WO2009012481A1 - Use of bacteriophage outer membrane breaching proteins expressed in plants for the control of gram-negative bacteria - Google Patents
Use of bacteriophage outer membrane breaching proteins expressed in plants for the control of gram-negative bacteria Download PDFInfo
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to methods for killing or suppressing growth of Gram-negative bacteria that infect, infest or cause disease in plants, including pathogenic, saprophytic and opportunistic microbes that cause disease in plants and food borne illness in people or in animal feed.
- Liberibacter asiaticus is a USDA Select Agent (potential bioterrorist agent; http: / /www.aph is.usda.qov/ programs /aq,_selectaqent/aq._,bjoterr.J:Qxinsl 1st. html) that was introduced into Florida in 2005 and has spread uncontrollably throughout Florida. This pathogen threatens world citrus production.
- the Gram negative bacterial pathogen Ralstonia solanacearum Race 3 Biovar 2 has been introduced into the U.S. numerous times and is such a serious threat to U.S. potato production that it is also a listed USDA Select Agent. This pathogen has been introduced into the U.S. by infecting geranium plants, but asymptomatically, so that detection of the pathogen is delayed.
- LPS lipopolysaccharide
- the LPS provides an effective defense to Gram negative bacteria against externally produced enzymes that can effectively degrade the bacterial cell wall (also called the murein layer), including the relatively thick but exposed cell walls of Gram-positive bacteria and fungi.
- lysozymes are antimicrobial agents found in mammalian cells, insects, plants, bacteria and viruses that break bacterial and fungal cell walls, specifically cleaving bonds between the amino sugars of the recurring muropeptides (C-I of N-acetylmuramic acid and C-4 of N- acetylglucosamine of microbial cell walls (Ibrahim et al. 2001 and references therein).
- Some lysozymes also are pleiotropically lytic proteins, meaning they are active in killing Gram-negative and Gram-positive bacteria, but this activity is not due to the enzymatic action of lysozyme, but specifically due to a short, linear peptide fragment that is a degradation product of some lysozymes; it is the linear degradation product of the lysozyme that penetrates the LPS barrier and the cell wall (but without harming either), reaching the inner membrane and permeabilizing the inner membrane, resulting in lysis (During et al, 1 999; Ibrahim et al. 2001 ). However, this linear peptide activity does not work well in plants (see below).
- cecropin B is rapidly degraded when incubated with intercellular plant fluid, with a half-life ranging from about three minutes in potato to about 25 hours in rice (Owens & Heutte, 1 997).
- Transgenic tobacco plants expressing cecropins have only slightly increased resistance to (Gram-negative) Pseudomanas syr/ngae pv. tabaci, the cause of tobacco wildfire (Huang et al 1 997).
- Synthetic cecropin analogs Shiva-1 and SB-37 expressed from transgenes in potato plants, only slightly reduced bacterial infection caused by (Gram-negative) Erwinia carotovora (Arce et al 1 999).
- Transgenic apple expressing the SB-37 peptide showed only slightly increased resistance to (Gram-negative) £ amylovora in field tests (Norelli et al 1 998).
- transgenic potatoes expressing attacin showed resistance to bacterial infection by £ carotovora (Arce et al 1 999) and transgenic pear and apple expressing attacin genes have also shown slightly enhanced resistance to £ amylovora (Norelli et al 1 994; Reynoird et al 1 999).
- Attacin E was also found to be rapidly degraded by plants (Ko et al 2000).
- Transgenic tobacco plants expressing a synthetic magainin analog that had been modified to be less sensitive to extracellular plant proteases were only slightly resistant to the bacterial pathogen £ carotovora (Li et al 2001 ).
- Bacteriophage T4 lysozyme has also been reported to slightly enhance resistance in transgenic potato against £ carotovora (During et al 1 993; Ahrenholz et al., 2000) and in transgenic apple plants against E amylovora (Ko 1 999).
- the action of lysozyme against Gram-negative bacteria is specifically due to a short lytic peptide fragment (Ibrahim et al. 2001 ) that is presumably sensitive to protease.
- Thaumatins exhibit the widest range of antimicrobial activity so far characterized, but also exhibit potent cytotoxic effects on eukaryotic cells (Taguchi et al 2000).
- Defensins produced by plants, mammals and insects, are characterized by complex ⁇ -sheet structures with several disulfide bonds that bind and disrupt microbial plasma membranes.
- a plant defensin from alfalfa gave robust resistance to a fungal pathogen (Guo et al 2000) and defensins from spinach were active in vitro against Gram positive and Gram negative bacteria (Segura et al. 1 998).
- human illnesses have resulted from both alfalfa and spinach infected with enteric bacteria; evidently these defensins are either not triggered by these bacteria or they are ineffective against these bacteria. More effective antibacterial agents are urgently needed to protect crop plants.
- Nonenzymatic, antimicrobial peptides are abundant in nature but of limited value in transgenic plants, primarily due to degradation by plant proteases.
- some Gram-negative bacteria are resistant to antimicrobial peptides even in culture media, due to variations in the chemical structure of the LPS (Gutsmann et al., 2005). This may help explain why plant pathogenic bacteria can overcome host plant defensins. To date, no antimicrobial peptide has proved more than marginally effective against Gram-negative bacteria when expressed in plants. More efficacious methods to control plant disease are urgently needed.
- [Para 1 2] By contrast with bacterial pathogens of animals, the vast majority of bacterial pathogens of plants are all Gram negative.
- the distinguishing feature of Gram-negative bacteria is the presence of the LPS, which forms an outer membrane that completely surrounds the cell wall. Mutations affecting the structure of the LPS of a (Gram-negative) bacterial plant pathogen of citrus caused the pathogen to die out very quickly on citrus, but not on bean (Kingsley et al., 1 993), indicating the importance of the LPS structure in evading specific plant phytochemical defenses.
- BPI is not toxic to Gram positive bacteria, fungi or animal cells, but rather attacks the LPS layer of Gram negative cells, disrupting its structure, and eventually attacking the inner membrane and causing lysis (Mannion et al., 1 990).
- a hallmark of BPI proteins is their strongly cationic, lysine rich nature and their opsonic or immune system activation ability (Levy et al., 2003).
- BPI protein family include lipopolysaccharide binding protein (LBP), lung specific X protein (LUNX), palate, lung and nasal epithelial clone (PLUNC) and parotid secretory protein (PSP), many of which have been identified by bioinformatics techniques with up to 43% identity between family members (Wheeler et al. 2003).
- LBP lipopolysaccharide binding protein
- LUNX lung specific X protein
- PUNC lung and nasal epithelial clone
- PSP parotid secretory protein
- holin and “holin-like” are not biochemically or even functionally accurate terms, but instead as used herein refer to any phage protein with at least one transmembrane domain that is capable of permeabilizing the inner membrane, thereby allowing molecules other than holins that are normally sequestered in the cyctoplasm by the inner membrane, including proteins such as endolysins, to breach or penetrate the inner membrane to reach the cell wall.
- the biochemical function(s) of holins is speculative; most, if not all of the current knowledge on holins is based on the ⁇ phage S protein (Haro et al. 2003).
- Holins are encoded by genes in at least 35 different families, having at least one transmembrane domain and classified into three topological classes (classes I, II, and III, with three, two and one transmembrane domains [TMD], respectively), all with no detected orthologous relationships (Grundling et al., 2001 ). At least two holins are known to be hemolytic and this hemolytic function has been hypothesized to play a role in the pathogenesis of certain bacteria towards insects and nematodes (Brillard et al., 2003). Only a few have been partially characterized in terms of in vivo function, leading to at least two very different theories of how they may function. The most widely accepted theory is that holins function to form oligomeric membrane pores (Graschopf & Blasi, 1 999; Young et al., 2000).
- endolysins Depolymerization of the murein layer is accomplished by lytic enzymes called endolysins. There are at least three functionally distinct classes of endolysins: 1 ) glucosaminidases (lysozymes) that attack the glycosidic linkages between the amino sugars of the peptidoglycan; 2) amidases that attack the N-acetylmuramoyl-L-alanine amide linkage between the glycan strand and the cross-linking peptide, and 3) endopeptidases that attack the interpeptide bridge linkages (Sheehan et al., 1 997).
- endolysins There are at least three functionally distinct classes of endolysins: 1 ) glucosaminidases (lysozymes) that attack the glycosidic linkages between the amino sugars of the peptidoglycan; 2) amidases that attack the N-acetylmuramoyl-L
- Endolysins are synthesized without an export signal sequence that would permit them access to the peptidoglycan (murein) layer, and they therefore usually accumulate in the cytoplasm of phage infected bacteria until they are released by the activity of holins (Young and Blasi, 1 995).
- Lysozymes have been suggested as useful antibiotics that can be used as external agents against both Gram-positive and Gram-negative bacteria because at least some of them are multifunctional (During et al., 1 999).
- endolysins Since most endolysins accumulate to high titers within the bacterial cell without causing lysis, endolysins other than certain lysozymes such as T4 would not be expected to attack Gram-negative bacteria if externally applied, since Gram-negative bacteria are surrounded with an outer membrane comprised of LPS and a lipid bilayer that would protect its murein layer from enzymatic attack just as effectively as its inner membrane does.
- U.S. patent 5,688,501 discloses a method for treating an infectious disease of animals using intact bacteriophage specific for the bacterial causal agent of that disease.
- U.S. patent 4,957,686 discloses a method for preventing dental caries by using intact bacteriophage specific for the bacterial causal agent of dental caries. Flaherty et al. (2000) describe a method for treating an infectious disease of plants using intact bacteriophage specific for the bacterial causal agent of that disease.
- the bacteriophage In all these cases and in similar cases using intact bacteriophage, the bacteriophage must attach to the bacterial host, and that attachment is highly host specific, limiting the utility of the phage to specific bacterial host species, and sometimes specific bacterial host strains. In addition, for attachment to occur, the bacteria must be in the right growth phase, and the phage must be able to gain access to the bacteria, which are often buried deep within tissues of either animals or plants, or shielded by bacterial biofilms, formed in part by the secretion of bacterial extracellular polysaccharides (EPS).
- EPS extracellular polysaccharides
- WO 01 /90331 and US 2002/0058027 disclose methods of preventing and treating Streptococcus infection of animals by use of a purified preparation consisting of a specific endolysin.
- the enzyme preparations must be purified, buffered, prepared for delivery to the target areas and preserved at the target site.
- the enzyme must be able to gain access to the infecting bacteria, and be present in sufficient quantity to kill the growing bacteria. None of these methods would be useful in the treatment of gram negative bacteria, because the endolysins could not penetrate the outer membrane of such bacteria.
