WO2000014260A1 - Procedes de lutte contre les maladies virales des plantes - Google Patents

Procedes de lutte contre les maladies virales des plantes Download PDF

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WO2000014260A1
WO2000014260A1 PCT/US1999/020455 US9920455W WO0014260A1 WO 2000014260 A1 WO2000014260 A1 WO 2000014260A1 US 9920455 W US9920455 W US 9920455W WO 0014260 A1 WO0014260 A1 WO 0014260A1
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plant
gene
cell
bigeminivirus
dna
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PCT/US1999/020455
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English (en)
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Dean W. Gabriel
Yong Ping Duan
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University Of Florida
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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
    • C12N15/63—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
    • C12N15/8283—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 virus 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems

Definitions

  • the present invention relates generally to the field of transgenic plants, and the control of viral disease in plants. More particularly, it concerns control of plant disease by engineering and expressing one or more bacterial avirulence genes in plants under the control of a promoter that regulates the expression of the gene(s) to prevent, control or limit the spread or infectivity of a viral disease in a transformed plant.
  • the hosts in which avirulence is observed always carry at least one resistance (R) gene that is genetically specific for a particular avr gene; this genetic requirement is often termed gene-for-gene ( ⁇ vr-for-R) specificity (for reviews, see Gabriel and Rolfe, 1990; Keen, 1992).
  • R resistance
  • ⁇ vr-for-R gene-for-gene
  • harpins protein products of hrp (nonhost hypersensitive response and host pathogenicity) genes, called harpins, were considered to be both necessary and sufficient for the nonhost HR (U. S. Patent 5,859,351; U. S.
  • the idea that a pathogen would possess one or more genes that encode avirulence, as opposed to virulence, has always been enigmatic. Indeed, several different working hypotheses have been proposed to explain this enigma (Gabriel and Rolfe, 1990).
  • avr genes have been shown to function also as pathogenicity (pth) genes, but only against specific plants that are hosts for the particular pathogen (Swarup et al, 1991, 1992; Yang et al, 1994; Gabriel et al, 1993).
  • This avr/plh gene family comprises the largest number of avr genes cloned and sequenced to date, and includes genes pthA and avrb ⁇ (Gabriel, 1997). Analyses (Yang and Gabriel, 1995a) of the published gene sequences and the predicted amino acid sequences encoded by avrb ⁇ .
  • pthA and other members of this avr/pth gene family revealed the presence of three stretches of basic residues with complete homology with the nuclear localization consensus sequences (K-R/K-X-R/K) found in many characterized nuclear localized proteins (Chelsky et al, 1989). These three putative nuclear localization sequences (NLSs) are located near the C-terminus of the proteins, at positions 1020-1024 (K-R-A-K-P), 1065-1069 (R-K-R-S-R), and 1 101-1 106 (R-V-K-R-P-R) in PthA.
  • NLSs nuclear localization sequences
  • NLS signals are found in the predicted polypeptide sequences of all functional Xanthomonas avr/pth gene family members identified to date (Yang and Gabriel, 1995a), and in several other avr genes.
  • Intl. Pat. Appl. Publ. No. WO 91/15585 (specifically incorporated herein by reference in its entirety) describes a method for protecting plants against microbial pathogens whereby a polynucleotide sequence of an avr gene that encodes a specific elicitor protein is incorporated into the genome of a plant containing a corresponding R gene. Unfortunately, the disclosed methods are not applicable for viral pathogens.
  • the avr genes are regulated in such a manner that the expression of the genes occurs upon triggering a defense or wound response of the plant, which activates a plant wound response promoter.
  • the publication does not describe the use of a pathogen-derived or artificial promoter responsive to a pathogen signal; the promoters used were either plant promoters or promoters responsive to plant wound or defense signals.
  • One limitation of this method was the requisite coexpression of a cognate plant R gene, that was either naturally present in the plant or simultaneously provided on the engineered plasmid expression construct. The use of non-host mediated responses has not been described.
  • the present invention seeks to overcome these and other limitations in the prior art by providing methods and compositions for the control of a viral pathogen in a plant or plant cell.
  • Viruses to be controlled by the present invention includes among others, the caulimoviruses, the Nanaviruses, the badnaviruses, the bigeminiviruses, the hybrigeminiviruses, and the monogeminiviruses.
  • viruses include, but are not limited to, Blueberry red ringspot caulimovirus, Carnation etched ring caulimovirus, Cauliflower mosaic caulimovirus, Dahlia mosaic caulimovirus, Figwort mosaic caulimovirus, Horseradish latent caulimovirus, Mirabilis mosaic caulimovirus, Peanut chlorotic streak caulimovirus, Soybean chlorotic mottle caulimovirus, Sweet potato caulimovirus, Thistle mottle caulimovirus, Banana bunchy top nanavirus, coconut foliar decay nanavirus, Faba bean necrotic yellows nanavirus, Milk vetch dwarf nanavirus, Subterranean clover stunt nanavirus, Banana streak badnavirus, Cacao swollen shoot badnavirus, Canna yellow mottle badnavirus, Commelina yellow mottle badnavirus, Dioscorea bacilliform badnavirus, Kalanchoe top
  • the methods of the present invention generally involve the introduction into a plant cell a genetic construct comprising a nucleic acid segment that encodes at least one avirulence/pathogenicity (Avr/Pth) polypeptide, wherein said segment is operably-linked to at least a first promoter element that is transcriptionally activated by a mechanism involving at least one virus-encoded polypeptide as a required component.
  • the nucleic acid segment comprised with this genetic construct encodes all, or substantially all of a bacterial Avr/Pth polypeptide.
  • the nucleic acid segment will comprise all, or substantially all of a gene that encodes such a polypeptide.
  • genes known to encode such polypeptides are found in Table 9, and include, but are not limited to, pthA, pthN, pthN2, pthA, pthJB, pthC, pthCBBl, avrBn, avrb ⁇ , avrB4, avrb7, avrBIn, avrBlOl, avr B 102, avrB103, avr B 104, avrB5, avrBs3, avrBs3-2, (avrBsP), avrxaS, avrXa/, avrXalO, avrXpl, avrPphA, avrPphBl.R3, avrPphD, avrPphEl.R2, avrPphF.Rl, avrPpiAl.R2,
  • Such genes are isolatable from a variety of bacterial genera, including, but not limited to, Cladosporium, Erwinia, Mayetiola, Pseudomonas, Salmonella, and Xanthomonas.
  • Table 9 identifies many of the known avr/pth genes that may be used in the practice of the invention including, but not limited to, C. fulvum, E. herbicola, M. destructor, P. syringae, S. typhimuriam, X. campestris, X. citri, and X. oryzae.
  • the gene may be full-length, substantially full-length, or may be truncated so as to encode only the portion of the polypeptide responsible for conferring producing the avirulence polypeptide in a plant expressing the nucleic acid segment.
  • the gene is operably linked to a promoter that is transcriptionally activated by a viral polypeptide.
  • promoters include, but are not limited to, an AC1, AC2, AIP and a BIP promoter.
  • the polynucleotide comprising the avirulence gene operably linked to a viral promoter may optionally comprise one or more targeting, localization, or enhancer sequences, and may optionally comprise one or more transcription termination sequences, or other gene regulatory sequences.
  • the nucleic acid segment may optionally comprise one or more 5' (or "upstream") sequences, and/or one or more 3' (or "downstream") sequences.
  • the promoter may comprise an early viral promoter.
  • the methods of the present invention may employ genetic constructs that comprise gene sequences isolated from a particular microorganism that are used to transform a target plant cell without prior modification, or alternatively the methods may employ one or more genetic constructs that have been altered, mutagenized, truncated, or otherwise modified by the hand of man prior to their use in transforming a particular target plant cell.
  • Such genetic constructs may be in the form of a recombinant vector that is stably maintained in a suitably transformed host cell, or alternatively, the genetic constructs of the present invention may comprises polynucleotide transgene sequences that are stably integrated into the genome of the host cell by homologous recombination.
  • Such genomically-integrated constructs are useful in the preparation of transgenic plants, that stably maintain the polynucleotide constructs and pass the genetic information to their decendants via standard Mendelian fashion.
  • the production of such transgenic plants that express the avirulence gene upon infection by a virus that produces a polypeptide that transcriptionally activates the heterologous viral promoter is a key aspect of the present invention.
  • Such transgenic plants are particularly desirable when planted in large populations, such as in commercial farming, or other crop plantings. Because expression of the avirulence gene constructs results in cell death of the host, viral infection of a plant comprising such a genetic construct is limited, because, upon infection, the transcriptional activation of the genetic construct begins to affect cell death in the infected plant.
  • This selective "suicide" by the infected plant is desirable because the dying plant is severely limited in its ability to reproduce the virus for subsequent infection of nearby plants, and the ultimate death of the plant itself, halts the ability of the infecting virus to "commandeer" the plant's metabolic processes in order to produce more virus particles.
  • initiating cell death of the plant via the presence of the genetic constructs of the invention in effect shuts down the viral "manufacturing plant” in the diseased plant. This reduces the titer of viral particles in the environment of the diseased plant, and thereby results in a reduced capacity of the viral organisms to subsequently infect neighboring susceptible plants.
  • the invention provides unique methods for reducing the infectivity of a virus in a plant population, controlling the spread of a virus in a plant population, decreasing the titer of virus produced within an infected plant, and limiting the number of plants in a population that are affected when a viral pathogen is introduced into the environment of the plant population.
  • nucleic acid segments when preparing or constructing the nucleic acid segments for introduction into the plant cells, it may be desirable to "plantize” or modify a given bacterially derived polynucleotide to alter particular nucleic acid residues in the primary sequence to facilitate better expression, or alter the activity of the gene sequence in the transformed plant.
  • Plantization of gene sequences is well known to those of skill in the plant molecular biological arts, and provides a means for preferentially altering the expression of a heterologous (or even homologous) gene in a transformed host cell. Methods for altering gene sequences to facilitate altered expression in a target host cell are described herein in Sections 4.0 to 4.21.
  • the subject invention concerns the use of natural or mutated viral promoters or synthetic promoters that are transcriptionally activated by one or more viral activators.
  • These promoters are operably linked to one or more microbial avirulence/pathogenicity (avr/pth) genes such that the promoter/gene construct is responsive to (transcriptionally activated or de-repressed by) one or more virus-encoded polypeptides.
  • avr/pth microbial avirulence/pathogenicity
  • Transcriptional activiation (or derepression of) the heterologous promoter causes an increase in the transcription of the avr/pth gene, and results in expression of the encoded polypeptide product in a host cell that comprises the genetic construct.
  • Infection of a transformed plant by a pathogenic virus that comprises the disclosed genetic constructs results in cascade of events: (1) the infecting virus expresses the unique viral activator or repressor in the plant host cell; (2) expression of the viral polypeptide initiates (or increases) expression of the viral promoter/avirulence gene fusion; (3) expression of the avr/pth gene activates a plant HR and cell death occurs; (4) cell death leads to death or decline of the infected plant; (5) the dying plant is unable to produce significant numbers of new viral particles; and (6) the infection of the virus into nearby healthy plants is reduced or arrested.
  • the selection of the avr gene used to induce the hypersensitive response (HR) cell death depends on the plant species to be protected, since the triggering of the HR in a particular plant species depends on a threshold level of production of the avr/pth gene. That threshold level, in turn, depends on the particular avr/pth gene. Several factors can affect the level of production of Avr protein, including the number of copies of the avr gene, the promoter strength and the position of the particular avr/pth gene following transformation into plants (position effect). The threshold level itself can be affected by the choice of the avr gene or mutant derivatives of the avr/pth gene.
  • a further aspect of the subject invention concerns use of mutations to weaken or strengthen an avr/pth gene ' s ability to quantitatively or qualitatively elicit an HR in the a particular plant, in order to adapt it for use to a particular viral promoter that may be "leaky".
  • leaky is meant having a low level of constitutive transcriptional activity in the plant, without promoter activation by the viral activator protein.
