17 this peak is absent (Figure IB).

c) Initial Characterization of the Bacterial Peptide

[00118] The BtNEB 17 compound was initially assessed for protein content via the

Bradford assay (Bradford 1976). Aliquots of 2 mL of PPBP with retention times of 80 - 82 min were lyopholized at -60 ⁰ C, under vacuum pressure. This was conducted using a Savant Modulyo Freeze-dryer fitted with a Savant Model VPOF oil pump and Savant Model VPL200 air pump. Two hundred μL of ddH ₂ 0 were added to samples and the Bradford assay was performed with samples being read for absorbance at 595 ran.

d) Inhibition Range and Activity Assessment

[00119] 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. In the present example, to assess the range of antimicrobial activity, 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). Other inhibited species/strains were Brevibacillus brevis, Geobacillus stearothermophilus, 2/2 Paenibacillus polymyxa and Escherichia coli MM294 (pBS42). Bacillus strains not inhibited included 0/3 B. subtilis plus the plant growth promoting strains listed (Table 1).

Table 1 : Antimicrobial spectrum of the bacterial peptide using the disk diffusion assay, where 15 μL of CFS were spotted on to sterilized 6 mm disks. The assay was done twice with two separate biological replicates, in duplicate (n=4).

Zone of

Inhibition in

Indicator Species Source* Diameters (mm)

Bacillus thuringiensis NEB 17 * SLC ¹ —

Bradyrhizobium japonicum 532C USDA ² —

Bradyrhizobium japonicum USDA 3 t USDA ² —

Bradyrhizobium japonicum USDA 110 USDA ² —

Escherichia coli MM294(pBS42) £ BGSC ³ 7.5

Escherichia coli JM83(pMK3) £ BGSC ³ —

Pseudomonas putida NRLL-B- 14688 ^f ARSCC ⁴ —

Ralstonia spp. Hl 6 ATCC 17699 * ATCC ⁵ —

Serratia proteomaculans 1-102 ' SLC ¹ —

Serratia proteomaculans 2-68R ' SLC ¹ —

Stenotrophomoas meiltophilia Alfa-nod KU ⁶ —

Staphylococcus epidermidis ATCC 12228 ^ ATCC ⁵ —

B. thuringiensis subsp. thuringiensis HD2 ^ BGSC ³ 10.5

B. thuringiensis subsp. kurstaki HDl ^ BGSC ³

B. thuringiensis subsp. sotto 4-1 * BGSC 10

Zone of Inhibition in

Indicator Species Source* Diameters (mm)

B. thuringiensis subsp. galleriae HD29 ^* BGSC ³ 10

B. thuringiensis subsp. canadensis HD224 ^ BGSC ³ 8

B. thuringiensis subsp. entomocidus HDlO ^ BGSC ³ 13

B. thuringiensis subsp. entomocidus HD9 ^ BGSC ³

B. thuringiensis subsp .subtoxicus HD 109 ' BGSC ³

B. thuringiensis subsp. morrisoni HD 12 ' BGSC ³ 8

B. thuringiensis subsp. darmstadiensis HD146 (103) ^ BGSC 9

B. thuringiensis subsp. pakistani HD395 * BGSC ³ 15

B. thuringiensis subsp. Indiana HD521 ^ BGSC ³ 9.5

B. thuringiensis subsp. tochigiensis HD868 (117-72) * BGSC ³ 7

B. thuringiensis subsp. cameroun 273B * BGSC ³ 11

B. thuringiensis serovar. xiaguangiensis 3397 * BGSC ³ 10

B. thuringiensis serovar. asturiensis EA 34594 ^ BGSC ³ 7

B. thuringiensis serovar. rongseni Scg04-02 ⁺ BGSC ³ 12

B. thuringiensis subsp. thuringiensis Btl627 * BGSC ³ 19

B. thuringiensis subsp. alesti HD4 * BGSC ³ 13.5

B. cereus T-HT ^f BGSC 13

Zone of Inhibition in

Indicator Species Source* Diameters (mm)

B. cereus T-HW3 BGSC

B. cereus 6A3 StrepR ^f BGSC ^J 18

B. cereus ATCC 14579 ^f BGSC ³ 14

B. licheniformis Alfa-Rhiz * USDA ²

B. licheniformis 9945A ^f BGSC ³ 9

B. licheniformis 749 ^ BGSC ³ 7.5

B. megaterium ATCC 19213 ^f BGSC ³ 18

B. megaterium QM B 1551 ^f BGSC ³ 14

B. pumilus ATCC 7061 ^f BGSC ³ 9

B. pumilus Biosubtyl * BGSC ³

B. sphaericus 1593 ^ BGSC

B. subtilis NEB 5 SLC

B. subtilis NEB 4 SLC ¹

5. subtilis subsp. subtilis 168 ^ BGSC ³

5αcz7/ws KTCC Bl KU ⁶ 12

Bacillus KTCC B2 KU ⁶ 13

Aneurinibacillus migulanus NRS-1137T ^ BGSC ³

Zone of Inhibition in Indicator Species ^a Source* Diameters (mm)

Brevibacillus brevis ATCC 8246 * BGSC ^ό 17

Geobacillus stearothermophilus 10 BGSC 11

Paenibacillus polymyxa ATCC 842 ^¥ BGSC ³ 10.5

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. Smith Laboratory Collection, Department of Plant Science, McGiIl University, Montreal, Quebec, Canada; USDA ² : United States Department of Agriculture, USA; 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.

[00120] 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 μL 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). Media types and components for the varying indicator strains consisted of King's Medium; YEM (Vincent 1970) consisting of: mannitol (10.0g L ^"1 ), K ₂ HPO ₄ (0.5 g L ^'1 ), MgSO ₄ (0.1954 g mL ^"1 ), NaCl (0.1 g U ¹ ), yeast extract (0.4 g L ^"1 ) and agar (15 %); MacConkey' s (Microbiology, Germany) agar prepared according to suppliers instructions, with 5 μg mL ^"1 of chloramphenicol for Escherichia coli MM294(pBS42) and 50 μg mL ^"1 of ampicillin for E. coli JM83(pMK3); 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 ).