- Lytic enzymes which form the basis for the methods disclosed in all of these PCT publications, are internally defined: "The present invention is based upon the discovery that phage lytic enzymes specific for bacteria infected with a specific phage can effectively and efficiently break down the cell wall of the bacterium in question. At the same time, the substrate for the enzyme is not present in mammalian tissues." (WO 01 /51073 paragraph 3, page 4). "The lytic enzymes produced by bacterial phages are specific and effective for killing select bacteria.” (paragraph 2, page 7).
- holin enzyme as used in Claim #3 of WO 01 /51 073 refers to the enzymes defined in Claim #1 as "the group consisting of lytic enzymes, modified lytic enzymes and combinations thereof." Similar references in the claims of WO 01 /82945, WO 01 /01 9385 and US 2002/01 871 36 and US 2002/01 2721 5 may be found. None of these patent applications disclose or claim the use of holin or other phage derived proteins that lack enzymatic activity in any manner, including the formulation of a compound or method of treatment of animal or plant diseases.
- WO 02/ 102405 discloses a method of preventing food poisoning in animals by inclusion of a purified preparation consisting of specific lytic enzymes and optionally, specific lytic "holin enzymes". Again, since holins are not known to exhibit enzymatic function, it is unclear as to what is taught or specified in the claims, other than a theoretical and undemonstrated enzyme defined by reference to a desirable characteristic or property.
- the prior art fails to teach or describe the identification or use of phage proteins with wide anti-microbial activity against Gram- negative bacteria.
- the prior art also fails to teach the use genes encoding phage proteins with wide anti-microbial activity against Gram-negative bacteria.
- the prior art fails to teach the use of phage proteins that are capable of destabilizing or permeabilizing the outer bacterial membrane (the bacterial lipopolysaccharide or LPS barrier) for the control of Gram negative bacterial infections of plants.
- the present invention provides a method for outer membrane (LPS barrier) destabilization and permeabilization based upon the action of a previously undescribed bacteriophage protein called herein a Bacteriophage Outer Membrane Breaching (BOMB) protein.
- LPS barrier Bacteriophage Outer Membrane Breaching
- the present invention is based, in part, on our discovery that BOMBs not only breach but destabilize the Gram negative bacterial outer membrane. This action occurs not only if the BOMB is synthesized from within the bacterial cell, but in addition, occurs if the BOMB is applied externally as well.
- This invention provides: 1 ) methods for the identification of broad-spectrum BOMBs with a high level of nonenzymatic activity to breach microbial outer membranes and thereby increase the efficacy of both natural plant defense compounds and artificially applied compounds; 2) conditions required for maintaining and increasing the anti-microbial and anti-pest efficacy of BOMBs in gene fusions; 3) methods for effective targeting of BOMBs expressed in plants through use of a xylem enhanced promoter and a leader peptide to direct the BOMB protein to the plant apoplast and xylem; 4) methods for the control of Gram negative bacterial diseases of plants by expression of gene fusions involving BOMBs and BOMB fragments, C- terminal additions and leader peptides, and optionally, endolysins and/or lipases; 5) methods for increasing the shelf-life of cut flowers; and 6) transgenic plants useful for the production of novel antimicrobial proteins based upon BOMBs and BOMB fragments.
- BOMB protein not only can have a direct inhibitory effect on Gram-negative bacteria in culture medium, but the effect is synergistic with enzymes that cause lysis or with compounds that are toxic. It has further been found that BOMB proteins compromise the integrity of the bacterial LPS barrier, but not the inner membrane.
- the present inventors have: 1 ) identified, cloned and expressed Xanthomonas pelargonii phage XpI 5 BOMB protein BC in E coir, 2) operably fused the bombBC gene separately to plant promoters in a gene expression cassette; 3) expressed functional BombBC in multiple different transgenic plants, both monocot and dicot, including tomato, tobacco, geranium, citrus and rice; 4) killed or inhibited growth of many different Gram-negative pathogens of said plants, conferring enhanced disease resistance or immunity to said plants.
- BombBC and more generally, BOMBs, may be functionally expressed in both monocot and dicot plants to enhance a plant's natural disease resistance mechanisms.
- This invention therefore provides a general method for strongly enhancing disease resistance in plants against Gram-negative bacteria, whether plant pathogens or not, comprising introducing into the plant a gene expression cassette operably fusing: 1 ) a promoter that functions in plants; 2) a BOMB gene or gene fragment that functions to express active BOMB protein in plants; 3) a transcriptional terminator region that functions in plants; and 4) obtaining expression of said gene for BOMB production in said plants.
- the above expression cassette containing a BOMB gene or gene fragment that functions to express active BOMB protein in plants has a plant secretion signal sequence that functions in plants, operably fused to the amino terminus of the BOMB gene or gene fragment.
- the present invention further provides nucleic acid molecules, operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules.
- the nucleic acid sequences of the present invention can be naturally produced or synthetically produced using methods well know to those skilled in the art of nucleic acid preparation.
- the invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a peptide, polypeptide or protein comprising the step of culturing a host cell transformed with a nucleic acid molecule of the invention under conditions in which the protein is expressed.
- This invention provides vectors comprising the nucleic acid constructs of the present invention, as well as host cells, recombinant cells and transgenic tissues and organisms comprising the vectors of the present invention. More particularly, this invention provides such cells and transgenic tissues and organisms that are hemizygotic, heterozygotic or homozygotic for the nucleic acid constructs, wherein if the organism is a plant it can be monoploid, diploid or polyploid. It is an object of the present invention to provide such cells and transgenic tissues and organisms wherein they express a single copy or multiple copies of one or more BOMB proteins, or BOMB-like ortholog protein products of the present invention.
- Cells or transgenic tissues and organisms which express multiple copies of one of the BOMB proteins, or BOMB-like proteins, mutant BOMB or BOMB- like proteins, or BOMB or BOMB-like ortholog proteins, or which express more than one of the BOMB or BOMB-like proteins, mutant BOMB or BOMB- like proteins, or BOMB or BOMB-like ortholog proteins, or which express a translational or transcriptional gene fusion carrying an BOMB or BOMB-like protein may be desirable, for example, to produce broad-spectrum resistance or tolerance to a variety of different Gram negative bacteria, whether pathogens, opportunistic or saprophytic.
- Gram-negative bacteria are in particular bacteria with an LPS, including but not limited to the following genera: Agrobacterium, Burkholderia, Candidatus Liberibacter, Erwinia, Escherichia, Pseudomonas, Ralstonia, Salmonella, Shigella, Xanthomonas and XyIeIIa.
- LPS LPS
- Gram-negative bacteria are in particular bacteria with an LPS, including but not limited to the following genera: Agrobacterium, Burkholderia, Candidatus Liberibacter, Erwinia, Escherichia, Pseudomonas, Ralstonia, Salmonella, Shigella, Xanthomonas and XyIeIIa.
- the present invention therefore also relates to a method for preparing transformed plant cells and plants, including seeds and all parts of plants, having increased resistance or immunity to Gram-negative bacterial infection or infestation, whether plant pathogenic or not.
- This method provides one or more BOMB genes, BOMB gene fusions, and the introduction of these genes and fusions into the genome of plant cells, followed by introduction of said genes into plant cells, regeneration of whole transformed plants from said cells, providing transgenic plants with resistance or immunity to disease, infection or infestation by Gram-negative bacteria.
- This invention describes the use of BOMB genes to control disease, infection and infestation in transgenic plants to: 1 ) control diseases otherwise affecting said transgenic plants, 2) to eliminate said transgenic plants from being carriers of diseases that affect other plants or animals (eg., nosocomial infestations or in animal feed), and 3) to prolong the shelf life of said transgenic plants if said plants are detached from roots (eg., cut flowers, grafting).
- plants denotes complete plants and also parts of plants, including seeds, tubers, cuttings, etc.
- the invention further provides nucleic acid probes for the detection of expression of the BOMB or BOMB-like proteins of the present invention, or mutants, or homologs, or orthologs thereof, in for example, plants which either have been genetically altered to express at least one of said proteins or which may naturally express BOMB or BOMB-like proteins, or mutants, or homologs, or orthologs thereof.
- This invention also provides the isolated nucleic acid sequence and its complement for Phage Pl 5 ORF "BC” (bombBC: SEQ ID No. 1 ) and its corresponding amino acid sequence (SEQ ID No. 2) encoding the BombBC peptide.
- BC Phage Pl 5 ORF
- the present invention also provides primers prepared from SEQ ID No. 1 that can be used to locate and identify homologs and orthologs in any prokaryotic or eukaryotic organism. The present invention also provides methods of using such primers to obtain and isolate such homologs and orthologs to SEQ ID No. 1 .
- the present invention also provides methods of using all or part of the sequence of SEQ ID No. 1 to identify homologs or orthologs by searching nucleic acid sequence data bases.
- Examples of such databases include but are not limited to the genomic sequence databases for corn, rice and Arabidopsis. Such sequence searching methods are well know to those skilled in the art.
- the present invention also provides any nucleic acid sequences that hybridize to SEQ ID No. 1 under stringent conditions. Such conditions are well known to those practiced in the art, using methods taught by, for example, Sambrook et al (1 989), but are normally a combination of temperature and salt concentration that is approximately 20 degrees Celsius below the calculated melting temperature (T m ) of the target molecule. The melting temperature is typically calculated using the formula of Bolton and McCarthy (1 962).
- the present invention further provides isolated nucleic acid molecules and their complements that encode a sequence with at least about 65% sequence identity to SEQ ID No. 1 , or at least about 70% sequence identity, or at least about 75% sequence identify, or at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 86% sequence identity, or at least about 87% sequence identity, or at least about 88% sequence identity, or at least about 89% sequence identity, or at least about 90% sequence identity, or at least about 91 % sequence identity, or at least about 92% sequence identity, or at least about 93% sequence identity, or at least about 94% sequence identity, or at least about 95% sequence identity, or at least about 96% sequence identity, or at least about 97% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity, or at least about 99.5% sequence identity, or at least about 99.9% sequence identity with SEQ ID No. 1 .
- the present invention also provides any such nucleic acids which encode a peptide or protein with BOMB
- the present invention further provides isolated amino acids that encode a sequence with at least about 65% sequence identity to SEQ ID No. 2, or at least about 70% sequence identity, or at least about 75% sequence identify, or at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 86% sequence identity, or at least about 87% sequence identity, or at least about 88% sequence identity, or at least about 89% sequence identity, or at least about 90% sequence identity, or at least about 91 % sequence identity, or at least about 92% sequence identity, or at least about 93% sequence identity, or at least about 94% sequence identity, or at least about 95% sequence identity, or at least about 96% sequence identity, or at least about 97% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity, or at least about 99.5% sequence identity, or at least about 99.9% sequence identity with SEQ ID No. 2.
- the present invention also provides the peptides and proteins encoded by such amino acid sequences including those with BOMB activity.
