WO2007016680A2 - Peptides de defense vegetaux a presentation a la surface des phages filamenteux, diriges contre phakopsora pachyrhizi et uromyces appendiculatus - Google Patents

Peptides de defense vegetaux a presentation a la surface des phages filamenteux, diriges contre phakopsora pachyrhizi et uromyces appendiculatus Download PDF

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WO2007016680A2
WO2007016680A2 PCT/US2006/030291 US2006030291W WO2007016680A2 WO 2007016680 A2 WO2007016680 A2 WO 2007016680A2 US 2006030291 W US2006030291 W US 2006030291W WO 2007016680 A2 WO2007016680 A2 WO 2007016680A2
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peptides
vector
seq
phage
peptide
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PCT/US2006/030291
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WO2007016680A3 (fr
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James T. English
Francis J. Schmidt
Gary Stacey
Zhiwel Fang
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University Of Missouri Board Of Curators
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Priority to CN2006800367777A priority Critical patent/CN101473226B/zh
Priority to BRPI0614523-0A priority patent/BRPI0614523A2/pt
Publication of WO2007016680A2 publication Critical patent/WO2007016680A2/fr
Publication of WO2007016680A3 publication Critical patent/WO2007016680A3/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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

  • This disclosure relates to the use of phage display technology to identify peptides that bind to pathogenic fungi and more particularly to pathogenic fungi of the genera Phytophthora, Phakapsora, and Uromyces.
  • Random peptide phage display libraries are constructed using degenerate oligonucleotides. Phage expressing the peptides on their surface are contacted with fungi at different life stages and those phage that bind are isolated, amplified and the peptides identified. Once identified, peptides can be screened for anti fungal activity and used to identify and characterize binding sites on fungi.
  • Phytophthora is an economically important disease causing organism in the United States causing large losses in many agronomically important crop species. Phytophthora sojae is the second most important pathogen of soybeans in the United States. (Doupnik, Plant Dis. 77:1170-1171, 1993). Phytophthora capsici has a broad host range and most notably limits production of high- value, solanaceous vegetable crops. Control of these pathogens is particularly difficult, often requiring treatment of entire fields with biocidal compounds. Although effective, increasing concern about the environmental and economic costs of such treatments require the need for alternative control methods. [0005] Phytophthora species are obligate parasites adapted to long-term survival in soil in the absence of host plants.
  • Oospores or chlamydospores exist in low densities in the soil and enable survival of the pathogen. In the presence of a susceptible plant, the pathogen progresses rapidly through a series of finely tuned developmental steps that produce cycles of infection and disease. Pathogen development from oospores or chlamydospores through zoospore release, encystment, germination and infection appear straight-forward at first glance. Yet, the procession of life stages is finely tuned to environmental signals, particularly those signals coming from a host plant. [0006] Zoospores are the life-stage of greatest importance for dispersal to root infection sites.
  • a major susceptible site is located just behind the apical meristem of the root where cells are elongating. Exudates released from elongating cells serve as signals that direct chemotactic movement of zoospores toward the site (Carlile, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983; Deacon and Donaldson, Mycol Res. 97:1153-1171, 1993).
  • the zoospore chemotactic response varies with the composition of root exudates and is species specific. For example, zoospores of P. capsici, P.
  • Encystment of zoospores of P. palmivora and other Phytophthora species can be influenced by local calcium ion concentrations (Griffith et al., Arch. Microbiol, 149:565-571, 1988; Warburton and Deacon, Fungal Genetics Biol, 25:54-62, 1998). Encystment can also be induced by high concentration of chemoattractants or by root cell wall components. For example, zoospores of P.
  • Haustoria are formed by some Phytophthora species, including P. infestans (Coffey and Wilson, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983), P. capsici (Jones et al., Phytopathology, 64:1084-1090, 1974), and P. sojae (Stossel et al., Can. J. Bot, 58:2594-2601, 1980). Both hyphae and haustoria establish close contact with host cell walls and membranes. Presumably, cell surface receptors are important in sensing plant signals, although direct evidence is lacking.
  • Fusion phage are filamentous bacteriophage vectors in which foreign peptides and proteins are cloned into a phage coat gene and displayed as part
  • a phage coat protein 15 of a phage coat protein.
  • the commonly used coat genes for the production of fusion phage are the pVIII gene and the pill gene. About 3900 copies of p VIII make up the major portion of the tubular virion protein coat. Each pVIII coat protein lies at a shallow angle to the long axis of the virion, with its C-terminus buried in the interior close to the DNA and its N-terminus exposed to the external environment. Five copies
  • phage 10 of the pill coat protein are located at the terminal end of each virion and are involved in attachment of the phage to pill of E. coli and for virus reassembly after infection and replication.
  • Peptides displayed as part of p VIII are constrained in the matrix of their display on the virion coat.
  • peptides displayed as part of pill are more flexible due to the terminal position of the pill proteins. Specific phage can be
  • phage display is useful for screening for rare peptides with desired binding characteristics.
  • Phage-displayed random peptide libraries have been used for isolating ligands to cell surface receptors on mammalian cells.
  • peptides have been isolated from phage-displayed libraries that bind the transmembrane integrin glycoproteins involved in cell-extracellular matrix and cell-cell interactions (O'Neil et al, Proteins, 14:509-515, 1992; Smith et al., J Biol Chem., 269:32788- 32795, 1994; Healy et al., Biochemistry, 34:3948-3955, 1995).
  • Phage-displayed random peptide libraries have also been used to select peptides that distinguish between brain and kidney tissue (Pasqualini and Ruoslahti, Nature, 380:364-366, 1996).
  • Phage display methods have been used almost exclusively to identify antibodies for plant virus diagnosis (Susi et al., Phytopathology, 88:230-233, 1998; Ziegler et al., Phytopathology, 88:1302-1305, 1998; Griep et al., J Plant Pathol., 105:147-156 1999; Toth et al., Phytopathology, 89:1015-1021, 1999). Phage display was used in a single instance to select antibodies with affinity to surface-exposed epitopes on germlings and spores of Phytophthora infestans (Gough et al., J. Immunol. Methods, 228:97-108, 1999).
  • Isolated phage- displayed, single-chain variable fragment (Fv) antibody fragments were not assessed for their potential to influence spore or germling behavior. Antibodies were tested for their antifungal activity with sporangia, but were found to have no detectable antifungal activity.
  • Phakopsora pachyrhizi is a fungus that causes a rust disease of soybean (Glycines max). The pathogen has spread from Asia to all other soybean production regions in the world. P. pachyrhizi arrived in the US during the fall of 2004. At present, there is no known durable resistance available in any soybean varieties. Uromyces appendiculatus is a fungus that causes rust on bean (Phaseolus vulgaris). Breeders are working to identify genes in bean that can be manipulated for rust resistance. U.S. soybean producers have anticipated the arrival of Phakopsora pachyrhizi, the fungus that causes soybean rust, since its reported occurrence in Brazil. The arrival of P. pachyrhizi in the U.S. last fall ended the anticipation, and farmers must now respond to the potential annual occurrence of this new disease. Farmer concerns have been based on reports of losses ranging from 10 to 80% in other regions of the world when control measures were not successfully implemented.
