US20180208938A1 - Compositions and Methods for Protecting Plants Against Bacterial Infections - Google Patents

Compositions and Methods for Protecting Plants Against Bacterial Infections Download PDF

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US20180208938A1
US20180208938A1 US15/412,420 US201715412420A US2018208938A1 US 20180208938 A1 US20180208938 A1 US 20180208938A1 US 201715412420 A US201715412420 A US 201715412420A US 2018208938 A1 US2018208938 A1 US 2018208938A1
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plant
chimeric protein
genetically altered
sequence
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Goutam Gupta
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Innate Immunity LLC
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Priority to BR112019015074-9A priority patent/BR112019015074A2/pt
Priority to AU2018210030A priority patent/AU2018210030B2/en
Priority to US16/479,860 priority patent/US20210054034A1/en
Priority to EP18742012.0A priority patent/EP3570664A4/fr
Priority to MA047320A priority patent/MA47320A/fr
Priority to PCT/US2018/014905 priority patent/WO2018136962A1/fr
Priority to CN201880018496.1A priority patent/CN110446721A/zh
Publication of US20180208938A1 publication Critical patent/US20180208938A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21064—Peptidase K (3.4.21.64)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to the treatment of plant diseases caused by the xylem-limited bacteria Xylella fastidiosa (Xf), such as Pierce's Disease of grapevine.
  • Antibiotics are commonly used to target specific genes of both gram-positive and gram-negative bacteria and clear them before they can cause physiological damage to an infected organism.
  • Xf xylem-limited bacteria
  • Antibiotics are commonly used to target specific genes of both gram-positive and gram-negative bacteria and clear them before they can cause physiological damage to an infected organism.
  • antibiotic resistance in the target microbial genes thereby severely limiting their clinical use (Peschel, 2002, Trends Microbiol. 10:179).
  • the clinical world witnessed an alarming trend in which several gram-positive and gram-negative have become increasingly resistant to commonly used antibiotics, such as penicillin and vancomycin, which target the enzymes involved in the formation and integrity of bacterial outer membrane.
  • cecropins and defensins have been evolutionarily conserved in invertebrates and vertebrates and constitute a major component of host innate immune defense (Boman, 2003, J. Int. Med. 254: 197-215; Raj & Dentino, FEMS Microbiol. Lett., 202, 9, 2002; Hancock The LANCET 1, 156, 201).
  • cecropin and defensin families have been isolated from plants, insects, and mammals.
  • Xylella fastidiosa (Xf) is a devastating bacterial pathogen that causes Pierce's Disease in grapevines (Davis et al., 1978, Science 199: 75-77), citrus variegated chlorosis (Chang et al., 1993, Curr. Microbiol. 27: 137-142), alfalfa dwarf disease (Goheen et al., 1973, Phytopathology 63: 341-345), and leaf scorch disease or dwarf syndromes in numerous other agriculturally significant plants, including almonds, coffee, and peach (Hopkins, 1989, Annu. Rev. Phytopathol.
  • Xf is acquired and transmitted to plants by leafhoppers of the Cicadellidae family and spittlebugs of the Cercropidae family (Purcell and Hopkins, 1996, Annu. Rev. Phytopathol. 34: 131-151). Once acquired by these insect vectors, Xf colonies form a biofilm of poorly attached Xf cells inside the insect foregut (Briansky et al., 1983, Phytopathology 73: 530-535; Purcell et al., 1979, Science 206: 839-841). Thereafter, the insect vector remains a host for Xf propagation and a source of transmission to plants (Hill and Purcell, 1997, Phytopathology 87: 1197-1201). In susceptible plants, Xf multiplies and spreads from the inoculation site into the xylem network, where it forms colonies that eventually occlude xylem vessels, blocking water transport.
  • Pierce's disease is an Xf-caused lethal disease of grapevines in North America through Central America, and has been reported in parts of northwestern South America. It is present in some California vineyards annually, and causes the most severe crop losses in Napa Valley and parts of the Central Valley. Pierce's Disease is efficiently transmitted by the glassy-winged sharpshooter insect vector. In California, the glassy-winged sharpshooter is expected to spread north into the citrus belt of the Central Valley and probably will become a permanent part of various habitats throughout northern California. It feeds and reproduces on a wide variety of trees, woody ornamentals and annuals in its region of origin, the southeastern United States. Crepe myrtle and sumac are especially preferred. It reproduces on Eucalyptus and coast live oaks in southern California.