- the invention also provides a DNA coding region of Claim 2, consisting of bombBC(SEQ ID No. 1 ) or any DNA sequence consisting of a stretch of 70% DNA sequence identity over a stretch of 50 base pairs. This is a practical standard that is used by the Food Allergy Research Resource Program to determine if a protein is likely to be similar to any known allergens, based either on protein or DNA coding sequences.
- the invention also provides a peptide fragment consisting of at least 8 contiguous amino acids of BombBC (SEQ ID No. 2), OR any peptide fragment or protein having 35% or greater similarity over 80 amino acids with BombBC (SEQ ID No. 2). This is a practical standard that is used by the Food Allergy Research Resource Program to determine if a protein is likely to be similar to any known allergens, based either on protein or DNA coding sequences.
- the present invention provides an isolated nucleic acid sequence comprising, consisting essentially of, or consisting of a nucleic acid sequence of SEQ ID No. 1 and conservative substitutions thereof; a nucleic acid sequence with at least 70% nucleic acid sequence identity to SEQ ID No. 1 ; a contiguous nucleic acid sequence with at least 70% nucleic acid sequence identity to a contiguous nucleic acid sequence of at least 50 base pairs of SEQ ID No. 1 ; a nucleic acid sequence which hybridizes to the nucleic acid sequence of SEQ ID No. 1 under stringent hybridization conditions; or encodes the amino acid sequence of SEQ ID No. 2.
- the present invention also provides nucleic acid constructs, vectors, plant cells, plant parts, plant tissues and whole plants comprising such nucleic acid sequences.
- the plant can be any plant, such as any monocotyledonous plant or any dicotyledonous plant. Examples of such plants useful in the present invention include but are not limited to a geranium, tobacco, citrus and rice.
- the present invention also provides methods of transforming a plant cell comprising introducing into the plant cell the isolated nucleic acid sequences of the present invention. [Para 54]
- the present invention may also find use in transforming or treating algae for bacterial infections, including by transforming algae with the sequences provided by the present invention.
- the present invention also provides methods for enhancing the resistance of a plant to infection or infestation by Gram-negative bacteria, whether pathogenic or not, comprising introducing into the plant genome of said plant the nucleic acid sequences of the present invention.
- the present invention also provides isolated peptides, polypeptides or proteins comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID No. 2; an amino nucleic acid sequence with at least 8 contiguous amino acids of SEQ ID No. 2; an amino acid sequence which hybridizes to the amino acid sequence of SEQ ID No. 2 under stringent hybridization conditions; or an amino acid sequence having 35% or greater amino acid sequence similarity over at least 80 amino acids with the amino acid sequence of SEQ ID No. 2.
- the present invention also provides isolated peptides, polypeptides or proteins which are derived from a bacteriophage; lack a bacterial secretion signal amino acid sequence; lack a transmembrane domain; that when expressed in a bacterium, does not cause lysis, but instead causes "quasilysis", whereby the optical density of the culture increases shortly after induction and thereafter declines to approximately the starting optical density; and that when expressed in a bacterium grown in the presence of a phytoalexin, it causes "quasilysis” and additional cell death, whereby the optical density of the culture increases shortly after induction and thereafter declines a level significantly below that of the starting optical density.
- the plant cells, plant parts, plant tissues or whole plants of the present invention can also cause insects and nematodes to fail to thrive or to avoid feeding on said plant cell, plant part, plant tissue or whole plant due to inhibition or killing of symbiotic Gram-negative bacteria that are important for digestion or survival of the insect or nematode.
- the present invention also provides methods of preventing, treating or reducing a Gram-negative bacterial infection or infestation of a plant cell, plant part, plant tissue or whole plant, said method comprising contacting the plant cell, plant part, plant tissue, or whole plant with the isolated peptide, polypeptide or protein of the present invention.
- the present invention also provides compositions comprising the isolated peptides, polypeptides or proteins of the present invention.
- compositions include but are not limited to seed treatments, such as seed coatings, and other forms of such compositions including but not limited to sprays, powders, slurries, dustings and the like.
- the present invention provides methods of preventing, treating or reducing microbial infection of an animal cell, animal tissue, or whole animal, said method comprising contacting the animal cell, animal tissue, or whole animal with the isolated peptides, polypeptides or proteins of the present invention.
- the peptides, polypeptides or proteins may be included in compositions used to treat such animals.
- compositions include but are not limited to sprays, powders, slurries, patches, implants and the like.
- the present invention provides methods of preventing, treating or reducing microbial infection of a surface or device, such as a countertop used to prepare food or a medical device, said methods comprising contacting the surface or device with the isolated peptides, polypeptides or proteins of the present invention.
- the peptides, polypeptides or proteins may be included in compositions used to treat such surfaces and devices. Examples of such compositions include but are not limited to paints, detergents, sprays, powders, slurries, patches, implants and the like.
- the present invention provides methods for enhancing the resistance of a plant cell, plant part, plant tissue or whole plant to infection or infestation by Gram-negative bacteria comprising introducing into the plant cell, plant part, plant tissue or whole plant an expression cassette comprising as operably linked components: a) a promoter region functional in plants; b) a nucleic acid sequence of claim 1 , claim 2 or claim 3; and c) a terminator region functional in plants; and then allowing expression of the expression cassette; thereby obtaining enhanced resistance of the plant cell, plant part, plant tissue or whole plant to infection or infestation by Gram- negative bacteria.
- Such methods can further comprise self-pollinating the whole plants with the introduced expression cassette or cross-pollinating the whole plants with the introduced expression cassette to a plant of its same species.
- such methods can even further comprise testing the whole plants obtained by introducing the expression cassette for the presence of the expression cassette or enhanced resistance to infection or infestation by Gram-negative bacteria prior to self- or cross-pollinating the whole plants.
- the methods can further comprise harvesting any seeds produced as a result of the self- or cross-pollinations.
- Such methods can even further comprise germinating the harvested seeds to produced seedlings and testing plant cells, plant parts, plant tissues or whole plants of the germinated seedlings for the presence of the expression cassette or enhanced resistance to infection or infestation by Gram-negative bacteria.
- the present invention also provides tissue cultures of the plant cells, plant parts, plant tissues or whole plants obtained by the methods of the present invention, wherein the so obtained plant cells, plant parts, plant tissues or whole plants contain the introduced expression cassette.
- the whole plants obtained according to the methods of the present invention which contain the introduced nucleic acid sequences can further be self- or cross-pollinated to another plant of the same species. Any resultant seeds can be harvested and used to produce further plants for self- and cross-pollination.
- the methods of the present invention can be used for both pathogenic and non-pathogenic Gram-negative bacteria.
- the methods of the present invention can further comprise introducing into the plant genome a second nucleic acid sequence coding for a second peptide, polypeptide or peptide which enhances the resistance of the plant to infection or infestation by a plant pathogen.
- the second peptide, polypeptide or protein can include but not be limited to a nonenzymatic lytic peptide, an enzymatic lytic peptide, or an enzymatic peptidoglycan degrading peptide.
- the second peptide, polypeptide or protein can be a lysozyme, an endolysin, a protease, a mureinolytic enzyme, an enzyme with transglycosylase activity, a lipase and an esterase.
- FIG. 68 Figure 1 shows purified BombBC protein (1 8 kDa) in lane 1 and molecular weight markers of indicated size in lane 2 of a polyacrylamide gel stained with Coomassie blue.
- Figure 2 shows PCR confirmation of transformation of four plant species using bombBC, including 3 plants each of Florist's geranium (Pelargonium X hortorum) cv. Avenida (Lanes 3, 4, 5), citrus ⁇ Citrus sinensis x Poncirus trifoliata) cv. Carizzo, tobacco (Nicotiana tobacum) cv. Xanthi, and rice (Oryza sativa japonica) cv. TP309.
- PCR primers used were IPG872 (5'-tca gcc act cga tgc cgt c) and IPG91 1 (5'-gca cga ttc aag agt agg).
- the expected PCR product in all cases is 974 bp.
- Figure 3 shows typical symptoms of bacterial blight on a nontransgenic Florist's geranium ⁇ Pelargonium X hortorum) cultivar "Avenida" leaf inoculated with X.
- pelargonii cells sprayed on the leaves at a concentration of 1 O 7 colony forming units per milliliter (cfu/ml) and also inoculated using scissors dipped in 10 9 cfu/ml of X. pelargonii cells to clip the leaves in several places. Following inoculation, plants were held at 32° C. The circled region was cut out, and contained ca. 1 O 5 cfu/cm 2 live X. pelargonii cells (for details, refer Example 1 1 below). Photo taken four weeks after inoculation.
- Figure 4 shows a transgenic Florist's geranium ⁇ Pelargonium X hortorum) cultivar "Avenida" leaf expressing BombBC and inoculated at the same time and in the same manner as that described in the legend of Figure 1 . Following inoculation, plants were held at 32° C. The circular cut out region contained no detected X. pelargonii cells. Photo taken four weeks after inoculation.
- Figure 5 shows growth of X. pelargonii strain CHSC inoculated on nontransgenic geranium (Pelargonium X hortorum) variety "Avenida” and rapid death of strain CHSC inoculated on transgenic variety "Avenida” expressing BombBC.
- Cell counts were taken daily for nine days by removing circular sections totaling 1 square centimeter (cm 2 ) using a cork borer from three inoculated leaves in the area most likely to contain pathogen cells (refer Figures 1 and 2).
- pelargonii cells were recovered from transgenic geranium variety "Avenida” plants after five days following inoculation ( Figure 3), and there was no evidence of symptoms of geranium blight caused by X. pelargonii. These plants were both immune to X. pe/argon// ⁇ nfect ⁇ on, and rapidly brought the artificially inoculated pathogen population to extinction.
- Figure 6 shows a comparison of nontransgenic Florist's geranium ⁇ Pelargonium X hortorum) cultivar "Avenida" leaf inoculated with R.
- solanacearum cells inoculated by syringe infiltration of 10 6 cfu/ml directly into the spongy mesophyl of leaves using the blunt end of a tuberculin syringe.
- these same syringe inoculated plants were also inoculated by adding 5 ml of a 1 O 7 cfu/ml liquid culture directly to the soil of the potted plants geranium plants. Following inoculation, plants were held at 32° C to encourage pathogen growth and symptom development.
- the present invention is based on our discovery that at least some bacteriophage encode previously unknown proteins called BOMB (Bacterial Outer Membrane Breaching) proteins that strongly inhibit growth of at least some bacteria in culture, evidently by degrading or affecting the structure of the LPS barrier of the bacteria. Furthermore, we discovered that: 1 ) surfactants, 2) enzymes that attack the peptidoglycan or cell wall, and 3) plant defense compounds increase the efficacy of expressed BOMBs against culture grown Gram-negative. Furthermore, we discovered that BombBC, from bacteriophage XpI 5 of X. pelargonii, had a lethal or inhibitory effect on multiple Gram-negative bacteria when expressed in various different transgenic plants, both monocots and dicots. Finally, we discovered that not only can at least some BOMBs, such as BombBC, be stably produced by plant cells without toxic effects to plants, but that said expression of BOMB genes in plants provides a novel means of protecting plants against Gram-negative bacteria.