  • Fungicides will likely be the front-line of defense for many years until new resistance genes or other forms of resistance are identified.
  • Fungicides have not traditionally been used in most soybean production. Consequently, there is limited information concerning the costs of this disease management practice and its likely economic viability. These concerns have led to variable estimates of the acreage in Missouri and other states that may be shifted from soybean to alternative crops.
  • U. appendiculatus and P. pachyrhizi belong to the order Uredinales, within the class Basidiomycetes.
  • U. appendiculatus produces five spore stages on a single host plant.
  • P. pachyrhizi reproduces predominantly by uredospores on a single host plant.
  • Uredospores are responsible for rapid spread of the fungus.
  • P. pachyrhizi can infect dozens of legume species, in addition to soybean.
  • Uredospores of U. appendicular penetrate through foliar stomatal openings. P.
  • pachyrhizi differs in that germinated uredospores penetrate directly through the leaf epidermal cell layer.
  • a uredospore that lands on a leaf surface germinates to produce an infection pad (appressorium) that adheres to the surface.
  • the appressorium produces a hyphal peg that penetrates the plant.
  • each fungus develops thread-like structures (hyphae) that grow inter-cellularly through leaf tissues. The hyphae enter host cells without killing them. There, they produce spherical structures (haustoria) that extract nutrients from the living leaf cells. Soon after infection each fungus forms uredia that produce additional spores.
  • Combinatorial phage-display libraries provide a vast array of random peptides from which to select ligands directed to proteins of interest (O 'Neil et al., 1992).
  • Phage-display peptide libraries are mixtures of filamentous phage clones, each of which displays a single foreign peptide sequence on the virion surface (Cwirla, 1990; Scott and Smith, 1990). The displayed peptide is physically linked with its coding DNA in the phage genome. Thus, the peptide can be easily and quickly identified and transferred to other vectors or display systems.
  • Typical libraries contain 10 9 random peptide variants.
  • Random peptide libraries are useful for isolating ligands of importance to cell-surface molecules of mammalian cells.
  • peptides with affinity for transmembrane glycoproteins, integrins have been isolated from libraries by biopanning against purified molecules (O.Neil et al., 1992; Smith et al., 1994; Healy et al., 1995). Some peptides were found to block cell adhesion to defined extracellular molecules and to other cells.
  • the present invention overcomes the problems outlined above and advances the art by providing resistance to pathogens in planta.
  • soybeans are made resistant to soybean rust where no durable resistance is currently available.
  • the same general techniques may provide Phaseoulus vulgaris that is resistant to common rust.
  • a method for identifying peptides having an affinity for the surface of a plant pathogen a library is constructed to include random peptides by providing degenerate oligonucleotides encoding peptides.
  • the oligonucleotides are inserted into an appropriate vector that expresses the encoded peptides on its surface and is capable of transfecting a host cell.
  • a host cell is transfected with the vector to amplify the vector in a infectious form to create a library of peptides on the vector.
  • the vector expressing the peptide library is then contacted with a target pathogen and allowed to bind to the pathogen.
  • Unbound vector is removed and vector that has bound to the pathogen eluted.
  • the eluted vector is then amplified in a suitable host cell and the inserted oligonucleotides isolated.
  • the oligonucleotides are then sequenced by any suitable method and the amino acid sequence of the peptides deduced from the sequence of the oligonucleotides.
  • One aspect of the disclosed instrumentalities concerns peptides, and the means for their selection, as new soybean and bean resistance factors.
  • the peptides are advantageously identified and selected without any knowledge of specific pathogenicity targets in the pathogen. Since these peptides do not necessarily occur in soybean or bean in nature, the pathogen has not been exposed to them before.
  • peptides may provide rust resistance in transformed plants when displayed as part of scaffold proteins.
  • these peptides may be selected according to the instrumentalities disclosed herein by binding affinity for infective structures of germlings, i.e., germinated spores, of U. appendiculatus and P. pachyrhizi.
  • cytokinin oxidase may be modified to serve as a scaffold protein for display of selected peptides in plants.
  • pathogen populations may be able to adapt to the presence of a selected resistance-conferring peptide.
  • a new defense peptide can be selected rapidly to meet the challenge of changing pathogen populations.
  • Particular advantages of the disclosed instrumentalities include speed and simplicity of peptide selection process and phenotype assessment, peptide selection does not required knowledge of pathogenicity targets in a pathogen, a high percentage of recovery of effective peptides, an ability to rapidly identify new defense peptides on demand, an ability to deploy scaffold-peptides to susceptible plant tissues, and an ability to modify scaffold-peptide constructs rapidly on demand.
  • Another aspect provides an antifungal composition
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 20
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO: 22
  • LTRCLVSTEMAARRP SEQ ID NO: 24
  • EFRKNYPSAAPLIPR SEQ ID NO: 31
  • Still another aspect is a recombinant nucleotide comprising a sequence encoding a peptide selected from the group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 20
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO: 22
  • LTRCLVSTEMAARRP SEQ ID NO: 24
  • Yet another aspect is a recombinant vector comprising a nucleotide sequence encoding a peptide selected from the group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 2O) 5 AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 2O
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO: 22
  • LTRCLVSTEMAARRP SEQ ID NO:
  • a further aspect is a cell transformed with a vector comprising a nucleotide sequence encoding a peptide selected from the group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 20
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO: 22
  • LTRCLVSTEMAARRP SEQ ID NO: 24
  • an expression cassette comprising as operatively linked components, a promoter; a nucleotide sequence encoding a peptide selected from the group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4); and a transcription termination signal sequence.
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 20
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO: 22
  • An additional aspect provides, a recombinant plant comprising an expression cassette comprising a promoter; a nucleotide sequence encoding a peptide selected from the group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4); and a transcription termination signal sequence.
  • ADPPRTVST SEQ ID NO: 7
  • ADRPSMSPT SEQ ID NO: 8
  • ADITDPMGA SEQ ID NO: 20
  • AVGTHTPDS SEQ ID NO: 21
  • AVSPNVHDG SEQ ID NO:
  • Another aspect provides a method for characterization of peptides having an affinity for the surface of plant pathogens comprising providing a library of random peptides made by providing degenerate oligonucleotides encoding peptides; inserting the oligonucleotides into an appropriate vector that expresses the peptides on its surface and is capable of transfecting a host cell; and transfecting an appropriate host cell with the vector to amplify the vector in an infectious form to create a library of peptides on the vector.
  • the vector expressing the peptide library is then contacted with a plant pathogen of interest and the vector allowed to bind to the pathogen.
  • the unbound vector is removed and the bound vector eluted from the pathogen.
  • the eluted vector is amplified in a suitable host cell and the inserted oligonucleotides in the eluted vectors isolated.
  • the peptides encoded by the oligonucleotides are then produced, contacted with plant pathogens of interest, and the effect on infectivity observed.
