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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8214—Plastid transformation
• • C—CHEMISTRY; METALLURGY
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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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—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 fungal resistance

Definitions

• FIELD OF INVENTION This application pertains to the field of genetic engineering of plant genomes, particularly plastids, and to methods of and engineered plants that express antimicrobial peptides that lead to and result in phytopathogenic bacteria resistance.
• Haynie in U. S. patent 5,847,047, entitled “Antimicrobial Composition of Polymer and a Peptide Forming Amphiphilic Helices of the Magainin-Type," offers a series of non-natural oligopeptides that share a common amino acid sequence referred to as the core oligopeptide.
• Such core oligopeptide has antimicrobial effects.
• the patent also provides N-addition analogues to the core oligopeptide that exhibit higher antimicrobial effects.
• Olsen et. al. in U. S. patent 6,143,498, entitled “Antimicrobial Peptide,” proposed a method of producing human antimicrobial peptides from the defensin superfamily through transformation of host cells. Olsen suggested the production of these defensin-related peptides through transformation of host cells with vectors containing the isolated DNA molecules of the peptides.
• a fusion gene - containing a basic anti icrovial peptide which- ligated directly or indirectly to a negatively charged acidic peptide having at least two cysteine residues - is cloned into an expression vector targeted toward microorganisms such as E. Coli.
• Plant diseases caused by bacterial pathogens have had a detrimental effect on global crop production for years. Between 1979 and 1980 India lost up to 60% of its rice crop due to bacterial rice blight. Between 1988 and 1990, there was a 10.1% loss of the global barley crop due to bacterial pathogens, worth $1.9 billion (Baker et al., 1997). In the United States, there was an estimated 44,600 metric ton reduction of soybean crops due to bacterial pathogens in 1994 (Wrath et al, 1996). On the average, pathogens are responsible for a 12-13% reduction of global crop production each year (Dempsey et al., 1998).
• Plant Defense against Pathogens Many of the pathways and products in the plant response to phytopathogens have been elucidated with the emergence of molecular biology.
• the plant defense response can be divided into 3 major categories, early defense (fast), local defense (fast/intermediate) and systemic defense (intermediate to slow) (Mourgues et al., 1998).
• Bacterial genes such as hrp (hypersensitive response and pathogenicity) or avr (avirulence) genes stimulate the plant defense mechanism (Baker et al., 1997).
• the most prominent early defense response is the HR (hypersensitive response), which leads to cellular death reducing further infection by the pathogen.
• Anti-fungal peptides produced by various organisms have been cloned and studied. However, although anti-fungal development has been promising, bacteria still maintain the ability to adapt to plant defenses. Those skilled in the art will be familiar with antimicrobial peptides. Examples of some of these substances include PGLa (frog skin), defensins (human phagocytes), cecropins (Silkmoth pupae or pig intestine), apidaecins (honeybee lymph), melittin (bee venom), bombinin (toad skin) and the magainins (frog skin).
• bactericidal peptides include large polypeptides such as lysozyme (MW 15000 daltons) and attacins (MW 20-23,000 daltons) as well as smaller polypeptides such as cecropin (MW 4000 daltons) and the magainins (MW 2500 daltons).
• large polypeptides such as lysozyme (MW 15000 daltons) and attacins (MW 20-23,000 daltons) as well as smaller polypeptides such as cecropin (MW 4000 daltons) and the magainins (MW 2500 daltons).
• the spectrum of biocidal activity of these peptides is somewhat correlated to size.
• the large polypeptides are active against limited types and species of microorganisms (e.g., lysozyme against only gram positive bacteria), whereas many of the smaller oligopeptides demonstrate a broad spectrum of antimicrobial activity, killing many species of both gram positive and gram negative bacteria.
