Methods of creating dwarf phenotypes in plants

Abstract

The invention is directed to the application of gene sequences which cause a dwarf phenotype in plants to the fields of forestry plants, ornamental horticultural plants, medicinal plants, and Nicotiana plants which are used for purposes other than for traditional tobacco products. The invention provides cDNAs identified by the polynucleotide sequences SEQ ID NO: 1-122 that may be used to create transfected or transgenic plants exhibiting a dwarf phenotype. The invention also provides methods of creating a transfected or transgenic plant exhibiting a dwarf phenotype by expressing in the plant DNA or mRNA identified by the sequences SEQ ID NO:1-122.

Description

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acids and amino acid sequences identified in multiple metabolic pathways that lead to dwarfism and stunting in plants and the use of these sequences to create dwarf varieties of any plant species. Particularly, this invention relates to the use of nucleic acids and amino acid sequences which cause dwarfing in the fields of forestry plants, ornamental horticultural plants, medicinal plants, and Nicotiana plants.

BACKGROUND OF THE INVENTION

[0003] The strategies for increasing the productivity of plants is dependent on rapid discovery of unknown gene sequences and their function through genomics research. These discoveries will provide fundamental information necessary to engineer plants for improved grain yields and resistance to drought, pests, salt, and other extreme environmental conditions. Such advances are critical for a world population expected to double by 2050. Moreover, this information may identify genes and products encoded by genes that are useful for human and animal healthcare such as pharmaceuticals.

[0004] There has been a massive accumulation of expressed sequence tags (ESTs) as a result of recent genome research. Potential use of this sequence information is enormous once gene function is determined. Knowledge of function allows engineering of commercial plants and seeds for forestry, ornamental and horticultural plants, including any plants used to produce pharmaceutical products, and particularly plants of the genus Nicotiana for purposes other than traditional tobacco products.

[0005] Use of these sequences to convey any number of desirable traits to pharmaceutical and fiber crops and thereby increase production and building materials, medicines and chemicals for other uses. For example, gene profiling in cottonwood may lead to an understanding of the types of genes and promoters that act primarily in fiber cells. The novel sequences derived from these profiling studies may be important in genetic engineering of cottonwood fiber for increased strength. In plant breeding, gene profiling coupled to physiological trait analysis can lead to the identification of predictive markers that will be increasingly important in marker assisted breeding programs. Mining the DNA sequence of a particular crop for genes important for yield, quality, health, appearance, color, taste, etc. are applications of obvious importance for crop improvement.

[0006] The Green Revolution crops, introduced in the late 1960s and early 1970s, produce several times as much grain as the traditional varieties they replaced, and they spread rapidly. They enabled India to double its wheat crop in seven years, dramatically increasing food supplies and averting widely predicted famine. The Green Revolution's leading research achievement was to hasten the perfection of dwarf spring wheat. Though it is conventionally assumed that farmers want a tall, impressive-looking harvest, in fact shrinking wheat and other crops has often proved beneficial. When bred for short stalks, plants expend less energy growing inedible column sections and more growing valuable grain. Stout, short-stalked wheat also neatly supports its kernels, whereas tall-stalked wheat may bend over at maturity, complicating reaping. Nature has favored genes for tall stalks, because in nature plants must compete for access to sunlight. However, in high-yield agriculture, equally short-stalked plants will receive equal sunlight. Researchers are actively seeking dwarf strains of rice and other crops in order to increase agronomic yields. The identification of genes and metabolic pathways that may be modified to create rapidly growing dwarf strains would greatly accelerate this effort. Furthermore, identification of these genes and metabolic pathways in food crops may lead to the development of dwarf strains in other plant types such as forest trees, ornamental species such as ornamental and turfgrass, and plants such as Nicotiana sp. grown as hosts for biopharmaceutical manufacturing.

SUMMARY OF THE INVENTION

[0007] The invention is directed to the application of gene sequences which cause a dwarf phenotype in plants to the fields of forestry plants, ornamental horticultural plants, medicinal plants, and Nicotiana plants which are used for purposes other than for traditional tobacco products.

[0008] The invention provides cDNAs identified by the polynucleotide sequences SEQ ID NO: 1-122 that may be used to create transfected or transgenic plants exhibiting a dwarf phenotype. These cDNAs have been identified by phenotypic screening of the Large Scale Biology's libraries over 8000 cDNAs from Arabidopsis, Nicotiana, Oryza and Papaver constructed in the GENEWARE® vector.

[0009] The invention provides methods of creating a transfected or transgenic plant exhibiting a dwarf phenotype comprising: expressing in the plant a cDNA (or its encoded mRNA) identified by a polynucleotide sequence chosen from the group consisting of SEQ ID NO: 1-122.

[0010] The invention also provides a method of creating a transfected or transgenic plant exhibiting a dwarf phenotype comprising the steps of: (a) providing a viral inoculum capable of infecting a plant comprising the cDNA (or its encoded mRNA) identified by a polynucleotide sequence chosen from the group of SEQ ID NO: 1-122; and (b) applying said viral inoculum to a plant; whereby the plant is infected and the cDNA (or its encoded mRNA) is expressed in the plant.

[0011] The methods of the invention provide for creating a transfected or transgenic plant exhibiting a dwarf phenotype in any plant type. Preferred embodiments of the invention provide methods for creating dwarf plants of ornamental and horticultural plants, medicinal plants or forest trees. A preferred embodiment provides methods for creating dwarf plants of Nicotiana sp. Another preferred embodiment provides methods for creating dwarf turfgrass.

[0012] The invention also provides methods for creating transfected or transgenic plants exhibiting a dwarf phenotype for use in biopharmaceutical manufacturing comprising: applying a viral inoculum capable of infecting a plant and comprising the DNA (or its encoded mRNA) identified by a polynucleotide sequence chosen from the group of SEQ. ID NO 1-122 to a plant that expresses a biopharmaceutical, whereby the plant is infected, exhibits a dwarf phenotype, and expresses the biopharmaceutical.

[0013] The invention also provides a transfected or transgenic plant exhibiting a dwarf phenotype made by the method comprising expressing in the plant a cDNA(or its encoded mRNA) identified by a polynucleotide sequence chosen from the group consisting of SEQ ID NO: 1-122. The invention provides for transfected or transgenic plants made by the use of this method with any plant type. Preferred embodiments are transfected or transgenic plants of ornamental and horticultural plants, medicinal plants or forest trees. Preferred embodiments include transfected or transgenic plants of Nicotiana sp and dwarf turfgrass.

[0014] The invention also provides methods of producing multiple crops of the transfected or transgenic plants expressing a cDNA(or its encoded mRNA) identified by a polynucleotide sequence chosen from the group consisting of SEQ ID NO: 1-122 and exhibiting a dwarf phenotype comprising the steps of: (a) planting a reproductive unit of the transfected or transgenic plant; (b) growing the planted reproductive unit under natural light conditions; (c) harvesting the plant; and (d) repeating steps (a) through (c) at least once in the year.

[0015] The invention provides a method of constructing and characterizing a normalized cDNA library in a viral vector. The invention further provides a method of constructing and characterizing of a normalized whole plant cDNA library in viral vectors.

[0016] The invention identifies cDNAs corresponding to genes in the trans-ketolase and carbohydrate metabolic pathways as useful for creating transfected or transgenic plants exhibiting a dwarf phenotype.

[0017] The invention also provides method of manufacturing a biopharmaceutical comprising:

DESCRIPTION OF THE INVENTION

[0018] Before the present proteins, nucleotide sequences, and methods are described, it should be noted that this invention is not limited to the particular methodology, protocols, plants, cell lines, vectors, and reagents described herein as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects of the invention, and is not intended to limit its scope which will be limited only by the appended claims.

[0019] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a host cell” includes a plurality of such host cells, reference to the “antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions

[0021] “Acylate” as used herein, refers to the introduction of an acyl group into into a molecule, i.e. acylation

[0022] “Adjacent” as used herein, refers to a position in a nucleotide sequence proximate to and 5′ or 3′ to a defined sequence. Generally, adjacent means within 2 or 3 nucleotides of the site of reference.

[0023] “Agonist”, as used herein, refers to a molecule which, when bound to a gene product of interest, increases the biological or immunological activity of that gene product. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to a gene product of interest.

[0024] “Alterations” in a polynucleotide sequence, as used herein, comprise any deletions, insertions, and point mutations in the polynucleotide sequence. Included within this definition are alterations to any genomic DNA sequence corresponding to the polynucleotide sequence.

[0025] “Amino acid sequence” as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. “Amino acid sequence” and like terms, such as “polypeptide” or “protein” as recited herein are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0026] “Amplification” as used herein refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0027] “Antibody” refers to intact molecules as well as fragments thereof which are capable of specific binding to the epitopic determinant. Antibodies that bind a polypeptide of interest can be prepared using intact polypeptides or fragments as the immunizing antigen. These antigens may be conjugated to a carrier protein, if desired.

[0028] “Antigenic determinant,” “determinant group,” or “epitope of an antigenic macromolecule” as used herein, refers to any region of the macromolecule with the ability or potential to elicit, and combine with, specific antibody. Determinants exposed on the surface of the macromolecule are likely to be immunodominant, i.e. more immunogenic than other (imunorecessive) determinants which are less exposed, while some (e.g. those within the molecule) are non-immunogenic (immunosilent). As used herein, antigenic determinant refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

[0029] “Antisense”, as used herein, refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term “antisense” or “(−) sense” is used in reference to the nucleic acid strand that is complementary to the “sense” or “(+) sense” strand. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, the transcript of this strand may hybridize to natural sequences to block either their further transcription or translation. In this manner, mutant phenotypes may be generated.

[0030] “Anti-Sense Inhibition” as used herein, refers to a type of gene regulation based on cytoplasmic, nuclear or organelle inhibition of gene expression due to the presence in a cell of an RNA molecule complementary to at least a portion of the mRNA being translated. It is specifically contemplated that DNA molecules may be from either an RNA virus or mRNA from the host cells genome or from a DNA virus.

[0031] “Antagonist” or “inhibitor”, as used herein, refer to a molecule which, when bound to a gene product of interest, decreases the biological or immunological activity of that gene product of interest. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to the gene product of interest.

[0032] “Biologically active”, as used herein, refers to a molecule having the structural, regulatory, or biochemical functions of a naturally occurring molecule.

[0033] “Cell Culture” as used herein, refers to a proliferating mass of cells which may be in either an undifferentiated or differentiated state, growing contiguously or non-contiguously.

[0034] “Chimeric plasmid” as used herein, refers to any recombinant plasmid formed (by cloning techniques) from nucleic acids derived from organisms which do not normally exchange genetic information (e.g. Escherichia coli and Saccharomyces cerevisiae).

[0035] “Chimeric Sequence” or “Chimeric Gene” as used herein, refers to a nucleotide sequence derived from at least two heterologous parts. The sequence may comprise DNA or RNA.

[0036] “Coding Sequence” as used herein, refers to a nucleic acid sequence which, when transcribed and translated, results in the formation of a cellular polypeptide or a ribonucleotide sequence which, when translated, results in the formation of a cellular polypeptide.

[0037] “Common Embryological Basis” as used herein, is intended to include all tissues which are derived from the same germinal layer, specifically the ectoderm layer, which forms during the gastrulation stage of embryogenesis. Such tissues include, but are not limited to, brain, epithelium, adrenal medulla, spinal chord, retina, ganglia and the like.

[0038] “Compatible” as used herein, refers to the capability of operating with other components of a system. A vector or plant viral nucleic acid which is compatible with a host is one which is capable of replicating in that host. A coat protein which is compatible with a viral nucleotide sequence is one capable of encapsidating that viral sequence.

[0039] “Complementary” or “Complementarity”, as used herein, refer to the Watson-Crick base-pairing of two nucleic acid sequences. For example, for the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementarity between two nucleic acid sequences may be “partial”, in which only some of the bases bind to their complement, or it may be complete as when every base in the sequence binds to it complementary base. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0040] “Complementation analysis” as used herein, refers to observing the changes produced in an organism when a nucleic acid sequence is introduced into that organism after a selected gene has been deleted or mutated so that it no longer functions fully in its normal role. A complementary gene to the deleted or mutated gene can restore the genetic phenotype of the selected gene.

[0041] “Constitutive expression” as used herein refers to gene expression which features substantially constant or regularly cyclical gene transcription. Generally, genes which are constitutively expressed are substantially free of induction from an external stimulus.

[0042] “Correlates with expression of a polynucleotide”, as used herein, indicates that the detection of the presence of ribonucleic acid that is similar to and indicative of the presence of an mRNA encoding a polypeptide in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.

[0043] “Deletion”, as used herein, refers to a change made in either an amino acid or nucleotide sequence resulting in the absence one or more amino acids or nucleotides, respectively.

[0044] “Differentiated cell” as used herein refers to a cell which has substantially matured to perform one or more biochemical or physiological functions.

[0045] “Dwarf Plant” as used herein, refers to a plant that is much below the height or size of its kind or related species.

[0046] “Encapsidation” as used herein, refers to the process during virion assembly in which nucleic acid becomes incorporated in the viral capsid or in a head/capsid precursor (e.g. in certain bacteriophages).

[0047] “Exon” as used herein, refers to a polynucleotide sequence in a nucleic acid that codes information for protein synthesis and that is copied and spliced together with other such sequences to form messenger RNA.

[0048] “Expression” as used herein is meant to incorporate one or more of transcription, reverse transcription and translation.

[0049] “Expressed sequence tag (EST)” as used herein refers to relatively short single-pass DNA sequences obtained from one or more ends of cDNA clones and RNA derived therefrom. They may be present in either the 5′ or the 3′ orientation. ESTs have been shown useful for identifying particular genes.

[0050] “Foreign gene” as used herein, refers to any sequence that is not native to the virus.

[0051] “Fusion protein” as used herein, refers to a protein containing amino acid sequences from each of two distinct proteins; it is formed by the expression of a recombinant gene in which two coding sequences have been joined together such that their reading frames are in phase. Hybrid genes of this type may be constructed in vitro in order to label the product of a particular gene with a protein which can be more readily assayed (e.g. a gene fused with lacZ in E. coli to obtain a fusion protein with β-galactosidase activity). Alternatively, a protein may be linked to a signal peptide to allow its secretion by the cell. The products of certain viral oncogenes are fusion proteins.

[0052] “Gene” as used herein, refers to a discrete nucleic acid sequence responsible for a discrete cellular product and/or performing one or more intercellular or intracellular functions. The term “gene”, as used herein, refers not only to the nucleotide sequence encoding a specific protein, but also to any adjacent 5′ and 3′ non-coding nucleotide sequence involved in the regulation of expression of the protein encoded by the gene of interest. These non-coding sequences include terminator sequences, promoter sequences, upstream activator sequences, regulatory protein binding sequences, and the like. These non-coding sequence gene regions may be readily identified by comparison with previously identified eukaryotic non-coding sequence gene regions. Furthermore, the person of average skill in the art of molecular biology is able to identify the nucleotide sequences forming the non-coding regions of a gene using well-known techniques such as a site-directed mutagenesis, sequential deletion, promoter probe vectors, and the like.

[0053] “Growth cycle” as used herein is meant to include the replication of a nucleus, an organelle, a cell, or an organism.

[0054] “Half-life” as used herein, refers to the time required for half of something to undergo a process (e.g. the time required for half the amount of a substance, such as a drug or radioactive tracer, in or introduced into a living system or ecosystem to be eliminated or disintegrated by natural processes.

[0055] “Heterologous” as used herein, refers to the association of a molecular or genetic element associated with a distinctly different type of molecular or genetic element.

[0056] “Host” as used herein, refers to a cell, tissue or organism capable of replicating a vector or plant viral nucleic acid and which is capable of being infected by a virus containing the viral vector or plant viral nucleic acid. This term is intended to include procaryotic and eukaryotic cells, organs, tissues or organisms, where appropriate.

[0057] “Homology” as used herein, refers to the degree of similarity between two or more nucleotide or amino-acid sequences. Homology may be partial or complete.

[0058] “Hybridization”, as used herein, refers to any process by which a strand of nucleic acid binds with a complementary or partially complementary strand through base pairing.

[0059] “Hybridization complex”, as used herein, refers to a complex formed between nucleic acid strands by virtue of hydrogen bonding, stacking or other non-covalent interactions between bases. A hybridization complex may be formed in solution or between nucleic acid sequences present in solution and nucleic acid sequences immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization).

[0060] “Immunologically active” refers to the capability of a natural, recombinant, or synthetic gene product of interest, or any oligopeptide thereof, to bind with specific antibodies and induce a specific immune response in appropriate animals or cells.

[0061] “Induction” and the terms “induce”, “induction” and “inducible” as used herein, refer generally to a gene and a promoter operably linked thereto which is in some manner dependent upon an external stimulus, such as a molecule, in order to actively transcribed and/or translate the gene.

[0062] “Infection” as used herein refers to the ability of a virus to transfer its nucleic acid to a host or introduce a viral nucleic acid into a host, wherein the viral nucleic acid is replicated, viral proteins are synthesized, and new viral particles assembled. In this context, the terms “transmissible” and “infective” are used interchangeably herein. The term is also meant to include the ability of a selected nucleic acid sequence to integrate into a genome, chromosome or gene of a target organism.

[0063] “Insertion” or “Addition”, as used herein, refers to the replacement or addition of one or more nucleotides or amino acids, to a nucleotide or amino acid sequence, respectively.

[0064] “In cis” as used herein, indicates that two sequences are positioned on the same strand of RNA or DNA.

[0065] “In trans” as used herein, indicates that two sequences are positioned on different strands of RNA or DNA.

[0066] “Intron” as used herein refers to a polynucleotide sequence in a nucleic acid that does not code information for protein synthesis and is removed before translation of messenger RNA.

[0067] “Isolated” as used herein refers to a polypeptide, polynucleotide molecules separated not only from other peptides, DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule but also from other macromolecules and preferably refers to a macromolecule found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a solution of the same. “Isolated” and “purified” do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.

[0068] “Kinase” as used herein, refers to an enzyme (e.g. hexokinase and pyruvate kinase) which catalyzes the transfer of a phosphate group from one substrate (commonly ATP) to another.

[0069] “Marker” or “Genetic Marker” as used herein, refers to a genetic locus which is associated with a particular, usually readily detectable, genotype or phenotypic characteristic (e.g., an antibiotic resistance gene).

[0070] “Metabolome” as used herein, indicates the complement of relatively low molecular weight molecules that is present in a plant, plant part, or plant sample, or in a suspension or extract thereof. Examples of such molecules include, but are not limited to: acids and related compounds; mono-, di-,and tri-carboxylic acids (saturated, unsaturated, aliphatic and cyclic, aryl, alkaryl); aldo-acids, keto-acids; lactone forms; gibberellins; abscisic acid; alcohols, polyols, derivatives, and related compounds; ethyl alcohol, benzyl alcohol, menthanol; propylene glycol, glycerol, phytol; inositol, furfuryl alcohol, menthol; aldehydes, ketones, quinones, derivatives, and related compounds; acetaldehyde, butyraldehyde, benzaldehyde, acrolein, furfural, glyoxal; acetone, butanone; anthraquinone; carbohydrates; mono-, di-, tri-saccharides; alkaloids, amines, and other bases; pyridines (including nicotinic acid, nicotinamide); pyrimidines (including cytidine, thymine); purines (including guanine, adenine, xanthines/hypoxanthines, kinetin); pyrroles; quinolines (including isoquinolines); morphinans, tropanes, cinchonans; nucleotides, oligonucleotides, derivatives, and related compounds; guanosine, cytosine, adenosine, thymidine, inosine; amino acids, oligopeptides, derivatives, and related compounds; esters; phenols and related compounds; heterocyclic compounds and derivatives; pyrroles, tetrapyrroles (corrinoids and porphines/porphyrins, w/w/o metal-ion); flavonoids; indoles; lipids (including fatty acids and triglycerides), derivatives, and related compounds; carotenoids, phytoene; and sterols, isoprenoids including terpenes.

[0071] “Modulate” as used herein, refers to a change or an alteration in the biological activity of a gene product of interest. Modulation may be an increase or a decrease in protein activity, a change in binding characteristics, or any other change in the biological, functional or immunological properties of the gene product of interest.

[0072] “Movement protein” as used herein refers to a noncapsid protein required for cell to cell movement of replicons or viruses in plants.

[0073] “Multigene family” as used herein refers to a set of genes descended by duplication and variation from some ancestral gene. Such genes may be clustered together on the same chromosome or dispersed on different chromosomes. Examples of multigene families include those which encode the histones, hemoglobins, immunoglobulins, histocompatibility antigens, actins, tubulins, keratins, collagens, heat shock proteins, salivary glue proteins, chorion proteins, cuticle proteins, yolk proteins, and phaseolins.

[0074] “Non-Native” as used herein refers to any RNA or DNA sequence that does not normally occur in the cell or organism in which it is placed. Examples include recombinant plant viral nucleic acids and genes or ESTs contained therein. That is, a RNA or DNA sequence may be non-native with respect to a viral nucleic acid. Such a RNA or DNA sequence would not naturally occur in the viral nucleic acid. Also, a RNA or DNA sequence may be non-native with repect to a host organism. That is, such a RNA or DNA sequence would not naturally occur in the host organism. Conversely, the term non-native does not imply that a RNA or DNA sequence must be non-native with respect to both a viral nucleic acid and a host organism concurrently. The present invention specifically contemplates placing a RNA or DNA sequence which is native to a host organism into a viral nucleic acid in which it is non-native.

[0075] “Nucleic acid sequence” as used herein refers to a polymer of nucleotides in which the 3′ position of one nucleotide sugar is linked to the 5′ position of the next by a phosphodiester bridge. In a linear nucleic acid strand, one end typically has a free 5′ phosphate group, the other a free 3′ hydroxyl group. Nucleic acid sequences may be used herein to refer to oligonucleotides, or polynucleotides, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. The term is intended to encompass all nucleic acids whether naturally occurring in a particular cell or organism or non-naturally occurring in a particular cell or organism.

[0076] “Operably Linked” refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control i.e., transcriptional and/or translational control, of the regulatory sequences.

[0077] “Organism” and “host organism” as used herein is specifically intended to include animals (including humans), plants, viruses, fungi, and bacteria.

[0078] “Origin of Assembly” as used herein, refers to a sequence where self-assembly of the viral RNA and the viral capsid protein initiates to form virions.

[0079] “Outlier Peak” as used herein, indicates a peak of a chromatogram of a test sample, or the relative or absolute detected response data, or amount or concentration data thereof. An outlier peak: 1) may have a significantly different peak height or area as compared to a like chromatogram of a control sample; or 2) be an additional or missing peak as compared to a like chromatogram of a control sample.

[0080] “Phenotype” or “Phenotypic Trait(s)” as used herein, refers to an observable property or set of properties resulting from the expression or suppression of a gene or genes.

[0081] “Plant” as used herein refers to any plant and progeny thereof, and to parts of plants including parts of plants, including seed, cuttings, tubers, fruit, flowers, branches, leaves, plant cells and other parts of any tree or other plant used in forestry, ornamental horticultural plants, medicinal plants including any plants used to produce pharmaceutical products, and plants of the genus Nicotiana which are used for purposes other than for traditional tobacco products.

[0082] “Plant Cell” as used herein, refers to the structural and physiological unit of plants, consisting of a protoplast and the cell wall.

[0083] “Plant Organ” as used herein, refers to a distinct and visibly differentiated part of a plant, such as root, stem, leaf or embryo.

[0084] “Plant Tissue” as used herein, refers to any tissue of a plant in planta or in culture. This term is intended to include a whole plant, plant cell, plant organ, protoplast, cell culture, or any group of plant cells organized into a structural and functional unit.

[0085] “Portion” as used herein, with regard to a protein (i.e. “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

[0086] “Positive-sense inhibition” as used herein refers to a type of gene regulation based on cytoplasmic inhibition of gene expression due to the presence in a cell of an RNA molecule substantially homologous to at least a portion of the mRNA being translated.

[0087] “Production Cell” as used herein, refers to a cell, tissue or organism capable of replicating a vector or a viral vector, but which is not necessarily a host to the virus. This term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms, such as bacteria, yeast, fungus and plant tissue.

[0088] “Promoter” as used herein, refers to the 5′-flanking, non-coding sequence substantially adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.

[0089] “Protoplast” as used herein, refers to an isolated plant cell without cell walls, having the potency for regeneration into cell culture or a whole plant.

[0090] “Purified” as used herein when referring to a peptide or nucleotide sequence, indicates that the molecule is present in the substantial absence of other biological macromolecular, e.g., polypeptides, polynucleic acids, and the like of the same type. The term “purified” as used herein preferably means at least 95% by weight, more preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 can be present). The term “pure” as used herein preferably has the same numerical limits as “purified” immediately above.

[0091] “Substantially purified” as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.

[0092] “Recombinant Plant Viral Nucleic Acid” as used herein, refers to a plant viral nucleic acid which has been modified to contain non-native nucleic acid sequences. These non-native nucleic acid sequences may be from any organism or purely synthetic, however, they may also include nucleic acid sequences naturally occurring in the organism into which the recombinant plant viral nucleic acid is to be introduced.

[0093] “Recombinant Plant Virus” as used herein, refers to a plant virus containing a recombinant plant viral nucleic acid.

[0094] “Regulatory region” or “Regulatory sequence” as used herein in reference to a specific gene refers to the non-coding nucleotide sequences within that gene that are necessary or sufficient to provide for the regulated expression of the coding region of a gene. Thus the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. Specific nucleotides within a regulatory region may serve multiple functions. For example, a specific nucleotide may be part of a promoter and participate in the binding of a transcriptional activator protein.

[0095] “Replication origin” as used herein, refers to the minimal terminal sequences in linear viruses that are necessary for viral replication.

[0096] “Replicon” as used herein, refers to an arrangement of RNA sequences generated by transcription of a transgene that is integrated into the host DNA that is capable of replication in the presence of a helper virus. A replicon may require sequences in addition to the replication origins for efficient replication and stability.

[0097] “Sample”, as used herein, is used in its broadest sense. A biological sample suspected of containing a nucleic acid or fragments thereof may comprise a tissue, a cell, an extract from cells, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), and the like.

[0098] “Silent mutation” as used herein, refers to a mutation which has no apparent effect on the phenotype of the organism.

[0099] “Site-directed mutagenesis” as used herein, refers to the in-vitro induction of mutagenesis at a specific site in a given target nucleic acid molecule.

[0100] “Specific binding” or “specifically binding”, as used herein, in reference to the interaction of an antibody and a protein or peptide, mean that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words, the antibody is recognizing and binding to a specific protein structure rather than to proteins in general.

[0101] “Stringent conditions”, as used herein, is the “stringency” which occurs within a range from about (Tm−5)° C. (i.e. 5 degrees below the melting temperature, Tm, of the probe) to about 20° to 25° C. below Tm. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. Also as known in the art, numerous equivalent conditions may be employed to comprise either low or high stringency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

[0102] “Subgenomic Promoter” as used herein, refers to a promoter of a subgenomic mRNA of a viral nucleic acid.

[0103] “Substantial Sequence Homology” as used herein, denotes nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting function of the gene products or an RNA coded for by such sequence.

[0104] “Substitution”, as used herein, refers to a change made in an amino acid of nucleotide sequence which results in the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0105] “Systemic Infection” as used herein denotes infection throughout a substantial part of an organism including mechanisms of spread other than mere direct cell inoculation but rather including transport from one infected cell to additional cells either nearby or distant.

[0106] “Transcription” as used herein, refers to the production of an RNA molecule by RNA polymerase as a complementary copy of a DNA sequence.

[0107] “Transcription termination region” as used herein, refers to the sequence that controls formation of the 3′ end of the transcript. Self-cleaving ribozymes and polyadenylation sequences are examples of transcription termination sequences.

[0108] “Transformation” as used herein, describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

[0109] “Transposon” as used herein refers to a nucleotide sequence such as a DNA or RNA sequence which is capable of transferring location or moving within a gene, a chromosome or a genome.

[0110] “Transgenic plant” as used herein refers to a plant which contains a foreign nucleotide sequence inserted into either its nuclear genome or organellar genome.

[0111] “Transcription” as used herein refers to the production of an RNA molecule by RNA polymerase as a complementary copy of a DNA sequence or subgenomic mRNA.

[0112] “Variants” of a gene product of interest, as used herein, refers to a sequence resulting when the gene product is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Variants may also include sequences with amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art.

[0113] “Vector” as used herein, refers to a self-replicating DNA or RNA molecule which transfers a DNA or RNA segment between cells.

[0114] “Virion” as used herein, refers to a particle composed of viral RNA and viral capsid protein.

[0115] “Virus” as used herein, refers to an infectious agent composed of a nucleic acid encapsidated in a protein. A virus may be a mono-, di-, tri- or multi-partite virus.

THE INVENTION

[0116] Identification and Analysis of cDNAs

[0117] The invention is based on the discovery of 122 cDNAs, identified by the polynucleotide sequences SEQ ID NO: 1-122, that may be used to create transfected or transgenic plants exhibiting a dwarf phenotype. Table 1 lists the source organism for all 122 cDNAs of the invention (as identified by its SEQ ID NO).

TABLE 1
		Sense or
SEQ ID		Antisense
NO.	Source	Configuration
1	Nicotiana benthamiana	A
2	Nicotiana benthamiana	A
3	Arabidopsis thaliana	S
4	Arabidopsis thaliana	S
5	Arabidopsis thaliana	S
6	Arabidopsis thaliana	S
7	Arabidopsis thaliana	S
8	Arabidopsis thaliana	A
9	Arabidopsis thaliana	A
10	Arabidopsis thaliana	A
11	Arabidopsis thaliana	A
12	Arabidopsis thaliana	A
13	Arabidopsis thaliana	A
14	Arabidopsis thaliana	A
15	Arabidopsis thaliana	A
16	Arabidopsis thaliana	A
17	Arabidopsis thaliana	A
18	Arabidopsis thaliana	A
19	Arabidopsis thaliana	A
20	Arabidopsis thaliana	A
21	Arabidopsis thaliana	A
22	Arabidopsis thaliana	A
23	Arabidopsis thaliana	A
24	Arabidopsis thaliana	A
25	Arabidopsis thaliana	A
26	Arabidopsis thaliana	A
27	Arabidopsis thaliana	A
28	Arabidopsis thaliana	A
29	Arabidopsis thaliana	A
30	Arabidopsis thaliana	A
31	Arabidopsis thaliana	A
32	Arabidopsis thaliana	A
33	Arabidopsis thaliana	A
34	Arabidopsis thaliana	A
35	Arabidopsis thaliana	A
36	Arabidopsis thaliana	A
37	Arabidopsis thaliana	A
38	Arabidopsis thaliana	A
39	Arabidopsis thaliana	A
40	Arabidopsis thaliana	A
41	Arabidopsis thaliana	A
42	Arabidopsis thaliana	A
43	Arabidopsis thaliana	A
44	Arabidopsis thaliana	A
45	Arabidopsis thaliana	A
46	Arabidopsis thaliana	A
47	Arabidopsis thaliana	A
48	Arabidopsis thaliana	A
49	Arabidopsis thaliana	A
50	Arabidopsis thaliana	A
51	Arabidopsis thaliana	A
52	Arabidopsis thaliana	A
53	Arabidopsis thaliana	A
54	Arabidopsis thaliana	A
55	Arabidopsis thaliana	A
56	Arabidopsis thaliana	A
57	Arabidopsis thaliana	A
58	Arabidopsis thaliana	A
59	Arabidopsis thaliana	A
60	Arabidopsis thaliana	A
61	Arabidopsis thaliana	A
62	Arabidopsis thaliana	A
63	Arabidopsis thaliana	A
64	Arabidopsis thaliana	A
65	Arabidopsis thaliana	A
66	Arabidopsis thaliana	A
67	Arabidopsis thaliana	A
68	Arabidopsis thaliana	A
69	Arabidopsis thaliana	A
70	Arabidopsis thaliana	A
71	Arabidopsis thaliana	A
72	Arabidopsis thaliana	A
73	Arabidopsis thaliana	A
74	Arabidopsis thaliana	A
75	Arabidopsis thaliana	A
76	Arabidopsis thaliana	A
77	Arabidopsis thaliana	A
78	Arabidopsis thaliana	A
79	Arabidopsis thaliana	A
80	Arabidopsis thaliana	A
81	Arabidopsis thaliana	A
82	Arabidopsis thaliana	A
83	Arabidopsis thaliana	A
84	Arabidopsis thaliana	A
85	Arabidopsis thaliana	A
86	Arabidopsis thaliana	A
87	Arabidopsis thaliana	A
88	Arabidopsis thaliana	A
89	Arabidopsis thaliana	A
90	Arabidopsis thaliana	A
91	Arabidopsis thaliana	A
92	Arabidopsis thaliana	A
93	Arabidopsis thaliana	A
94	Arabidopsis thaliana	A
95	Arabidopsis thaliana	A
96	Arabidopsis thaliana	A
97	Arabidopsis thaliana	S
98	Arabidopsis thaliana	A
99	Arabidopsis thaliana	n.d.
100	Arabidopsis thaliana	n.d.
101	Arabidopsis thaliana	n.d.
102	Arabidopsis thaliana	n.d.
103	Arabidopsis thaliana	n.d.
104	Arabidopsis thaliana	n.d.
105	Arabidopsis thaliana	n.d.
106	Arabidopsis thaliana	n.d.
107	Arabidopsis thaliana	n.d.
108	Arabidopsis thaliana	n.d.
109	Arabidopsis thaliana	n.d.
110	Arabidopsis thaliana	n.d.
111	Arabidopsis thaliana	n.d.
112	Arabidopsis thaliana	A
113	Nicotiana benthamiana	A
114	Nicotiana benthamiana	A
115	Nicotiana benthamiana	A
116	‘Nicotiana benthamiana	S
117	Oryza japonica	S
118	Oryza japonica	S
119	Oryza indica	S
120	Oryza indica	S
121	Papaver rhoeas	S
122	Oryza japonica	S

[0118] The 122 cDNAs of the invention were identified by phenotypic screening and bioinformatic analysis of libraries of over 8000 cDNAs from Arabidopsis, Nicotiana, Oryza and Papaver constructed in the GENEWARE® vector. Table 1 lists whether the cDNA insert is in the sense (S) or antisense (A) configuration in the GENEWARE® vector used for the phenotypic screening. The use of the GENEWARE® vector in the field of genomics has been described in PCT WO 99/36516 published Jul. 22, 1999, which is herein incorporated by reference for all purposes. The general phenotypic screening method (described in greater detail below) involves constructing a GENEWARE® viral nucleic acid vector from each clone of a normalized cDNA library of interest. Each GENEWARE® vector is then used to create an infectious viral unit which is applied to the individual plants of interest. Inoculation with GENEWARE® viral nucleic acid vectors results in a high rate of systemic infection of plants. The TMV based viral vector identified as PBSG1057 which has the ablility to transfect plants has been deposited under the Budapest Treaty at the AFCC and is designated ATCC #203981. Infected (and uninfected) plants are grown under identical conditions and an automated visual phenotypic analysis is conducted of each plant. The phenotypic data including descriptive of various parts of each plant is entered into a matrix-style database created using LIMS software. Once in the database, the phenotypic results are linked to the sequence data and bioinformatic analysis associated with each of the GENEWARE® vector (i.e. each cDNA in the library).