  • the isolated oligonucleotides are sequenced, the amino acid sequence of the peptides deduced from the nucleotide sequence, and the peptides produced by chemical synthesis.
  • peptides are produced by inserting the isolated oligonucleotides into an expression vectors which is used to transform a suitable host cell. The transformed host cells are then maintained under conditions suitable for expression of the peptides.
  • Combinatorial libraries have also been screened against intact cells for recovery of peptides that bind to populations of undefined surface proteins. Peptides recovered by this type of screening can be evaluated for a phenotype of interest. For instance, we selected peptides with affinity to motile zoospores of the oomycete plant pathogen, Phytophthora. Phenotype screens produced a sub- collection of peptides that disrupted normal development by inducing premature encystment of the spores (Bishop-Hurley et al., 2002). We also showed that affinity- selected peptides disrupt zoospore development when displayed either in a phage- display format or when synthesized as free molecules (Laskey et al., 2001; Bishop- Hurley et al., 2002).
  • FIG. 1 shows the organization of f8 peptide sequences into six families. On the left is the dendogram and on the right are the names and sequences of the peptides.
  • FIG. 2 shows encystment of P. capsici zoospores in response to contact with the indicated f8 phage-displayed peptides at the various concentrations given. Percentage values represent the means of two experiments. The percentage encystment for the control zoospore population that contained no phage varied between 0 and 10%.
  • FIG. 3 shows encystment of P. capsici zoospores in response to contact with the indicated f8 and f88-4 phage-displayed peptides at the various concentrations given.
  • FIG. 4 shows binding of the indicated phage-displayed peptides to 5 P. capici zoospsores. Values represent the mean of three experiments.
  • FIG. 5 shows the binding specificity of the indicated phage- displayed peptides to P. capici. Values represent the mean of three experiments.
  • FIG. 6 is a map of plasmid pJE-7.
  • AOX-P is the alcohol oxidase promoter
  • Mat- ⁇ . is the mat-alpha secretory sequence
  • CKXl is the cytokinin oxidase [0 1 sequence
  • Pc87 is an exemplary peptide
  • AOX-TT is the alcohol oxidase termination sequence.
  • FIG. 7 shows the amino acid sequence (SEQ ID NO: 48) of the exemplary insert contained in pJE-7 where the underlined nucleotides are the mat- alpha secretory sequence (cleavage by the Kex2 enzyme occurs after the last underlined arginine), followed by the cytokinin oxidiase 1 sequence.
  • the two double- underlined amino acids (KL) were added to aid in construction of the fusion protein.
  • the amino acid sequence of the exemplary peptide Pc87 is shown in bold type.
  • FIG. 8 is a process schematic diagram showing one method for identifying plant defense peptides that may be used to transform planta and impart resistance to rust pathogens.
  • FIG. 9 shows encystment of zoospores around the apical region of .5 a transformed root secreting cytokinin oxidase (CKX) that displays an affinity- selected selected peptide (left) and normal zoospore encystment pattern around a wild-type root tip (right).
  • CKX transformed root secreting cytokinin oxidase
  • FIG. 10 shows inhibition of U. appendicular germling growth by affinity-selected phage-peptide clone 19 (left), and normal uredospore germination 50 and growth in the presence of non-selected phage library (right).
  • “Secretion sequence” means a sequence that directs newly synthesized secretory or membrane proteins to and through membranes of the endoplasmic reticulum, or from the cytoplasm to the periplasm across the inner membrane of bacteria, or from the matrix of mitochondria into the inner space, or from the stroma of chloroplasts into the thylakoid. Fusion of such a sequence to a gene that is to be expressed in a heterologous host ensures secretion of the recombinant protein from the host cell.
  • “Germling” means a newly germinated cyst (5-8 hr post germination) that bears an emergent germ tube.
  • TBS Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl).
  • a "recombinant polynucleotide” means a polynucleotide that is free of one or both of the nucleotide sequences which flank the polynucleotide in the naturally-occurring genome of the organism from which the polynucleotide is derived.
  • the term includes, for example, a polynucleotide or fragment thereof that is incorporated into a vector or expression cassette; into an autonomously replicating plasmid or virus; into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule independent of other polynucleotides. It also includes a recombinant polynucleotide that is part of a hybrid polynucleotide, for example, one encoding a polypeptide sequence.
  • IPTG is isopropylthiogalactoside.
  • ju means transducing unit.
  • NAP buffer is 80 mM NaCl, 50 mM NH 4 H 2 PO 4 , pH adjusted to 7.0 with NH 4 OH.
  • NZY-Tc is a bacterial growth medium containing 1% NZ amine A (a typtone-type medium; Humko-Sheffield Chemical, Norwich, N. Y.), 0.5% yeast extract, 0.5% NaCl, pH 7.0 adjusted with NaOH.
  • PCR means polymerase chain reaction.
  • polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric (2 or more monomers) form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Although nucleotides are usually joined by phosphodiester linkages, the term also includes polymeric nucleotides containing neutral amide backbone linkages composed of aminoethyl glycine units. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA.
  • internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), those containing pendant moieties, such as, for example, proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.
  • pendant moieties such
  • Polynucleotides include both sense and antisense strands.
  • Sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide. _ . . -
  • Protein “Protein” and “Polypeptide” are used interchangeably and mean a compound that consists of two or more amino acids that are linked by means of peptide bonds.
  • Recombinant protein means that the protein, whether comprising a native or mutant primary amino acid sequence, is obtained by expression of a gene carried by a recombinant DNA molecule in a cell other than the cell in which that gene and/or protein is naturally found. In other words, the gene is heterologous to the host in which it is expressed. It should be noted that any alteration of a gene, including the addition of a polynucleotide encoding an affinity purification moiety, makes that gene unnatural for the purposes of this definition, and thus that gene cannot be "naturally” found in any cell.
  • non-immunoglobulin peptide means a peptide which is not an immunoglobulin, a recognized region of an immunoglobulin, or contains a region of an immunoglobulin. For example, a single chain variable region of an immunoglobulin would be excluded from this definition.
  • substantially pure or substantially purified means that the substance is free from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism.
  • Purity may be assayed by standard methods, and will ordinarily be at least about 40% pure, more ordinarily at least about 50% pure, generally at least about 60% pure, more generally at least about 70% pure, often at least about 75% pure, more often at least about 80% pure, typically at least about 85% pure, more typically at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in even more preferred embodiments, at least 99% pure.
  • the analysis may be weight or molar percentages, evaluated, e.g., by gel staining, spectrophotometry, or terminus labeling etc.
  • a program of development accordingly, identifies peptides that bind to infective P. pachyrhizi spores and germlings, evaluates the effectiveness of binding peptides for inhibition of spore germination and fungal growth, incorporates and tests candidate defense peptides in plants for resistance potential.
  • the rust pathogen produces spores (urediniospores) that infect soybean leaves, stems, and seed pods.
  • the spores are blown by wind and land on the plant where they germinate to produce a germ tube (defined as the germling phase) that penetrates plant tissues.
