WO2000014260A1 - Methods for controlling viral diseases in plants - Google Patents

Abstract

Disclosed are methods for controlling a viral infection in a plant. In particular, genetic constructs are provided comprising an inducible promoter operably linked to a bacterially-derived avirulence/pathogenicity gene, that, when expressed in a suitably-transformed plant cell, produces cell death and renders the plant unable to reproduce the virus, thus preventing spread of the viral infection to nearby plants.

Description

DESCRIPTION

METHODS FOR CONTROLLING VIRAL DISEASES IN PLANTS

1.0 BACKGROUND OF THE INVENTION

The present application claims priority from U. S. Provisional Application Serial No.

60/099,033, filed September 3, 1998; the entire contents of which is specifically incorporated herein by reference in its entirety. The United States government has certain rights to the present invention pursuant to Grant Number 96-35303-3254 from the Department of Agriculture.

1.1 FIELD OF THE I VENTION

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.

1.2 DESCRIPTION OF RELATED ART Avirulence (avr) genes are of microbial origin and induce a rapid plant cell death response (a hypersensitive response or HR) when expressed inside most nonhost plant cells (DeFeyter et al, 1998; Gopalan et al, 1996; Scofield et al, 1996; Tang et al, 1996; Van den Ackerveken et al, 1996). avr genes have been demonstrated in many different microbial plant pathogens, avr genes were originally described in 1942 (Flor, 1942) and have been identified in at least 27 different plant host/parasite systems, including diseases caused by rusts, smuts, bunts, mildews, scabs, nematodes, insects, bacteria, viruses, and even by other plants (Day, 1974). Race variation in microbial pathogenic species or "pathovar" (pathogenic variant) is determined by avr genes, which act as negative factors to limit the growth of strains within a pathovar or species to a subset of hosts within the range of the pathovar or species.

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 (αvr-for-R) specificity (for reviews, see Gabriel and Rolfe, 1990; Keen, 1992). It has historically been recognized that avr genes require specific plant resistance (R) genes to function for avirulence, and their role in eliciting the nonhost HR is unknown in most cases. In fact, the 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. Patent 5,859,324; U. S. Patent 5,858,786; U. S. Patent 5,850,015; U. S. Patent 5,849,868; U. S. Patent 5,776,889; U. S. Patent 5,708,139; U. S. Patent 5,650,387; Wei et al, 1992; He et al, 1993, each of which is specifically incorporated herein by reference in its entirety). 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). An entire family of avr genes has 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 avrbό (Gabriel, 1997). Analyses (Yang and Gabriel, 1995a) of the published gene sequences and the predicted amino acid sequences encoded by avrbό. 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. 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.

1.3 DEFICIENCIES IN THE PRIOR ART

Plant viruses do not generally trigger wound responses, nor activate plant promoters. Therefore, prior methods designed to control microbial pathogens are not useful in controlling plant viruses. In addition, the requirement to provide expression of a cognate plant R gene is not part of the present invention.

2.0 SUMMARY OF THE INVENTION 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. Exemplary 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-spotting badnavirus, Rice tungro bacilliform badnavirus, Schefflera ringspot badnavirus, Sugarcane bacilliform badnavirus, Abutilon mosaic bigemini virus, Ageratum yellow vein bigeminivirus, Bean calico mosaic bigeminivirus, Bean golden mosaic bigeminivirus, Bhendi yellow vein mosaic bigeminivirus, Cassava African mosaic bigeminivirus, Cassava Indian mosaic bigeminivirus, Chino del tomate bigeminivirus, Cotton leaf crumple bigeminivirus, Cotton leaf curl bigeminivirus, Croton yellow vein mosaic bigeminivirus, Dolichos yellow mosaic bigeminivirus, Euphorbia mosaic bigeminivirus, Horsegram yellow mosaic bigeminivirus, Jatropha mosaic bigeminivirus, Lima bean golden mosaic bigeminivirus, Melon leaf curl bigeminivirus, Mung bean yellow mosaic bigeminivirus, Okra leaf-curl bigeminivirus, Pepper hausteco bigeminivirus, Pepper Texas bigeminivirus, Potato yellow mosaic bigeminivirus, Rhynchosia mosaic bigeminivirus, Serrano golden mosaic bigeminivirus, Squash leaf curl bigeminivirus, Tobacco leaf curl bigeminivirus, Tomato Australian leafcurl bigeminivirus, Tomato golden mosaic bigeminivirus, Tomato Indian leafcurl bigeminivirus, Tomato leaf crumple bigeminivirus, Tomato mottle bigeminivirus, Tomato yellow leaf curl bigeminivirus, Tomato yellow mosaic bigeminivirus, Watermelon chlorotic stunt bigeminivirus, Watermelon curly mottle bigeminivirus, Beet curly top hybrigeminivirus, Chloris striate mosaic monogeminivirus, Digitaria striate mosaic monogeminivirus, Digitaria streak monogeminivirus, Maize streak monogeminivirus, Miscanthus streak monogeminivirus, Panicum streak monogeminivirus, Paspalum striate mosaic monogeminivirus, Sugarcane streak monogeminivirus, Tobacco yellow dwarf monogeminivirus, and Wheat dwarf monogeminivirus.

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. Preferably, the nucleic acid segment comprised with this genetic construct encodes all, or substantially all of a bacterial Avr/Pth polypeptide. In certain embodiments, the nucleic acid segment will comprise all, or substantially all of a gene that encodes such a polypeptide. Exemplary 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, avrbό, 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, avrPpiBl.R3, avrPpiC, avrPpiD.R5, avrPpiE, avrPmaAl, avrD, avrRpt2, avr P to, avrE, avr A, avrB, avrC and hrpN. 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.

Preferably the gene is operably linked to a promoter that is transcriptionally activated by a viral polypeptide. Exemplary 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. In one illustrative embodiment, 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. In this way, when a single plant in a transgenic plant population is infected by a pathogenic virus, 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. Thus, 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.

In certain embodiments, 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. As described above, 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. 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. Such expression brings about rapid cell death of the host cell, and, if the host cells are present in a plant, leads to decline and death of the entire plant itself. This programmed "suicide" of the infected plant provides a novel control mechanism for reducing or limited the spread of viral diseases in susceptible plants. 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. Therefore, 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". By "leaky" is meant having a low level of constitutive transcriptional activity in the plant, without promoter activation by the viral activator protein.

In another embodiment, 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. Such binding, however, 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. In an illustrative embodiment, 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. Such a recombinant vector may then be introduced into a target plant cell (as described above for the promoter/avirulence gene construct) thereby providing to the cell a two-component genetic construct: (a) a first construct comprising a virally-inducible promoter operably to a bacterially derived avirulence gene, and (b) a second construct comprising a constitutive promoter operably linked to a nucleic acid sequence that encodes an aptamer peptide sequence operably linked to a DNA binding protein sequence. Such constructs may be provided on a single polynucleotide segment, or alternatively, the genetic constructs may be provided to a host cell on distinct polynucleotide segments. For genomic integration of such segments, the constructs may be provided to a cell on a single vector, or alternatively, provided to the cell on distinct vectors. In a preferred embodiment, the present invention relates to a recombinant vector that comprises a constitutive promoter operatively linked to a gene fusion comprising an aptamer coding sequence translationally-fused to a transcriptional activator coding sequence such that upon expression of the gene, a hybrid protein is produced comprising the aptamer fused with the transcriptional activator. Said aptamer binds to a viral-encoded protein. Also provided are methods for the construction of synthetic genes encoding protein aptamers (selected such that they bind to specific viral repressor proteins or other proteins) fused with transcriptional activators to provide RNA polymerase II-based transcription at the TATA box. For example, one such activator is the transcription activation domain of Gal4.

A further embodiment of the invention concerns construction of artificial promoters that include DNA aptamer sequences upstream of the TATA box of a minimal promoter, such that the DNA aptamer sequence recognizes and binds to any viral protein, much as a repressor protein binds to target DNA. A further aspect of the present invention provides a method of expressing an avr gene- containing nucleic acid segment in a cell. The method generally involves transforming said cell with a vector comprising an avr gene-containing nucleic acid segment operatively linked to an inducible promoter and culturing the cell under conditions effective to express the avr gene from the promoter.

Preferably, the cell is a plant cell and, in particular, a monocotyledonous or dicotyledonous plant cell. Of course, in certain embodiments, particularly in the preparation of recombinant vectors and the like, it may be desirable to prepare the constructs of the present invention for use in bacterial cells, such as E. coli, or in yeast. In a preferred embodiment, the present invention relates to a recombinant vector comprising an inducible promoter operatively linked to an avirulence gene-containing nucleic acid segment, in such an orientation as to control expression of said segment. 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.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

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. 3 illustrates suppression by aptamers of the citrus canker phenotype normally elicited by pthA in citrus leaves two weeks after inoculation using A. tumefaciens GV2260/pYD40.1 (top left). Note the nearly complete loss of yellow canker symptoms in the leaves inoculated with GV2260/pGZ8.1 (bottom left; labeled YP aptamer) or with GV2260/pGZ8.3 (right middle; labeled HP aptamer). The rounded, raised areas of the leaf tissue are due to the inoculation method. Inoculation was by syringe infiltration as described in Yang and Gabriel, 1994. FIG. 4 illustrates the HR normally elicited on tobacco by expression of pthA is blocked by transgenic tobacco strongly expressing YΫv.uidA. The control leaf to the left is inoculated with tumefaciens-delivered pthA, which is transiently expressed (left side) and with A. tumefaciens-deliveτed vector only (right side). Note that transiently expressed pthA elicits a strong HR by 48 hours after inoculation. The transgenic leaf to the right is identically inoculated. Note that transiently expressed pthA does not elicit an HR 48 hours after inoculation. Expression oϊYVv.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.