  • the subject invention also concerns the use of natural or mutated viral promoters or synthetic promoters that bind to viral repressors or other proteins, fused to microbial avr genes such that the fusion is responsive to (transcriptionally-activated by) a combination of a DNA virus encoded protein(s) and a synthetic aptamer/activator fusion polypeptide encoded by a distinct nucleic acid segment, in order to control viral diseases of plants. Infection by the virus results in expression of the unique viral repressor protein that binds to the promoter.
  • binding does not result in the expression of the avr gene until a second molecule, the aptamer/activator, provided by the second gene construct, binds to the repressor. Binding of the aptamer/activator to the repressor protein positions the activator in such a way that it can interact with the RNA polymerase II basal apparatus. This results in (increased) expression of the avr gene and activation of the plant HR and cell death.
  • the present invention also provides a recombinant vector, comprising a constitutive promoter operatively linked to a gene fusion comprising an aptamer coding sequence translationally fused to a DNA binding protein coding sequence such that upon expression of the gene, a hybrid protein is produced comprising the aptamer fused with the DNA binding protein.
  • This aptamer binds to a viral-encoded protein.
  • transcriptional activators for example, one such activator is the transcription activation domain of Gal4.
  • the recombinant vector may be a plasmid, a cosmid, a phage, a phagemid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or other suitable vector, including viral vectors and the like, for delivery of the avirulence gene polynucleotide into a target host cell.
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • FIG. 1 illustrates the general structure of pthA and all members of the avrBs3/pthA gene family.
  • FIG. 2 illustrates suppression by aptamers of the hypersensitive response (HR) normally elicited by pthA in P. vulgaris (common bean) cv. California Light Red leaf after inoculation using A. tumefaciens GV2260/pYD40.1 (top center leaf). Note the nearly complete loss of HR symptoms in the leaves inoculated with GV2260/pGZ8.1 (left leaf; YP aptamer) or with GV2260/pGZ8.3 (right leaf; HP aptamer). Inoculation was by vacuum infiltration as described in Yang and Gabriel, 1994.
  • FIG. 1 illustrates suppression by aptamers of the hypersensitive response (HR) normally elicited by pthA in P. vulgaris (common bean) cv. California Light Red leaf after inoculation using A. tumefaciens GV2260/pYD40.1 (top center leaf). Note the nearly complete loss of HR symptoms in the leaves inoculated with
  • FIG. 4 illustrates the HR normally elicited on tobacco by expression of pthA is blocked by transgenic tobacco strongly expressing Y ⁇ v.uidA.
  • the control leaf to the left is inoculated with tumefaciens-delivered pthA, which is transiently expressed (left side) and with A. tumefaciens-delive ⁇ ed vector only (right side).
  • transiently expressed pthA elicits a strong HR by 48 hours after inoculation.
  • the transgenic leaf to the right is identically inoculated.
  • transiently expressed pthA does not elicit an HR 48 hours after inoculation.
  • Expression o ⁇ YVv.uidA was confirmed by Gus assays. Inoculation was by syringe infiltration as described in Yang and Gabriel, 1994.
  • FIG. 5 shows a hypersensitive response (HR) elicited by pthA in Phaseolus vulgaris (common bean) cv. California Light Red leaf after inoculation using Agrobacterium tumefaciens GV2260/pYD40.1. Note the strong HR at the top of the leaf and the relative lack of a plant response to the vector alone at the bottom.
  • HR hypersensitive response
  • FIG. 6 shows a schematic illustration of a promoter/ ⁇ vr construct in plant nucleus, wherein the infecting virus makes the Activator protein that binds to both the viral promoter(s) that it normally binds to and binds to the portion of the viral promoter that has been engineered as a fusion to drive transcription of an avr gene. Expression of the Avr protein above an empirically determined basal level by the viral activator results in host cell death and the HR.
  • FIG. 7 shows a schematic illustration of promoter/ ⁇ vr construct in PI and FI plant nuclei and aptamer/activator and aptamer/DNA binding construct(s) in P2 and FI plant nuclei, wherein the infecting virus makes any unique protein, such as, but not limited to, the coat protein.
  • the coat protein is recognized by two aptamer/fusions, both of which are expressed constitutively by the transgenic plant.
  • the first aptamer/fusion binds to both the coat protein because of the aptamer, and to the promoter region of the promoter/avr gene construct. Such binding does not activate transcription until the viral coat protein is present as a result of viral infection.
  • the second aptamer/fusion also binds to the coat protein because of the aptamer, and this brings the activator part of this aptamer/fusion in a position to activate transcription of the avr gene, which results in host cell death and the HR.
  • FIG. 8 shows a schematic illustration of a promoter/ ⁇ vr construct in PI and FI of plant nuclei and aptamer/activator construct in P2 and FI plant nuclei, wherein the infecting virus makes the Repressor protein that binds to both the viral promoter(s) that it normally binds to and binds to a recognized DNA sequence motif positioned appropriately near the TATA box on a minimal promoter fused to an avr gene. Such binding does not activate transcription until a second protein molecule, the Aptamer/Activator, binds to the viral repressor that has bound to the avr gene promoter.
  • the Activator portion of the protein Upon binding, the Activator portion of the protein is in a position to activate transcription of the ⁇ vr gene, which results in host cell death and the HR.
  • the Promoter/ ⁇ vr gene construct is engineered into one parental line (PI), while the Aptamer/Activator construct is engineered into a second parental line (P2), and that the invention is not fully realized until the two parental lines are crossed to produce FI seed, plants and derivatives.
  • FIG. 9 shows polynucleotide constructs used in a transient expression assay.
  • the pYD12 series were delivered into cells of intact, detached orange leaves via particle bombardment.
  • pYD40.1 and the pGZ series were delivered into cells of intact, attached orange leaves via Agrobacterium- mediated transfer.
  • the left and right border sequences of T- DNA are represented by T L and T R .
  • FIG. 10 shows an uninfected tomato cultivar "Rio Grande" plant inoculated with GV2260/ pYD63.7, GV2260/pYD40.2, GV2260/pYD40.1 andGV2260/pYD63.1.
  • the HR appears only with GV2260/pYD40.1, in which pthA is constitutively expressed.
  • FIG. 11 shows a tomato cultivar "Rio Grande” plant infected with TMoV, as described by Duan et al, (1997) and inoculated with GV2260/ pYD63.7, GV2260/pYD40.2, GV2260/ pYD40.1 andGV2260/pYD63.1. Note the HR in all panels except the control panel labeled "vector" (GV2260/pYD40.2).
  • ⁇ vrb ⁇ 5 is a member of the avrBs3/pthA gene family that is 97% identical to pthA and used as a control that does not give cankers on citrus.
  • the aptamers are fused to uidA to both stabilize the aptamer and to test for gene expression of the aptamer inplanta.
  • the present invention provides methods for the prevention, alteration, or amelioration of viral infection and disease in selected plants.
  • the invention concerns the creation of transgenic plants that are resistant to one or more viral diseases.
  • the invention provides methods for transforming a selected plant cell with at least one genetic construct comprising all, or substantially all of a gene segment that encodes one or more bacterially derived avirulence genes that is transcriptionally activated or, alternatively, repressed by a DNA virus peptide or polypeptide.
  • the polynucleotide encoding such avr/pth gene(s) are operably linked to one or more plant-expressible promoters that promote expression of the gene in the transformed plant cell to produce the polypeptide in the transformed plant cell.
  • Such promoters may be inducible, constitutative, native bacterial promoters, native plant promoters, or any other native or genetically engineered promoter that is sufficient to promote the expression of the gene product in the transformed plant cell.
  • pathogenicity protein signal alone that causes the primary disease symptoms
  • Pth protein signal produced by all three pathogens is nearly identical, each causes pathogenic symptoms only on specific (host) plants (Gabriel, 1999).
  • the signal that causes citrus cankers only causes such cankers in citrus.
  • the signal that causes bean blight only causes such blights in bean, and the signal that causes rice blight only causes such blights in rice.
  • the signal proteins in all three cases are of nearly identical structure and sequence.
  • the pathogenic symptoms directly elicited by pthA expression in citrus cells are host- specific.
  • transient expression assays of pthA in tobacco, bean, poplar and cotton no canker phenotype was observed. Instead, a rapid plant defense reaction known as a hypersensitive response (HR) phenotype is observed.
  • HR hypersensitive response
  • Pathogen genes that elicit an HR are called avirulence (avr) genes.
  • pthA is therefore a citrus-specific pathogenicity (pth) gene as well as an avr gene in plants other than citrus.
  • pthA is a member of a large and highly conserved Xanthomonas gene family, called the avrBs3/pthA gene family (Gabriel, 1999; Leach and White, 1996). At least 27 members of this gene family have been cloned (Gabriel, 1999); all are >90% identical at the nucleotide sequence level (Gabriel, 1999; Gabriel, 1997; Leach and White, 1996). These genes have the general structure shown in FIG. 1.
  • pthA expression inside plant cells is sufficient to cause citrus canker disease, what is the role of the pathogen?
  • a functional type III protein secretion system encoded by hrp (hypersensitive response and pathogenicity) genes is required (Alfano and Collmer, 1997).
  • Function of pthA in X. citri for either pathogenicity or avirulence is hrp dependent (Yang and Gabriel, 1995).
  • the type III secretion system is a host cell contact- dependent, protein injection device (Silhavy, 1997).
  • PthA is a signal molecule injected by citri into citrus cells, where it functions as a signal to trigger cell division and cell death.
  • rice blight symptoms are known to be enhanced by avrXa7, another known member of this same gene family (Leach and White, 1996).
  • PthA appears to be secreted from X. citri and functions inside the plant cell to cause cankers on citrus and the HR on all other plants.
  • PthF and AvrXa7 are analogously secreted from X. phaseoli and X. oryzae, respectively, and function inside plant cells to cause blight on bean and rice (respectively) and the HR on all other plants.
  • the transient expression assays illustrated here on citrus and bean in FIG. 2 and FIG. 3 and the transgenic tobacco in FIG. 4 provide the experimental basis for the novel idea that blocking these signals would control these diseases.
  • the present invention also demonstrates activation of an avirulence gene by any unique protein produced by a virus and not found in uninfected plants, including both RNA and DNA viruses.
  • the novel promoters are transcriptionally activated by virus-specific transcriptional activator protein(s) and/or other novel promoter/activator proteins to control viral diseases in plants. This is accomplished by activating avirulence genes introduced into a plant to cause immediate plant cell death.
  • Cell type-specific and gene-specific activator proteins bind enhancer DNA sequences, often as a complex, and upstream of the TATA box, to initiate transcription.
  • Promoters sequences and the transcription factors that modulate promoter activity are both modular in nature, and chimeric promoters may be constructed that take advantage of recent detailed understanding of promoter activities. Repressors may be turned into activators (Moore et al, 1998).
  • One aspect of this invention is a method of tightly regulating a synthetic promoter that is activated by a repressor protein of viral origin that binds to a viral DNA sequence.
  • Moore et al, (1998) teach use of a repressor protein that is fused to a transcriptional activator.
  • a critical part of the present invention is the use of genes encoding "aptamers” (described below), selected for binding repressors of viral origin, which are fused with genes encoding proteins such as the transcription activation domain of Gal4, to provide RNA polymerase Il-based transcription at the TATA box. Transcriptional activation cannot occur without the virus-encoded repressor, and activation occurs in the presence of repressor, resulting in cell death.
  • Nucleic acids and proteins often carry the ability to bind to other molecules with a level of affinity and degree of molecular specificity similar to that exhibited by antibodies.
  • An entirely new genetic technology is developing around the ability to isolate extremely rare nucleic acid sequences with specific ligand binding properties (similar to antibodies) from very large pools of random sequences.
  • the process used is an iterative selection and amplification scheme, sometimes called systematic evolution of ligands by exponential enrichment (SELEX) (see e.g., Tuerk and Gold, 1990; Gold, 1995).
  • SELEX systematic evolution of ligands by exponential enrichment
  • the selected molecules with specific ligand binding properties are called "aptamers” (from the Latin aptus, meaning "to fit") (see e.g., Szostak, 1992).
  • aptamer was used to describe nucleic acid molecules, it is also applied to proteins as well (Tuerk and Gold, 1990; Colas et al, 1996).