  • the fungus develops further by producing a filamentous growth that spreads inter and intracellularly through tissues (Bonde et al., 1976; Koch and Hoppe, 1988). With time, tissue colonization leads to production of additional infective spores that are released in wind and moved to other plants.
  • a surface protein selected for this use may, for example, be an enzyme that is needed for cell wall formation and vegetative growth or perhaps, a protein that controls uptake of minerals that are critical for growth.
  • the methods disclosed herein, by way of illustration, may effectively screen pathogens against diverse peptide collections that contain one billion or more random peptide combinations. The technique is specifically applied to defeat P. pachyrhizi. and related pathogens.
  • washing may remove all peptides that do not stick strongly to spores and germlings.
  • the spores and germlings may be treated to release the adhering peptides, increase their numbers, and repeat the screening process.
  • recovery is certain of numerous peptides that stick very strongly to proteins and other components of spore and germ tube surfaces. In one example, 3 or 4 sequential screening steps are sufficient to provide this result.
  • cytokinin oxidase has been used successfully for this purpose. This protein is produced naturally by plants, and it is involved in the regulation of the plant growth hormone, cytokinin. Defense peptides may be attached to this protein, and used to
  • Defense peptides may be fused to cytokinin oxidase and other proteins that are naturally secreted by cells into the intercellular space where they can come into contact with the colonizing rust pathogen.
  • the fusions may include proteins that are components of plant cell walls.
  • proteomic databases provide a wide variety of gene sequences for candidate peptide-carrier proteins that may be used as an alternative to CICX. These carrier protein structures may be analyzed using conventional structural proteomic algorithms for optimal presentment or display of the defense peptide..
  • Candidate protein-peptide constructs may be expressed and secreted initially in the yeast, Pichia. This host facilitates concentrating and purifying a protein or poly peptide, and testing the same for inhibition of pathogen spore germination or germling growth.
  • peptide libraries are constructed by the insertion of nucleic acid sequences encoding peptides of six to 15 amino acids in length into suitable vectors, although sequences encoding longer peptides can be used.
  • the peptides encoded by the nucleotides can be completely random in nature or can be constrained in their composition to meet structural or functional requirements. For example, and without limitation, a cysteine bridge can be inserted into the peptide.
  • the nucleic acid sequence does not encode an immunoglobulin (antibody) or a recognized immunoglobulin region such as a variable region. Any vector which will express the inserted oligonucleotides can be used.
  • a vector is used which will result in expression of the peptide library on the surface of a cell or virus or on the surface of an intracellular compartment or organelle of a cell or virus.
  • the expressed peptides will be available to interact with potential target molecules or cells that come in contact with the surface containing the peptides.
  • the potential target must also reside intracellularly or the organelle or intracellular compartment must be exposed to the external environment by, for example, lysis of the cell.
  • oligonucleotides are synthesized on a solid support using the phosphite triester method of Beaucage and Caruthers (Tetrahedron Lett. 22:1859-1862, 1981; also see, U.S. Pat. Nos. 4,973,679 and 4,458,066). Numerous solid supports are available including controlled pore glass beads, polystyrene copolymers, silica gel and cellulose paper.
  • oligonucleotide begins with the linkage of the 3'-hydroxyl group of the first nucleoside to the solid support.
  • Solid supports containing nucleotides are available from commercial sources.
  • the oligonucleotide is synthesized from the 3' to 5' direction and the chain is elongated by nucleophilic attack of the 5'-hydroxyl of the immobilized oligonucleotide on the activated 3' phosphate or phosorphramidite of a soluble 5'- protected building block.
  • the intermediate dinucleoside phosphite formed must next be oxidized to the more stable phosphate before chain extension. The process is repeated until the desired number of nucleotides has been added.
  • Any vector system capable of expressing the peptides of the peptide library may be used in the practice hereof and numerous vector systems are known in the art (See e.g., Wilson and Findlay, Can. J Microbiol, 44:313-329, 1998).
  • suitable phage systems include type 8, type 88, and type 8+8.
  • suitable phage systems include type 3, type 33 and type 3+3.
  • suitable phage systems included type 6, type 66 and type 6+6.
  • phage T7 and phage 8 vector systems can be used.
  • the peptides of the library are expressed fused to a coat protein of a filamentous bacteriophage so that the peptides are expressed on the surface of the virion and so are available to interact with target molecules or cell surface receptors.
  • the f8-l library is used in which random 8-mer peptides are fused to the pVIII coat protein.
  • the f88-4 library is used in which random 15-mer peptides are fused to the pVIII coat protein. Phage in the f88-4 library display peptides without bias toward the occurrence of any amino acid. Phage in the f8-l library are unbiased with the exception of alanine at the first position and one of four residues at the second position. All other positions are randomly occupied by any amino acid.
  • the first amino acid is an alanine (A) and the second amino acid is a valine (V), alanine (A) 5 aspartate (D), glutamate (E) or glycine (G).
  • the remainder of the amino acids in the peptide are completely randomized.
  • One pVIII is the recombinant displaying the foreign 15-mer peptide, while the other is the wild-type pVIII normally present on the phage. Expression of the recombinant pVIII gene is driven by the IPTG inducible tac promoter/operator. Because of the presence of two pVIII genes, the f88 virion consists of a mosaic pattern of wild-type and recombinant pVIII subunits.
  • the oligonucleotide sequence used for the 15-mer amino acid inserts is (NNK) 15 , where N is A, T 5 C, or G and K designates G or T.
  • the region surrounding the 15-mer insert is: LVPMLSFA(X) 15 PAEGDDPAKA (SEQ ID NO: 1), where X is any amino acid encoded by the codon NNK.
  • the phage particles can be used to screen the random peptides expressed on the virion for their ability to bind to compounds and cells of interest.
  • the phage-displayed peptide library is used to screen for peptides that bind to plant pathogens.
  • the peptides are screened for their ability to bind to pathogenic fungi.
  • phage-displayed peptides are screened for their ability to bind to members of the genus Phytophthora.
  • the organism After incubation, the organism is subject to multiple washes in order to remove unbound and weakly bound peptides.
  • washing is done using a solution of approximately 50 niM LiCl.
  • bound phage-displayed peptides are eluted, preferably at low pH, and the eluted phage amplified in a suitable host.
  • the host is starved K91 E. coll. Methods for the amplification of bacteriophage in E. coli are well known in the art and can be found, for example, in Smith and Scott, Methods in Enzymology, 217:228- 257, 1993; Ausubel et al.
  • the screening procedure is repeated at least once in order to enrich high-affinity phage displayed peptides. In another embodiment, the screening process is repeated three times.
  • the phage are amplified, preferably in E. coli, and the phage DNA isolated using standard methods such as those found in, for example Smith and Scott, Methods in Enzymology, 217:228-257, 1993; Ausubel et al. eds., Short Protocols in Molecular Biology, 2nd ed., Wiley & Sons, 1995; and Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989.
  • the inserted oligonucleotides can be cleaved from the DNA using the same restriction enzymes used to insert the oligonucleotides, and the restriction enzyme fragments separated from the remainder of the DNA.