FIG. 6 shows a schematic illustration of a promoter/αvr 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/αvr 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.

SUBSTITUTESHEET RtJLE 26 FIG. 8 shows a schematic illustration of a promoter/αvr 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. Upon binding, the Activator portion of the protein is in a position to activate transcription of the αvr gene, which results in host cell death and the HR. Note that the Promoter/αvr 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 . Nos: nopaline synthase; S: translational enhancer region from tobacco mosaic virus.

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).

FIG. 12 shows the general structural features of the polynucleotide encoding the PthA polypeptide. aa, amino acid; LZ, leucine zipper; NLSs, nuclear localization signal sequences; TA, transcriptional activator. (From 14-23, 102 bp, leucine-rich tandem repeats - determine specificity and plant response phenotype). FIG. 13 illustrates DNA constructions used in the transient expression assays. The plasmids were delivered into plant cells via Agrobacterium-mediated transfer. The left and right border sequences of T-DNA are represented by T_L and T_R. Nos, Nopaline Synthase. S, translational enhancer region from tobacco mosaic virus. uidA is a Gus reporter gene. αvrb<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.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides methods for the prevention, alteration, or amelioration of viral infection and disease in selected plants. In particular, 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. In this method, 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.

4.1 SOME A VR/PTH GENES ELICIT PLANT CELL DEATH IN A WIDE RANGE OF PLANT SPECIES

Although eliciting symptoms often seems irrelevant or even counterproductive to a parasitic lifestyle, ability to elicit certain symptoms greatly enhances the growth rate, fitness or epidemiological spread of pathogens. For example, cankers rupture the epidermal surface of citrus, resulting in release of X. citri (Swarup et al, 1991), and blights can cause increased release of Xanthomonas to the leaf surface as well (Yang et al, 1994). Release of these pathogens is critical for epidemic spread. Despite the severity of the plant diseases caused by the xanthomonads that cause cankers and blights, these pathogens are highly restricted in host range. X. citri causes cankers only on citrus; X. phaseoli causes blight only on bean, and X. oryzae causes blight only on rice.

The specificity of these pathogens for target plant hosts is due in large part to each pathogen's ability to inject a single pathogenicity (Pth) protein signal into almost any plant cell that it contacts, and it is that protein signal alone that causes the primary disease symptoms (Duan et al, 1999). Although the 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. Surprisingly, 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. In 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. 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. In fact, 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.

Since pthA expression inside plant cells is sufficient to cause citrus canker disease, what is the role of the pathogen? For either pathogenicity or avirulence phenotypes to be elicited by most bacterial plant pathogens, 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.

Since the citrus canker phenotype could be elicited by expression of pthA in host cells, it indicated that other diseases associated with other members of the avrBs3/pthA family might also be elicited by transient expression in host cells (Gabriel, 1997). This was directly tested by cloning gene pthF from X. phaseoli (92% identical based on partial DNA sequence), and transiently expressing this gene in bean. pthF elicited blight symptoms on bean, and an HR on other plants. More importantly, the results with pthF indicated that blight symptoms, as with canker symptoms, can be caused by expression of a single pth gene in plants. By extension, rice blight symptoms are known to be enhanced by avrXa7, another known member of this same gene family (Leach and White, 1996). Once it was understood that all members of this avr/pth gene family encoded nearly identical proteins, it became clear that any one of them may potentially be used to elicit hypersensitive cell death in a wide range of plant species.

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. In addition, 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. These proteins alone are likely sufficient signals for the primary symptoms of each disease. Once it was understood that all of the members of this avr/pth gene family encoded nearly identical proteins, it became clear that if the function of these essential, disease-causing, effector proteins could be blocked, disease control might be achieved.

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.

All eukaryotic cells must have a means to respond to external stimuli. It is generally accepted that this control is achieved at the transcription level (Verrijzer and Tjian, 1996). Transcriptional control begins with a highly conserved, almost ubiquitous structural feature of eukaryotic promoters called a TATA box (Lewin, 1997). Much of the transcriptional machinery ("the basal apparatus") required for initiating transcription sits poised on the TATA box, awaiting activation. Without activation, the gene is not transcribed or transcribed at such low levels as to be undetectable in vivo (i.e. it is "silent"). 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. Although natural viral genomes do not provide such gene fusions, 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). The selected molecules with specific ligand binding properties are called "aptamers" (from the Latin aptus, meaning "to fit") (see e.g., Szostak, 1992). Although originally the term 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. One large group of plant DNA viruses is the 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). Additionally, 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). Finally, 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. In many cases, 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.

Many, if not most, 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. For example, pthA (GenBank Accession No. U28802) was cloned from the citrus canker pathogen X. citri, and is critical for pathogenicity on citrus; however, when expressed inside plant cells other than citrus, it induces a rapid host cell death or HR (FIG. 5). Microbial avr genes that cause a rapid host cell death or HR when expressed inside plant cells are another key component of the subject invention.

In an overall and general sense, 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).

Since only a small amount of the Avr polypeptide is needed to elicit such cell death either the promoter must be stringently regulated, the Avr polypeptide must be expressed at low levels or the Avr polypeptide must be mutated to weaken its effects. For example, site- directed mutations in one or more nuclear localizing signal sequences (NLSs) present on pthA have been developed that quantitatively decreased the nonhost HR reaction (see FIG. 7 and Table 7). PCR primers were used to create site-directed mutations that eliminated each of the three NLSs individually in bothpthA and avrbό. The three NLSs, named in order towards the C terminus, are nlsA, nlsB and nlsC. 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. In each case, 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. tumefaciens strain GV2260 using triparental matings as described (Kapila et al, 1997), and inoculated into nonhost bean and cotton plants. 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. For example, 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). However, 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. The term "aptamer" as used herein refers to a nucleic acid or polypeptide having the ability to bind with a high degree of affinity and specificity to a target polypeptide molecule. Methods of obtaining aptamers are known in the art. For example, methods include iterative selection and amplification, sometimes called "biopanning". In biopanning, a random aptamer library consisting of eight amino acids is literally displayed externally on the coat protein of Ml 3 phage (New England Biolabs). The basic process is to screen a random pool of aptamers in a large "library" for those sequences that stick to a target molecule affixed to a substrate. Then wash away everything that does not stick and then amplify the sequences that do stick. (For example, allow the Ml 3 phage to replicate after panning). Then repeat, this time using the amplified library that is enriched with sequences that stuck to the target the first time around. Aptamers may be selected by screening the entire target protein, such as AL1, or only a portion of AL1 that is expected to be exposed after AL1 binds to its DNA target.

In one embodiment of this technology, 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. Alternatively, short fragments may be synthesized without use of a MAP resin. In a second embodiment of this technology, 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. In 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 NH₃ -terminal end of domain II of GAL4.

The majority of plant viruses are not DNA viruses, but rather RNA viruses. Most 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). There are other bacterial promoters that would likely work as well. 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). As detailed in previous example using a repressor, 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. In a preferred embodiment, different aptamers that bind different regions of the coat protein of the virus (whether RNA or DNA) 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 are then 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. 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/αvr 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/αvr 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.

It may be desirable to verify that plants transformed with the synthetic promoter fused with the avr gene are not damaged in any way by the construct or the transformation process, and it may also be important to verify that the synthetic promoter is mostly, or absolutely, silent. Therefore, the synthetic promoter/ovr gene fusion is transformed into one parental line, while the Aptamer/Activator and or Aptamer/DNA binding protein constructs are engineered into a second parental line. Likewise, various methods may be utilized to verify that the Aptamer/Activator and Aptamer/DNA binding protein constructs are expressed, and that plants transformed with the construct are not damaged in any way by the construct or the transformation process. It is desirable that both constructs are stably inherited and present as a single copy in each plant line.

Another aspect of the invention is realized when the two parental lines are crossed to produce FI plants. At this point, F2 plants may be selected in e.g., self-fertile plant systems for true breeding plants homozygous for both engineered traits. As such, 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. Additionally, 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/αvr 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.

4.2 PLANT DNA VIRUSES

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.

In one particular example, 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. 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.

4.3 RECOMBINANT HOST CELLS

The nucleotide sequences of the subject invention can introduce an avirulence gene- inducible promoter construct of interest into a wide variety of microbial and eukaryotic hosts. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. 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. Among eukaryotes are 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. 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.