• magainin, cecropins, and bombininin oligopeptides form similar secondary structures described as an amphiphilic helix (Kaiser et al. Arum. Rev. Biophys. Biophys. Chem 16, 561-581, 1987). These peptides with a-helical structures are ubiquitous and found in many organisms. They are believed to participate in the defense against potential microbial pathogens.
• One of the first biocidal oligopeptides to be isolated from natural sources was bombinin and is described by Csordas et al. (Proc. Int. Symp. Anim. Plant Toxins, 2, 515-523, (1970)). Csordas teaches significant sequence homology between bombinin and melittin, another antimicrobial peptide, isolated from bee venom.
• magainins from Xenopus laevis Africann frog
• its analogues have been investigated by Zasloff et al. (WO 9004408) as pharmaceutical compositions such as a broad- spectrum topical agent, a systemic antibiotic; a wound-healing stimulant; and an anticancer agent (Jacob and Zasloff, 1994).
• Cuervo et al. (WO 9006129) describe the preparation of deletion analogues of magainin I and II for use as pharmaceutical compositions. They disclose a general scheme for the synthetic preparation of compounds with magainin-like activity and structure.
• magainin-type antimicrobial peptides has not yet been explored.
• Plastid Transformation To date, plastid transformation, particularly has enabled generation of herbicide (Daniell et al, 1998), insect resistant crops (Kota et al., 1999; McBride et al., 1995; DeCosa et al., 2000) and production of pharmaceutical proteins (Guda et al., 2000; Staub et al., 2000). Plastid transformation was selected because of several advantages over nuclear transformation (Daniell, 1999 A, B; Bogorad, 2000; Heifetz, 2000).
• plastid expressed genes are maternally inherited in most crops. Gene containment is possible when foreign genes are engineered via the plastid genome, which prevents pollen transmission in crops that maternally inherit the plastid genome. Because a majority of crop plants inherit their plastid genes maternally, the foreign genes do not escape into the environment. Although pollen from plants that exhibit maternal inheritance contain metabolically active plastids, the plastid DNA is lost during pollen maturation (Helfetz, 2000). Despite the potential advantage of plastid reproduction of AMPs, it was not obvious that AMPs would be produed in this manner.
• small peptides are not stable inside living cells and are highly susceptible to proteolytic degradation. For this reason, small peptides are usually produced as fusion proteins with larger peptides in biological systems. Megainin type peptides are chemically synthesized and never made in biological systems for that reason. Therefore, it was not obvious to express a small peptide of a few amino acids within plastids. Successful expression of this antimicrobial peptide was not anticipated but this invention opens the door for expression of several small peptides within plastids, including hormones.
• This invention provides a new option in the battle against phytopathogenic bacteria through transformation of the plant plastid genome.
• the present invention is applicable to all plastids of plants. These include chro oplasts which are present in the fruits, vegetables and flowers; amyloplasts which are present in tubers like the potato; proplastids in roots; leucoplasts and etioplasts, both of which are present in non-green parts of plants. All known methods of transformation can be used to introduce the vectors of this invention into target plant plastids including bombardment, PEG Treatment, Agrobacterium, microinjection, etc.
• This invention provides plastid expression constructs which are useful for genetic engineering of plant cells and which provide for enhanced expression of a foreign peptide in plant cell plastids.
• the transformed plant is preferably a metabolically active plastid, such as the plastids found in green plant tissues including leaves and cotyledons.
• the plastid is preferably one which is maintained at a high copy number in the plant tissue of interest.
• the plastid expression constructs for use in this invention generally include a plastid promoter region and a DNA sequence of interest to be expressed in transformed plastids.
• the DNA sequence may contain one or a number of consecutive encoding regions, one of which preferably encoding an antimicrobial peptide of the magainin family.
• Plastid expression construct of this invention is linked to a construct having a DNA sequence encoding a selectable marker which can be expressed in a plant plastid. Expression of the selectable marker allows the identification of plant cells comprising a plastid expressing the marker.