[00121] 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 μL 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 (μg L ^"1 ) was determined and compared with the AU.

e) Mode of Action Assessment

[00122] The mode of action was assessed following the methods of Ahern et al.

(2003). Briefly, subculture strains of B. thuringiensis ssp. thuringiensis BtI 627 and B. cereus ATCC 45679 were grown in King's medium (Atlas 1995) to an O.D. ₆₀ o _nm of 0.35 - 0.40. At this time, cultures were diluted, with sterilized medium, to an O.D. _6O o _nm of 0.27 - 0.30. Cultures were then divided into 10 mL aliquots and placed into 30 mL flasks. Volumes of 0, 100, 300 and 600 μL of PPBP (0.066 μg μl ^"1 ) were added to the cultures. B. 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 Ultrospec™ 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 μL 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 μg μL ^'1 ).

[00123] An assessment of the mode of action confirmed that the bacterial peptide is both bactericidal and bacteristatic (Figures 2A-C). From the onset of exposure, cell

density of both B. thuringiensis spp. thuringiensis Bt 1627 (Figure 2C) and B. cereus ATCC 14579 (Figure 2B) decreased continually and cell lysis eventually occurred. The static effect was observed on B. cereus ATCC 14579 (Figure 2B), while B. thuringiensis spp. thuringiensis Bt 1627 (Figure 2C) was able to recover showing a later increase in growth. B. thuringiensis NEB 17 (Figure 2A) served as a negative control and there was no effect on the producer strain. Results were consistent between experiments using the CFS and the PPBP.

f) SDS-PAGE and Direct Inhibition

[00124] Samples of PPBP and CFS were run on a SDS-PAGE gel and compared against a protein standard (1.4 - 26.6 kDa, Bio-Rad catalogue #161-0326). Purified medium (passed through HPLC purification and collected at 80-82 min), centrifuged medium, and 6x loading dye were controls. An aliquot of either PPBP or CFS was diluted in a 1 :1 (vol/vol) SDS/sample buffer and denatured by heating for 5 min at 100 ⁰ C. For direct detection of BtNEB 17 peptide activity, PPBP, CFS, purified medium and centrifuged medium (all without added sample buffer and loading dye) were run on the same gel. Samples were run on a 22% polyacrylamide gel using a tris-glycine running buffer at pH 8.3, 200 V, and 50 niA gel ^'1 (changed after 15 min to 30 niA gel ^'1 ). After electrophoresis, the gel was fixed for 30 min in 25% isopropanol and 10% acetic acid and sliced vertically. The first half was stained with Coomassie Blue (100 mL acetic acid, 900 mL ddH ₂ O:methanol (1 : 1), 2.5 g Coomassie blue G-250) for Ih. It was then destained with two washings of 100 mL acetic acid and 900 mL ddH ₂ O:methanol (1:1), then left to destain overnight. The second half of the gel was soaked in several changes of distilled water for overnight and overlaid with soft agar in a Petri dish. Direct detection of the BtNEB 17 peptide was determined using the indicator strain, Bacillus thuringiensis ssp. thuringiensis Bt 1267. Briefly, 300 μL of culture containing the indicator strain was inoculated onto the plate. The Petri plate was maintained at 27 ⁰ C for at least 48 h.

[00125] 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).

g) Enzyme Degradation/ Heat and pH stability assays

[00126] Separate samples of the PPBP and CFS samples were digested with

Proteinase K (from Tritirachium album, Sigma No. P-2308), Protease (from Streptomyces griseus, Sigma No. P-6911), α-amylase (from barley malt VIII-A, Sigma No. A-2771) and catalase (from Corynebacterium glutamicum, Sigma No. 02071). Upon enzymatic digestion the PPBP was then tested for antimicrobial activity. Working buffer solutions and pH levels were as follows: proteinase K - 100 mol L ^"1 Tris-HCL, pH 7.5 at 20°C; protease - 0.04 mol L ^"1 potassium phosphate (equal volumes of monobasic/dibasic), pH 7.5 at 20°C; a-amylase - 0.02 mol L ^"1 sodium phosphate (monobasic) plus 0.06 mol L ^"1 NaCl, pH 6.9 at 20 °C; catalase - 0.05 mol L ^"1 potassium phosphate (monobasic), pH 7.0 at 20°C. For proteinase K, protease and α-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.

[00127] Heat stability assays were conducted on the CFS and PPBP. A 250 μL sample of material was heated for 30 min at the following temperatures: 40, 60, 75, 100, and 121 °C 250 μL were also exposed to -20 °C for 30 days, 4 °C for 30 days and 22 °C for 24 h. To assess an effective pH range, 10 mL of CFS were subjected to a range of pH

from 1.00 - 13.75 (modified from Oscariz et al 1999). As large volumes of material were needed for this assay the CFS were used to determine pH stability, instead of the PPBP. Centrifuged medium adjusted to the same pH was a control. 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).

[00128] The biological activity of the PPBP and the CFS disappeared completely when exposed to 2 mg mL ^"1 of protease and almost completely when treated with proteinase K (data not shown). Exposure to 1 mg mL ^"1 of proteinase K and protease resulted in partial loss of activity (data not shown). However, no loss of activity was seen when treated with either 1 or 2 mg mL ^"1 of α-amylase. Catalase when added at 40 000 U ml ^"1 or 60 000 U ml ^"1 had no effect on the activity of the PPBP and the CFS. To ensure that any degradation and/or denaturation resulting in loss of bioactivity was not due to heat treatment used in the enzyme assay, controls were run, wherein PPBP and CFS were exposed to 37 ⁰ C for 2 h and 37 ⁰ C for 2 h plus 100 ⁰ C for 2 min (Table 2). No loss in bioactivity was found when the PPBP and CFS were exposed to these conditions, ensuring that the loss of activity during the proteinase K and protease digestion assays were actually due to the enzyme degradation.