  • the oligonucleotides can then be sequenced using any standard method. Sequencing can be carried out by any suitable method, for example, dideoxy sequencing (Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977), chemical sequencing (Maxam and Gilbert, Proc. Natl. Acad. Sci. USA, 74:560-564, 1977) or any variation thereof, including the use of automatic sequencers.
  • sequencing is accomplished using an ABI Prism 377 automated sequencer (Applied Biosystems, Foster City, Calif.). Once the sequence of the oligonucleotides is known, the amino acid sequences of the peptides encoded can be readily deduced using the genetic code. [0089] High-affinity binding, phage displayed peptides can be further screened for their ability to alter the development, growth and/or infectivity of pathogens. In this embodiment, phage-displayed peptides are incubated with a target pathogen for a time sufficient to allow binding. Following binding, the pathogen is observed for alterations in its development or ability to infect a host.
  • approximately 200 zoospores of a member of the genus Phytophthora are combined with a phage-displayed peptide in distilled water in two-fold serial dilutions at constant volume in petri dishes.
  • the range of phage concentrations can vary, but generally ranges between 1 to 10x10 9 virion/ ⁇ l.
  • a negative control containing no phage is included in each screening. After an incubation period of usually about 20 minutes at room temperature, the number of zoospores encysted at each phage concentration is determined.
  • the present method can also be used to characterize peptide binding receptors on the surface of plant pathogens.
  • peptide displaying phage that have been labeled (test phage) are incubated with cells of different organisms and at different stages of development.
  • the relative binding affinity of the labeled phage can then be determined by competitive binding and Scatchard analysis.
  • a competitive binding analysis a constant concentration of test phage is allowed to bind to a target pathogen and then unlabeled challenge phage is added over a range of concentrations.
  • the challenge phage may be the same as the test phage or it may be different.
  • the target pathogen is then washed to remove non- specifically or weakly bound phage and the amount of test phage bound is determined by measuring the amount of label present on the target cells.
  • the degree of competition can be measured as the concentration of challenge phage required to inhibit test phage binding by 50% (IC 50 ).
  • Results from competition assays can be use to determine changes in the number, type and affinity of cell surface receptors over time.
  • oligonucleotides within the scope of the disclosed instrumentalities are recombinant oligonucleotides, discovered by the method taught herein, encoding peptides having antifungal activity. These recombinant oligonucleotides can be used to produce recombinant polynucleotides which are commonly used as cloning or expression vectors although other uses are possible.
  • a cloning vector is a self-replicating DNA molecule that serves to transfer a DNA segment into a host cell. The three most
  • cloning vectors 5 common types are bacterial plasmids, phages, and other viruses.
  • An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into a protein.
  • Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells.
  • this sequence is generally one that enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences.
  • origins of replication Various bacterial and viral origins of replication are well known to those skilled in the art and include, but are not limited to the pBR322 plasmid origin, the 2 ⁇ plasmid origin, and the SV40, polyoma,
  • oligonucleotide sequences of the present disclosure may be used to produce antifungal peptides by the use of recombinant expression vectors containing the oligonucleotide sequence.
  • Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV 40
  • bacterial plasmids bacterial plasmids
  • phage DNA phage DNA
  • yeast plasmids vectors derived from combinations of plasmids and phage DNA
  • viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector that is replicable and viable in the host may be used.
  • nucleotide sequence of interest may be inserted into the vector
  • sequence is inserted into an appropriate restriction endonuclease site(s) using procedures commonly known to those skilled in the art and detailed in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, (1989) and Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., John Wiley & Sons
  • the sequence of interest is operably linked to a suitable expression control sequence or promoter recognized by the host cell to direct mRNA synthesis.
  • Promoters are untranslated sequences located generally 100 to 1000 base pairs (bp) upstream from the start codon of a structural gene that regulate the transcription and translation of nucleic acid sequences under their control. Promoters are generally classified as either inducible or constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in the environment, e.g. the presence or absence of a nutrient or a change in temperature. Constitutive promoters, in contrast, maintain a relatively constant level of transcription.
  • a nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide;
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is achieved by ligation at restriction enzyme sites.
  • Common promoters used in expression vectors include, but are not limited to, LTR or SV40 promoter, the E. coli lac or trp promoters, and the phage lambda PL promoter.
  • Useful inducible plant promoters include heat-shock promoters (Ou-Lee et al. (1986) Proc. Natl Acad. Set USA 83: 6815; Ainley et al. (1990) Plant MoI. Biol. 14: 949), a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al. (l991)Plant MoI Biol.
  • Expression vectors may also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may also contain sequences useful for the amplification of gene expression. [0098] Expression and cloning vectors can, and usually do, contain a selection gene or selection marker.
  • this gene encodes a protein necessary for the survival or growth of the host cell transformed with the vector.
  • suitable markers include dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cells and tetracycline or ampicillin resistance for E. coli.
  • Selection markers in plants include resistance to bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, methotrexate, phleomycin, phosphinotricin, spectinomycin, streptomycin, sulfonamide and sulfonylureas. Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995, p. 39.
  • expression vectors can also contain marker sequences operatively linked to a nucleotide sequence for a protein that encode an additional protein used as a marker.
  • the result is a hybrid or fusion protein comprising two linked and different proteins.
  • the marker protein can provide, for example, an immunological or enzymatic marker for the recombinant protein produced by the expression vector. Suitable markers include, but are not limited to, alkaline phosphatase (AP), myc, hemagglutinin (HA), ⁇ -glucuronidase (GUS), luciferase, and green fluorescent protein (GFP).
  • the polynucleotide sequences of the present disclosure may also be part of an expression cassette that at a minimum comprises, operably linked in the 5' to 3' direction, a regulatory sequence such as a promoter, a polynucleotide encoding a peptide of the present disclosure, and a transcriptional termination signal sequence functional in a host cell.
  • the promoter can be of any of the types discussed herein, for example, a tissue specific promoter, a developmentally regulated promoter, an organelle specific promoter, a seed specific promoter, a plastid specific promoter, etc.
  • the expression cassette can further comprise an operably linked targeting, transit, or secretion peptide coding region capable of directing transport of the protein produced.
  • the expression cassette can also further comprise a nucleotide sequence encoding a selectable marker and/or a purification moiety.
  • the present disclosure includes recombinant constructs comprising an isolated polynucleotide sequence encoding the antifungal peptides of the present disclosure.
  • the constructs can include a vector, such as a plasmid or viral vector, into which the sequence has been inserted, either in the forward or reverse orientation.
  • the recombinant construct can further comprise regulatory sequences, including, for example, a promoter operatively linked to the sequence. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available.
  • a further embodiment of the present disclosure relates to transformed host cells containing constructs comprising the oligonucleotide sequences of the present disclosure.
  • the host cell can be a higher eukaryotic cell, such as a mammalian or plant cell, or a lower eukaryotic cell such as a yeast cell, or the host can be a prokaryotic cell such as a bacterial cell.