  • One embodiment comprises a chimeric protein with a first domain, a second domain, and a third domain, wherein said first domain comprises either i) a recognition element comprising a Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence, said second domain comprises either i) a recognition element comprising a sequence selected from Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence wherein the second domain is an element that is different from the element of the first domain and said third domain comprises a linker; wherein said linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity, and said linker ranges in length.
  • the first domain is located at the amino terminus of said chimeric protein and has an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3, 8-10, 12 or a homologue thereof.
  • the second domain is located at the carboxyl terminus of said chimeric protein and has the amino acid sequence set forth in SEQ ID NOs 14-16 are a homologue thereof.
  • the third domain of the chimeric protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 17-23 or a homologue thereof.
  • the first domain may be located at the amino terminus of said chimeric protein and wherein said second domain may be located at the carboxyl terminus of said chimeric protein.
  • Each chimeric protein comprises a lysis element and a recognition element.
  • the chimeric protein may comprise SEQ ID NOs 4-7 or a homologue thereof.
  • Another embodiment comprises a polynucleotide of a nucleic acid sequence encoding the chimeric protein.
  • Another embodiment includes an expression vector comprising this polynucleotide operably linked to a promoter.
  • a genetically altered plant or parts thereof and its progeny comprising this polynucleotide operably linked to a promoter, wherein said plant or parts thereof and its progeny produce said chimeric protein is yet another embodiment.
  • seeds and pollen contain this polynucleotide sequence or a homologue thereof, a genetically altered plant cell comprising this polynucleotide operably linked to a promoter such that said plant cell produces said chimeric protein.
  • Another embodiment comprises a tissue culture comprising a plurality of the genetically altered plant cells.
  • Another embodiment provides for a method for constructing a genetically altered plant or part thereof having increased resistance to bacterial infections compared to a non-genetically altered plant or part thereof, the method comprising the steps of: introducing a polynucleotide encoding a chimeric protein into a plant or part thereof to provide a genetically altered plant or part thereof, wherein said chimeric protein comprising a first domain, a second domain, and a third domain, wherein said first domain comprises either i) a recognition element comprising a Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence, said second domain comprises either i) a recognition element comprising a sequence selected from Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence wherein an element of the second domain is a different one than the element of the first domain and said third domain comprises a linker; wherein said link
  • a polynucleotide encoding the chimeric protein is introduced via introgression or transforming said plant with an expression vector comprising said polynucleotide operably linked to a promoter.
  • the first domain may be located at the amino terminus of said chimeric protein and includes an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NOs:1-3, 8-10, or 12 and wherein said second domain may be located at the carboxyl terminus of said chimeric protein and includes an amino acid sequence set forth in SEQ ID NO: 14-16.
  • Another embodiment provides for a method of enhancing a wild-type plant's resistance to bacterial diseases comprising transforming a cell from said wild-type plant with a polynucleotide encoding a chimeric protein to generate a transformed plant cell; and growing said transformed plant cell to generate a genetically altered plant wherein said chimeric protein comprises a first domain, a second domain, and a third domain; wherein said first domain comprises a thionin or pro-thionin, said second domain comprises Proteinase K or pro-Proteinase K, and said third domain comprises a peptide linker; wherein said peptide linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity; wherein said peptide linker ranges in length between three amino acids and approximately forty-four amino acids; wherein said genetically altered plant or part thereof produces said chimeric protein; and wherein said chimeric protein kills bacteria that cause said bacterial diseases;
  • the first domain may be located at the amino terminus of said chimeric protein and can include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 8-10, or 12 and wherein said second domain may be located at the carboxyl terminus of said chimeric protein and can include the amino acid sequence set forth in SEQ ID NOs: 14-16.
  • another embodiment comprises a genetically altered plant, for example a grape, or part thereof that is produced by this method.
  • a polynucleotide that encodes for the chimeric protein may be introduced to the plant using an expression vector for agrobacterium for the transformation.