A large number of 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. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Bacillus (including the species and subspecies B. thuringiensis v. kurstaki HD-1,

B. thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensis v. ber liner, B. thuringiensis v. thuringiensis, B. thuringiensis v. tolworthi, B. thuringiensis v. dendrolimus, B. thuringiensis v. alesti, B. thuringiensis v. galleriae, B. thuringiensis v. aizawai, B. thuringiensis v. subtoxicus, B. thuringiensis v. entomocidus, B. thuringiensis v. tenebrionis and B. thuringiensis v. san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,

Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and

Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,

Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium

Of particular interest are such phytosphere bacterial species as 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

X. campestris; Rhizobium spp. including R. melioti; Alcaligenes spp. including A. eutrophus; and Azotobacter spp. including A. vinlandii. Also of particular interest are the phytosphere fungal species, and in particular, the yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. difβuens, C. laurentii, Saccharomyces rosei,

S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and

Aureobasidium pollulans.

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. 4.4 POLYNUCLEOTIDE SEQUENCES

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. For example, 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.

Vectors, plasmids, phagemids, cosmids, viral vectors, shuttle vectors, baculovirus vectors, BACs (bacterial artificial chromosomes), PACs (plant artificial chromosomes), YACs (yeast artificial chromosomes) and DNA segments for use in transforming cells with a nucleic acid construct of interest, will, of course, generally comprise at least one inducible promoter disclosed herein, or alternatively, will comprise at least one or more promoters which have at least about 80% or 85% or 90% or 95% sequence identity to the promoters disclosed herein. These DNA constructs may contain a cDNA, or one or more genes which one desires to introduce into the cells. These 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. Alternatively, the nucleic acid constructs may contain antisense constructs, or ribozyme-encoding regions when delivery or introduction of such nucleic acid constructs is desirable.

4.4.1 METHODS FOR PREPARING MUTAGENIZED POLYNUCLEOTIDES

In certain circumstances, it may be desirable to modify or alter one or more nucleotides in one or more of the promoter sequences disclosed herein for the purpose of altering or changing the transcriptional activity or other property of the promoter region. In general, 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. Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular promoter region. In particular, site-specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. 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. Typically, 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. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, 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.

In general, 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. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. 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. A genetic selection scheme has been devised to enrich for clones incorporating the mutagenic oligonucleotide (Kunkel et al, 1987). Alternatively, the use of PCR™ 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. The PCR™-mediated mutagenesis procedures of Tomic et al, (1990) and Upender et al, (1995) provide two examples of such protocols. A PCR™ employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected promoter-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of DNA sequences may be obtained. For example, recombinant vectors encoding the desired promoter sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation that result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" also is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well- known rules of complementary base pairing (Watson, 1987). Typically, 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).

A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U. S. Patents 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, 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. By raising and lowering the temperature of the reaction mixture, 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. Preferably a reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U. S. Patent 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase (QβR) 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. In this method, 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 (SDA) 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. A similar approach is used in SDA. Still other amplification methods have been described (British Patent No. 2202328;

Intl. Pat. Appl. Publ. No. PCT/US89/01025, each of which is specifically incorporated herein by reference in its entirety). These amplification methods may also be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR™ like, template and enzyme dependent synthesis. These primers may be modified by labeling with a capture moiety (e.g., biotin), and/or a detector moiety (e.g., an enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact and may then be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence. Other 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. In NASBA, 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. These amplification techniques involve annealing a primer that has sequences specific for the target gene sequence to be modified. Following polymerization, 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. In an isothermal cyclic reaction, 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.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). 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). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" (Frohman, 1990), and "one-sided PCR™" (Ohara, 1989) which are well known to those of skill in the art.

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu and Dean, 1996, incorporated herein by reference in its entirety), may also be used in the amplification of DNA sequences of the present invention. 4.4.2 REGULATORY ELEMENTS

Preferred genetic constructs will include at least a first promoter-avirulence gene polynucleotide composition of the present invention, and may optionally include one or more 3'-end polynucleotide sequences that acts as a signal to terminate transcription and allow for the poly-adenylation of the resultant mRNA.

As the DNA sequence between the transcription initiation site and the start of the coding sequence, i.e. the untranslated leader sequence, can influence gene expression, one may also wish to employ particular leader sequences, in addition to the described "operator" motifs and/or DNA binding motifs that are critical parts of the described invention. Preferred 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. The choice of such 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.

4.5 POST-TRANSCRIPTIONAL EVENTS AFFECTING EXPRESSION OF TRANSGENES IN PLANTS In many instances, 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 inventors contemplate that in certain instances it may be desirable to alter the transcription and/or expression of the avirulence gene constructs of the present invention to increase, decrease, or otherwise regulate or control these constructs in particular host cells and/or transgenic plants. 4.5.1 EFFICIENT INITIATION OF PROTEIN TRANSLATION

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.

In most eukaryotic mRNAs, the point of translational initiation occurs at the AUG codon closest to the 5' cap of the transcript. Comparison of plant mRNA sequences and site directed mutagenesis experiments have demonstrated the existence of a consensus sequence surrounding the initiation codon in plants, 5'-UAAACAAUGGCU-3' (SEQ ID NO:4) (Joshi, 1987; Lutcke et al, 1987). However, consensus sequences will be apparent amongst individual plant species. For example, a compilation of sequences surrounding the initiation codon from 85 maize genes yields a consensus of 5'-(C/G)AUGGCG-3' (Luehrsen et al, 1994). In tobacco protoplasts, transgenes encoding β-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). When producing chimeric transgenes (i.e. transgenes comprising DNA segments from different sources operably linked together), often the 5^'-UTL of plant viruses is used. The 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 (Ω) 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. The 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-Ω (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). However, 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).

These 5 '-UTLs had different effects in tobacco. In contrast to maize, the TMV Ω 5'- UTL and the AMV coat protein 5'-UTL enhanced expression in tobacco, whereas the glutelin, maize PEP-carboxylase and maize ribulose- 1,5-bisphosphate carboxylase 5'-UTLs did not show enhancement relative to the native luciferase 5^'-UTL (Koziel et al, 1996). Only the CaMV 35S 5'-UTL region enhanced luciferase expression in both maize and tobacco (Koziel et al, 1996). Furthermore, the TMV and BMV coat protein 5'-UTLs were inhibitory in both maize and tobacco protoplasts (Koziel et al, 1996).

4.5.2 USE OF INTRONS TO INCREASE EXPRESSION

Including one or more 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. Likewise, the first intron of the alcohol dehydrogenase 1 (Adhl) has been shown to increase expression of a genomic clone of Adhl comprising the endogenous promoter in transformed maize cells (Callis et al, 1987; Dennis et al, 1984). Other introns that are also able to increase expression of transgenes which contain them include 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 α/„ 1992).

Generally, to achieve optimal expression, 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).

4.5.3 USE OF SYNTHETIC GENES TO INCREASE EXPRESSION OF HETEROLOGOUS GENES IN PLANTS When introducing a prokaryotic gene into a eukaryotic host, or when expressing a eukaryotic gene in a non-native host, the sequence of the gene must often be altered or modified to allow efficient translation of the transcript(s) derived from the gene. Significant experience in using synthetic genes to increase expression of a desired protein has been achieved in the expression of B. thuringiensis-devived genes in plants. Native B. thuringiensis genes are expressed only at low levels in dicots and not at all in monocots (Koziel et al, 1996). 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. For example, synthetic B. thuringiensis δ-endotoxin encoding genes have resulted in significant improvements in expression of the δ-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).

In a similar fashion the inventors contemplate that the genetic constructs of the present invention, because they comprise at least one avirulence or pathogenicity gene of bacterial origin, 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. Using 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.

4.5.4 CHLOROPLAST SEQUESTERING AND TARGETING

Another approach for increasing expression of A+T rich genes in plants has been demonstrated in tobacco chloroplast transformation. High-level expression of an unmodified B. thuringiensis δ-endotoxin gene in tobacco has been reported by McBride et al. (1995).

Additionally, methods of targeting proteins to the chloroplast have been developed. This technique, utilizing the pea chloroplast transit peptide, has been used to target the enzymes of the polyhydroxybutyrate synthesis pathway to the chloroplast (Nawrath et al, 1994). Also, this technique negated the necessity of modification of the coding region other than to add an appropriate targeting sequence.

U. S. Patent 5,576,198 (specifically incorporated herein by reference) 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. Also included are 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.

4.5.5 EFFECTS OF 3' REGIONS ON TRANSGENE EXPRESSION The 3'-end regions of transgenes have been found to have a large effect on transgene expression in plants (Ingelbrecht et al, 1989). In this study, different 3' ends were operably linked to the neomycin phosphotransferase II (Nptll) reporter gene and expressed in transgenic tobacco. The different 3' ends used were obtained from the octopine synthase gene, the 2S seed protein from Arabidopsis, the small subunit of rbcS from Arabidopsis, extension form carrot, and chalcone synthase from Antirrhinum. In stable tobacco transformants, there was about a 60-fold difference between the best-expressing construct (small subunit rbcS 3' end) and the lowest expressing construct (shalcone synthase 3' end). TABLE 1 PLANT PROMOTERS

Promoter Reference"

Viral

Figwort Mosaic Virus (FMV) U. S. Patent 5,378,619

Cauliflower Mosaic Virus (CaMV) U. S. Patent 5,530,196

U. S. Patent 5,097,025

U. S. Patent 5,110,732

Plant

Elongation Factor U. S. Patent 5,177,011

Tomato Polygalacturonase U. S. Patent 5,442,052

Arabidopsis Histone H4 U. S. Patent 5,491,288

Phaseolin U. S. Patent 5,504,200

Group 2 U. S. Patent 5,608,144

Ubiquitin U. S. Patent 5,614,399

P119 U. S. Patent 5,633,440 α-amylase U. S. Patent 5,712,112

Wheat starch branching enzyme U. S. Patent 5,866,793

Osmotin U. S. Patent 5,874,626

Viral enhancer Plant promoter

CaMV 35S enhancer/mannopine synthase promoter U. S. Patent 5,106,739

Εach reference is specifically incorporated herein by reference in its entirety.