  • Plant DNA viruses usually do not turn on all of their genes at once in order to reproduce upon plant infection. Instead, plant cells transcribe "early” viral genes and the products of these genes induce expression (transcription) of additional viral genes required later in the infection or reproduction process. Among such "early” viral genes are unique transcriptional activators and repressors. The plant hosts do not make such viral activators and repressors. Otherwise the virus could not maintain control of its own transcription program. These viral-encoded, DNA binding proteins (present only during viral infection), are key components of the present invention. All plant viruses, whether DNA viruses or RNA viruses, produce unique proteins upon infection. These include the coat protein and cell movement proteins.
  • geminiviruses which infect both monocotyledonous and dicotyledonous plants. Geminiviruses replicate in the nuclei of plant cells. Subgroup I geminiviruses (including wheat dwarf virus and maize streak virus) carry replication initiator proteins that appear to be in the myb-related class of plant transcriptional activators and their binding site on the virus DNA has been identified (Hofer et al, 1992). Subgroup II and III geminiviruses (including beet curly top, tomato yellow leaf curl, tomato golden mosaic and African cassava mosaic viruses) carry L2 and AL2 genes that encode transcriptional activator proteins of other viral genes (Sunter and Bisaro, 1997).
  • a DNA binding repressor element (protein) coding region was isolated to a 300-bp DNA fragment of tomato golden mosaic virus (Sunter and Bisaro, 1997).
  • a DNA binding domain has been identified on a protein encoded by the rice tungro bacilliform virus (Jacquot et al, 1997). Therefore most, if not all, plant DNA viruses produce proteins upon infection of plant cells that are likely to be involved in transcriptional regulation of other viral genes.
  • the promoter fragment and/or the DNA sequence motif that is bound has been identified on the viral genome.
  • the viral promoter fragment and or DNA binding motif can be used to create promoters that are responsive to viral activator proteins, or that bind to viral repressor proteins. These viral promoter fragments and/or DNA binding motifs represent a key component of the present invention.
  • avr genes have been found to encode proteins that are "injected” or otherwise delivered inside the plant cell. Expression of these avr genes inside the plant cell causes the death of plant cells that are non-hosts.
  • pthA GenBank Accession No. U28802
  • HR rapid host cell death
  • a natural viral infection would result in expression of the viral transcriptional activator, which in turn causes transcription of the engineered plant cell carrying the viral response promoter fused to the avr gene, and a rapid host cell death of the infected cell(s) results, thus limiting viral infection (FIG. 6).
  • nlsA was changed from K-R-A-K-P to H-R-A-I-P; nlsB was changed from R-K-R-S-R to R-H-R-S-I, and nlsC was changed from R-V-K-R-P-R to R- V-H-R-P-I.
  • lysine was changed to histidine, and a second lysine or arginine was changed to isoleucine (from a basic amino acid to a neutral one) on both genes.
  • the mutant genes were sequenced to confirm that only the desired mutations were present. These clones were then used to replace pthA in pYD40.1, which were mobilized into A.
  • FIG. 6 A further aspect of the simplest form of the subject invention illustrated in FIG. 6 is the use of site directed mutations as described in this paragraph or other means (such as screening for natural mutations (Yang and Gabriel, 1995b) to weaken the effect of the Avr polypeptide in nonhost plants.
  • Another preferred form of the subject invention is to utilize minimal promoters consisting of a TATA-box and specific binding sites for viral encoded repressors that are not present in plants.
  • the AL1 replication protein of tomato golden mosaic virus (TGMV) binds to a 13-bp DNA sequence (5'-GGTAGTAAGGTAG-3') SEQ ID NO:5 and appears to act as a repressor.
  • This binding site if placed upstream of the TATA-box (which lacks intrinsic transcriptional activity), would allow AL1 to bind.
  • Multiple binding sites may be provided in tandem array upstream of the TATA box (FIG. 8), since the strength of the promoter activation depends upon the affinity of the DNA binding protein for the target DNA sequence (Messing, 1998).
  • AL1 would not likely activate the promoter. Activation requires an activator polypeptide, such as domain-II of GAL4 (residues 768-881) (Moore et al, 1998).
  • the activator polypeptide by itself cannot bind to AL1, but could be made to bind to AL1 by fusing it with a polypeptide aptamer that was selected for ability to bind to ALL Strength of promoter activation would depend upon the affinity of the aptamer for the target polypeptide sequence.
  • An important element to the repressor method is the identification of aptamer sequences that can be used to synthesize gene fusions with the activator, such as GAL4.
  • target fragments of AL1 may be synthesized on a polylysine core resin to produce a multiple antigenic peptide (MAP).
  • the MAP immunogen is composed of multiple copies of a single target epitope attached to a small, non-immunogenic, polylysine core.
  • MAP resins typically have four or eight peptide arms branching out of a polylysine core matrix.
  • short fragments may be synthesized without use of a MAP resin.
  • DNA aptamers may be screened and iteratively amplified that bind to any target viral polypeptide that is produced in infection, and the DNA sequence of the selected aptamer included in the upstream promoter element of the minimal promoter fused to an avirulence gene.
  • this method it is not necessary to identify a repressor polypeptide, but rather any viral polypeptide that will bind to a DNA aptamer.
  • Short peptides such as polypeptide aptamers, are unstable and will be degraded if expressed alone in plant cells. However, when attached to polypeptides such as GAL4, they are stabilized. In a preferred embodiment, in order to stabilize the short peptides, they can be translationally fused to the NH 3 -terminal end of domain II of GAL4.
  • RNA viruses don't make DNA binding polypeptides, as DNA viruses do, but they do make unique polypeptides, such as the coat protein, that are not found in plants. Therefore a further embodiment of the subject invention is to create transgenic plants that carry three genetic constructs: 1) an artificial, stringently regulated promoter fused to a gene; 2) a constitutively expressed DNA binding protein/aptamer that binds to a DNA sequence that is part of the artificial promoter, and 3) a constitutively expressed activator/aptamer as detailed in the example above.
  • the aptamers in each case must recognize and bind the unique polypeptide made by the virus, such as the coat protein (FIG. 7).
  • the DNA binding protein may be artificial or natural, but it should be one that is not found in plants that binds to a known DNA sequence motif that is also not found in plants.
  • a good example of a preexisting DNA binding protein that binds to a known DNA sequence motif that is not found in plants is the lac repressor that binds to the lac operator (Moore et al., 1998).
  • lac repressor that binds to the lac operator
  • the operator sequence is fused to the minimal promoter consisting of the TATA box and transcription initiation site to form the stringently regulated, artificial promoter exactly as described (Moore et al, 1998).
  • this promoter would be then be fused to an avirulence gene such as pthA.
  • Aptamers would then be selected exactly as described above for the unique viral repressor protein, with care taken that the aptamers do not bind to plant proteins, but only to viral protein.
  • different aptamers that bind different regions of the coat protein of the virus are selected and tested pair-wise in competitive binding assays. Those that do not appear to interfere with the binding of another are selected and then sequenced as described above.
  • F2 plants may be selected in e.g., self-fertile plant systems for true breeding plants homozygous for both engineered traits.
  • the invention concerns both the making of transgenic plants carrying the viral or artificial promoter fused to at least one avr/pth gene, the generation of fertile transgenic offspring (or progeny) that inherit the engineered trait(s) and also, the subsequent propagation and recovery of seeds that may be subsequently planted to obtain progeny plants demonstrating the desired phenotype.
  • the invention also concerns the making of transgenic plants having the Aptamer/Activator and Aptamer/DNA binding protein genes in one plant line, the artificial promoter/ ⁇ vr gene in a second plant line, and all components together in FI hybrid seed and lines and subsequent generations of plants carrying one or both of the engineered traits.
  • Plant DNA viruses usually do not turn on all of their genes at once in order to reproduce upon plant cell infection. Instead, plant cells transcribe "early" genes and the products of these genes induce expression of vital genes required later in the reproduction process. Often, these early vital genes encode transcriptional activators. avr genes are of microbial origin and induce a rapid plant cell death response when expressed inside plant cells (DeFeyter et al, 1998; Gopalan et al, 1996; Scofield et al, 1996; Tang et al, 1996; Van den Ackerveken et al, 1996). Upon virus infection, the vital transcriptional activator causes transcription of the engineered plant cell carrying the response promoter fused to the avr gene, and a rapid host cell death of the infected cell(s) results, thus limiting viral infection.
  • the DNA virus used was tomato mottle geminivirus (TMoV), a bipartite geminivirus. All bipartite geminiviruses share many common features such as genome organization and replication processes. Expression of the TMoV AVI coat protein gene and BVl movement protein gene depends on the AC2 (syn. AL2 or C2) transcriptional activator gene (Abouzid et al, 1992; Sunter and Bisaro, 1997). The promoters of AVI and BVl are therefore inducible. This feature permits the engineering and expression of an avirulence (avr) gene under the control of an AVI or BVl promoter in a transformed plant.
  • avr avirulence
  • Activation of the avirulence gene due to the presence of the TMoV virus resulted in production of the avirulence protein product, which is a signal molecule that induces very rapid (hypersensitive) plant resistance response and host cell death, resulting in elimination of the virus. If the infected plant cells die, the virus cannot spread systematically in the plant, and infection is aborted.
  • Illustrative prokaryotes both Gram-negative and Gram-positive, include Enter obacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae.
  • Enter obacteriaceae such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus
  • Bacillaceae Rhizobiceae, such as Rhizobium
  • Spirillaceae such as photobacterium, Zymomonas, Serratia,
  • fungi such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
  • yeast such as Saccharomyces and Schizosaccharomyces
  • Basidiomycetes yeast such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
  • Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the genetic constructs of the present invention and the avirulence gene into the host cell, availability of expression systems, efficiency of expression, stability of the gene of interest in the host, and the presence of auxiliary genetic capabilities.
  • microorganisms known to inhabit the phylloplane (the surface of the plant leaves) and or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops may also be desirable host cells for manipulation, propagation, storage, delivery and/or mutagenesis of the disclosed genetic constructs.
  • microorganisms include bacteria, algae, and fungi.
  • microorganisms such as bacteria, e.g., genera Bacillus (including the species and subspecies B. thuringiensis v. kurstaki HD-1,
  • fungi particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
  • phytosphere bacterial species include Pseudomonas spp. including P. syringae, P. cepacia, and P. fluorescens; Serratia spp. including S. marcescens; Acetobacter spp. including A. xylinum; Agrobacterium spp. including A. tumefaciens;
  • Rhodobacter spp. including R. sphaeroides and R. capsulatus; Xanthomonas spp. including
  • Rhizobium spp. including R. melioti
  • Alcaligenes spp. including A. eutrophus
  • Azotobacter spp. including A. vinlandii.
  • yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. dif ⁇ uens, C. laurentii, Saccharomyces rosei,
  • Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing a selected genetic construct into the host, availability of expression systems, efficiency of expression, stability of the polynucleotide in the host, and the presence of auxiliary genetic capabilities. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
  • Virtually any DNA composition may be used for delivery of the genetic constructs of the present invention to selected recipient plant cells to ultimately produce transformed plants and plant cell lines in accordance with the present invention.
  • polynucleotides in the form of vectors and plasmids, or linear nucleic acid fragments, in some instances containing only the particular polynucleotide to be expressed in the animal, and the like, may be employed.
  • DNA constructs can include structures such as promoters, enhancers, polylinkers, or regulatory genes as desired.
  • the DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the animal cell.
  • the nucleic acid constructs may contain antisense constructs, or ribozyme-encoding regions when delivery or introduction of such nucleic acid constructs is desirable.
  • the means and methods for mutagenizing a DNA segment such as one comprising an inducible promoter region are well known to those of skill in the art. Modifications to such promoter regions may be made by random, or site-specific mutagenesis procedures.
  • the promoter region may be modified by altering its structure through the addition or deletion of one or more nucleotides from the sequence that encodes the corresponding un-modified promoter region.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. Such phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired promoter region or peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • a genetic selection scheme has been devised to enrich for clones incorporating the mutagenic oligonucleotide (Kunkel et al, 1987).
  • PCRTM with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector.
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided in U. S. Patent 4,237,224 (specifically incorporated herein by reference in its entirety).