  • another embodiment comprises a composition comprising a chimeric protein as disclosed herein as a topical treatment of plants at risk for infection with xf and a method of treating plants at risk for infection with xf comprising applying the composition to the plants at risk of infection with xf.
  • One aspect of one embodiment of the present invention provides for a chimera protein having a recognition domain and lysis domain from the grape proteome.
  • the recognition domain and the lysis domain are each specific for Xf.
  • One aspect of one embodiment of the present invention provides for transgenic plant lines expressing the chimera protein.
  • Another aspect of the present invention provides for a transgenic plant line that is Xf resistant.
  • Another aspect of one embodiment of the present invention is a transgenic plant line that is expected to show high efficacy against Xf infection in plants and have no toxicity. Another embodiment provides for the plant to be a grape plant.
  • Another aspect of the present invention provides for a chimera protein made of grape innate immune proteins useful to create a transgenic plant with innate immunity to Xf caused disease.
  • Another aspect of the present invention provides for synergy of Xf recognition and lysis to facilitate rapid clearance of Xf bacteria in a plant.
  • Another aspect of the present invention provides for a method of enhancing a plant's resistance to bacterial diseases by transforming a plant (or otherwise altering the DNA of plant) with one or more polynucleotides encoding one or more chimeric proteins described herein such that the plant containing the heterologous DNA produces the chimeric protein, and the chimeric protein kills bacteria that cause bacterial diseases after the bacteria infect the plant.
  • FIG. 1 A typical three-layers membrane of a gram-negative bacterium such as Xf is shown.
  • the outer-membrane protein mopB and LPS are possible recognition targets.
  • mopB is targeted by grape proteinase K where LPS is targeted by grape BPI/LBP.
  • Grape gamma thionins with selectivity for gram-negative bacteria such as Xf are chosen as lysis domains.
  • FIG. 4 Amino acid sequences of the chimeras shown in FIGS. 2 and 3 are illustrated in ribbon structure.
  • Proteinase K has been chosen as the recognition (cleavage) domain over HNE due to its higher cleavage activity on mopB.
  • BPI/LBP family proteins are conserved in human, animal, and plant. Grape BPI/LBP consists of two similar domains that can bind LPS and penetrate outer-membrane of gram-negative bacteria. They are joined by a proline-rich linker. One such BPI/LBP domain and the same linker is chosen when BPI/LBP is on the N-terminal of the chimera.
  • GSTAPPA linker When grape defensin on the N-terminal of the chimera, GSTAPPA linker is used join both BPI/LBP and proteinase K. We have also designed chimeras by extending the grape defensin sequence beyond the last cysteine by VFDEKto increase activity and lower toxicity.
  • FIG. 5 Chimera peptides are illustrated according to one embodiment of the present invention having a first domain, second domain and a third domain.
  • isolated refers to material that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced.
  • purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • a nucleic acid or particular bacteria that are the predominant species present in a preparation is substantially purified.
  • purified denotes that a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel.
  • isolated nucleic acids or proteins have a level of purity expressed as a range.
  • the lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.
  • nucleic acid refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, “nucleic acid” polymers occur in either single- or double-stranded form, but are also known to form structures comprising three or more strands.
  • nucleic acid includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • DNA “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp).
  • kb kilobases
  • bp base pairs
  • Estimates are typically derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide are well known in the art. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide.
  • Table 3 provides a list of exemplary conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • Oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known in the art. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells may express genes that are not found within the native (non-recombinant or wild-type) form of the cell or express native genes that are otherwise abnormally expressed—over-expressed, under-expressed or not expressed at all.
  • transgenic “transformed”, “transformation”, and “transfection” are similar in meaning to “recombinant”. “Transformation”, “transgenic”, and “transfection” refer to the transfer of a polynucleotide into the genome of a host organism or into a cell. Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra-chromosomally (not integrated into the chromosome of the cell).
  • Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow.
  • Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certain growth or environmental conditions in order for the expression vector to remain in the cell or the cell's progeny. Further, for expression to occur the organism or cell may need to be kept under certain conditions.
  • Host organisms or cells containing the recombinant polynucleotide can be referred to as “transgenic” or “transformed” organisms or cells or simply as “transformants”, as well as recombinant organisms or cells.