[0119] Out of over 8000 Nicotiana benthamiana plants infected by the GENEWARE®, 111 were discovered that exhibited a dwarf phenotype. Sequence analysis of these cDNAs (as described in greater detail below) yielded the identifying nucleic acid sequences SEQ. ID. NOs. 1-111. Bioinformatic analysis of these sequences using BLAST and other methods (described in greater detail below) yielded E.C. annotations for a large number of these sequences.

[0120] Further bioinformatic analysis of the 111 polynucleotide sequences identified an additional 34 cDNAs that may also function to cause dwarf phenotype in plants. Pfam analysis (described in greater detail below) of the 111 cDNAs identified SEQ ID NO:95 and 102 as members of the transketolase functional family, and the pfkb carbohydrate kinase family, respectively. Using this information, the 11 additional sequences (identified by SEQ ID NO: 112-122) were discovered in the LSBC GENEWARE® libraries that are either a member of the transketolase having the same metabolic activity as SEQ ID NO. 95, or a member pfkb carbohydrate kinase families having the same metabolic activity as SEQ ID NO. 102.

[0121] Following the identification of plants exhibiting the dwarf phenotype, biochemical analyses of tissue may be carried out in order to ascertain further details of the expressed cDNAs function. Methods including GC/MS analysis and Maldi-TOF analysis of the tissue have been carried out (described in greater detail below) and yield information on the profile of metabolites and proteins present in the infected plant's tissue. The results of these biochemical analyses are linked to the phenotype, sequence, and other bioinformatic data associated with each of the GENEWARE® vector. Using these biochemical analysis methods, and associated data processing techniques, the identification of at least one variation in the metabolome of an infected (versus an uninfected) plant may ascribe a function to the cDNA of interest.

[0122] According to the present invention, the dwarf phenotype may be created in a wide variety of plants or plant cell systems using the cDNAs identified by SEQ ID NO:1-122 and the various transformation methods described. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, monocotyledonous and dicotyledonous plants, including horticultural and ornamental plants (e.g., the grass and turfgrass species, and flowering plants such as petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine, fir, spruce species, and including Abies sp., Acer glabrum, Pinus sp., Alnus sp., Arbutus arizonica, Betula occidentalis, Cedrus sp., Cryptomeriajaponica, Cupressus sp., Eucalyptus sp., Ginkgo biloba, Juniperus sp., Libocedrus decurrens, Liriodendron tulipifera, Lithocarpus densiflora, Metasequoia glyptostroboides, P. ponderosa var. scopulorum, Picea sp., Platanus sp., Populus sp., Pseudotsuga sp., Purshia tridentata, Quercus sp., Sequoia sp., Taxus brevifolia, Thuja sp., Torreya californica, Tsuga heterophylla, Umbellularia californica); plants used in phytoremediation (e.g., heavy metal accumulating plants), medicinal plants (e.g. Solanaceae, Atropa belladonna, Duboisia myoporides, Hyoscymus niger, Scopolina atropoides, Solanum tuberosum, Eschscholtzia californica, Berberis stolonifera, Papaver somniferum) and plants used for experimental purposes (e.g., Arabidopsis thaliana, Nicotiana sp.).

[0123] For a more complete listing of medicinal plants see Table 2. Another treatment of medicinal herbs can be found in, “1999 PDR for Herbal Medicines” 2nd edition, editors, Joerg Gruenwald et al.,, Medical Economics Company, Montvale, N.J., which is herein incorporated by reference for all purposes.

TABLE 2
Medicinal Plant             Medicinal Plant
Abies lasiocarpa            Juglans major
Abies excelsa               Juniperus communis
Abronia wootonii            Juniperus monosperma
Acacia arabica              Juniperus sibirica
Acacia catechu              Kallstroemia grandiflora
Acacia constricta           Kallstroemia spp.
Acacia greggii              Kalmia angustifolia
Acacia senegal              Kalmia latifolia
Acalypha californica        Kalmia microphylla
Acalypha lindheimeri        Kalmia polifolia
Achillea lanulosa           Karwinskia humboldtiana
Achillea millefolium        Krameria grayi
Achlys triphylla            Krameria lanceolata
Aconitum columbianum        Krameria parvifolia
Acorus calamus              Lactuca serriola
Actaea alba                 Lamium amplexicaule
Actea rubra                 Larrea tridentata
Adiantum capillus-veneris   Ledum glandulosum
Adiantum jordanii           Ledum groenlandicum
Adiantum pedatum            Leonurus cardiaca
Adoxa moschatellina         Leonurus sibirica
Aesculus californica        Lepechinia calycina
Aesculus glabra             Lepidium montanum
Aesculus hippocastanum      Lespedeza violacea
Aesculus pavia              Leucophyllum frutescens
Agastache urticifolia       Levisticum ligusticum
Agave chisoensis            Lewisia rediviva
Agave parryi                Liatris punctata
Agrimonia gryposepala       Liatris squarrosa
Agrimonia striata           Ligusticum filicinum
Agropyron repens            Ligusticum grayi
Alchemilla mollis           Ligusticum porteri
Alchemilla vulgaris         Lilium grayi
Aletris farinosa            Lilium philadelphicum
Alhagi camelorum            Linaria canadensis
Allium cernuum              Linaria dalmatica
Allium geyeri               Linaria vulgaris
Allium schoenoprasum        Linnaea borealis
Alnus incana                Linum lewisii
Aloe spp.                   Linum medium
Aloe vera                   Linum usitatissimum
Althea officinalis          Liquidambar orientalis
Amaranthus hybridus         Liquidamber styraciflua
Ambrosia ambrosioides       Lithospermum arvense
Ambrosia artemisiifolia     Lithospermum multiflorum
Ambrosia trifida            Lithospermum ruderale
Amelanchier alnifolia       Lobelia cardinalis
Amsinckia intermedia        Lobelia cardinalis,
Amsonia hirtella            Lobelia cardinalis,
Amygdalus persica           Lobelia inflata
Anaphalis margaritacea      Lobelia kalmii
Anemone deltoidea           Lobelia siphilitica
Anemone globosa             Lomatium cous
Anemone halleri             Lomatium dissectum
Anemone occidentalis        Lophocereus (Pachycereus)
Anemone patens              Lycium fremontii
Anemone patens,             Lycium pallidum
Anemone quinquefolia        Lycopodium clavatum
Anemone tuberosa            Lycopus americanus
Anemopsis californica       Lycopus asper
Anethum graveolens          Lycopus uniflorus
Angelica sp.                Lycopus virginicus
Angelica archangelica       Lysichitum americanum
Angelica arguta             Lythrum salicaria
Angelica dawsonii           Macromeria viridiflora
Angelica genuflexa          Magnolia grandiflora
Angelica grayi              Mahonia aquifolia
Angelica hendersonii        Mahonia fremontii
Angelica lineariloba        Mahonia haematocarpa
Angelica pinnata            Mahonia nervosa
Angelica venenosa           Mahonia repens
Antennaria howellii         Mahonia trifoliata
Antennaria rosea            Mahonia wilcoxii
Apocynum androsaemifolium   Malus sylvestris
Apocynum cannabinum         Malva neglecta
Apocynum medium             Mammillaria arizonica
Aquilegia caerulea          Marah gilensis
Aquilegia chrysantha        Marrubium vulgare
Aralia californica          Matricaria chamomilla
Aralia nudicaulis           Matricaria matricarioides
Aralia racemosa             Medicago sativa
Aralia spinosa              Melampyrum lineare
Arbutus menziesii           Melilotus albus
Arctium minus               Menispermum canadense
Arctostaphylos pungens      Mentha arvensis
Arctostaphylos uva-ursi     Mentha pulegium
Argemone corymbosa          Mentha spicata
Argemone mexicana           Menyanthes trifoliata
Argemone platyceras         Mertensia ciliata
Argemone polyanthemos       Mimulus guttatus
Arisaema atrorubens         Mirabilis longiflora
Arisaema dracontium         Mirabilis multiflorum
Arisaema stewardsonii       Mitchella repens
Arisaema triphyllum         Monarda citriodora
Aristolochia californica    Monarda didyma
Aristolochia serpentaria    Monarda fistulosa
Aristolochia watsonii       Monarda media
Arnica angustifolium        Monarda menthaefolia
Arnica cordifolia           Monarda mollis
Arnica latifolia            Monarda pectinata
Arnica mollis               Monarda punctata
Arnica montana              Monardella villosa
Artemisia douglasiana       Moneses uniflora
Artemisia filifolia         Monotropa hypopitys
Artemisia franserioides     Monotropa uniflora
Artemisia frigida,          Mortonia scabrella
Artemisia frigida           Myrica californica
Artemisia ludoviciana       Myrica cerifera
Artemisia tridentata        Myristica fragrans
Artemisia vulgaris          Nelumbo lutea
Asarum canadense            Nepeta cataria
Asarum caudatum             Nicotiana attenuata
Asclepias albicans          Nicotiana glauca
Asclepias asperula          Nicotiana repanda
Asclepias brachystephana    Nicotiana tabacum
Asclepias erosa             Nicotiana trigonophylla
Asclepias fascicularis      Nuphar luteum
Asclepias speciosa          Nymphaea odorata
Asclepias subulata          Ocimum basilicum
Asclepias syriaca           Oenothera biennis
Asclepias texana            Oenothera hookeri
Asclepias tuberosa          Oplopanax horridum
Asclepas viridis            Opuntia erinacea
Asclepias viridis           Opuntia phaeacantha
Asparagus officinale        Orobanche fasciculata
Aspidium filix-mas          Orobanche ludoviciana
Astragalus gummifer         Orobanche uniflora
Astragalus americanus       Osmorhiza obtusa
Astragalus membranaceus     Osmorrhiza longistylis
Atriplex canescens          Osmorrhiza occidentalis
Avena fatua                 Ourouparia gambir
Avena sativa                Oxalis cymosa
Balsamorhiza sagittata      Oxalis oregana
Baptisia australis          Oxalis metcalfei
Baptisia leucantha          Paeonia brownii
Baptisia leucophaea         Paeonia californica
Baptisia sphaerocarpa       Panax quinquefolium
Baptisia tinctoria          Panax trifolium
Buddleya sp.                Papaver rhoeas
Berberis fendleri           Papaver somniferum
Berberis vulgaris           Parthenium incanum
Berberis -                  Parthenocissus inserta
Besseya wyomingensis        Parthenocissus quinquefolia
Bidens frondosa             Passiflora foetida
Bidens pilosa               Passiflora incarnata
Bignonia capreolata         Passiflora lutea
Bouvardia ternifolia        Passiflora sanguinea
Brassica arvensis           Paullinia cupana
Brickellia amplexicaulis    Pedicularis bracteosa
Brickellia californica      Pedicularis canadensis
Brickellia grandiflora      Pedicularis contorta
Brugmansia sp.              Pedicularis densiflora
Bryonia alba                Pedicularis grayii
Bupleurum americanum        Pedicularis groenlandica
Bursera microphylla         Pedicularis lanceolata
Bursera odorata             Pedicularis parryi
Cacalia decomposita         Pedicularis racemosa
Caesalpinia gilliessii      Peganum harmala
Caesalpinia pulcherrima     Peniocereus greggii
Caffea arabica              Penstemon cobaea
Calendula officinalis       Penstemon eatoni
Callirhoe involucrata       Penstemon lyallii
Caltha biflora              Perezia nana
Caltha leptosepala          Perezia wrightii
Caltha palustris            Perideridia gairdneri
Calypso bulbosa             Perilla frutescens
Camassia quamash            Petasites frigidus
Camissonia (Oenothera)      Petasites frigidus,
Campsis radicans            Petasites sagittatus
Cannabis sativa             Philadelphus lewisii
Capsella bursa-pastoris     Phoradendron flavescens
Capsicum annuum             Phoradendron juniperinum
Capsicum frutescens         Physalis crassifolia
Cardamine cordifolia        Physocarpus monogynus
Carnegia gigantea           Physostigma venenosum
Cassia angustifolia         Phytolacca americana
Cassia covesii              Picea engelmanni
Cassia fasciculata          Pinus contorta
Cassia fistula              Pinus edulis
Cassia leptocarpa           Pinus palustris
Cassia marilandica          Pinus ponderosa
Cassia senna                Pinus strobus
Cassia wislizenii           Pinus taeda
Castanopsis chrysophylla    Piper sp
Castela emoryi              Piper cubeba
Castilleja sp.              Plantago lanceolata
Castilleja miniata          Plantago major
Caulophyllum thalictrioides Plantago patagonica
Ceanothus americanus        Plantago rugeli
Ceanothus cuneatus          Pluchea camphorata
Ceanothus fendleri          Podophyllum peltatum
Ceanothus greggii           Polygala alba
Ceanothus herbaceum         Polygala lutea
Ceanothus spinosus          Polygala obscura
Ceanothus velutinus         Polygala paucifolia
Celastrus scandens          Polygala senega
Celtis occidentalis         Polygonatum biflorum
Centaurium venustum         Polygonatum canaliculatum
Cephaelis ipecacuanha       Polygonum bistortioides
Cephalanthus occidentalis   Polymnia spp
Cerastium arvense           Polymnia canadensis
Cercis occidentalis         Polypodium glycyrriza
Cercocarpus sp.             Polystichum munitum
Cetraria islandica          Populus balsamifera
Chamaelirium luteum         Populus fremontii
Chelidonium majus           Populus tremulioides
Chelone glabra              Portulaca oleracea
Chelone lyoni               Potentilla diversifolia
Chenopodium ambrosioides    Potentilla fruticosa
Chilopsis linearis          Potentilla palustris
Chimaphila umbellata        Potentilla strigosa
Chimaphila umbellata,       Potentilla tridentata
Chionanthus virginiana      Proboscidea parviflora
Chlorogalum pomeridianum    Prosopis juliflora
Chondrus crispus            Prunella vulgaris
Choisya arizonica           Prunus americana
Chrysanthemum leucanthemum  Prunus avium
Chrysanthemum parthenium    Prunus laurocereus
Cichorium intybus           Prunus serotina
Cicuta douglasii            Prunus virginiana
Cimicifuga arizonica        Pseudotsuga menziesii
Cimicifuga elata            Psoralea esculenta
Cimicifuga racemosa         Ptelea pallida
Cinchona succirubra         Ptelea trifoliata
Cinnamomum camphora         Pulsatilla ludoviciana
Cirsium undulatum           Punica granatum
Citrullus colocynthis       Purshia tridentata
Citrus sinensis             Pyrola asarifolia
Claviceps purpurea          Pyrola minor
Claytonia lanceolata        Pyrola rotundifolia
Clematis columbiana         Pyrola secunda
Clematis hirsutissima       Prola virens
Clematis ligusticifolia     Quercus alba
Clematis pseudoalpina       Quercus gambelii
Clematis viorna             Quillaja saponaria
Clematis virginiana         Ratibida columnaris
Cleome serrulata            Rhamnus alnifolia
Cocculus sp.                Rhamnus betulifolia
Cola nitida                 Rhamnus californica
Colchicum autumnale         Rhamnus frangula
Collinsonia canadensis      Rhamnus purshiana
Commandra umbellata         Rheum officinale
Conium maculatum            Rhus choriophylla
Conopholis alpina           Rhus glabra
Conopholis americana        Rhus microphylla
Convallaria majus           Rhus (Toxicodendron)
Convolvulus arvensis        Rhus trilobata
Convolvulus scammonia       Ribes aureum
Conyza canadense            Ricinus communis
Copaiba langsdorffii        Romneya coulteri
Coptis groenlandica         Rosa acicularis
Coptis laciniata            Rosa humilis
Coptis occidentalis         Rosa virginiana
Corallorhiza maculata       Rosa woodsii
Corallorrhiza striata       Rubus idaeus
Cordia boissieri            Rubus odoratus
Cornus canadensis           Rubus parviflorus
Cornus florida              Rudbeckia hirta
Cornus stolonifera          Rudbeckia laciniata
Corydalis aureus            Ruellia ciliosa
Corydalis sempervirens      Rumex acetosella
Crataegus spp.              Rumex crispus
Crataegus columbiana        Rumex hymenosepalus
Crataegus douglasii         Ruta graveolens
Crataegus mollis            Sabal texana
Crataegus rivularis         Sabatia angularis
Crataegus succulenta        Sabatia campestris
Cucurbita foetidissima      Sabatia stellaris
Cupressus arizonica         Sagittaria cuneata
Cupressus macrocarpa        Sagittaria latifolia
Curcuma sp.                 salix sp.
Cuscuta gronovi             Salix discolor
Cymopterus fendleri         Salvia apiana
Cynanchum nigrum            Salvia azurea
Cynara sp.                  Salvia clevelandii
Cynoglossum officinale      Salvia columbariae
Cypripedium sp.             Salvia greggii
Cypripedium acaule          Salvia henryi
Cypripedium arietinum       Salvia lemmonii
Cypripedium calceolus       Salvia leucophylla
Cypripedium montanum        Salvia mellifera
Cypripedium parviflorum     Salvia regla
Cypripedium reginae         Salvia reflexa
Cytisus scoparius           Salvia spathaceae
Dalea formosa               Sambucus canadensis
Darlingtonia californica    Sambucus mexicana
Datura ferox                Sambucus racemosa
Datura metelioides          Sanguinaria canadensis
Datura wrightii             Sanguisorba canadensis
Daucus carota               Sanicula marilandica
Delphinium barbeyi          Santalum album
Delphinium elongatum        Sanvitalia abertii
Dendromecon rigida          Sapindus saponaria
Dicentra canadensis         Saponaria officinalis
Dicentra cucullaria         Sarracenia psittacina
Dicentra formosa            Sarracenia purpurea
Dicentra spectabilis        Sarracenia rubra
Digitalis purpurea          Sassafras IL
Dionaea muscipula           Satureja douglasii
Dioscorea villosa           Saururus cernuus
Dipsacus sylvestris         Scopola carniolica
Dipsacus fullonum           Scrophularia californica
Dodecathion pulchellum      Scrophularia lanceolata
Dracocephalum moldavica     Scutellaria brittonii
Dracocephalum parviflorum   Scutellaria californica
Drosera linearis            Scutellaria drummondii
Drosera rotundifolia        Scutellaria epilobiifolia
Dyssodia papposa            Scuteliaria galericulata
Ecballium elaterium         Scutellaria incana
Echevaria rusbyi            Scutellaria integrifolia
Echinacea angustifolia      Scutellaria latiflora
Echinacea pallida           Scutellaria resinosa
Echinacea purpurea          Scutellaria serrata
Echinacea tennessiensis     Scutellaria tesselata
Elettaria carmamomum        Scutellaria wrightii
Encelia farinosa            Sedum rhodanthum
Ephedra californica         Sedum roseum
Ephedra nevadensis          Selenicereus spp.
Ephedra torreyana           Senecio aureus
Ephedra trifurca            Senecio cineraria
Ephedra viridis             Sequoia sempervirens
Epifagus virginianum        Serenoa repens
Epigaea repens              Shephardia argentea
Epilobium angustifolium     Shephardia canadensis
Epilobium hirsutum          Sida hederacea
Epipactis gigantea          Sidalcea neomexicana
Epipactis helleborine       Sidalcea malvaeflora
Equisetum arvense           Silphium laciniata
Equisetum pratense          Silphium perfoliatum
Eremocarpus setigerus       Silphium terebinthinaceum
Eriodictyon angustifolia    Silybum marianum
Eriodictyon californica     Simmondsia chinensis
Eriodictyon crassifolium    Smilacina racemosa
Eriodictyon glutinosa       Smilacina stellata
Eriogonum leptophyllum      Smilacina trifolia
Eriogonum umbellata         Smilax spp.
Eriogonum wrightii          Smilax californica
Erodium cicutarium          Smilax glauca
Eryngium leavenworthii      Smilax herbacea
Eryngium lemmonii           Smilax rotundifolia
Eryngium yuccafolium        Solanum carolinense
Erysimum capitatum          Solanum dulcamara
Erythronium grandiflorum    Solanum eleagnifolium
Erythronium montanum        Solanum nodiflorum
Erythroxylon coca           Solidago canadensis
Eschscholtzia californica   Sophora secundiflora
Eschscholtzia mexicana      Sorbus scopulina
Eschscholtzia minutiflora   Spartium junceum
Eucalyptus sp.              Sphaeralcea ambigua
Euonymus occidentalis       Sphaeralcea angustifolia
Eupatorium coelestinum      Sphaeralcea coccinea
Eupatorium greggii          Sphaeralcea fendleri
Eupatorium herbaceum        Sphaeralcea parviflora
Eupatorium maculatum        Sphenosciadium capitellatum
Eupatorium perfoliatum      Spigelia marilandica
Eupatorium purpureum        Spiraea alba
Eupatorium rugosum          Spiraea tomentosa
Eustoma grandiflorum        Stachys albens
Eysenhardtia polystachya    Stachys palustris
Fallugia paradoxa           Stachys rigida
Ferula foetida              Stellaria media
Ferula galbaniflua          Stenocereus thurberi
Flourensia cernua           Sticta PH
Fouquieria splendens        Stillingia sylvatica
Fragaria glauca             Streptopus amplexifolius
Fragaria ovalis             Strychnos nux-vomica
Fragaria virginiana         Swertia radiata
Frankenia grandiflora       Symphytum officinalis
Frankenia palmeri           Symplocarpus foetidus
Fraxinus ornus              Tanacetum huronense
Fremontia californica       Tanacetum parthenium
Fritillaria atropurpurea    Tanacetum vulgare
Fritillaria pudica          Taraxacum sp.
Fucus vesiculosus           Taxus brevifolia
Fumaria officinalis         Tecoma stans
Gaillardia pinnatifida      Teucrium laciniatum
Galium aparine              Thalictrum fendleri
Galium borealis             Thamnosma texana
Garcinia hanburyi           Thamnosma montana
Garrya spp.                 Thelesperma gracile
Garrya elliptica            Tephrosia virginiana
Garrya flavescens           Thermopsis montana
Garrya wrightii             Thuja plicata
Gaultheria procumbens       Thymus vulgaris
Gaultheria shallon          Tillandsia recurvata
Gaura lindheimeri           Tillandsia usnioides
Gaura parviflora            Toluifera balsamum
Gaylussacia brachycera      Toluifera pereirae
Gelsemium sempervirens      Toxicodendron radicans
Gentiana affinis            Toxicodendron vernix
Gentiana algida             Tradescantia occidentalis
Gentiana andrewsi           Tragopogon dubius
Gentiana calycosa           Trautvettaria carolinensis
Gentiana crinata            Tribulus terrestrus
Gentiana heterosepala       Trichostema lanatum
Gentiana parryi             Trifolium pratense
Gentiana saponaria          Trillium erectum
Gentiana simplex            Trillium grandiflorum
Gentiana thermalis          Trillium ovatum
Gentianella (Gentian)       Trillium sessile
Geranium maculatum          Trillium undulatum
Geranium richardsonii       Trollius laxus
Geranium viscosissimum      Tsuga mertensiana
Geum rivale                 Turnera diffusa
Geum triflorum              Umbellularia californica
Gigartina mamillosa         Urginea maritima
Gillenia trifoliata         Urtica dioica
Glecoma hederacea           Usnea barbata
Glycyrriza glabra           Usnea hirsutissima
Glycyrrhiza lepidota        Vaccinium corymbosum
Gnaphallium sp.             Vaccinium myrtillus
Goodyera spp.               Vaccinium ovatum
Gossypium thurberi          Vaccinium oxycoccos
Grindelia aphanactis        Vaccinium parvifolium
Grindelia squarrosa         Vaccinium scoparium
Guaiacum angustifolium      Vaccinium tenellum
Guaiacum coulteri           Vaccinium uliginosum
Guaiacum sanctum            Vaccinium vitis-idaea
Gutierrezia sarothrae       Valeriana acutiloba
Habenaria blephariglottis   Valeriana arizonica
Habeneria fimbriata         Valeriana edulus
Habenaria (Plantanthera)    Valeriana officinalis
Hagenia abyssinica          Valeriana occidentalis
Hamamelis virginiana        Valeriana sitchensis
Haplopappus laricifolius    Vancouveria hexandra
Hedeoma hyssopifolium       Veratrum californicum
Hedeoma oblongifolia        Veratrum viride
Hedysarum alpinum           Verbascum blattaria
Helenium (Dugaldia)         Verbascum thapsus
Heliotropium convolvulaceum Verbena bipinnatifida
Heracleum lanatum           Verbena bracteata
Heterotheca grandiflora     Verbena canadensis
Heterotheca psammophylla    Verbena ciliata
Heterotheca subaxillaris    Verbena gooddingii
Heuchera americanus         Verbena hastata
Heuchera micrantha          Verbena macdougalii
Heuchera parvifolia         Verbena stricta
Heuchera sanguinea          Verbena wrightii
Hibiscus moscheutos         Verbesina encelioides
Hibiscus oculiroseus        Veronica americana
Hierochloe odorata          Veronica chamaedrys
Holodiscus dumosus          Veronicastrum IM
Humulus americanus          Viburnum acerifolium
Humulus lupulus             Viburnum americanum
Hydrastis canadensis        Viburnum cassinoides
Hydrocotyle bonariensis     Viburnum edule
Hydrophyllum capitatum      Viburnum ellipticum
Hyocyamus niger             Viburnum opulus
Hypericum ascyron           Viburnum prunifolium
Hypericum aureum            Viburnum rufidulum
Hypericum formosum          Vigueria dentata
Hypericum perforatum        Vinca major
Hyptis emoryi               Viola sp
Hyssopus officinalis        Viola canadensis
Ilex vomitoria              Viola pedata
Impatiens biflora           Viola tricolor
Impatiens capensis          Vitex agnus-castus
Impatiens pallida           Xanthium spinosum
Indigofera sphaerocarpa     Xanthium strumarium
Inula helenium              Xerophyllum tenax
Ipomea arborescens          Yucca baccata
Ipomea jalapa               Yucca baileyi
Ipomea leptophylla          Yucca elata
Iris missouriensis          Yucca schottii
Iris prismatica             Zanthoxylum fagaria
Iris versicolor             Zauschneria latifolia
Jateorhiza palmata          Zigadenus elegans
Jatropha cardiophylla       Zigadenus venenosus
Jatropha dioica             Zingiber sp.
Jatropha macrorhiza         Zizia aptera
Jeffersonia diphylla

[0124] The dwarf phenotype may be created using the cDNAs of the present invention in conjunction with a wide variety of plant virus expression vectors. The plant virus selected may depend on the plant system chosen and its known susceptibility to viral infection. Preferred embodiments of the plant virus expression vectors include, but are not limited to those in Table 3.

TABLE 3
Plant Viruses                    Plant Viruses
Abelia latent tymovirus          Lucerne transient streak
Abutilon mosaic bigeminivirus    Lychnis ringspot hordeivirus
Ahlum waterborne carmovirus      Maclura mosaic macluravirus
Alfalfa 1 alphacryptovirus       Maize dwarf mosaic potyvirus
Alfalfa 2 betacryptovirus        Maize streak monogeminivirus
Alfalfa mosaic alfamovirus       Maracuja mosaic tobamovirus
Alsike clover vein mosaic virus  Marigold mottle potyvirus
Alstroemeria ilarvirus           Melandrium yellow fleck
Alstroemeria mosaic potyvirus    Melilotus mosaic potyvirus
Alstroemeria streak potyvirus    Melon Ourmia ourmiavirus
Amaranthus leaf mottle potyvirus Melothria mottle potyvirus
Amaryllis alphacryptovirus       Milk vetch dwarf nanavirus
Amazon lily mosaic potyvirus     Mulberry latent carlavirus
Apple mosaic ilarvirus           Muskmelon vein necrosis carlavirus
Apple stem grooving capillovirus Myrobalan latent ringspot nepovirus
Arabis mosaic nepovirus          Nandina mosaic potexvirus
Arracacha A nepovirus            Narcissus late season yellows
Arracacha A nepovirus            Narcissus latent macluravirus
Arracacha B nepovirus            Narcissus mosaic potexvirus
Arracacha Y potyvirus            Narcissus tip necrosis carmovirus
Artichoke Italian latent nepovirus Narcissus tip necrosis carmovirus
Artichoke latent potyvirus       Narcissus yellow stripe potyvirus
Artichoke latent S carlavirus    Neckar River tombusvirus
Artichoke mottled crinkle        Nerine potyvirus
Artichoke vein banding nepovirus Nicotiana velutina mosaic furovirus
Artichoke yellow ringspot        Oat blue dwarf marafivirus
Asparagus 1 potyvirus            Oat blue dwarf marafivirus
Asparagus 2 ilarvirus            Oat golden stripe furovirus
Asparagus 3 potexvirus           Odontoglossum ringspot
Aster chlorotic stunt carlavirus Okra leaf-curl bigeminivirus
Asystasia gangetica mottle       Okra mosaic tymovirus
Aucuba ringspot badnavirus       Olive latent 1 sobemovirus
Barley stripe mosaic hordeivirus Olive latent 2 ourmiavirus
Barley stripe mosaic hordeivirus Onion mite-borne latent potexvirus
Barley yellow dwarf luteovirus   Onion yellow dwarf potyvirus
Barley yellow streak mosaic virus Orchid fleck rhabdovirus
Bean calico mosaic bigeminivirus Panicum mosaic sobemovirus
Bean common mosaic potyvirus     Papaya mosaic potexvirus
Bean distortion dwarf            Papaya ringspot potyvirus
Bean leaf roll luteovirus        Paprika mild mottle tobamovirus
Bean pod mottle comovirus        Parietaria mottle ilarvirus
Bean yellow mosaic potyvirus     Parsnip leafcurl virus
Beet curly top hybrigeminivirus  Parsnip mosaic potyvirus
Beet leaf curl rhabdovirus       Parsnip yellow fleck sequivirus
Beet mild yellowing luteovirus   Passiflora ringspot potyvirus
Beet mosaic potyvirus            Passionfruit woodiness potyvirus
Beet necrotic yellow vein furovirus Patchouli mosaic potyvirus
Beet pseudo-yellows closterovirus Pea early browning tobravirus
Beet soil-borne furovirus        Pea enation mosaic enamovirus
Beet western yellows leuteovirus Pea mild mosaic comovirus
Beet yellows closterovirus       Pea mosaic potyvirus
Belladonna mottle tymovirus      Pea seed-borne mosaic potyvirus
Bidens mosaic potyvirus          Pea streak carlavirus
Black raspberry necrosis virus   Peach enation nepovirus
Blueberry leaf mottle nepovirus  Peach rosette mosaic nepovirus
Blueberry necrotic shock ilarvirus Peanut chlorotic streak caulimovirus
Bramble yellow mosaic potyvirus  Peanut clump furovirus
Broad bean mottle bromovirus     Peanut mottle potyvirus
Broad bean necrosis furovirus    Peanut stunt cucumovirus
Broad bean stain comovirus       Peanut yellow spot tospovirus
Broad bean true mosaic comovirus Pelargonium flower break
Broad bean wilt fabavirus        Pelargonium line pattern
Brome mosaic bromovirus          Pelargonium vein clearing
Burdock yellow mosaic potexvirus Pelargonium zonate spot
Cacao necrosis nepovirus         Pepino mosaic potexvirus
Cacao swollen shoot badnavirus   Pepper Indian mottle potyvirus
Cacao yellow mosaic tymovirus    Pepper mild mosaic potyvirus
Cactus 2 carlavirus              Pepper mild mottle tobamovirus
Cactus X potexvirus              Pepper Moroccan tombusvirus
Canavalia maritima mosaic        Pepper mottle potyvirus
Caper latent carlavirus          Pepper ringspot tobravirus
Caraway latent nepovirus         Pepper severe mosaic potyvirus
Carnation rhabdovirus            Pepper Texas bigeminivirus
Carnation rhabdovirus            Pepper veinal mottle potyvirus
Carnation 1 alphacryptovirus     Petunia asteroid mosaic
Carnation 2 alphacryptovirus     Physalis mild chlorosis luteovirus
Carnation etched ring caulimovirus Physalis mosaic tymovirus
Carnation Italian ringspot       Pineapple chlorotic leaf streak
Carnation latent carlavirus      Pineapple wilt-associated
Carnation mottle carmovirus      Pittosporum vein yellowing
Carnation mottle carmovirus      Plantain 6 carmovirus
Carnation necrotic fleck         Plantain 7 potyvirus
Carnation ringspot dianthovirus  Plantain X potexvirus
Carnation vein mottle potyvirus  Plum American line pattern ilarvirus
Carnation yellow stripe necrovirus Plum pox potyvirus
Carrot mosaic potyvirus          Poinsettia mosaic tymovirus
Carrot mottle mimic umbravirus   Poplar mosaic carlavirus
Carrot mottle umbravirus         Poplar vein yellowing
Carrot yellow leaf closterovirus Potato 14R tobamovirus
Cassava African mosaic           Potato A potyvirus
Cassava brown streak potyvirus   Potato Andean latent tymovirus
Cassava brown streak-associated  Potato Andean mottle comovirus
Cassava Caribbean mosaic         Potato aucuba mosaic potexvirus
Cassava Colombian symptomless    Potato black ringspot nepovirus
Cassava common mosaic            Potato leafroll luteovirus
Cassava green mottle nepovirus   Potato M carlavirus
Cassava Indian mosaic            Potato mop-top furovirus
Cassava Ivorian bacilliform      Potato mop-top furovirus
Cassava Ivorian bacilliform      Potato T trichovirus
Cassava X potexvirus             Potato U nepovirus
Cassia mild mosaic carlavirus    Potato V potyvirus
Cassia severe mosaic closterovirus Potato X potexvirus
Celery latent potyvirus          Potato Y potyvirus
celery mosaic potyvirus          Potato yellow dwarf
Cherry leaf roll nepovirus       Primula mosaic potyvirus
Chickpea bushy dwarf potyvirus   Primula mottle potyvirus
Chickpea chlorotic dwarf         Prune dwarf ilarvirus
Chickpea distortion mosaic       Prunus necrotic ringspot ilarvirus
Chicory yellow mottle nepovirus  Radish mosaic comovirus
Chilli veinal mottle potyvirus   Raspberry ringspot nepovirus
Chino del tomat, bigeminivirus   Red clover mottle comovirus
Citrus leaf rugose ilarvirus     Red clover necrotic mosaic
Citrus ringspot virus            Red clover vein mosaic carlavirus
Clover mild mosaic virus         Rhynchosia mosaic bigeminivirus
Clover wound tumor phytoreovirus Ribgrass mosaic tobamovirus
Clover wound tumor phytoreovirus Rice hoja blanca tenuivirus
Clover yellow mosaic potexvirus  Rice stripe necrosis furovirus
Clover yellow vein potyvirus     Rice stripe tenuivirus
Colocasia bobone disease         Rose tobamovirus
Commelina X potexvirus           Rubus Chinese seed-borne
Cowpea chlorotic mottle          saguaro cactus carmovirus
Cowpea mild mottle carlavirus    Scrophularia mottle tymovirus
Cowpea mosaic comovirus          Shallot latent carlavirus
Cowpea mosaic comovirus          Shallot mite-borne latent potexvirus
Cowpea mottle carmovirus         Shallot yellow stripe potyvirus
Cowpea severe mosaic comovirus   Silene X potexvirus
Cowpea severe mosaic comovirus   Sint-Jan's onion latent carlavirus
Croton yellow vein mosaic        Sitke waterborne tombusvirus
Cucumber green mottle mosaic     Solanum apical leaf curling
Cucumber leaf spot carmovirus    Solanum nodiflorum mottle
Cucumber mosaic cucumovirus      Solanum nodiflorum mottle
Cucumber mosaic cucumovirus      Sonchus cytorhabdovirus
Cucumber necrosis tombusvirus    Sonchus yellow net
Cycas necrotic stunt nepovirus   Sorghum mosaic potyvirus
Cymbidium ringspot tombusvirus   Sowbane mosaic sobemovirus
Cynara nucleorhabdovirus         Soybean crinkle leaf bigeminivirus
Dahlia mosaic caulimovirus       Soybean dwarf luteovirus
Dandelion yellow mosaic          Soybean mild mosaic virus
sequivirus                       Soybean mosaic potyvirus
Daphne Y potyvirus               Spinach latent ilarvirus
Dasheen bacilliform badnavirus   Spinach temperate alphacryptovirus
Dasheen mosaic potyvirus         Spring beauty latent bromovirus
Datura Colombian potyvirus       Statice Y potyvirus
Datura distortion mosaic potyvirus Strawberry latent ringspot
Datura innoxia Hungarian mosaic  Subterranean clover red leaf
Datura mosaic potyvirus          Sugarcane mosaic potyvirus
Datura necrosis potyvirus        Sunflower ringspot ilarvirus
Datura shoestring potyvirus      Sunn-hemp mosaic tobamovirus
Datura yellow vein               Sweet clover latent
Desmodium mosaic potyvirus       Sweet clover necrotic mosaic
Dioscorea green banding mosaic   Sweet potato feathery mottle
Dioscorea latent potexvirus      Sweet potato latent potyvirus
Dogwood mosaic nepovirus         Sweet potato mild mottle
Dulcamara mottle tymovirus       Sweet potato ringspot nepovirus
Eggplant green mosaic potyvirus  Sweet potato sunken vein
Eggplant mild mottle carlavirus  Tamarillo mosaic potyvirus
Eggplant mottled crinkle         Tamus latent potexvirus
Eggplant mottled dwarf           Telfairia mosaic potyvirus
Eggplant severe mottle potyvirus Tobacco etch potyvirus
Elderberry carlavirus            Tobacco leaf curl bigeminivirus
Elderberry latent carmovirus     Tobacco mild green mosaic
Elm mottle ilarvirus             Tobacco mosaic satellivirus
Epirus cherry ourmiavirus        Tobacco mosaic tobamovirus
Erysimum latent tymovirus        Tobacco mottle umbravirus
Eucharis mottle nepovirus        Tobacco necrosis necrovirus
Euphorbia mosaic bigeminivirus   Tobacco necrosis satellivirus
Foxtail mosaic potexvirus        Tobacco necrotic dwarf luteovirus
Foxtail mosaic potexvirus        Tobacco rattle tobravirus
Foxtail mosaic potexvirus        Tobacco ringspot nepovirus
Frangipani mosaic tobamovirus    Tobacco streak ilarvirus
Furcraea necrotic streak         Tobacco stunt varicosavirus
Galinsoga mosaic carmovirus      Tobacco vein mottling potyvirus
Garlic common latent carlavirus  Tobacco vein-distorting luteovirus
Glycine mottle carmovirus        Tobacco wilt potyvirus
Grapevine A trichovirus          Tobacco yellow dwarf
Grapevine ajinashika disease     Tobacco yellow net luteovirus
Grapevine Algerian latent        Tobacco yellow vein umbravirus
Grapevine B trichovirus          Tobacco yellow vein assistor
Grapevine Bulgarian latent       Tomato aspermy cucumovirus
Grapevine chrome mosaic          Tomato Australian leafcurl
Grapevine chrome mosaic          Tomato black ring nepovirus
Grapevine corky bark-associated  Tomato black ring nepovirus
Grapevine fanleaf nepovirus      Tomato bushy stunt tombusvirus
Grapevine fleck virus            Tomato golden mosaic
Grapevine leafroll-associated    Tomato mild mottle potyvirus
Grapevine line pattern ilarvirus Tomato mosaic tobamovirus
Grapevine stem pitting associated Tomato mottle bigeminivirus
Grapevine stunt virus            Tomato Peru potyvirus
Groundnut chlorotic spot         Tomato ringspot nepovirus
Groundnut rosette umbravirus     Tomato spotted wilt tospovirus
Guar top necrosis virus          Tomato top necrosis nepovirus
Habenaria mosaic potyvirus       Tomato yellow leaf curl
Helenium S carlavirus            Tropaeolum 1 potyvirus
Henbane mosaic potyvirus         Tropaeolum 2 potyvirus
Heracleum latent trichovirus     Tulare apple mosaic ilarvirus
Hibiscus latent ringspot nepovirus Tulip chlorotic blotch potyvirus
Hippeastrum mosaic potyvirus     Tulip halo necrosis virus
Honeysuckle latent carlavirus    Tulip X potexvirus
Hop American latent carlavirus   Turnip crinkle carmovirus
Hop latent carlavirus            Turnip mosaic potyvirus
Humulus japonicus ilarvirus      Turnip rosette sobemovirus
Hydrangea mosaic ilarvirus       Turnip yellow mosaic tymovirus
Impatiens latent potexvirus      Ullucus mild mottle tobamovirus
Impatiens necrotic spot tospovirus Ullucus mosaic potyvirus
Iris fulva mosaic potyvirus      Vallota mosaic potyvirus
Ivy vein clearing cytorhabdovirus Vanilla necrosis potyvirus
Johnsongrass mosaic potyvirus    Viola mottle potexvirus
Kalanchoe isometric virus        Viola mottle potexvirus
Konjak mosaic potyvirus          Watercress yellow spot virus
Kyuri green mottle mosaic        Watermelon mosaic 1 potyvirus
Lamium mild mottle fabavirus     Watermelon mosaic 2 potyvirus
Lato River tombusvirus           Weddel waterborne carmovirus
Leek yellow stripe potyvirus     Welsh onion yellow stripe
Lettuce big-vein varicosavirus   Wheat soil-borne mosaic furovirus
Lettuce infectious yellows       Wheat streak mosaic rymovirus
Lettuce mosaic potyvirus         White clover mosaic potexvirus
Lettuce necrotic yellows         Wild cucumber mosaic tymovirus
Lettuce speckles mottle umbravirus Wild potato mosaic potyvirus
Lilac chlorotic leafspot capillovirus Wild potato mosaic potyvirus
Lilac ring mottle ilarvirus      Wineberry latent virus
Lily X potexvirus                Wisteria vein mosaic potyvirus
Lisianthus necrosis necrovirus   Yam mosaic potyvirus
Lucerne Australian latent        Zygocactus Montana X potexvirus
nepovirus
Lucerne Australian symptomless
Lucerne enation nucleorhabdovirus