  • PCRTM polymerase chain reaction
  • the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Polymerase chain reaction methodologies are well known in the art.
  • Qbeta Replicase described in Intl. Pat. Appl. Publ. No. PCT/US87/00880 (specifically incorporated herein by reference in its entirety) may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ -thio]triphosphates in one strand of a restriction site (Walker et al, 1992, incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids that involves multiple rounds of strand displacement and synthesis, i.e. nick translation.
  • a similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
  • RCR Repair Chain Reaction
  • a similar approach is used in SDA. Still other amplification methods have been described (British Patent No. 2202328;
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al, 1989; Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer that has sequences specific for the target gene sequence to be modified.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second protein-specific primer, followed by polymerization.
  • the double stranded DNA molecules are then multiply-transcribed by a polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate sequences that are specific for the selected gene sequence.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • leader sequences are contemplated to include those that include sequences predicted to direct optimum expression of the attached gene, i.e. to include a preferred consensus leader sequence that may increase or maintain mRNA stability and prevent inappropriate initiation of translation.
  • sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in animals, and in particular humans, will be most preferred.
  • the level of transcription of a particular transgene in a given host cell is not always indicative of the amount of protein being produced in the transformed host cell. This is often due to post-transcriptional processes, such as splicing, polyadenylation, appropriate translation initiation, and RNA stability that affect the ability of a transcript to produce protein. Such factors may also affect the stability and amount of mRNA produced from the given transgene. As such, it is often desirable to alter the post-translational events through particular molecular biology techniques.
  • the 5 '-untranslated leader (5'-UTL) sequence of eukaryotic mRNA plays a major role in translational efficiency.
  • Many early chimeric transgenes using a viral promoter used an arbitrary length of viral sequence after the transcription initiation site and fused this to the AUG of the coding region. More recently studies have shown that the 5'-UTL sequence and the sequences directly surrounding the AUG can have a large effect in translational efficiency in host cells and particularly certain plant species and that this effect can be different depending on the particular cells or tissues in which the message is expressed.
  • transgenes encoding ⁇ -glucuronidase (GUS) and bacterial chitinase showed a 4-fold and an eight-fold increase in expression, respectively, when the native sequences of these genes were changed to encode 5'-ACCAUGG-3' (Gallie et al, 1987b; Jones et al, 1988).
  • GUS ⁇ -glucuronidase
  • bacterial chitinase showed a 4-fold and an eight-fold increase in expression, respectively, when the native sequences of these genes were changed to encode 5'-ACCAUGG-3' (Gallie et al, 1987b; Jones et al, 1988).
  • chimeric transgenes i.e. transgenes comprising DNA segments from different sources operably linked together
  • alfalfa mosaic virus (AMV) coat protein and brome mosaic virus (BMV) coat protein 5'- UTLs have been shown to enhance mRNA translation 8-fold in electroporated tobacco protoplasts (Gallie et al, 1987a; 1987b).
  • a 67-nucleotide derivative ( ⁇ ) of the 5'-UTL of tobacco mosaic virus RNA (TMV) fused to the chloramphenicol acetyltransferase (CAT) gene and GUS gene has been shown to enhance translation of reporter genes in vitro (Gallie et al, 1987a; 1987b; Sleat et al, 1987; Sleat et al, 1988).
  • Electroporation of tobacco mesophyll protoplasts with transcripts containing the TMV leader fused to reporter genes CAT, GUS, and LUC produced a 33-, 21-, and 36-fold level of enhancement, respectively (Gallie et al, 1987a; 1987b; Gallie et al, 1991). Also in tobacco, an 83-nt 5'-UTL of potato virus X RNA was shown to enhance expression of the neomycin phosphotransferese II (Nptll) 4-fold (Poogin and Skryabin, 1992). The effect of a 5'-UTL may be different depending on the plant, particularly between dicots and monocots.
  • TMV 5'-UTL has been shown to be more effective in tobacco protoplasts (Gallie et al, 1989) than in maize protoplasts (Gallie and Young, 1994). Also, the 5'-UTLs from TMV- ⁇ (Gallie et al, 1988), AMV-coat (Gehrke et al, 1983; Jobling and Gehrke, 1987), TMV-coat (Goelet et al, 1982), and BMV-coat (French et al, 1986) worked poorly in maize and inhibited expression of a luciferase gene in maize relative to its native leader (Koziel et al, 1996).
  • the 5'-UTLs from the cauliflower mosaic virus (CaMV) 35S transcript and the maize genes glutelin (Boronat et al. 1986), PEP-carboxylase (Hudspeth and Grula, 1989) and ribulose biphosphate carboxylase showed a considerable increase in expression of the luciferase gene in maize relative to its native leader (Koziel et al, 1996).
  • introns in the transcribed portion of a gene has been found to increase heterologous gene expression in a variety of plant systems (Callis et al, 1987; Maas et al, 1991; Mascerenhas et al, 1990; McElroy et al, 1990; Vasil et al, 1989), although not all introns produce a stimulatory effect and the degree of stimulation varies.
  • the enhancing effect of introns appears to be more apparent in monocots than in dicots. Tanaka et al, (1990) has shown that use of the catalase intron 1 isolated from castor beans increases gene expression in rice.
  • Adhl alcohol dehydrogenase 1
  • introns 2 and 6 of Adhl (Luehrsen and Walbot, 1991), the catalase intron (Tanaka et al, 1990), intron 1 of the maize bronze 1 gene (Callis et al, 1987), the maize sucrose synthase intron 1 (Vasil et al, 1989), intron 3 of the rice actin gene (Luehrsen and Walbot, 1991), rice actin intron 1 (McElroy et al, 1990), and the heat shock protein HSP70 (U. S. Patent 5,859,347, specifically incorporated herein by reference in its entirety). Similar results may also be obtained using sequences from certain exons, for example, the maize ubiquitin exon 1 (Christensen et ⁇ / repeat 1992).
  • the selected intron(s) should be present in the 5' transcriptional unit in the correct orientation with respect to the splice junction sequences (Callis et al, 1987; Maas et al, 1991; Mascerenhas et al, 1990; Oard et al, 1989; Tanaka et al, 1990; Vasil et al, 1989).
  • Intron 9 of Adhl has been shown to increase expression of a heterologous gene when placed 3' (or downstream of) the gene of interest (Callis et al, 1987).
  • Codon usage in the native genes is considerably different from that found in typical plant genes, which have a higher G+C content.
  • Strategies to increase expression of these genes in plants generally alter the overall G+C content of the genes.
  • synthetic B. thuringiensis ⁇ -endotoxin encoding genes have resulted in significant improvements in expression of the ⁇ -endotoxins in various crops including cotton (Perlak et al, 1990; Wilson et al, 1992), tomato (Perlak et al, 1991), potato (Perlak et al, 1993), rice (Cheng et al, 1998), and maize (Koziel et al, 1993).
  • the genetic constructs of the present invention may in certain circumstances be altered to increase the expression of these prokaryotic-derived genes in particular eukaryotic host cells and/or transgenic plants that comprise such constructs.
  • molecular biology techniques that are well known to those of skill in the art, one may alter the coding or non coding sequences of the particular avr or pth gene(s) to optimize or facilitate its expression in transformed plant cells at levels suitable for preventing the spread of viral pathogens in such plants.
  • U. S. Patent 5,576,198 discloses compositions and methods useful for genetic engineering of plant cells to provide a method of controlling the timing or tissue pattern of expression of foreign DNA sequences inserted into the plant plastid genome.
  • Constructs include those for nuclear transformation that provide for expression of a viral single subunit RNA polymerase in plant tissues, and targeting of the expressed polymerase protein into plant cell plastids.
  • plastid expression constructs comprising a viral gene promoter region which is specific to the RNA polymerase expressed from the nuclear expression constructs described above and a heterologous gene of interest to be expressed in the transformed plastid cells.
  • Cauliflower Mosaic Virus (CaMV) U. S. Patent 5,530,196
  • an expression vector comprising at least one avirulence/pathogenicity gene-containing polynucleotide operably linked to an inducible promoter.
  • an expression vector is an isolated and purified DNA molecule comprising an avirulence/pathogenicity coding region operably linked to a promoter that expresses the gene, which coding region is operatively linked to a transcription- terminating region, whereby the promoter drives the transcription of the coding region.
  • the promoter of the present invention is operatively linked to a coding region that encodes a functional RNA.
  • a functional RNA may encode for a polypeptide (mRNA), be a tRNA, have ribozyme activity, or be an antisense RNA.
  • mRNA polypeptide
  • tRNA tRNA
  • ribozyme activity or be an antisense RNA.
  • operatively linked means that a promoter is connected to a nucleic acid region encoding functional RNA in such a way that the transcription of that functional RNA is controlled and regulated by that promoter.
  • Means for operatively linking a promoter to a nucleic acid region encoding functional RNA are well known in the art.
  • RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
  • mRNA messenger RNA
  • a variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein.
  • the ability of such nucleic acid probes to specifically hybridize to all or portions of one or more avirulence/pathogenicity genes lends them particular utility in a variety of embodiments.
  • the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample, and in the identification of new species or genera of avirulence/pathogenicity genes from a variety of host organisms.
  • oligonucleotide primers it is advantageous to use oligonucleotide primers.
  • the sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of avirulence/pathogenicity genes from a sample using PCRTM technology. Segments of related avirulence/pathogenicity genes from other species may also be amplified by PCRTM using such primers.
  • a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of an avirulence/pathogenicity gene sequence.
  • a size of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patent 4,683,195, and U. S. Patent 4,683,202, (each specifically incorporated herein by reference in its entirety), or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.
  • nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to. a 14 nucleotide long contiguous DNA segment of one or more avirulence/pathogenicity genes will find particular utility.
  • nucleic acid probes While the ability of such nucleic acid probes to specifically hybridize to avirulence/pathogenicity gene sequence makes them ideal for use in detecting the presence of complementary sequences in a given sample, other uses are also envisioned, including the use of the sequence information for the preparation of mutant species primers, synthetic gene sequences, gene fusions, and/or primers for use in preparing other avirulence/pathogenicity genetic constructs.
  • hybridization probe of about 14 or so nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having contiguous complementary sequences over stretches of about 15, 16, 17, 18, 19, or 20 or more bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion.
  • Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,195 and U. S. patent 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • nucleic acid reproduction technology such as the PCRTM technology of U. S. Patent 4,683,195 and U. S. patent 4,683,202 (each incorporated herein by reference)
  • the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.
  • one may desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating particular DNA segments.
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • specific hybridization is detected, or even quantitated, by means of the label.
  • a bacterial cell, a yeast cell, or a plant cell transformed with an avirulence/pathogenicity gene-containing expression vector of the present invention also represents an important aspect of the present invention.
  • transgenic plants and the progeny and seeds derived from such a transformed or transgenic plant are also important aspects of this invention.
  • Such transformed host cells are often desirable for use in the expression of the various DNA gene constructs disclosed herein.
  • Such methods are routine to those of skill in the molecular genetic arts.
  • various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA in the particular transformed host cell.
  • the initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal.
  • the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region.
  • This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the expression construct during introduction of the DNA into the host.
  • the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host.
  • the avirulence/pathogenicity-encoding gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region.
  • This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host.
  • one or more markers may be present, which have been described previously.
  • the plasmid will desirably include a sequence homologous with the host genome.
  • the left and right T-DNA borders from the Ti plasmid may be used when integration is desired using A.
  • transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for presence of the genetic construct.
  • Genes or other nucleic acid segments, as disclosed herein, can be inserted into host cells using a variety of techniques that are well known in the art. Five general methods for delivering a nucleic segment into cells have been described: (1) chemical methods (Graham and VanDerEb, 1973); (2) physical methods such as micro injection (Capecchi, 1980), electroporation (U. S. Patent 5,472,869; Tomes et al, 1990; Wong and Neumann, 1982; Fromm et al, 1985), microprojectile bombardment (Wang et al, 1988; Vain et al, 1990; U. S. Patent 5,874,265.
  • the vectors comprise, for example, plasmids (such as pBR322, pUC series, M13mp series, pACYC184, etc), cosmids, phage, and/or phagemids and the like.