- BOMB Bacterial Outer
- the present invention is also based on our discovery that at least some plant secretion signal peptides may be used as a means for targeting the antimicrobial effect of BOMBs to the plant apoplast and xylem, where they accumulate, providing a novel means of protecting plants against a wide variety of gram negative bacteria. Furthermore, we have discovered that transgenic plants expressing BOMBs may be used to produce crude or purified extracts of antimicrobial compounds. [Para 77] The following exemplary embodiments are intended to illustrate the present invention in greater detail:
- [Para 78] 1 To identify BOMB and/or BOMB-like genes, it is first necessary to isolate and purify a DNA bacteriophage that has very strong antimicrobial activity against a variety of target organisms. This is accomplished by first obtaining bacteriophage that attack target Gram negative bacteria. Bacteriophage that attack a specific bacterium may be isolated with ease from raw sewage, pond water, or drainage from greenhouse complexes using well publicized methods known to those skilled in the art. Secondly, a variety of bacteriophage plaques are evaluated by size of the plaques formed after plating the bacteriophage with a gram negative host bacterium using methods known to those skilled in the art.
- bacteriophage are selected by their ability to lyse or inhibit additional gram negative bacteria that they are incapable of infecting. This is accomplished by a series of infection assays and overlay assays. Finally, phage nucleic acid is isolated and treated with DNAse and separately with RNAse using methods known to those skilled in the art. Only DNA based phage are selected. [Para 79] 2. Following phage purification, the bacteriophage DNA is fragmented and fully sequenced, as exemplified by Phage 1 5 sequence deposited in GenBank as Accession NC_007024.
- sequencing may be accomplished by shot-gun library sequencing or by subcloning, restriction mapping and sequencing using primer walking techniques.
- Phage genomic regions expressing BOMBs from gram negative bacteria may not be readily clonable in E ⁇ ?//and are recognized by the fact that they can only be cloned either without their native promoters or cloned downstream of fully repressed promoters. These regions may be sequenced directly from phage DNA.
- BOMBs and/or BOMB-like genes are identified by examining every LFG of the phage, starting with those found in any DNA fragment that is not sub-clonable.
- BOMB characteristically are: 1 ) small (20 kD or less) LFGs with 2) multiple helix-loop-helix-loop domains, 3) no transmembrane domains and 4) no leader sequences. LFGs with these characteristics are then selected for further testing using a functional gene expression assay.
- the predicted peptide coding regions of the putative BOMB genes are amplified by polymerase chain reaction (PCR) from the phage DNA and cloned without promoters in a suitable vector.
- PCR polymerase chain reaction
- coding regions are then operably fused with strongly regulated, repressible promoters in suitable bacterial expression vectors. Repression of the promoter operably fused with the putative BOMB genes is then released, which should result in a noticeable reduction or termination of growth of the E.coli strains carrying the clones. Any such clones are then further tested for their effect on other bacteria.
- Any DNA clones that, on induction, cause a noticeable reduction or termination of growth of the E coli strains carrying the clones are further evaluated by measuring the optical density OD at 600 nanometers (nm) of the cultures over a 24 hour period of time starting with a low, but measurable OD at the time of induction. These measurements are taken in the presence and in the absence of a phytoalexin such as berberine or a detergent such as Silwet L77. Observations are made for evidence of cell lysis or lack thereof. Any DNA clones that, upon induction, cause a continuous decline in cell density over time (up to 24 hrs) are likely BOMB candidate genes.
- Such clones may be further confirmed as BOMB genes if the effect of added phytoalexin, such as berberine chloride, or wetting agent, such as Silwet L77 is synergistic with the DNA clone in reducing cell culture density continuously over time (up to 24 hrs).
- phytoalexin such as berberine chloride
- wetting agent such as Silwet L77
- Said BOMB clone is operably fused within a plant gene expression cassette, minimally comprising a promoter that is functional in plants, followed by the BOMB clone and followed by a plant terminator in a plant expression vector that may be used for transient gene expression in plants.
- plant promoters and promoters from plant viruses that are functional in plants are widely available for use to functionally express a foreign gene in plants in transient expression assays, for example, the CaMV promoter found in the pCAMBIA series of plant expression vectors (Cambia, Canberra, Australia).
- Several plant terminators are also available, including the widely available NOS terminator, also found in the pCAMBIA plant expression vector series.
- the plant expression vectors may optionally also contain T-DNA borders and ability to replicate in Agrobacterium tumefaciens, Rhizobium spp., Sinorhizobium spp. or Mesorhizobium spp., which are subsequently used to transfer the DNA region between the T-DNA borders into plants.
- an intron may be optionally used to increase gene expression
- lntrons are known to be required for abundant expression of many genes in plants, including both dicots and ornamental plants and especially monocots, possibly by enhancing transcript stability or facilitating mRNA maturation (CaIMs et al., 1 987; Mun, J. H. et al. 2002; Rose & Beliakoff, 2000; Rose, 2002, Simpson & Filipowicz, 1 996).
- a plant secretion signal is added to the BOMB coding region.
- Some plant stress-associated and/or disease- associated proteins have been found to accumulate preferentially and most abundantly in the xylem of plants, presumably requiring a specific secretion signal sequence. Only a very few proteins are found in the xylem; it is unclear how they are secreted through the plant cell wall to reach the xylem. Such proteins have secretion signal peptides that are useful for targeting antimicrobial compounds to the plant apoplast and xylem; we call these "xylem secretion signal peptides”.
- the xylem secretion signal peptide sequence is amplified from an appropriate plant source by PCR and cloned upstream of the BOMB sequence.
- One embodiment is a 24 amino acid plant signal peptide derived from one such protein, Pl 2 (GenBank Accession # AFOl 5782; Ceccardi et al., 1 998).
- BOMB protein levels in the tissues inoculated with the BOMB clone are compared with BOMB levels in the tissues inoculated with the empty vector control.
- the most active DNA constructs are then tested in host plant transient expression challenge assays using appropriate pathogenic species of Gram negative bacteria; for example, Xanthomonas pelargonii inoculated into geranium or Ralstonia solanacearum inoculated into tobacco, geranium, tomato or pepper.
- Nonhost plant transient expression challenge assays may also be used, provided the nonhost plant produces a visible hypersensitive response (HR) to the challenge pathogen.
- HR visible hypersensitive response
- the prophylactic and therapeutic treatment of a variety of diseases caused by various species and pathovars of Xanthomonas, Pseudomonas, Erwinia, Ag ro bacterium, Ca. Liberibacter, XyIeIIa, Ralstonia and Burkholderia is achieved.
- Transgenic plants are created using plants that are hosts of the indicated pathogen genus, said host plants carrying one or more BOMB, or BOMB-like peptides fused with a xylem secretion signal peptide, operably linked with a plant promoter such that the BOMB-like peptides are made by the plants.
- the prophylactic and therapeutic elimination of fecal bacteria that can infect fresh vegetables such as spinach and bean sprouts and cause a variety of intestinal diseases, including Escherichia, Shigella and Salmonella is achieved.
- Transgenic plants are created using plants that are hosts of the indicated pathogen genus, said host plants carrying one or more BOMB or BOMB-like peptides fused with a xylem secretion signal peptide, operably linked with a plant promoter such that the BOMB-like peptides are made by the plants.
- transgenic plants are created that are hosts of the indicated genus, said host plants carrying one or more BOMB or BOMB-like peptides fused with a xylem secretions signal peptide together with an esterase, a lytic peptide or lytic enzyme, all operably linked with plant promoters such that the BOMB and/or BOMB-like peptides and lytic enzymes are made by the plant hosts.
- Lytic peptides or enzymes may be linear or compact and globular, and include but are not limited to lysozymes, cecropins, attacins, magainins, holins, permeability increasing proteins, etc.
- transgenic plants It is a further object of the invention to prevent or to dampen epidemics or plagues by planting these transgenic plants as "trap" plants in an environment such that populations of infectious bacteria, fungi, nematodes or insects are reduced by feeding upon the transgenic plants.
- Such an environment may include commercial crops, including nontransgenic crops of the same or different plant species as the transgenic trap plants, gardens and inside buildings.
- livestock feeds may incorporate or consist of transgenic whole plants, transgenic plant parts or a crude, semi-pure or pure extract of transgenic plants expressing BOMB and/or BOMB-like enzymes or peptide fragments.
- human foods such as eggs or sprouts may be treated with a spray preparation of BOMBs and or BOMB-like enzymes or peptide fragments made from transgenic plants.
- BOMB refers inclusively to any bacteriophage derived protein: 1 ) without a bacterial secretion signal sequence; 2) without a transmembrane domain, and 3) with the capacity to negatively affect, breach, permeabilize or degrade the outer LPS barrier of Gram negative bacteria.
- Expression of a BOMB protein in E coli causes "quasilysis” — upon induction, the optical density of the cell culture continues to increase for one to two hours in a manner similar to that of an uninduced culture, but then the optical density drops back to the starting level at the time of induction. BOMBs lack the capacity to cause lysis — upon induction, the optical density of the cell culture drops abruptly.
- BOMBs also lack the capacity to disrupt the inner membrane of bacteria in a manner similar to that of holins when produced or overproduced inside a bacterial cell. Disruption of the inner membrane of a bacterium is assayed by expression of both a BOMB gene and an endolysin gene simultaneously inside a bacterial cell; over-expression of a BOMB gene and endolysin simultaneously will not result in cell lysis within several hours or less.
- the term "holin” refers to any bacteriophage derived protein with at least one transmembrane domain with the capacity to disrupt the inner membrane of bacteria when produced without a leader inside a bacterial cell.
- endolysin refers to any enzyme capable of depolymerization of the murein or peptidoglycan cell wall.
- the term includes: 1 ) glucosaminidases (lysozymes) that attack the glycosidic linkages between the amino sugars of the peptidoglycan; 2) amidases that attack the N-acetylmuramoyl-L-alanine amide linkage between the glycan strand and the cross-linking peptide, and 3) endopeptidases that attack the interpeptide bridge linkages (Sheehan et al., 1 997).
- Endolysins are synthesized without an export signal sequence that would permit them access to the peptidoglycan (murein) layer, and they therefore usually accumulate in the cytoplasm of phage infected bacteria until they are released by the activity of holins.
- the term “quasilysis” means that upon induction, the optical density of the cell culture continues to increase for one to two hours in a manner similar to that of an uninduced culture, but then the optical density drops back to the starting level at the time of induction.
- the term “lysis” means that upon induction, the optical density of the cell culture drops abruptly.