[0125] A further listing of plants and plant viruses that may used with the methods of the invention is shown in Table 4. Additional examples of virus infections of plant species can be found at: http://image.fs.uidaho.edu/vide/. Additional virus accessions can be retrieved at: http://www.atcc.org.

TABLE 4
Plant or Virus Name              Plant or Virus Name
Cryptomeria japonica             Tulip band-breaking
Eucalyptus grandis               potyvirus
Eucalyptus nitens                Tulip breaking potyvirus
Eucalyptus urophylla             Tulip chlorotic blotch
Picea abies                      potyvirus
Picea glauca                     Tulip halo necrosis (?) virus
Pinus albicaulis                 Tulip X potexvirus
Pinus aristata                   Linum usitatissimum
Pinus armandii                   Synonyms:
Pinus attenuata                  Linum crepitans; Linum
Pinus ayacahuite                 humile; Linum usitatissimum ssp.
Pinus balfouriana                transitorium; Linum usitatissimum
Pinus brutia                     var. humile
Pinus bungeana                   Common names:
Pinus canariensis                Flax; Linseed; Lino
Pinus cembroides                 Susceptible to:
Pinus contorta                   Alfalfa mosaic alfamovirus
Pinus culminicola                Beet curly top
Pinus durangensis                hybrigeminivirus
Pinus echinata                   Beet pseudo-yellows (?)
Pinus edulis                     closterovirus
Pinus elliottii                  Oat blue dwarf marafivirus
Pinus engelmannii                Tobacco rattle tobravirus
Pinus flexilis                   Hibiscus
Pinus gerardiana                 Susceptible to:
Pinus griffithii                 Abutilon mosaic
Pinus halepensis                 bigeminivirus
Pinus hartwegii                  Cotton leaf crumple
Pinus jefferyi                   bigeminivirus
Pinus koraiensis                 Hibiscus yellow mosaic (?)
Pinus lambertiana                tobamovirus
Pinus lumholtzii                 Hibiscus cannabinus
Pinus massoniana                 Common names:
Pinus monticola                  Deccan-hemp; Indian-hemp;
Pinus mugo                       Kenaf
Pinus palustris                  Susceptible to:
Pinus pinaster                   Cotton anthocyanosis (?)
Pinus pinceana                   luteovirus
Pinus ponderosa                  Cotton leaf crumple
Pinus pungens                    bigeminivirus
Pinus radiata                    Cotton leaf curl
Pinus resinosa                   bigeminivirus
Pinus roxburghii                 Hibiscus chlorotic ringspot
Pinus sabiniana                  carmovirus
Pinus serotina                   Hibiscus latent ringspot
Pinus strobus                    nepovirus
Pinus sylvestris                 Kenaf vein-clearing (?)
Pinus tabulaeformis              rhabdovirus
Pinus taeda                      Malva vein clearing
Pinus thunbergii                 potyvirus
Pinus torreyana                  Okra mosaic tymovirus
Pinus virginiana                 Ficus carica
Pinus wangii                     Common names:
Pinus yunnanensis                Fig; Higo
Populus deltoides                Susceptible to:
Populus tremuloides              Fig (?) potyvirus
Cryptomeria japonica             Fig S carlavirus
Eucalyptus grandis               Morus alba
Eucalyptus nitens                Synonyms:
Eucalyptus urophylla             Morus alba f. tatarica;
Picea abies                      Morus alba var.
Picea glauca                     constantinopolitana; Morus alba
Pinus albicaulis                 var. multicaulis; Morus indica;
Pinus aristata                   Morus multicaulis
Pinus armandii                   Common names:
Pinus attenuata                  White mulberry; Mora
Pinus ayacahuite                 Susceptible to:
Pinus balfouriana                Citrus enation- woody gall
Pinus brutia                     (?) luteovirus
Pinus bungeana                   Mulberry latent carlavirus
Pinus canariensis                Mulberry ringspot
Pinus cembroides                 nepovirus
Pinus contorta                   Mirabilis jalapa
Pinus culminicola                Common names:
Pinus durangensis                Common four-o'clock
Pinus echinata                   Susceptible to:
Pinus edulis                     Mirabilis mosaic
Pinus elliottii                  caulimovirus
Pinus engelmannii                Fraxinus excelsior
Pinus flexilis                   Synonyms:
Pinus gerardiana                 Fraxinus excelsior var.
Pinus griffithii                 pendula
Pinus halepensis                 Common names:
Pinus hartwegii                  European ash
Pinus jefferyi                   Susceptible to:
Pinus koraiensis                 Arabis mosaic nepovirus
Pinus lambertiana                Jasminum officinale
Pinus lumholtzii                 Common names:
Pinus massoniana                 Poet's jasmine; Common
Pinus monticola                  jasmine; Jessamine
Pinus mugo                       Susceptible to:
Pinus palustris                  Arabis mosaic nepovirus
Pinus pinaster                   Ligustrum vulgare
Pinus pinceana                   Synonyms:
Pinus ponderosa                  Ligustrum insulare;
Pinus pungens                    Ligustrum insulense
Pinus radiata                    Common names:
Pinus resinosa                   Common privet
Pinus roxburghii                 Susceptible to:
Pinus sabiniana                  Arabis mosaic nepovirus
Pinus serotina                   Petunia asteroid mosaic
Pinus strobus                    tombusvirus
Pinus sylvestris                 Olea europaea
Pinus tabulaeformis              Common names:
Pinus taeda                      Olive; Aceituna
Pinus thunbergii                 Susceptible to:
Pinus torreyana                  Cherry leaf roll nepovirus
Pinus virginiana                 Olive latent ringspot
Pinus wangii                     nepovirus
Pinus yunnanensis                Olive latent 1 (?)
Populus deltoides                sobemovirus
Populus tremuloides              Olive latent 2 (?)
Populus trichocarpa              ourmavirus
Pseudotsuga menziesii            Oenothera biennis
Taxus brevifolia                 Synonyms:
Ulmus parvifolia                 Oenothera biennis ssp.
Chamaecyparis lawsoniana         sulfurea; Oenothera chicagoensis;
Common names:                    Oenothera muricata; Oenothera
Port Orford-cedar; Ginger-       suaveolens; Onagra biennis
pine; Oregon-cedar; Lawson's     Common names:
cypress                          Common evening-primrose;
Susceptible to:                  German rampion
Arabis mosaic nepovirus          Insusceptible to:
Eucalyptus cloeziana             Carnation vein mottle
Common names:                    potyvirus
Cloeziana gum; Gympie            Cymbidium
messmate                         Susceptible to:
Populus balsamifera              Cymbidium mosaic
Susceptible to:                  potexvirus
Poplar mosaic carlavirus         Cymbidium ringspot
Poplar vein yellowing (?)        tombusvirus
nucleorhabdovirus                Cymbidium alexanderi
Populus candicans                Susceptible to:
Synonyms:                        Odontoglossum ringspot
Populus balsamifera ssp.         tobamovirus
balsamifera; Populus tacamahacca Odontoglossum grande
Common names:                    Synonyms:
Balsam poplar; Tacamahac         Rossioglossum grande
poplar; Balm of Gilead           Susceptible to:
Susceptible to:                  Odontoglossum ringspot
Poplar mosaic carlavirus         tobamovirus
Populus deltoides subspecies     Cocos nucifera
angulata, monilifera,            Common names:
missouriensis                    Coconut; Coconut palm;
Susceptible to:                  Copra; Khopra; Nariyal; Coco
Poplar mosaic carlavirus         Susceptible to:
Ulmus americana                  Coconut foliar decay
Common names:                    nanavirus
American elm                     Papaver nudicaule
Susceptible to:                  Synonyms:
Cherry leaf roll nepovirus       Papaver miyabeanum
Ulmus glabra                     Common names:
Synonyms:                        Iceland poppy; Arctic poppy
Ulmus montana; Ulmus             Susceptible to:
scabra                           Beet curly top
Common names:                    hybrigeminivirus
Scotch elm; Wych elm             Tobacco mosaic
Susceptible to:                  tobamovirus
Elm mottle ilarvirus             Tomato spotted wilt
Ulmus minor                      tospovirus
Synonyms:                        Turnip mosaic potyvirus
Ulmus campestris; Ulmus          Papaver somniferum
carpinifolia; Ulmus carpinifolia Common names:
var. suberosa; Ulmus foliacea    Opium poppy
Ulmus foliacea var. suberosa;    Susceptible to:
Ulmus glabra var.                Bean yellow mosaic
suberosa; Ulmus nitens;          potyvirus
Ulmus suberosa                   Papaver rhoeas
Susceptible to:                  Common names:
Elm mottle ilarvirus             Corn poppy; Shirley poppy;
Subject: turf                    Field poppy
Agropyron cristatum              Susceptible to:
Festuca arizonica                Beet western yellows
Agropyron cristatum x            clostreovirus
desertorum                       Sesamum indicum
Festuca arundinacea              Synonyms:
Agropyron dasystachyum           Sesamum orientale
Festuca duriuscula               Common names:
Agropyron desertorum             Sesame; Benne seed
Festuca eliator                  Susceptible to:
Agropyron elongatum              Abelia latent tymovirus
Festuca eliator                  Apple stem pitting virus
arundinacea                      Arracacha A nepovirus
Agropyron inerme                 Asparagus 3 potexvirus
Festuca idahoensis               Asystasia gangetica mottle (?)
Agropyron intermedium            potyvirus
Festuca longifolia               Blackgram mottle (?)
Agropyron riparium               carmovirus
Festuca megalura                 Cassia yellow spot
Agropyron sibericum              potyvirus
Festuca ovina                    Cherry leaf roll nepovirus
Agropyron smithii                Citrus ringspot virus
Festuca rubra                    Lisianthus necrosis (?)
Agropyron spicatum               necrovirus
Festuca rubra var.               Malva veinal necrosis (?)
commutata                        potexvirus
Agropyron spicatum x             Melothria mottle (?)
repens                           potyvirus
Festuca rubra var. rubra         Mulberry latent carlavirus
Agropyron trachycaulum           Mulberry ringspot
Hordeum brachyantherum           nepovirus
Agropyron trichophorum           Okra mosaic tymovirus
Koeleria cristata                Patchouli mottle (?)
Agrostis alba                    potyvirus
Lolium multiflorum               Pea stem necrosis virus
Agrostis palustris               Peach enation (?) nepovirus
Lolium perenne                   Peanut green mosaic
Agrostis tenuis                  potyvirus
Oryzopsis hymenoides             Peanut mottle potyvirus
Alopecurus arundinaceus          Peanut stunt cucumovirus
Phalaris arundinacea             Satsuma dwarf (?)
Alopecurus pratensis             nepovirus
Phleum alpinum                   Soybean mild mosaic virus
Arcatagrostis latifolia          Sweet potato yellow dwarf
Phleum pratense                  (?) ipomovirus
Beckmannia syzigachne            Tobacco ringspot nepovirus
Phragmites australis             Watermelon mosaic 2
Bromus biebersteinii             potyvirus
Poa alpina                       Phytolacca americana
Bromus carinatus                 Synonyms:
Poa ampla                        Phytolacca decandra
Bromus catharticus               Common names:
Poa bulbosa                      Pokeweed; Poke;
Bromus inermis                   Pigeonberry
Poa canbyi                       Susceptible to:
Bromus marginatus                Alfalfa mosaic alfamovirus
Poa compressa                    Bean yellow mosaic
Bromus mollis                    potyvirus
Poa glauca                       Beet curly top
Dactylis glomerata               hybrigeminivirus
Poa palustris                    Beet mosaic potyvirus
Deschampsia caespitosa           Carnation mottle
Poa pratensis                    carmovirus
Viruses for Graminae:            Carnation ringspot
Maize streak monogeminivirus     dianthovirus
Wheat streak mosaic rymovirus    Cucumber mosaic
Barley yellow dwarf luteovirus   cucumovirus
Barley stripe mosaic hordeivirus Cymbidium ringspot
Sugarcane mosaic potyvirus       tombusvirus
Beet western yellows luteovirus  Pepper veinal mottle
Maize dwarf mosaic potyvirus     potyvirus
Foxtail mosaic potexvirus        Pokeweed mosaic potyvirus
Johnsongrass mosaic potyvirus    Red clover necrotic mosaic
Panicum mosaic (?) sobemovirus   dianthovirus
Rice stripe tenuivirus           Tobacco rattle tobravirus
Rice hoja blanca tenuivirus      Tobacco ringspot nepovirus
Wheat yellow leaf closterovirus  Tomato black ring
Brome mosaic bromovirus          nepovirus
Ribgrass mosaic tobamovirus      Turnip mosaic potyvirus
Wheat soil-borne mosaic furovirus Plantago major
Deschampsia caespitosa (L.)      Common names:
Beauv. ssp. Beringensis          Common plantain;
Poa sandbergii                   Broadleaf plantain; Great plantain
Elymus angustus                  Susceptible to:
Poa trivialis                    Carnation vein mottle
Elymus canadensis                potyvirus
Puccinellia distans              Cherry rasp leaf nepovirus
Elymus cinereus                  Plantago 4 (?) caulimovirus
Secale cereale                   Plantago mottle tymovirus
Elymus dahuricus                 Ribgrass mosaic
Sitanion hystrix                 tobamovirus
Elymus glaucus                   Phlox drummondii
Stipa comata                     Common names:
Elymus junceus                   Drummond phlox; Annual
Stipa viridula                   phlox
Elymus triticoides               Susceptible to:
Triticum aestivum, spp.          Apple mosaic ilarvirus
WARM SEASON GRASSES              Arabis mosaic nepovirus
Andropogon geradii               Beet curly top
Distichlis stricta               hybrigeminivirus
Andropogon hallii                Beet western yellows
Panicum virgatum                 luteovirus
Bouteloua curtipendula           Carnation ringspot
Schizachyrium scoparium          dianthovirus
Bouteloua gracillis              Cherry leaf roll nepovirus
Sorghastrum nutans               Cymbidium ringspot
Buchloe dactyloides              tombusvirus
Sporobolus airoides              Dogwood mosaic (?)
Calamovilfa longifolia           nepovirus
Sporobolus crypatandrus          Elm mottle ilarvirus
Cynodon dactylon                 Melon Ourmia ourmiavirus
LEGUMES                          Okra mosaic tymovirus
Astragalus cicer                 Poplar mosaic carlavirus
Onobrychis viciaefolia           Prune dwarf ilarvirus
Coronilla varia                  Ribgrass mosaic
Trifolium hybridum               tobamovirus
Hedysarum boreale                Spinach latent ilarvirus
Trifolium pratense               Strawberry latent ringspot
Lotus corniculatus               (?) nepovirus
Trifolium repens                 Sweet potato mild mottle
Lupinus spp.                     ipomovirus
Trifolium repens L.              Tobacco ringspot nepovirus
Medicago sativa                  Tobacco streak ilarvirus
Vicia villosa                    Tomato spotted wilt
Melilotus officinalis            tospovirus
Tritolium ambigium               Polypodium vulgare
Astragalus glycyphyllos          Susceptible to:
Common names:                    Fern (?) potyvirus
Liquorice milk-vetch             rimula malacoides
Susceptible to:                  Susceptible to:
Alfalfa mosaic alfamovirus       Carnation mottle
Astragalus sinicus               carmovirus
Susceptible to:                  Hydrangea ringspot
Bean leaf roll luteovirus        potexvirus
Milk vetch dwarf nanavirus       Primula mottle (?) potyvirus
Soybean dwarf luteovirus         Sweet potato mild mottle
Subterranean clover red leaf     ipomovirus
luteovirus                       Viola mottle potexvirus
Subterranean clover stunt        Pteris ‘Childsii’
nanavirus                        Susceptible to:
Watermelon mosaic 2              Harts tongue fern (?)
potyvirus                        tobravirus
Coronilla varia                  Ranunculus repens
Synonyms:                        Common names:
Securigera varia                 Creeping buttercup
Common names:                    Susceptible to:
Crown-vetch; Trailing            Arabis mosaic nepovirus
crown-vetch                      Ranunculus repens
Susceptible to:                  symptomless (?) rhabdovirus
Peanut stunt cucumovirus         Malus domestica
Trifolium hybridum               Synonyms:
Common names:                    Malus malus; Pyrus malus
Alsike clover; Swedish           Common names:
clover; Trefle-hybride; Trefle-  Apple; Common apple
batard; Schwedenklee;            Susceptible to:
Bastardklee; Trevo-hibrido;      Apple mosaic ilarvirus
Trebol-hibrido                   Insusceptible to:
Susceptible to:                  Plum pox potyvirus
Alfalfa mosaic alfamovirus       Malus platycarpa
Alsike clover vein mosaic        Susceptible to:
virus                            Apple chlorotic leaf spot
Bean leaf roll luteovirus        trichovirus
Bean yellow mosaic               Apple stem pitting virus
potyvirus                        Malus sylvestris
Beet curly top                   Common names:
hybrigeminivirus                 Crab apple; Wild apple
Beet yellows closterovirus       Susceptible to:
Broad bean mottle                Apple chlorotic leaf spot
bromovirus                       trichovirus
Broad bean stain comovirus       Apple stem grooving
Clover mild mosaic virus         capillovirus
Clover yellow mosaic             Apple stem pitting virus
potexvirus                       Cherry rasp leaf nepovirus
Clover yellow vein               Horseradish latent
potyvirus                        caulimovirus
Cucumber mosaic                  Tomato ringspot nepovirus
cucumovirus                      Tulare apple mosaic
Muskmelon vein necrosis          ilarvirus
carlavirus                       Prunus avium
Pea early browning               Synonyms:
tobravirus                       Cerasus avium var.
Pea enation mosaic               aspleniifolia; Prunus avium var.
enamovirus                       aspleniifolia; Prunus cerasus var.
Pea streak carlavirus            avium
Peanut stunt cucumovirus         Common names:
Red clover mottle                Mazzard cherry; Sweet
comovirus                        cherry
Red clover vein mosaic           Susceptible to:
carlavirus                       Arabis mosaic nepovirus
Soybean dwarf luteovirus         Cherry leaf roll nepovirus
Subterranean clover red leaf     Cherry mottle leaf (?)
luteovirus                       trichovirus
Tomato ringspot nepovirus        Cherry rasp leaf nepovirus
Turnip mosaic potyvirus          Epirus cherry ourmiavirus
White clover mosaic              Myrobalan latent ringspot
potexvirus                       nepovirus
Lotus corniculatus               Petunia asteroid mosaic
Synonyms:                        tombusvirus
Lotus corniculatus ssp.          Prunus domestica
major; Lotus corniculatus var.   Common names:
major; Lotus major               Plum
Common names:                    Susceptible to:
Bird's-foot trefoil              Apple chlorotic leaf spot
Susceptible to:                  trichovirus
Cucumber mosaic                  Arabis mosaic nepovirus
cucumovirus                      Citrus enation-woody gall
Lupinus albus                    (?) luteovirus
Common names:                    Petunia asteroid mosaic
White lupine; Egyptian           tombusvirus
lupine                           Plum American line pattern
Susceptible to:                  ilarvirus
Alfalfa mosaic alfamovirus       Plum pox potyvirus
Amaranthus leaf mottle           Prune dwarf ilarvirus
potyvirus                        Sowbane mosaic
Bean common mosaic               sobemovirus
potyvirus                        Strawberry latent ringspot
Bean yellow mosaic               (?) nepovirus
potyvirus                        Prunus persica
Beet western yellows             Synonyms:
luteovirus                       Amygdalus persica;
Bidens mosaic potyvirus          Amygdalus persica var.
Broad bean mottle                camelliiflora; Amygdalus persica
bromovirus                       var. densa; Persica vulgaris;
Broad bean true mosaic           Prunus persica var. camelliiflora;
comovirus                        Prunus persica var. densa
Carnation yellow stripe (?)      Common names:
necrovirus                       Peach; Melocotonero;
Cassia mild mosaic (?)           Abridor; Durazno
carlavirus                       Susceptible to:
Chicory yellow mottle            Apple chlorotic leaf spot
nepovirus                        trichovirus
Cowpea chlorotic mottle          Arabis mosaic nepovirus
bromovirus                       Cherry leaf roll nepovirus
Cucumber mosaic                  Cherry mottle leaf (?)
cucumovirus                      trichovirus
Dogwood mosaic (?)               Cherry rasp leaf nepovirus
nepovirus                        Myrobalan latent ringspot
Epirus cherry ourmiavirus        nepovirus
Glycine mottle (?)               Peach enation (?) nepovirus
carmovirus                       Peach rosette mosaic
Lucerne Australian latent        nepovirus
nepovirus                        Peach yellow leaf (?)
Lucerne transient streak         closterovirus
sobemovirus                      Plum American line pattern
Pea enation mosaic               ilarvirus
enamovirus                       Plum pox potyvirus
Pea streak carlavirus            Prune dwarf ilarvirus
Peanut mottle potyvirus          Prunus necrotic ringspot
Peanut stunt cucumovirus         ilarvirus
Pepper Moroccan                  Strawberry latent ringspot
tombusvirus                      (?) nepovirus
Plum pox potyvirus               Tomato ringspot nepovirus
Prunus necrotic ringspot         Pyrus communis
ilarvirus                        Synonyms:
Ribgrass mosaic                  Pyrus asiae-mediae; Pyrus
tobamovirus                      balansae; Pyrus bourgaeana;
Soybean dwarf luteovirus         Pyrus domestica; Pyrus elata;
Soybean mild mosaic virus        Pyrus medvedevii
Soybean mosaic potyvirus         Common names:
Subterranean clover red leaf     Pear; Pera
luteovirus                       Susceptible to:
Turnip mosaic potyvirus          Apple chlorotic leaf spot
Watermelon mosaic 2              trichovirus
potyvirus                        Apple stem pitting virus
Wisteria vein mosaic             Rosa
potyvirus                        Susceptible to:
Medicago sativa                  Apple mosaic ilarvirus
Synonyms:                        Arabis mosaic nepovirus
Medicago caerulea var.           Citrus enation - woody gall
pauciflora; Medicago             (?) luteovirus
karatschaica; Medicago lavrenkoi; Prunus necrotic ringspot
Medicago pauciflora; Medicago    ilarvirus
sativa var. pilifera             Rose (?) tobamovirus
Susceptible to:                  Strawberry latent ringspot
Alfalfa 1 alphacryptovirus       (?) nepovirus
Alfalfa 2 (?) betacryptovirus    Rubus fruticosus
Alfalfa mosaic alfamovirus       Synonyms:
Bean leaf roll luteovirus        Rubus plicatus; Rubus
Bean yellow mosaic               affinis
potyvirus                        Common names:
Beet curly top                   Blackberry; Bramble;
hybrigeminivirus                 European blackberry
Broad bean mottle                Susceptible to:
bromovirus                       Black raspberry necrosis
Carnation mottle                 virus
carmovirus                       Raspberry leaf curl (?)
Carrot mosaic (?) potyvirus      luteovirus
Cassia mild mosaic (?)           Strawberry latent ringspot
carlavirus                       (?) nepovirus
Chickpea distortion mosaic       Rubus idaeus
potyvirus                        Synonyms:
Clover yellow mosaic             Rubus buschii; Rubus
potexvirus                       idaeus var. vulgatus; Rubus
Clover yellow vein               vulgatus var. buschii
potyvirus                        Common names:
Cucumber mosaic                  European red raspberry;
cucumovirus                      Red raspberry
Lucerne Australian latent        Susceptible to
nepovirus                        Arabis mosaic nepovirus
Lucerne Australian               Black raspberry necrosis
symptomless (?) nepovirus        virus
Lucerne enation (?)              Cherry leaf roll nepovirus
nucleorhabdovirus                Cole latent (?) carlavirus
Lucerne transient streak         Raspberry bushy dwarf
sobemovirus                      idaeovirus
Milk vetch dwarf nanavirus       Raspberry leaf curl (?)
Narcissus mosaic potexvirus      luteovirus
Pea enation mosaic               Raspberry ringspot
enamovirus                       nepovirus
Pea seed-borne mosaic            Raspberry vein chlorosis (?)
potyvirus                        nucleorhabdovirus
Pea streak carlavirus            Rubus yellow net (?)
Peanut stunt cucumovirus         badnavirus
Red clover mottle                Strawberry latent ringspot
comovirus                        (?) nepovirus
Red clover necrotic mosaic       Thimbleberry ringspot virus
dianthovirus                     Tomato ringspot nepovirus
Red clover vein mosaic           Citrus limon
carlavirus                       Synonyms:
Subterranean clover stunt        Citrus limonum; Citrus
nanavirus                        medica var. limon
Tobacco ringspot nepovirus       Common names:
Tobacco streak ilarvirus         Lemon; Limonero;
Tobacco yellow dwarf             Limoniere; Citronnier;
monogeminivirus                  Zitronenbaum
Watermelon mosaic 2              Susceptible to:
potyvirus                        Citrus enation - woody gall
White clover mosaic              (?) luteovirus
potexvirus                       Citrus leaf rugose ilarvirus
Melilotus albus                  Citrus ringspot virus
Synonyms:                        Citrus tatter leaf capillovirus
Melilotus albus var. annuus;     Citrus tristeza closterovirus
Melilotus leucanthus             Citrus variegation ilarvirus
Common names:                    Citrus paradisi
White sweet-clover; White        Common names:
melilot; Hubam                   Grapefruit; Pomelo; Toronja
Susceptible to:                  Susceptible to:
Alfalfa mosaic alfamovirus       Citrus enation - woody gall
Apple mosaic ilarvirus           (?) luteovirus
Bean common mosaic               Citrus leaf rugose ilarvirus
potyvirus                        Citrus ringspot virus
Bean yellow mosaic               Citrus tristeza closterovirus
potyvirus                        Pepper veinal mottle
Beet curly top                   potyvirus
hybrigeminivirus                 Citrus sinensis
Broad bean mottle                Synonyms:
bromovirus                       Citrus aurantium var.
Broad bean necrosis              sinensis; Citrus macracantha
furovirus                        Common names:
Broad bean stain comovirus       Sweet orange; Naranja
Broad bean true mosaic           Susceptible to:
comovirus                        Citrus enation - woody gall
Clover yellow mosaic             (?) luteovirus
potexvirus                       Citrus leaf rugose ilarvirus
Clover yellow vein               Citrus leprosis (?)
potyvirus                        rhabdovirus
Cucumber mosaic                  Citrus ringspot virus
cucumovirus                      Citrus tatter leaf capillovirus
Galinsoga mosaic                 Citrus tristeza closterovirus
carmovirus                       Sambucus canadensis
Milk vetch dwarf nanavirus       Common names:
Muskmelon vein necrosis          American elder; American
carlavirus                       elderberry; Sweet elder
Pea enation mosaic               Susceptible to:
enamovirus                       Elderberry carlavirus
Pea mild mosaic comovirus        Elderberry latent (?)
Pea streak carlavirus            carmovirus
Peanut clump furovirus           Dodonaea viscosa
Peanut stunt cucumovirus         Common names:
Plum pox potyvirus               Hop shrub
Prune dwarf ilarvirus            Susceptible to:
Prunus necrotic ringspot         Dodonaea yellows-
ilarvirus                        associated virus
Red clover mottle                Antirrhinum majus
comovirus                        Common names:
Red clover vein mosaic           Snapdragon
carlavirus                       Susceptible to:
Subterranean clover stunt        Alfalfa mosaic alfamovirus
nanavirus                        Arabis mosaic nepovirus
Sweet clover latent (?)          Asystasia gangetica mottle
nucleorhabdovirus                (?) potyvirus
Sweet clover necrotic            Broad bean wilt fabavirus
mosaic dianthovirus              Carnation mottle
Tobacco etch potyvirus           carmovirus
Tobacco rattle tobravirus        Carnation ringspot
Tobacco ringspot nepovirus       dianthovirus
Tobacco streak ilarvirus         Cherry leaf roll nepovirus
Turnip mosaic potyvirus          Clover yellow vein
Watermelon mosaic 2              potyvirus
potyvirus                        Cowpea mosaic comovirus
White clover mosaic              Cucumber mosaic
potexvirus                       cucumovirus
Trifolium dubium                 Cymbidium ringspot
Synonyms:                        tombusvirus
Trifolium filiforme var.         Dogwood mosaic (?)
dubium; Trifolium minus;         nepovirus
Trifolium parviflorum; Trifolium Elm mottle ilarvirus
procumbens                       Groundnut eyespot
Common names:                    potyvirus
Small hop clover; Suckling       Maracuja mosaic (?)
clover; Lesser yellow trefoil; Low tobamovirus
hop clover; Yellow clover;       Marigold mottle potyvirus
Shamrock                         Papaya mosaic potexvirus
Susceptible to:                  Pea streak carlavirus
Alfalfa mosaic alfamovirus       Peanut clump furovirus
Bean leaf roll luteovirus        Pepper Moroccan
Peanut stunt cucumovirus         tombusvirus
Soybean dwarf luteovirus         Plantago mottle tymovirus
Subterranean clover stunt        Poplar mosaic carlavirus
nanavirus                        Prune dwarf ilarvirus
WETLAND - RIPARIAN               Prunus necrotic ringspot
Agrostis alba                    ilarvirus
Glyceria occidentalis            Red clover necrotic mosaic
Alopecurus arundinaceus          dianthovirus
Glyceria striata                 Red clover vein mosaic
Alopecurus pratensis             carlavirus
Hordeum brachyantherum           Rubus Chinese seed-borne
Beckmannia syzigachne            (?) nepovirus
Phalaris arundinacea             Scrophularia mottle
Deschampsia caespitosa           tymovirus
Poa palustris                    Soybean mild mosaic virus
WILDFLOWERS AND                  Soybean mosaic potyvirus
FORBES                           Spinach latent ilarvirus
Achillea millefolium             Strawberry latent ringspot
Lupinus albicalus                (?) nepovirus
Cheiranthus allionii             Tamus latent (?) potexvirus
Lupinus perennis                 Tobacco necrosis necrovirus
Coreopsis lanceolata             Tobacco rattle tobravirus
Papaver rhoeas                   Tobacco ringspot nepovirus
Echinacea purpurea               Tobacco streak ilarvirus
Ratibida columnaris              Tomato black ring
Eschscholtzia californica        nepovirus
Rudbeckia hirta                  Tomato bushy stunt
Linum lewisii                    tombusvirus
Lupinus luteus                   Viola mottle potexvirus
Common names:                    White clover mosaic
European yellow lupine;          potexvirus
Yellow lupine                    Scrophularia nodosa
Susceptible to:                  Common names:
Bean yellow mosaic               Figwort; Figwort herb
potyvirus                        Susceptible to:
Clover yellow vein               Scrophularia mottle
potyvirus                        tymovirus
Dogwood mosaic (?)               Capsicum annuum
nepovirus                        Synonyms:
Peanut stunt cucumovirus         Capsicum cordiforme
Cheiranthus cheiri               Common names:
Synonyms:                        Pimiento; Bell pepper;
Erysimum cheiri                  Cayenne pepper; Chili pepper;
Common names:                    Common garden pepper; Green
Wallflower                       pepper; Mango pepper; Paprika
Susceptible to:                  pepper
Alfalfa mosaic alfamovirus       Susceptible to:
Beet western yellows             Alfalfa mosaic alfamovirus
luteovirus                       Bean distortion dwarf (?)
Chicory yellow mottle            bigeminivirus
nepovirus                        Beet western yellows
Cucumber mosaic                  luteovirus
cucumovirus                      Cassia mild mosaic (?)
Tobacco rattle tobravirus        carlavirus
Tobacco ringspot nepovirus       Celery latent (?) potyvirus
Tomato spotted wilt              Chilli veinal mottle (?)
tospovirus                       potyvirus
Turnip crinkle carmovirus        Chino del tomat,
Turnip mosaic potyvirus          bigeminivirus
Turnip yellow mosaic             Cucumber mosaic
tymovirus                        cucumovirus
Coreopsis lanceolata             Datura distortion mosaic
Susceptible to:                  potyvirus
Bidens mosaic potyvirus          Eggplant mosaic tymovirus
Papaver rhoeas                   Eggplant mottled dwarf
Common names:                    nucleorhabdovirus
Corn poppy; Shirley poppy;       Eggplant severe mottle (?)
Field poppy                      potyvirus
Susceptible to:                  Henbane mosaic potyvirus
Beet western yellows             Marigold mottle potyvirus
clostrovirus                     Melon Ourmia ourmiavirus
Linum grandiflorum               Paprika mild mottle
Synonyms:                        tobamovirus
Linum rubrum                     Peanut stunt cucumovirus
Common names:                    Pelargonium vein clearing (?)
Flowering flax                   cytorhabdovirus
Susceptible to:                  Pepper hausteco
Beet pseudo-yellows (?)          bigeminivirus
closterovirus                    Pepper Indian mottle
Oat blue dwarf marafivirus       potyvirus
Linum usitatissimum              Pepper mild mosaic (?)
Synonyms:                        potyvirus
Linum crepitans; Linum           Pepper mild mottle
humile; Linum usitatissimum ssp. tobamovirus
transitorium; Linum usitatissimum Pepper mild tigr, (?)
var. humile                      bigeminivirus
Common names:                    Pepper Moroccan
Flax; Linseed; Lino              tombusvirus
Susceptible to:                  Pepper mottle potyvirus
Alfalfa mosaic alfamovirus       Pepper ringspot tobravirus
Beet curly top                   Pepper severe mosaic
hybrigeminivirus                 potyvirus
Beet pseudo-yellows (?)          Pepper Texas bigeminivirus
closterovirus                    Pepper veinal mottle
Oat blue dwarf marafivirus       Potyvirus
Tobacco rattle tobravirus        Physalis mosaic tymovirus
ORNAMENTAL GRASSES               Pittosporum vein yellowing
Acorus Gramineus                 nucleorhabdovirus
Acorus Calamus                   Potato aucuba mosaic
Acorus Gramineus                 potexvirus
Alopecurus Pratensis             Potato mop-top furovirus
Andropogon Scoparius             Potato Y potyvirus
Andropogon Gerardii              Red pepper 1 (?)
Arrhenatherum Elatius            alphacryptovirus
Arundo Formosana                 Red pepper 2 (?)
Briza Media                      alphacryptovirus
Calamagrostis Acutiflora         Ribgrass mosaic
Calamagrostis Arundinacea        tobamovirus
Calamagrostis Acutiflora         Serrano golden mosaic
Calamagrostis Acutiflora         bigeminivirus
Carex Glauca                     Sweet potato ringspot (?)
Carex Siderostica                nepovirus
Carex Albula                     Tobacco etch potyvirus
Carex Nigra                      Tobacco leaf curl
Carex Muskingumensis             bigeminivirus
Carex Riparia                    Tobacco mild green mosaic
Carex Evergold                   tobamovirus
Carex Comans                     Tobacco mosaic satellivirus
Cortaderia Selloana              Tobacco rattle tobravirus
Cortaderia Selloana Rosea        Tobacco streak ilarvirus
Deschampsia Cespitosa            Tomato bushy stunt
Elymus Arenarius                 tombusvirus
Erianthus Ravennae               Tomato mosaic tobamovirus
Ovina Gigantea                   Tomato Peru potyvirus
Ovina Glauca                     Tomato spotted wilt
Glyceria Maxima                  tospovirus
Hakonechloa Macra                Lycopersicon esculentum
Hakonechloa Macra                Common names:
Helictotrichon Sempervirens      Tomato; Tomate
Holcus Variegated                Susceptible to:
Hystrix Patula                   Abelia latent tymovirus
Imperata Red Baron               Abutilon mosaic
Juncus Effusus                   bigeminivirus
Juncus Ensifolius                Alfalfa mosaic alfamovirus
Juncus Filiformis                Arabis mosaic nepovirus
Juncus Inflexus                  Arracacha A nepovirus
Koeleria Cristata                Arracacha B (?) nepovirus
Koeleria Glauca                  Beet curly top
Luzula Sylvatica                 hybrigeminivirus
Melica Ciliata                   Beet western yellows
Melica Nutans                    luteovirus
Miscanthus Sinensis              Blueberry leaf mottle
Molinia Caerulea                 nepovirus
Virgatum Rotstrahlbusch          Brinjal mild mosaic (?)
Pennisetum Alopecuroides         potyvirus
Pennisetum Ruppelianum           Carnation mottle
Pennisetum Alopecuroides         carmovirus
Pennisetum Alopecuroides         Carrot mosaic (?) potyvirus
Pennisetum Alopecuroides         Cassava green mottle
Pennisetum Setaceum              nepovirus
Pennisetum Setaceum              Cassia mild mosaic (?)
Pennisetum Cassian               carlavirus
Phalaris Arundinacea             Chickpea chlorotic dwarf (?)
Phalaris Arundinacea             monogeminivirus
Phalaris Arundinacea             Chino del tomat,
Sesleria Autumnalis              bigeminivirus
Sesleria Caerulea                Clover wound tumor
Sporobolus Helerolepsis          phytoreovirus
Stipa Capillata                  Commelina X potexvirus
Stipa Extremiorientalis          Cowpea mild mottle (?)
Stipa Gigantea                   carlavirus
Stipa Tenuissima                 Croton yellow vein mosaic
Stipa Grandis                    bigeminivirus