  • a genetically altered organism is any organism with any change to its genetic material, whether in the nucleus or cytoplasm (organelle).
  • a genetically altered organism can be a recombinant or transformed organism.
  • a genetically altered organism can also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has changes in its DNA caused by the one or more mutagens, as compared to the wild-type organism (i.e, organism not subjected to the mutagens).
  • an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.
  • the organism is a plant.
  • vector refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host.
  • the polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature; etc.
  • vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.
  • An expression vector is nucleic acid capable of replicating in a selected host cell or organism.
  • An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome.
  • an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette”.
  • a “cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of the cassettes assists in the assembly of the expression vectors.
  • An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
  • a polynucleotide sequence is operably linked to an expression control sequence(s) (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence.
  • an expression control sequence e.g., a promoter and, optionally, an enhancer
  • Transformation and generation of genetically altered monocotyledonous and dicotyledonous plant cells is well known in the art. See, e.g., Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc. (1995); and Wang, et al. Acta Hort. 461:401-408 (1998).
  • the choice of method varies with the type of plant to be transformed, the particular application and/or the desired result.
  • the appropriate transformation technique is readily chosen by the skilled practitioner.
  • Exemplary transformation/transfection methods available to those skilled in the art include, but are not limited to: direct uptake of foreign DNA constructs (see, e.g., EP 295959); techniques of electroporation (see, e.g., Fromm et al., Nature 319:791 (1986)); and high-velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (see, e.g., Kline, et al., Nature 327:70 (1987) and U.S. Pat. No. 4,945,050).
  • Specific methods to transform heterologous genes into commercially important crops are published for rapeseed (De Block, et al., Plant Physiol.
  • One exemplary method includes employing Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transforming agent to transfer heterologous DNA into the plant.
  • Agrobacterium tumefaciens -meditated transformation techniques are well described in the scientific literature. See, e.g., Horsch, et al. Science 233:496-498 (1984), and Fraley, et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
  • a plant cell, an explant, a meristem or a seed is infected with Agrobacterium tumefaciens transformed with the expression vector/construct which contains the heterologous nucleic acid operably linked to a promoter.
  • the transformed plant cells are grown to form shoots, roots, and develop further into genetically altered plants.
  • the heterologous nucleic acid can be introduced into plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens .
  • the Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens , and is stably integrated into the plant genome. See, e.g., Horsch, et al. (1984), and Fraley, et al. (1983).
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the desired transformed phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, in Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof.
  • Such regeneration techniques are described generally in Klee, et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).
  • This invention utilizes routine techniques in the field of molecular biology.
  • Basic texts disclosing the general methods of use in this invention include Green and Sambrook, 4th ed. 2012, Cold Spring Harbor Laboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual (1993); and Ausubel et al., eds., Current Protocols in Molecular Biology, 1994—current, John Wiley & Sons. Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology maybe found in e.g., Benjamin Lewin, Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632); Krebs, et al.
  • plant includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs), and suspensions of plant cells.
  • Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like).
  • the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to the molecular biology and plant breeding techniques described herein, specifically angiosperms (monocotyledonous (monocots) and dicotyledonous (dicots) plants including eudicots. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
  • the genetically altered plants described herein can be monocot crops, such as, sorghum, maize, wheat, rice, barley, oats, rye, millet, and triticale.
  • the genetically altered plants described herein can also be dicot crops, such as apple, grape, pear, peach, plum, orange, lemon, lime, grapefruit, pomegranate, olive, peanut, tobacco, etc.
  • the genetically altered plants (or plants with altered genomic DNA) can be horticultural plants such as rose, marigold, primrose, dogwood, pansy, geranium, etc.
  • the genetically altered plants are citrus plants.
  • the genetically altered plants are N. benthamiana or tobacco plants.
  • a genetically altered plant Once a genetically altered plant has been generated, one can breed it with a wild-type plant and screen for heterozygous F1 generation plants containing the genetic change present in the parent genetically altered plant. Then F2 generation plants can be generated which are homozygous for the genetic alteration. These heterozygous F1 generation plants and homozygous F2 plants, progeny of the original genetically altered plant, are considered genetically altered plants, having the altered genomic material from the genetically altered parent plant.