[00129] Both the CFS and PPBP were stable over a wide heat range, and were resistant to degradation when exposed to 100 ⁰ C for 15 min (Table 2). They were also stable when kept at -20 ⁰ C for 30 days, at 4 ⁰ C for 30 days and resistant to lypholization. The pH stability was between 1.00 and 9.25. At higher pH levels, the biological activity disappeared, and the peptide was not effective as a bacteriocin. Results from the activity assessment show the CFS had 32 AU (0.071 μg μL ^"1 ). This was repeated on a new culture of bacteria and another CFS was generated and results were similar, 64 AU (0.059 μg μL ^"1 ).

[00130] Table 2: Characterization of the PPBP in response to varying temperature and pH levels.

PH Duration % Activity *

1.00 3h 50

1.25 3h 91

1.50 3h 100

1.75 3h 100

2.00 3h 100

4.00 3h 100

6.00 3h 100

8.00 3h 100

9.00 3h 73

9.25 3h 50

9.50 3h 0

Temp. (C°) Duration % Activity**

-20 30 days 100

4 30 days 100

22 24 h 100

37 2h 100

45 30min 100

60 30min 100

80 30min 94

100 2 min 100

37 + 100 2 h + 2 min | 100

100 15 min 95

121 5 min 0

* Activity was determined via disk diffusion assay on one separate biological replicate of the CFS and on one separate biological replicate of PPBP ** f Samples were incubated at 37 ⁰ C for 2 h, followed by incubation at 100°C for 2 min. Tested on the indicator strain, B. thuringiensis ssp. thuringiensis Bt 1627 and/or B. cereus ATCC 14579.

h) Mass Spectroscopy

[00131 ] Prior to analysis, PPBP samples were lyopholized as described above. Analysis was conducted at the Genome Quebec and McGiIl University Innovation Center, using an Ultima MALDI (Matrix Assisted Laser Desorption Ionization) - QTOF (Quadrapole Time of Flight) mass spectrophotometer (Waters Corp., Milford, Massachusetts). For comparison of methods, additional mass spectrometry analysis was also conducted on a fast-atom bombardment mass spectrometry (FAB-MS) in positive mode in a JEOL-SX/SX102A mass spectrometer (JOEL Inc., Toyko, Japan). Both types of mass spectrometry analysis were conducted on material isolated from different BtNEB 17 cultures, grown at different times.

[00132] As discussed above, SDS-PAGE provided an estimate of the bacterial peptide's molecular weight. MALDI mass spectrometry data confirmed these results. A strong mass peak from MALDI analysis is observed at 3162.3 Da (Figure 4). Additional testing, using FAB mass spectrometry, yielded similar results (data not shown).

Example 2

Peptide Sequence and Production of Tl 7 by NEBl 7

a) Bacterial Strains and Tl 7 Isolation

[00133] 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. ₆ oo _nm of at least 1.4 (or approximately 5.5 log CFU (colony forming units) cells per mL) as determined using spectrophotometry Ultrospec™ 4050 Pro UV/ Visible Spectrophotometer LKB (Cambridge, England).

[00134] 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.

[00135] For HPLC analysis samples were centrifuged on a Sorvall Biofuge Pico™

(Mandel Scientific) at 13,000 g for 13 minutes, and the supernatent collected for chromatography. The HPLC (Waters, MA, USA) had a Vydac Cl 8 reversed-phase column and the gradients of acetonitrile:water during the fractionation were as follows: 45 min. at 18% acetonitrile, 45 to 110 min. 18 to 60.4% acetonitrile; 110 to 115 min, 60.7 to 100% acetonitrile, then finally, 115 to 120 min at 100 to 18% acetonitrile. The material was collected at 1 min intervals (Bai et al. 2002b), as it has been previously shown that Tl 7 elutes between 80 and 85 min. (Gray et al. 2006a).

b) Protein Sequencing

[00136] Protein sequencing was conducted at McGiIl University and at the Virginia

Bioinformatics Institute. Edman degradation for N-terminal sequencing was conducted on a Procise Applied Biosystems 492 gas-phase/pulsed-liquid automated sequencer. PTH (phenylthiohydantoin) derivatized amino acid residues were then analyzed on a C: 18 HPLC column. The amino acid sequence was then assigned using the software program Model 610A. Sequencing was conducted one time each on two separate biological replicates of material from NEB 17. However, there was a sudden stop in the sequence after the 18 ^th cycle during each run.

[00137] Attempts to digest the peptide with carboxypepsidase Y were unsuccessful, along with digestion attempts with trypsin (added at a concentration of 2μg/ 200μL of peptide), which gave sufficient amounts of activity of chromotrypsin. However, a successful combination of carboxypepsidase Y and W allowed the generation of a C-terminal ladder, in which two additional amino acids were cleaved and the possibility of a third, though the signal was not as strong. The sequence then stopped at that point.

[00138] Sequence determination of the thuricin 17 peptide was conducted via a combination of Edman Degradation based N-terminal sequencing and tandem mass spectrometry. The data showed the N-terminal sequence as follows: WTCWSCLVCAACSVELL (SEQ ID NO: 1). During the Edman Degradation the analysis stopped after the 18 ^th cycle and did this consistently in both repetitions. The positions of cysteines within the sequence were not determined by N-terminal sequencing since this amino acid is degraded during the Edman sequencing reaction. Instead they were determined by ms/ms fragment analysis of the parent ion, of mass 3051 Da (Fig. 5). In determining the sequence, the 3061 Da ion was used. 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.

[00139] 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. They proposed the presence of cysteine in the sequence that was obtained, as no signal could be determined at some points during the Edman degradation. Furthermore, the sequence information for Plantaricin S, a bacteriocin produced from Lactobacillus plantarum LPCOlO requiring the complementary action of two peptides, was obtained up to amino acid residues 26 and 24; a PTM may have prevented further sequencing (Jimenez-Diaz et al. 1995).

[00140] 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. The activity of T7, from B. thuringiensis BMGl.7, was inhibited by proteinase K, but not with trypsin (Cherif et al. 2001). 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.

c) Amino Acid Analysis

[00141] Amino acid analysis was performed at the University of Virginia Health Center. Briefly, the peptide was hydrolysed in 6N HCL vapor for 24 h at 11O ⁰ C, to free the amino acids. During this assay, glycoproteins, containing a carbohydrate (CHO) moiety, will often turn black, as carbon is oxidized. This can indicate the presence of a CHO on the protein. Derivatization occurred; this yields phenylthiocarbamyl (PTC) amino acids that are analyzed via HPLC, where the instrument is fitted with a C: 18 reverse phase column.