TABLE 2 TISSUE SPECIFIC PLANT PROMOTERS

Tissue Specific Tissue(s) Reference³

Promoter

Blec Epidermis U. S. Patent 5,646,333

Malate synthase Seeds; seedlings U. S. Patent 5,689,040

Isocitrate lyase Seeds; seedlings U. S. Patent 5,689,040

Patatin Tuber U. S. Patent 5,436,393

ZRP2 Root U. S. Patent 5,633,363

ZRP2(2.0) Root U. S. Patent 5,633,363

ZRP2(1.0) Root U. S. Patent 5,633,363

RB7 Root U. S. Patent 5,459,252

Root U. S. Patent 5,401,836

Fruit U. S. Patent 4,943,674

Meristem U. S. Patent 5,589,583

Guard cell U. S. Patent 5,538,879

Stamen U. S. Patent 5,589,610

SodAl Pollen; middle layer; Van Camp et al, 1996 stomium of anthers

SodA2 Vascular bundles; Van Camp et l, 1996 stomata; axillary buds; pericycle; stomium; pollen

CHS 15 Flowers; root tips Faktor et α/., 1996

Psam-1 Phloem tissue; cortex; Vander et α/., 1996 root tips

ACT1 1 Elongating tissues i and Huang et al, 1997 organs; pollen; ovules

ZmGBS Pollen; endosperm Russell and Fromm, 1997 zmZ27 Endosperm Russell and Fromm, 1997

OsAGP Endosperm Russell and Fromm, 1997 osGTl Endosperm Russell and Fromm, 1997 Tissue Specific Tissue(s) Reference"

Promoter

RolC Phloem tissue; bundle Graham et al, 1997 sheath; vascular parenchyma

Sh Phloem tissue Graham et al, 1997

CMd Endosperm Grosset et al, 1997

Bnml Pollen Treacy et al, 1997 rice tungro Phloem Yin et al, 1997a; 1997b bacilliform virus

S2-RNase Pollen Ficker et α/., 1998

LeB4 Seeds Baumlein et α/., 1991 gf-2.8 Seeds; seedlings Berna and Bernier, 1997

"Each reference is specifically incorporated herein by reference in its entirety.

The ability to express genes in a tissue specific manner in plants has led to the production of male and female sterile plants. Generally, the production of male sterile plants involves the use of anther-specific promoters operably linked to heterologous genes that disrupt pollen formation (U. S. Patents 5,689,051; 5,689,049; 5,659,124, each specifically incorporated herein by reference). U. S. Patent 5,633,441 (specifically incorporated herein by reference) discloses a method of producing plants with female genetic sterility. The method comprises the use of style-cell, stigma-cell, or style- and stigma-cell specific promoters that express polypeptides that, when produced in the cells of the plant kill or significantly disturbs the metabolism, functioning or development of the cells.

TABLE 3 INDUCIBLE PLANT PROMOTERS Promoter Reference"

Heat shock promoter U. S. Patent 5,447,858

Em U. S. Patent 5,139,954

Adhl Kyozuka et al, 1991

HMG2 U. S. Patent 5,689,056

Cinnamyl alcohol dehydrogenase U. S. Patent 5,633,439

Asparagine synthase U. S. Patent 5,595,896

GST-II-27 U. S. Patent 5,589,614

"Each reference is specifically incorporated herein by reference in its entirety.

4.6 EXPRESSION VECTORS

The present invention also provides an expression vector comprising at least one avirulence/pathogenicity gene-containing polynucleotide operably linked to an inducible promoter. Thus, in one embodiment 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.

In another embodiment, 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. As used herein, the term "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.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depend directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the functional RNA to which it is operatively linked. 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).

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.

4.7 DNA SEGMENTS AS HYBRIDIZATION PROBES AND PRIMERS

In another aspect, 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. Most importantly, 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.

In certain embodiments, 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 PCR™ technology. Segments of related avirulence/pathogenicity genes from other species may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the present invention, 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. One will generally prefer to design nucleic acid molecules having gene- complementary stretches of 14 to 20 nucleotides, or even longer where desired. 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 PCR™ 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.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate avirulence/pathogenicity gene sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In addition to the use in directing the expression of functional RNA of the present invention, the 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. Longer contiguous identical or complementary sequences, e.g., those of about 20, 21, 22, 23, 24, etc., 30, 31, 32, 33, 34, etc., 40, 41, 42, 43, 44, etc., 50, 51, 52, 53, 54, etc., 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including all intermediate lengths and up to and including full-length sequences will also be of use in certain embodiments. 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.

The use of a 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. One will generally prefer to design nucleic acid molecules having gene- complementary stretches of about 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more contiguous nucleotides in length where desired. Of course, 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 PCR™ 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.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one may desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, 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. Detection of DNA segments via hybridization is well known to those of skill in the art, and the teachings of U. S. Patent 4,965,188 and U. S. Patent 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al, 1994; Segal 1976; Prokop and Bajpai, 1991; and Kuby, 1994, are particularly relevant.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate an avirulence/pathogenicity gene from a related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of 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.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) 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.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

4.8 TRANSFORMED HOST CELLS AND TRANSGENIC PLANTS

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. Furthermore, 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. In some aspects of the invention, it is often desirable to modulate, regulate, or otherwise control the expression of the gene segments disclosed herein. Such methods are routine to those of skill in the molecular genetic arts. Typically, when increased or over-expression of a particular gene is desired, 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.

Typically, the initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, 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. Where no functional replication system is present, 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. Desirably, the avirulence/pathogenicity gene- promoter construct will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that an avirulence/pathogenicity gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

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. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome. Alternatively, the left and right T-DNA borders from the Ti plasmid may be used when integration is desired using A. tumefaciens vectors for plant transformation. 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 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. specifically incorporated herein by reference in its entirety), "gene gun" (Hilber et al, 1994; Yang et al, 1990); (3) viral vectors (Clapp, 1993; Danos and Heard, 1992; Eglitis and Anderson, 1988); (4) receptor-mediated mechanisms (Curiel et al, 1991 ; Wagner et al, 1992); and (5) bacterial-mediated delivery such as A. tumefaciens transformation (Smith and Hood, 1995). For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher organisms, including plants. The vectors comprise, for example, plasmids (such as pBR322, pUC series, M13mp series, pACYC184, etc), cosmids, phage, and/or phagemids and the like. Accordingly, 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. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. 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.

Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain.

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. In certain embodiments, 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. Likewise, 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.

Intermediate vectors cannot replicate themselves in agrobacteria. The intermediate vector can be transferred into A. tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E coli and in agrobacteria. They comprise a selection marker gene and a linker or polylinker that are framed by the right and left T-DNA border regions. They can be transformed directly into agrobacteria (Holsters et al, 1978). The agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional t-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. 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. If, for example, 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. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in Εur. Pat. Appl. No. ΕP 120 516; Hockema (1985); An et al, 1985, Herrera-Εstrella et al, (1983), Bevan et al, (1983), and Klee et al, (1985). A particularly useful Ti plasmid cassette vector for transformation of dicotyledonous plants consists of the enhanced CaMV35S promoter (EN-35S) and the 3' end including polyadenylation signals from a soybean gene encoding the '-subunit of β-conglycinin.

Between these two elements is a multilinker containing multiple restriction sites for the insertion of genes of interest.

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.

Optionally, the enhanced CaMV35S promoter may be replaced with the 1.5 kb mannopine synthase (MAS) promoter (Velten et al, 1984). After incorporation of a DNA construct into the vector, it is introduced into A. tumefaciens strain ACO that contains a disarmed Ti plasmid. Cointegrate Ti plasmid vectors are selected and subsequently may be used to transform a dicotyledonous plant.

A. tumefaciens ACO is a disarmed strain similar to pTiB6SE described by Fraley et al, (1985). For construction of ACO 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 remainder of the T-DNA extending to a point just beyond the right border was replaced with a novel piece of DNA including (from left to right) a segment of pBR322, the oriV region from plasmid RK2, and the kanamycin resistance gene from Tn601. The pBR322 and oriV segments are similar to these segments and provide a region of homology for cointegrate formation.

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. 4.8.1 ELECTROPORATION

The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. 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.

The introduction of DNA by electroporation is well-known to those of skill in the art (see e.g., U. S. Patent 5,324,253). In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mechanical wounding. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells (U. S. Patent 5,484,956; U. S. Patent 5,886,244), or embryogenic callus (U. S. Patent 5,405,765), or alternatively, one may transform immature embryos or other organized tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. 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.