Stipa Pennata                    Cucumber mosaic
Stipa Ucrainica                  cucumovirus
Impatiens                        Cymbidium ringspot
Impatiens necrotic spot tospovirus tombusvirus
Carnation mottle carmovirus      Datura distortion mosaic
Helenium S carlavirus            potyvirus
Impatiens latent (?) potexvirus  Datura innoxia Hungarian
Aster chlorotic stunt (?) carlavirus mosaic (?) potyvirus
Dasheen mosaic potyvirus         Datura mosaic (?) potyvirus
Aglaonema                        Datura necrosis potyvirus
Alocasia                         Datura yellow vein
Amorphophallus                   nucleorhabdovirus
Arisaema                         Dogwood mosaic (?)
Caladium hortulanum              nepovirus
Chenopodium amaranticolor        Dulcamara mottle
Chenopodium ambrosioides         tymovirus
Chenopodium quinoa               Eggplant green mosaic
Colocasia esculenta              potyvirus
Cryptocoryne                     Eggplant mosaic tymovirus
Cyrtosperma                      Eggplant mottled dwarf
Dieffenbachia picta              nucleorhabdovirus
Nicotiana benthamiana            Eggplant severe mottle (?)
Philodendron selloum             potyvirus
Philodendron verrucosum          Elderberry latent (?)
Richardia                        carmovirus
Saponaria vaccaria               Elm mottle ilarvirus
Spathiphyllum                    Epirus cherry ourmiavirus
Tetragonia tetragonioides        Foxtail mosaic potexvirus
Xanthosoma caracu                Groundnut eyespot
Zantedeschia (no species name    potyvirus
provided)                        Henbane mosaic potyvirus
Zantedeschia elliottiana         Lettuce necrotic yellows
Colocasia bobone disease (?)     cytorhabdovirus
rhabdovirus                      Maracuja mosaic (?)
Dasheen bacilliform (?)          tobamovirus
badnavirus                       Marigold mottle potyvirus
Dasheen mosaic potyvirus         Melilotus mosaic (?)
Colocasia esculenta              potyvirus
Konjak mosaic (?) potyvirus      Melon Ourmia ourmiavirus
Philodendron                     Nerine X potexvirus
oxycardium                       Okra leaf-curl bigeminivirus
Philodendron selloum             Ononis yellow mosaic
Abelia latent tymovirus          tymovirus
Abelia grandiflora               Parietaria mottle ilarvirus
Abelmoschus esculentus           Parsnip yellow fleck
Acer palmatum                    sequivirus
Amaranthus caudatus              Pea streak carlavirus
Atropa belladonna                Peanut clump furovirus
Brassica campestris ssp.         Peanut stunt cucumovirus
pekinensis                       Pelargonium line pattern (?)
Catharanthus roseus              carmovirus
Celosia argentea                 Pelargonium zonate spot
Chenopodium amaranticolor        ourmiavirus
Chenopodium murale               Pepino mosaic potexvirus
Chenopodium quinoa               Pepper Indian mottle
Datura metel                     potyvirus
Datura stramonium                Pepper mild tigr, (?)
Glycine max                      bigeminivirus
Gomphrena globosa                Pepper Moroccan
Gossypium hirsutum               tombusvirus
Hordeum vulgare                  Pepper mottle potyvirus
Lobelia erinus                   Pepper ringspot tobravirus
Lycopersicon esculentum          Pepper severe mosaic
Momordica balsamina              potyvirus
Nicotiana clevelandii            Pepper Texas bigeminivirus
Nicotiana glutinosa              Pepper veinal mottle
Nicotiana rustica                potyvirus
Petunia x hybrida                Physalis mosaic tymovirus
Physalis peruviana               Pittosporum vein yellowing
Sesamum indicum                  nucleorhabdovirus
Solanum melongena                Plantain X potexvirus
Solanum tuberosum                Plum pox potyvirus
Tetragonia tetragonioides        Potato 14R (?) tobamovirus
Tithonia speciosa                Potato Andean latent
Torenia fournieri                tymovirus
Vicia faba                       Potato Andean mottle
Allium                           comovirus
Susceptible to:                  Potato aucuba mosaic
Onion yellow dwarf               potexvirus
potyvirus                        Potato black ringspot
Allium ampeloprasum var.         nepovirus
holmense                         Potato leafroll luteovirus
Garlic common latent (?)         Potato M carlavirus
carlavirus                       Potato mop-top furovirus
Allium ampeloprasum var.         Potato U nepovirus
sectivum                         Potato V potyvirus
Susceptible to:                  Potato Y potyvirus
Sint-Jan's onion latent (?)      Potato yellow mosaic
carlavirus                       bigeminivirus
Allium cepa                      Raspberry ringspot
Synonyms:                        nepovirus
Allium ascalonicum; Allium       Red clover necrotic mosaic
cepa var. aggregatum; Allium     dianthovirus
cepa var. solaninum              Ribgrass mosaic
Common names:                    tobamovirus
Onion; Shallot; Tama-negi;       Rose (?) tobamovirus
Eschalot; Potato onion; Multiplier Rubus Chinese seed-borne (?)
onion; Cebolla; Spanish onion    nepovirus
Susceptible to:                  Serrano golden mosaic
Leek yellow stripe potyvirus     bigeminivirus
Onion mite-borne latent (?)      Solanum apical leaf curling (?)
potexvirus                       bigeminivirus
Onion yellow dwarf               Soybean crinkle leaf (?)
potyvirus                        bigeminivirus
Pepper venial mottle             Soybean mild mosaic virus
potyvirus                        Strawberry latent ringspot (?)
Shallot latent carlavirus        nepovirus
Shallot mite-borne latent (?)    Sunflower ringspot (?)
potexvirus                       ilarvirus
Shallot yellow stripe (?)        Sweet potato mild mottle
potyvirus                        ipomovirus
Sint-Jan's onion latent (?)      Tamarillo mosaic potyvirus
carlavirus                       Tamus latent (?) potexvirus
Tobacco rattle tobravirus        Tobacco etch potyvirus
Welsh onion yellow stripe (?)    Tobacco leaf curl
potyvirus                        bigeminivirus
Amaranthaceae                    Tobacco mild green mosaic
Susceptible to:                  tobamovirus
Apple stem grooving              Tobacco mosaic satellivirus
capillovirus                     Tobacco mosaic
Insusceptible to:                tobamovirus
Voandzeia necrotic mosaic        Tobacco mottle umbravirus
tymovirus                        Tobacco necrosis necrovirus
Amaranthus bicolor               Tobacco necrotic dwarf
Insusceptible to:                luteovirus
Onion mite-borne latent (?)      Tobacco rattle tobravirus
potexvirus                       Tobacco ringspot nepovirus
Amaranthus caudatus              Tobacco streak ilarvirus
Synonyms:                        Tobacco stunt varicosavirus
Amaranthus caudatus ssp.         Tobacco vein-distorting (?)
mantegazzianus; Amaranthus       luteovirus
caudatus var. alopecurus;        Tobacco vein mottling
Amaranthus dussii; Amaranthus    potyvirus
edulis; Amaranthus               Tobacco yellow dwarf
mantegazzianus                   monogeminivirus
Common names:                    Tobacco yellow net (?)
Inca wheat; Love-lies-           luteovirus
bleeding; Tassel-flower; Kiwichi; Tobacco yellow vein
Coimi                            assistor (?) luteovirus
Susceptible to:                  Tobacco yellow vein (?)
Abelia latent tymovirus          umbravirus
Alfalfa mosaic alfamovirus       Tomato aspermy
Amaranthus leaf mottle           cucumovirus
potyvirus                        Tomato Australian leafcurl
Amaranthus mosaic (?)            bigeminivirus
potyvirus                        Tomato black ring
Arracacha A nepovirus            nepovirus
Arracacha B (?) nepovirus        Tomato bushy stunt
Bean yellow mosaic               tombusvirus
potyvirus                        Tomato chlorotic spot (?)
Beet curly top                   tospovirus
hybrigeminivirus                 Tomato golden mosaic
Beet mosaic potyvirus            bigeminivirus
Cactus X potexvirus              Tomato infectious chlorosis (?)
Carnation mottle                 closterovirus
carmovirus                       Tomato mild mottle (?)
Carnation ringspot               potyvirus
dianthovirus                     Tomato mosaic tobamovirus
Carnation vein mottle            Tomato mottle
potyvirus                        bigeminivirus
Celery latent (?) potyvirus      Tomato Peru potyvirus
Chicory yellow mottle            Tomato pseudo curly top (?)
nepovirus                        hybrigeminivirus
Clover yellow mosaic             Tomato ringspot nepovirus
potexvirus                       Tomato spotted wilt
Clover yellow vein               tospovirus
potyvirus                        Tomato top necrosis (?)
Cucumber mosaic                  nepovirus
cucumovirus                      Tomato vein clearing
Cymbidium ringspot               nucleorhabdovirus
tombusvirus                      Tomato yellow leaf curl
Dahlia mosaic caulimovirus       bigeminivirus
Elderberry carlavirus            Tomato yellow mosaic
Grapevine fanleaf nepovirus      bigeminivirus
Heracleum latent trichovirus     Tulip chlorotic blotch
Humulus japonicus ilarvirus      potyvirus
Iris fulva mosaic potyvirus      Tulip X potexvirus
Lamium mild mottle               Turnip crinkle carmovirus
fabavirus                        Ullucus mild mottle
Lettuce mosaic potyvirus         tobamovirus
Maclura mosaic                   White clover mosaic
macluravirus                     potexvirus
Marigold mottle potyvirus        Wild potato mosaic
Peanut stunt cucumovirus         potyvirus
Plantain X potexvirus            Wineberry latent virus
Potato 14R (?) tobamovirus       Nicotiana benthamiana
Potato Andean latent             Susceptible to:
tymovirus                        Ahlum waterborne (?)
Potato black ringspot            carmovirus
nepovirus                        Alstroemeria (?) ilarvirus
Potato leafroll luteovirus       Alstroemeria mosaic
Red clover necrotic mosaic       potyvirus
dianthovirus                     Alstroemeria streak (?)
Ribgrass mosaic                  potyvirus
tobamovirus                      Amazon lily mosaic (?)
Telfairia mosaic potyvirus       potyvirus
Tobacco etch potyvirus           Apple mosaic ilarvirus
Tobacco necrosis necrovirus      Arracacha Y potyvirus
Tobacco rattle tobravirus        Artichoke latent potyvirus
Tobacco ringspot nepovirus       Artichoke latent S (?)
Tobacco streak ilarvirus         carlavirus
Tomato black ring                Artichoke mottled crinkle
nepovirus                        tombusvirus
Tomato spotted wilt              Artichoke vein banding (?)
tospovirus                       nepovirus
Turnip mosaic potyvirus          Asparagus 3 potexvirus
Ullucus mild mottle              Asystasia gangetica mottle
tobamovirus                      (?) potyvirus
Viola mottle potexvirus          Barley yellow streak mosaic
Watermelon mosaic 2              virus
potyvirus                        Bean calico mosaic
Zygocactus Montana X (?)         bigeminivirus
potexvirus                       Bean common mosaic
Amaranthus tricolor              potyvirus
Synonyms:                        Beet curly top
Amaranthus gangeticus;           hybrigeminivirus
Amaranthus gangeticus var.       Blueberry leaf mottle
melancholicus; Amaranthus        nepovirus
mangostanus; Amaranthus          Blueberry necrotic shock
polygamus; Amaranthus            ilarvirus
tricolor ssp. mangostanus;       Caper latent carlavirus
Amaranthus tricolor ssp. tristis Caraway latent (?)
Common names:                    nepovirus
Chinese amaranth;                Carrot mottle mimic
Tampala; Ganges amaranth         umbravirus
Susceptible to:                  Carrot mottle umbravirus
Amaranthus leaf mottle           Carrot yellow leaf (?)
potyvirus                        closterovirus
Amaranthus mosaic (?)            Cassava African mosaic
potyvirus                        bigeminivirus
Apple mosaic ilarvirus           Cassava brown streak-
Amaryllis                        associated (?) carlavirus
Susceptible to:                  Cassava brown streak
Amaryllis (?)                    potyvirus
alphacryptovirus                 Cassava Caribbean mosaic (?)
Narcissus                        potexvirus
Susceptible to:                  Cassava Colombian
Narcissus yellow stripe          symptomless (?) potexvirus
potyvirus                        Cassava common mosaic (?)
Insusceptible to:                potexvirus
Silene X (?) potexvirus          Cassava green mottle
Narcissus jonquilla              nepovirus
Common names:                    Cassava Indian mosaic
Jonquil                          bigeminivirus
Susceptible to:                  Cassava Ivorian bacilliform
Strawberry latent ringspot       ourmiavirus
(?) nepovirus                    Cassava X potexvirus
Insusceptible to:                Cherry leaf roll nepovirus
Ornithogalum mosaic              Chickpea bushy dwarf
potyvirus                        potyvirus
Narcissus poeticus               Chickpea chlorotic dwarf (?)
Common names:                    monogeminivirus
Narcissus; Pheasant's-eye;       Chickpea distortion mosaic
Poet's narcissus                 potyvirus
Susceptible to:                  Chicory yellow mottle
Narcissus tip necrosis (?)       nepovirus
carmovirus                       Chino del tomat,
Narcissus pseudonarcissus        bigeminivirus
Common names:                    Citrus ringspot virus
Daffodil; Common daffodil        Cowpea chlorotic mottle
Susceptible to:                  bromovirus
Arabis mosaic nepovirus          Croton yellow vein mosaic
Narcissus late season            bigeminivirus
yellows (?) potyvirus            Cucumber necrosis
Narcissus latent                 tombusvirus
macluravirus                     Cymbidium ringspot
Narcissus mosaic potexvirus      tombusvirus
Narcissus tip necrosis (?)       Cynara (?)
carmovirus                       nucleorhabdovirus
Raspberry ringspot               Dandelion yellow mosaic
nepovirus                        sequivirus
Tobacco rattle tobravirus        Dasheen mosaic potyvirus
Tomato black ring                Desmodium mosaic
nepovirus                        potyvirus
Yucca                            Dioscorea green banding
Susceptible to:                  mosaic potyvirus
Furcraea necrotic streak (?)     Dioscorea latent (?)
dianthovirus                     potexvirus
Chlorophytum comosum             Dogwood mosaic (?)
Common names:                    nepovirus
Spider plant; Spider-ivy;        Eggplant green mosaic
Ribbon plant                     potyvirus
Insusceptible to:                Eggplant mottled dwarf
Onion mite-borne latent (?)      nucleorhabdovirus
potexvirus                       Eggplant severe mottle (?)
Shallot mite-borne latent (?)    potyvirus
potexvirus                       Elderberry latent (?)
Sint-Jan's onion latent (?)      carmovirus
carlavirus                       Epirus cherry ourmiavirus
Tradescantia-Zebrina             Euphorbia mosaic
potyvirus                        bigeminivirus
Catharanthus roseus              Grapevine A (?) trichovirus
Synonyms:                        Grapevine Algerian latent
Ammocallis rosea;                tombusvirus
Lochnera rosea; Vinca rosea      Grapevine Bulgarian latent
Common names:                    nepovirus
Bright-eyes; Madagascar          Grapevine chrome mosaic
periwinkle; Old-maid; Rose       nepovirus
periwinkle; Rosy periwinkle      Grapevine fanleaf nepovirus
Susceptible to:                  Groundnut chlorotic spot (?)
Abelia latent tymovirus          potexvirus
Alfalfa mosaic alfamovirus       Groundnut rosette
Apple mosaic ilarvirus           umbravirus
Bean pod mottle comovirus        Hibiscus latent ringspot
Beet curly top                   nepovirus
hybrigeminivirus                 Hydrangea mosaic ilarvirus
Belladonna mottle                Ivy vein clearing (?)
tymovirus                        cytorhabdovirus
Cacao yellow mosaic              Kalanchoe isometric virus
tymovirus                        Lato River tombusvirus
Carnation mottle                 Lettuce big-vein
carmovirus                       varicosavirus
Cassava green mottle             Lettuce mosaic potyvirus
nepovirus                        Lilac chlorotic leafspot
Cherry leaf roll nepovirus       capillovirus
Citrus leaf rugose ilarvirus     Lily X potexvirus
Citrus ringspot virus            Lucerne Australian
Clover wound tumor               symptomless (?) nepovirus
phytoreovirus                    Maracuja mosaic (?)
Clover yellow mosaic             tobamovirus
potexvirus                       Melon Ourmia ourmiavirus
Cowpea severe mosaic             Melothria mottle (?)
comovirus                        potyvirus
Cucumber mosaic                  Nandina mosaic (?)
cucumovirus                      potexvirus
Dogwood mosaic (?)               Narcissus latent
nepovirus                        macluravirus
Dulcamara mottle                 Narcissus tip necrosis (?)
tymovirus                        carmovirus
Elm mottle ilarvirus             Neckar River tombusvirus
Erysimum latent tymovirus        Nerine potyvirus
Foxtail mosaic potexvirus        Nicotiana velutina mosaic (?)
Humulus japonicus ilarvirus      furovirus
Lilac ring mottle ilarvirus      Oat golden stripe furovirus
Nandina mosaic (?)               Okra mosaic tymovirus
potexvirus                       Olive latent 1 (?)
Narcissus mosaic potexvirus      sobemovirus
Okra mosaic tymovirus            Olive latent 2 (?)
Pea seed-borne mosaic            ourmiavirus
potyvirus                        Paprika mild mottle
Peach enation (?) nepovirus      tobamovirus
Peanut stunt cucumovirus         Parsnip yellow fleck
Pepper ringspot tobravirus       sequivirus
Pepper veinal mottle             Passiflora ringspot
potyvirus                        potyvirus
Plum American line pattern       Peanut chlorotic streak
ilarvirus                        caulimovirus
Poplar mosaic carlavirus         Peanut clump furovirus
Potato 14R (?) tobamovirus       Peanut green mosaic
Potato black ringspot            potyvirus
nepovirus                        Peanut yellow spot
Potato T trichovirus             tospovirus
Prune dwarf ilarvirus            Pelargonium vein clearing (?)
Prunus necrotic ringspot         cytorhabdovirus
ilarvirus                        Pepper Moroccan
Scrophularia mottle              tombusvirus
tymovirus                        Pepper mottle potyvirus
Spring beauty latent             Pepper ringspot tobravirus
bromovirus                       Pepper Texas bigeminivirus
Tobacco mosaic satellivirus      Pepper veinal mottle
Tobacco necrosis necrovirus      potyvirus
Tobacco rattle tobravirus        Physalis mosaic tymovirus
Tobacco ringspot nepovirus       Pittosporum vein yellowing
Tobacco streak ilarvirus         nucleorhabdovirus
Tobacco stunt varicosavirus      Plantain 6 (?) carmovirus
Tomato spotted wilt              Plantain 7 (?) potyvirus
tospovirus                       Plantain X potexvirus
Tulare apple mosaic              Plum American line pattern
ilarvirus                        ilarvirus
Turnip crinkle carmovirus        Plum pox potyvirus
Watermelon mosaic 2              Poinsettia mosaic (?)
potyvirus                        tymovirus
Wild cucumber mosaic             Potato 14R (?) tobamovirus
tymovirus                        Potato Andean latent
Hedera helix                     tymovirus
Common names:                    Potato Andean mottle
English ivy                      comovirus
Susceptible to:                  Potato black ringspot
Ivy vein clearing (?)            nepovirus
cytorhabdovirus                  Potato mop-top furovirus
sparagus officinalis             Potato T trichovirus
Synonyms:                        Prune dwarf ilarvirus
Asparagus longifolius            Prunus necrotic ringspot
Common names:                    ilarvirus
Garden asparagus;                Red clover necrotic mosaic
Asparagus; Esparrag              dianthovirus
Susceptible to:                  Rice stripe necrosis (?)
Arabis mosaic nepovirus          furovirus
Asparagus 1 potyvirus            Rubus Chinese seed-borne (?)
Asparagus 2 ilarvirus            nepovirus
Strawberry latent ringspot (?)   Silene X (?) potexvirus
nepovirus                        Sitke waterborne (?)
Tobacco streak ilarvirus         tombusvirus
Dryopteris filix-mas             Solanum apical leaf curling (?)
Common names:                    bigeminivirus
Male fern                        Solanum nodiflorum mottle
Susceptible to:                  sobemovirus
Fern (?) potyvirus               Sonchus yellow net
Polystichum falcatum             nucleorhabdovirus
Susceptible to:                  Soybean mosaic potyvirus
Harts tongue fern (?)            Sweet potato feathery
tobravirus                       mottle potyvirus
Phyllitis scolopendrium          Sweet potato latent (?)
Synonyms:                        potyvirus
Asplenium scolopendrium          Sweet potato mild mottle
Common names:                    ipomovirus
Hart's-tongue fern               Sweet potato ringspot (?)
Susceptible to:                  nepovirus
Harts tongue fern (?)            Sweet potato sunken vein (?)
tobravirus                       closterovirus
Aucuba japonica                  Tamus latent (?) potexvirus
Synonyms:                        Telfairia mosaic potyvirus
Aucuba japonica var.             Tobacco mosaic satellivirus
variegata                        Tobacco mosaic
Common names:                    tobamovirus
Spotted-laurel; Japanese-        Tobacco rattle tobravirus
laurel                           Tobacco streak ilarvirus
Susceptible to:                  Tobacco stunt varicosavirus
Aucuba ringspot (?)              Tomato Australian leafcurl
badnavirus                       bigeminivirus
Cycas necrotic stunt             Tomato bushy stunt
nepovirus                        tombusvirus
Begonia elatior                  Tomato golden mosaic
Susceptible to:                  bigeminivirus
Carnation mottle                 Tomato mild mottle (?)
carmovirus                       potyvirus
Begonia x tuberhybrida           Tomato mosaic tobamovirus
Common names:                    Tomato mottle
Hybris tuberous begonia          bigeminivirus
Insusceptible to:                Tomato ringspot nepovirus
Aster chlorotic stunt (?)        Tomato yellow leaf curl
carlavirus                       bigeminivirus
Catalpa bignonioides             Tomato yellow mosaic
Synonyms:                        bigeminivirus
Catalpa bignonioides f.          Tropaeolum 1 potyvirus
aurea                            Tropaeolum 2 potyvirus
Common names:                    Tulip chlorotic blotch
Catawba; Common catalpa;         potyvirus
Indian-bean; Southern catalpa;   Tulip halo necrosis (?) virus
Cigartree; Smoking-bean          Tulip X potexvirus
Susceptible to:                  Ullucus mild mottle
Scrophularia mottle              tobamovirus
tymovirus                        Ullucus mosaic potyvirus
Acer palmatum                    Vanilla necrosis potyvirus
Abelia latent tymovirus          Watercress yellow spot
Betula                           virus
Susceptible to:                  Watermelon mosaic 2
Cherry leaf roll nepovirus       potyvirus
Ceiba pentandra                  Weddel waterborne (?)
Synonyms:                        carmovirus
Bombax pentandrum; Ceiba         Wild potato mosaic
casearia; Eriodendron            potyvirus
anfractuosum                     Yam mosaic potyvirus
Common names:                    Nicotiana tabacum
Ceiba; Kapok; Silk-cotton-       Synonyms:
tree; White silk-cotton-tree;    Nicotiana chinensis;
Kapokbaum; Kapokier; Arbe-       Nicotiana tabacum var.
kapok                            macrophylla
Susceptible to:                  Common names:
Cacao swollen shoot              Tobacco
badnavirus                       Susceptible to:
Cacao yellow mosaic              Abutilon mosaic
tymovirus                        bigeminivirus
Okra mosaic tymovirus            Alfalfa mosaic alfamovirus
Myosotis sylvatica               Alstroemeria (?) ilarvirus
Synonyms:                        Alstroemeria mosaic
Myosotis alpestris;              potyvirus
Myosotis oblongata               Amaranthus leaf mottle
Common names:                    potyvirus
Garden forget-me-not;            Arabis mosaic nepovirus
Wood forget-me-not               Arracacha A nepovirus
Susceptible to:                  Arracacha B (?) nepovirus
Arabis mosaic nepovirus          Arracacha Y potyvirus
Carnation ringspot               Artichoke Italian latent
dianthovirus                     nepovirus
Cymbidium ringspot               Artichoke yellow ringspot
tombusvirus                      nepovirus
Tobacco rattle tobravirus        Asparagus 2 ilarvirus
Tobacco ringspot nepovirus       Asparagus 3 potexvirus
Tomato black ring                Asystasia gangetica mottle
nepovirus                        (?) potyvirus
Ananas comosus                   Barley stripe mosaic
Synonyms:                        hordeivirus
Ananas duckei; Ananas            Bean distortion dwarf (?)
sativus; Ananas sativus var.     bigeminivirus
duckei; Bromelia ananas;         Bean yellow mosaic
Bromelia comosa                  potyvirus
Common names:                    Beet curly top
Pineapple; Pina                  hybrigeminivirus
Susceptible to:                  Beet pseudo-yellows (?)
Pineapple chlorotic leaf         closterovirus
streak (?) nucleorhabdovirus     Belladonna mottle
Pineapple wilt-associated        tymovirus
(?) closterovirus                Bidens mosaic potyvirus
Tomato spotted wilt              Blueberry leaf mottle
tospovirus                       nepovirus
Buxus sempervirens               Blueberry necrotic shock
Synonyms:                        ilarvirus
Buxus colchica                   Bramble yellow mosaic (?)
Common names:                    potyvirus
Boxwood; Common                  Broad bean wilt fabavirus
boxwood; Turkish boxwood         Burdock yellow mosaic (?)
Susceptible to:                  potexvirus
Arabis mosaic nepovirus          Cacao necrosis nepovirus
Cactaceae family                 Cacao yellow mosaic
Including:                       tymovirus
Austrocylindropuntia cylindrica  Carnation ringspot
Cactaceae                        dianthovirus
Carnegiea gigantea (syn. Cereus  Cassava African mosaic
giganteus)                       bigeminivirus
Saguaro; Giant cactus            Cassava green mottle
Cereus                           nepovirus
Chamaecereus sylvestrii          Cassava Indian mosaic
Echinocereus procumbens          bigeminivirus
Echinopsis                       Cassava Ivorian bacilliform
Epiphyllum                       ourmiavirus
Ferocactus acanthodes (syn.      Cassia mild mosaic (?)
Echinocactus acanthodes)         carlavirus
Opuntia engelmannii              Cassia severe mosaic (?)
Opuntia vulgaris (syn. Cactus    closterovirus
monacanthos; Opuntia             Celery latent (?) potyvirus
monacantha)                      Cherry leaf roll nepovirus
Prickly-pear cactus; Tuna;       Chickpea chlorotic dwarf (?)
Prickly-pear; Drooping prickly-  monogeminivirus
pear                             Chicory yellow mottle
Pereskia saccharosa              nepovirus
Schlumbergera bridgesii          Chilli veinal mottle (?)
Zygocactus                       potyvirus
Zygocactus truncatus             Chino del tomat,
Zygocactus x Schlumbergera       bigeminivirus
Susceptible to:                  Citrus ringspot virus
Cactus X potexvirus              Clover wound tumor
Cactus 2 carlavirus              phytoreovirus
Lobelia erinus                   Clover yellow vein
Common names:                    potyvirus
Edging lobelia                   Commelina X potexvirus
Susceptible to:                  Cowpea chlorotic mottle
Abelia latent tymovirus          bromovirus
Arabis mosaic nepovirus          Cowpea mosaic comovirus
Carnation ringspot               Cowpea mottle (?)
dianthovirus                     carmovirus
Cherry leaf roll nepovirus       Cowpea severe mosaic
Elm mottle ilarvirus             comovirus
Peanut stunt cucumovirus         Croton yellow vein mosaic
Strawberry latent ringspot       bigeminivirus
(?) nepovirus                    Cucumber green mottle
Tobacco rattle tobravirus        mosaic tobamovirus
Tomato black ring                Cucumber mosaic
nepovirus                        cucumovirus
Humulus japonicus                Cucumber necrosis
Synonyms:                        tombusvirus
Humulus scandens                 Cymbidium ringspot
Common names:                    tombusvirus
Japanese hop                     Datura Colombian potyvirus
Susceptible to:                  Datura distortion mosaic
Hop latent carlavirus            potyvirus
Humulus japonicus ilarvirus      Datura innoxia Hungarian
Lonicera                         mosaic (?) potyvirus
Susceptible to:                  Datura mosaic (?) potyvirus
Eggplant mottled dwarf           Datura necrosis potyvirus
nucleorhabdovirus                Datura shoestring potyvirus
Pittosporum vein yellowing       Datura yellow vein
nucleorhabdovirus                nucleorhabdovirus
Insusceptible to:                Dioscorea latent (?)
Tomato yellow leaf curl          potexvirus
bigeminivirus                    Dogwood mosaic (?)
Lonicera americana               nepovirus
Susceptible to:                  Eggplant green mosaic
Honeysuckle latent               potyvirus
carlavirus                       Eggplant mild mottle (?)
Carica papaya                    carlavirus
Synonyms:                        Eggplant mottled crinkle
Carica peltata; Carica           tombusvirus
posoposa; Papaya carica          Eggplant mottled dwarf
Common names:                    nucleorhabdovirus
Papaya; Pawpaw                   Eggplant severe mottle (?)
Susceptible to:                  potyvirus
Croton yellow vein mosaic        Elderberry latent (?)
bigeminivirus                    carmovirus
Papaya mosaic potexvirus         Elm mottle ilarvirus
Papaya ringspot potyvirus        Epirus cherry ourmiavirus
Watermelon mosaic 1              Eucharis mottle (?)
potyvirus                        nepovirus
Dianthus barbatus                Foxtail mosaic potexvirus
Common names:                    Frangipani mosaic
Sweet William                    tobamovirus
Susceptible to:                  Galinsoga mosaic
Alfalfa mosaic alfamovirus       carmovirus
Arabis mosaic nepovirus          Grapevine Bulgarian latent
Beet curly top                   nepovirus
hybrigeminivirus                 Grapevine chrome mosaic
Beet mosaic potyvirus            nepovirus
Carnation latent carlavirus      Grapevine fanleaf nepovirus
Carnation mottle                 Guar top necrosis virus
carmovirus                       Henbane mosaic potyvirus
Carnation necrotic fleck         Hibiscus latent ringspot
closterovirus                    nepovirus
Carnation (?) rhabdovirus        Hippeastrum mosaic
Carnation ringspot               potyvirus
dianthovirus                     Hop American latent
Carnation vein mottle            carlavirus
potyvirus                        Humulus japonicus ilarvirus
Carnation yellow stripe (?)      Ivy vein clearing (?)
necrovirus                       cytorhabdovirus
Clover wound tumor               Kalanchoe isometric virus
phytoreovirus                    Kyuri green mottle mosaic
Melon Ourmia ourmiavirus         tobamovirus
Okra mosaic tymovirus            Lamium mild mottle
Peanut stunt cucumovirus         fabavirus
Pelargonium line pattern (?)     Lilac chlorotic leafspot
carmovirus                       capillovirus
Potato black ringspot            Lilac ring mottle ilarvirus
nepovirus                        Lisianthus necrosis (?)
Potato M carlavirus              necrovirus
Silene X (?) potexvirus          Lucerne Australian latent
Strawberry latent ringspot (?)   nepovirus
(?) nepovirus                    Lucerne Australian
Tobacco ringspot nepovirus       symptomless (?) nepovirus
Tomato bushy stunt               Lucerne transient streak
tombusvirus                      sobemovirus
Viola mottle potexvirus          Lychnis ringspot
Dianthus caryophyllus            hordeivirus
Common names:                    Maclura mosaic
Carnation; Clavel                macluravirus
Susceptible to:                  Maracuja mosaic (?)
Alfalfa mosaic alfamovirus       tobamovirus
Arabis mosaic nepovirus          Marigold mottle potyvirus
Beet curly top                   Melandrium yellow fleck
hybrigeminivirus                 bromovirus
Carnation 1                      Melilotus mosaic (?)
alphacryptovirus                 potyvirus
Carnation 2 (?)                  Melon Ourmia ourmiavirus
alphacryptovirus                 Milk vetch dwarf nanavirus
Carnation etched ring            Myrobalan latent ringspot
caulimovirus                     nepovirus
Carnation Italian ringspot       Narcissus latent
tombusvirus                      macluravirus
Carnation latent carlavirus      Neckar River tombusvirus
Carnation mottle                 Nerine potyvirus
carmovirus                       Nicotiana velutina mosaic (?)
Carnation necrotic fleck         furovirus
Closterovirus                    Odontoglossum ringspot
Carnation (?) rhabdovirus        tobamovirus
Carnation ringspot               Okra leaf-curl bigeminivirus
dianthovirus                     Olive latent 1 (?)
Carnation vein mottle            sobemovirus
Potyvirus                        Olive latent 2 (?)
Carnation yellow stripe (?)      ourmiavirus
necrovirus                       Orchid fleck (?) rhabdovirus
Lettuce infectious yellows       Paprika mild mottle
(?) closterovirus                tobamovirus
Melandrium yellow fleck          Parietaria mottle ilarvirus
bromovirus                       Parsnip yellow fleck
Potato M carlavirus              sequivirus
Tobacco stunt varicosavirus      Passionfruit woodiness
Gypsophila elegans               potyvirus
Common names:                    Patchouli mosaic potyvirus
Baby's-breath                    Pea early browning
Susceptible to:                  tobravirus
Belladonna mottle                Pea mosaic potyvirus
tymovirus                        Pea streak carlavirus
Lychnis ringspot                 Peach enation (?) nepovirus
hordeivirus                      Peach rosette mosaic
Tobacco etch potyvirus           nepovirus
Tobacco necrosis necrovirus      Peanut chlorotic streak
Tobacco rattle tobravirus        caulimovirus
Tobacco ringspot nepovirus       Peanut clump furovirus
Tomato bushy stunt               Peanut stunt cucumovirus
tombusvirus                      Pelargonium line pattern (?)
Euonymus europaeus               carmovirus
Synonyms:                        Pelargonium vein clearing
Euonymus vulgaris                (?) cytorhabdovirus
Common names:                    Pelargonium zonate spot
European spindletree;            ourmiavirus
Spindletree                      Pepino mosaic potexvirus
Susceptible to:                  Pepper Indian mottle
Arabis mosaic nepovirus          potyvirus
Strawberry latent ringspot (?)   Pepper mild mosaic (?)
nepovirus                        potyvirus
Euonymus japonica                Pepper mild mottle
Susceptible to:                  tobamovirus
Euonymus fasciation (?)          Pepper Moroccan
rhabdovirus                      tombusvirus
Euonymus (?) rhabdovirus         Pepper mottle potyvirus
Beta vulgaris                    Pepper ringspot tobravirus
Common names:                    Pepper severe mosaic
Beet                             potyvirus
Susceptible to:                  Pepper Texas bigeminivirus
Alfalfa mosaic alfamovirus       Pepper veinal mottle
Arabis mosaic nepovirus          potyvirus
Arracacha A nepovirus            Physalis mosaic tymovirus
Asparagus 2 ilarvirus            Pittosporum vein yellowing
Asparagus 3 potexvirus           nucleorhabdovirus
Barley stripe mosaic             Plantain X potexvirus
hordeivirus                      Plum American line pattern
Beet 1 alphacryptovirus          ilarvirus
Beet 2 alphacryptovirus          Plum pox potyvirus
Beet 3 alphacryptovirus          Poinsettia mosaic (?)
Beet curly top                   tymovirus
hybrigeminivirus                 Poplar mosaic carlavirus
Beet distortion mosaic virus     Potato 14R (?) tobamovirus
Beet leaf curl (?)               Potato A potyvirus
rhabdovirus                      Potato Andean mottle
Beet mild yellowing              comovirus
luteovirus                       Potato aucuba mosaic
Beet mosaic potyvirus            potexvirus
Beet necrotic yellow vein        Potato black ringspot
furovirus                        nepovirus
Beet pseudo-yellows (?)          Potato mop-top furovirus
closterovirus                    Potato T trichovirus
Beet soil-borne furovirus        Potato U nepovirus
Beet western yellows             Potato V potyvirus
luteovirus                       Potato X potexvirus
Beet yellow net (?)              Potato Y potyvirus
luteovirus                       Potato yellow dwarf
Beet yellow stunt                nucleorhabdovirus
closterovirus                    Primula mosaic potyvirus
Beet yellows closterovirus       Primula mottle (?) potyvirus
Broad bean wilt fabavirus        Prune dwarf ilarvirus
Butterbur mosaic (?)             Radish mosaic comovirus
carlavirus                       Raspberry ringspot
Cacao necrosis nepovirus         nepovirus
Cacao yellow mosaic              Red clover necrotic mosaic
tymovirus                        dianthovirus
Cactus X potexvirus              Red clover vein mosaic
Caraway latent (?)               carlavirus
nepovirus                        Rhynchosia mosaic
Carnation latent carlavirus      bigeminivirus
Carnation mottle                 Ribgrass mosaic
carmovirus                       tobamovirus
Carnation vein mottle            Rose (?) tobamovirus
potyvirus                        Rubus Chinese seed-borne
Celery latent (?) potyvirus      (?) nepovirus
Cherry leaf roll nepovirus       Silene X (?) potexvirus
Chickpea chlorotic dwarf         Solanum nodiflorum mottle
(?) monogeminivirus              sobemovirus
Chicory yellow blotch (?)        Sonchus cytorhabdovirus
carlavirus                       Sowbane mosaic
Clover yellow mosaic             sobemovirus
potexvirus                       Soybean crinkle leaf (?)
Clover yellow vein               bigeminivirus
potyvirus                        Soybean mild mosaic virus
Cowpea chlorotic mottle          Soybean mosaic potyvirus