- the term “esterase” refers inclusively to any enzyme categorized as either a carboxylic-ester hydrolase (EC 3.1 .1 .1 ) or a triacylglycerol acylhydrolase (EC 3.1 .1 .3).
- carboxylic-ester hydrolase (EC 3.1 .1 .1 ), refers to a “carboxylesterase” and catalyzes the reaction of a carboxylic ester + H2O to an alcohol plus a carboxylate.
- carboxylic-ester hydrolase ali-esterase; B-esterase; monobutyrase; cocaine esterase; procaine esterase; methylbutyrase; vitamin A esterase; butyryl esterase; carboxyesterase; carboxylate esterase; carboxylic esterase; methylbutyrate esterase; triacetin esterase; carboxyl ester hydrolase; butyrate esterase; methylbutyrase; carboxylesterase; propionyl esterase; nonspecific carboxylesterase; esterase D; esterase B; esterase A; serine esterase; carboxylic acid esterase; cocaine esterase.
- lipase refers to any triacylglycerol acylhydrolase (EC 3.1 .1 .3), commonly called “triacylglycerol lipase” and catalyzing the reaction of triacylglycerol plus H2O to diacylglycerol plus a carboxylate.
- lipase Other common names for lipase are: tributyrase; butyrinase; glycerol ester hydrolase; tributyrinase; Tween hydrolase; steapsin; triacetinase; tributyrin esterase; Tweenase; amno N-AP; Takedo 1 969-4-9; Meito MY 30; Tween esterase; GA 56; capalase L; triglyceride hydrolase; triolein hydrolase; tween-hydrolyzing esterase; amano CE; cacordase; triglyceridase; triacylglycerol ester hydrolase; amano P; amano AP; PPL; glycerol-ester hydrolase; GEH; meito Sangyo OF lipase; hepatic lipase; lipazin; post-heparin plasma protamine-resistant lipase
- Gram-negative bacterium refers to any bacterium producing lipopolysaccharide (LPS).
- LPS lipopolysaccharide
- Disease resistance refers to any reduction in disease symptoms or pathogen numbers in the plant or material tested caused by the treatment, as compared with the most susceptible phenotypic symptoms or pathogen numbers known in comparable tests of untreated plants or materials.
- the term “resistance” to bacteria refers to any reduction in bacterial numbers in the plant or material tested caused by the treatment, as compared with untreated plants or materials.
- the term “immunity” to bacteria refers to elimination of detectable bacterial cell counts in the plant or material tested caused by the treatment, as compared with untreated plants or materials.
- the term “allele” refers to any of several alternative forms of a gene.
- amino acid refers to the aminocarboxylic acids that are components of proteins and peptides.
- amino acid abbreviations are as follows: A (Ala); C (Cys); D (Asp); E (GIu); F (Phe); G (GIy); H (His); I (Iso); K (Lys); L (Leu); M (Met); N (Asn); P (Pro); Q (GIn); R (Arg); S (Ser); T (Thr); V (VaI); W (Trp), and Y (Tyr).
- “Homologous” refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules.
- a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
- the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half ⁇ e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
- a first oligonucleotide anneals with a second oligonucleotide with "high stringency” or "under high stringency conditions” if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 60%, more preferably at least about 65%, even more preferably at least about 70%, yet more preferably at least about 80%, and preferably at least about 90% or, more preferably, at least about 95% complementary anneal with one another.
- the stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known.
- Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et a/., 1 989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
- NCBI National Center for Biotechnology Information
- NLM National Library of Medicine
- NIH National Institutes of Health
- BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI "blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. [Para 1 1 1 ]
- Gapped BLAST can be utilized as described in Altschul et a/. (1 997, Nucleic Acids Res. 25:3389-3402).
- PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules ⁇ id.) and relationships between molecules which share a common pattern.
- the default parameters of the respective programs ⁇ e.g., XBLAST and NBLAST) can be used as available on the website of the National Center for Biotechnology Information of the National Library of Medicine at the National Institutes of Health.
- nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
- isolated nucleic acid refers to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule ⁇ e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
- crop plant refers to any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food or food additives, smoking products, pulp production and wood production.
- cross pollination or “cross-breeding” refer to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.
- cultivar refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.
- dotyledon and “dicot” refer to a flowering plant having an embryo containing two seed halves or cotyledons.
- Examples include citrus; geranium; tobacco; tomato; the legumes, including peas, alfalfa, clover and soybeans; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets and buttercups.
- ER retention signal refers to an amino acid sequence (the ER retention signal peptide) attached to a polypeptide which causes the polypeptide to be retained and accumulated in the endoplasmic reticulum (ER).
- female plant refers to a plant that produces ovules.
- Female plants generally produce seeds after fertilization.
- a plant designated as a "female plant” may contain both male and female sexual organs.
- the "female plant” may only contain female sexual organs either naturally ⁇ e.g., in dioecious species) or due to emasculation ⁇ e.g., by detasselling).
- filial generation refers to any of the generations of cells, tissues or organisms following a particular parental generation.
- the generation resulting from a mating of the parents is the first filial generation (designated as “Fl " or "Fi"), while that resulting from crossing of Fl individuals is the second filial generation (designated as "F2" or "F 2 ").
- the term “gamete” refers to a reproductive cell whose nucleus (and often cytoplasm) fuses with that of another gamete of similar origin but of opposite sex to form a zygote, which has the potential to develop into a new individual. Gametes are haploid and are differentiated into male and female.
- the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.
- Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
- the term "genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.
- hemizygous refers to a cell, tissue or organism in which a gene is present only once in a genotype, as a gene in a haploid cell or organism, a sex-linked gene in the heterogametic sex, or a gene in a segment of chromosome in a diploid cell or organism where its partner segment has been deleted.
- heterologous polynucleotide or a “heterologous nucleic acid” or an “exogenous DNA segment” refer to a polynucleotide, nucleic acid or DNA segment that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
- a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified.
- the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
- heterologous trait refers to a phenotype imparted to a transformed host cell or transgenic organism by an exogenous DNA segment, heterologous polynucleotide or heterologous nucleic acid.
- heterozygote refers to a diploid or polyploid individual cell or plant having different alleles (forms of a given gene) present at least at one locus.
- heterozygous refers to the presence of different alleles (forms of a given gene) at a particular gene locus.
- homolog or “homologue” refer to a nucleic acid or peptide sequence which has a common origin and functions similarly to a nucleic acid or peptide sequence from another species.
- homozygote refers to an individual cell or plant having the same alleles at one or more loci.
- homozygous refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.
- hybrid refers to any individual cell, tissue or plant resulting from a cross between parents that differ in one or more genes.
- inbred or “inbred line” refers to a relatively true-breeding strain.
- line is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s).
- a plant is said to "belong” to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry ⁇ e.g., via inbreeding or selfing).
- TO primary transformant
- locus refers to any site that has been defined genetically.
- a locus may be a gene, or part of a gene, or a DNA sequence that has some regulatory role, and may be occupied by different sequences.
- lytic protein refers to any enzyme, in whole or in part, or lytic peptide that: 1 ) degrades or penetrates the peptidoglycan or murein layer that forms the bacterial cell wall of both Gram positive or Gram negative bacteria, and 2) has the ability to permeabilize or disrupt the bacterial inner membrane.
- Said proteins may be linear, partially degraded or compact and globular, and include but are not limited to lysozymes, cecropins, attacins, magainins, permeability increasing proteins, etc.
- male plant refers to a plant that produces pollen grains.
- the “male plant” generally refers to the sex that produces gametes for fertilizing ova.
- a plant designated as a "male plant” may contain both male and female sexual organs. Alternatively, the “male plant” may only contain male sexual organs either naturally ⁇ e.g., in dioecious species) or due to emasculation ⁇ e.g., by removing the ovary).
- mass selection refers to a form of selection in which individual plants are selected and the next generation propagated from the aggregate of their seeds.
- the term "monocotyledon” or “monocot” refer to any of a subclass (Monocotyledoneae) of flowering plants having an embryo containing only one seed leaf and usually having parallel-veined leaves, flower parts in multiples of three, and no secondary growth in stems and roots. Examples include lilies; orchids; rice; corn, grasses, such as tall fescue, goat grass, and Kentucky bluegrass; grains, such as wheat, oats and barley; irises; onions and palms.
- mutant or “mutation” refer to a gene, cell, or organism with an abnormal genetic constitution that may result in a variant phenotype.
- nucleic acid or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof ⁇ e.g. degenerate codon substitutions) and complementary sequences 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 (Batzer eta/. (1 991 ) Nucleic Acid Res. 1 9:5081 ; Ohtsuka eta/. (1 985) J. Biol. Chem. 260:2605-2608; Cassol eta/. (1 992); Rossolini eta/. (1 994) MoI. Cell. Probes 8:91 -98).
- nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
- nucleic acid also encompasses polynucleotides synthesized in a laboratory using procedures well known to those skilled in the art.
- a DNA segment is referred to as "operably linked" when it is placed into a functional relationship with another DNA segment.
- DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
- DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase.
- enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
- open pollination refers to a plant population that is freely exposed to some gene flow, as opposed to a closed one in which there is an effective barrier to gene flow.
- open-pollinated population or “open-pollinated variety” refer to plants normally capable of at least some cross-fertilization, selected to a standard, that may show variation but that also have one or more genotypic or phenotypic characteristics by which the population or the variety can be differentiated from others.
- a hybrid which has no barriers to cross-pollination, is an open-pollinated population or an open-pollinated variety.
- ortholog and “orthologue” refer to a nucleic acid or peptide sequence which functions similarly to a nucleic acid or peptide sequence from another species. For example, where one gene from one plant species has a high nucleic acid sequence similarity and codes for a protein with a similar function to another gene from another plant species, such genes would be orthologs.
- phenotype refers to the observable characters of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual's genetic makeup ⁇ i.e., genotype) and the environment.
- phytoalexin refers to any antimicrobial chemical compound made by a plant, whether preformed or made in response to presence of a microbe.
- plant line is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s).
- a plant is said to "belong” to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry ⁇ e.g., via inbreeding or selfing).
- TO primary transformant
- the term “pedigree” denotes the lineage of a plant, e.g. in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
- plant tissue refers to any part of a plant.
- plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath.
- promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
- protein refers to amino acid residues and polymers thereof. Unless specifically limited, the terms encompass amino acids containing known analogues of natural amino acid residues that have similar binding properties as the reference amino acid and are metabolized in a manner similar to naturally occurring amino acid residues. Unless otherwise indicated, a particular amino acid sequence also implicitly encompasses conservatively modified variants thereof ⁇ e.g. conservative substitutions) as well as the sequence explicitly indicated.
- polypeptide also encompasses polypeptides synthesized in a laboratory using procedures well known to those skilled in the art.
- recombinant refers to a cell, tissue or organism that has undergone transformation with recombinant DNA.