[00142] Amino acid analysis results coincided, for the most part, with the sequence data. The amino acid analysis detected the presence of 1- Asx, 1-Glx, 3-Ser, 1-Gly, 4-His, 2-Thr, 7-Ala, 3-Val and 4-Leu, which yields an estimated molecular weight of 3242 + 1

H ₂ O, for a total of 3260 Da. Interestingly these provide an overestimate of the molecular weight by 100 Da. This may be explained in that the configuration of amino acids in the presumed PTM(s) is not known. This suggests a PTM of 100 Da that was undetected during the initial mass spectrometry analysis. Furthermore, in digesting the peptide some amino acids could be counted more than once. It was thought that perhaps the remaining weight was due to a CHO (carbohydrate) moiety, which was the PTM blocking the Edman degradation. However, the presence of a CHO moiety results in a black color upon acid hydrolysis and this did not occur, making the presence of CHO unlikely. Thus, it was confirmed that Tl 7 does not contain a CHO component and its molecular weight is probably composed entirely of amino acids, perhaps with a 100 Da PTM.

[00143] BLAST searches on the sequence show some homology to other bacterial peptides such as Sinorhizobium meliloti 1021 complete chromosome; segment 6/12 Identities = 8/13 (61%), Positives = 10/13 (76%), Xanthomonas campestris pv. campestris str. ATCC 33913, Identities = 7/13 (53%), Positives = 11/13 (84%), Rhodobacter sphaeroides, Identities = 10/16 (62%), Positives = 10/16 (62%), Neisseria meningitidis MC58, Identities = 8/12 (66%), Positives = 11/12 (91%). However, 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.

d) Thuricin 17 Production

[00144] 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 Ultrospec™ 4300 Pro UV/Visible Spectrophotometer. The CFU was determined by diluting subsamples, taken each hour, in 0.9% NaCl solution. Fifty μL 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 μL 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. thuringiensis HD2 (Favret and Yousten 1989) and thuricin 7, B. thuringiensis BMG1.7 (Cherif et al 2001). Initial traces of T17 were found at an O.D. _ό oo _nm of 1.3 (Figure 6B). The concentration then continued to increase as the stationary phase continued. The AU was calculated as the reciprocal of the highest dilution that gave a visible inhibition zone. It has been previously shown that bacteriocin production occurs in the mid-logarithmic growth phase; these include entomocin 9, B. thuringiensis ssp. entomocidus HD9 (Cherif et al. 2003) and tochicin, B. thuringiensis HD868 (Paik et al. 1997).

Example 2A

[00145] Further DNA sequencing and sequencing analysis was performed to characterize thuricin 17 in greater detail.

[00146] 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).

[00147] Partial N- and C-terminal amino acid sequencing was previously used to determine the N-terminal peptide sequence of thuricin- 17 (Gray et al, 2005). Using known codon preferences for Bacillus thuringiensis and Bacillus weihenstephanensis, sequencing primers NEB-R and NEB-F2 were designed to obtain the nucleotide sequence of the region upstream and downstream of the thuricin- 17-encoding gene. The resulting

sequences were of very low quality, presumably due to the presence of multiple primer binding sites in this region. Downstream sequence analysis revealed a homologue of the albA gene of B. 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. This genetic region, including primer binding sites, is shown in Figure 7.A. 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.

Example 3

[00148] To determine whether or not thuricin 17 plays a role in plant growth enhancement by NEB 17, this study determined the effect of thuricin 17 on soybean photosynthesis and growth under controlled environment conditions.

a) Isolation of thuricin 17

[00149] Bacillus thuringiensis NEB17 was cultured in King's Medium B (Altas,

1995). 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. Subcultures were separated by differential centrifugation (Sorvall RC 5C Plus, Mandel Scientific Co., USA) for 20 min at 2,800 x g and 4°C. Butanol, to 60% the final volume, was added and the mixture was shaken at 4 ⁰ C overnight. The mixture was then allowed to stand for 2 h, while the two phases separated, after which the upper butanol layer was collected. The butanol was removed by rotary evaporation (Yamota RE500, Yamato, USA) at 5O ⁰ C under vacuum. 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). The HPLC was equipped a C ₁₈ reverse-phase column (Vydac218TP54, 300 nm, 5 μm, 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.

Bacteriocin Properties ofThuricin 17

[00150] To ensure that the material being tested was the bacteriocin thuricin 17, bacterial strains were tested for their inhibition by thuricin 17. This was done via the disk diffusion assay with strains cultured on King's Medium B (Atlas, 1995), solidified with 15% agar (Figure 9). Petri plates containing medium were inoculated with strains susceptible to thuricin 17 (Gray et al, 2006a) and 15 μL of thuricin 17 (10 ^"7 M) was spotted onto sterilized disks (6 mm diameter) (Figures 9C and D). Plates were then maintained at 28 ⁰ C for at least 48 h. Controls were the producer strain, B. thuringiensis NEB 17 and partially purified media (Figures 9 A and B).

b) Plant Bioanalysis ofThuricin 17

[00151 ] The 110 collected fractionations were initially assayed to assess their plant biological activity. In the first step, 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 Bayfϊeld. The active

fractions selected in the first step (81-90 minute fractions) 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.

[00152] Once the bioactivity of thuricin 17 had been established, the 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. As the molecular weight of this compound has been determined (Gray et al., 2006a) concentrations of thuricin 17 are given in Molar and the applied concentrations were 0, 5 x 10 ^"11 , 5 x 10 ^"10 and 5 x 10 ^"9 M, (water control, T17-1, T17-2 and T17-3, respectively) (Figure 10C). The germination assay was conducted as described above and the entire experiment conducted twice.

c) Greenhouse Experiments

[00153] Based on results from the germination assay, 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. Seven days after seeding, at the VE (emergent) stage (Fehr et al., 1971), seedlings were transplanted into 13 cm pots, each containing 100 g of sterilized dry vermiculite, at a rate of 1 seedling per pot. Four days after transplanting, healthy seedlings

were inoculated with B. japonicum 532C (BJ 532C).