bromovirus                       Spinach latent ilarvirus
Cowpea mild mottle (?)           Strawberry latent ringspot (?)
carlavirus                       nepovirus
Croton yellow vein mosaic        Sunn-hemp mosaic
bigeminivirus                    tobamovirus
Cucumber mosaic                  Sweet clover necrotic
cucumovirus                      mosaic dianthovirus
Cucumber soil-borne              Sweet potato latent (?)
carmovirus                       potyvirus
Cycas necrotic stunt             Sweet potato mild mottle
nepovirus                        ipomovirus
Cymbidium ringspot               Sweet potato ringspot (?)
tombusvirus                      nepovirus
Dogwood mosaic (?)               Tamarillo mosaic potyvirus
nepovirus                        Telfairia mosaic potyvirus
Elderberry carlavirus            Tobacco etch potyvirus
Elderberry latent (?)            Tobacco leaf curl
carmovirus                       bigeminivirus
Elm mottle ilarvirus             Tobacco mild green mosaic
Epirus cherry ourmiavirus        tobamovirus
Foxtail mosaic potexvirus        Tobacco mosaic satellivirus
Grapevine Bulgarian latent       Tobacco mosaic
nepovirus                        tobamovirus
Grapevine fanleaf nepovirus      Tobacco mottle umbravirus
Groundnut eyespot                Tobacco necrosis necrovirus
potyvirus                        Tobacco necrosis
Helenium S carlavirus            satellivirus
Heracleum latent trichovirus     Tobacco necrotic dwarf
Humulus japonicus ilarvirus      luteovirus
Impatiens latent (?)             Tobacco rattle tobravirus
potexvirus                       Tobacco ringspot nepovirus
Lettuce infectious yellows       Tobacco streak ilarvirus
(?) closterovirus                Tobacco stunt varicosavirus
Lettuce mosaic potyvirus         Tobacco vein-distorting (?)
Lettuce speckles mottle          luteovirus
umbravirus                       Tobacco vein mottling
Lilac chlorotic leafspot         potyvirus
capillovirus                     Tobacco wilt potyvirus
Marigold mottle potyvirus        Tobacco yellow dwarf
Mulberry latent carlavirus       monogeminivirus
Odontoglossum ringspot           Tobacco yellow net (?)
tobamovirus                      luteovirus
Parsnip leafcurl virus           Tobacco yellow vein
Parsnip yellow fleck             assistor (?) luteovirus
sequivirus                       Tobacco yellow vein (?)
Pea seed-borne mosaic            umbravirus
potyvirus                        Tomato aspermy
Peanut clump furovirus           cucumovirus
Peanut stunt cucumovirus         Tomato Australian leafcurl
Pelargonium line pattern (?)     bigeminivirus
carmovirus                       Tomato black ring
Pepper ringspot tobravirus       nepovirus
Physalis mild chlorosis (?)      Tomato bushy stunt
luteovirus                       tombusvirus
Potato 14R (?) tobamovirus       Tomato golden mosaic
Potato black ringspot            bigeminivirus
nepovirus                        Tomato mild mottle (?)
Potato M carlavirus              potyvirus
Potato mop-top furovirus         Tomato mosaic tobamovirus
Potato T trichovirus             Tomato mottle
Potato U nepovirus               bigeminivirus
Radish mosaic comovirus          Tomato Peru potyvirus
Raspberry ringspot               Tomato ringspot nepovirus
nepovirus                        Tomato spotted wilt
Red clover necrotic mosaic       tospovirus
dianthovirus                     Tomato top necrosis (?)
Ribgrass mosaic                  nepovirus
tobamovirus                      Tomato yellow leaf curl
Rubus Chinese seed-borne         bigeminivirus
(?) nepovirus                    Tomato yellow mosaic
Sowbane mosaic                   bigeminivirus
sobemovirus                      Tulare apple mosaic
Soybean dwarf luteovirus         ilarvirus
Spinach latent ilarvirus         Tulip chlorotic blotch
Strawberry latent ringspot       potyvirus
(?) nepovirus                    Tulip halo necrosis (?) virus
Subterranean clover red leaf     Turnip mosaic potyvirus
luteovirus                       Turnip rosette sobemovirus
Sunn-hemp mosaic                 Ullucus mild mottle
tobamovirus                      tobamovirus
Sweet potato mild mottle         Ullucus mosaic potyvirus
ipomovirus                       Watermelon mosaic 2
Tobacco etch potyvirus           potyvirus
Tobacco mosaic                   Wild potato mosaic
tobamovirus                      potyvirus
Tobacco necrosis necrovirus      Wisteria vein mosaic
Tobacco rattle tobravirus        potyvirus
Tobacco ringspot nepovirus       Petunia x hybrida
Tobacco streak ilarvirus         Common names:
Tobacco stunt varicosavirus      Common garden petunia;
Tobacco yellow dwarf             Garden petunia
monogeminivirus                  Susceptible to:
Tomato black ring                Abelia latent tymovirus
nepovirus                        Alfalfa mosaic alfamovirus
Tulip halo necrosis (?) virus    Alstroemeria (?) ilarvirus
Tulip X potexvirus               Alstroemeria mosaic
Turnip mosaic potyvirus          potyvirus
Viola mottle potexvirus          Amaranthus leaf mottle
Spinacia oleracea                potyvirus
Common names:                    Amaranthus mosaic (?)
Spinach                          potyvirus
Susceptible to:                  Aquilegia (?) potyvirus
Alfalfa mosaic alfamovirus       Arabis mosaic nepovirus
Amaranthus leaf mottle           Arracacha A nepovirus
potyvirus                        Arracacha B (?) nepovirus
Arabis mosaic nepovirus          Artichoke latent potyvirus
Asparagus 3 potexvirus           Artichoke vein banding (?)
Barley stripe mosaic             nepovirus
hordeivirus                      Artichoke yellow ringspot
Bean yellow mosaic               nepovirus
potyvirus                        Asparagus 2 ilarvirus
Beet curly top                   Bean yellow mosaic
hybrigeminivirus                 potyvirus
Beet leaf curl (?)               Beet curly top
rhabdovirus                      hybrigeminivirus
Beet mild yellowing              Beet western yellows
luteovirus                       luteovirus
Beet mosaic potyvirus            Bidens mottle potyvirus
Beet necrotic yellow vein        Black raspberry necrosis
furovirus                        virus
Beet pseudo-yellows (?)          Brinjal mild mosaic (?)
closterovirus                    potyvirus
Beet soil-borne furovirus        Broad bean V (?) potyvirus
Beet western yellows             Broad bean wilt fabavirus
luteovirus                       Butterbur mosaic (?)
Beet yellows closterovirus       carlavirus
Black raspberry necrosis         Cacao necrosis nepovirus
virus                            Caper latent carlavirus
Broad bean wilt fabavirus        Carnation mottle
Canavalia maritima mosaic        carmovirus
(?) potyvirus                    Cassava green mottle
Carnation mottle                 nepovirus
carmovirus                       Cassava Indian mosaic
Carnation ringspot               bigeminivirus
dianthovirus                     Cassava Ivorian bacilliform
Carnation vein mottle            ourmiavirus
potyvirus                        Celery latent (?) potyvirus
Celery latent (?) potyvirus      Cherry leaf roll nepovirus
Cherry leaf roll nepovirus       Chicory yellow mottle
Clover yellow mosaic             nepovirus
potexvirus                       Chrysanthemum B
Clover yellow vein               carlavirus
potyvirus                        Citrus ringspot virus
Cowpea mild mottle (?)           Cowpea chlorotic mottle
Carlavirus                       bromovirus
Cowpea mosaic comovirus          Cowpea mosaic comovirus
Croton yellow vein mosaic        Cowpea severe mosaic
bigeminivirus                    comovirus
Cumcumber leaf spot              Croton yellow vein mosaic
carmovirus                       bigeminivirus
Cucumber mosaic                  Cucumber leaf spot
cucumovirus                      carmovirus
Cycas necrotic stunt             Cymbidium ringspot
nepovirus                        tombusvirus
Cymbidium ringspot               Datura distortion mosaic
tombusvirus                      potyvirus
Dandelion yellow mosaic          Datura innoxia Hungarian
sequivirus                       mosaic (?) potyvirus
Daphne Y potyvirus               Datura mosaic (?) potyvirus
Dogwood mosaic (?)               Dogwood mosaic (?)
nepovirus                        nepovirus
Elderberry latent (?)            Eggplant green mosaic
carmovirus                       potyvirus
Elm mottle ilarvirus             Eggplant mosaic tymovirus
Epirus cherry ourmiavirus        Eggplant mottled dwarf
Foxtail mosaic potexvirus        nucleorhabdovirus
Galinsoga mosaic                 Elderberry latent (?)
carmovirus                       carmovirus
Habenaria mosaic (?)             Elm mottle ilarvirus
potyvirus                        Epirus cherry ourmiavirus
Heracleum latent trichovirus     Galinsoga mosaic
Lettuce infectious yellows       carmovirus
(?) closterovirus                Grapevine chrome mosaic
Lettuce mosaic potyvirus         nepovirus
Lettuce necrotic yellows         Grapevine fanleaf nepovirus
cytorhabdovirus                  Groundnut eyespot
Lettuce speckles mottle          potyvirus
umbravirus                       Guar top necrosis virus
Lucerne Australian latent        Henbane mosaic potyvirus
nepovirus                        Hibiscus latent ringspot
Lucerne Australian               nepovirus
symptomless (?) nepovirus        Hibiscus yellow mosaic (?)
Lucerne transient streak         tobamovirus
sobemovirus                      Hippeastrum mosaic
Lychnis ringspot                 potyvirus
hordeivirus                      Honeysuckle latent
Melon Ourmia ourmiavirus         carlavirus
Melothria mottle (?)             Humulus japonicus ilarvirus
potyvirus                        Kyuri green mottle mosaic
Milk vetch dwarf nanavirus       tobamovirus
Mulberry latent carlavirus       Lamium mild mottle
Nandina mosaic (?)               fabavirus
potexvirus                       Lettuce infectious yellows
Nicotiana velutina mosaic        (?) closterovirus
(?) furovirus                    Lettuce necrotic yellows
Oat blue dwarf marafivirus       cytorhabdovirus
Okra mosaic tymovirus            Lilac chlorotic leafspot
Parietaria mottle ilarvirus      capillovirus
Parsnip leafcurl virus           Lilac mottle carlavirus
Parsnip mosaic potyvirus         Lisianthus necrosis (?)
Parsnip yellow fleck             necrovirus
sequivirus                       Lucerne Australian
Patchouli mosaic potyvirus       symptomless (?) nepovirus
Pea early browning               Lucerne transient streak
tobravirus                       sobemovirus
Pea streak carlavirus            Lychnis ringspot
Peanut chlorotic streak          hordeivirus
caulimovirus                     Marigold mottle potyvirus
Peanut clump furovirus           Melandrium yellow fleck
Peanut mottle potyvirus          bromovirus
Peanut stunt cucumovirus         Melilotus mosaic (?)
Pelargonium flower break         potyvirus
carmovirus                       Melon Ourmia ourmiavirus
Pelagonium line pattern (?)      Narcissus mosaic potexvirus
carmovirus                       Neckar River tombusvirus
Pepper Moroccan                  Olive latent ringspot
tombusvirus                      nepovirus
Pepper ringspot tobravirus       Olive latent 2 (?)
Petunia asteroid mosaic          ourmiavirus
tombusvirus                      Paprika mild mottle
Physalis mild chlorosis (?)      tobamovirus
luteovirus                       Parietaria mottle ilarvirus
Potato 14R (?) tobamovirus       Parsnip yellow fleck
Potato T trichovirus             sequivirus
Potato U nepovirus               Passionfruit Sri Lankan
Radish mosaic comovirus          mottle (?) potyvirus
Raspberry ringspot               Passionfruit woodiness
neprovirus                       potyvirus
Red clover necrotic mosaic       Pea early browning
dianthovirus                     tobravirus
Ribgrass mosaic                  Pea seed-borne mosaic
tubamovirus                      potyvirus
Rose (?) tobamovirus             Peach enation (?) nepovirus
Sowbane mosaic                   Peanut chlorotic streak
sobemovirus                      caulimovirus
Soybean mild mosaic virus        Peanut clump furovirus
Spinach latent ilarvirus         Peanut green mosaic
Spinach temperate                potyvirus
alphacryptovirus                 Peanut stunt cucumovirus
Statice Y potyvirus              Peanut yellow spot
Strawberry latent ringspot       tospovirus
(?) nepovirus                    Pelargonium line pattern (?)
Sunflower ringspot (?)           carmovirus
ilarvirus                        Pelargonium vein clearing (?)
Sunn-hemp mosaic                 cytorhabdovirus
tobamovirus                      Pepper mild mottle
Sweet potato mild mottle         tobamovirus
ipomovirus                       Pepper Moroccan
Tobacco necrosis necrovirus      tombusvirus
Tobacco necrotic dwarf           Pepper ringspot tobravirus
luteovirus                       Pepper severe mosaic
Tobacco rattle tobravirus        potyvirus
Tobacco ringspot nepovirus       Pepper veinal mottle
Tobacco streak ilarvirus         potyvirus
Tobacco stunt varicosavirus      Petunia asteroid mosaic
Tomato black ring                tombusvirus
nepovirus                        Petunia vein clearing (?)
Tomato bushy stunt               caulimovirus
tombusvirus                      Physalis mosaic tymovirus
Tomato spotted wilt              Pittosporum vein yellowing
tospovirus                       nucleorhabdovirus
Tulip halo necrosis (?) virus    Plantago mottle tymovirus
Tulip X potexvirus               Plantain X potexvirus
Turnip mosaic potyvirus          Plum American line pattern
Vallota mosaic potyvirus         ilarvirus
Viola mottle potexvirus          Plum pox potyvirus
Watermelon mosaic 2              Poplar mosaic carlavirus
potyvirus                        Potato 14R (?) tobamovirus
Wineberry latent virus           Potato Andean latent
Wisteria vein mosaic             tymovirus
potyvirus                        Potato aucuba mosaic
Cleome spinosa                   potexvirus
Synonyms:                        Potato black ringspot
Cleome hassleriana; Cleome       nepovirus
arborea; Cleome pungens          Potato mop-top furovirus
Common names:                    Potato U nepovirus
Spider-flower                    Potato yellow mosaic
Susceptible to:                  bigeminivirus
Turnip yellow mosaic             Primula mosaic potyvirus
tymovirus                        Prune dwarf ilarvirus
Gloriosa rothschildiana          Prunus necrotic ringspot
Synonyms:                        ilarvirus
Gloriosa superba; Gloriosa       Raspberry ringspot
abyssinica; Gloriosa homblei;    nepovirus
Gloriosa hybrid; Gloriosa simplex; Ribgrass mosaic
Gloriosa speciosa; Gloriosa      tobamovirus
virescens                        Rose (?) tobamovirus
Common names:                    Rubus Chinese seed-borne
Flame lily; Glory lily;          (?) nepovirus
Climbing lily; Creeping lily     Solanum nodiflorum mottle
Susceptible to:                  sobemovirus
Gloriosa fleck (?)               Sonchus cytorhabdovirus
nucleorhabdovirus                Soybean crinkle leaf (?)
Tradescantia zebrina             bigeminivirus
Synonyms:                        Soybean mild mosaic virus
Tradescantia pendula;            Soybean mosaic potyvirus
Zebrina pendula                  Spinach latent ilarvirus
Common names:                    Sunflower ringspot (?)
Wandering-jew                    ilarvirus
Susceptible to:                  Sunn-hemp mosaic
Tradescantia-Zebrina             tobamovirus
potyvirus                        Sweet potato mild mottle
Chrysanthemum morifolium         ipomovirus
Synonyms:                        Tamarillo mosaic potyvirus
Dendranthema x                   Tobacco etch potyvirus
grandiflorum; Anthemis           Tobacco leaf curl
grandiflorum; Anthemis           bigeminivirus
stipulacea; Chrysanthemum        Tobacco mild green mosaic
sinense; Chrysanthemum           tobamovirus
stipulaceum;                     Tobacco rattle tobravirus
Dendranthema x                   Tobacco ringspot nepovirus
morifolium; Matricaria morifolia Tobacco streak ilarvirus
Common names:                    Tobacco stunt varicosavirus
Florist's chrysanthemum;         Tobacco yellow vein (?)
Mum; Chrisanthemum               umbravirus
Susceptible to:                  Tomato black ring
Chrysanthemum B                  nepovirus
carlavirus                       Tomato bushy stunt
Cucumber mosaic                  tombusvirus
cucumovirus                      Tomato golden mosaic
Oat blue dwarf marafivirus       bigeminivirus
Tomato aspermy                   Tomato infectious chlorosis (?)
cucumovirus                      closterovirus
Helianthus annuus                Tomato mosaic tobamovirus
Synonyms:                        Tomato mottle
Helianthus annuus var.           bigeminivirus
macrocarpus; Helianthus          Tomato Peru potyvirus
lenticularis                     Tomato ringspot nepovirus
Common names:                    Tomato spotted wilt
Common annual sunflower;         tospovirus
Sunflower; Hopi sunflower;       Tomato top necrosis (?)
Common sunflower; Girasol        nepovirus
Susceptible to:                  Tomato vein clearing
Alfalfa mosaic alfamovirus       nucleorhabdovirus
Artichoke curly dwarf (?)        Tomato yellow mosaic
potexvirus                       bigeminivirus
Artichoke latent potyvirus       Tulip chlorotic blotch
Beet western yellows             potyvirus
luteovirus                       Tulip halo necrosis (?) virus
Bidens mosaic potyvirus          Turnip mosaic potyvirus
Bidens mottle potyvirus          Ullucus mild mottle
Cassia mild mosaic (?)           tobamovirus
carlavirus                       Ullucus mosaic potyvirus
Cherry leaf roll nepovirus       White clover mosaic
Citrus ringspot virus            potexvirus
Clover yellow mosaic             Wisteria vein mosaic
potexvirus                       potyvirus
Clover yellow vein               Theobroma cacao
potyvirus                        Synonyms:
Cucumber mosaic                  Theobroma sativa
cucumovirus                      Common names:
Cymbidium ringspot               Cacao; Chocolate-tree
tombusvirus                      Susceptible to:
Elm mottle ilarvirus             Cacao necrosis nepovirus
Galinsoga mosaic                 Cacao swollen shoot
carmovirus                       badnavirus
Humulus japonicus ilarvirus      Cacao yellow mosaic
Lettuce infectious yellows       tymovirus
(?) closterovirus                Cowpea mild mottle (?)
Maracuja mosaic (?)              carlavirus
tobamovirus                      Okra mosaic tymovirus
Melandrium yellow fleck          Tetragonia tetragonioides
bromovirus                       Susceptible to:
Patchouli mosaic potyvirus       Abelia latent tymovirus
Peanut stunt cucumovirus         Alfalfa mosaic alfamovirus
Pepper veinal mottle             Alstroemeria (?) ilarvirus
potyvirus                        Alstroemeria mosaic
Physalis mosaic tymovirus        potyvirus
Prune dwarf ilarvirus            Alstroemeria streak (?)
Prunus necrotic ringspot         potyvirus
ilarvirus                        Amaranthus leaf mottle
Red clover necrotic mosaic       potyvirus
dianthovirus                     Apple stem pitting virus
Sunflower crinkle (?)            Arabis mosaic nepovirus
umbravirus                       Arracacha A nepovirus
Sunflower mosaic (?)             Arracacha B (?) nepovirus
potyvirus                        Arracacha latent (?)
Sunflower ringspot (?)           carlavirus
ilarvirus                        Arracacha Y potyvirus
Sunflower yellow blotch (?)      Asparagus 1 potyvirus
umbravirus                       Asparagus 3 potexvirus
Tobacco necrosis necrovirus      Asystasia gangetica mottle
Tobacco rattle tobravirus        (?) potyvirus
Tobacco streak ilarvirus         Bean common mosaic
Tomato black ring                potyvirus
nepovirus                        Bean yellow mosaic
Tomato spotted wilt              potyvirus
tospovirus                       Beet leaf curl (?)
Tropaeolum 2 potyvirus           rhabdovirus
Convolvulus arvensis             Beet mild yellowing
Common names:                    luteovirus
Field bindweed                   Beet mosaic potyvirus
Insusceptible to:                Beet necrotic yellow vein
Carnation vein mottle            furovirus
potyvirus                        Beet western yellows
Cornus florida                   luteovirus
Common names:                    Beet yellows closterovirus
Flowering dogwood;               Broad bean necrosis
American-boxwood                 furovirus
Susceptible to:                  Cacao necrosis nepovirus
Cherry leaf roll nepovirus       Cacao yellow mosaic
Dogwood mosaic (?)               tymovirus
nepovirus                        Carnation mottle
Synonyms:                        carmovirus
Corylus avellana f. aurea;       Carnation ringspot
Corylus avellana f. contorta;    dianthovirus
Corylus avellana f. fusco-rubra; Carnation vein mottle
Corylus avellana f. heterophylla; potyvirus
Corylus avellana f.              Cassava green mottle
pendula; Corylus avellana        nepovirus
var. aurea; Corylus avellana var. Cassava Ivorian bacilliform
contorta; Corylus avellana var.  ourmiavirus
fusco-rubra; Corylus avellana var. Cassia mild mosaic (?)
heterophylla;                    carlavirus
Corylus avellana var.            Celery latent (?) potyvirus
pendula; Corylus heterophylla    Chickpea distortion mosaic
Common names:                    potyvirus
European filbert; European       Chrysanthemum B
hazel; Avellana; Hazelnut        carlavirus
Susceptible to:                  Clover wound tumor
Tulare apple mosaic              phytoreovirus
ilarvirus                        Clover yellow vein
Kalanchoe blossfeldiana          potyvirus
Synonyms:                        Commelina X potexvirus
Kalanchoe globulifera var.       Cowpea mild mottle (?)
coccinea                         carlavirus
Susceptible to:                  Cucumber mosaic
Kalanchoe latent carlavirus      cucumovirus
Kalanchoe mosaic (?)             Cycas necrotic stunt
potyvirus                        nepovirus
Kalanchoe top-spotting           Cymbidium ringspot
badnavirus                       tombusvirus
Brassica napus var. napus        Dasheen mosaic potyvirus
Synonyms:                        Dioscorea latent (?)
Brassica campestris f.           potexvirus
annua; Brassica campestris f.    Dogwood mosaic (?)
biennis; Brassica napus f. annua; nepovirus
Brassica napus f. biennis; Brassica Eucharis mottle (?)
napus ssp. oleifera;             nepovirus
Brassica napus var. annua;       Foxtail mosaic potexvirus
Brassica napus var. biennis;     Groundnut eyespot
Brassica napus var. oleifera     potyvirus
Common names:                    Habenaria mosaic (?)
Rape; Colza; Bird rape;          potyvirus
Canola                           Helenium S carlavirus
Susceptible to:                  Heracleum latent trichovirus
Watercress yellow spot           Hibiscus latent ringspot
virus                            nepovirus
Brassica nigra                   Hypochoeris mosaic (?)
Synonyms:                        furovirus
Brassica nigra var.              Impatiens latent (?)
abyssinica; Sinapis nigra        potexvirus
Common names:                    Iris mild mosaic potyvirus
Black mustard                    Kalanchoe isometric virus
Susceptible to:                  Kalanchoe latent carlavirus
Beet western yellows             Lamium mild mottle
luteovirus                       fabavirus
Ribgrass mosaic                  Lettuce big-vein
tobamovirus                      varicosavirus
Turnip mosaic potyvirus          Lettuce mosaic potyvirus
Turnip yellow mosaic             Lilac chlorotic leafspot
tymovirus                        capillovirus
Citrullus vulgaris               Lily X potexvirus
Synonyms:                        Lisianthus necrosis (?)
Citrullus lanatus var.           necrovirus
lanatus; Citrullus aedulis; Citrullus Lucerne Australian latent
lanatus var. caffer; Colocynthis nepovirus
citrullus; Cucurbita citrullus   Lychnis ringspot
Common names:                    hordeivirus
Watermelon                       Maclura mosaic
Susceptible to:                  macluravirus
Cucumber green mottle            Malva veinal necrosis (?)
mosaic tobamovirus               potexvirus
Cucumber vein yellowing          Marigold mottle potyvirus
virus                            Melandrium yellow fleck
Telfairia mosaic potyvirus       bromovirus
Watermelon chlorotic stunt       Melilotus mosaic (?)
bigeminivirus                    potyvirus
Wild cucumber mosaic             Melon Ourmia ourmiavirus
tymovirus                        Narcissus latent
Cucurbita maxima                 macluravirus
Common names:                    Narcissus mosaic potexvirus
Squash; Pumpkin                  Narcissus tip necrosis (?)
Susceptible to:                  carmovirus
Apple mosaic ilarvirus           Nerine potyvirus
Bean yellow mosaic               Nerine X potexvirus
potyvirus                        Odontoglossum ringspot
Beet curly top                   tobamovirus
hybrigeminivirus                 Okra mosaic tymovirus
Cherry leaf roll nepovirus       Ornithogalum mosaic
Clover yellow mosaic             potyvirus
potexvirus                       Parietaria mottle ilarvirus
Cucumber leaf spot               Parsnip leafcurl virus
carmovirus                       Parsnip yellow fleck
Cucumber mosaic                  sequivirus
cucumovirus                      Patchouli mottle (?)
Daphne X potexvirus              potyvirus
Elm mottle ilarvirus             Pea early browning
Eucharis mottle (?)              tobravirus
nepovirus                        Pea mosaic potyvirus
Grapevine fanleaf nepovirus      Pea seed-borne mosaic potyvirus
Humulus japonicus ilarvirus      Peach enation (?) nepovirus
Kyuri green mottle mosaic        Peanut clump furovirus
tobamovirus                      Peanut green mosaic
Lettuce infectious yellows       potyvirus
(?) closterovirus                Peanut stunt cucumovirus
Lisianthus necrosis (?)          Pelargonium flower break
necrovirus                       carmovirus
Maracuja mosaic (?)              Pelargonium line pattern (?)
tobamovirus                      carmovirus
Melandrium yellow fleck          Pepino mosaic potexvirus
bromovirus                       Pepper ringspot tobravirus
Melon leaf curl                  Plantago mottle tymovirus
bigeminivirus                    Poplar mosaic carlavirus
Melothria mottle (?)             Potato 14R (?) tobamovirus
potyvirus                        Potato black ringspot
Papaya ringspot potyvirus        nepovirus
Pea seed-borne mosaic            Potato mop-top furovirus
potyvirus                        Potato U nepovirus
Peanut stunt cucumovirus         Primula mosaic potyvirus
Poplar mosaic carlavirus         Red clover necrotic mosaic
Prune dwarf ilarvirus            dianthovirus
Prunus necrotic ringspot         Ribgrass mosaic
ilarvirus                        tobamovirus
Radish mosaic comovirus          Solanum nodiflorum mottle
Sowbane mosaic                   sobemovirus
sobemovirus                      Soybean dwarf luteovirus
Squash leaf curl                 Spinach latent ilarvirus
bigeminivirus                    Strawberry latent ringspot
Squash mosaic comovirus          (?) nepovirus
Strawberry latent ringspot       Sweet clover necrotic
(?) nepovirus                    mosaic dianthovirus
Sunflower ringspot (?)           Sweet potato mild mottle
ilarvirus                        ipomovirus
Tobacco necrosis necrovirus      Sweet potato ringspot (?)
Tobacco ringspot nepovirus       nepovirus
Tobacco streak ilarvirus         Tamus latent (?) potexvirus
Tomato bushy stunt               Telfairia mosaic potyvirus
tombusvirus                      Tobacco etch potyvirus
Watermelon curly mottle          Tobacco necrosis necrovirus
bigeminivirus                    Tobacco ringspot nepovirus
Watermelon mosaic 1              Tobacco stunt varicosavirus
potyvirus                        Tomato black ring
Watermelon mosaic 2              nepovirus
potyvirus                        Tomato bushy stunt
Wild cucumber mosaic             tombusvirus
tymovirus                        Tomato vein clearing
Zucchini yellow fleck            nucleorhabdovirus
potyvirus                        Tulip chlorotic blotch
Zucchini yellow mosaic           potyvirus
potyvirus                        Tulip halo necrosis (?) virus
Cycas revoluta                   Tulip X potexvirus
Common names:                    Turnip crinkle carmovirus
Sago cycas; Sotesu-nut           Turnip mosaic potyvirus
Susceptible to:                  Ullucus C comovirus
Cycas necrotic stunt             Ullucus mild mottle
nepovirus                        tobamovirus
Dioscorea alata                  Ullucus mosaic potyvirus
Synonyms:                        Vallota mosaic potyvirus
Dioscorea rubella                Viola mottle potexvirus
Common names:                    Watermelon mosaic 2
Yam; Greater yam; Water          potyvirus
yam; Winged yam; White yam;      Wineberry latent virus
Guyana arrowroot; Ten-months     Wisteria vein mosaic
yam; Name-de-Agna                potyvirus
Susceptible to:                  Camellia japonica
Dioscorea alata potyvirus        Synonyms:
Dioscorea trifida (?)            Camellia japonica var.
potyvirus                        hortensis; Camellia japonica var.
Yam internal brown spot (?)      hozanensis; Camellia japonica var.
badnavirus                       spontanea; Thea japonica
Yam mosaic potyvirus             Common names:
Vaccinium corymbosum             Common camellia
Synonyms:                        Susceptible to:
Vaccinium constablaei            Camellia yellow mottle (?)
Common names:                    varicosavirus
Highbush blueberry;              Thunbergia alata
Blueberry; American blueberry;   Common names:
Swamp blueberry                  Black-eyed-Susan-vine;
Susceptible to:                  Ojitos-negros
Blueberry leaf mottle            Susceptible to:
nepovirus                        Datura yellow vein
Blueberry necrotic shock         nucleorhabdovirus
ilarvirus                        Prune dwarf ilarvirus
Blueberry red ringspot           Daphne cneorum
caulimovirus                     Common names:
Blueberry scorch carlavirus      Rose daphne; Garland
Blueberry shoestring             flower
sobemovirus                      Susceptible to:
Croton bonplandianus             Daphne S (?) carlavirus
Synonyms:                        Daphne X potexvirus
Croton sparsiflorus              Daphne Y potyvirus
Susceptible to:                  Corchorus olitorius
Croton yellow vein mosaic        Common names:
bigeminivirus                    Nalta jute; Tossa jute; Tussa
Euphorbia marginata              jute
Synonyms:                        Susceptible to:
Euphorbia variegata              Okra mosaic tymovirus
Common names:                    Tropaeolum majus
Snow-on-the-mountain             Common names:
Susceptible to:                  Garden nasturtium; Indian-
Beet curly top                   cress; Mastuerzo
hybrigeminivirus                 Susceptible to:
Dulcamara mottle                 Alfalfa mosaic alfamovirus
tymovirus                        Apple mosaic ilarvirus
Poinsettia mosaic (?)            Arabis mosaic nepovirus
tymovirus                        Beet curly top
Watermelon mosaic 2              hybrigeminivirus
potyvirus                        Beet western yellows
Quercus velutina                 luteovirus
Common names:                    Broad bean wilt fabavirus
Black oak                        Cherry leaf roll nepovirus
Susceptible to:                  Clover mild mosaic virus
Oak ringspot virus               Cucumber mosaic
Eustoma russellianum             cucumovirus
Synonyms:                        Cymbidium mosaic
Bilamista grandiflora;           potexvirus
Eustoma grandiflorum;            Cymbidium ringspot
Lisianthius russellianus         tombusvirus
Common names:                    Lamium mild mottle
Bluebells; Prairie-gentian       fabavirus
Susceptible to:                  Lettuce infectious yellows
Bean yellow mosaic               (?) closterovirus
potyvirus                        Melandrium yellow fleck
Lisianthus necrosis (?)          bromovirus
necrovirus                       Nasturtium mosaic (?)
Pelargonium peltatum             potyvirus
Synonyms:                        Okra mosaic tymovirus
Geranium peltatum                Pea early browning
Common names:                    tobravirus
Ivy geranium; Hanging            Poplar mosaic carlavirus
geranium                         Red clover necrotic mosaic
Susceptible to:                  dianthovirus
Pelargonium flower break         Ribgrass mosaic
carmovirus                       tobamovirus
Pelargonium line pattern (?)     Strawberry latent ringspot
carmovirus                       (?) nepovirus
Pelargonium vein clearing        Sunn-hemp mosaic
(?) cytorhabdovirus              tobamovirus
Pelargonium x domesticum         Tobacco rattle tobravirus
Insusceptible to:                Tobacco ringspot nepovirus
Aster chlorotic stunt (?)        Tomato black ring
carlavirus                       nepovirus
Carnation vein mottle            Tomato spotted wilt
potyvirus                        tospovirus
Chrysanthemum B                  Tropaeolum 2 potyvirus
carlavirus                       White clover mosaic
Saintpaulia ionantha             potexvirus
Common names:                    Anethum graveolens
African violet; Usambara         Synonyms:
violet                           Anethum sowa;
Susceptible to:                  Peucedanum graveolens
Carnation ringspot               Common names:
dianthovirus                     Dill; Dill seed; Garden dill;
Saintpaulia leaf necrosis (?)    Eneldo; Aneto; Fenouil-batard;
rhabdovirus                      Endro
Ribes nigrum                     Susceptible to:
Common names:                    Artichoke yellow ringspot
Black currant; Cassis            nepovirus
Susceptible to:                  Carrot mottle umbravirus
Strawberry latent ringspot (?)   Carrot red leaf luteovirus
nepovirus                        Celery mosaic potyvirus
Hypericum perforatum             Heracleum latent trichovirus
Common names:                    Parsnip yellow fleck
Common St. John's-wort;          sequivirus
Klamathweed; St. John's-wort;    Foeniculum vulgare
Goatweed                         Common names:
Insusceptible to:                Fennel; Florence fennel;
Carnation ringspot               Finocchio; Hinojo
dianthovirus                     Susceptible to:
Hyacinthus orientalis            Coriander feathery red vein
Common names:                    nucleorhabdovirus
Common hyacinth                  Insusceptible to:
Susceptible to:                  Celery yellow spot (?)
Hyacinth mosaic potyvirus        luteovirus
Crocus vernus                    Heracleum latent trichovirus
Susceptible to:                  Parsnip yellow fleck
Iris severe mosaic potyvirus     sequivirus
Freesia refracta                 Valeriana officinalis
Synonyms:                        Common names:
Freesia leichtlinii; Gladiolus   Common valeriana; Garden-
refractus                        heliotrope
Susceptible to:                  Susceptible to:
Freesia leaf necrosis            Watermelon mosaic 2
varicosavirus                    potyvirus
Freesia mosaic potyvirus         Verbena hybrida
Gladiolus                        Common names:
Susceptible to:                  Garden verbena; Florist's
Artichoke Italian latent         verbena
nepovirus                        Susceptible to:
Bean yellow mosaic               Carnation ringspot
potyvirus                        dianthovirus
Cycas necrotic stunt             Melilotus mosaic (?)
nepovirus                        potyvirus
Narcissus latent                 Viola odorata
macluravirus                     Common names:
Iris                             English violet; Sweet violet;
Susceptible to:                  Garden violet
Iris mild mosaic potyvirus       Susceptible to:
Iris severe mosaic potyvirus     Tulip X potexvirus
Juglans regia                    Viola mottle potexvirus
Synonyms:                        Vitis vinifera
Juglans duclouxiana;             Common names:
Juglans fallax; Juglans kamaonica; European grape; Wine
Juglans orientis; Juglans regia ssp. grape; Vid
kamaonica; Juglans regia var.    Susceptible to:
orientis; Juglans                Arabis mosaic nepovirus
regia var. sinensis; Juglans     Artichoke Italian latent
sinensis                         nepovirus
Common names:                    Grapevine A (?) trichovirus
English walnut; Persian          Grapevine ajinashika
walnut; Nogal                    disease (?) luteovirus
susceptible to:                  Grapevine Algerian latent
Cherry leaf roll nepovirus       tombusvirus
Leguminosae                      Grapevine B (?) trichovirus
Insusceptible to:                Grapevine Bulgarian latent
Voandzeia necrotic mosaic        nepovirus
tymovirus                        Grapevine chrome mosaic
Mimosa pudica                    nepovirus
Common names:                    Grapevine corky bark-
Sensitive-plant; Touch-me-       associated (?) closterovirus
not; Shame plant                 Grapevine fanleaf nepovirus
Insusceptible to:                Grapevine fleck virus
Mimosa mosaic virus              Grapevine leafroll-
Soybean mosaic potyvirus         associated (?) closteroviruses
Lilium                           Grapevine line pattern (?)
Susceptible to:                  ilarvirus
Lily mottle potyvirus            Grapevine stem pitting
Tomato aspermy                   associated closterovirus
cucumovirus                      Grapevine stunt virus
Tulip breaking potyvirus         Petunia asteroid mosaic
Tulipa                           tombusvirus
Susceptible to:                  Strawberry latent ringspot
Arabis mosaic nepovirus          (?) nepovirus
Tobacco rattle tobravirus        Zingiber officinale
Tomato black ring                Synonyms:
nepovirus                        Amomum zingiber
Tomato bushy stunt               Common names:
tombusvirus                      Ginger; Jengibre
                                 Susceptible to:
                                 Ginger chlorotic fleck (?)
                                 sobemovirus