  • MARTI Marker Assisted Rapid Trait Introgression
  • an F1 plant is be backcrossed to the elite parent, producing BC1F1 which genetically produces 50% wild-type and 50% heterozygote chimeric protein.
  • PCR using the polynucleotide probe is performed to select the heterozygote genetically altered plants containing polynucleotide encoding the chimeric protein.
  • Selected heterozygotes are then backcrossed to the elite parent to perform further introgression.
  • This process of MARTI is performed for another four cycles.
  • the heterozygote genetically altered plant is self-pollinated by bagging to produce BC6F2 generation.
  • the BC6F2 generation produces a phenotypic segregation ratio of 3 wild-type parent plants to 1 chimeric protein genetically altered plant.
  • the application of MARTI using PCR with a polynucleotide probe significantly reduces the time to introgress the chimeric protein genetic alteration into elite lines for producing commercial hybrids.
  • the final product is an inbred plant line almost identical (99%) to the original elite in-bred parent plant that is the homozygous for the polynucleotide encoding the chimeric protein.
  • bacterium includes both a single bacterium and a plurality of bacteria.
  • the recognition sites MopB and Lipopolysaccharide on the surface of the gram-negative Xff are identified.
  • a chimera protein having a recognition domain to target both mopB and lipopolysaccharide (LPS) on the Xf membrane.
  • the recognition domain can be located at the amine (N)-terminus of the protein chimera or at the carboxy (C)-terminus of the chimera.
  • the recognition domains are joined to a grape defensin [11] (thionin) to form the Xf-killing chimera.
  • the defensin can be located at either the amine terminus or the carboxy terminus and is connected to the recognition domain by a linker.
  • the recognition domain can be a proteinase K which targets mopB since it has higher cleavage activity on mopB than HNE [12].
  • the proteinase K is a grape homolog rather than a human homolog.
  • the recognition domain can also be the bactericidal permeability-increasing protein (BPI)/Lipopolysaccharide-binding protein (LBP) protein [13].
  • BPI/LBP can be a grape homolog for Xf LPS recognition.
  • BPI/LBP can increase the permeability of the chimera thereby also increasing the membrane pore forming ability of the defensin.
  • the linker can be a plurality of amino acids or other linker type.
  • the linker can be between 2-10, 10-20, 20-40, 40-100, or 100-200 or more amino acids.
  • the linker can be a non-amino acid linker.
  • Panel A illustrates a chimera having defensin on the N-terminal and proteinase K on the C-terminal.
  • Panel B illustrates a chimera with proteinase K on the N-terminal and defensin on the C-terminal.
  • the proteinase K is a grape homolog which belongs to the subtilisin family [14].
  • Panel A illustrates a chimera with defensin on the N-terminal and BPI/LBP on the C-terminal.
  • Panel B illustrates a chimera with BPI/LBP on the N-terminal and defensin on the C-terminal.
  • the amino acid sequence of four different embodiments of chimera proteins are illustrated. Note that in addition to the sequences of the active chimera with recognition domain, linker, and lysis domain, the upstream signal sequence is also included. The signal sequence facilitates the secretion of the chimera in the xylem (the site of Xf colonization). In one embodiment, both the recognition (BPI/LBP and proteinase K) and lysis (defensin) domains are chosen from the grape proteome.
  • the chimera proteins as described herein are expected to be more active than HNE and insect Cecropin B chimera proteins previously described.
  • thionin proteinase K or BPI/LBP is located at the amino terminus of the chimeric protein, it is encoded as a pro-protein (pro-thionin or pro-proteinase K or pro-BPI/LBP) which contains an amino acid signal sequence (the exact number of amino acids in the signal sequence can vary by organism). The signal sequence assists in the trafficking of the chimeric protein to the endoplasmic reticulum or a cellular vesicle.
  • the signal sequence is cleaved off pro-protien prior to, during, or after passage of pro-protein through the lipid membrane to yield mature protein. See, Romero, et al., Eur. J. Biochem. 243:202-8 (1997).
  • thionin proteinase K or BPI/LBP is located at the carboxyl terminus of the chimeric protein
  • thionin proteinase K or BPI/LBP does not contain an amino acid signal sequence.