• transformation vectors for transfer of the construct into a plant cell include means for inserting the expression and selection constructs into the plastid genome. This preferably comprises regions of homology to the target plastid genome which flank the constructs.
• the plastid vector or constructs of the invention preferably include a plastid expression vector which is capable of importing phytopathogenic bacteria resistance to a target plant species which comprises an expression cassette which is described further herein.
• a vector generally includes a plastid promoter region operative in said plant cells' plastids, a DNA sequence which encode at least an antimicrobial peptide of the magainin family.
• expression of one or more DNA sequences of interest will be in the transformed plastids.
• the preferred embodiment of the invention provides a universal plastid vector comprising a DNA construct.
• the DNA construct includes a 5' part of a plastid spacer sequence; a promoter, such as Prrn, which is operative in the plastid of the target plant cells; a heterologous DNA sequence encoding at least one antimicrobial peptide of the magainin family; a gene that confers resistance to a selectable marker such as the aadA gene; a transcription termination region functional in the target plant cells; and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome.
• a promoter such as Prrn
• the vector may further comprise a ribosome binding site (rbs), a 5' untranslated region (5'UTR).
• rbs ribosome binding site
• a promoter such as psbA, accD or 16srRNA, is to be used in conjunction with the 5'UTR.
• the heterologous DNA sequence of the DNA construct may also include other genes whose expression are desired.
• non-universal plastid vectors such as pUC, pBlueScript, pGEM may be used as the agent to insert the DNA construct
• This invention provides transformed crops, like solanaceous, monocotyledonous and dicotyledonous plants, that are resistant to phytopathogenic bacteria.
• the plants are edible for mammals, including humans.
• These plants express an antimicrobial peptide at levels high enough to provide upwards of 96% inhibition of growth against Pseudomonas syringae, a major plant pathogen.
• the transformed plants do not differ morphologically from untransformed plants.
• FIG. 1 Phenotype of T 0 and Titransgenic plants. Plantsl-3 are To transgenic plants while plant 4 is untransformed. Plants 5-7 are T, transgenic plants. Seedlings germinated on MSO+500 ⁇ g/ml spectinomycin (B). Three Ti transgenic lines (1-3) and Control (4).
• FIG. 3 (A) Primers, 8P and 8M used to confirm integration of foreign genes via PCR. 8P anneals with the 5'end of the aadA gene and 8M anneals with the 3'end of the 16S rDNA gene. PCR analysis of DNA extracted from T 0 (B), Ti (C) and T 2 (D) plants run on a 0.8% agarose gel. To (B) Lane 1 lkb ladder, 2 through 5 transgenic lines, 6 MSI-99 plasmid. T, (C) Lane 1, lkb ladder, 2 through 4 transgenic, lane 5 plasmid control and lane 6 untransformed plant DNA. T 2 (C) lane 1, lkb ladder, 2 through 5 transgenic, lane 6 plasmid control and lane 7 untransformed plant DNA.
• FIG. 4 Southern analysis of To and Ti generations.
• A Probe used to confirm integration of foreign genes. The 2.3kb probe fragment was cut with BamHI and Notl containing the flanking sequence.
• B Lane 2-6 To transgenic lines, lane 1 untransformed and Lane 7 plasmid DNA.
• C Lanes 2-7 Ti transgenic lines, Lane 1 untransformed and Lane 8 plasmid DNA.
• Figure 5. In situ bioassays. 5 to 7mm areas of To transformants and untransformed Petit Havana leaves were scraped with fine grain sandpaper. Ten ⁇ l of 8xl0 5 , 8xl0 4 , 8xl0 3 and 8xl0 2 cells from an overnight culture of P. syringae were added to each prepared area.