[0126] Overview of Bioinformatics Methods

[0127] A. Phred, Phrap and Consed

[0128] Phred, Phrap and Consed are a set of programs which read DNA sequencer traces, make base calls, assemble the shotgun DNA sequence data and analyze the sequence regions that are likely to contribute to errors. Phred is the initial program used to read the sequencer trace data, call the bases and assign quality values to the bases. Phred uses a Fourier-based method to examine the base traces generated by the sequencer. The output files from Phred are written in FASTA, phd or scf format. Phrap is used to assemble contiguous sequences from only the highest quality portion of the sequence data output by Phred. Phrap is amenable to high-throughput data collection. Finally, Consed is used as a “finishing tool” to assign error probabilities to the sequence data. Detailed description of the Phred, Phrap and Consed software and its use can be found in the following references which are hereby incorporated herein by reference: Ewing, B., Hillier, L., Wendl, M. C. and Green, P. (1998) “Base-calling of automated sequencer traces using Phred. I. Accuracy assessment.” Genome Res. 8: 175-178; Ewing, B. and Green, P. (1998) “Base-calling of automated sequencer traces using Phred. II. Error probabilities.” Genome Res. 8:186-194; Gordon, D., Abajian, C. and Green, P. (1998) “Consed: a graphical tool for sequence finishing.” Genome Res. 8: 195-202.

[0129] B. BLAST

[0130] The BLAST (“Basic Local Alignment Search Tool”) set of programs may be used to compare the large numbers of sequences and obtain homologies to known protein families. These homologies provide information regarding the function of newly sequenced genes. Detailed description of the BLAST software and its uses can be found in the following references which are hereby incorporated herein by reference: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) “Basic Local Alignment Search Tool.” J. Mol. Biol. 215: 403-410; Altschul, S. F. (1991) “Amino acid subsitution matrices from an informatics theoretic perspective.” J. Mol. Biol. 219: 555-565.

[0131] Generally, BLAST performs sequence similarity searching and is divided into 5 basic programs: (1) BLASTP compares an amino acid sequence to a protein sequence database; (2) BLASTN compares a nucleotide sequence to a nucleic acid sequence database; (3) BLASTX compares translated protein sequences done in 6 frames to a protein sequence database; (4) TBLASTN compares a protein sequence to a nucleotide sequence database that is translated into all 6 reading frames; (5) TBLASTX compares the 6 frame translated protein sequence to the 6-frame translation of a nucleotide sequence database. Programs (3)-(5) may be used to identify weak similarities in nucleic acid sequence.

[0132] The BLAST program is based on the High Segment Pair (HSP), two sequence fragments of arbitrary but equal length whose alignment is locally maximized and whose alignment meets or exceeds a cutoff threshold. BLAST determines multiple HSP sets statistically using “sum” statistics. The score of the HSP is then related to its expected chance of frequency of occurrence, E. The value, E, is dependent on several factors such as the scoring system, residue composition of sequences, length of query sequence and total length of database. In the output file will be listed these E values, these are typically in a histogram format, and are useful in determining levels of statistical significance at the user's predefined expectation threshold. Finally, the Smallest Sum Probability, P(N) is the probability of observing the shown matched sequences by chance alone and is typically in the range of 0-1.

[0133] BLAST measures sequence similarity using a matrix of similarity scores for all possible pairs of residues and these specify scores for aligning pairs of amino acids. The matrix of choice for a specific use depends on several factors: the length of the query sequence and whether or not a close or distant relationship between sequences is suspected. Several matrices are available including PAM40, PAM120, PAM250, BLOSUM 62 and BLOSUM 50. Altschul et al. (1990) found PAM120 to be the most broadly sensitive matrix (i.e. point accepted mutation matrix per 100 residues). However, in some cases the PAM120 matrix may not find short but strong or long but weak similarities between sequences. In these cases, pairs of PAM matrices may be used, such as PAM40 and PAM 250, and the results compared. Typically, PAM 40 is used for database searching with a query of 9-21 residues long, while PAM 250 is used for lengths of 47-123.

[0134] The BLOSUM (Blocks Substitution Matrix) series of matrices are constructed based on percent identity between two sequence segments of interest. Thus, the BLOSUM62 matrix is based on a matrix of sequence segments in which the members are less than 62% identical. BLOSUM62 shows very good performance for BLAST searching. However, other BLOSUM matrices, like the PAM matrices, may be useful in other applications. For example, BLOSUM45 is particularly strong in profile searching.

[0135] C. FASTA

[0136] The FASTA suite of programs permits the evaluation of DNA and protein similarity based on local sequence alignment. The FASTA search algorithm utilizes Smith/Waterman- and Needleman/Wunsch-based optimization methods. These algorithms consider all of the alignment possibilities between the query sequence and the library in the highest-scoring sequence regions. The search algorithm proceeds in four basic steps:

[0137] 1). The identities or pairs of identities between the two DNA or protein sequences are determined. The ktup parameter, as set by the user, is operative and determines how many consecutive sequence identities are required to indicate a match.

[0138] 2). The regions identified in step 1 are re-scored using a PAM or BLOSUM matrix. This allows conservative replacements and runs of identities shorter than that specified by ktup to contribute to the similarity score.

[0139] 3). The region with the single best scoring initial region is used to characterize pairwise similarity and these scores are used to rank the library sequences.

[0140] 4). The highest scoring library sequences are aligned using the Smith-Waterman algorithm. This final comparison takes into account the possible alignments of the query and library sequence in the highest scoring region.

[0141] Further detailed description of the FASTA software and its use can be found in the following reference which is hereby incorporated herein by reference: Pearson, W. R. and Lipman, D. J. (1988) “Improved tools for biological sequence comparison.” Proc.Natl.Acad. Sci. 85: 2444-2448.

[0142] D. Pfam

[0143] Despite the large number of different protein sequences determined through genomics-based approaches, relatively few structural and functional domains are known. Pfam is a computational method that utilizes a collection of multiple alignments and profile hidden Markov models of protein domain families to classify existing and newly found protein sequences into structural families. Detailed description of the Pfam software and its uses can be found in the following references which are hereby incorporated herein by reference: Sonhammer, E. L. L., Eddy, S. R. and Durbin, R. (1997) “Pfam: a comprehensive database of protein domain families based on seed alignments.” Proteins: Structure, Function and Genetics 28: 405-420; Sonhammer, E. L. L., Eddy, S. R. Bimey, E., Bateman, A. and Durbin, R. (1998) “Pfam: multiple sequence alignments and HMM-profiles of protein domains.” Nucleic Acids Res. 26: 320-322; Bateman, A., Birney, E., Durbin, R., Eddy, S. R. Finn, R. D. and Sonhammer, E. L. L. (1999) Nucleic Acids Res. 27: 260-262.

[0144] Pfam 3.1, the latest version, includes 54% of proteins in SWISS_PROT and SP-TrEMBL-5 as a match to the database and includes expectation values for matches. Pfam consists of parts A and B. Pfam-A, contains a hidden Markov model and includes curated families. Pfam-B, uses the Domainer program to cluster sequence segments not included in Pfam-A. Domainer uses pairwise homology data from Blastp to construct aligned families.

[0145] Alternative protein family databases that may be used include PRINTS and BLOCKS, which both are based on a set of ungapped blocks of aligned residues. However, these programs typically contain short conserved regions whereas Pfam represents a library of complete domains that facilitates automated annotation. Comparisons of Pfam profiles may also be performed using genomic and EST data with the programs, Genewise and ESTwise, respectively. Both of these programs allow for introns and frameshifting errors.

[0146] E. BLOCKS

[0147] The determination of sequence relationships between unknown sequences and those that have been categorized can be problematic because background noise increases with the number of sequences, especially at a low level of similarity detection. One recent approach to this problem has been tested that efficiently detects and confirms weak or distant relationships among protein sequences based on a database of blocks. The BLOCKS database provides multiple alignments of sequences and contains blocks or protein motifs found in known families of proteins.

[0148] Other programs such as PRINTS and Prodom also provide alignments, however, the BLOCKS database differs in the manner in which the database was constructed. Construction of the BLOCKS database proceeds as follows: one starts with a group of sequences that presumably have one or more motifs in common, such as those from the PROSITE database. The PROTOMAT program then uses a motif finding program to scan sequences for similarity looking for spaced triplets of amino acids. The located blocks are then entered into the MOTOMAT program for block assembly. Weights are computed for all sequences. Following construction of a BLOCKS database one can use BLIMPS to perform searches of the BLOCKS database. Detailed description of the construction and use of a BLOCKS database can be found in the following references which are hereby incorporated herein by reference: Henikoff, S. and Henikoff, J. G. (1994) “Protein family classification based on searching a database of blocks.” Genomics 19: 97-10; Henikoff, J. G. and Henikoff, S. (1996) “The BLOCKS database and its applications.” Meth. Enz. 266: 88-105.

[0149] F. PRINTS

[0150] The PRINTS database of protein family fingerprints can be used in addition to BLOCKS and PROSITE. These databases are considered to be secondary databases because they diagnose the relationship between sequences that yield function information. Presently, however, it is not recommended that these databases be used alone. Rather, it is strongly suggested that these pattern databases be used in conjunction with each other so that a direct comparison of results can be made to analyze their robustness.

[0151] Generally, these programs utilize pattern recognition to discover motifs within protein sequences. However, PRINTS goes one step further, it takes into account not simply single motifs but several motifs simultaneously that might characterize a family signature. Other programs, such as PROSITE, rely on pattern recognition but are limited by the fact that query sequences must match them exactly. Thus, sequences that vary slightly will be missed. In contrast, the PRINTS database fingerprinting approach is capable of identifying distant relatives due to its reliance on the fact that sequences do not have match the query exactly. Instead they are scored according to how well they fit each motif in the signature. Another advantage of PRINTS is that it allows the user to search both PRINTS and PROSITE simultaneously. A detailed description of the use of PRINTS can be found in the following references which are hereby incorporated herein by reference:Attwood, T. K., Beck, M. E., Bleasly, A. J., Degtyarenko, K., Michie, A. D. and Parry-Smith, D. J. (1997) Nucleic Acids Res. 25: 212-216.

[0152] Related, Variant, Altered and Extended Nucleic Acid Sequences

[0153] In one embodiment, the invention provides a polypeptide comprising the amino acid sequence encoded by a cDNA identified by a polynucleotide sequence chosen from the group consisting of SEQ ID NO: 1-122. The invention also encompasses variant polypeptides which retain the functional activity of causing a dwarf phenotype in a plant. A preferred variant is one having at least 80%, more preferably 90%, and most preferably 95% amino acid sequence identity to the original polypeptide sequence.

[0154] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding the same polypeptide, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence, and all such variations are to be considered as being specifically disclosed.

[0155] It may be advantageous to produce nucleotide sequences encoding polypeptide or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding a polypeptide and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

[0156] The invention also encompasses production of DNA sequences having the function of causing a dwarf phenotype in a plant, or portions thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into such a sequence or any portion thereof.

[0157] Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the polynucleotide sequences shown in SEQ ID NO: 1-122, under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at a defined stringency.

[0158] Altered nucleic acid sequences causing a dwarf phenotype in a plant which are encompassed by the invention include deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that is functionally equivalent. The encoded polypeptide may also contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and consequently remains functionally equivalent. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the functional activity is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

[0159] Also included within the scope of the present invention are alleles of the genes encoded by cDNAs identified by the polynucleotide sequences SEQ ID NO: 1-122. As used herein, an “allele” or “allelic sequence” is an alternative form of the gene which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0160] Methods for DNA sequencing which are well known and generally available in the art may be used to practice any embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE® (US Biochemical Corporation, Cleveland, Ohio), TAQ® polymerase (U.S. Biochemical Corporation, Cleveland, Ohio), thermostable T7 polymerase (Amersham Pharmacia Biotech, Chicago, Ill.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE® amplification system (Life Technologies, Rockville, Md.). Preferably, the process is automated with machines such as the MICROLAB® 2200 (Hamilton Company, Reno, Nev.), PTC200 DNA Engine thermal cycler (MJ Research, Watertown, Mass.) and the ABI 377™ DNA sequencer (Perkin Elmer).

[0161] The nucleic acid sequences of the invention may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed, “restriction-site” PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

[0162] Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO 4.06 primer analysis software (National Biosciences Inc., Plymouth, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

[0163] Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR.

[0164] Another method which may be used to retrieve unknown sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER™ DNA Walking Kits libraries (Clontech, Palo Alto, Calif.) to walk in genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

[0165] When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into the 5′ and 3′ non-transcribed regulatory regions.

[0166] Capillary electrophoresis systems which are commercially available (e.g. from PE Biosystems, Inc., Foster City, Calif.) may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera. Output/light intensity may be converted to electrical signal using appropriate software (e.g. GENOTYPER® and SEQUENCE NAVIGATOR® from PE Biosystems, Foster City, Calif.) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

[0167] Vectors, Engineering, and Expression of Sequences

[0168] In another embodiment of the invention, cDNA sequences or fragments thereof which have the function of causing a dwarf phenotype in a plant, or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotide sequences which encode substantially the same or a functionally equivalent polypeptide also may be produced and these sequences may be used to clone and express the polypeptide of interest.

[0169] As will be understood by those of skill in the art, it may be advantageous to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0170] The polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter their polypeptide encoding sequences for a variety of reasons, including but not limited to, introducing alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.

[0171] In another embodiment of the invention, natural, modified, or recombinant polynucleotide sequences having the function of causing a dwarf phenotype in a plant may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of the dwarf phenotype, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the wild-type coding sequence and the heterologous protein sequence, so that the wild-type polypeptide may be cleaved and purified away from the heterologous moiety.

[0172] In another embodiment, polynucleotide sequences having the function of causing a dwarf phenotype in a plant may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the polypeptide product may be produced using chemical methods to synthesize the amino acid sequence. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A™ peptide synthesizer (PE Corporation, Norwalk, Conn.).

[0173] The newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (see, e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; or Creighton, supra). Additionally, the amino acid sequence, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

[0174] In order to express a biologically active polypeptide, the encoding nucleotide sequences or their functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

[0175] Methods which are well known to those skilled in the art may be used to construct expression vectors containing nucleic acid sequences and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, both of which are hereby incorporated by reference herein.

[0176] A variety of expression vector/host systems may be utilized to contain and express sequences having the function of causing a dwarf phenotype in a plant. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV; brome mosaic virus) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0177] The “control elements” or “regulatory sequences” are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ translated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT® phagemid (Stratagene, La Jolla, Calif.) or PSPORT1™ plasmid (Life Technologies, Inc., Rockville, Md.) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

[0178] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the resulting gene product. For example, when large quantities of gene product are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifinctional E.coli cloning and expression vectors such as BLUESCRIPT® phagemid (Stratagene, La Jolla, Calif.), in which a sequence may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEMX™ vectors (Promega Corporation, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0179] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0180] In cases where plant expression vectors are used, the expression of sequences having the function of causing a dwarf phenotype in a plant may be driven by any of a number of promoters. In a preferred embodiment, plant vectors are created using a recombinant plant virus containing a recombinant plant viral nucleic acid, as described in PCT publication WO 96/40867 which is hereby incorporated herein by reference. Subsequently, the recombinant plant viral nucleic acid which contains one or more non-native nucleic acid sequences may be transcribed or expressed in the infected tissues of the plant host and the product of the coding sequences may be recovered from the plant, as described in WO 99/36516, which is hereby incorporated herein by reference.

[0181] An important feature of this embodiment is the use of recombinant plant viral nucleic acids which contain one or more non-native subgenomic promoters capable of transcribing or expressing adjacent nucleic acid sequences in the plant host and which result in replication and local and/or systemic spread in a compatible plant host. The recombinant plant viral nucleic acids have substantial sequence homology to plant viral nucleotide sequences and may be derived from an RNA, DNA, cDNA or a chemically synthesized RNA or DNA. A partial listing of suitable viruses is described below.

[0182] The first step in producing recombinant plant viral nucleic acids according to this particular embodiment is to modify the nucleotide sequences of the plant viral nucleotide sequence by known conventional techniques such that one or more non-native subgenomic promoters are inserted into the plant viral nucleic acid without destroying the biological function of the plant viral nucleic acid. The native coat protein coding sequence may be deleted in some embodiments, placed under the control of a non-native subgenomic promoter in other embodiments, or retained in a further embodiment. If it is deleted or otherwise inactivated, a non-native coat protein gene is inserted under control of one of the non-native subgenomic promoters, or optionally under control of the native coat protein gene subgenomic promoter. The non-native coat protein is capable of encapsidating the recombinant plant viral nucleic acid to produce a recombinant plant virus. Thus, the recombinant plant viral nucleic acid contains a coat protein coding sequence, which may be native or a nonnative coat protein coding sequence, under control of one of the native or non-native subgenomic promoters. The coat protein is involved in the systemic infection of the plant host.

[0183] Some of the viruses which meet this requirement include viruses from the tobamovirus group such as Tobacco Mosaic virus (TMV), Ribgrass Mosaic Virus (RGM), Cowpea Mosaic virus (CMV), Alfalfa Mosaic virus (AMV), Cucumber Green Mottle Mosaic virus watermelon strain (CGMMV-W) and Oat Mosaic virus (OMV) and viruses from the brome mosaic virus group such as Brome Mosaic virus (BMV), broad bean mottle virus and cowpea chlorotic mottle virus. Additional suitable viruses include Rice Necrosis virus (RNV), and geminiviruses such as tomato golden mosaic virus (TGMV), Cassava latent virus (CLV) and maize streak virus (MSV). However, the invention should not be construed as limited to using these particular viruses, but rather the method of the present invention is contemplated to include all plant viruses at a minimum.

[0184] Other embodiments of plant vectors used for the expression of sequences having the function of stunting a plant include, for example, viral promoters such as the 35S and 19S promoters of CaMVused alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0185] An insect system may be used to express the polypeptides of the invention. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the gene product may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the gene product may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0186] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the nucleic acid sequences of the invention may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the relevant gene product in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0187] Specific initiation signals may also be used to achieve more efficient translation of the nucleic acid sequences of the invention. Such signals include the ATG initiation codon and adjacent sequences. In cases where a sequence, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

[0188] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

[0189] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express a specific gene product may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

[0190] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk− or aprt− cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection; for example, dhfr, which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0191] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if a nucleic acid sequence of the invention is inserted within a marker gene sequence, recombinant cells containing that specific sequence can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0192] Alternatively, host cells which contain a nucleic acid sequence of the invention and which express its gene product may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

[0193] The presence of polynucleotide sequences of the invention can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of polynucleotide sequence of interest. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences of interest to detect transformants containing the relevant DNA or RNA. As used herein “oligonucleotides” or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.

[0194] A variety of protocols for detecting and measuring the expression of a cDNA, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

[0195] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to the polynucleotide sequences of the invention include oligonucleotide labeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits from Pharmacia & Upjohn (Kalamazoo, Mich.), Promega Corporation (Madison, Wis.) and U.S. Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0196] Host cells transformed with a polynucleotide sequence of the invention may be cultured under conditions suitable for the expression and recovery of protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of its corresponding polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join polynucleotide sequences of the invention to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS™ extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (available from Invitrogen, San Diego, Calif.) between the purification domain and polypeptide of interest may be used to facilitate purification. One such expression vector provides for expression of a fusion protein comprising a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif 3: 263-281,) while the enterokinase cleavage site provides a means for purifying polypeptide of interest from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0197] In addition to recombinant production, a fragment of a polypeptide of the invention may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Various peptide fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

[0198] In additional embodiments, the nucleotide and amino acid sequences of the present invention may be incorporated into any molecular biology techniques yet to be developed, provided these new techniques rely on properties of nucleotide and amino acid sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

[0199] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. The examples are intended specifically to illustrate the various methods used to identify and characterize the cDNAs of the present invention and the method by which they can be used to cause a dwarf phenotype in a plant.