4.8.2 MICROPROJECTILE BOMBARDMENT

A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Cristou et al, 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that 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. For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, 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. If desired, 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.

In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust several of the bombardment parameters in small-scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

4.8.3 AGROBACTERIUM-MEΌI AΎEΌ TRANSFER

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 α , 1985; Rogers et α , 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 (Spielmann et αl, 1986; Jorgensen et α/., 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 αl, 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. In addition, 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. However, inasmuch as use of the word "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.

More preferred is a 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.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two or more independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Backcrossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

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 α/., 1985; Uchimiya et al, 1986; Callis et al, 1987; Marcotte et al, 1988).

Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al, 1985; Toriyama et al, 1986; Yamada et al, 1986; Abdullah et al, 1986).

To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1988). In addition, "particle gun" or high-velocity microprojectile technology can be utilized (Vasil, 1992).

Using that latter technology, 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.

4.9 GENE EXPRESSION IN PLANTS Although great progress has been made in recent years with respect to preparation of transgenic plants that express bacterially derived proteins such as avirulence/pathogenicity gene-products and DNA binding proteins such as the lac operator, the results of expressing native bacterial genes in plants are often disappointing. Unlike microbial genetics, little was known by early plant geneticists about the factors that affected heterologous expression of foreign genes in plants. In recent years, however, several potential factors have been implicated as responsible in varying degrees for the level of protein expression from a particular coding sequence. For example, scientists now know that maintaining a significant level of a particular mRNA in the cell is indeed a critical factor. Unfortunately, the causes for low steady state levels of mRNA encoding foreign proteins are many. First, 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. Second, 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. In addition, every 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. Unfortunately, it is impossible to predict, and nearly impossible to determine, the structure of any RNA (except for tRNA) in vitro or in vivo. However, it is likely that dramatically changing the sequence of an RNA will have a large effect on its folded structure. It is likely that structure per se or particular structural features also have a role in determining RNA stability. To overcome these limitations in foreign gene expression, researchers have identified particular sequences and signals in RNAs that have the potential for having a specific effect on RNA stability. In certain embodiments of the invention, therefore, there is a desire to optimize expression of the disclosed nucleic acid segments in planta. One particular method of doing so is by alteration of the bacterial gene to remove sequences or motifs that decrease expression in a transformed plant cell. The process of engineering a coding sequence for optimal expression in planta is often referred to as "plantizing" a DNA sequence.

Particularly problematic sequences are those that are A+T rich. Unfortunately, since many bacterial species have genomes that are rich in A+T sequences, 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. They further showed that a pentamer of ATTTA had a profound destabilizing effect on a stable message, and that this signal could exert its effect whether it was located at the 3 '-end or within the coding sequence. However, the number of ATTTA sequences and/or the sequence context in which they occur also appears to be important in determining whether they function as a destabilizing sequence. The addition of a polyadenylation sequence to the 3 '-end is common to most eukaryotic mRNAs, both plant and animal. The currently accepted view of poly A addition is that the nascent transcript extends beyond the mature 3 '-terminus. Contained within this transcript are signals for polyadenylation and proper 3 '-end formation. This processing at the 3'-end involves cleavage of the mRNA and addition of polyA to the mature 3'-end. By searching for consensus sequences near the polyA tract in both plant and animal mRNAs, it has been possible to identify 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. In animal cells, some variants of this sequence that are functional have been identified; in plant cells there seems to be an extended range of functional sequences (Wickens and Stephenson, 1984; Dean et al, 1986). Because all of these consensus sequences are variations on AATAAA, they all are A+T rich sequences. 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.

In naturally occurring mRNAs that are normally polyadenylated, it has been observed that disruption of this process, either by altering the polyA signal or other sequences in the mRNA, profound effects can be obtained in the level of functional mRNA. This has been observed in several naturally occurring mRNAs, with results that are gene-specific so far.

It has been shown that in natural mRNAs proper polyadenylation is important in mRNA accumulation, and that disruption of this process can affect mRNA levels significantly. However, insufficient knowledge exists to predict the effect of changes in a normal gene. In a heterologous gene, it is even harder to predict the consequences. However, it is possible that the putative sites identified are dysfunctional. That is, these sites may not act as proper polyA sites, but instead function as aberrant sites that give rise to unstable mRNAs.

In animal cell systems, 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.

TABLE 4 POLYADENYLATION SITES IN PLANT GENES

PA AATAAA Major consensus site

P1A AATAAT Major plant site

P2A AACCAA Minor plant site

P3A ATATAA

P4A AATCAA

P5A ATACTA

P6A ATAAAA

P7A ATGAAA

P8A AAGCAT

P9A ATTAAT

P10A ATACAT

P11A AAAATA

P12A ATTAAA Minor animal site

P13A AATTAA

P14A AATACA

P15A CATAAA

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. As described above, 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. In particular, 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. Indeed, most of the known bacterial genes which have been expressed in plants are also on the order of 40-50% A+T. Therefore, for optimizaing the expression of selected bacterial avr/pth genes in a tranasformed plant host cell, it may be desirable in many instances to prepare synthetic gene sequences, or otherwise alter all or a portion of the particular coding sequence to be introduced into the target plant.

Due to the degeneracy of the genetic code and the limited number of codon choices for any amino acid, most of the "excess" A+T of the structural coding sequences of most bacterial genes are found in the third position of the individual triplet codons. That is, genes of some bacterial species have A or T as the third nucleotide in many codons. Thus, A+T content in part can determine codon usage bias. In addition, it is clear that genes evolve for maximum function in the organism in which they evolve. This means that particular 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.

Typically, to obtain high-level expression of selected bacterial genes in plants, existing structural coding sequence ("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. For purposes of the present invention 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. In replacing the ATTTA sequences and polyadenylation signals, 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. Although the absence of five or more consecutive A or T nucleotides eliminates most plant polyadenylation signals, if there are more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then 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.

It is also preferred that 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.

4.10 SYNTHETIC OLIGONUCLEOTIDES FOR MUTAGENESIS

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, EcøRI, 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. In most cases, a minimum of about 5 to about 8 base pairs of homology to the template DΝA on both ends of the synthesized fragment are maintained to insure proper hybridization of the primer to the template. The 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%.

TABLΕ 5

PREFERRED CODOΝ USAGE IN PLANTS

Amino Acid Codon Percent Usage in Plants

ARG CGA 7

CGC 11

CGG 5

CGU 25

AGA 29

AGG 23

LEU CUA 8

CUC 20

CUG 10

CUU 28

UUA 5

UUG 30 Amino Acid Codon Percent Usage in Plants

SER UCA 14

UCC 26

UCG 3

UCU 21

AGC 21

AGU 15

THR ACA 21

ACC 41

ACG 7

ACU 31

PRO CCA 45

CCC 19

CCG 9

CCU 26

ALA GCA 23

GCC 32

GCG

GCU 41

GLY GGA 32

GGC 20

GGG 1 1

GGU 37

ILE AUA 12

AUC 45

AUU 43 Amino Acid Codon Percent Usage in Plants

VAL GUA 9 GUC 20 GUG 28 GUU 43

LYS AAA 36 AAG 64

ASN AAC 72

AAU 28

GLN CAA 64 CAG 36

HIS CAC 65 CAU 35

GLU GAA 48 GAG 52

ASP GAC 48 GAU 52

TYR UAC 68

UAU 32

CYS UGC 78 UGU 22

PHE UUC 56 Amino Acid Codon Percent Usage in

Plants

UUU 44

MET AUG 100

TRP UGG 100

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).

Alternatively, 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.

4.11 "PLANTIZED" GENE CONSTRUCTS

The expression of a plant gene that exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region that adds polyadenylated nucleotides to the 3' end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter." The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA.

A number of 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).

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. For example, 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. For purposes of this description, 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. Furthermore, 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.

The DNA construct also contains a 3' non-translated region. The 3' non-translated regions contain a polyadenylation signal which functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the viral RNA. Examples of suitable 3' regions are (1) the 3' transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein (7S) genes and the small subunit of the RuBP carboxylase (E9) gene.

4.12 METHODS FOR CONTROLLING VIRAL INFECTION IN TRANSGENIC PLANTS By transforming a suitable host cell, such as a plant cell, with a recombinant avirulence/pathogenicity gene-containing segment, the expression of the encoded avirulence/pathogenicity gene under the control of an inducible promoter can result in the formation of transgenic plants in which the spread of viral infection can be controlled.

By way of example, one may utilize an expression vector containing a coding region for a bacterial avirulence/pathogenicity polynucleotide and an appropriate selectable marker to transform a suspension of embryonic plant cells, such as wheat or corn cells using a method such as particle bombardment (Maddock et al, 1991 ; Vasil et al, 1992) to deliver the DNA coated on microprojectiles into the recipient cells. 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 αl., 1983). Alternatively, 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 α/., 1987; Benbrook et α/., 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 regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al, 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al, 1983).

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. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, as discussed before. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art. Such plants can form germ cells and transmit the transformed trait(s) to progeny plants. Likewise, transgenic plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. 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.