• Figure 7 In vitro bioassays for P. aeruginosa. Five ⁇ l of bacterial cells from an overnight culture were diluted to (A 60 o 0.1-0.3) and incubated for 2 hours at 25 °C with lOO ⁇ g of total protein extract from Ti plants. One ml of LB broth was added to each sample. Samples were incubated overnight at 37°C. Absorbance at 600nm was recorded. Data was analyzed using GraphPad Prism. Negative control was an untransformed plant extract. Buffer only was added as a control and stock culture was used as a reference point. Figure 8. Five ⁇ l of an overnight culture of P.
• syringae diluted to (A 60 o 0.1-0.3) was mixed with lOO ⁇ g total protein extract from T2 lines 11A and 13A (germinated in the absence of spectinomycin). After 2-hour incubation, 1ml of LB broth was added to the mixture and incubated over night at 27°C. The following morning absorbance at 600 nm was recorded (A). In parallel, 50 ⁇ l of each mix was plated onto LB plates and incubated overnight at 27°C. The next morning a count of viable CFUs were made using the Bio Rad Gell Dock (B).
• This invention demonstrates the confering of phytopathogenic resistance in plants through plastid transformation.
• This invention includes the use of all plastids in plants, including chloroplasts, chloroplasts which are present in fruits, vegetables and flowers, amyloplasts which are present in tubers, proplastids in roots, lencoplasts in non-green parts of plants, hi a preferred embodiment of the invention, the chloroplast genome is used. Plastid transformation and expression vectors comprising heterologous DNA encoding magainin and its analogues are provided .
• the anti-microbial peptide (AMP) used in this invention is an amphipathic alpha-helix molecule that has an affinity for negatively charged phospholipids commonly found in the outer-membrane of bacteria. Upon contact with these membranes, individual peptides aggregate to form pores in the membrane, resulting in bacterial lysis. Because of the concentration dependent action of the AMP, it was expressed via the plastid genome to accomplish high dose delivery at the point of infection. PCR products and Southern blots confirmed plastid integration of the foreign genes and homoplasmy. Growth and development of the transgenic plants was unaffected by expression of the AMP within the plastids.
• cationic peptides such as MSI-99 are a net positive charge, an affinity for negatively charged prokaryotic membrane phospholipids over neutral-charged eukaryotic membranes, and the ability to form aggregates that disrupt the bacterial membrane (Houston et al., 1997; Matsuzaki et al, 1999; Biggin and Sansom, 1999).
• the outer membrane is an essential and highly conserved part of all bacterial cells, it is highly unlikely that bacteria would be able to adapt (as they have against antibiotics) and to resist the lytic activity of these peptides.
• the thylakoid membrane consists of primarily glycolipids and galactolipids instead of phospholipids.
• MGDG Monogalactosyldiacylglycerol
• DGDG digalactosyldiacylglycerol
• An object of this invention is to compartmentalize the expression of the MSI-99 within the plastid. Compartmentalization of lytic enzymes is a natural occurrence in plants. Compartmentalization serves two purposes: to increase the yield of the peptide and to deliver the peptide at the site of the infection. Due to the high copy number associated with plastid expression, a larger amount of the peptide is produced. The higher yield is important due to the concentration- dependent action of the anti-microbial peptide.
• the peptide would be released at the site of infection during the HR response.
• HR response occurs, cells are lysed. This disrupts the osmotic balance and causes plastids to lyse. This would release the peptide at high concentration resulting in aggregation and formation of pores in the outer membrane of bacteria. This aids in the prevention of the spread of infection by bacteria.
• a high level of AMP expression can be expected due to the following reasons. The nature of plastids to move from a somatically unstable heteroplasmic state to a state of homoplasmy itself lends to high expression (Brock and Hagemann, 2000).
• the A+T % of MSI-99 is 51.39%, which is compatible with the Nicotiana tobacum plastid 61% A+T content (Bogorad et al., 1991; Shimada et al., 1991). Also, published reports from our lab report expression of Cry2A operon (A+T content of 65%) at levels as high as 46% total soluble protein (DeCosa et al., 2000). MSI-99 was most effective against P. syringae, evidenced by total inhibition of 1000 P. syringae cells with only l ⁇ g/1000 bacteria (Smith et al. unpublished data).