EXAMPLES

[0200] I. Construction and Characterization of a Normalized Arabidopsis cDNA library in GENEWARE® Vectors

[0201] A. Plant Tissue Generation:

[0202] Arabidopsis thaliana ecotype Columbia (0) seeds were sown and grown on PEAT LITE MIX (Speedling Inc., Sun City, Fla.) supplemented with NUTRICOTE fertilizer (Plantco Inc., Ontario, Canada). Plants were grown under a 16-hour light/8-hour dark cycle in an environmental controlled growth chamber. The temperature was set at 22° C. for daytime and 18° C. for nighttime. The entire plant, root, leaves and all aerial parts were collected 4 weeks post sowing. Tissue was washed in deionized water and frozen in liquid nitrogen.

[0203] B. RNA Extraction:

[0204] High quality total RNA is isolated using a hot borate method. All solutions were made in DEPC-treated, double-deionized water and autoclaved. All glassware, mortars, pestles, spatulas, and glass rods were baked at 400° C. for four hours. All plasticware was DEPC-treated for at least three hours and then autoclaved.

[0205] Thirty-five milliliters of XT buffer (0.2 M Na borate decahydrate, 30 mM EGTA, 1% SDS (w/v), 1% deoxycholate, sodium) per 10 grams of tissue was dispensed into 50 milliliter Falcon tubes. PVP-40, 000 was added to a final concentration of 2% (w/v). NP-40 was added to a final concentration of 1% (w/v). Tubes were placed in an 80° C. water bath. The mortar and pestles were then pre-cooled in liquid nitrogen. Proteinase K (0.5 mg/ml XT buffer) was dispensed into 250 ml centrifuge bottles and the bottles were then placed on ice.

[0206] The tissue was added to the pre-chilled mortar and pestle and ground to a fine powder. Working as quickly as possible, the tissue was transferred to a glass beaker using a spatula chilled in liquid nitrogen. DTT (1.54 mg/ml XT buffer) was added to the XT buffer/PVP/NP-40 buffer and was immediately added to the ground tissue. The tissue was homogenized using a polytron at level 5 for one minute. The homogenate was decanted into the 250 ml centrifuge bottle containing the proteinase K. The homogenate was incubated at 42° C., 100 rpm for 1.5 hours. Eighty microliters of 2M KCl/ml of XT buffer was added to the homogenate and gently swirled until mixed. The samples were then incubated on ice for one hour. The samples were centrifuged at 12,000× G in a BECKAN® JA-14 rotor (Beckman Instruments, Inc., Fullerton, Calif.) for 20 minutes at 4° C. to remove debris. The supernatant was then filtered through a funnel lined with sterile miracloth into a sterile 250 ml centrifuge bottle. Eight molar LiCl was added to a final concentration of 2M LiCl and the samples were incubated on ice overnight.

[0207] Precipitated RNA was pelleted by centrifugation at 12,000× G in a BECKMAN® JA-14 rotor for 20 minutes (Beckman Instruments, Inc., Fullerton, Calif.) and the supernatant was discarded. The RNA pellet was washed in 5 milliliters of cold 2M LiCl in 30 ml centrifuge tubes. Glass rods and gentle vortexing were used to break and disperse the RNA pellet. The pellets were centrifuged in a Beckman JA-20 rotor for 10 krpm at 4° C. for 10 minutes. The supernatant was decanted. This wash step was repeated 3 times until the supernatant was relatively colorless. The RNA pellet was resuspended in 5 milliliters of 10 Tris-Cl (pH 7.5). The insoluble material was pelleted in a JA-17 at 10 k rpm for 10 minutes at 4° C. The supernatant was transferred to another 30 ml centrifuge tube and 0.1× volume of 2M K-acetate (pH 5.5) was added. The samples were incubated on ice for 15 minutes and centrifuged in a BECKMAN® JA-17 rotor (Beckman Instruments, Inc., Fullerton, Calif.) at 10 k rpm, 4° C., for 10 minutes to remove polysaccharides and insoluble material. The supernatant was transferred to a sterile 30 ml centrifuge tube and RNA was precipitated by adding 2.5× volumes of 100% ethanol. The RNA was precipitated overnight at −20° C. The precipitated RNA was pelleted by centrifugation at 9 krpm, 4° C. for 30 minutes in a JA-17 rotor. The RNA pellet was washed with 5 milliliters of cold 70% ethanol and centrifuged in a JA-17 rotor at 9 k rpm, 4° C. for 10 minutes. The residual ethanol was removed using a BECKMAN® speed vac (Beckman Instruments, Inc., Fullerton, Calif.). The RNA pellet was resuspended in 3 milliliters of DEPC-ddH2O+1 mM EDTA. The RNA was precipitated with 0.1× volumes of 3M Na-acetate pH 6.0 and 2× volumes of cold 100% ethanol. The RNA was put at -80° C. for storage. A BECKMAN® spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) was used to measure absorbance (A) at A260 and A280. The A260 was used to determine concentration (40 μg RNA/ml=1 A260 absorbance unit) and the A260/A280 ratio was used to determine the initial quality of the RNA (1.8 to 2.0 is good).

[0208] The yield of total RNA from 60 g of tissue is ˜15 mg. Then, mRNA was isolated from total RNA using oligo (dT)25 DYNABEADS® (Dynal, Inc., Lake Success, N.Y.). Typically, 1% of total RNA population can be recovered as mRNA in Arabidopsis thaliana whole plant and from 5 μg of poly A+ RNA, approximate 4.5 μg of single strand cDNA and 6.7 μg of double strand cDNA was synthesized.

[0209] C. cDNA Synthesis:

[0210] Poly A+ RNA was purified from total RNA using the oligo (dT)25 DYNABEADS® kit (Dynal, Inc., Lake Success, N.Y.) according to manufacturer's instructions. Briefly, DYNABEADS® was resuspended by mixing on a roller and transfer 600 μl to an RNase free tube. The beads were further equilibriated with 2× binding buffer (20 mM Tris-HCl, pH 7.5, 1M LiCl, 2 mM EDTA) twice and resuspended in 200 μl of 2× binding buffer. Total RNA 1 mg (200 μl) was heated at 70° C. for 5 minutes and incubated with the above oligo (dT)25 DYNABEADS® for 10 min at RT. The supernatant containing unbound rRNA and tRNA was subsequently removed by magnetic stand and washed twice with 1× wash buffer (10 mM Tris-HCl, pH 7.5, 0.15M LiCl, 1 mM EDTA). The mRNA was eluted from the DYNABEADS® in ddH2O and used as the starting material for double strand cDNA synthesis.

[0211] Double strand cDNA was synthesized either with NotI-(dT)25 primer or on oligo (dT)25 DYNABEADS® based on the manufacturer's instruction (Gibco-BRL superscript system). Typically, 5 μg of poly A+ RNA was annealed and reverse transcribed at 37° C. with SUPERSCRIPT II reverse transcriptase (Stratagene, La Jolla, Calif.). For the non-normalized cDNA library, double stranded cDNAs were ligated to a 500 to 1000-fold molar excess SalI adaptor, restriction enzyme NotI digested and size-selected by column fractionation. Those cDNAs were then cloned directionally into the XhoI-NotI sites of the TMV expression vector, 1057 N/P.

[0212] D. Normalization Procedure:

[0213] For the normalized cDNA preparation, the supernatant was removed from the DYNABEADS® and the cDNA containing beads were washed twice with 1× TE buffer. To carry out the normalization process, the second strand cDNA were eluted from the beads. 100 μl of TE buffer was added to the beads and heated at 95° C. for 5 min and the supernatant was then collected on magnetic stand. The above procedure was repeated once to ensure complete elution. The yield of second strand cDNA was quantitated using a UV spectrophotometer.

[0214] First strand cDNA beads is combined with second strand cDNA in 4× SSC, 5× Denhardt's and 0.5% SDS for multiple rounds of short hybridization. Since the second strand cDNA was synthesized using the first strand cDNA as the template, approximately the same amount of first and second strand cDNAs were present in the hybridization reaction. Nine μg of second strand cDNA in 200 μl of 1× TE buffer was added to the cDNA driver (first strand cDNA on beads) in a screw cap tube. The reaction was heated at 95° C. for 5 min, then 60 μl of 20× SSC, 30 μl of 50× Denhardt's (1% of Ficoll, 1% of polyvinylpyrrolidone and 1% of bovine serum albumin) and 15μl of 10% SDS were added and the reaction was brought to 65° C. for 8 hours.

[0215] The beads and supernatant were separated at 65° C. by magnet. The supernatant was transferred to a fresh tube and kept at 65° C. The beads were regenerated by adding 200 μl of ddH2O and heated at 95° C. for 5 min. We collected the beads for the next round of hybridization and kept the solution containing the bound second strand cDNA for further analysis. The partially normalized second strand cDNA solution was added back to the regenerated beads and a return to another round of hybridization of 8 hours. This procedure was repeated 4-5 times.

[0216] E. Slot Blot Analysis:

[0217] To follow the process of cDNA normalization a rapid slot blot procedure was developed. Following sequencing of 960 cDNAs, 46 cDNAs were selected to follow the representation of various classes of cDNAs through the normalization procedure. Based on their frequency of appearance in the sequence, these clones represent transcripts of different expression levels (high, moderate and low). Ten nanograms of each cDNA were deposited onto a HYBOND™-N+ membrane (Amersham Pharmacia Biotech, Chicago, Ill.) along with control vector (pBS) and water controls. DNA was denatured, neutralized, and subsequently crosslinked into the membrane using UV-STRATALINKER™ 2400 (Stratagene, La Jolla, Calif.).

[0218] cDNAs from either the non-normalized or normalized pool were labelled with 32P and hybridized on the slot blot membrane overnight at 65° C. in 1% bovine serum albumin, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5 M sodium phosphate (pH 7.2), and 7% sodium dodecyl sulfate (SDS). Then, blots were washed once in 1× SSC/0.2% SDS for 20 min at room temperature followed by two washes in 0.2× SSC/0.2% SDS for 20 min. at 65° C. The resulting membranes were then developed using a PHOSPHORIMAGER™ (Amersham Pharmacia Biotech, Chicago, Ill.) and quantitated using available software.

[0219] F. Conversion of Single-Stranded Normalized cDNAs to Double-Stranded Form:

[0220] Second strand normalized cDNA in hybridization solution was purified by QIAQUICK™ column (QIAGEN GmbH, Hilden, Germany) and eluted in 88 μl of ddH2O (total 1.2 μg of DNA is recovered). One μl (3 μg) of NotI-oligo dT primer was added and heated at 95° C. for 5 min followed by cool down to 37° C. The first strand cDNA was extended with T7 DNA polymerase (Amersham Pharmacia Biotech, Chicago, Ill.) in the presence of dNTP in 120 μl reaction at 37° C. for 1 hour. T4 DNA polymerase (NEB) was then used to polish the ends following the extension reaction for 5 min at 16° C. The resulting double strand cDNA was ethanol precipitated and ligated with 500- to 1 000-fold molar excess of SalI adaptor followed by NotI digestion. The resulting cDNAs were size-fractionated using a Clontech spin column 400 and the first two fractions that contained the cDNAs were pooled and used for the subsequent cloning process.

[0221] G. Construction of cDNA Libraries in GENEWARE® Vectors:

[0222] (+) Sense cDNA clones were prepared as follows. The Tobacco Mosaic Virus expression vector, 1 056GTN-AT9 was linearized with NotI and XhoI and a 900 bp stuffer DNA was removed. The presence of the stuffer DNA in between those two sites is to ensure the complete digestion by restriction enzymes and thus achieve the high cloning efficiency. The digested vector was gel purified and then used to set up ligation reaction with normalized cDNA SalI-NotI fragments to generate (+) sense cDNA clones.

[0223] (−) Sense cDNA clones were prepared as follows. The Tobacco Mosaic Virus expression vector 1057 NP also linearized with NotI and XhoI and a stuffer DNA fragment was removed. The digested vector was gel purified and used to set up ligation reaction to generate (−) sense strand library.

[0224] Each ligation was transformed into chemically competent E. coli cells, DH5 α according to manufacturer's instruction (Life Technologies, Rockville, Md.). Preliminary analysis of cloning efficiency was measured by plating of a small portion of the transformation, while archiving the majority for future applications. Vector-only ligations gave ˜2×104 cfu/μg vector and ligations with cDNA insertions gave ˜5×105 cfu/μg.

[0225] H. Analysis of Normalized cDNA Populations:

[0226] With each successive round of kinetic re-association, the total cDNA population is depleted thereby confirming the removal of a population of the cDNA from the mixture at each step. To further understand the consequences of this depletion and measure the relative normalization in cDNA representation following various stages of the kinetic re-association method, slot blots of 46 genes of varying representations were hybridized with probes made from non-normalized and normalized cDNA preparations. The resulting blots were then analyzed for representation by PHOSPHORIMAGER® analysis. The hybridization pattern of non-normalized cDNA to the gene array reveals a quite asymmetric representation with some genes hybridizing with great intensity while others showing no hybridization at all. The variance among hybridization intensities for each spot within the filter was measured by standard deviation and found to be 649. In order to analyze the cDNA fraction depleted from the mixture, the first strand magnetic bead matrix was eluted, a radioactive probe was generated and hybridized to a replica of the slot blot described above. The resulting hybridization intensities indicated that primarily those cDNAs of higher copy number were bound and removed from the normalized cDNA population, confirming that the depletion phenomenon correlated with removal of primarily high copy number cDNAs. The cDNA population not bound to first strand magnetic beads after 5 serial passages was collected, radioactive probe was generated and hybridized to a replica slot blot of known gene set described above. The resulting hybridization pattern (i.e. the relative intensity of the slots on the blot) was in striking contrast to that of the non-normalized cDNA and to that of the bound cDNA fraction. Assuming that the majority of the hybridization signal to the slot blot for the non-normalized cDNA blot results from hybridization to high abundance genes, an initial comparison can be made between the number of bound counts on the normalized versus non-normalized slot blots. This comparison is possible since each probe added to the blots was derived from the same quantity of cDNA material and an equal number of probe counts were applied to the blots. The non-normalized blot contained 17,898 counts while the normalized blot contained only 1494 counts. This represents a 12-fold reduction in overall signal indicating a significant reduction in high gene copy number in the normalized cDNA population.

[0227] When the hybridization intensity of the non-normalized cDNA probe to each gene is plotted against the relative number of counts (following subtraction of the pBS vector control intensity from each sample), there is almost a 4-log difference in sequence representation in the cDNA population and an overall variance in standard deviation of 649-fold. In contrast, the hybridization of normalized cDNA probe to each gene revealed an average of only 32-fold difference. This represents both a reduction in high copy cDNAs and an increased representation in low copy cDNAs by >3 logs. The variance between the most highly represented cDNA and lowest represented cDNA within the normalized cDNA population was ˜1.5 logs. The above values characterizing the degree of library normalization are equivalent to those achieved by Soares, et al. (1994).

[0228] I. Analysis of GENEWARE® Clones:

[0229] To ascertain the cloning efficiency of normalized cDNA into each vector and the average insert size, 96 random colonies were picked and grown by standard methods. DNA was isolated from bacteria using a BIOROBOT™ 9600 (QIAGEN GmbH, Hilden, Germany). DNA was digested with Not I and BsiWI restriction endonucleases (recognition sites flank the cDNA insertion). The digestions were separated on agarose gels and visualized by ethidium bromide staining. The digestions revealed a vector religation background of ˜4%. Ligations giving >75% insertions were passed as to quality control and more colonies were picked. Approximently 600 independent clones were analyzed by restriction digestion as described above. Interestingly, a similar percentage of vector background was detected ˜4% and the average insert size in the vector was ˜1 kb, with many inserts with 2 kb or greater sized inserts. Following analysis of DNA by restriction mapping, DNA was subjected to sequencing and further analysis.

[0230] J. Sequence Analysis of the Normalized Arabidopisis Library in GENEWARE®:

[0231] Initial analysis of non-normalized Arabidopsis cDNA library required the sequencing of 1709 independent clones. Three 96-well plates of randomly picked normalized Arabidopsis library in GENEWARE® [(−) sense] were initially sequenced by primer TP6 to yield 262 5′ sequences and passed sequence quality control. Initially, internal cluster analysis was performed to identify identical sequences in this sequence subset. Analysis using BLASTN algorithm showed that of the 262 sequences analyzed, 252 were unique and only 10 were found to cluster into five two-member clusters. We then identified the redundancy of the sequences against the larger public databases. For cluster analysis, we used a very low BLASTX score criteria (e=10−6) and compared all sequences against the GENBANK® nr database (United States Department of Health and Human Services). In this manner, we could derive the most information concerning the redundancy, gene type found and open reading frame status of all clones simultaneously. The low BLASTX score was used to allow all possible protein homologues to be identified. The clustering analysis revealed that of the 262 sequences there were 252 single member sequence clusters and five two-gene clusters. This represents 96% singletons from this sample size. The genes appearing more than once in the library varied from two different chlorophyll a/b binding proteins, lipid transport proteins to ferrodoxin-thioredoxin reductases. This result compares quite favorably to the 4 redundant clones (of one gene type) identified by Soares, et al. (1994) from 187 randomly picked clones from one normalized library.

[0232] Further analysis of the sequences from the GENEWARE® normalized cDNA library revealed that of the 262 sequences subjected to BLASTX search of the GENBANK® nr database, 29% of the sequences failed to show significant homology to any characterized protein or open reading frame (ORF). Of the 252 singletons in the library, 179 showed single hit to an identified ORF, while 73 showed no hit. These results suggest that, in spite of the well characterized nature of the sequence database quality libraries can still contain a high proportion of new expressed sequences.

[0233] The excellent representation and extremely low redundancy observed in these initial plates of normalized Arabidopsis cDNAs in GENEWARE® prompted us to sequence additional clones. This was important because there is often a significant bias in small sample sizes with regard to representation. A total of 1,151 sequences passed sequence quality control. Internal cluster analysis showed that ˜260 multi-sequence clusters were present, with the highest representation at 6 members and the majority with only 2 members (˜150). About 600 unique clusters were identified from the total of 856 clusters from the 1151 sequences. Therefore, from the 1151 sequences analyzed, 1,010 unique genes were identified, or a 87.7% gene discovery rate. In contrast, internal cluster analysis of the non-normalized Arabidopsis cDNA sequences revealed ˜840 multi-gene clusters with the highest represented cluster containing 27 members. Cluster analysis of the 1709 non-normalized Arabidopsis cDNAs revealed clusters of 27 members and many other highly populated clusters, a dramatic difference from the normalized cDNAs.

[0234] Further comparison of 1,151 randomly chosen non-normalized sequences for redundancy with the results from the 1,151 normalized population clearly indicated the positive effects of normalization and the greater number of unique genes identified from this normalized population. Many genes that have representations of >12 in the non-normalized library have been reduced to 1-4 members in the normalized population. One chlorophyll a/b binding protein gene exhibited a reduction from 15 members in the non-normalized population to 1 in the normalized library, whereas a gene encoding a distinct chlorophyll a/b binding protein showed less reduction in the normalized gene population. This observation is consistent with the conclusion that certain genes do not undergo the same degree of normalization compared with other genes.

[0235] Additional sequences from the normalized Arabidopsis library were obtained by sequence analysis. BLASTN analysis of the 1,343 normalized sequences revealed that 858 were represented in the Arabidopsis EST database, while the remaining 485 sequences were apparently unique, with no obvious homologue in the database. Of those sequences showing BLASTN hits, 43.6% showed coverage of the first through tenth base in the longest EST in the database. Furthermore, 242 of the 858 (28%) showed 5′ sequences that were at the first base of the longest EST or longer. These data show that the cDNAs cloned into GENEWARE® are of significant quality and represent, in many cases, the longest 5′ sequences obtained to date. To further ascertain the proportion of cDNAs containing full-length protein open reading frames, we employed the ORF finder program used to analyze the ABRC library for sense clones. This algorithm checks for ATG sequences in the first 70 bases of a sequence and then scans for sequences lacking an in-frame stop codon for at least 300 nt downstream in the same frame. To understand the number of quality ORFs in a library, we used the ABRC library as a benchmark. Analysis of 11,957 sequences within the ABRC library with the ORF finder program revealed 3,207 hits (26.8%) with putative open reading frames. From the 1,343 sequences of the normalized Arabidopsis cDNA library in GENEWARE®, 907 (67.5%) were hits using the ORF finder program. Coupling the number of cDNAs that represent near the 5′ end of the known RNA sequence (43.6%) with the number of clones that contain putative intact ORFs (67.5%) testifies to the quality and integrity of the cDNAs in the GENEWARE® vector. These data clearly indicate a high proportion of full-length clones.

[0236] K. Quantity of Normalized Arabidopsis cDNAs Cloned into GENEWARE® Vectors:

[0237] As previously described, the normalized Arabidopsis cDNA population was cloned into GENEWARE® vectors in both the positive (+) and negative (−) sense direction to allow for both overexpression and gene knockout analysis. The total number of clones in the 1057 PN vector in negative orientation was 20,160. These were arrayed into 210 96-well glycerol stock plates. Likewise, 20,160 clones from the ligation of normalized Arabidopsis cDNA in sense orientation into 1056 GTN vector have been arrayed in 210 96-well glycerol stock plates. These numbers clearly show that the GENEWARE® vectors can be used as primary cloning vectors and that very complex libraries can be obtained in two orientations from a single pool on non-amplified normalized cDNA.

[0238] II. Construction of Tissue-Specific N. benthamiana cDNA Libraries

[0239] A. mRNA Isolation:

[0240] Leaf, root, flower, meristem, and pathogen-challenged leaf cDNA libraries were constructed. Total RNA samples from 10-5 μg of the above tissues were isolated by TRIZOL reagent (Life Technologies, Rockville, Md.). The typical yield of total RNA was 1 mg. PolyA+RNA was purified from total RNA by DYNABEADS® oligo (T)25. Purified mRNA was quantified by UV absorbance at OD260. The typical yield of mRNA was 2% of total RNA. The purity was also determined by the ratio of OD260/OD280. The integrity of the samples has OD values of 1.8-2.0.

[0241] B. cDNA Synthesis:

[0242] cDNA was synthesized from mRNA using the SUPERSCRIPT® plasmid system (Life Technologies, Rockville, Md.) with cloning sites of NotI at the 3′ end and SalI at the 5′ end. After fractionation through a gel column to eliminate adapter fragments and short sequences, cDNA was cloned into both GENEWARE® vector p1057 NP and phagemid vector PSPORT™ in the multiple cloning region between NotI and XhoI sites. Over 20,000 recombinants were obtained for all of the tissue-specific libraries.

[0243] C. Library Analysis:

[0244] The quality of the libraries was evaluated by checking the insert size and percentage from representative 24 clones. Overall, the average insert size was above 1 kb, and the recombinant percentage was >95%.

[0245] III. Construction of Normalized N. benthamiana cDNA Library in GENEWARE® Vectors

[0246] A. cDNA Synthesis.

[0247] A pooled RNA source from the tissues described above was used to construct a normalized cDNA library. Total RNA samples were pooled in equal amounts first, then polyA+RNA was isolated by DYNABEADS® oligo (dT)25. The first strand cDNA was synthesized by the Smart III system (Clontech, Palo Alto, Calif.). During the synthesis, adapter sequences with Sfi1a and Sfi1b sites were introduced by the polyA priming at the 3′ end, and 5′ end by the template switch mechanism (Clontech, Palo Alto, Calif.). Eight μg first strand cDNA was synthesized from 24 μg mRNA. The yield and size were confirmed by UV absorbance and agarose gel electrophoresis.

[0248] B. Construction of Genomic DNA Driver.

[0249] Genomic DNA driver was constructed by immobilizing biotinylated DNA fragments onto streptavidin-coated magnetic beads. Fifty μg genomic DNA was digested by EcoR1 and BamH1 followed by fill-in reaction using biotin-21-dUTP. The biotinylated fragments were denatured by boiling and immobilized onto DYNABEADS® by the conjugation of streptavidin and biotin.

[0250] C. Normalization Procedure.

[0251] Six μg of the first strand cDNA was hybridized to 1 μg of genomic DNA driver in 100 μl of hybridization buffer (6× SSC, 0.1% SDS, 1× Denhardt's buffer) for 48 hours at 65° C. with constant rotation. After hybridization, the cDNA bound on genomic DNA beads was washed 3 times by 20 μl 1× SSC/0.1% SDS at 65° C. for 15 min and one time by 0.1× SSC at room temperature. The bounded cDNA on the beads was then eluted in 10μl of fresh-made 0.1N NaOH from the beads and purified by using a QIAGEN DNA purification column (QIAGEN GmbH, Hilden, Germany), which yielded 110 ng of normalized cDNA fragments. The normalized first strand cDNA was converted to double strand cDNA in 4 cycles of PCR with Smart primers annealed to the 3′ and 5′end adapter sequences.

[0252] D. Evaluation of Normalization Efficiency.

[0253] Ninety-six non-redundant cDNA clones selected from a randomly sequenced pool of 500 clones of a previously constructed whole seedling library were used to construct a nylon array. One hundred ng of the normalized cDNA fragments vs. the non-normalized fragments were radioactively labeled by 32P and hybridized to DNA array nylon filters. Hybridization images and intensity data were acquired by a PHOSPHORIMAGER® (Amersham Pharmacia Biotech, Chicago, Ill.). Since the 96 clones on the nylon arrays represent different abundance classes of genes, the variance of hybridization intensity among these genes on the filter were measured by standard deviation before and after normalization. These results indicated that by using this type of normalization approach, we could achieve a 1 000-fold reduction in variance among this set of genes.

[0254] E. Cloning of Normalized cDNA into GENEWARE® Vector.

[0255] The normalized cDNA fragments were digested by Sfi1 endonuclease, which recognizes 8-bp sites with variable sequences in the middle 4 nucleotides. After size fractionation, the cDNA was ligated into GENEWARE® vector p1057 NP in antisense orientation and transformed into DH5α cells. Over 50,000 recombinants were obtained for this normalized library. The percentage of insert and size were evaluated by Sfi digestion of randomly picked 96 clones followed by electrophoresis on 1% of agarose gel. The average insert size was 1.5 kb, and the percentage of insert was 98% with vector only insertions of >2%.

[0256] F. Sequence Analysis of Normalized cDNA Library.

[0257] As of the date of this report, 2 plates of 96 randomly picked clones have been sequenced from the 5′ end of cDNA inserts. One hundred ninety-two quality sequences were obtained after trimming of vector sequences and other standard quality checking and filtering procedure, and subjected to BLASTX search in DNA and protein databases. Over 40% of these sequences had no hit in the databases. Clustering analysis was conducted based on accession numbers of BLASTX matches among the 112 sequences that had hits in the databases. Only three genes (tumor-related protein, citrin, and rubit) appeared twice. All other members in this group appeared only once. This was a strong indication that this library is well-normalized. Sequence analysis also revealed that 68% of these 192 sequences had putative open reading frames using the ORF finder program (as described above), indicating possible full-length cDNA.

[0258] IV. DNA Preparation

[0259] A. High Throughput Clone Preparation.

[0260] Arraying of the ABRC library into GENEWARE® vectors occurred as previously discussed to obtain ˜5,000 antisense and ˜3,000 sense clones with minimal redundancy. The ligations were between highly purified and quality controlled GENEWARE® cloning vector plasmids and the corresponding fragments from each individual pool of ABRC clones. Cloning efficiencies were in the range of 1×105 to 5×105 per μg of plasmid. Colonies were picked using a Flexys Colony Picker (The Sanger Centre, England) and manual methods. Colonies were applied to deep-well cell growth blocks (DWBs) and grown from 18-26 hours at 37° C. at ˜500 rpm in the presence of ampicillin concentrations of 500 μg/ml. From the almost 9,000 colonies picked by the Flexys, >97% of the cultures successfully grew. DNA was prepared using the QIAGEN BIOROBOT 9600 DNA robots and QIAGEN 96-well manifolds (manual preparation) at a rate of 2,000 DNA preparations per day. The final throughput, during campaign production, estimated for each system was ˜20 plates of 96 samples per day, per production line—robotic or manual. Such throughput could be sustained to generate 20-40,000 samples in a matter of one to two weeks of effort. During one ten day period, one hundred four (140) 96-well plates of DNA were produced.

[0261] B. Quality Control Methods:

[0262] DNA samples were subjected to quality control (QC) analysis by at least one of two methods: 1) restriction endonuclease digestion and analysis by agarose gel electrophoresis (all plates) or 2) UV spectroscopy to determine DNA quantitation for all 96 samples of a plate (statistical sampling of each days output). For UV analysis, an aliquot of the DNA samples from each plate was taken and measured using a Molecular Dynamics UV spectrometer in 96-well format (Molecular Dynamics, Sunnyvale, Calif.). DNA concentrations of 0.05-0.2 μl with OD 260/280 ratios of 1.7+0.2 are expected. For DNA sequencing purposes (a downstream method to be used to analyze all “hit” samples), DNA quantity of 0.04-0.2 μg/μl is desired. In general, plates that contain >25% of samples not conforming to this metric are rejected and new DNA for the plate must be generated once again. For conformation of the presence of insertions and full-length GENEWARE® vector, agarose gel electrophoresis of restriction endonuclease fragments was used. Aliquots of sixteen samples from each 96-well DNA plate were targeted for restriction digestion using Nco I and BstE II restriction endonucleases. Samples were separated on 1% agarose gels. Generally, plates that showed >25% of samples that were not full length or did not contain insertions were rejected. From a total of 140 96-well DNA plates prepared, 112 passed QC and were made available for generation of infectious units.

[0263] V. High-Throughput DNA Sequencing and Sequence Analysis Protocols

[0264] A. Generation of Raw Sequence Data and Filtering Protocols:

[0265] High-throughput sequencing was carried out using the PCT200® and TETRAD® PCR machines (MJ Research, Watertown, Mass.) in 96-well plate format in combination with two ABI 377™ automated DNA sequencers (PE Corporation, Norwalk, CT). The throughput at present is six 96-well plates per day.

[0266] The electropherogram generated from sequencer by ABI Sequencing Analysis (version 3.3) was used to generate sequence in the text format using “Phred,” which also gives a confidence score for each base call that reflect the error probability and the quality for that base. Cross_match was used to mask the vector sequence. The low quality portion of the sequence (i.e. phred score lower than 20) was removed. The vector and the polyA or polyT were also removed from the raw sequence. The high quality, processed sequences with the processing information were stored in the database. Sequences were used for further bioinformatic analysis.

[0267] B. Sequence Data Analysis and Bioinformatics:

[0268] Once the filtering and the vector sequence removal steps are completed, the resulting sequences are subjected to database search. First, low sensitivity methods such as BLASTN and BLASTX can be used. For those sequences that have no hit, more sensitive methods, such as Blimps and Pfam can be used. To speed up the analysis process, appropriate filters may be used. For example, for EST sequences from a given cDNA library sequenced from the 5′ end, an ATG filter can be used to make sure that only full-length cDNA will be analyzed. The filtered sequence can be translated in one frame rather than six frames for Pfam analysis.

[0269] The results from the database search are stored in the relational database and can be used for further analysis. For example, all the BLAST results can be stored in a relational table that contains Query, Score, pValue, Hit, Length, Annotation, Frame, Identity, Homology, Query Length, Subject Length, Database Queried and Method used to analyze. Any result can be queried and analyzed by the fields mentioned. A database link between the analysis result database and the laboratory information management system (LIMS) has been created so that the analysis result can be related to the experimental data.

[0270] C. Metabolic Pathway Analysis:

[0271] Many metabolic pathway databases have been constructed that group proteins based on their roles in a metabolic pathway. The basic identifiers for these proteins are E.C. numbers; therefore, the position of a given enzyme in a metabolic pathway may be determined based on its E.C. number. The E.C. number of a protein can be obtained by its Genbank ID. This approach can be used to assign the corresponding E.C. number to the hits found for each cDNA sequence. By querying the metabolic pathway using the E.C. number of a hit, a potential link between this cDNA sequence and the metabolic pathway may be established. Each link can be used as a building block for a plant metabolic pathway. This potential link between cDNA sequence and metabolic pathway provides a starting point to analyze the gene's role in a metabolic pathway.

[0272] In addition, we have created an interactive, queriable relational prokaryotic and eukaryotic metabolic pathway database. This metabolic pathway database was created by accessing all public sequences that have associated E.C. numbers, running HMMs (hidden Markov models) and other proprietary LSBC algorithms against these sequences, and classifying these sequences into protein families based on conserved domains (Pfam database assignments). Pfam is a database of multiple alignments of protein domains or conserved protein regions. It is assumed that they represent some evolutionary conserved structure which has implications for the protein's function. Pfam is actually formed in two separate ways. Pfam-A are accurate human crafted multiple alignments whereas Pfam-B is an automatic clustering of the rest of SWISSPROT and TrEMBL derived from the Prodom (http://www.toulouse.inra.fr/prodom.html) database. Each protein family has the following data: 1). A seed alignment which is a hand edited multiple alignment representing the domain; 2). A Hidden Markov Model (HMM) derived from the seed alignment which can be used to find new members of the domain and also take a set of sequences to realign them to the model; 3). A full alignment which is a automatic alignment of all the examples of the domain using the HMM to find and then align the sequences; and 4). An annotation file which contains a brief description of the domain, some parameters for Pfam methods, and links to other databases.

[0273] We have run HMMs and other LSBC algorithms against the LSBC Sequence Database and classified these sequences into protein families based on conserved domains, and relate these sequences back to public sequences for E.C. mapping to metabolic pathways. We have run HMMs and other LSBC algorithms against all sequenced microbial genomes and classified these sequences into protein families based on conserved domains, and relate these sequences back to public sequences for E.C. mapping to metabolic pathways. We further related the Arabidopsis, N. benthamiana, and Oryza clones to specific sites on metabolic pathways.

[0274] D. Sequence Analysis of Library Created from GENEWARE® Vectors:

[0275] Five hundred sixty-eight (568) independent clones were sequenced from the virus expression library and the clones from this library were analyzed by vector, N filters and BLAST analysis. Of the 568 initial sequences submitted for analysis, 131 were eliminated by the N-filter indicating that ˜15% of the sequence were undetermined Ns. The remaining 437 sequences were then subjected to analysis for duplication within each set of submitted plates. Fifty-five (55) sequences were removed due to this duplication filter. These sequences were BLASTN searched against 539 sequences from the AtwpLNLH library in Lambda Zap II. Thirty percent (30%) of the sequences (i.e., 132 sequences) found a match in both libraries. From the original set of GENEWARE® clones, 305 were found to be unique with respect to the Lambda Zap II library. These sequences were then BLASTX-searched against non-redundant GENBANK . From the 305 submitted sequences, 173 sequences found solid hits in protein coding sequence as determined by hit criteria and 132 were found to be unique. Further BLASTN analysis showed a range of sequence homology, but many represented hits to BAC or chromosomal sequences. A wide range of sequences were found including, ribosomal proteins, photosystem reaction center proteins, fumarase and other general metabolism proteins, transcription factors, kinase homologs, omega-6 fatty acid desaturase and various hypothetical proteins. These results strongly suggest that little or no bias is introduced during the construction of cDNA libraries in GENEWARE®.