4.13 VIRAL PATHOGENESIS IN PLANTS AND COMMERCIAL CROPS

Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more viral pests. 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, turnip, swiss chard, horseradish, tomatoes, kale, turnips, and a variety of other plants are sensitive to viral infection by one or more pathogens.

Fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears₅ 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. Even field crops such as cereals, canola/rape seed, evening primrose, meadow foam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, legumes such as beans, soybeans and the like; sunflowers, potatoes and other tuberous plants; and tobacco are often targets for viral infection and disease. Bedding plants, flowers, ornamentals, vegetables and container stock are also frequently attacked by microbial pathogens, as are forests, fruit, ornamental, and nut- bearing trees, as well as shrubs and other nursery stock. Even turf and ornamental grasses are often attacked by pests such as armyworm, sod webworm, and tropical sod webworm.

Because crops of commercial interest are often the targets of viral attack, environmentally sensitive methods for controlling or eradicating viral infestation are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and for use in commercial and residential areas where the control of viral populations using eco-friendly compositions is desirable and economically indicated.

4.14 RECOMBINANT VECTORS EXPRESSING A VR/PTH GENES 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. Such 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. In preferred embodiments, 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. The 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.

In another embodiment, 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. One can provide for DNA constructs that comprise (a) transcriptional and/or translational regulatory signals for expression of the gene, (b) the gene under their regulatory control, and (c) a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

The transcriptional initiation signals will preferably include at least a first promoter and at least a first transcriptional initiation start site. In some instances, 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. For example, 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.

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. 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. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, 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.

By 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. Preferably, 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. For example, genes expressing metal chelating agents, e.g., siderophores may be introduced into the host along with the structural gene expressing the Avr/Pth polypeptide. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the host that produces the Avr/Pth polypeptide, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment.

Where no functional replication system is present, 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. In this way, the probability of legitimate recombination is enhanced, so that the transgene will be integrated into the host DNA and stably maintained by the host. Desirably, 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. Therefore, in the event that the transgene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct. A large number of 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 λ_L and λ_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. Patents 4,332,898; 4,342,832; and 4,356,270 (each of which is specifically incorporated herein by reference). 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.

Where stable episomal maintenance or integration is desired, a plasmid will be employed 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. Patents 4,356,270; 4,362,817; 4,371,625, and 5,441,884, each incorporated specifically herein by reference. The selected 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. In addition, one or more markers may be present, which have been described previously. Where integration is desired, 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).

In accordance with the present invention, nucleic acid sequences include and are not limited to DNA, including and not limited to cDNA and genomic DNA, genes; RNA, including and not limited to mRNA and tRNA; peptide nucleic acids (PNAs), ribozymes, antisense sequences, nucleosides, and suitable nucleic acid sequences such as those sequences encoding one or more of the Avr/Pth polypeptides disclosed in Table 9.

As such the present invention also concerns 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. Likewise, 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.

Included within the term "DNA segment", "nucleic acid segment" "polynucleotide" and "sequence region" are nucleic acid segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, 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. In this respect, 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.

Particularly preferred are 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. It will also be understood that 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.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

For certain application, relatively small contiguous nucleic acid sequences are preferable, such as those which are about 14 or 15 or 16 or 17 or 18 or 19, or 20, or 30-50, 51-80, 81-100 or so nucleotides in length. Alternatively, in some embodiments, and particularly those involving preparation of recombinant vectors, transformation of suitable host cells, and preparation of transgenic plant cell, longer nucleic acid segments are preferred, particularly those that include the entire coding region of one or more avr/pth genes. As such, the preferred segments may include those that are up to about 20,000 or so nucleotides in length, or alternatively, shorter sequences such as those about 19,000. about 18,000, about 17,000, about 16,000, about 15,000, about 14,000, about 13,000, about 12,000, 1 1,000, about 10,000, about 9,000, about 8,000, about 7,000, about 6,000, about 5,000. about 4,500, about 4,000, about 3,500, about 3,000, about 2,500, about 2,000, about 1 ,500. about 1,000, about 500, or about 200 or so base pairs in length. Of course, these numbers are not intended to be exclusionary of all possible intermediate lengths in the range of from about 20,000 to about 15 nucleotides, as all of these intermediate lengths are also contemplated to be useful, and fall within the scope of the present invention. It will be readily understood that "intermediate lengths", in these contexts, 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. In one embodiment, 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.

The 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. Alternatively, 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. If desired, one may also prepare 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 PCR™ technology, in connection with the compositions disclosed herein.

4.15 VECTORS, HOST CELLS, AND PROTEIN EXPRESSION

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding an Avr/Pth polypeptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of 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). In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of selected peptides or epitopic core regions, such as may be used to generate antibodies, also falls within the scope of the invention. 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.

4.16 TRANSFORMED HOST CELLS AND TRANSGENIC PLANTS

In one embodiment, 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.

In yet another aspect, 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. In general, 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. Such 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. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant protein expressed in a particular transgenic cell, 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. The use of 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.

As used herein, the term "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.

It is contemplated that in some instances the genome of a 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. In some instances, 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. In certain situations, it may be desirable to have one, two, three, four, or even more Avr/Pth proteins (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic 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.

Means for transforming a plant cell and the preparation of a transgenic cell line are well known in the art, and are discussed herein. 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. These 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. In a related aspect, 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.

4.17 ISOLATING HOMOLOGOUS GENE AND GENE FRAGMENTS The 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.

It should be apparent to a person skilled in this art that 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. For example, 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.

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. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. 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. In the use of the nucleotide segments as probes, 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 ³²P, ¹²⁵1, ³⁵S, 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. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. 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.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, 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. Thus, 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. In addition it has been shown that 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). Thus, 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. Further, 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.

4.18 RIBOZYMES

Ribozymes are enzymatic RNA molecules that cleave particular mRNA species. In certain embodiments, 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.

Six basic varieties of naturally occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target-binding portion of an enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target 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.

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. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, 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. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al, 1992). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif. Examples of 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. Patent 5,631,359; an example of the hepatitis δ virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et al, (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991 ; Collins and Olive, 1993); and an example of the Group I intron is described by Cech et al, (U.S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the 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. Alternatively, 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) 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. Alternatively, 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). Those skilled in the art realize that 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 et al, Int. Pat. Appl. Publ. No. 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. Alternatively, 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.

4.19 PEPTIDE NUCLEIC ACID COMPOSITIONS

In certain embodiments, the inventors contemplate the use of peptide nucleic acids (PNAs) in the practice of the methods of the invention. PNAs are DNA analogs that mimic the structure of the polynucleotide, in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, 1997). PNAs can be utilized in a number of methods that traditionally have used RNAs or DNAs (U. S. Patent 5,786,461 ; U. S. Patent 5,773,571, U. S. Patent 5,766,855; U. S. Patent 5,736,336; U. S. Patent 5,719,262; and U. S. Patent 5,539,082, each specifically incorporated herein by reference in its entirety). Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. Methods of making, and using PNAs are also found in Corey (1997).

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). Other applications of 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).

4.20 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. In particular embodiments of the invention, 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.

TABLE 6

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic Acid Asp D GAC GAU

Glutamic Acid Glu E GAA GAG

Phenylalanine Phe F UUC uuu

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Tip w UGG

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.

In making such changes, 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).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101, specifically incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U. S. Patent 4,554,101 , the following 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).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, 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.

4.21 DEFINITIONS

In accordance with the present invention, nucleic acid sequences include and are not limited to DNA (including and not limited to genomic or extragenomic DNA), genes, RNA

(including and not limited to mRNA and tRNA), nucleosides, and suitable nucleic acid segments either obtained from native sources, chemically synthesized, modified, or otherwise prepared by the hand of man. The following words and phrases have the meanings set forth below.

A, an: In accordance with long standing patent law convention, the words "a" and

"an" when used in this application, including the claims, denotes "one or more". 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.

Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1- INDUCTION OF RESISTANCE RESPONSE BY TMoV

In this example, 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 avrbό under the control of the AVI and BVl promoters. 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.

The inducible promoters AIP and BIP were PCR-amplified from the ToMV DNA extracts from infected tomato tissues using two pairs of primers. For AIP, DG 74 (5'- AGAATTCGGGGCATTTTTGTAATAAG-3')' (SEQ ID NO:l ) and DG75 (5'- AGGATCCATTTTGAGTTAAAGAC-3'; SEQ ID NO:2) were used. For BIP, DG74 and DG76 (5'-AGGATCCATAGTCAAACACTTAAC-3'; SEQ ID NO:3). The PCR™ products were cloned into pGEM-T vector (Promega). The AIP or BIP promoter sequences, flanked by EcøRI and BamΑl sites, were used to replace the CaMV 35S² promoter of pKYLX71 :35S² (Duan et al, 1997a; 1997b) to generate two new plasmids pYD54 and pYD55. The avirulence genes avrbό and pthA were cloned into the new vectors by using BamRl and Hindlll. This formed clones pYD63.1, pYD63.5, pYD63.7, and pYD63.9 (FIG. 9), which were mobilized into A. 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).