• MSI-99 The synthetic peptide used in this invention (MSI-99), is an analogue of the naturally occurring 23 amino acid peptide, magainin II.
• MSI-99 is a 22 amino acid sequence with an overall charge of +6 as shown in Figure 1.
• the gene cassette used for transformation consisted of the 16S rRNA promoter, the aadA gene, which confers resistance to spectinomycin, the MSI-99 gene and the psbA (photosynthetic binding protein) terminator.
• the gene construct may contain, in addition to the MSI-99 gene, another heterologous DNA sequence coding for a gene of interest.
• Flanking sequences are from the petunia plastid genome as shown in Figure 1A. Transformation efficiency was much lower (7%) than that observed using the pLD vector (91%), which contains tobacco homologous flanking sequences.
• Other vectors that are capable of plastid transformation may be used to deliver the gene cassette into the plastid genome of the target plant cells. Such vectors do include plastid expression vectors such as pUC, pBlueScript, pGEM, and all others identified by Daniell in US patents number 5,693,507 and 5,932,479. These publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
• the vectors preferably include a ribosome binding site (rbs) and a 5' untranslated region (5'UTR). A promoter operably in green or non-green plastids is to be used in conjunction with the 5'UTR)
• the number of transformants from the total number of shoots determined percent of transformants. Out of 55 spectinomycin resistant shoots screened, only 4 were transformants with the MSI-99 gene and the rest were mutants. All transformants grew healthy with no apparent morphological effects to To and Tj, generations as shown in Figure 2A. Ti, seeds germinated in the presence of spectinomycin produced healthy green seedlings, while control seedlings were bleached as shown in Figure 2B.
• PCR was performed by landing one primer on the 5'end of the aadA coding sequence, not present in native plastid and the 3 'end of the 16S rDNA ( Figure 3 A). PCR products of T 0 , Ti, and T 2 generations yielded the same size product as the plasmid (MSI-99) as shown in Figure 3B,C,D confirming integration of the foreign genes.
• the probe used for the Southern analysis was a 2.3kb fragment from the 5'end of the tail (BamHI) to the 3'end of the 16SrDNA (Notl) ( Figure 4A). The plant DNA was digested with BamHI.
• DNA from untransformed plants produced a 3.269kb fragment and transformed plant DNA produced a 4.65kb fragment.
• Southern analysis confirmed integration of foreign genes for To and Ti, as shown in Figure 4B,C. Untransformed DNA showed a 3.2kb fragment while the transformed contained a 4.65kb fragment. Presence of some wild type fragments in To transgenic samples indicated some heteroplasmy as shown in Figure 4B. However, DNA from Ti, generation produced only the 4.65Kb fragment confirming homoplasmy. As shown in Figure 4C. A cell is said to be homoplasmic when all of the plastid are uniformly transformed. If only a fraction of the genomes was transformed, the copy number should be less than 10,000 (Bendich, 1987). By confirming that the MSI-99 integrated genome is the only one present in transgenic plants (homoplasmy), one could estimate that the MSI-99 gene copy number could be as many as 10,000 per cell.
• the initial low rate of transformation was most likely due to less than 100% homology between the petunia flanking sequences and the tobacco plastid genome. This is not surprising because very low transformation efficiency was also observed when tobacco plastid flanking sequences were used to transform potato plastid genome (Sidorov et al., 1999). Also, other projects in our lab that use the pLD vector (has tobacco flanking sequences) obtained transformation efficiency of 91% transformants to mutants. To and T] transgenic plants were healthy and showed no morphological or developmental abnormalities. Retention of lytic activity was evident in the sharp decrease in bacterial growth in the in vitro bioassays (84 to 96%). When comparing Southern blots to lytic activity, lytic activity increased as homoplasmy was reached.