- the original recombinant is designated as “RO” or “Ro.”
- Selfing the RO produces a first transformed generation designated as “Rl “ or “Ri .”
- secretion signal refers to an amino acid sequence (the secretion signal peptide) attached to a N-terminus of a polypeptide, which is needed for secretion of the mature polypeptide from the cell.
- self pollinated or “self-pollination” means the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of the same or a different flower on the same plant.
- transcript refers to a product of a transcription process.
- transformation refers to the transfer of nucleic acid ⁇ i.e., a nucleotide polymer) into a cell.
- genetic transformation refers to the transfer and incorporation of
- DNA especially recombinant DNA, into a cell.
- transformant refers to a cell, tissue or organism that has undergone transformation.
- the original transformant is designated as “TO” or “To.”
- Selfing the TO produces a first transformed generation designated as “Tl “ or “Ti .”
- transgene refers to a nucleic acid that is inserted into an organism, host cell or vector in a manner that ensures its function.
- transgenic refers to cells, cell cultures, organisms (e.g., plants), and progeny which have received a foreign or modified gene by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the organism receiving the foreign or modified gene.
- transposition event refers to the movement of a transposon from a donor site to a target site.
- the term “variety” refers to a subdivision of a species, consisting of a group of individuals within the species that are distinct in form or function from other similar arrays of individuals.
- the terms “untranslated region” or “UTR” refer to any part of a mRNA molecule not coding for a protein ⁇ e.g., in eukaryotes the poly(A) tail).
- the term "vector” refers broadly to any plasmid or virus encoding an exogenous nucleic acid.
- the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like.
- the vector may be a viral vector that is suitable as a delivery vehicle for delivery of the nucleic acid, or mutant thereof, to a cell, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma eta/. (1 997, Proc.
- viral vectors include, but are not limited to, a recombinant vaccinia virus, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno- associated virus, a recombinant avian pox virus, and the like (Cranage et a/., 1 986, EMBO J. 5:3057-3063; International Patent Application No. WO94/1 7810, published August 1 8, 1 994; International Patent Application No. WO94/23744, published October 27, 1 994).
- non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
- expression units or expression vectors or systems
- Methods for generating expression units/systems/vectors for use in plants are well known in the art and can readily be adapted for use in the instant invention.
- a skilled artisan can readily use any appropriate plant/vector/expression system in the present methods following the outline provided herein.
- the expression control elements used to regulate the expression of the protein can either be the expression control element that is normally found associated with the coding sequence (homologous expression element) or can be a heterologous expression control element.
- Transcription initiation regions can include any of the various opine initiation regions, such as octopine, mannopine, nopaline and the like that are found in the Ti plasmids of Ag ro bacterium tumefacians.
- plant viral promoters can also be used, such as the cauliflower mosaic virus 1 9S and 35S promoters (CaMV 1 9S and CaMV 35S promoters, respectively) to control gene expression in a plant (U.S. Patent Nos. 5,352,605; 5,530,1 96 and 5,858,742 for example).
- Enhancer sequences derived from the CaMV can also be utilized (U.S. Patent Nos. 5,1 64,31 6; 5,1 96,525; 5,322,938; 5,530,1 96; 5,352,605; 5,359,1 42; and 5,858,742 for example).
- plant promoters such as RUBISCO small and large subunit promoters, prolifera promoter, fruit-specific promoters, Ap3 promoter, heat shock promoters, seed-specific promoters, etc. can also be used.
- Either a gamete-specific promoter, a constitutive promoter (such as the CaMV or Nos promoter), an organ-specific promoter (such as the E8 promoter from tomato) or an inducible promoter is typically ligated to the protein or antisense encoding region using standard techniques known in the art.
- the expression unit may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.
- the expression units will typically contain, in addition to the protein sequence, a plant promoter region, a transcription initiation site and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the expression unit are typically included to allow for easy insertion into a preexisting vector.
- the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
- the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
- the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct.
- Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen eta/., EMBOy 3:835-846 (1 984)) or the nopaline synthase signal (Depicker et a/., MoI. and Appl. Genet.
- the resulting expression unit is ligated into or otherwise constructed to be included in a vector that is appropriate for higher plant transformation.
- the vector may also contain a selectable marker gene by which transformed plant cells can be identified in culture.
- Replication sequences, of bacterial or viral origin are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included.
- a selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as ampicillin, kanamycin or tetracycline.
- sequences of the present invention can also be fused to various other nucleic acid molecules such as Expressed Sequence Tags (ESTs), epitopes or fluorescent protein markers.
- ESTs Expressed Sequence Tags
- epitopes epitopes
- fluorescent protein markers fluorescent protein markers
- ESTs are gene fragments, typically 300 to 400 nucleotides in length, sequenced from the 3' or 5' end of complementary-DNA (cDNA) clones. Nearly 30,000 Arab/dops/s thaUana ESTs have been produced by a French and an American consortium (Delseny eta/., FEBS Lett. 405(2): 1 29- 1 32 (1 997); Arabidopsis thaliana Database, http://genome.www.stanford.edu/Arabidopsis). For a discussion of the analysis of gene-expression patterns derived from large EST databases, see, e.g., M. R. Fannon, TIBTECH 14:294-298 (1 996).
- Homologous recombination permits site- specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. Homologous recombination and site-directed integration in plants are discussed in, for example, U.S. Patent Nos. 5,451 ,51 3; 5,501 ,967 and 5,527,695.
- Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; Agrobacterium-, Rhizobium-, Mesorhizobium- and S/nor/?/zob/um-med ⁇ ated transformation. See, for example, U.S. Patent Nos.
- Agrobacterium tumefaciens is a naturally occurring bacterium that is capable of inserting its DNA (genetic information) into plants, resulting in a type of injury to the plant known as crown gall. It can also insert foreign DNA into plants through the use of its modified or "disarmed" natural DNA insertion system, but without forming crown gall disease. Most species of plants can now be transformed using this method.
- Rhizobium spp. Mesorhizobium spp. and Sinorhizobium spp. are naturally occurring bacteria that are also capable of inserting foreign DNA (genetic information) into plants. Many species of plants can now be transformed using this method. See, for example, Broothaerts et al., Nature 433:629-633 (2005).
- Microprojectile bombardment is also known as particle acceleration, biolistic bombardment, and the gene gun (Biolistic ® Gene Gun).
- the gene gun is used to shoot pellets that are coated with genes ⁇ e.g., for desired traits) into plant seeds or plant tissues in order to get the plant cells to then express the new genes.
- the gene gun uses an actual explosive (.22 caliber blank) to propel the material. Compressed air or steam may also be used as the propellant.
- the Biolistic ® Gene Gun was invented in 1 983-1 984 at Cornell University by John Sanford, Edward Wolf, and Nelson Allen. It and its registered trademark are now owned by E. I. du Pont de Nemours and Company.
- WHISKERSTM is an alternative to other methods of inserting DNA into plant cells ⁇ e.g., the Biolistic ® Gene Gun, Agrobacterium tumefaciens, the "Shotgun” Method, etc.); and it consists of needle-like crystals ("whiskers") of silicon carbide.
- the fibers are placed into a container along with the plant cells, then mixed at high speed, which causes the crystals to pierce the plant cell walls with microscopic "holes" (passages). Then the new DNA (gene) is added, which causes the DNA to flow into the plant cells.
- the plant cells then incorporate the new gene(s); and thus they have been genetically engineered.
- the essence of the WHISKERSTM technology is the small needle- like silicon carbide "whisker” (0.6 microns in diameter and 5-80 microns in length) which is used in the following manner.
- a container holding a "transformation cocktail” composed of DNA ⁇ e.g., agronomic gene plus a selectable marker gene), embryogenic corn tissue, and silicon carbide "whiskers” is mixed or shaken in a robust fashion on either a dental amalgam mixer or a paint shaker.
- the fibrous, needle-like "whiskers" form of silicon carbide is a pulmonary health hazard and therefore must be handled much differently from non-fibrous silicon carbide powders that contain no whiskers.
- the two silicon carbide forms, powder and fibrous whiskers are regulated much differently, with the British Columbian (Canadian) Occupational Health and Safety (OHS) regulating the fibrous form the same as asbestos at 0.1 fiber per cc (f/cc) exposure limit, whereas the ordinary, non-fibrous form has an exposure limit of 3-1 0 mg/ cubic meter.
- Silicon carbide whiskers were shown to generate mutagenic reactive hydroxyl radicals in a manner similar to asbestos and to cause DNA strand breakage; silicon carbide powder did not cause such effects (Svensson et al., 1 997).
- Si carbide powder did not cause such effects (Svensson et al., 1 997).
- [Para 1 85] Breaching the plant cell wall using silicon carbide powder does not direct any DNA associated with the powder to the plant nucleus, although this will happen at a low frequency. This problem can be overcome if the DNA is directed to the nucleus, as occurs in natural infections of A tumefaciens or by certain viruses. Nuclear localization signal sequences (NLSs) guide the protein and any associated nucleic acid to the plant nucleus.
- NLSs Nuclear localization signal sequences
- Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (see, for example, Messing & Vierra, Gene 1 9: 259- 268 (1 982); Bevan et a/., Nature 304: 1 84-1 87 (1 983)), the bar gene which confers resistance to the herbicide phosphinothricin (White eta/., Nucl Acids Res 1 8: 1062 (1 990), Spencer eta/, Theor Appl Genet 79: 625-631 (1 990)), and the dhfr gene, which confers resistance to methotrexate (Bourouis et a/, EMBO J. 2(7): 1 099-1 1 04 (1 983)).
- a transgenic plant formed using Agrobacterium, Rhizobium, Mesorhizobium or Sinorhizobium transformation methods typically contains a single gene on one chromosome, although multiple copies are possible. Such transgenic plants can be referred to as being hemizygous for the added gene. A more accurate name for such a plant is an independent segregant, because each transformed plant represents a unique T-DNA integration event (U.S. Patent No. 6,1 56,953).
- a transgene locus is generally characterized by the presence and/or absence of the transgene.
- a heterozygous genotype in which one allele corresponds to the absence of the transgene is also designated hemizygous (U.S. Patent No. 6,008,437).
- Open-Pollinated Populations The improvement of open- pollinated populations of such crops as rye, many maizes and sugar beets, herbage grasses, legumes such as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil palm and some rubber, depends essentially upon changing gene-frequencies towards fixation of favorable alleles while maintaining a high (but far from maximal) degree of heterozygosity. Uniformity in such populations is impossible and trueness-to-type in an open-pollinated variety is a statistical feature of the population as a whole, not a characteristic of individual plants. Thus, the heterogeneity of open- pollinated populations contrasts with the homogeneity (or virtually so) of inbred lines, clones and hybrids.