[00154] 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. Cell density was estimated by spectrophotometry at 620 ran (Bhuvaneswari et al., 1980) and the broth was diluted with sterilized tap water to A ₆₂₀ = 0.08 (approximately 10 ⁸ cells mL ^"1 ), and the inoculation dose was 10 ⁸ cells per seedling (Zhang and Smith, 1994).

[00155] 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. For the largest plants this was equivalent to 1 mL per plant, with smaller amounts for smaller (earlier stage of development) plants. For soil irrigation, 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.

[00156] During the growth period, plants were watered daily with half strength nitrogen- free Hoagland's solutions (Hoagland and Arnon, 1950), in which the Ca(NO ₃ ) ₂ and KNO ₃ were replaced with 0.5 mM CaCl ₂ , 0.5 mM K ₂ HPO ₄ , and 0.5 mM KH ₂ PO ₄ to provide nitrogen free nutrient solution. The greenhouse temperature was 25 ± 2°C, relative humidity was 75% and a 16 h photoperiod was created by supplemental lighting from high-pressure sodium lamps. At each harvest, data were collected on plant height, leaf greenness (SPAD-502, Minolta, Japan), leaf area (Delta-T Devices, Cambridge, UK), nodule number and nodule dry weight, shoot and root dry weight (Zhang and Smith,

1995). Shoot, root and nodule tissues were air-dried at 6O ⁰ C for 5 days for determination of dry weight. Nitrogen content and photosynthesis were measured using an NC 2500 Elemental Analyzer (CE Instrument Inc., Italy) and Li-Cor 6400 (Li-Cor Inc, USA), respectively.

d) Statistical Analysis

[00157] 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.

[00158] Initial purification of thuricin 17 began with analysis of the crude extract of bacterial medium in which the PGPR strain Bacillus thuringiensis NEB 17 had been grown. The crude extract showed a peak that corresponded to thuricin 17 (Figure 8A). HPLC partial purification of thuricin 17 showed a distinctive peak on the chromatogram at approximately 85 minutes (Figure 8B). This peak was not present for control medium that had not grown B. thuringiensis NEB 17 (Figure 8C). The distinctive thuricin 17 peak facilitates isolation and recognition during the purification process. The thuricin 17 isolated and used in this experiment was bactericidal to the closely related strains B. cereus ATCC 14579, (Figure 9C), and Brevibacillus brevis ATCC 8246, (Figure 9D), confirming its bacteriocin nature. However, thuricin 17 did not inhibit the growth of B. thuringiensis NEB 17 (Figure 9A), the thuricin producer, or B. japonicum 532C.

[00159] The germination assay showed that material in fractions collected at 85-88 min caused the greatest stimulation of germination (Figures 8A and 8B). This HPLC retention time corresponds to that of thuricin 17. The concentration of thuricin 17 that caused the greatest stimulation of germination, relative to the medium extract control, was 10- ¹⁰ M (Figure 10).

[00160] hi greenhouse studies there were no interactions between cultivar and

application method, cultivar and concentration, and among cultivar, application method and concentration. However, there was an interaction between application method and concentration of thuricin 17, hence means are presented for this interaction. When applied as a leaf spray thuricin 17, treatment T 17-2 increased leaf photosynthetic rates about 6% over the control (from 13.75 to 14.55 μmol 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).

[00161] When applied to roots, all three thuricin 17 treatments increased photosynthetic rates, as compared with the control and T 17-2 had the greatest effect. Leaf greenness (SPAD reading) was increased by all thuricin 17 treatments, with Tl 7-1 having the greatest effect. Leaf area (cm ² plant ^"1 ) was also increased by thuricin 17 (Table 3). The greatest increase was due to treatment T17-1, being 173.0, followed by T17-2, 169.9, as compared with the control at 155.9 (Table 3). Plant dry weight increased from 1.147 (control) to 1.278 for T17-1 and 1.250 g for T17-2. Application of thuricin 17 did not affect plant height (Table 3).

[00162] 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

Shoot Root Total

μmol cm s ' cm plant cm gplanf

Leaf spray

Control ³ 13.75 c ^c 27.3 d 154.3 d 13.9 0.817 c 0.320 1.137 c

T17-r 13.86 c 28.1 c 163.6 be 14.1 0.860 be 0.327 1.187 be

T 17-2 14.55 a 29.2 a 175.2 a 14.5 0.942 a 0.362 1.304 a

T17-3 14.41 ab 28.7 abc 171.1 ab 14.3 0.928 a 0.357 1.285 a

Root irrigation

Control 13.63 c 27.3 d 155.9 cd 14.1 0.830 c 0.317 1.147 c

T17-1 14.43 ab 29.3 a 173.0 a 14.3 0.920 a 0.358 1.278 a

T17-2 14.44 a 29.0 ab 169.9 ab 14.2 0.893 ab 0.357 1.250 ab

T 17-3 14.03 be 28.4 be 156.6 cd 14.1 0.838 c 0.350 1.188 be

"Means were based on leaf spray treatments containing the surfactant Tween 20, while treatments for root irrigation did not. ^b 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. ^c Means within the same column and factor followed by the same letter are not different (P <0.05) by an ANOVA-protected LSD test. When letters are absent, ANOVA indicated no difference among means (n = 4).

[00163] Direct application of thuricin 17 to leaf tissue (Table 4) increased nodule number (P < 0.05). T17-2 increased nodule number to 103.6 nodules plant ^"1 , an 18% over the control plants. However, application of thuricin 17 to leaves did not affect nodule dry weight. Nitrogen concentration (mg g ^"1 dry weight) in shoot tissue was increased by

thuricin 17 treatments T 17-2 and T 17-3. However, thuricin 17 did not affect root N concentrations. The pattern of effects was similar for total fixed N (mg plant ^"1 ), in that there were effects of leaf spray with 45.58 and 45.51 mg of fixed N per plant for T 17-2 and Tl 7-3, respectively, versus 35.16 mg fixed N plant ^"1 for the control (Table 4). Root irrigation with solutions containing thuricin 17 also increased nodule number for all three treatments, as compared with the control. T 17-2 caused the greatest increase, at 21% more than the control. As with the leaf spray, thuricin 17 treatment did not affect nodule dry weight.