  • Introduction of the construct into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, Polybrene, protoplast fusion, liposomes, direct microinjection into the nuclei, scrape loading, and electroporation.
  • transformation/expression vectors into plant protoplasts, cells, callus tissue, leaf discs, meristems, etc., to generate transgenic plants.
  • methods include, for example, Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome- mediated transformation, etc. (reviewed in Potrykus (1991) Annu. Rev. Plant Physiol Plant MoI. Biol. 42: 205).
  • Peptides produced by expression of the polynucleotides of the present disclosure can be obtained by transforming a host cell by any of the previously described methods, growing the host cell under appropriate conditions, inducing expression of the polynucleotide and isolating the protein(s) of interest. If the protein in retained within the host cell, the protein can be obtained by lysis of the host cells, while if the protein is a secreted protein, it can be isolated from the culture medium. Several methods are available for purification of proteins and are known to those of ordinary skill in the art.
  • peptides encoded by the polynucleotides of the present disclosure can be produced by chemical synthesis using either solid-phase peptide synthesis or by classical solution peptide synthesis also known as liquid-phase peptide synthesis.
  • oligomer-supported liquid phase synthesis the growing product is attached to a large soluble polymeric group.
  • the product from each step of the synthesis can then be separated from unreacted reactants based on the large difference in size between the relatively large polymer-attached product and the unreacted reactants. This permits reactions to take place in homogeneous solutions, and eliminates tedious purification steps associated with traditional liquid phase synthesis.
  • Oligomer-supported liquid phase synthesis has also been adapted to automatic liquid phase synthesis of peptides. [0105]
  • solid-phase peptide synthesis the procedure entails the sequential assembly of the appropriate amino acids into a peptide of a desired sequence while the end of the growing peptide is linked to an insoluble support.
  • the carboxyl terminus of the peptide is linked to a polymer from which it can be liberated upon treatment with a cleavage reagent.
  • an amino acid is bound to a resin particle, and the peptide generated in a stepwise manner by successive additions of protected amino acids to produce a chain of amino acids. Modifications of the technique described by Merrifield are commonly used (see, e.g., Merrifield, J. Am. Chem. Soc. 96: 2989-93, 1964).
  • peptides are synthesized by loading the carboxy-terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethylphenylacetamidomethyl), which is covalently attached to an insoluble polystyrene resin cross-linked with divinyl benzene.
  • organic linker e.g., PAM, 4-oxymethylphenylacetamidomethyl
  • the terminal amine may be protected by blocking with t-butyloxycarbonyl. Hydroxyl- and carboxyl-groups are commonly protected by blocking with O-benzyl groups.
  • the blocking groups are removed typically by using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods (e.g., Bergot and McCurdy, Applied Biosystems Bulletin, 1987). Following cleavage and purification, a yield of approximately 60 to 70% is typically produced.
  • Purification of the product peptides is accomplished by, for example, crystallizing the peptide from an organic solvent such as methyl-butyl ether, then dissolving in distilled water, and using dialysis (if the molecular weight of the subject peptide is greater than about 500 daltons) or reverse high-pressure liquid chromatography (e.g., using a Cl 8 column with 0.1% trifluoroacetic acid and acetonitrile as solvents) if the molecular weight of the peptide is less than 500 daltons.
  • Purified peptide may be lyophilized and stored in a dry state until use. Analysis of the resulting peptides may be accomplished using the common methods of analytical high pressure liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS).
  • HPLC high pressure liquid chromatography
  • ES-MS electrospray mass spectrometry
  • transgenic plants comprising cells containing polynucleotides of the present disclosure can be produced by any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant that expresses the protein(s) encoded by the polynucleotides of the present disclosure at a desired level.
  • Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature (Gasser and Fraley, Science 244:1293, 1989; Fisk and Dandekar, Scientia Horticulturae 55:5, 1993; and the references cited therein).
  • plants are transformed with recombinant polynucleotides encoding the antifungal peptides of the present disclosure which result in the peptides being secreted by the plant.
  • the antifungal peptides are secreted by the roots of the transformed plant. Plants secreting antifungal peptides can be constructed by the
  • plants can be transformed with a nucleotide sequence encoding a fusion protein constructed from the antifungal peptides of the present disclosure and a protein which is normally secreted by the plant.
  • a fusion protein can be produced between an
  • Cytokinin oxidase is a protective enzyme that acts to degrade exogenous cytokinins that could interfere with plant growth control.
  • the antifungal peptides By fusing the antifungal peptides to the region of the cytokinin oxidase gene controlling secretion, the antifungal peptide would be secreted by the transformed plant, thus providing protection from pathogenic fungi.
  • fusion proteins containing antifungal peptides can be screened for activity using the phage display method of the present disclosure.
  • a fusion protein can be construction containing, an antifungal peptide; the secretory control portion of a protein, such as cytokinin oxidase; and the pVIII or pill phage coat protein. Phage displayed fusion proteins so
  • .5 constructed can then be screened using the method of the present disclosure to select those fusion proteins that bind to a target pathogenic fungus and result in alternations which limit pathogenicity.
  • the fungal strains used were P. caps ⁇ ci (ATCC 15399); P. sojae (strain 7-6-1, race 25) (A. F. Schmitthenner, Ohio State University); and Phytophthora parasitica. All cultures were maintained as mycelia on lima bean agar plates (P. sojae) or corn meal agar plates (Difco, USA) (P. capsici and P. parasitica) at 15° C. Mycelium copies were made by transferring plugs of mycelium (5 mmx5 mm) to agar plates containing clarified 10% V8® vegetable juice (Campbell Soup Co., USA). Three plugs per plate were grown for three to six days at 25° C.
  • P. capsici by trimming the plates and incubating at 25° C. with light. After one to two days, zoospore release was induced by flooding the plates with sterile water for 20 to 30 minutes. P. parastica zoospore production was identical to that of P. capsici except that the plates were washed with sterile water for two minutes prior to incubating at 25° C. with light. Zoospore release was induced from P. sojae sporangia by flooding the plates four times in sterile water at 30 minute intervals. Zoospores were released within two to four hours.
  • zoospores were filtered through four layers of cheesecloth to remove sporangial cases and mycelial fragments. A sample of the suspension was vortexed for 30 seconds to induce encystment and the cysts counted under a microscope in a hemacytometer.
  • TU tetracyclin transducing units
  • the phage were titered as transducing units (TU) in E. coli K91BluKan starved cells (prepared as above). Phage were analytically titered using TBS/gelatin as the diluent. Ten microliters of each phage dilution were deposited as a droplet on the inner wall of a 15 ml sterile disposable tube held at a 10° angle from
  • the superbroth also contained 1 mM IPTG to induce recombinant pVIII expression.
  • the infected cells were then spread (200 ml per plate) on Luria-Bertani (LB) plates containing 40 mg/ml tetracyclin. The plates were then incubated for ⁇ 24 hr at 37° C.
  • the bound phage were eluted with 200 ⁇ of elution buffer (0.1 N HCl, glycine sufficient to bring pH to 2.2, 1 mg/ml bovine serum albumin).