  • plasmids such as pBR322, pUC series, M13mp series, pACYC184, etc
  • cosmids phage, and/or phagemids and the like.
  • the disclosed polynucleotides can be inserted into a given vector at a suitable restriction site.
  • the resulting plasmid may be used, for example, to transform bacterial cells such as E. coli or A. tumefaciens.
  • the bacterial cells are then cultivated in a suitable nutrient medium, harvested and lysed.
  • the plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis.
  • each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary.
  • Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al, 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc.
  • acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
  • Technology for introduction of DNA into cells is well known to those of skill in the art, and described hereinbelow in detail.
  • a large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T- DNA using A.
  • tumefaciens or A. rhizogenes as transformation agent, fusion, injection, or electroporation as well as other possible methods. If agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA.
  • the Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.
  • Plant explants can advantageously be cultivated with A. tumefaciens or A. rhizogenes for the transfer of the DNA into the plant cell.
  • Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
  • the plants so obtained can then be tested for the presence of the inserted DNA.
  • No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.
  • T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in ⁇ ur. Pat. Appl. No. ⁇ P 120 516; Hockema (1985); An et al, 1985, Herrera- ⁇ strella et al, (1983), Bevan et al, (1983), and Klee et al, (1985).
  • the vector preferably contains a segment of pBR322 which provides an origin of replication in E. coli and a region for homologous recombination with the disarmed T-DNA in Agrobacterium strain ACO; the oriV region from the broad host range plasmid RK1; the streptomycin/spectinomycin resistance gene from Tn7; and a chimeric NPTII gene, containing the CaMV35S promoter and the nopaline synthase (NOS) 3' end, which provides kanamycin resistance in transformed plant cells.
  • a segment of pBR322 which provides an origin of replication in E. coli and a region for homologous recombination with the disarmed T-DNA in Agrobacterium strain ACO
  • the oriV region from the broad host range plasmid RK1
  • the streptomycin/spectinomycin resistance gene from Tn7
  • a chimeric NPTII gene containing the CaMV35S promoter and the nopaline synthase
  • the enhanced CaMV35S promoter may be replaced with the 1.5 kb mannopine synthase (MAS) promoter (Velten et al, 1984).
  • MAS mannopine synthase
  • A. tumefaciens ACO is a disarmed strain similar to pTiB6SE described by Fraley et al, (1985).
  • the starting Agrobacterium strain was the strain A208 that contains a nopaline-type Ti plasmid.
  • the Ti plasmid was disarmed in a manner similar to that described by Fraley et al, (1985) so that essentially all of the native T-DNA was removed except for the left border and a few hundred base pairs of T-DNA inside the left border.
  • the inserted DNA Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia.
  • the individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
  • Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
  • pectolyases pectolyases
  • Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.
  • a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters or solid culture medium.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene.
  • the number of cells in a focus which express the exogenous gene product 48 h post-bombardment often range from 1 to 10 and average 1 to 3.
  • TRFs trauma reduction factors
  • Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
  • the use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et ⁇ , 1985; Rogers et ⁇ , 1988). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements.
  • the region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Shmann et ⁇ l, 1986; Jorgensen et ⁇ /., 1987).
  • Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al, 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide-coding genes.
  • the vectors described (Eichholtz et ⁇ l, 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes.
  • Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
  • Agrobacterium-mediated transformation of leaf disks and other tissues such as cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al. 1987). Therefore, commercially important cereal grains such as rice, corn, and wheat must usually be transformed using alternative methods (see e.g., U. S. Patent 5,610,042).
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene.
  • heterozygous usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene segregates independently during mitosis and meiosis.
  • transgenic plant that is homozygous for the added structural gene; i.e. a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al, 1985; Lorz et al, 1985; Fromm et ⁇ /., 1985; Uchimiya et al, 1986; Callis et al, 1987; Marcotte et al, 1988).
  • DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al, 1987; Klein et al, 1988a; 1988b; McCabe et al, 1988).
  • the metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.
  • full length RNA synthesis may not occur at a high frequency. This could, for example, be caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription.
  • full length RNA may be produced in the plant cell, but then processed (splicing, polyA addition) in the nucleus in a fashion that creates a nonfunctional mRNA. If the RNA is not properly synthesized, terminated and polyadenylated, it cannot move to the cytoplasm for translation. Similarly, in the cytoplasm, if mRNAs have reduced half-lives (which are determined by their primary or secondary sequence) insufficient protein product will be produced. In addition, there is an effect, whose magnitude is uncertain, of translational efficiency on mRNA half-life.
  • RNA molecule folds into a particular structure, or perhaps family of structures, which is determined by its sequence.
  • the particular structure of any RNA might lead to greater or lesser stability in the cytoplasm. Structure per se is probably also a determinant of mRNA processing in the nucleus.
  • tRNA a determinant of mRNA processing in the nucleus.
  • tRNA RNA
  • it is likely that dramatically changing the sequence of an RNA will have a large effect on its folded structure.
  • structure per se or particular structural features also have a role in determining RNA stability.
  • researchers have identified particular sequences and signals in RNAs that have the potential for having a specific effect on RNA stability.
  • Particularly problematic sequences are those that are A+T rich.
  • native bacterial gene sequences must often be modified for optimal expression in eukaryotes, and particularly in a transformed plant.
  • Many short-lived mRNAs have A+T rich 3' untranslated regions, and these regions often have the ATTTA sequence, sometimes present in multiple copies or as multimers (e.g., ATTT ATTTA).
  • Shaw and Kamen showed that the transfer of the 3' end of an unstable mRNA to a stable RNA (globin or VA1) decreased the stable RNA's half-life dramatically.
  • This processing at the 3'-end involves cleavage of the mRNA and addition of polyA to the mature 3'-end.
  • consensus sequences that apparently are involved in polyA addition and 3'-end cleavage.
  • the same consensus sequences seem to be important to both of these processes.
  • These signals are typically a variation on the sequence AATAAA.
  • AATAAA sequence AATAAA
  • This sequence is typically found 15 to 20 bp before the polyA tract in a mature mRNA. Studies in animal cells indicate that this sequence is involved in both polyA addition and 3 '-maturation. Site-directed mutation in this sequence may disrupt these functions (Conway and Wickens, 1988; Wickens et al, 1987). However, it has also been observed that sequences up to 50 to 100 bp 3' to the putative polyA signal are also required; i.e., a gene that has a normal AATAAA but has been replaced or disrupted downstream does not get properly polyadenylated (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; McDevitt et al, 1984). That is, the polyA signal itself is not sufficient for complete and proper processing. It is not yet known what specific downstream sequences are required in addition to the polyA signal, or if there is a specific sequence that has this function. Therefore, sequence analysis can only identify potential polyA signals.
  • AATAAA is by far the most common signal identified in mRNAs upstream of the polyA, but at least four variants have also been found (Wickens and Stephenson, 1984). In plants, not nearly so much analysis has been done, but it is clear that multiple sequences similar to AATAAA can be used.
  • the plant sites in Table 4 called major or minor refer only to the study of Dean et al, (1986) which analyzed only three types of plant gene.
  • the designation of polyadenylation sites as major or minor refers only to the frequency of their occurrence as functional sites in naturally occurring genes that have been analyzed. In the case of plants this is a very limited database. It is hard to predict with any certainty that a site designated major or minor is more or less likely to function partially or completely when found in a heterologous gene such as those encoding the avirulence polypeptides of the present invention.
  • the present invention provides a method for preparing synthetic avirulence genes that express their polypeptide product at sufficiently high levels in a transformed plant, so as to bring about death of the transformed plant when infected with a viral pathogen.
  • the expression of native bacterially derived genes in plants is often problematic.
  • the nature of the coding sequences of many bacterial genes distinguishes them from plant genes as well as many other heterologous genes expressed in plants.
  • many bacterial genes may be very rich (>60%) in adenine (A) and thymine (T) residues, while most plant genes are on the order of 45-55% A+T.
  • most of the known bacterial genes which have been expressed in plants are also on the order of 40-50% A+T.
  • nucleotide sequences found in a gene from one organism where they may play no role except to code for a particular stretch of amino acids, have the potential to be recognized as gene control elements in another organism (such as transcriptional promoters or terminators, polyA addition sites, intron splice sites, or specific mRNA degradation signals). It is perhaps surprising that such misread signals are not a more common feature of heterologous gene expression, but this can be explained in part by the relatively homogeneous A+T content ( ⁇ 50%) of many organisms. This A+T content plus the nature of the genetic code put clear constraints on the likelihood of occurrence of any particular oligonucleotide sequence. Thus, a gene from E. coli with a 50% A+T content is much less likely to contain any particular A+T rich segment than a gene from an organism such as B. thuringiensis, which has a >62% A+T content.
  • structural gene that codes for the Avr/Pth polypeptides are modified by removal of ATTTA sequences and putative polyadenylation signals by site directed mutagenesis of the DNA comprising the structural gene. It is most preferred that substantially all the polyadenylation signals and ATTTA sequences are removed although enhanced expression levels are observed with only partial removal of either of the above identified sequences. Alternately if a synthetic gene is prepared which codes for the expression of the subject protein, codons are selected to avoid the ATTTA sequence and putative polyadenylation signals.
  • putative polyadenylation signals include, but are not necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA.
  • codons are preferably utilized which avoid the codons that are rarely found in plant genomes.
  • the selected DNA sequence is scanned to identify regions with greater than four consecutive adenine (A) or thymine (T) nucleotides.
  • the A+T regions are scanned for potential plant polyadenylation signals.
  • the nucleotide sequence of this region is preferably altered to remove these signals while maintaining the original encoded amino acid sequence.
  • the second step is to consider the about 15 to about 30 or so nucleotide residues surrounding the A+T rich region identified in step one. If the A+T content of the surrounding region is less than 80%, the region should be examined for polyadenylation signals. Alteration of the region based on polyadenylation signals is dependent upon (1) the number of polyadenylation signals present and (2) presence of a major plant polyadenylation signal.
  • the extended region is examined for the presence of plant polyadenylation signals.
  • the polyadenylation signals are removed by site-directed mutagenesis of the DNA sequence.
  • the extended region is also examined for multiple copies of the ATTTA sequence that are also removed by mutagenesis.
  • regions comprising many consecutive A+T bases or G+C bases are disrupted since these regions are predicted to have a higher likelihood to form hairpin structure due to self-complementarity. Therefore, insertion of heterogeneous base pairs would reduce the likelihood of self-complementary secondary structure formation that is known to inhibit transcription and/or translation in some organisms. In most cases, adverse effects may be minimized using sequences that do not contain more than five consecutive A+T or G+C residues.
  • oligonucleotides When oligonucleotides are used in the mutagenesis, it is desirable to maintain the proper amino acid sequence and reading frame, without introducing common restriction sites such as Bglll, Hindlll, Sacl, Kpnl, Ec ⁇ RI, Ncol, Pstl and Sail into the modified gene. These restriction sites are found in poly-linker insertion sites of many cloning vectors. Of course, the introduction of new polyadenylation signals, ATTTA sequences or consecutive stretches of more than five A+T or G+C, should also be avoided.
  • the preferred size for the oligonucleotides is about 40 to about 50 bases, but fragments ranging from about 18 to about 100 bases have been utilized.
  • oligonucleotides should avoid sequences longer than five base pairs A+T or G+C. Codons used in the replacement of wild- type codons should preferably avoid the TA or CG doublet wherever possible. Codons are selected from a plant preferred codon table (such as Table 5 below) so as to avoid codons which are rarely found in plant genomes, and efforts should be made to select codons to preferably adjust the G+C content to about 50%.
  • Regions with many consecutive A+T bases or G+C bases are predicted to have a higher likelihood to form hairpin structures due to self-complementarity. Disruption of these regions by the insertion of heterogeneous base pairs is preferred and should reduce the likelihood of the formation of self-complementary secondary structures such as hairpins which are known in some organisms to inhibit transcription (transcriptional terminators) and translation (attenuators).