[00164] 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.

Treatment Nodule Nodule dry N concentration Fixed N number weight

Shoot Root Shoot Root Total plant ¹ mg plant ' mgg f' dw mg plant ¹

Leaf spray

Control ³ 88.1 <f 0.107 43.0 e 29.1 35.16 b 9.28 44.44 c

T17-l ^b 94.4 c 0.112 44.0 de 29.9 37.91 b 9.75 47.66 c

T 17-2 103.6 ab 0.119 48.3 a 30.4 45.58 a 10.99 56.57 a

T 17-3 101.1 b 0.117 49.1 a 31.1 45.51 a 11.07 56.58 a

Root irrigation

Control 87.6 d 0.107 42.8 e 29.0 35.57 b 9.16 44.73 c

T17-1 103.8 ab 0.117 46.2 bed 29.8 42.51 a 10.68 53.19 ab

T17-2 106.1 a 0.117 47.0 abc 30.4 42.05 a 10.85 52.90 ab

T 17-3 102.0 b 0.113 45.2 cd 29.9 37.93 b 10.51 48.44b c

"Means were based on leaf spray treatments containing the surfactant Tween 20, while treatments for root irrigation did not. ^b 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. ^c Means within the same column and factor followed by the same letter are not different (P <0.05) by an ANOVA-protected LSD test. When letters are absent, ANOVA indicated no difference among means (n = 4).

[00165] Root irrigation with thuricin 17 solution also increased N concentration

(mg g ^"1 dry weight) in shoot tissue with Tl 7-2 having the greatest effect (Table 4). As with the leaf spray, there was no difference among treatments for N concentration in root tissue. Both shoot and total fixed N per plant were increased by T 17-2, as compared to the control, whereas there was no difference for the amount of fixed N in root tissues. Values for shoot and total fixed N for Tl 7-2 were 42.05 and 52.90 mg, respectively, while control values were 35.57 and 44.73 mg, respectively. Collectively these data show that the bacteriocin thuricin 17 directly enhances soybean growth.

Example 4

a) Production of Thuricin 17 (Tl 7)

[00166] 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.

(b) Purification of Thuricin 17

[00167] 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.

[00168] Two liters of bacterial culture of Bacillus thuringiensis strain NEB 17 were extracted with 800 mL of n-butanol. The butanol-soluble material was evaporated and re-dissolved in 25 mL of 20% acetonitrile. The HPLC analysis showed the presence of thuricin 17 at a concentration of 5.42 mg mL ^"1 in this solution (Figure 1 IA). These 25 mL (containing 135.5 mg of bacteriocin) aliquots were loaded on PrepSep Cl 8 cartridge. The retained compounds were eluted with 50 mL of 35, 43 and 100% acetonitrile. 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.

Example 5

Determination of T17 promotion of seedling emergence and earth growth of corn supplied ^■ with and without fertilizer (Hoagland's solution)

[00169] Seeds of corn (Zea mays hybrid var. MZ 310) were surface sterilized with

50% 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 Tl 7 (10 ^"9 , 10 ^'10 , 10 ^'11 M) or control (dH ₂ O) solutions for 30 minutes prior to transfer into individual Petri plates (Figure 12). Ten seeds of corn 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 Tl 7 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 T17 solution or dH ₂ O. 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 μmoles 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).

[00170] In total, there were 40 pots with 5 pots per treatment. Corn plants were watered daily (50 mL) with their respective Tl 7 solution without HS or dH ₂ O for the control. After 1 week of growth, the treatments of Tl 7 solutions with HS were started. The control treatments were only given dH ₂ O or HS. Corn seedlings began to emerge after 3 days. Emergence for corn was considered when seedlings were 2 or 3 mm above the medium (Figure 12). Plants were harvested after 14 days of growth. Data were collected on plant height and leaf area. Corn 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.

[00171] Table 5: Growth measurements of corn plants after 14 days of Thuricin 17 treatments with and without fertilizer ( ¹ A concentration of Hoagland's solution). Values are means ± SE (in parentheses) of n = 4-5 replicates. Tl 7 treatments without fertilizer were supplied with distilled water only.

„ .. , , Leaf area Dry weight (g)

Treatment Height (cm) . _2\

^(cm > Shoot Root Total

Without fertilizer

Control 188 (± 09) 216 (±14) Oil (±001) 020 (±001) 031 (±001)

THlO- ⁹ M 212 (± 05) 242 (±11) 021 (±001) 020 (±001) 041 (±002)

TV? 10" ¹⁰ M 200 (±05) 216 (+14) 024 (±001) 020 (±001) 044 (±002)

THlO- ¹¹ M 197 (±06) 181 (± 18) 014 (±001) 019 (±001) 033 (±002)

With fertilizer

Control 259(±06) 347(±25) 024(±002) 023(±000) 048(±002)

T1710 ^"9 M 255 (±08) 340 (±10) 028 (±002) 021 (±001) 050 (±002)

THlO- ¹⁰ M 259 (±05) 337 (±03) 036 (±001) 024 (±001) 060 (±002)

THlO- ¹¹ M 260 (±05) 297 (±19) 025 (±004) 022 (±001) 048 (+004)

[00172] Corn treated with thuricin 17 solutions of 10 ^"9 , 10 ^~10 and 10 ^"11 M had higher emergence rates from 72 to 80 h after seeding than the control plants, which were only given distilled water (Figure 12). Furthermore, the higher emergence rates contributed to higher shoot and total plant dry weights of corn plants supplied with and without Hoagland's solution at 14 days of growth as compared to the control plants (Table 5).