5.2 EXAMPLE 2 - TRANSFORMING PLANTS WITH DNAs ENCODING ANTIBODIES AGAINST AVR OR PTH POLYPEPTIDES

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. For example, 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.

5.3 EXAMPLE 3 - OPTIMIZATION OF CODON USAGE

In order to express polypeptide aptamers in plants, 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 NH₃-terminal end of domain II of GAL4. TABLE 7 NLS SITE-DIRECTED KNOCKOUT MUTATIONS IN PTHA

In Cotton* In Bean*

Vector Only Null Null pthA Strong HR Strong HR pthA with 1 NLS mutation Reduced HR Reduced HR

pthA with 2 NLS mutations Weak HR Weak HR

pthA with 3 NLS mutations Almost Null; very Almost Null; very weak weak HR HR

*In both cotton and bean, the results were similar. The "null" and the "strong HR" phenotypes are illustrated in FIG. 5. The "reduced", "weak" and "very weak" HR phenotypes refer to macroscopically visible symptoms observed relative to the "strong HR" phenotype.

TABLE 8 EVALUATION OF THE INDUCIBLE PROMOTERS FROM TMoV

Tomato* I Innffe< cted Tomato Cotton pYD40.1 (35S::pthA) Strong HR Strc HR Strong HR pYD40.2 (control; no avr gene) - - pYD63.1 (AIP '::avrb6) - HR pYD63.5 (Bl? .avrbό) - HR pYD63.7 (ALP-.φthA) - HR pYD63.9 (BI? :pthA) - HR

* The results for tomato scored on 24 h after inoculation, for infected tomato scored at 30 h after inoculation; for cotton at 48 h after inoculation. - , no HR phenotype. The HR phenotypes on tomato may be seen in FIG. 12. (uninfected tomato) and FIG. 10 (infected tomato).

5.4 EXAMPLE 4 - EXEMPLARY AVIRULENCE/PATHOGENICITY GENES In addition to the specific examples described above, the inventors contemplate the use of a variety of avirulence and/or pathogenicity genes in the practice of the present invention. In particular, the genes described in Table 9 are contemplated to be useful in the preparation of inducible promoter-gene constructs that may be used to transform plant cells to provide means for controlling viral infection and spread in such transgenic plants. TABLE 9 EXEMPLARY AVIRULENCE/PATHOGENICITY GENES FOR USE IN THE PRACTICE OF THE PRESENT INVENTION

ORGANISM GENE GENBANK REFERENCE DESIGNATION ACCESSION NO.

Cladosporium fulvum avr4 X78829 Nature, 367:6461 :384-386, 1994

Cladosporium fulvum avr4 Y08356 Plant Cell, 9:1-13, 1997

Cladosporium fulvum avr9 M55289 Mol. Plant Microbe Interact, 4(l):52-59, 1991

Cladosporium fulvum avr9 X60284 J. Plant, 2(3):359-366, 1992

Erwinia herbicola pv. gypsophilae avr AF071231 GenBank

Mayetiola destructor 0PG15-1 AF051559 Genome, 41(5):702-8, 1998

Pseudomonas syringae avrA M15194 J. Bacleriol, 169:572-578, 1987

Pseudomonas syringae avrB M21965 J. Bacteriol, 170:4846-4854, 1988

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Pseudomonas syringae pv. apii avrD AF083919 GenBank

ORGANISM GENE GENBANK REFERENCE DESIGNATION ACCESSION NO.

Pseudomonas syringae pv. cilantro avrD AF083918 GenBank Pseudomonas syringae pv. lachrymans avrD L11334 Mol. Plant Microbe Interact, 7(1): 131 - 139, 1994 Pseudomonas syringae pv. phaseolicola avrD L11336 Mol. Plant Microbe Interact. , 7(1): 131-139, 1994 Pseudomonas syringae pv. phaseolicola avrPphE AJ224433 Mol. 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. Plant Microbe Interact. , 8(l):49-57, 1995 Pseudomonas syringae pv. tomato avrP L20425 Mol. Gen. Genet., 239:6-16, 1993 Salmonella typhimurium avrA AF013573 Proc. Natl. Acad. Sci. USA, 94(18):9887-9892, 1997 Xanthomonas campestris aurBsl M32142 J03672 Mol. Plant Microbe Interact, 1 : 191-198, 1988 Xanthomonas campestris aurBsp M80803 Mol. Gen. Genet., 218: 127-136, 1989 Xanthomonas campestris avrBs3 X16130 Mol. Gen. Genet., 218(1 ): 127-136, 1989 Xanthomonas campestris avrBs3-2 X68781 Mol. Gen. Genet. , 238(l-2):261-269, 1993 Xanthomonas campestris pv. malvacearum avrbό L06634 Mol. Plant Microbe Interact. , 6(2):225-237, 1993 Xanthomonas campestris avrRxv L20423 Mol. Plant Microbe Interact. , 6:616-627, 1993 Xanthomonas campestris pthN AFO 16221 Phytopathology, X:X-X, 1997 Xanthomonas campestris avr M99059 GenBank

ORGANISM GENE GENBANK REFERENCE

DESIGNATION ACCESSION NO.

Xanthomonas citri pthA U28802 S48727 J. Bacteriol, 177(17):4963-4968, 1995

S48730

Xanthomonas oryzae avrXalO U50552 Mol Plant Microbe Interact. , 5(6):451 -459, 1992

5.5 EXAMPLE 5 - PREPARATION OF APTAMER-ENCODING DNA SEQUENCES

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). The basic process is to screen the random pool for those sequences that stick to a target molecule affixed to a substrate. Then wash away everything that does not stick and then amplify the sequences that do stick. The procedure is repeated a second time using the amplified library that is enriched with sequences that stuck to the target during the first round. The selected molecules with specific ligand binding properties are called "aptamers" (from the Latin aptus, to fit) (Szostak, 1992). Although originally the term aptamer 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). The processes for generating and selecting aptamers are well known in the art, as described by Tuerk and Gold (1990); Szostak (1992); Gold (1995); Colas et al, (1996) and Conrad et al, (1996); each of which is incorporated herein by reference.

A series of three short (eight amino acids in length) polypeptide aptamers were identified following four cycles of enrichment using a random aptamer library displayed on M13 phage (New England Biolabs). Selection was against the last 85 amino acids at the carboxyl terminal end of the PthA protein (downstream from the NLS region). All three aptamers bound tightly to PthA and to PthF in ELISA tests. Because the predicted protein identities of the carboxyl terminus of other members of the avrBs3/pthA gene family (Gabriel, 1999; Leach and White, 1996) are so perfectly preserved, it is highly likely that these same aptamers will also bind to AvrXa7. 5.6 EXAMPLE 6 - ANALYSIS OF APTAMER BLOCKING IN TRANSIENT EXPRESSION ^~ ASSAYS IN BEAN AND CITRUS

Several methods may be used to induce disease symptoms on plants. 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). These 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."

When the HP and YP aptamers were simultaneously expressed in plants with PthA, both partially blocked function of PthA to elicit symptoms, whether cankers on citrus or an HR on other plants, such as bean (FIG. 2 and FIG. 3). Cankers on citrus were greatly reduced in appearance, but not fully eliminated. The HR, which normally occurs 48 hours after inoculation, was delayed by an additional 48 hours and was greatly reduced. Similar results were obtained in blocking the blight function of pthF in bean. In all cases tested, the YP aptamer appeared to give better control of symptoms than the HP aptamer.

5.7 EXAMPLE 7 - USE OF APTAMER BLOCKING METHOD IN PERMANENTLY TRANSFORMED PLANTS

After determining that transiently expressed aptamers could block symptoms elicted by pthA and pthF on bean and citrus, studies were undertaken to permanently transform plants. Tobacco was chosen for transformation with the aptamers because pthA elicits a good HR on tobacco in transient expression assays identical to the one shown on bean in FIGr2, above. The results using the YP aptamer confirmed results obtained with the transient expression assays and are shown in FIG. 4. The YP aptamer, expressed in transgenic tobacco, delayed but did not eliminate the HR elicited on tobacco. The HR appeared some 48 hours later, and was much weaker. Some transgenic plants, which exhibited qualitatively weak Gus reporter activity, delayed the HR by only 12 hours, and the HR appeared reduced, but not weak. This indicated a quantitative effect of the aptamer.

5.8 EXAMPLE 8 - TRIPARTITE GENETIC CONSTRUCTS 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. In one embodiment, one may utilize a DNA binding protein that binds to an artificial promoter, such as the one described by Moore et al. (1998), based on the bacterial lac gene repressor binding to the lac operator. One may make a fusion of the lac repressor with an aptamer that binds to the coat protein or any other protein of any given virus, whether RNA or DNA. 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.

Likewise, 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. An exemplary such 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). 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. In a preferred embodiment, different aptamers that bind different regions of the coat protein of the virus (whether RNA or DNA) 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.

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/αvr 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.

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7.0 BRIEF DESCRIPTION OF OLIGONUCLEOTIDE SEQUENCES SEQ ID NO:l is PCR™ primer DG 74: 5'-AGAATTCGGGGCATTTTTGTAATAAG-3'

SEQ ID NO:2 is PCR™ primer DG75: 5*-AGGATCCATTTTGAGTTAAAGAC-3'

SEQ ID NO:3 is PCR™ 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'

All of the 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.