  • a completely synthetic gene for a given amino acid sequence can be prepared, with regions of five or more consecutive A+T or G+C nucleotides being avoided. Codons are selected avoiding the TA and CG doublets in codons whenever possible. Codon usage can be normalized against a plant preferred codon usage table (such as Table 5) and the G+C content preferably adjusted to about 50%. The resulting sequence should be examined to ensure that there are minimal putative plant polyadenylation signals and ATTTA sequences. Restriction sites found in commonly used cloning vectors are also preferably avoided. However, placement of several unique restriction sites throughout the gene is useful for analysis of gene expression or construction of gene variants.
  • promoters that are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of A. tumefaciens), the Cauliflower Mosaic Virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide) and the mannopine synthase (MAS) promoter (Velten et al, 1984 and Velten and Schell, 1985). All of these promoters have been used to create various types of DNA constructs that have been expressed in plants (see e.g., Int. Pat. Appl. Publ. No. WO 84/02913).
  • NOS nopaline synthase
  • OCS octopine synthase
  • Promoters that are known or are found to cause transcription of RNA in plant cells can be used in the present invention.
  • Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the CaMV35S promoter and promoters isolated from plant genes such as ssRUBISCO genes. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of protein.
  • the promoters used in the DNA constructs (i.e. chimeric plant genes) of the present invention may be modified, if desired, to affect their control characteristics.
  • the CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots.
  • the resulting chimeric promoter may be used as described herein.
  • the phrase "CaMV35S" promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc.
  • the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression.
  • the RNA produced by a DNA construct of the present invention also contains a 5' non-translated leader sequence.
  • This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.
  • the 5' non-translated regions can also be obtained from viral RNA's, from suitable eukaryotic genes, or from a synthetic gene sequence.
  • the present invention is not limited to constructs, as presented in the following examples. Rather, the non-translated leader sequence can be part of the 5' end of the non-translated region of the coding sequence for the virus coat protein, or part of the promoter sequence, or can be derived from an unrelated promoter or coding sequence. In any case, it is preferred that the sequence flanking the initiation site conform to the translational consensus sequence rules for enhanced translation initiation reported by Kozak (1984).
  • the DNA constructs of the present invention may also contain one or more modified or fully synthetic structural coding sequences which have been changed to enhance the performance of the gene in plants.
  • the structural genes of the present invention may optionally encode a fusion protein comprising an amino-terminal chloroplast transit peptide or secretory signal sequence.
  • Transgenic plants are then regenerated from transformed embryonic calli that express the encoded polypeptide.
  • the formation of transgenic plants may also be accomplished using other methods of cell transformation that are known in the art such as Agrobacterium-mediated DNA transfer (Fraley et ⁇ l., 1983).
  • DNA can be introduced into plants by direct DNA transfer into pollen (U. S. Patent 5,629,183; Zhou et al, 1983; Hess. 1987; Luo et al, 1988), by injection of the DNA into reproductive organs of a plant (Pena et al, 1987), or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos (Neuhaus et ⁇ /., 1987; Benbrook et ⁇ /., 1986). Methods for the regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants are well known in the art (Weissbach and Weissbach, 1988).
  • This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots.
  • a transgenic plant of this invention thus has an increased amount of a coding region that encodes the avirulence/pathogenicity polypeptide of interest.
  • a preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny.
  • a more preferred transgenic plant is homozygous for that gene, and transmits that gene to each of its offspring on sexual mating.
  • Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants.
  • the progeny from these plants become true breeding lines that are evaluated for, by way of example, increased resistance to viral infection, preferably in the field, under a range of environmental conditions.
  • the inventors contemplate that the present invention will find particular utility in the creation of transgenic plants of commercial interest including various grains, grasses, fibers, tubers, legumes, ornamental plants, cacti, succulents, fruits, berries, and vegetables, as well as a number of nut- and fruit-bearing trees and plants.
  • Vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets, bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, peas, Chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach, green onions, squash, greens, sugar beets, sweet potatoes, turn
  • Fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears 5 plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blackberries, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, grapes, avocados, bananas, kiwi, organe, lemon, lime, grapefruit, persimmons, pomegranate, pineapple, mango, and other tropical fruits are also susceptible to viral pathogens.
  • One important embodiment of the invention is a recombinant vector that comprises a nucleic acid segment encoding one or more of the avrlpth genes disclosed herein.
  • a vector may be transferred to and replicated in a prokaryotic or eukaryotic host, with bacterial cells being particularly preferred as prokaryotic hosts, and plant cells being particularly preferred as eukaryotic hosts.
  • the recombinant vector comprises a nucleic acid segment encoding one or more of the Avr/Pth polypeptides disclosed in Table 9. Highly preferred nucleic acid segments are those that comprise all, or substantially all of the coding regions that encode these polypeptides.
  • GenBank accession numbers for such exemplary polynucleotides are also given in Table 9.
  • Another important embodiment of the invention is a transformed host cell that expresses one or more of these recombinant vectors.
  • the host cell may be either prokaryotic or eukaryotic, and particularly preferred host cells are those that express the nucleic acid segment(s) comprising the recombinant vector which encode one or more of the Avr/Pth polypeptides disclosed in Table 9.
  • Bacterial cells are particularly preferred as prokaryotic hosts, and plant cells are particularly preferred as eukaryotic hosts.
  • the invention encompasses a method of using a nucleic acid segment that encodes one or more of the Avr/Pth polypeptides disclosed in Table 9.
  • the method generally comprises the steps of: (a) preparing a recombinant vector in which the gene is positioned under the control of a promoter; (b) introducing the recombinant vector into a host cell; (c) culturing the host cell under conditions effective to allow expression of the protein encoded by the gene; and (d) obtaining the expressed Avr/Pth protein or peptide so produced.
  • a wide variety of ways are available for introducing a selected avrlpth gene into the microorganism host under conditions that allow for stable maintenance and expression of the gene.
  • the transcriptional initiation signals will preferably include at least a first promoter and at least a first transcriptional initiation start site.
  • it may be desirable to provide for regulative expression of the polypeptide where expression of the polypeptide will only occur after transformation into a suitable host cell, such as a transformed plant cell. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the cell comprising the nucleic acid construct.
  • a temperature sensitive regulatory region may be employed, where the cells may be cultured in the laboratory without expression of the bacterially derived avr/pth gene, but upon release into the environment, expression would begin.
  • the transcriptional and translational termination region may preferably comprise one or more stop codon(s), terminator region(s), and optionally, one or more polyadenylation signal(s).
  • a hydrophobic "leader" sequence may be employed at the amino terminus of the translated polypeptide sequence in order to promote secretion of the protein across the inner membrane.
  • the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region.
  • This sequence as a double strand may be used by itself for transformation of a selected host cell, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the Avr/Pth expression construct during introduction of the DNA into the host.
  • a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed.
  • the marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like.
  • complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field.
  • One or more markers may be employed in the development of the constructs, as well as for modifying the host.
  • the organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field.
  • the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, more preferably at least about 1000 bp, and usually not more than about 2000 bp of a sequence homologous with a sequence in the host.
  • bp basepairs
  • the transgene will be integrated into the host DNA and stably maintained by the host.
  • the transgene will be in close proximity to region of the host DNA where the integration is desired, thus providing for more efficient complementation as well permitting the stable integration of the transgene into the genome of the transformed host.
  • transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, virus and the like.
  • Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the ⁇ L and ⁇ R promoters, the tac promoter, the naturally- occurring promoters associated with the bacterial avr/pth gene, where functional in the host. See for example, U. S.
  • the termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.
  • a plasmid which has a replication system that is functional in the selected host cell.
  • the replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus that is stable in the host.
  • a large number of standard cloning/expression plasmids are available, and their use to one of skill in the molecular biological arts in the preparation of transgenes and the like are well known. See for example, Olson et al. (1982); Bagdasarian et al. (1981), Baum et al, 1990, and U. S.
  • avr/pth gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region.
  • This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host.
  • one or more markers may be present, which have been described previously.
  • the plasmid will desirably include a sequence homologous with the host genome.
  • the transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for activity. If desired, unwanted or ancillary DNA sequences may be selectively removed from the recombinant bacterium by employing site-specific recombination systems, such as those described in U. S. Patent 5,441,884 (specifically incorporated herein by reference).
  • DNA segments that are free from total genomic DNA and that comprise one or more of the avr/pth genes disclosed herein.
  • DNA segments encoding Avr/Pth polypeptide species may be obtained from native bacterial sources, or synthesized either partially or entirely in vitro using methods that are well known to those of skill in the art.
  • genes may be used that comprise all, or substantially all of a sequence that encodes an Avr/Pth polypeptide that retains its ability to produce cell death in a suitably transformed host cell, when said cell is contacted with the appropriate viral stimulus.
  • a DNA segment comprising an isolated or purified gene refers to a DNA segment which may include in addition to polypeptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, operon sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case, a gene encoding a bacterial Avr/Pth protein, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.
  • DNA sequences that encode one or more of the polypeptides disclosed in Table 9, and accordingly, sequences that have between about 70% and about 15% or between about 75% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91%) or 92% or 93% and about 97% or 98% or 99% amino acid sequence identity or functional equivalence to the amino acid sequences disclosed in Table 9 will be highly desirable sequences for use in the practice of the present invention.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences that encodes such and Avr/Pth polypeptide, so long as the sequence meets the criteria set forth above, including the maintenance of biological activity where expression of a functional polypeptide in a host cell is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • intermediate lengths means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, 24, 25, 26, 27, 28, 29, etc. ; 30, 31 , 32, 33, 34, 35, 36, etc. ; 40, 41, 42, 43, 44, etc., 50, 51, 52, 53, etc.; 60, 61, 62, 63, etc., 70, 80, 90, 100, 110, 120, 130, etc.; 200, 210, 220, 230, 240, 250, etc.; including all integers in the entire range from about 14 to about 10,000, including those integers in the ranges 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000 and the like.
  • a preferred polynucleotide will comprise a sequence of from about 1800 to about 18,000 base pair in length that encodes one or more native, mutagenized, or modified bacterially-derived Avr/Pth polypeptides, which when expressed in a transformed plant in the presence of a viral pathogen, brings about cell death and plant death to such a transformed plant, so that the spread of the viral pathogen to other nearby plants is curtailed, reduced, altered, slowed, or otherwise decreased in a manner that is desirable for protecting a crop against further viral spread.
  • DNA segments of the present invention encompass biologically functional, equivalent peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the activity of the protein, its expression, production, or persistence in a particular transformed host cell or to impart other desirable or beneficial characteristics to the mutagenized polypeptide.
  • fusion proteins and peptides e.g., where the peptide- coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • Recombinant vectors form further aspects of the present invention.
  • Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full-length protein, a substantially full-length or even a truncated or smaller polypeptide, is positioned under the control of at least a first promoter.
  • the promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology; for example, see Sambrook et al (1989).
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
  • Pichia expression vector system Pharmacia LKB Biotechnology
  • DNA segments that encode peptide antigens from about 8, 9, 10, or 11 or so amino acids, and up to and including those of about 30, 40, or 50 or so amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful.
  • the invention provides a transgenic plant having incorporated into its genome a transgene that encodes an Avr or Pth polypeptide.
  • a further aspect of the invention is a transgenic plant having incorporated into its genome a transgene that encodes such a polypeptide.
  • Other embodiments of the invention also concern the progeny of such a transgenic plant, as well as its seed, the progeny from such seeds, and seeds arising from the second and subsequent generation plants derived from such a transgenic plant.
  • the invention also discloses and claims host cells, both native, and genetically engineered, which express one or more avr/pth genes to produce the encoded polypeptide(s) in a suitably transformed host cell, and in particular, in a transformed plant cell.
  • the present invention provides methods for producing a transgenic plant that expresses such a nucleic acid segment.
  • the process of producing transgenic plants is well known in the art.
  • the method comprises transforming a suitable host cell with one or more DNA segments that contain a promoter operatively linked to a coding region that encodes one or more Avr/Pth polypeptides.
  • a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo.
  • the invention also provides for the expression of an antisense oligonucleotide or other nucleic acid sequences that are complementary to the mRNA that encodes the expressed polypeptide.
  • antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well known in the art.
  • transgenic plant is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non- transformed plant but which one desires to either genetically engineer or to have altered expression.