Example 6

Determination of Tl 7 promotion of seedling emergence and early growth of tomato supplied with fertilizer (Hoagland's solution)

[00173] Seeds of tomato {Lycopersion esculentum L. Fl hybrid var. Veronica) were surface sterilized with 50% 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 T17 (10 ^"9 , 10 ^"10 , 10 ^"11 M) dissolved in Hoagland's solution (HS, Ji strength) or control (HS)

solutions for 30 minutes prior to transfer into individual Petri plates. Ten seeds of tomato were placed in previously surface sterilized 400 mL pots containing a Whatman filter paper (A4) and 400 mL of fine vermiculite. The seeds were watered with 180 mL of the respective Tl 7 solution or HS for the control. The pots were placed in a growth chamber under these conditions: 25/22 ⁰ C (day/night), 16 h photoperiod, and with a light intensity of 340 μmoles m ^"2 s ^'1 . In total, there were 20 pots with 5 pots per treatment. 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.

[00174] Table 6: Growth measurements of tomato plants after 23 days of T17 treatments with fertilizer ('A concentration of Hoagland's solution). Values are means ± SE (in parentheses) of n = 4-5 replicates.

, , Leaf area ^ ₁ ^^ < ^g )

Treatment Height (cm) ₂

^[cm ) Shoot Root Total

Control 18. 6 (± 0.3) 42. 6 (±1.3) 0. 25 (±0. 02) 0. 08 (±0. 00) 0. 33 (±0. 02)

T17 10 ^'9 M [ 20. 1 (+0.4) 45. 9 (±1.6) 0. 27 (±0. 02) 0. 08 (±0. 01) 0. 34 (±0. 02)

T17 lθ ^'10 M [ 20. 2 (±0.1) 56. 9 (±0.8) 0. 30 (±0. 01) 0. 08 (±0. 00) 0. 38 (±0. 01)

T1710 ^" " M 20.2 (±0.6) 48.4 (±0.9) 0.29 (±0.02) 0.08 (±0.00) 0.37 (±0.02)

[00175] 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). Yet at 23 days of growth, 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).

Example 7

Determination of Bacthuricin F4 (BF4) promotion of seedling emergence and early growth of soy bean.

[00176] 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 BF4 (10 ^"9 , 10 ^'10 , 10 ^"n 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 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 μmoles 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.

[00177] Table 7: Seedling emergence (%) after 96 h and growth measurements of soybean plants after 15 days after Bacthuricin F4 treatments. Values are means ± SE (in parentheses) of n = 3 replicates.

_ . . Seedling „ , . . , _λ Leaf area Dry weight®

Treatment * Height (cm) emergence (%) (an ² ) shoot Root

Control 64 (± 2.4) 13.0 (±0.3) 42.5 (±3.0) 0.24 (± 0.01) 0.08 (± 0.01)

BF410 ^"9 M 58 (±4.9) 14.0 (±0.7) 44.5 (±27) 0.24 (± 0.01) 0.09 (± 0.01)

BF410 ^" '°M 62 (± 7.3) 13.4 (±0.1) 39.5 (± 25) 0.26 (± 0.01) 0.09 (± 0.03)

BF410 ^"π M 70 (± 7.8) 13.8 (±0.3) 47.7 (±4.4) 0.28 (± 0.01) 0.09 (± 0.01)

[00178] Soybean plants treated with BF4 at 10 ^"10 and 10 ^' " M had higher shoot dry weights at 15 days of growth as compared to the control plants.

Example 8

Determination of isolated bacteriocin (C85) produced by Bacillus cereus UW85 on promotion of seedling emergence and early growth of soybean.

[00179] 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. 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 μmoles 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 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).

Example 9

Effect ofchitin hexamer and Thuricin 17 (Tl 7) on liginification-related and antioxidative enzymes of soybean plant

(a) Plant material

[00180] 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 Vermiculite™ (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).

(b) Treatments

[00181] Treatments of chitin hexamer and thuricin 17 were applied when the seedling reached the first trifoliate stage (~2 weeks old). Chitin hexamer and thuricin 17 treatments were applied through cut stems, as described by Orozco-Cardenas and Ryan (1999) . The plants were excised at the base of the stem with a sharp scalpel and promptly placed in 2 mL Eppendorff™ tubes containing 0.5 mL of 100 μmol 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 μmol-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.

(c) Determination of PAL and TAL

[00182] Leaf samples (300 mg fresh weight) were extracted in 4 mL of buffer (50 mM Tris pH 8.5, 14.4 mmol L ^"1 2-mercaptoethanol, 1% w/v insoluble polyvinyl- polypyrrorolidone) and centrifuged at 6,000 g for 10 min at 4 °C. The total protein concentration in soluble enzyme extracts was determined using the Bradford (1976) assay.

[00183] The method of Beaudoin-Eagan and Thorpe (1985) was used to estimate phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) activities. 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 μL 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 μL 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 .

(d) Determination of total phenolics

[00184] Total phenolic content was determined by the Folin-Ciocalteu method

(Singleton and Rossi, 1965). The assay mixture contained 50 μL 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).

(e) Determination of POD and SOD activities

[00185] The activity of peroxidase (POD) was assessed using the method of Chance and Maehly (1955). The reaction mixture consisted with 50 μL 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 μL 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 μmol of tetraguaiacol per minute.

[00186] The activity of superoxide dismutase (SOD) was determined by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) following the method of Giannopolitis and Ries (1977). The reaction mixture (3.0 mL) consisted of 63 μM NBT (nitroblue tetrazolium), 1.3 μM riboflavin, 13 mM methionine, 0.1 mM EDTA, 50 mM Tris-HCl (pH 8.0), and 50 μL extract. The mixture was held in a test tube and placed for 20 min under light at 78 μmol 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.

(f) Detection of antioxidant enzymes

[00187] For active staining of POD after separation through 12.5 % polyacrylamide gel electrophoresis (PAGE), the gels was soaked for 10 min in 50 mM Tris buffer (pH 8.0)

then incubated with 0.46% (v/v) guaiacol, and 13 mM H ₂ O ₂ in the same buffer at room temperature until red bands appeared; these were subsequently fixed in water/methanol/acetic acid (6.5: 2.5: 1, v/v/v) (Caruso et al., 1999).