• Plastid expression in crops such as tobacco should allow for mass production of the peptide at a lower cost compared to chemical synthesis or production in E. coli. This invention thus demonstrates another option in the on going battle against pathogenic bacteria.
• the invention is exemplified by the following non-limiting example.
• Example 1 Plant transformation For plant transformation, Nicotiana tabacum var. Petit Havana seeds were germinated on MSO media at 27°C with photoperiods of 16 hour light and 8 hour dark. Sterile leaves were bombarded using the Bio-Rad Helium driven PDS-1000/He System. After bombardment, leaves were wrapped and kept in the dark for 48 hours. Leaves were then cut into 1cm 2 squares and placed on a petri dish containing RMOP media with 500 ⁇ g/ml spectinomycin (first round of selection). Four to six weeks later, shoots were transferred to fresh media and antibiotic (second round of selection). Shoots that appeared during the second selection were transferred to bottles containing MSO and spectinomycin (500 ⁇ g ml).
• Plants were screened via PC for transformation. Those that were PCR positive for the presence of the MSI-99 gene were transferred to pots and grown in chambers at 27°C with photoperiods of 16-hour light and 8-hour dark. After flowering, seeds were harvested and sterilized with a solution of I-part bleach and 2-part water with 1 drop of tween-20. Seeds were vortexed for 5 minutes then washed 6 times with 500 ⁇ l of dH 2 0 and dried in speed vac. T 1 , and T 2 seeds were germinated on MSO + 500 ⁇ g/ml spectinomycin. Untransformed Petit Havana seeds were germinated on the same media as a control to ensure the spectinomycin was active.
• PCR conformation Plant DNA extraction on To, T_, and T 2 was performed using the QIAGEN DNeasy Mini Kit on putative transgenic samples and untransfon-ned plants.
• PCR primers were designed using Primer Premier software and made by GIBCO BRL. Primer
• PCR was carried out using the Gene Amp PCR system 2400 (Perkin-Elmer). Samples were run for 29 cycles with the following sequence: 94°C for 1 minute, 65°C for 1 minute and 72°C for 3 minutes. The cycles were proceeded by a 94°C denaturation period and followed by a 72°C final extension period. A 4°C hold followed the cycles. PCR products were separated on agarose gels.
• leaf tissue minus mid-rib
• phosphate buffer pH5.5 with 5mM PMSF and 5mM with a plastic pestle 50 mg was grounded in a micro-centrifuge containing 150 ⁇ l of phosphate buffer pH5.5 with 5mM PMSF and 5mM with a plastic pestle. Samples were centrifuged for 5 minutes at 10,000x g at 4°C. Supernatant was transferred to a fresh tube and kept on ice. Protein concentration was determined by Bradford assay. One hundred ⁇ g of total plant protein was mixed with 5 ⁇ l of bacteria from overnight culture in a falcon tube. Initial absorbency ranged from 0.1 to 0.3 (Aeoo)- Tubes were incubated for 2 hours at 25°C on a rotary shaker at 125rpm.
• a serial dilution was prepared from the starting bacterial culture (Absorbance ⁇ oO) 0.1-0.3) used for the In vitro bioassay. Fifty ⁇ lof each dilution was plated on LB medium and incubated overnight at 27°C. The following morning, CFUs were counted using the Bio Rad Gel Dock and the amount of cells used in the bioassay was calculated. The minimum inhibitory concentration of I ⁇ g/1000 P.syringae cells was used to determine antimicrobial peptide concentration in lOO ⁇ g of cell free plant extracts.
• In situ bioassay P. syringae was cultured overnight prior to the assay. Five to seven mm areas of To transformants and untransformed Petit Havana leaves were scraped with fine grain sandpaper. Ten ⁇ l of 8xl0 5 , 8xl0 4 , 8XI0 3 and 8x10 2 cells from an overnight culture of P. syringae were added to each prepared area. Photos were taken 5 days after inoculation.

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