  • transgenic plant of the present invention will have been augmented through the stable introduction of one or more transgenes, either native, synthetically modified, or mutated.
  • more than one transgene will be incorporated into the genome of the transformed host plant cell. Such is the case when more than one DNA segment is incorporated into the genome of such a plant.
  • a preferred gene that may be introduced includes, for example, a DNA sequence from bacterial origin that encodes an avirulence/pathogenicity polypeptide, and particularly one or more of those described herein which are obtained from the species disclosed in Table 9.
  • Vectors, plasmids, cosmids, bacterial artificial chromosomes (BACs), plant artificial chromosomes (PACs), yeast artificial chromosomes (YACs), and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed Avr/Pth proteins.
  • DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences that have positively- or negatively-regulating activity upon the particular genes of interest as desired.
  • the DNA segment or gene may encode either a native or modified protein, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant
  • Such transgenic plants may be desirable for controlling the spread of a viral infection in a population of monocotyledonous or dicotyledonous plants.
  • Particularly preferred plants include grains such as corn, wheat, rye, rice, barley, and oats; legumes such as beans, soybeans; tubers such as potatoes; fiber crops such as flax and cotton; turf and pasture grasses; ornamental plants; shrubs; trees; vegetables; berries; citrus crops, including oranges, tangerines, grapefruit, limes, lemons, and the like; fruits, cacti, succulents, and other commercially-important crops including greenhouse, garden and houseplants.
  • the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and a seed produced by the progeny of the original transgenic plant, produced in accordance with the above process.
  • Such progeny and seeds will have one or more Avr/Pth-encoding transgene(s) stably incorporated into its genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding one or more Avr/Pth proteins or polypeptides are aspects of this invention.
  • genes and polypeptide-encoding DNA sequences according to the subject invention include not only full-length sequences but also fragments of these sequences, (including e.g., fusion proteins), which retain the antiviral activity of the sequences specifically exemplified herein.
  • the antiviral activity of various genetic constructs can be identified and obtained through several means.
  • the specific inducible promoter sequences, as well as the avirulence/pathogenicity genes, or portions thereof, may be obtained from a culture depository, or constructed synthetically, for example, by use of a gene machine. Variations of these genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal 1 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes or gene fragments that encode biologically active polypeptides may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these constructs.
  • Equivalent polypeptides and/or polynucleotides encoding these equivalent polypeptides can also be isolated from DNA libraries using the teachings provided herein.
  • antibodies to the polypeptides disclosed and claimed herein can be used to identify and isolate other similar or related polypeptides from a mixture of proteins. These antibodies can then be used to specifically identify equivalent polypeptides possessing the desired characteristics by a variety of methodologies including, e.g., immunoprecipitation, enzyme linked immunoassay (ELISA), and/or Western blotting.
  • ELISA enzyme linked immunoassay
  • a further method for identifying the polypeptides and polynucleotides of the subject invention is through the use of oligonucleotide probes.
  • These probes are nucleotide sequences having a detectable label.
  • the probe's detectable label provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying genes of the subject invention.
  • the nucleotide segments that are used as probes according to the invention may be synthesized by use of nucleic acid synthesizers using standard procedures.
  • the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.
  • Typical radioactive labels include 32 P, 125 1, 35 S, or the like.
  • a probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs.
  • Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives.
  • the probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
  • the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, by methods currently known to an ordinarily skilled artisan, and perhaps by other methods which may become known in the future.
  • the potential variations in the probes listed are due, in part, to the redundancy of the genetic code. Because of the redundancy of the genetic code, i.e. more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins. Therefore different nucleotide sequences can code for a particular amino acid.
  • the amino acid sequences of the disclosed polypeptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art.
  • proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984).
  • the subject invention includes mutants of the amino acid sequence depicted herein that do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained.
  • the invention also includes mutants of organisms hosting all or part of one or more of the DNA constructs of the invention. Such mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, such mutants may include asporogenous host cells that also can be prepared by procedures well known in the art.
  • Ribozymes are enzymatic RNA molecules that cleave particular mRNA species.
  • the inventors contemplate the selection and utilization of ribozymes capable of cleaving the RNA segments of the present invention, and their use to reduce activity of target mRNAs in particular cell types or tissues.
  • RNA Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
  • the enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif.
  • hammerhead motifs are described in Rossi et al, (1992); examples of hairpin motifs are described by Hampel et al, (Eur. Pat. EP 0360257), Hampel and Tritz (1989), Hampel et al, (1990) and Cech et al., (U. S.
  • ribozyme constructs need not be limited to specific motifs mentioned herein.
  • the invention provides a method for producing a class of enzymatic cleaving agents that exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.
  • Small enzymatic nucleic acid motifs e.g., ribozymes of the hammerhead or hairpin variety
  • ribozymes of the hammerhead or hairpin variety may be used for exogenous delivery into selected plant cells (see e.g., Perriman et al, 1995).
  • the simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure.
  • catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon et al, 1991; Kashani-Sabet et al, 1992; Dropulic et al, 1992; Weerasinghe et al, 1991 ; Ojwang et al, 1992; Chen et al, 1992; Sarver et al, 1990).
  • eukaryotic promoters e.g., Scanlon et al, 1991; Kashani-Sabet et al, 1992; Dropulic et al, 1992; Weerasinghe et al, 1991 ; Ojwang et al, 1992; Chen et al, 1992; Sarver et al, 1990.
  • any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector.
  • the activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper
  • WO 93/23569 and Sullivan et al, Int. Pat. Appl. Publ. No. WO 94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al, 1992; Taira et al, 1991 ; Ventura et al, 1993).
  • Hammerhead or hairpin ribozymes may be individually analyzed by computer folding (Jaeger et al, 1989) to assess whether the ribozyme sequence folds into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Ribozymes may be designed as described in Draper et al, (Int. Pat. Appl. Publ. No. WO 93/23569), or Sullivan et al, (Int. Pat. Appl. Publ. No. WO 94/02595), and may be chemically synthesized.
  • the method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al, (1987) and in Scaringe et al, (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'- end, and phosphoramidites at the 3'-end. Average stepwise coupling yields are typically >98%.
  • Hairpin ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992). Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C- allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high-pressure liquid chromatography and suspended in water. Ribozyme activity can be optimized by altering the length of the ribozyme binding arms.
  • ribozymes with modifications that prevent their degradation by serum ribonucleases may be chemically synthesized (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al.,, 1990; Pieken et al, 1991 ; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Patent 5,334,711 ; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.
  • a means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al, 1993: Zhou et al, 1990). Ribozymes expressed from such promoters can function in mammalian cells (e.g., Kashani-Saber et al, 1992; Ojwang et al, 1992; Chen et al. 1992; Yu et al, 1993; L'Huillier et al, 1992; Lisziewicz et al, 1993). Such transcription units can be incorporated into a variety of vectors for introduction into selected target cells, including but not restricted to, plasmid vectors, viral vectors and such like.
  • PNAs when delivered within cells have the potential to be general sequence-specific regulators of gene expression. Reviews of PNAs and their use as antisense and anti-gene agents exist (Nielsen et al, 1993b; Hanvey et al, 1992; and Good and Nielsen, 1997).
  • PNAs include use in DNA strand invasion (Nielsen et al, 1991), antisense inhibition (Hanvey et al, 1992), mutational analysis (Orum et al, 1993), enhancers of transcription (Mollegaard et al, 1994), nucleic acid purification (Orum et al, 1 95), isolation of transcriptionally active genes (Boffa et al, 1995), blocking of transcription factor binding (Vickers et al, 1995), genome cleavage (Veselkov et al, 1996), biosensors (Wang et al, 1996), in situ hybridization (Thisted et al, 1996), and in an alternative to Southern blotting (Perry-O'Keefe, 1996).
  • BIOLOGICAL FUNCTIONAL EQUIVALENTS Modification and changes may be made in the structure of the avirulence/pathogenicity genes, promoters, genetic constructs, plasmids, and/or polypeptides of the present invention and still obtain functional molecules that possess the desirable biologically-active characteristics.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • mutated polynucleotides and/or polypeptides are contemplated to be useful for increasing the avirulence activity of the polypeptide, and consequently increasing the activity and/or expression of the recombinant avirulence/pathogenicity transgene in a plant cell.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 6.
  • Tyrosine Tyr Y UAC UAU For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine ( ⁇ 1.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1) glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • nucleic acid segments including and not limited to mRNA and tRNA
  • nucleosides including and not limited to mRNA and tRNA
  • suitable nucleic acid segments either obtained from native sources, chemically synthesized, modified, or otherwise prepared by the hand of man.
  • Expression The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.
  • Promoter A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
  • Regeneration The process of growing a plant from a plant cell (e.g., plant protoplast or explant).
  • Structural gene A gene that is expressed to produce a polypeptide.
  • Transformation A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.
  • Transformed cell A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.
  • Transgenic cell Any cell derived or regenerated from a transformed cell or derived from a transgenic cell.
  • exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
  • Transgenic plant A plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA molecule not originally present in a native, non-transgenic plant of the same strain.
  • the terms "transgenic plant” and “transformed plant” have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast as being a transgenic plant, and that usage will be followed herein.
  • a plasmid is an exemplary vector.
  • the DNA virus used was tomato mottle geminivirus (TMoV), a bipartite geminivirus. All bipartite geminiviruses share many common features such as genome organization and replication processes. Expression of the TMoV AVI coat protein gene and BVl movement protein gene depends on the AC2 (syn. AL2 or C2) transcriptional activator gene (Abouzid et al, 1992; Sunter and Bisaro 1997). The promoters of AVI and BVl are therefore inducible. This feature allowed the engineering and expression in plants pthA and avrb ⁇ under the control of the AVI and BVl promoters.
  • TMV tomato mottle geminivirus
  • Activation of the avirulence gene due to the presence of the TMoV virus resulted in production of the avirulence protein product, which is a signal molecule that induces very rapid (hypersensitive) plant resistance response and host cell death, resulting in elimination of the virus.
  • tumefaciens GV2260 using triparental matings (Kapila et al, 1997), and inoculated into uninfected tomato and cotton, and tomato infected with TMoV. Results of the inoculations are shown in FIG. 10, FIG. 11, and Table 7. Tomato plants infected with TMoV and inoculated with either avr gene under the control of either the BIP or AIP promoters exhibited a rapid cell death characteristic of an HR (note browning in FIG. 11), while tomato plants not infected with TMoV and inoculated with the same constructs did not show an HR (FIG. 10).
  • the polynucleotides that encode aptamers can be fused with other gene fragments and can be used to transform plants and plant tissue using standard materials and methods.
  • A. tumefaciens has been widely used to transform plants with heterologous DNA (reviewed in Smith and Hood, 1995).
  • Heterologous DNA is incorporated into the Ti-plasmid or a portion of the Ti plasmid of A. tumefaciens and the plasmid is introduced back into the bacterium.
  • the plant to be transformed is then infected with bacteria, typically by inoculating a wound site on the plant or plant tissue.
  • the Ti-plasmid containing the heterologous DNA is then transferred into the nucleus of the plant cell where the transferred DNA is integrated into the host cell genome.
  • Plants and plant tissue can also be transformed using methods such as protoplast uptake of heterologous DNA (Lorz et al. 1985) and by particle bombardment using high velocity microprojectiles that have been coated with the DNA that is to be introduced into the plant (Klein et al 1988a; 1988b), a method which is particularly well suited and widely used for monocot transformation, especially transformation of the Graminae.
  • the polypeptide sequence of the aptamers is used to obtain the codon usage typical for the target plant. Tables of codon preferences by individual species may be obtained from a variety of sources. An exemplary list is found on the World Wide Web (http://www.dna.affrc.go.jp/%)7Enakamura/CUTG.html. ).