- Mass Selection In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and there is no control over pollination, mass selection amounts to a form of random mating with selection. As stated above, the purpose of mass selection is to increase the proportion of superior genotypes in the population.
- Synthetics A synthetic variety is produced by crossing inter se a number of genotypes selected for good combining ability in all possible hybrid combinations, with subsequent maintenance of the variety by open pollination. Whether parents are (more or less inbred) seed-propagated lines, as in some sugar beet and beans ( V/c/a) or clones, as in herbage grasses, clovers and alfalfa, makes no difference in principle. Parents are selected on general combining ability, sometimes by test crosses or topcrosses, more generally by polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfing or sib crossing).
- Hybrids A hybrid is an individual plant resulting from a cross between parents of differing genotypes. Commercial hybrids are now used extensively in many crops, including corn (maize), sorghum, sugarbeet, sunflower and broccoli. Hybrids can be formed in a number of different ways, including by crossing two parents directly (single cross hybrids), by crossing a single cross hybrid with another parent (three-way or triple cross hybrids), or by crossing two different hybrids (four-way or double cross hybrids).
- Hybrids may be fertile or sterile depending on qualitative and/or quantitative differences in the genomes of the two parents.
- Heterosis, or hybrid vigor is usually associated with increased heterozygosity that results in increased vigor of growth, survival, and fertility of hybrids as compared with the parental lines that were used to form the hybrid. Maximum heterosis is usually achieved by crossing two genetically different, highly inbred lines.
- Example 1 Use of a plant pathogen to isolate bacteriophage capable of infecting a Gram negative plant pathogen. Xanthomonas pelarqonii.
- Example 2 Use of agar plate overlay assays to characterize phage host range and to identify phage with an ability to kill bacterial hosts that they cannot infect.
- PYGM plates were overlaid with X. pelargonii XHSC and droplets of various purified phage samples obtained from Example 1 were added to the plates and incubated at 30° C for 48 hours. All phage were able to infect CHSC and cause clear zones of lysis. Cell suspensions of overnight broth cultures of X. citri B21 .2, X. campestris 528 and R. solanacearum G2 were added to 0.7% water agar as described in Example 1 and individually overlayed on the phage infected CHSC plates.
- the Pl 5 DNA was digested with £ ⁇ ?RV, yielding eleven fragments, ranging in size from 1 2.4 kb to 357 bp. Most of the fragments were cloned; some were not cloned, despite repeated attempts, most likely due to the presence of restriction endonucleases and holins.
- the cloned DNA fragments were used directly for sequencing, using vector-based primers initially, and primer walking thereafter until each fragment was completed. Fragments that were not cloned were sequenced using Pl 5 genomic DNA. Fragment assembly was accomplished using Pl 5 genomic DNA and primers extending outside each fragment in both directions. Pl 5 has a double stranded DNA genome which is 55,770 bp in length (GenBank NC.007024).
- ORF analysis of the sequenced phage was done using a combination of several programs including PromScan, Terminator (GCG), GeSTer (Unniraman et al. 2001 , 2002), Glimmer, Genie, Codon preference (GCG), ORF finder (NCBI) and Blast (NCBI) analyses. Potential Shine-Delgarno sequences were identified manually by examining the sequence. Using default Glimmer settings, only 32 ORFs were identified; none of these ORFs corresponded to functional genes later identified as holins or BOMB by functional analyses, although lysY, predicted to encode an endolysin, was identified.
- Example 4 Use of a phvtoalexin and inducible gene expression systems to identify candidate genes encoding proteins with ability to kill from the outside.
- bacteriophage are known to encode proteins that are able to degrade the bacterial cell wall (endolysins) and proteins that are able to degrade or breach the bacterial inner membrane (holins).
- endolysins proteins that are able to degrade or breach the bacterial inner membrane
- bacteriophage proteins with ability to degrade or breach the bacterial outer membrane ie., "BOMB” proteins
- LysY Induced expression of the endolysin, LysY, caused a slow reduction in cell density (not shown), and by contrast with the effects of HoIZ expressed alone, cell lysis debris was apparent in these cultures. Since LysY was cloned without a leader sequence, this endolysin appeared to behave similarly to lysozyme, and exhibited some ability to penetrate or permeabilize the bacterial inner membrane, allowing it to reach and degrade the bacterial cell wall, causing lysis.
- berberine chloride a commercially prepared, plant derived, antimicrobial compound (a "phytoalexin") worked synergistically with BombBC to reduce culture density. This synergistic effect was not seen with either a holin nor an endolysin. Berberine may be used to assay for defects in the LPS barrier and/or efflux pumping ability of phytopathogenic bacteria (Reddy et al., 2007). Bacteria are sensitive to berberine in a concentration dependent manner.
- berberine that leaks through the LPS must be actively pumped out (effluxed) for bacterial survival; if either the LPS is breached or the efflux pumps are disabled, bacteria are unable to grow in the presence of berberine.
- berberine 5,6-dihydro-9,l 0- dimethoxybenzo-1 ,3-benzodioxoloquino-lizimium, an alkaloid DNA intercalating agent; Schmeller et al., 1 997), was added (5 micrograms/ml) to cells carrying bombBC and grown in liquid culture in these experiments, cell death was much more rapid when BombBC was expressed.
- Example 5 Use of P3rpoH::lacZ reporter to confirm effect of BOMB protein on bacterial LPS.
- £ coli strains ADA41 0 carries a P3rpoH::lacZ reporter gene that is selectively activated when the LPS or outer membrane of the cells are damaged (Shapiro and Baneyx, 2002).
- the bombBC coding region was recloned into the pMAL expression vector (New England Biolabs, Ipswich, MA), overexpressed in £ ⁇ ?// BL21 DE3 cells, and purified (Fig. 1 ).
- the CaMV promoter from pBI221 (Clontech, Palo Alto, CA) was enzymatically recloned into the polylinker cloning site of pCAMBIA0390 (Cambia, Canberra, AU), which has a left T-DNA border, the polylinker site, a NOS transcriptional terminator and right T-DNA borders, creating plPG700.
- the phage Pl 5 bombBC gene was enzymatically recloned into plPG700 downstream from the CaMV promoter and upstream from the NOS terminator, creating plPG780.
- a 24 amino acid plant signal peptide derived from a protein known to accumulate in the citrus xylem, Pl 2 was used to create a xylem secretion signal leader (SEQ ID No. 3 and SEQ ID No. 4).
- the xylem secretion signal peptide sequence was amplified from Citrus sinensis (sweet orange) by PCR and cloned upstream of the bombBC gene and resulting in a translational gene fusion between Pl 2 and BombBC (SEQ ID No. 5) on plPG780.
- pCAMBIA2301 carries the kanamycin resistance gene driven by a dual CaMV promoter for plant selection. plPG786 was used for transformation and regeneration of tobacco and citrus, while plPG787 was used for transformation of geranium and rice.
- Example 7 Use of transient expression of bombBC ' m sweet pepper plants to demonstrate enhanced resistance to Xanthomonas and Ralstonia.
- the plant transformation and expression vector plPG780 was moved into A. tumefaciens strain GV2260 by either electroporation or bacterial conjugation as described (Kapila et al., 1 997).
- GV2260 carrying plPG780 was used for transient expression in pepper and geranium plants as described (Kapila et al. 1 997; Duan et al., 1 999; Wroblewski et al. 2005).
- Cultures of Agrobacterium harboring the constructs of interest were grown in minimal medium in the presence of acetosyringone to induce the Agrobacterium ⁇ //rgenes.
- the optical density of the cultures was maintained at 0.008 for pepper and at 0.25 for geranium.
- Strain GV2260 carrying plPG780 or empty vector control was first flooded into the apoplastic spaces of sweet pepper ⁇ Capsicum) leaves through open stomata by injection using a tuberculin syringe without a needle. An area of from 2 to 1 0 cm 2 of leaf was flooded and the area inoculated was then circled with a permanent marker.
- Sweet pepper is a nonhost of both pathogens.
- Plants that are attacked in nature are considered to be “hosts” of the indicated pathogens. All other plants are considered to be “nonhosts” of the indicated pathogens.
- R resistance
- HR hypersensitive response
- plPG780 was inoculated on one half of the leaf and the empty vector control was inoculated on the other half of the same leaf.
- HR symptoms elicited on the control side of the inoculated leaf by either X. pelargonii or R. solanacearum were abolished in the presence of transiently expressed BombBC on plPG780.
- Example 8 Use of transient expression of bombBCXn geranium (Pelargonium X hortorum) plants to demonstrate enhanced resistance to Ralstonia.
- bombBCXn geranium Pieris X hortorum
- assays similar to those described in Example 7 above were performed, this time using Florists' geranium (Pelargonium X hortorum). This was done in order to confirm that the killing or disabling of this pathogen's ability to elicit an HR on nonhosts also extended to pathogens of susceptible host plants.
- Example 9 Use of transient expression of bombBCXn citrus plants to demonstrate enhanced resistance to Xanthomonas citri.
- assays similar to those described in Examples 7 and 8 above were performed, this time using grapefruit ⁇ Citrus paradisi) plants inoculated with X. citri, causal agent of citrus canker disease. This agent is a regulated pathogen, and such inoculations had to be performed under strict quarantine.
- Example 1 0 Creation of transgenic geranium ⁇ Pelargonium X hortorum) using bomb BC.
- transgenic geraniums were achieved using either A. tumefaciens (Robichon et al., 1 995. Approximately 9% PCR positive geranium petiole explants were confirmed (of the 360 total petioles subjected to the transformation protocols. A total of 33 transgenic geranium were obtained, based on PCR amplification of the bombBC gene ( Figure 2). Selected plants were asexually reproduced and challenge inoculated with different pathogens as described below.
- bombBC gene shown to be expressed in transient expression assays, could be stably transformed and presumably expressed in geraniums at efficiencies equivalent to those obtained using empty vector or another gene construct, indicating that BombBC was not detrimental to geranium plants.
- Example 1 1 Creation of transgenic tobacco ⁇ Nicotiana tabaccum) using bombBC.
- Transgenic Nicotiana tabaccum cv. Xanthi plants were created using Agrobacterium tumefaciens and Rhizobium spp. using the bombBC gene cloned into plPG786. The most efficient methods for production of transgenic tobacco were achieved using the leaf disc method with A. tumefaciens as described (Horsch et al. 1 985). Transformants were selected on MS media (Murashige and Skoog 1 962) containing kanamycin at 1 00 ⁇ g/ml.
- Example 1 2 Creation of transgenic citrus (Citrus sinensis x Poncirus trifoliata) using bombBC Transgenic citrus ⁇ Citrus sinensis x Poncirus trifoliata) cv.
- Carizzo plants were created using Agrobacterium tumefaciens and Rhizobium spp. using bombBC gene cloned into plPG786. The most efficient methods for production of transgenic citrus were achieved using A. tumefaciens applied to etiolated citrus stem sections as described (Moore et al., 1 992). Approximately 6% PCR positive citrus stem explants were confirmed (of the 650 total stem sections subjected to the transformation protocols.