  • the chimeric protein still can contain an amino acid signal sequence at the amino terminus of the chimeric protein as described below.
  • the thionin (or pro-thionin) can be a thionin (or pro-thionin) that exists in a plant (and more specifically in a grape plant), or an optimized thionin (or optimized pro-thionin) as described below.
  • the proteinase K (or pro-proteinase K) can be a proteinase K (or pro-proteinase K) that exists in a plant (and more specifically in a grape plant), or an optimized proteinase K (or pro-proteinase K) as described below.
  • the BPI/LBP (or pro-BPI/LBP) can be a BPI/LBP (or pro-BPI/LBP) that exists in a plant (and more specifically in a grape plant), or an optimized BPI/LBP (or pro-BPI/LBP) as described below.
  • the third domain, the peptide linker can be a selected from SEQ ID NOs: 17-26 or a variant thereof or a non-amino acid linker.
  • the chimera of the invention may be produced using any of a number of systems to obtain the desired quantities of the protein.
  • There are many expression systems well known in the art. See, e.g., Gene Expression Systems, Fernandes and Hoeffler, Eds. Academic Press, 1999; Ausubel, supra.
  • the polynucleotide that encodes the chimera or component thereof is placed under the control of a promoter that is functional in the desired host cell.
  • promoters are available, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active.
  • expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included.
  • Constructs that include one or more of these control sequences are termed “expression cassettes” or “constructs”. Accordingly, the nucleic acids that encode the joined polypeptides are incorporated for high level expression in a desired host cell.
  • CHAMPs as secreted proteins in plant, insect and mammalian expression systems
  • active components of the chimera will typically require various post-translational modifications to produce correctly-folded, biologically active polypeptides.
  • defensins contain up to four disulfide bridges that are required for functional activity, and SRDs may contain glycosylation sites and disulfide bonds
  • expression of SRD/defensin chimeras as secreted proteins is preferred in order to take advantage of the robust structural integrity rendered by these post-translational modifications.
  • insect cells possess a compartmentalized secretory pathway in which newly synthesized proteins that bear an N-terminal signal sequence transit from the endoplasmic reticulum (ER), to the Golgi apparatus, and finally to the cell surface via vesicular intermediates.
  • the compartments of the secretory pathway contain specialized environments that enhance the ability of proteins that pass through to fold correctly and assume a stable conformation.
  • the ER supports an oxidizing environment that catalyzes disulfide bond formation, and both the ER and Golgi apparatus contains glycosylation enzymes that link oligosaccharide chains to secretory proteins to impart stability and solubility.
  • secreted proteins receive these modifications as a way of stabilizing protein structure in the harsher environment of the cell surface, in the presence of extracellular proteases and pH changes.
  • an insect expression system that may be used to express the chimeras of the invention is a Bacculovirus expression system (see below). The use of a Bacculovirus expression system to express a prototype SRD/defensin chimera is illustrated in Example 3, infra.
  • chimeras may be expressed in a Baculovirus system as follows. Briefly, DNA expressing a chimera are cloned into a modified form of the Baculovirus transfer vector pAcGP67B (Pharmingen, San Diego, Calif.). This plasmid contains the signal sequence for gp67, an abundant envelope surface glycoprotein on Autographa californica nuclear polyhedrosis virus (AcNPV) that is essential for the entry of Baculovirus particles into target insect cells. Insertion of the chimera gene into this vector will yield expression of a gp67 signal peptide fusion to the chimera, under the control of the strong Baculovirus polyhedrin promoter. The signal peptide will direct the entire protein through the secretory pathway to the cell surface, where the signal peptide is cleaved off and the chimera protein can be purified from the cell supernatant.
  • pAcGP67B Baculovirus transfer vector
  • This plasmid contains the signal sequence
  • the Baculovirus transfer vector pAcGP67B may be modified by inserting a myc epitope and 6.times.His tag at the 3′ end of the multiple cloning region for identification and purification purposes (pAcGP67B-MH).
  • Chimera genes inserted into pAcGP67B-MH may be co-transfected with Baculogold DNA into Sf21 cells using the Baculogold transfection kit (Pharmingen).