  • a methionine codon is added to the amino terminal end of the aptamer and also a "hinge" region comprised of three glycines and a serine is added to the carboxy terminus, followed by the codon adjusted coding sequence for the NH 3 -terminal end of domain II of GAL4. TABLE 7 NLS SITE-DIRECTED KNOCKOUT MUTATIONS IN PTHA
  • Plant Microbe Interact. 7(6):726-739, 1994 Pseudomonas syringae pv. phaseolicola avrPphE U16817 Mol. Plant Microbe Interact , 7(6):726-739, 1994 Pseudomonas syringae pv. pisi avrRps4 L43559 Mol. Plant Microbe Interact., 9(1):55-61, 1996 Pseudomonas syringae pv. tomato avrE U16118 Mol. Plant Microbe Interact, 8(l):49-57, 1995 Pseudomonas syringae pv. tomato avrE U16119 Mol.
  • Nucleic acids and polypeptides often carry the ability to bind other molecules with a high degree of affinity and molecular specificity similar to that exhibited by antibodies.
  • An entirely new genetic technology is developing around the ability to isolate extremely rare nucleic acid sequences with specific ligand binding properties (similar to antibodies) from very large pools of random sequences.
  • the process used is an iterative selection and amplification scheme, sometimes called SELEX (for Systematic Evolution of Ligands by Exponential Enrichment (Tuerk and Gold, 1990; Gold, 1995), and sometimes called “biopanning” (New England Biolabs).
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • Biopanning New England Biolabs
  • aptamers from the Latin aptus, to fit
  • the selected molecules with specific ligand binding properties are called "aptamers” (from the Latin aptus, to fit) (Szostak, 1992).
  • aptamers was used to describe nucleic acid molecules, it has also been applied to proteins as well (Tuerk and Gold, 1990; Colas et al, 1996). The technology had broad application to various areas of pharmaceutics as well as medical diagnostics (Gold, 1995).
  • Symptoms may be induced transiently, and without the presence of the pathogen, as illustrated in a recent publication using pthA expressed in citrus (Duan et al, 1999). Cankers on citrus are induced by pthA, blights on bean are induced by pthF and an HR on all other plants is induced by these same genes.
  • the transient expression assays were performed by cloning the avr/pth genes from X. citri and X. phaseoli on a fragment of DNA that was delivered into plant cells by particle bombardment (biolistically) using superfine tungsten particles coated with the DNA or by A. tumefaciens delivery (Duan et al, 1999).
  • transient expression assay methods reliably reproduce cankers, blight or HR without the presence of the bacterial pathogen. These methods do not permanently transform the plant, but allow the protein signals to be made transiently inside the plant cell. These methods are useful for assays of aptamer blocking proteins that cause a reduction in the timing or intensity of the plant response phenotype in these transient expression assays.
  • the transient expression assays illustrated here involve the same protocols as recently reported (Duan et al, 1999), using the DNA constructs used to test pthA, illustrated in FIG. 13.
  • the plasmids were used to express pthA and aptamers separately and simultaneously in citrus (where pthA elicits cankers) and in bean (where pthA elicits an HR).
  • Two aptamer clones are illustrated, named "HP apt" and "YP apt.”
  • RNA viruses The majority of plant viruses are RNA viruses. Most RNA viruses don't make DNA binding proteins, as DNA viruses do, but they do make unique proteins, such as the coat protein, that are not found in plants.
  • a second fusion is then constructed that comprises an aptamer that binds to a different part of the coat protein of the target virus to an activator, such as the GAL4 activator.
  • a further example of the subject invention is to create transgenic plants that carry three genetic constructs: 1) an artificial, stringently regulated promoter fused to an avr gene; 2) a constitutively expressed DNA binding protein/aptamer that binds to a DNA sequence that is part of the artificial promoter, and 3) a constitutively expressed activator/aptamer as detailed in the example above.
  • the aptamers in each case must recognize and bind the unique protein made by the virus.
  • the DNA binding protein may be artificial or natural, but it is preferably one that is not found in plants that binds to a known DNA sequence motif that is also not found in plants.
  • DNA binding protein that binds to a known DNA sequence motif that is not found in plants is the lac repressor that binds to the lac operator (Moore et al, 1998).
  • lac repressor that binds to the lac operator
  • Other bacterial promoters are also contemplated to be useful, and in certain instances the operator sequence may be fused to a minimal promoter comprising a TATA box and a transcription initiation site to form the stringently regulated, artificial promoter in a manner as described by Moore et al. (1998). As detailed herein, such a promoter is then fused to an avirulence gene such as pthA.
  • Aptamers are then selected as described above for the unique viral repressor protein, with care taken that the aptamers do not bind to plant proteins, but only to viral protein.
  • different aptamers that bind different regions of the coat protein of the virus are selected and tested pair-wise in competitive binding assays. Those that do not appear to interfere with the binding of another are selected and then sequenced as described above.
  • Translational gene fusions may be created with one member of the pair, such that when the gene encoding the fusion is constitutively expressed in the plant of interest, aptamers that recognize one part of the viral protein are attached to the DNA binding protein, such as the lac repressor.
  • the DNA sequence of the second member of the selected aptamer pair is also used to create a translational gene fusion with an activator protein, such as GAL4, exactly as described in the above example.
  • the aptamer/activator When expressed in the absence of virus in the plant, neither the aptamer/activator nor the aptamer/DNA binding protein should bind to any plant proteins, and although the aptamer/DNA binding protein should bind to the artificial promoter/avr gene, the artificial promoter/avr gene remains silent. However, upon viral infection, the viral coat protein (or other selected target protein) is made, and this binds to the aptamer/DNA binding protein that binds to the artificial promoter/ ⁇ vr gene. Also, the aptamer/activator binds to the same viral coat protein (but not the same location). This localizes the aptamer/activator in the correct position to initiate transcription of the avr gene, resulting in host cell death and limitation of the viral infection.
  • Faktor Kooter, Dixon, Lamb
  • Faktor Kooter, Dixon, Lamb
  • Plant Mol. Biol 32(5):849-859
  • Ficker Kirch, Eij lander, Jacobsen, Thompson
  • Multiple elements of the S2-RNase promoter from potato Solanum tuberosum L.
  • Flor “Inheritance of pathogenicity in Melampsora lini,” Phytopathology, 32:653-669, 1942.
  • Gallie and Young "The regulation of expression in transformed maize aleurone and endosperm protoplasts," Plant Physiol, 106:929-939, 1994.
  • Gallie, Feder, Schimke, Walbot "Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression," Mol. Gen. Genet., 228:258-264, 1991.
  • Gallie, Sleat, Turner, Wilson "Mutational analysis of the tobacco mosaic virus 5'-leader for altered ability to enhance translation," Nucl. Acids Res., 16:883-893, 1988.
  • Gallie, Sleat, Watts, Turner, Wilson "A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo," Nucl. Acids Res., 15:8693-8711, 1987b.
  • Gallie, Sleat, Watts, Turner, Wilson "The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo," Nucl. Acids
  • RNA moiety of ribonuclease P is the catalytic subunit of the enzyme," Cell, 35(3 Pt 2):849-857, 1983.
  • PNAs protein nucleic acids
  • Velten and Schell "Selection-expression plasmid vectors for use in genetic transformation of higher plants," Nucl. Acids Res., 13(19):6981-6998, 1985. Velten et al. , EMBO J. , 3 :2723-2730, 1984. Ventura, Wang, Ragot, Perricaudet, Saragosti, "Activation of HIV-specific ribozyme activity by self-cleavage," Nucl. Acids Res., 21(14):3249-3255, 1993. Verrijzer and Tjian, "TAFs mediate transcriptional activation and promoter selectivity, "
  • HIV-1 infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme
  • J. Virol, 65(10):5531-5534, 1991 conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme
  • SEQ ID NO:l is PCRTM primer DG 74: 5'-AGAATTCGGGGCATTTTTGTAATAAG-3'
  • SEQ ID NO:2 is PCRTM primer DG75: 5*-AGGATCCATTTTGAGTTAAAGAC-3'
  • SEQ ID NO:3 is PCRTM primer DG76: 5'-AGGATCCATAGTCAAACACTTAAC-3'
  • SEQ ID NO:4 is an initiation codon sequence in plants: 5'-UAAAC AAUGGCU-3 '
  • SEQ ID NO:5 is the tomato golden mosaic virus AL1 replication protein binding site: 5'-GGTAGTAAGGTAG-3'
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented are as described in the claims below.

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Abstract

L'invention concerne des procédés de lutte contre l'infection virale d'une plante, et notamment des constructions génétiques comprenant un promoteur pouvant être induit, et lié de manière fonctionnelle à un gène à pouvoir pathogène/avirulent dérivé d'une bactérie, lequel, lorsqu'il est exprimé dans une cellule végétale transformée de manière appropriée, produit la mort des cellules et rend la plante incapable de reproduire le virus, empêchant ainsi l'infection virale de se propager aux plantes voisines.
PCT/US1999/020455 1998-09-03 1999-09-03 Procedes de lutte contre les maladies virales des plantes WO2000014260A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2000023593A2 (fr) * 1998-10-16 2000-04-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
WO2009149191A2 (fr) 2008-06-03 2009-12-10 University Of Rochester Procédés de traitement de maladie intestinale inflammatoire et gestion des symptômes de celle-ci
CN101792774A (zh) * 2010-04-12 2010-08-04 浙江师范大学 农杆菌介导的盾叶薯蓣转基因方法
CN106008675A (zh) * 2009-01-12 2016-10-12 乌拉·博纳斯 模块式dna结合结构域及使用方法
CN106967798A (zh) * 2017-03-17 2017-07-21 湖南农业大学 辣椒和番茄细菌性疮痂病菌mlst分子分型方法
CN116477992A (zh) * 2023-04-23 2023-07-25 山东省博兴县薪利生物质能有限公司 一种以木醋液为原料的液体肥料及其制备方法

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WO1996036697A1 (fr) * 1995-05-18 1996-11-21 Board Of Trustees Of The University Of Kentucky Sequences et procedes de regulation de la transcription
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WO1991015585A1 (fr) * 1990-04-02 1991-10-17 Rijkslandbouwuniversiteit Wageningen Procede de protection des plantes contre les pathogenes
WO1996036697A1 (fr) * 1995-05-18 1996-11-21 Board Of Trustees Of The University Of Kentucky Sequences et procedes de regulation de la transcription
WO1996039806A1 (fr) * 1995-06-07 1996-12-19 Cleveland Clinic Foundation Plantes transgeniques exprimant conjointement un systeme fonctionnel 2-5a humain

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TOBIAS,C.M., ET AL.: "plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto", THE PLANT JOURNAL, vol. 17, no. 1, January 1999 (1999-01-01), pages 41 - 50, XP002127994 *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023593A2 (fr) * 1998-10-16 2000-04-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
WO2000023593A3 (fr) * 1998-10-16 2000-07-27 Fraunhofer Ges Forschung Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
WO2009149191A2 (fr) 2008-06-03 2009-12-10 University Of Rochester Procédés de traitement de maladie intestinale inflammatoire et gestion des symptômes de celle-ci
EP2296683A2 (fr) * 2008-06-03 2011-03-23 University Of Rochester Procédés de traitement de maladie intestinale inflammatoire et gestion des symptômes de celle-ci
EP2296683A4 (fr) * 2008-06-03 2012-03-07 Univ Rochester Procédés de traitement de maladie intestinale inflammatoire et gestion des symptômes de celle-ci
US8703152B2 (en) 2008-06-03 2014-04-22 University Of Rochester Methods of treating inflammatory intestinal disease and managing symptoms thereof
CN106008675A (zh) * 2009-01-12 2016-10-12 乌拉·博纳斯 模块式dna结合结构域及使用方法
CN106008675B (zh) * 2009-01-12 2021-03-09 乌拉·博纳斯 模块式dna结合结构域及使用方法
CN101792774A (zh) * 2010-04-12 2010-08-04 浙江师范大学 农杆菌介导的盾叶薯蓣转基因方法
CN106967798A (zh) * 2017-03-17 2017-07-21 湖南农业大学 辣椒和番茄细菌性疮痂病菌mlst分子分型方法
CN106967798B (zh) * 2017-03-17 2021-11-26 湖南农业大学 辣椒和番茄细菌性疮痂病菌mlst分子分型方法
CN116477992A (zh) * 2023-04-23 2023-07-25 山东省博兴县薪利生物质能有限公司 一种以木醋液为原料的液体肥料及其制备方法

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