- Example 1 3 Creation of transgenic rice (Orvza sativa iaponica) using bomb BC. Transgenic rice ⁇ Oryza sativa] a ponica) cv.
- Example 14 Use of asexuallv reproduced progeny of transgenic geranium, citrus and tobacco plants to obtain cloned bombBC plants.
- Transgenic geranium, citrus and tobacco plants were obtained as set forth in Examples 10, 1 1 and 1 2.
- the transgenic geranium, citrus and tobacco plants were asexually propagated to produce progeny clones using techniques well known to one skilled in the art of geranium, citrus or tobacco propagation.
- a "scion” cutting is taken from a transgenic stem section with leaves and grafted or spliced onto a nontransgenic rootstock, such that the roots and lower main stem are comprised of the nontransgenic rootstock, while the upper main stem and shoots are comprised of the transgenic scion.
- the scion cuttings were in all cases genetically identical to the mother plant (ie., 1 00% PCR positive for BombBC); the genetic modifications performed in the mother plant were stable. These results demonstrated that the genetic modifications performed in the mother plant were stable through at least one asexual generation.
- Example 1 Use of sexually reproduced progeny of transgenic rice and tobacco plants to obtain cloned bombBC plants.
- Transgenic diploid rice and tobacco plants were obtained as set forth in Examples 1 1 and 1 3.
- the transgenic (To generation) rice and tobacco plants were self-pollinated and the seed (Ti generation) was harvested from the self-pollinated plants, processed, planted, and progeny plants grown from the self-pollinated- seed.
- PCR assays were used to determine that the Ti progeny plants all had a classical genetic 3: 1 ratio, wherein 3/4 of the plants (1 /4 homozygous transgenic and 1 /2 heterozygous transgenic plants) were found to be transgenic by PCR tests, and 1 /4 of the plants were nontransgenic.
- Example 1 Use of BombBC expressed in transgenic geranium ⁇ Pelargonium X hortorum) host plants to confer resistance to Xanthomonas pel argon ii and Ralstonia solanacearum. Pathogen challenge inoculations of transgenic Florist's geranium ⁇ Pelargonium X hortorum) plants expressing active BombBC and of asexually propagated Florist's geranium plants expressing active BombBC were conducted using X. pelargonii and R. solanacearum. The transgenic parental or asexually produced progeny clones obtained from the transgenic parental plants reduced disease symptoms.
- pelargonii cells were recovered from transgenic geranium variety "Avenida” plants after five days following inoculation ( Figure 3), and there was no evidence of symptoms of geranium blight caused by X. pelargonii. These plants were immune to X. pelargonii infection.
- R. solanacearum strain Rsp673 originally isolated from geranium and known to be strongly pathogenic to geranium, was inoculated by syringe infiltration of 1 O 6 cfu/ml directly into the spongy mesophyl of leaves using the blunt end of a tuberculin syringe.
- these same syringe inoculated plants were also inoculated by adding 5 ml of a liquid culture containing 1 O 7 cfu/ml of cells directly to the soil of the potted geranium plants (refer Figure 4). Following inoculation, plants were held at 32° C to encourage pathogen growth and symptom development.
- Example 1 Use of BombBC expressed in transgenic tobacco host plants to confer resistance to Ralstonia solanacearum. Pathogen challenge inoculations of transgenic tobacco ⁇ Nicotiana tabaccum cv. Xanthi) plants expressing BombBC were conducted using R. solanacearum. Both sexually propagated (seeded, Tl generation from Example 1 5; Exp 3 in Table below) and asexually propagated (cuttings, TO generation from Example 1 1 ; Exp. 1 and 2 in Table below)) tobacco plants were inoculated and compared, since the method of asexual propagation provides a healed over, but still significantly enlarged cut surface beneath the soil line that might facilitate entry by the soil-born pathogen. [Para 239] R.
- solanacearum strain Rsp446, strongly pathogenic to tobacco was In inoculated by adding 5 ml of a liquid culture containing 5X1 O 7 to 2 X 10 8 cfu/ml of cells directly to the soil of the potted tobacco plants. Following inoculation, plants were held at 32° C to encourage pathogen growth and symptom development. Plants were examined daily and wilted plants exhibiting black vein symptoms were noted and discarded. The results, recorded as number of survivors / total tested, after 68 days were as follows:
- Example 1 Use of BombBC expressed in transgenic citrus and tobacco host plants to confer resistance to Candidatus Liberibacter asiaticus.
- Citrus greening disease, or Huanglongbin is caused by Ca. Liberibacter asiaticus. This uncultured bacterial pathogen is a USDA Select Agent. It is known to attack tobacco plants, which may be used as a proxy host to test genes for resistance against the bacterium in transgenic tobacco (Francischini et al., 2007). Cuscuta spp. (dodder) was used to transmit greening from a known positively infected source, a sweet orange plant, to each of 6 healthy plants of Nicotiana tabacum L. cv. Xanthi.
- Example 1 Use of BombBC expressed in transgenic citrus host plants to confer resistance to citrus canker disease.
- Example 20 Use of transgenic rice plants to express enzvmaticallv active BombBC.
- Transgenic rice plants expressing BombBC were created using Ag ro bacterium tumefaciens (Hiei et al., 1 997) carrying the bombBC gene cloned into plPG787. It is anticipated that these plants will be resistant to Gram negative bacterial pathogens, including X. oryzae and X. oryzicola.
- Example 21 Method of Using Bomb Proteins Expressed in Transgenic Plants to Extend the Shelf-Life of Cut Flowers.
- Bomb proteins when produced in transgenic plants that are typically marketed as cut flowers, such as roses, carnations, chrysanthemums, gladiolas, etc., will enhance longevity of the cut transgenic flowers by suppressing bacterial growth in the vase water caused by opportunistic or soft-rotting bacteria such as Erwinia carotovora and Erwinia chrysanthemi.
- Transgenic plants that will later be marketed as cut flowers will be produced by methods described in the above examples.
- Example 22 Method of Using Bomb Proteins as an Additive to Extend the Shelf Life of Cut Flowers and Animal Feed.
- Bomb proteins possibly in combination with lytic proteins, when added to the vase or shipping container water of nontransgenic plants that are typically marketed as cut flowers, such as roses, carnations, chrysanthemums, gladiolas, etc., will enhance longevity of the cut transgenic flowers by suppression of fungal and bacterial growth in the vase water.
- Typical microbial species that shorten the shelf life of cut flowers are Erwinia carotovora and Erwinia chrysanthemi.
- Bomb proteins will most likely be produced in transgenic plants. Crude extracts of protein will be harvested, and either dried using a granular additive or suspended in an appropriate liquid and packaged. In another example, when the dried protein is added to animal feed, it will control microbial contamination, including those microbes that may cause food poisoning. A dry or liquid preparation of Bomb proteins could be added to animal feed during factory preparation or afterwards by the animal owner by mixing. Either way, the result will be a longer shelf life of the feed and reduced opportunity for growth of microbes that can result in food poisoning.
- Example 23 Method of Using Bomb Proteins in Transgenic Plants to Control Gram-Negative Bacteria, Whether Disease Agents of Plants or Not.
- transgenic plants producing Bomb proteins possibly in combination with production of a lytic protein, are planted in field situations, they will exhibit resistance not only to Gram negative bacterial diseases of said plants through killing or inhibiting growth of these Gram negative bacteria, but also they will kill or inhibit growth of Gram negative bacteria such as E coli, Shigella spp. and Salmonella spp. that may infect said plants, but without causing plant disease.
- Such transgenic plants may become part of a food security program aimed at reducing the possibility of spread of human diseases by food supply contamination.
- the opsXlocus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide.
- Norelli JL et al. 1 998 Effect of cercropin-type transgenes on fire blight resistance of apple. Acta Hort 489:273-278.
- Norelli JL et al. 1 999 Genetic transformation for fire blight resistance in apple. Acta Hort 489:295-296.
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EP2687097A1 (en) * | 2012-07-16 | 2014-01-22 | Universite D'angers | Potentiating agents for protecting plants from fungal infections |
WO2016123425A1 (en) * | 2015-01-29 | 2016-08-04 | Altria Client Services Llc | Endolysin from bacteriophage against geobacillus and methods of using |
US9781929B2 (en) | 2015-01-29 | 2017-10-10 | Altria Client Services Llc | Bacteriophage and methods of using |
US11566260B2 (en) | 2012-07-16 | 2023-01-31 | Universite D'angers | Potentiating agents for protecting plants from fungal infections |
CN116333075A (en) * | 2023-03-29 | 2023-06-27 | 海南省农业科学院蔬菜研究所 | Antibacterial peptide and application thereof |
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CN108207368A (en) * | 2017-11-24 | 2018-06-29 | 桂阳金盾南方苹果有限公司 | The sick control method of southern apple fire |
CN112143747B (en) * | 2020-09-09 | 2022-09-13 | 昆明理工大学 | Phage lyase, gene thereof, gene recombination expression vector and application |
CN113151192B (en) * | 2021-03-05 | 2023-11-24 | 菲吉乐科(南京)生物科技有限公司 | Xanthomonas phage capable of cross-species lysis, composition, kit and application thereof |
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EP2687097A1 (en) * | 2012-07-16 | 2014-01-22 | Universite D'angers | Potentiating agents for protecting plants from fungal infections |
WO2014012766A1 (en) * | 2012-07-16 | 2014-01-23 | Université d'Angers | Potentiating agents for protecting plants from fungal infections |
US10405550B2 (en) | 2012-07-16 | 2019-09-10 | Universite D'angers | Potentiating agents for protecting plants from fungal infections |
US11566260B2 (en) | 2012-07-16 | 2023-01-31 | Universite D'angers | Potentiating agents for protecting plants from fungal infections |
WO2016123425A1 (en) * | 2015-01-29 | 2016-08-04 | Altria Client Services Llc | Endolysin from bacteriophage against geobacillus and methods of using |
US9781929B2 (en) | 2015-01-29 | 2017-10-10 | Altria Client Services Llc | Bacteriophage and methods of using |
US10357056B2 (en) | 2015-01-29 | 2019-07-23 | Altria Client Services Llc | Endolysin from bacteriophage against Geobacillus and methods of using |
US11096414B2 (en) | 2015-01-29 | 2021-08-24 | Altria Client Services Llc | Endolysin from bacteriophage against geobacillus and methods of using |
CN116333075A (en) * | 2023-03-29 | 2023-06-27 | 海南省农业科学院蔬菜研究所 | Antibacterial peptide and application thereof |
CN116333075B (en) * | 2023-03-29 | 2023-08-18 | 海南省农业科学院蔬菜研究所 | Antibacterial peptide and application thereof |
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