  • Recombinant viruses formed by homologous recombination are amplified, and the protein purified from a final amplification in High Five cells (Invitrogen, Carlsbad, Calif.), derived from Trichoplusia ni egg cell homogenates. High Five cells have been shown to be capable of expressing significantly higher levels of secreted recombinant proteins compared to Sf9 and Sf21 insect cells.
  • transgenic plant expression systems may also be utilized for the generation of the chimera proteins of the invention, including without limitation tobacco and potato plant systems (e.g., see Mason et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5335-5340).
  • a bioreactor such as the CELLine 350 bioreactor (Integra Biosciences).
  • This particular bioreactor provides for culturing the plant cells within a relatively low-volume, rectangular chamber (5 ml), bounded by an oxygen-permeable membrane on one side, and a protein-impermeable, 10 kD molecular weight cut-off membrane on the other side, separating the cell compartment from the larger (350 ml) nutrient medium reservoir.
  • the use of such a bioreactor permits simple monitoring of protein concentrations in the cell chamber, as a function of time, and simple characterization of proteins secreted into the medium using SDS-PAGE.
  • bioreactors also facilitate the expression of heterologous proteins in plant expression systems.
  • Various other bioreactor and suspension-culture systems may be employed. See, for example, Decendit et al., 1996, Biotechnol. Lett. 18: 659-662.
  • Genes encoding the anti-Xf chimeras of the invention may be introduced into grapevines using several types of transformation approaches developed for the generation of transgenic plants (see, for example, Szankowski et al., 2003 Plant Cell Rep. 22: 141-149). Standard transformation techniques, such as Agrobacterium -mediated transformation, particle bombardment, microinjection, and elecroporation may be utilized to construct stably-transformed transgenic plants (Hiatt et al., 1989, Nature 342: 76-78). In addition, recombinant viruses which infect grapevine plants may be used to express the heterologous chimera protein of interest during viral replication in the infected host (see, for example, Kumagai et al., 1993, Proc. Natl. Acad. Sci. USA 90: 427-430).
  • FIG. 9 Vectors capable of facilitating the expression of a transgene in embryogenic cells of grapevine plants are known, several of which are shown in FIG. 9 by way of illustration, not limitation (see, for example, Verch et al., 2004, Cancer Immunol. Immunother. 53: 92-99; Verch et al., 1998, J. Immunol. Methods 220: 69-75; Mason et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5335-5340). See, also, Szankowski et al., 2003, Plant Cell Rep. 22: 141-149.
  • transgenic grape plants expressing a test protein in the plant's xylem can be generated using standard methodologies.
  • the genetic information necessary to express an anti-Xf chimera may be introduced into grapevine embryonic cells to generate transgenic grapevines expressing the chimera using standard transgenic methodologies.
  • DNA encoding the chimera is fused to a xylem targeting sequence or a secretion leader peptide from a xylem-expressed plant protein or precursor.
  • pear PGIP see Example 4, supra
  • a specific embodiment utilizes the PGIP secretion leader peptide:
  • Another example of a secretion leader which may be employed is the rice alpha-amylase leader:
  • Another embodiment provides for treating infected plants with topical chimera protein as described herein.
  • the signal sequence at the N-terminus will not be present in the protein chimera for topical use.
  • Topical treatment will clear Xf from infected plants according to one embodiment.
  • the ability to produce the chimeras on a large scale will also allow topical delivery to cure grapevines already infected with Xf and block PD.
  • the anti- Xylella fastidiosa chimeras of the invention may be used for the treatment of Pierce's Disease in grapevines.
  • Candidate chimeras may be initially evaluated using cell survival assays capable of assessing Xf killing.
  • Chimeras showing activity in such in vitro assay systems may be further evaluated in plant assay systems.
  • Chimeras demonstrating Xf killing in these systems may be used for the therapeutic treatment of symtomatic or asymptomatic grapevines or for the prophylactic treatment of vines exposed to Xf or at risk of being exposed to Xf.
  • an anti-Xf chimera is administered to the affected plant in a manner that permits the chimera to gain access to the xylem, where Xf colonies are located.
  • the chimera may be administered directly to the xylem system, for example, via microinjection into the plant (e.g., stem, petiole, trunk).
  • anti-Xf chimera composition is injected directly into an infected grapevine, in one embodiment via a plugged, approximately 0.5 cm hole drilled into the vine, through which a syringe containing the composition may be inserted to deliver the composition to the xylem.