  • elution buffer 0.1 N HCl, glycine sufficient to bring pH to 2.2, 1 mg/ml bovine serum albumin.
  • the eluted phage were amplified by infection of starved E. coli K91BluKan 50 cells as described above.
  • the amplified phage were then purified by precipitation with polyethylene glycol as described below, and resuspended in TBS buffer as described by Smith and Scott ⁇ Methods in En ⁇ ymology, 217:228-257, 1993).
  • the yield of phage eluted from the zoospores after each round of screening was between 10 "4 to 10 "5 % indicating that this procedure was successful in selecting for phage binding to the zoospores.
  • the zoospores were intact and spherical after the washing steps, showing that little or no encystment had occurred during the selection process.
  • E. coli K91 BluKan cells infected with phage were grown overnight in 20 ml superbroth (containing 40 mg/ml tetracyclin) at 37° C. and 170 rpm. The culture was centrifuged to pellet the E. coli cells (containing phage) in a SS34 rotor for 10 min at 5,000x g. The supernatant was removed and placed in a new Oak Ridge tube and PEG/NaCl (16.7% polyethylene glycol/3.3 M NaCl) was added at a rate of 150 1 per ml supernatant to precipitate the phage. The phage were precipitated overnight at 4° C.
  • TBS Tris-buffered saline
  • DNA used for sequencing was isolated from individual phage clones according to the method of Smith and Scott ⁇ Methods in Enzymology ⁇ 217:228- 257, 1993). Single-stranded DNA was sequenced from the 3' end using an ABI Prism 377 automated sequencer (Applied Biosystems, Foster City, Calif.) following the manufacturer's protocol.
  • the primer used for f8 clones was 5'- GGAGCCTTTAATTGTATCGG-S 1 (SEQ ID NO: 2).
  • the primer used for f88 clones was 5'-AGT AGC AGA AGC CTG AAG A-3 1 (SEQ ID NO: 3).
  • DNA sequences were translated using the "translate" program of the ExPASy Molecular Biology Server (website http://www.expasv.ch/). Sequences were compared with nucleic acid and protein sequences stored in sequence databases (GenBank, EMBL, dbEST, SwissProt, PIR) using standard algorithms (i.e.) FASTA (Lipman and Person, Science, 227:1435-1441, 1985) and BLAST (Altschul et al. 5 J Molecular Biol, 215:403-410, 1990) commands. Peptide sequences were aligned using ClustalW (Thompson et al., Nuc.
  • the dendogram constructed from the aligned peptides, indicated that the f8-mer peptide sequences could be grouped into six broad family groups as depicted in FIG. 1 and Table 1. Selected sequences from the f88- 4/15 mer library are shown in Table 2.
  • Selected phage clones were isolated according to Smith and Scott (Methods in Enzymology, 217:228-257, 1993), and twice purified using polyethylene glycol as described above with the exception that phage were resuspended in distilled water instead of TBS.
  • the virion concentration was calculated by measuring the absorbance at A 269 (Smith and Scott, Methods in Enzymology, 217:228-257, 1993).
  • parastica zoospores were incubated with phage- bearing peptides at 1x10 10 virion/ ⁇ l, a concentration that resulted in almost 100% encystment for P. capsici zoospores. Similar results were obtained with f88-4 15 mer peptides. The ability of representative 15 mer peptides to cause premature encystment in comparison to the f-8 clone Pc87 is shown in FIG. 3.
  • Phage-displayed peptides with high and low encystment induction abilities were compared for their ability to bind to P. capsici zoospores.
  • Phage clones Pc87 and Pc45 were randomly selected as the representative clones that induced high and low levels of encystment, respectively (cf. FIG. 2).
  • Phage vector was included as a control treatment. Phage clones were amplified by E. coli infections and purified as described above. For each binding reaction, 5x10 10 TU of phage were incubated with 200,000 P. capsici zoospores. The binding reaction and washes were performed as described in Example 3 for phage selection.
  • Phage eluted from the zoospore population were titered in E. coli K91BluKan cells and expressed as total transducing units. A similar procedure was used to determine whether the selected phage bound to P. capsici cysts.
  • Phage vector and clones Pc45 and Pc87 bound differentially to P. capsici zoospores. More than 10 7 TU of phage Pc87 were eluted from zoospores after 30 minutes incubation, while only about 50,000 TU of phage Pc45 or phage vector were eluted under the same conditions (FIG. 4). Moreover, binding was specific for the zoospore stage: less than 10 4 Pc87 TU were eluted from cysts-about the same background binding observed with control vector phage (FIG. 5).
  • Carboxy-terminal DNA fusions were constructed to encode for the peptides of interest by ligation of synthetic oligonucleotides into the restriction enzyme sites, HindIII and Xbal, of the carrier vector pJE-6.
  • the plasmid vector pJE-6 was constructed from the Pichia pastoris expression construct pROM-46 derived from the plasmid pPICZ-alpha (Invitrogen, Carlsbad, Calif.), previously described (Cregg et al., Bio/Technology, 11 : 905-910, 1993; Rosenfeld, Methods in Enzymology, 306:154-169, 1999).
  • Plasmid pROM-46 was digested with restriction endonuclease, HindIII, filled-in with Klenow enzyme and dNTP's, and re-ligated withT4 DNA ligase. These steps eliminated a HindIII restriction site present within the pPICZ- alpha plasmid sequence, and the plasmid was designated pJE-4. The sequence at the 3' end of the coding sequence was mutagenized by PCR to replace the stop codon with the restriction site HindIII. This plasmid was designated pJE-6.
  • Synthetic oligonucleotides encoding for an exemplary peptide (Pc87, ADRPSMSPT, SEQ ID NO: 8), were ligated into the plasmid pJE-6, digested with HindIII and Xbal. This plasmid designated pJE-7 (FIG. 6). The pJE-7 plasmid was sequenced to confirm the insert and the results are in shown in FIG. 7. TABLE l
  • protein scaffolds may be designed for display of peptides when the peptides are transformed into plants.
  • cytokinin oxidase CKX
  • a member of CKX may be used as a peptide-delivery scaffold.
  • CKX family derived from maize (Morris, 1997) may, for example, be used as a delivery molecule.
  • CKX is endogenously produced, possesses a peptide signal sequence for secretion from cells and is sufficiently glycosolated to provide stability in the presence of proteolytic enzymes in the intercellular region (Morris et al., 1999). [0124] Based on the known three-dimensional structure of CKX, as
  • CKX may be engineered to display peptides at an exposed C- terminus.
  • the scaffold constructs ay, for example, be expressed and secreted from yeast.
  • the inhibitory ability of selected peptides was dramatic, and 80-90% zoospore encystment was obtained.
  • Phytophthora zoospore encystment was 25% or less (Fang et al., 2004).
  • 15 hairy roots may, for example, be produced using these constructs, and in doing so it was found that the scaffold-peptides are secreted into the rhizosphere where they induce zoospore encystment before they accumulate at the root surface, thus blocking infection. This is shown in Fig. 9.