[00188] For the catalase activity (C AT) staining after 12.5 % PAGE, the gel was incubated with 3.2 mM H ₂ O ₂ for 20 min, and a treatment with a solution containing 1% FeCl ₃ and 1% K ₃ Fe(CN) ₆ for 10 min, as described by Racchi et al (2001).

[00189] 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 μM 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.

[00190] Chitin hexamer elicited increases in PAL, TAL, total phenolic compounds,

POD and CAT but SOD activity was not induced. Thuricin 17 elicited PAL, TAL, total phenolic compounds, POD and SOD, but CAT activity was not induced.

[00191 ] Changes in lignification related enzymes were apparent by 72 h after chitin hexamer and/or thuricin 17 treatment of soybean leaves (Figure 16). PAL activity in Tl 7 treated leaves increased until 60 h after treatment and thereafter decreased (Figure 16A). PAL activity in chitin hexamer treated leaves increased continuously throughout experiment period, while PAL in chitin hexamer and thuricin 17 treated leaves did not increase above the control level. At 60 h, PAL activity increased by 61.8% in thuricin 17 treated leaves and 8.4% in chitin hexamer treated leaves, compared with control. At 72 h, P AL activity was 11.5 and 18.1%, respectively, greater than the control in Tl 7 and chitin hexamer treated leaves. Vander et al. (1998) found that chitin oligomers (degree of polymerization 4-10) did not elicit PAL activities at 24 h after injection into intercellular spaces of wheat leaves whereas, deacetylation levels of 35, 50 and 60% were determined, indicating PAL induction. 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).

[00192] 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. At 48 h, 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. At 72 h, TAL activity was increased by 5.0% in T17 and 23.8% in leaves of chitin hexamer treated plants, respectively, compared with the control.

[00193] The concentration of total phenolic compounds in soybean leaves was determined at 12, 36 and 72 h after chitin hexamer and T17 treatments (Figure 17). At 36 h, total phenolics increased by 15.3% in chitin hexamer treated leaves, by 8.0% following T17 treatment, and by 19.3% in chitin hexamer and Tl 7 treated leaves, compared with the control. At 72 h, total phenolics increased by 23.2% in T17 treated leaves and by 19.0% in chitin hexamer and Tl 7 treated leaves, but by only 1.4% in chitin hexamer treated leaves, as compared with the control. Treatment of insoluble mycelial walls of a fungus, Chaetomium globosum, stimulated the induction of PAL and the accumulation of phenolic acids in cultured carrot cells (Kurosaki et al., 1986). Chitin and chitosan have been shown to be effective elicitors in the hypersensitive lignification response of intact (Vander et al., 1998) and wounded (Barber et al., 1989; Pearce and Ride, 1982) plants. Also, the elicitation of lignification-related enzyme activity not only depends on the chain length but also on the abundance of the chitin oligomers (Pearce and Ride, 1982).

[00194] POD and SOD activity in soybean leaves was measured at 24, 48 and 72 h after chitin hexamer and Tl 7 treatment (Figure 18). At 24 h, POD activity increased by 31.9% in chitin hexamer and Tl 7 treated leaves (Figure 18A). At 48 h, POD activity was increased by 74.6% in T17 treated leaves. At 72 h, POD activity increased by 40.3% in chitin hexamer and by 81.2% in T 17, but by only 3.4% in chitin hexamer and Tl 7 treated leaves, compared with control leaves. At 48 h, SOD activity increased by 24.9% in chitin hexamer and by 79.9% in Tl 7 treated leaves, compared with control leaves (Figure 18B).

Chitin oligomers (degree of polymerization 7-10) induced POD activities at 24 h after injection into intercellular spaces of wheat leaves whereas POD induction increased dramatically with DAs of 50 and 60% (Vander et al., 1998).

[00195] After polyacryamide gel electrohoresis (P AGE) activities of POD, CAT and SOD were measured to detect possible changes in isozyme levels of soybean leaves (Figure 19). At 60 h, two bands (40 and 31 kDa) stained for POD activity in leaves treated with thuricin 17 (Figure 19A). Activity of the 31 kDa isoenzyme was induced stronger in Tl 7 treated leaves than control treatment. One band (59 kDa) from leaves treated with the chitin hexamer stained for CAT activity (Figure 19B). The electrophoretic pattern of SODs in leaves showed six bands (25, 23, 20, 18, 15 and 13 kDa) of activity, which were identified as Fe-SODs, since they were inhibited by H ₂ O ₂ (Figure 19C(b)) and were activated by KCN (Figure 19C(c)). Two major Fe-SOD bands of these were induced stronger in leaves treated with Tl 7 and chitin hexamer + Tl 7 than control treatment (Figure 19C(a)). Plants generally contain Fe-SOD and Cu/Zn-SOD in chloroplasts, Cu/Zn-SOD in the cytosol and Mn-SOD in the mitochondrial matrix and proxisomes (Bower et al., 1994). An increase in peroxisomal Mn-SOD activity has been reported to occur under stress as a specific defense against oxidative stress in pea plants (Palma et al., 1987).

Example 10

[00196] This example describes transformation of corn plants with a thuricin-17 coding sequence by particle bombardment.

[00197] 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).

[00198] 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 μM 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.

[00199] 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.

[00200] Five micrograms of circular plasmid DNA containing thuricin 17 gene with a selectable marker gene precipitated onto 1.0 μ gold particles are used for particle bombardment. The particle bombardment is performed with a PDS 1000/He gun (BioRad). The embroygenic corn cultures are bombarded 5-7 days after subculturing.

[00201 ] Cultures are transferred to medium supplemented with 0.6 M mannitol for

2-λ h prior to bombardment and are removed to culture medium 24 h post-bombardment. The cultures are transferred to fresh nutrient media every two weeks containing a selection compound that selects for a co-transformed marker of choice until vigorous callus cultures are produced.

[00202] For embryo maturation, 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 μE πT ² s ^"1 ) for plant development.

[00203] 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.

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[00204] All documents referred to herein are fully incorporated by reference.

[00205] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention, unless defined otherwise.

[00206] As can be understood by one skilled in the art, many modifications to the exemplary embodiments described herein are possible. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.