  • a method of treating Pierce's Disease in a Vitus vinifera plant infected with Xf comprises spraying the Vitus vinifera plant with an adherent composition containing an anti-Xf chimera.
  • adherent compositions are known, and typically are formulated in liquid for ease of application with a sprayer. Adherent powders or semi-liquids may also be employed.
  • a related embodiment is a method of preventing the development of Pierce's Disease in a Vitus vinifera plant, and comprises spraying the Vitus vinifera plant with an adherent composition containing an anti-Xf chimera.
  • an expressible gene encoding the chimera may be introduced into a plant virus capable of infecting grapevine plants, and the recombinant virus used to infect the plant, resulting in the expression of the chimera in the plant.
  • the use of xylem secretory signals may be used to target the chimera product to the infected plant's xylem.
  • the chimera may also be administered to the plant via the root system, in order to achieve systemic administration and access to primary xylem chambers. Similarly, the chimera may be administered to vine trunks, directly into primary xylem chambers, in order to deliver the chimera to upstream xylem throughout the plant.
  • the treatment of Pierce's Disease using the chimeras of the invention may also target the insect vectors responsible for the spread of Pierce's Disease.
  • anti-Xf chimeras are introduced into the insect vector itself, so that the chimera can kill the Xf colonies residing in the insect, thereby inhibiting the further spread of the pathogen.
  • plants susceptible to feeding by a Xf vector insect e.g., glassy winged sharpshooter
  • a composition that comprises the chimera and a carrier capable of adhering to the surface of the vine plants are sprayed with a composition that comprises the chimera and a carrier capable of adhering to the surface of the vine plants.
  • the insect thereby mediates the injection of the composition into the plant's xylem sap as it feeds on the plant. Accordingly, the anti-microbial composition then has the opportunity to inhibit the development of Xf colonies in the newly infected plant by killing bacteria at the feeding insertion site. Additionally, the ingestion of the composition by the insect also provides an opportunity to target and kill Xf colonies residing inside the vector insect, thereby inhibiting further spread.
  • a composition comprising an anti-Xf chimera of the invention, an insect food source, and/or a biological or chemical insect attractant may be placed locally in regions at risk for, or known to be susceptible to, insect-vectored Xf (e.g., vineyards, groves).
  • a composition comprises an anti-Xf chimera solubilized in a sucrose solution.
  • the anti-Xf composition may be solubilized or suspended in a sap or sap-containing solution, preferably using sap from the insect vector's natural food sources.
  • the composition may be exposed to the insect vector in any number of ways, including for example by placing appropriate feeder vessels in susceptible vineyards, adjacent crop areas, inhabited groves or in breeding habitats.
  • the glassy-winged sharpshooter inhabits citrus and avocado groves and some woody ornamentals in unusually high numbers. At immediate risk are vineyards near citrus orchards.
  • diseases caused by Xf may be prevented or inhibited using the chimeras of the invention in a prophylactic treatment approach, using the same or similar methods as described above.
  • plants which are not susceptible to Xf infection and/or Xf-caused disease, but which are used by Xf insect vectors to breed or feed may be sprayed with a composition containing an anti-Xf chimera of the invention. Insect vectors feeding upon such plants, for example, will ingest the composition, which is then available to kill Xf present in the insect vector, thereby preventing the spread of new infections to susceptible or carrier plants.
  • Human proteinase K [17] is very well characterized and the cleavage analysis shows that it is more active on Xf mopB than HNE.
  • Proteainase K homologs in grape belong to subtilisin family and they have 43% sequence similarity to human proteinase K.
  • the gamma thionin family members [18] appear to be more effective on gram-negative bacteria such as Xf.
  • the human BPI/LBP protein has been shown to have activities on the outer-membrane of gram-negative bacteria.
  • the members of grape BPI/LBP family members have similar domain structures as the human homolog and show 43% sequence similarity.
  • linkers are chosen: for example a synthetic GSTAPPA (SEQ ID NO: 18) linker and another a natural linker, RANATTLPKYYQNSRHPVSCTDPSK (SEQ ID NO 19), that joins the two similar domains of BPI/LBP.

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