  • 15-mer peptides were selected from combinatorial libraries that bind strongly to uredospores of U. appendiculatus. This fungus was used as a surrogate or selected analog for P. pachyrhizi, where access to P. pachyrhizi is closely controlled for containment of this pathogen.
  • These screens identified a number of peptides that inhibit growth after initial spore germination, for example, as shown in Fig. 10.
  • .5 peptides are effective at micromolar concentrations. Deployment of defense peptides in CKX may enable delivery to the surface of fungal hyphae that penetrate the intercellular space of leaves.
  • FIG. 8 is a process schematic diagram showing a protocol 800 for peptide selection.
  • a starting phage-display peptide library may be mixed in step 802 with plant extracts to remove peptides that interact with plant proteins and other factors.
  • the output with such peptides removed is designated Sub-library 1.
  • Sub-library 1 is mixed 804 with germinated uredospores or germlings by what is commonly known as a biopanning technique. Bound phage-
  • each recovered phage-display peptide is increased by amplification 808 in E. coli.
  • the phage-display peptides are recovered 810 after amplification and mixed 812 with plant extracts to again ensure removal of peptides that interact with plant proteins and other factors.
  • the output with such peptides removed is designated Sub-library 2.
  • Step 814 determines whether the above steps have been performed a sufficient number of times to assure stringency of selection of candidate plant defense peptides.
  • Sub-libraries 3, 4 and 5 are generated by successive iterations n in which steps 806 through 812 are repeated.
  • Each iteration entails mixing the most recently generated Sub-library, e.g., Sub-library
  • Bound phage-display peptides are recovered from the germlings. The concentration of each recovered phage-display peptide is increased by amplification in E. coli.
  • Candidate peptides that show successful inhibition may be provided 818 as fused gene constructs combining the plant defense peptides with a plant surface protein gene, such as CKX, amplified 820 in an interim host, such as Pichia, and eventually used to transform 822 soybean or field bean to impart rust
  • Part A entails the preparation of Sub-library 1 by removal of peptides that interact with plant components. A 3.0 g quantity of Pinto leaf is mixed
  • Part B involves a first round of biopanning. A approximately 1.5 million spores from a selected pathogen are incubated in 0.01 % Tween 20 and 25 ppm B-ionone for 5 hours at 20 C. Add 10 12 TU phages. The mixture is divided into 2 microtubes totaling 3 ml in volume. The microtubes including the spore/phage mixture are incubated at room temperature of about 23° C) on an orbital shaker ( ⁇ 500 rpm) for 30 minutes. The incubated mixture is centrifuged, supernatant is removed, 0.5 ml of water is added, and the remaining contents are combined into one micro tube.
  • Part C entails the preparation of Sub-library 2.
  • Sub-library 2 is produced using the amplified Sub-library 1 from the first round of biopanniing in Part B. This is done by using Sub-library 1 instead of original f88-4/15-mer library of Part A, but otherwise following Part A of the protocol.
  • Part D entails the generation of additional Sub-libraries by use of further rounds of biopanning including rounds 2, 3 and 4. Part B is repeated three times:
  • Sub-library are used to produce Sub-library 3; 2. For biopanning round 3, 5.6 x 10 ⁇ TU phages amplified from the 2nd biopanning are used to produce Sub-library 4; and 3. For biopanning round 4, 5.1 x l ⁇ " TU phages amplified from the 3rd biopanning are used to produce Sub-library 5. Colonies from biopanning-round 4 are picked, sequenced, and tested for inhibition of germling growth.
  • Part E entails testing for inhibition of uredospore growth. Incubate 30 ul of germinated spores ( ⁇ 250) with 3 x 10 12 at 20 C overnight. Assess fungal germ tube growth (i.e. germling growth) and compare with water and f88, i.e. phage only without any display peptide, as controls.
  • Tables 3 and 4 show peptides that may be used to inhibit growth of germinated uredospores (germlings) from these pathogens.
  • cytokinin oxidase as a protein scaffold for delivery of select peptides to points of U. appendiculatus infection of bean ( ⁇ haseolus vulgaris) tissues.
  • CKX is modified as described above to fuse the defense peptides of Tables 3 and 4.
  • An agrobacterium-mediated transformation of Phaseolus vulgaris is performed to provide a plant expressing each peptide. The transformed plants are exposed to U. appendiculatus. The rate and severity of infection are compared to a 5 control plant to confirm efficacy of the defense peptides against the pathogen.
  • cytokinin oxidase as a protein scaffold for delivery of select peptides to points of P. pachyrhizi. infection of soybean ⁇ Glycine Max) tissues.
  • CKX is modified as described above to fuse the defense peptides of 10 Tables 3 and 4.
  • An agrobacterium-mediated transformation of soybean is performed to provide a plant expressing each peptide. The transformed plants are exposed to P. pachyrhizi.. The rate and severity of infection are compared to a control plant to confirm efficacy of the defense peptides against the pathogen.
  • GPIIb/IIIa from a conformationally constrained phage display library. Proteins 14:509-515.

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Abstract

L'invention concerne une méthode destinée à l'identification de peptides présentant une affinité pour la surface de champignons, ainsi qu'une méthode destinée à l'identification de peptides capables de modifier le développement d'un champignon. L'invention concerne également des compositions comprenant les peptides identifiés à l'aide de la méthode de la présente invention. L'invention concerne également des polynucléotides isolés, des vecteurs, des cassettes d'expression et des cellules transformées capables d'exprimer les peptides identifiés à l'aide de la méthode de la présente invention.
PCT/US2006/030291 2005-08-02 2006-08-02 Peptides de defense vegetaux a presentation a la surface des phages filamenteux, diriges contre phakopsora pachyrhizi et uromyces appendiculatus WO2007016680A2 (fr)

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CN2006800367777A CN101473226B (zh) 2005-08-02 2006-08-02 针对豆薯层锈菌和疣顶单胞锈菌的噬菌体展示植物防御肽
BRPI0614523-0A BRPI0614523A2 (pt) 2005-08-02 2006-08-02 método para identificação de peptìdeos, polinucleotìdeo, vetor recombinante, célula, planta e métodos para seleção de peptìdeos e para conferir em uma planta a capacidade de resistir à infecção causada por fungos

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CN111235034B (zh) * 2020-02-17 2021-11-30 中国农业大学 一种区分玉米南方锈菌和普通锈菌孢子的方法

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US20010029024A1 (en) * 2000-02-11 2001-10-11 Kodadek Thomas J. Selection of peptides with antibody-like properties

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014165066A3 (fr) * 2013-03-13 2014-11-27 E. I. Dupont De Nemours & Company Identification d'effecteurs protéiques de p. pachyrhizi et leur utilisation dans le cadre de la production de plantes résistantes à la rouille asiatique du soja
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EP3263592A1 (fr) * 2013-03-13 2018-01-03 E. I. du Pont de Nemours and Company Identification d'effecteurs protéiques de p. pachyrhizi et leur utilisation dans le cadre de la production de plantes résistantes à la rouille asiatique du soja

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