Claims

CLAIMS ~

1. A nucleic acid segment comprising at least one bacterial avirulence gene operably linked to an inducible promoter that is transcriptionally activated by a viral polypeptide, wherein transcriptional activation induces expression of said avirulence gene in a host cell transformed with said segment.

2. The nucleic acid segment of claim 1, wherein said gene is transcriptionally activated by an early viral polypeptide.

The nucleic acid segment of claim 1 or 2, wherein said early viral polypeptide is selected from the group consisting of an AC 1, AC2, AIP and a BIP promoter.

The nucleic acid segment of any preceding claim, wherein said avirulence gene is selected from the group consisting of a pth gene, an hrp gene, and an ╬▒vr gene.

5. The nucleic acid segment of any preceding claim, wherein said avirulence gene is selected from the group consisting of pthA, pthN, pthN2, pthA, pthB, pthC, pthCBBl, αvrBn, αvrbό, αvrB4, αvrb7, αvrBIn, αvrBlOl, αvrB102, αvrB103, αvrB104, αvrB5, αvrBs3, αvrBs3-2, αvrBsP, αvrxαS, αvrXα/, αvrXαlO, αvrXpl, αvrPphA, αvrPphBl.R3, αvrPphD, αvrPphEl.R2, αvrPphF.Rl, αvrPpiAl.R2, αvrPpiBl.R3, αvrPpiC, αvrPpiD.R5, αvrPpiE, αvrPmαAl, αvrD, αvrRpt2, αvr P to, αvrE, αvr A, αvrB, αvrC and hrpN.

6. The nucleic acid segment of any preceding claim, wherein said avirulence gene is isolated from Cladosporium, Erwinia, Mayetiola, Pseudomonas, Salmonella, or Xanthomonas.

The nucleic acid segment of any preceding claim, wherein said avirulence gene is isolated from C. fulvum, E. herbicola, M. destructor, P. syringae, S. typhimuriam, X. campestris, X. citri, or X. oryzae.

8. The nucleic acid segment of any preceding claim, wherein said avirulence gene is transcriptionally activated by a polypeptide from a virus selected from the group consisting of 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-spotting badnavirus, Rice tungro bacilliform badnavirus, Schefflera ringspot badnavirus, Sugarcane bacilliform badnavirus, Abutilon mosaic bigeminivirus, Ageratum yellow vein bigeminivirus, Bean calico mosaic bigeminivirus, Bean golden mosaic bigeminivirus, Bhendi yellow vein mosaic bigeminivirus, Cassava African mosaic bigeminivirus, Cassava Indian mosaic bigeminivirus, Chino del tomate bigeminivirus, Cotton leaf crumple bigeminivirus, Cotton leaf curl bigeminivirus, Croton yellow vein mosaic bigeminivirus, Dolichos yellow mosaic bigeminivirus, Euphorbia mosaic bigeminivirus, Horsegram yellow mosaic bigeminivirus, Jatropha mosaic bigeminivirus, Lima bean golden mosaic bigeminivirus, Melon leaf curl bigeminivirus, Mung bean yellow mosaic bigeminivirus, Okra leaf-curl bigeminivirus, Pepper hausteco bigeminivirus, Pepper Texas bigeminivirus, Potato yellow mosaic bigeminivirus, Rhynchosia mosaic bigeminivirus, Serrano golden mosaic bigeminivirus, Squash leaf curl bigeminivirus, Tobacco leaf curl bigeminivirus,

Tomato Australian leafcurl bigeminivirus, Tomato golden mosaic bigeminivirus, Tomato Indian leafcurl bigeminivirus, Tomato leaf crumple bigeminivirus, Tomato mottle bigeminivirus, Tomato yellow leaf curl bigeminivirus. Tomato yellow mosaic bigeminivirus, Watermelon chlorotic stunt bigeminivirus, Watermelon curly mottle bigeminivirus, Beet curly top hybrigeminivirus, Chloris striate mosaic monogeminivirus, Digitaria striate mosaic monogeminivirus, Digitaria streak monogeminivirus, Maize streak monogeminivirus, Miscanthus streak monogeminivirus, Panicum streak monogeminivirus, Paspalum striate mosaic monogeminivirus, Sugarcane streak monogeminivirus, Tobacco yellow dwarf monogeminivirus, and Wheat dwarf monogeminivirus.

9. The nucleic acid segment of claim 8, wherein said virus causes metstreak disease of maize, Hoja blanca (white tip) disease, bunchy top disease, sugarcane mosaic disease, sugarbeet yellowing disease, citrus quick decline disease, swollen shoot disease, plum pox disease, or barley yellow dwarf disease.

10. The nucleic acid segment of any preceding claim, further comprising an aptamer coding sequence translationally fused to a DNA binding protein coding sequence, said nucleic acid segment operably positioned under the control of a constitutive promoter capable of expressing the encoded translationally fused polypeptide in a host cell.

11. The nucleic acid segment of claim 11, wherein said aptamer coding sequence is capable of binding to said viral polypeptide.

12. The nucleic acid segment of any preceding claim, wherein said viral polypeptide is a repressor protein.

13. A recombinant vector comprising the nucleic acid segment of any one of claims 1 to 12.

14. The recombinant vector of claim 13, wherein said vector is a plasmid, a cosmid a phagemid, an artificial chromosome, a baculovirus, or a viral vector.

15. The recombinant vector of claim 13 or 14, wherein said vector is replicable in a plant cell.

16. The recombinant vector of any one of claims 13 to 15, wherein said vector is replicable in an Agrobacterium cell.

17. A transformed host cell comprising the nucleic acid segment of any one of claims 1 to 12 or the vector of any one of claims 13 to 16.

18. The transformed host cell of claim 17, wherein said host cell is a bacterial or a plant cell.

19. The transformed host cell of claim 17 or 18, wherein said cell is an Agrobacterium or an Escherichia cell.

20. The transformed host cell of claim 17 or 18, wherein said cell is a monocotyledonous or a dicotyledonous plant cell.

21. The transformed host cell of claim 20, wherein said plant cell is a monocotyledonous plant cell.

22. The transformed host cell of claim 20 or 21 , wherein said plant cell is selected from the group consisting of an oat, wheat, rice, barley, sorghum, maize, sugarcane, asparagus, Bermuda grass, bluegrass, bent grass, St. Augustine grass and canola plant cell.

23. The transformed host cell of claim 20, wherein said plant cell is a dicotyledonous plant cell.

24. The transformed host cell of claim 20 or 23, wherein said plant cell is selected from the group consisting of a tomato, potato, soybean, cotton, sunflower, tobacco, cucumber, citrus, apple, pear, peach, plum, cherry, common bean, cassava, watermelon, cantelope, almond, pecan, hazelnut, pistachio, walnut, macademia nut, alfalfa, papaya, peanut, coffee, hops, sunflower, pea, sugarbeats, tea, brassicas, taro, banana, cacao and plum cell.

25. The transformed host cell of claim 24, wherein said plant cell is a citrus or bean cell.

26. The transformed host cell of claim 25, wherein said citrus cell is selected from the group consisting of orange, grapefruit, lime, lemon, tangerine, and tangelo cell.

27. The transformed host cell of any one of claims 17 to 26, wherein said cell is a pluripotent cell.

28. The transformed host cell of any one of claims 17 to 27, wherein said cell is comprised within a plant.

29. A transgenic plant comprising the nucleic acid segment of any one of claims 1 to F2, the recombinant vector of any one of claims 13 to 16, or the transformed host cell of any one of claims 17 to 28.

30. A progeny of any generation of the plant of claim 29, wherein said seed or progeny comprises said selected nucleic acid segment.

31. A seed of any generation of the transgenic plant of claim 29.

32. A seed of any generation of the progeny of claim 30.

33. A plant grown from the seed of claim 31 or 32.

34. Use of the nucleic acid segment of any one of claims 1 to 12, the recombinant vector of any one of claims 13 to 16, or the transformed host cell of any one of claims 17 to

28 in the preparation of a pluripotent plant cell.

35. Use of the nucleic acid segment of any one of claims 1 to 12, the recombinant vector of any one of claims 13 to 16, or the transformed host cell of any one of claims 17 to

28 in the preparation of a transgenic plant.

36. Use of the nucleic acid segment of any one of claims 1 to 12, the recombinant vector of any one of claims 13 to 16, or the transformed host cell of any one of claims 17 to

28 in the preparation of a transgenic seed.

37. A method of inducing cell death upon viral infection in a plant cell, comprising providing to a plant cell at least one bacterial avirulence gene operatively linked to a promoter that is transcriptionally activated upon infection by a virus, wherein transcriptional activation induces expression of said avirulence gene thereby inducing cell death in said plant cell.

38. A method of controlling the extent of viral infection in a plant population, comprising providing to a subpopulation of said plant population a nucleic acid segment that comprises at least one bacterial avirulence gene operably linked to a promoter that is transcriptionally activated upon infection by a virus, wherein transcriptional activation induces expression of said avirulence gene thereby inducing death in cells of said plant subpopulation, and thereby controlling the extent of viral infection in the remaining plant population.