[0276] VI. Preparation of Infectious Units

[0277] DNA plates that pass QC testing were then moved to the next stage of the cycle, the generation of infectious units. In vitro RNA transcriptions have been optimized to produce maximal amounts of RNA in smaller volumes to reduce costs and increase the lifetime of a DNA preparation. A transcription mixture containing a 6-to-1 RNA cap structure-to-rGTP ratio, Ambion mMessage Machine buffer and enzyme mix (Ambion, Inc., Austin, Tex.) is delivered to a 96-well plate by the TECAN liquid handling robot (TECAN, Research Triangle Park, N.C.). To this reaction mix, the Robbins Scientific HYDRA 96-sample pipeting robot (Robbins Scientific, Sunnyvale, Calif.) delivers 2 μl of DNA solution. This final transcription reaction is incubated at 37° C. for 1.5 hours. Following incubation, the TECAN robot delivers 95 μl of a 100 mM Na/K PO4 buffer containing TMV coat protein (devoid of all infectious RNA) to the transcription plate and it is incubated overnight. This incubation generates encapsidated transcripts, which are very stable at room temperature or 4° C. and amplified with regard to number of infectious units per μg of RNA transcript. The generation of infectious materials is measured by inoculation of GFP-expressing virus to systemic host or Nicotiana tabacum NN lines, incubation at permissive temperatures and counting of developing local lesions on inoculated leaves. Before addition of the TMV coat protein mixture, 0.5 μl from 8 wells of each transcription plate is removed and analyzed by agarose gel electrophoresis. The presence of an RNA band of ˜1.6 to 3.5 kb is strong evidence for a successful transcription. If >25% contain only lower molecular weight RNA bands, or if the band is diffuse <500 bp of dsDNA marker, the transcription plate is considered to have failed and removed from the stream of plates prepared for inoculation. During a two week period, 112 plates were transcribed and 108 plates were passed for plant inoculation in growth rooms and in the field.

[0278] VII. Plant Inoculation with Encapsidated RNA Transcripts

[0279] In order to prepare for plant inoculation, 90 μl of each encapsidated RNA transcript sample and 90 μl of FES transcript inoculation buffer (0.1 M glycine, 0.06 M K2HPO4, 1% sodium pyrophosphate, 1% diatomaceous earth and 1% silicon carbide) were combined in the wells of a new 96-well plate. The 96 well plate was then placed on ice.

[0280] Nicotiana benthamiana plants 14 days post sowing were removed from the greenhouse and brought into the laboratory. Humidity domes were placed over the plants to retain moisture. The RNA transcript sample was mixed by pipetting the solution prior to application to ensure that the silicon carbide and the diatomaceous earth were resuspended. The entire sample, 180 μl, was drawn up and pipetted in equal aliquots (approximately 30 μl), onto the first two true leaves of three separate Nicotiana benthamiana plants. The mixture was spread across the leaf surface using a Texwipe™ Cleanfoam™ swab (The Texwipe Co, Upper Saddle River, N.J.). The wiping action caused by the swab together with the silicon carbide in the buffer sufficiently abrades the leaves so as to allow the encapsidated RNA transcript to enter the plant cell structure. Other methods used for inoculation have included pipeting of encapsidation-FES mixture onto leaves and rubbing by hand, cotton swab or nylon inoculation wand. Alternatively, nylon inoculation wands may be incubated in the transcript-FES mixture for ˜30 min to soak up ˜15 μl and then rubbed directly onto the leaves.

[0281] Once an entire 32 plant flat was inoculated, the plants were misted with deionized water and the humidity domes were replaced over them. The inoculated plants were retained in the laboratory for 6 hours and then returned to the greenhouse. Once in the greenhouse, the humidity domes were removed and the plants were misted a second time with deionized water.

[0282] VIII. Inoculated Plant Growth

[0283] Plants inoculated with encapsidated virus were grown in a greenhouse. Day length was set to 16 hours and shade curtains (33% transmittance) were used to reduce solar intensity. Whenever ambient light fell below 250 μmol m2s−1, a 50:50 mixture of metal halide and sodium halide lamps (Sylvania), delivering an irradiance of approximately 250 μmol m2s−1, were used to provide supplemental lighting. Evaporative cooling and steam heat were used to regulate temperature, with a daytime set point of 27° C. and a nighttime set point of 22° C. The plants were irrigated with Hogland's fertilizer mix as required. Drainage water was collected and treated with 0.5% sodium hypochlorite for 10 minutes before discharging into the municipal sewer.

[0284] To allow space for increased plant size, the inoculated N. benthamiana were repositioned at seven days post-inoculation (dpi) so that they occupied twice their original area. At 13 dpi, the plants were examined visually for symptoms of TMV infection and were assigned a numerical score to indicate the extent of viral infection (0=no infection, 1=possible infection, 2=limited/late infection, 3=typical infection, 4=severe infection). At the same time, the plants were assigned a fate for harvest (typically the highest quality plant in each triplicate was assigned to metabolic screens and the second highest quality plant was assigned to focused screens). In cases where plant symptoms deviated substantially from those of plants inoculated with control vectors, a description of plant phenotype was recorded (as described below). At 14 dpi infected plants were harvested.

[0285] IX. Infectivity Analysis

[0286] The method to measure the infectivity of the transcript encapsidations was to inoculate a set of 96-well plates from both positive and negative sense clones and look for systemic virus movement and phenotype development. Of the 8,352 plants inoculated with unique encapsidated transcriptions, 6,266 became systemically infected for an infection rate of 76%. Overall, the majority of plates generated showed very good infection rates. As shown in a graph of the number of systemically infectious constructs per each individual plate plotted against plate number. The majority of plates had systemic rates >70% with one at 100%. Approximately 25 plates had infection rates ranging between 40 and 70% while only 6% (>5 plates) showed infection rates <45%.

[0287] A population of constructs did not show systemic infection on Nicotiana benthamiana plants. Analysis using the LIMS revealed a substantial correlation between a subset of inoculators and the transcription plates showing poor infection rates. These results strongly suggest that inoculation technique is critical for good infectivity although other possible causes could include poor DNA or transcription quality, or simply inoculation error. In some cases the constructs may be restricted to inoculated leaves by way of adverse influence of the gene insertion on virus replication and movement. For example, one observed healthy inoculated Nicotiana benthamiana plant exhibited clear chlorotic spots on inoculated leaves, yet no systemic symptoms. Other plants, not scored as infected in our LIMS, were observed to have subliminal infections in source tissues. It was clear that the properties of the genetic insertion had differing effects on virus phenotypic symptoms. Eighty-two of those constructs exhibiting poor systemic infection were re-inoculated into Nicotiana tobacum NN plants to test for local lesions. The presence of local lesions indicated infectious viral vectors. From this data, a statistical calculation can be made to determine the percentage of non-systemic infective constructs that are locally infectious. Plants were scored 6 days post-inoculation for the presence of localized necrotic lesions resulting from infection and localized movement of virus vectors on the inoculated leaves of the plants. Of the 82 constructs analyzed, 50 showed local lesions indicating the presence of infectious viral vectors. Based on the infection rate observed in Nicotiana benthamiana and NN tobacco plants, we estimate that 1,181 (˜61%) of the constructs not showing systemic infection on Nicotiana benthamiania plants were still infectious and amenable to biochemical analysis.

[0288] X. Phenotypic Evaluation

[0289] At 13 dpi a visual examination was made to identify plants whose phenotype deviates substantially from plants infected with a GENEWARE® control. The phenotypically different plants were divided into regions (for example: shoot apical region, infected phloem source leaves, stem) and descriptive terms were applied to each region to document the visual observation. Additionally, a confirmation was made as to whether or not the operator considered the plant to be a “hit” and a numerical score was applied to document the phytotoxic/herbicide effect of the RNA insert (1=possible effect, 2=mild, 3=moderate, 4=severe).

[0290] A matrix-style phenotypic database was created using the LIMS software. The LIMS software allows all descriptive terms to be used for any major part of the plant and the capacity of sub-parts to be described. Notable phenotypic events are captured by description of individual plant parts. The matrix is configured in a Web-based page that allows one to score infection and phenotyping using a graphic replicated of the physical arrangement of plants in the growth room. This approach is rapid, allowing 96 plants to be described in detail as being infected, not infected with a detailed phenotype in ˜15 min. Editing of output files can occur rapidly in MS Excel if desired. The output file is then loaded as CSV files into the LIMS where it is immediately available to Boolean query as to phenotype descriptors with “and, or, not” statements. Images of infected plants are linked to the SeqIDs in the database so that the plant tray bar code (for infection), well position, SeqID, phenotype and picture all link together when a query is made. This is linked back to the sequence database for sequence annotation data. Using this system, 8,352 phenotypic observations were made in the period of two days and entered into the LIMS. Hundreds of interesting visual phenotypes were observed.

[0291] XI. Field-Scale Genomics

[0292] The effects of gene overexpression and gene silencing in plants may have dramatic differences when grown under different conditions. The Kentucky field test plots available to Biosource provides an opportunity to subject plants to substantially different growth conditions and thereby broaden the chances of detecting various types of “hits” in a genomics screen. To compare the ability of virus vectors to be applied under field conditions and under controlled growth room conditions, we inoculated, in duplicate, 960 positive-sense constructs on Nicotiana benthamiana plants grown in the field test plot in Owensboro, Ky. This activity was concurrent with inoculations and screens performed in Vacaville, Calif. Complete encapsidated transcription reactions were prepared at Large Scale Biology Corporation in Vacaville, Calif. and following incubation with TMV coat protein, FES buffer was added to each well. All samples in column 12 of each plate contained encapsidated transcripts of 1057 vector containing the GFP gene. The mixture was then overnight-mailed to Owensboro, Ky. where it was inoculated onto 4-5 week post-sowing plants by rubbing cotton swabs, pre-wetted by incubation with encapsidated transcript-FES mixture, on plant leaves. Plants were inoculated in duplicate. Plants were allowed to remain in the field for 4 weeks post-inoculation and then subjected to phenotypic analysis. Photographic documentation of the plants both pre- and post-inoculation was prepared. Plants were scored by visual evaluation as to number of infected plants compared with total number of plants inoculated. Of the 1920 plants inoculated, 1,712 (88%) showed systemic infections. More than 100 new phenotypes were noted in the field. Each was compared with the phenotype of the same construct inoculated into plants in Vacaville, Calif. growth rooms. Two new phenotypes are particularly noteworthy: two independent plants showed survival phenotypes under anaerobic conditions, whereas all neighbors had succumbed to root rot in a low spot in the field.

[0293] In order to evaluate the effect of gene silencing in Nicotiana tabacum plants, mRNA from Arabidopsis thaliana whole plants was subjected to fragment normalization such that small cDNA fragments were produced. The cDNA population showed high degree of normalization by hybridizations with known genes of variable expression and by comparison with non-normalized cDNA fragments. The average size of the normalized fragments in the GENEWARE® vectors was between 400-500 bp allowing facile movement of the recombinant viruses systemically in field Nicotiana tabacum c.v. MD609 plants. A total of 11 plates of DNA constructs (1056) were prepared, transcribed and encapsidated with GFP constructs integrated at every 12th position. These were mixed with FES and overnight-mailed to Owensboro, Ky. These 1056 constructs were inoculated in duplicate (2112 total) on MD609 tobacco plants 11 weeks post-sowing. One set of the replicates (1056 plants) were scored by visual evaluation as to number of infected plants compared with total number of plants inoculated. Of the 1056 plants inoculated, 808 showed systemic infections, or 76.5% infection rate. “Hits” were determined by unusual visual symptoms and corresponding constructs will be characterized by DNA sequencing.

[0294] An uncharacterized GENEWARE® library comprised of 20,000 Arabidopsis thaliana normalized fragment cDNAs and 10,000 of Nicotiana benthamiana genomic DNA fragments was prepared and sprayed as a population on Nicotiana tabacum c.v. MD609 plants. The Arabidopsis cDNA library, ˜10,000, was constructed by ligation into prepared GENEWARE® vectors and purified from pooled bacterial transformants and followed by pooled transcription. The remaining 10,000 cDNA fragments were individual clones prepared and transcribed independently and then mixed in a pooled encapsidation. The Nicotiana library was a prototype cell-free cloning library from restriction endonuclease fragmented gDNA of <500 bp in size. The number of clones corresponds to an approximation of the amount of DNA undergoing complete ligation. Transcriptions from each non-encapsidated library were inoculated separately into Nicotiana tabacum protoplasts and allowed to incubate for three days. Cells were lysed and libraries combined. The pool of cell lysates and encapsidated transcriptions containing viral libraries were shipped to Owensboro, KY where they were inoculated onto Nicotiana tabacum c.v. MD609 plants at 1, {fraction (1/10)}, {fraction (1/100)} and {fraction (1/000)} dilution of the mixed virion preparation (using 60 ml, 6 mls, 0.6 mls and 0.06 mls of the library respectively). Eight hundred (800) plants were spray-inoculated with each library virion dilution. Plants were visually scored and of the 3,200 plants inoculated, 1,304 showed visual symptoms 3 weeks post-infection. The infectivity rate varied from ˜60% for the most concentrated inoculum to ˜20% for the most dilute as would be expected due to dilution. Analysis will continue to define “Hits” by unusual visual symptoms and PCR amplification and DNA sequencing will characterize corresponding construct.

[0295] XII. GC/MS Metabolite Analysis

[0296] A. Harvest and Preparation of Tissues for Metabolic Screening

[0297] Fourteen dpi infected plants to be harvested were moved from the greenhouse to the laboratory. Plants were scanned and identified by a bar-code that linked the infected plant to the tissue sample. The infected tissue was cut off of the plant and placed in a corresponding centrifuge tube. A tungsten carbide ball was placed on top of the infected tissue sample. The tungsten carbide ball facilitates pulverization of plant tissue. The tubes and sample were stored on dry ice during the harvesting procedure. The samples were then stored at −70° C. Before conducting a metabolic screen, the tissue samples must be pulverized. The sample tubes were loaded into a KLECO pulverizer and pulverized to create a fine powder of the tissue sample. The tissue sample powder was then weighed out into a metabolic extraction vial.

[0298] B. FAME Analysis Procedure for FAME Screen.

[0299] Nicotiana benthamiana plants expressing genes of interest in RNA vectors were grown for 14 dpi as described above. Three leaf disks (0.5 cm in diameter) were placed in cell wells of a borosilicate 96-deepwell plate (Zinsser). 500 μl of heptane was added to each well using a Biomek 2000 Laboratory Automation Workstation. The heptane/tissue samples were stirred on a Bodine magnetic stirrer. After 30 minutes, 50 μl of 0.5N sodium methoxide in methanol was added to each well using the Biomek 2000. After 30 minutes of stirring, 10 μl of water was added to each well. Injections were made directly from the 96-deepwell plate into a Hewlett Packard gas chromatograph (GC) using a LEAP auto injector. The GC method involved a 2 μl injection into a split/splitless injection port using a DB 23 narrow bore column (15 M, 0.25 I.D.). The oven temperature was isothermic at 170° C. The injector temperature was 230° C. and the detector (flame ionization) temperature was 240° C. The run time was 5 minutes, with an equilibration time of 0.5 minutes. The split ratio was 20:1 and the helium flow rate was held at a constant pressure of 19 psi. This GC method allowed for separation and quantification of fatty acid methyl esters which included C16:0,C16:1,C18:0,C18:1,C18:2,and C18:3. Using a dual column GC, four 96-well plates could be sampled in less than 24 hours.

[0300] The following sequences exhibited a positive FAME result (had altered levels of the fatty acids assayed): SEQ ID NOs: 7, 53, and 92. The result of the FAME analysis for SEQ ID NO:92 is shown in Table 5. Table 5 shows the relative percent amounts of fatty acids found in plants transfected with a viral vector comprising SEQ ID NO: 92. An increase in 16:0 fatty acids was observed in 3 of the 5 samples assayed. Table 6 shows the relative percent amounts of fatty acids found in plants transfected with SEQ ID NOs: 7 and 53.

TABLE 5
FAME Profile
Sample	16:0	16:1	unk	16:3	unk	18:0	18:1	18:2	18:3	unk
1	24.7	3.4	1.1	3.2	2.6	2.6	3.3	9.2	47.8	2.0
2	20.1	2.9	0.8	4.6	2.9	3.5	7.1	9.2	46.7	2.3
3	17.6	1.8	1.0	3.5	2.9	2.2	6.0	11.8	50.4	2.7
4	23.3	1.9	1.0	3.1	4.6	3.8	8.9	10.6	37.6	5.3
5	23.0	2.6	0.7	3.5	1.6	2.3	3.8	8.1	52.9	1.6
control	19.6	2.8	1.1	3.3	1.8	1.8	3.1	12.0	53.6	1.0
control	18.4	2.7	1.1	3.3	1.7	1.7	3.1	11.3	55.4	1.3

[0301]

TABLE 6
FAME Profile
Sample	16:0	16:1	unk	16:3	unk	18:0	18:1	18:2	18:3	unk
SEQ ID	23.0	3.5	1.9	2.6	1.7	2	3.3	11.7	49.1	1.3
NO: 53
SEQ ID	25.7	3.4	1.3	1.8	0.8	2.3	2.1	8	54.7	0
NO: 7
control	18.7	2.8	1.2	3.8	1.4	1.5	4.2	10.7	55	0.6

[0302] C. Insect Control Bioassays.

[0303] Nicotiana benthamiana plants expressing genes of interest in RNA viral vectors were grown for 14 dpi as described previously. Fresh leaf tissue (sample size ˜2.5 cm diameter) was excised from the base of infected leaves using a scalpel and placed in insect-rearing tray (Bio RT32, C-D International) wells containing 3 ml of 2% agar. Using a small paintbrush to handle insects, 2 first-instar larvae of tobacco hornworm (Manduca sexta) were placed in each well and trays were sealed using vented covers. Trays were then incubated at 28 C with 48% humidity for 72 hours with a 12-hour photoperiod. Following incubation, samples were scored for mortality and leaf damage according to the following criteria: mortality, 0=0 dead/2 alive; 1=1 dead/1 alive; 2=2 dead/0 alive; leaf damage, 0=0 to 20% leaf consumed; 1=21 to 40% leaf consumed; 2=41 to 60% leaf consumed; 3=61 to 80% leaf consumed; and 4=81 to 100% leaf consumed. Following scoring, insects were weighed on an analytical balance and photographed using a digital camera.

[0304] The following sequences exhibited a positive insect control phenotype: SEQ ID NOs: 3, 5, 7, 27, 32, 37, 59, 80, 92, 103, 106, 108, 109, 110, and 111.

[0305] D. Carbohydrate Screen.

[0306] The dry residue was transferred from the extracting cartridge (10-20 mg) into a 100×13 mm glass tube containing 0.5 ml of 0.5 N HCI in methanol and 0.12 ml of methyl acetate and then sealed (Teflon coated screw cap) under nitrogen and heated for 16 hours at 80° C. The liquid phase was then transferred using an 8-channel pipetter (Matrix) to a glass insert supported by a 96 well aluminum block plate (Modem Metal Craft) and evaporated to dryness (Concentrator Evaparray). The methyl-glycosides and methyl-glycoside methyl esters were silylated in 0.1 ml pyridine and 0.1 ml BSTFA+1% TMCS at room temperature for one hour. The sample generated was analyzed on a DB 1 capillary column (15 meters) with an 11 minute program temperature (from 160° C. to 190° C. at 5° C./min and 190° C. to 298° C. at 36° C./minute and hold 2 minutes) and 3 minutes equilibration time. The following components of the plant cell wall were identified in the tobacco sample: arabinose, rhamnose, xylose, galactose, galacturonic acid, mannose, glucuronic acid and glucose.

[0307] E. GC/MS Metabolite Analysis:

[0308] A 3 mm tungsten carbide ball bearing was placed into each well of a 96-well deep well block and 300 μl of grinding buffer (2 mM NaOH, 1 mM PMSF, 10 mM beta-mercaptoethanol, and deuterium-labeled compounds) was added to each well. A 13 mm circle (˜20 mg) leaf disc plug from ˜4 week old Nicotiana benthamiana (2 week post-inoculation) apical leaves were placed into the 96-well microtiter deepwell plate. The plate was tightly sealed and placed on a mechanical shaker (paint mixer, up to four at a time) for 2 min, then rotated 180° and shaken for an additional 2 min. Subsequently, the samples were spun for 10 min at 3200 RPM in a refrigerated (15° C.) centrifuge equipped for microtiter plates. Following centrifugation, the 96-well plate containing the homogenized samples was placed on a TECAN GENESIS RSP 200 (TECAN, Research Triangle Park, N.C.) liquid handler/robotics system. Both Logic and Gemini software were used to control the TECAN liquid handler. Approximately 200 μl was transferred to a pre-conditioned (1 ml MeOH followed by 1 ml of distilled deionized H2O) Waters 96-well Oasis HLB solid phase extraction (SPE) plate by the TECAN liquid handler for metabolite analysis by GC/MS. The Waters Extraction Plate Manifold Kit and a vacuum not greater than 5 mm Hg was used to aspirate plant samples from SPE plate into a waste reservoir. The SPE plate was then washed with 1 ml of 5% MeOH in H2O by aspirating into waste reservoir and compounds eluted from SP resin with 350 μl of MeOH into a 96-well collection plate. Samples were then transferred to GC autosampler vials, capped and stored in the freezer at 80° C. for metabolite analysis.

[0309] An internal standard solution was prepared by making a stock solution at a concentration of 1 μl (using compound density). Grinding buffer (2 mM NaOH above) with the internal standard was prepared at a concentration of 10 ng/μl for each (3,000 ng/300 μl) to yield a concentration equivalent of approximately 150 ng/mg wet weight of plant tissue. Following extraction of plant material, this solution was transferred to the SPE plate by the TECAN liquid handler and extracted with 350 μl of MeOH. Approximately 20 μl of the sample will be injected onto a 30 m×0.32 mm DB-WAX (1 μm film thickness) GC column with a large volume injector during the preliminary study. The GC column oven was temperature held at 35 C for 5 min, then programmed at 2.5° C./min to 250° C. and held for 15 min.

[0310] Samples that contained peaks that were present in altered levels relative to control samples as identified from chromatograms were further analysis using mass spectroscopy. Samples that were transfected with the following nucleic acid sequences were found to have altered metabolic profiles: SEQ ID NO: 43, 50, 81, 85, and 92. Table 7 shows the retention time and % change in peaks relative to controls for several sequences. Table 7 also shows the identity of the peaks as determined by mass spectroscopy.

TABLE 7
Metabolic Profiles
SEQ ID NO	RT (MIN)	% Change	Compound
43	10.68	+130	Malic Acid
43	11.63	+250	Ribonic Acid; Gamma-
			lactone
43	12.93	+260	Quinic Acid
43	14.12	+120	Inositol
81	10.67	+300	Malic Acid
81	10.87	+150	L-Aspartic Acid
81	10.92	+80	5-Oxo-L-Proline
			(pyroglutamic)
81	12.48	+100	Ribonic Acid
81	12.64	+800	Citric Acid
81	16.44	+60	Sucrose
92 FA	9.31	−95	Dodecanoic Acid (12:0)
92 FA	10.28	−90	Myristic Acid (14:0)
92 FA	11.20	+500	Hexadecenoic Acid (16:1)
92 FA	11.96	+200	Oleic Acid (18:1)
92	10.68	+700	Malic Acid
92	11.63	+300	Ribonic Acid; Gamma-
			lactone
92	12.33	+300	Phosphoric Acid
92	12.65	−1400	Citric Acid
92	12.93	+500	Quinic Aci
92	14.12	+800	Inositol
50	11.0	New
50	11.7	New

[0311] A 3 mm tungsten carbide ball bearing was placed into each well of a 96-well deep well block and 300 μl of grinding buffer (2 mM NaOH, 1 mM PMSF, 10 mM beta-mercaptoethanol, and deuterium-labeled compounds) was added to each well. A 13 mm circle (˜20 mg) leaf disc plug from ˜4 week old Nicotiana benthamiana (2 week post-inoculation) apical leaves were placed into the 96-well microtiter deepwell plate. The plate was tightly sealed and placed on a mechanical shaker (paint mixer, up to four at a time) for 2 min, then rotated 180° and shaken for an additional 2 min. Subsequently, the samples were spun for 10 min at 3200 RPM in a refrigerated (15° C.) centrifuge equipped for microtiter plates. Following centrifugation, the 96-well plate containing the homogenized samples was placed on a TECAN GENESIS RSP 200 (TECAN, Research Triangle Park, N.C.) liquid handler/robotics system. Both Logic and Gemini software were used to control the TECAN liquid handler. Approximately 200 μl was transferred to a pre-conditioned (1 ml MeOH followed by 1 ml of distilled deionized H2O) Waters 96-well Oasis HLB solid phase extraction (SPE) plate by the TECAN liquid handler for metabolite analysis by GC/MS. The Waters Extraction Plate Manifold Kit and a vacuum not greater than 5 mm Hg was used to aspirate plant samples from SPE plate into a waste reservoir. The SPE plate was then washed with 1 ml of 5% MeOH in H2O by aspirating into waste reservoir and compounds eluted from SP resin with 350 μl of MeOH into a 96-well collection plate. Samples were then transferred to GC autosampler vials, capped and stored in the freezer at −80° C. for metabolite analysis.

[0312] XIII. Protein Profiling by MALDI-TOF

[0313] Approximately 14 days post-inoculation, 960 different N. benthamiana leaf plugs transfected with encapsidated virion from a GENEWARE® expression library from growth rooms and 38 from N. benthamiana infected in Owensboro, Ky. were collected and the soluble proteins extracted with a high throughput micro-extraction technique described below. An aliquot of this solution was automatically diluted with matrix by a liquid handler in preparation for analysis by MALDI-TOF mass spectrometry for proteins.

[0314] A. Sample Preparation by High Throughput Micro-Extraction:

[0315] A 3 mm tungsten carbide ball bearing was placed into each well of a 96-well deep well block and 300 μl of grinding buffer (2 mM NaOH, 1 mM PMSF, 10 mM beta-mercaptoethanol, and deuterium-labeled compounds-GC/MS analysis) was added to each well. A 13 mm circle (˜20 mg) leaf disc plug from ˜4 week old Nicotiana benthamiana (2 week post-inoculation) apical leaves were placed into the 96-well microtiter deepwell plate. The plate was tightly sealed and placed on a mechanical shaker (paint mixer, up to four at a time) for 2 min, then rotated 180° and shaken for an additional 2 min. Subsequently, the samples were spun for 10 min at 3200 RPM in a refrigerated (15° C.) centrifuge equipped for microtiter plates. Following centrifugation, the 96-well plate containing the homogenized samples was placed on a TECAN GENESIS RSP 200 (TECAN, Research Triangle Park, N.C.) liquid handler/robotics system. Both Logic and Gemini software were used to control the TECAN liquid handler. Samples were diluted by the TECAN liquid handler in a round bottom 96-well plate for MALDI-TOF analysis by adding 18 μl of sinapinic acid matrix and 2 μl of plant extract to each well. Samples were mixed well by aspirating/dispensing 10 μl volumes five times. A 2 μl aliquot of each sample was spotted onto a 100 sample MALDI plate. In addition, a 5.0 μl aliquot of each sample was transferred to a 96-well microtiter plate for PCR and/or MALDI backup analysis and stored at −80° C. Two plant trays containing 96 individually infected each were extracted each day for 5 days.

[0316] B. MALDI-TOF Mass Spectrometry Analysis:

[0317] An aliquot of the homogenized plant samples were diluted 1 :10 with sinapinic acid (Aldrich, Milwaukee, Wis.) matrix, 2 μl applied to a stainless steel MALDI plate surface and allowed to air dry for analysis. The sinapinic acid was prepared at a concentration of 10 mg/ml in 0.1% TFA/acetonitrile (70/30) by volume. MALDI-TOF mass spectra were obtained with a PerSeptive Biosystems Voyager DE-PRO operated in the linear mode. A pulsed nitrogen laser operating at 337 nm was used in the delayed extraction mode for ionization. An acceleration voltage of 25 kV with a 90% grid voltage and a 0.1% guide wire voltage was used. Approximately 150 scans were acquired and averaged over the mass range of 2000-156,000 Da. with a low mass gate of 2000. Ion source and mirror pressures were approximately 2.2×10−7 and 8×10−8 Torr, respectively. All spectra were mass calibrated with a single-point fit using horse apomyoglobin (16,952 Da).

[0318] C. Results:

[0319] This study describes a method that was developed using the high-throughout capabilities of MALDI-TOF MS to detect changes in total protein profiles of crude plant extracts derived from a GENEWARE® cDNA library. As many as 192 samples per day were extracted and analyzed for protein profiling using MALDI-TOF mass spectrometry. In addition, the method has been optimized in house for detection of a wide range of protein masses from one MALDI-TOF scan. More than 50 proteins were routinely detected in a MALDI profile spectrum ranging from approx. 3,000 to 110,000 Da. In addition to the coat protein (˜17,500 Da), both small (˜14,500 Da) and large (˜52,750 Da) subunits of RuDP carboxylase were routinely detected in the plant samples. Several other proteins were common to most of the plants analyzed. The most abundant proteins were observed at around 3,386, 3,970, 4,408, 5,230, 7,280 (doubly charged ion for small sub-unit of RuDP carboxylase), 8,334, 9,350, 10,450 (most abundant protein overall), 14,020, 18,006, 19,628, 20,286, 21,173, 24,014, 25,124 and 29,140 (dimer of small sub-unit) daltons. A series of less abundant proteins were also detected. Up-regulated or novel proteins were detected in 17.3% of the 960 spectra that were analyzed. This data was entered into the LIMS database.

[0320] XIV. ABRC Library Construction in GENEWARE Expression Vectors

[0321] Expressed sequence tag (EST) clones were obtained from the Arabidopsis Biological Resource Center (ABRC; The Ohio State University, Columbus, Ohio 43210). These clones originated from Michigan State University (from the labs of Dr. Thomas Newman of the DOE Plant Research Laboratory and Dr. Chris Somerville, Carnegie Institution of Washington) and from the Centre National de la Recherche Scientifique Project (CNRS project; donated by the Groupement De Recherche 1003, Centre National de la Recherche Scientifique, Dr. Bernard Lescure and colleagues). The clones were derived from cDNA libraries isolated from various tissues of Arabidopsis thaliana var Columbia. A clone set of 11,982 clones was received as glycerol stocks arrayed in 96 well plates, each with an ABRC identifier and associated EST sequence.

[0322] An ORF finding algorithm was performed on the EST clone set to find potential full-length genes. Approximately 3,200 full-length genes were found and used to make GENEWARE constructs in the sense orientation. Five thousand of the remaining clones (not full-length) were used to make GENEWARE constructs in the antisense orientation.

[0323] Full-length clones used to make constructs in the sense orientation were grown and DNA was isolated using Qiagen (Qiagen Inc., Valencia, Calif. 91355) mini-preps. Each clone was digested with NotI and Sse 8387 eight base pair enzymes. The resultant fragments were individually isolated and then combined. The combined fragments were ligated into pGTN P/N vector (with polylinker extending from PstI to NotI −5′ to 3′). For each set of 96 original clones approximately 192 colonies were picked from the pooled GENEWARE ligations, grown until confluent in deep-well 96-well plates, DNA prepped and sequenced. The ESTs matching the ABRC data was bioinformatically checked by BLAST and a list of missing clones was generated. Pools of clones found to be missing were prepared and subjected to the same process. The entire process resulted in greater than 3,000 full-length sense clones.

[0324] The negative sense clones were processed in the same manner, but ligated into pGTN N/P vector (with polylinker extending from NotI to PstI −5′ to 3′). For each set of 96 original clones approximately 192 colonies were picked from the pooled geneware ligations and DNA prepped. The DNA from the GENEWARE ligations was subjected to RFLP analysis using TaqI 4 base cutter. Novel patterns were identified for each set. The RFLP method was applied and only applicable for comparison within a single ABRC plate. This procedure resulted in greater than 6,000 negative sense clones.

[0325] The identified clones were re-arrayed, transcribed, encapsidated and used to inoculate plants.

[0326] XV. Inoculation of Plants

[0327] A. Plant Growth.

[0328] N. benthamiana seeds were sown in 6.5 cm pots filled with Redi-earth medium (Scotts) that had been pre-wetted with fertilizer solution (prepared by mixing 147 kg Peters Excel 15-5-15 Cal-Mag (The Scotts Company, Marysville Ohio), 68 kg Peters Excel 15-0-0 Cal-Lite (15% Ca), and 45 kg Peters Excel 10-0-0 MagNitrate (10% Mg) in hot tap water to 596 liters total volume and then injecting this concentrate into irrigation water using an injection system (H. E. Anderson, Muskogee Okla.), at a ratio of 200:1). Seeded pots were placed in the greenhouse for 1 d, transferred to a germination chamber, set to 27° C., for 2 d (Carolina Greenhouses, Kinston, N.C.), and then returned to the greenhouse. Shade curtains (33% transmittance) were used to reduce solar intensity in the greenhouse and artificial lighting, a 1:1 mixture of metal halide and high pressure sodium lamps (Sylvania) that delivered an irradiance of approximately 220 μmol m2s−1, was used to extend day length to 16 h and to supplement solar radiation on overcast days. Evaporative cooling and steam heat were used to regulate greenhouse temperature, maintaining a daytime set point of 27° C. and a nighttime set point of 22° C. At approximately 7 days post sowing (dps), seedlings were thinned to one seedling per pot and at 17 to 21 dps, the pots were spaced farther apart to accommodate plant growth. Plants were watered with Hoagland nutrient solution as required. Following inoculation, waste irrigation water was collected and treated with 0.5% sodium hypochlorite for 10 minutes to neutralize any viral contamination before discharging into the municipal sewer.

[0329] B. Innoculation.

[0330] For each GENEWARE™ clone, 180 μL of inoculum was prepared by combining equal volumes of encapsidated RNA transcript and FES buffer (0.1M glycine, 0.06 M K2HPO4, 1% sodium pyrophosphate, 1% diatomaceous earth (Sigma), and either 1% silicon carbide (Aldrich), or 1% Bentonite (Sigma)). The inoculum was applied to three greenhouse-grown Nicotiana benthamiana plants at 14 or 17 days post sowing (dps) by distributing it onto the upper surface of one pair of leaves of each plant (30 μL per leaf). Either the first pair of leaves or the second pair of leaves above the cotyledons was inoculated on 14 or 17 dps plants, respectively. The inoculum was spread across the leaf surface using one of two different procedures. The first procedure utilized a Cleanfoam swab (Texwipe Co, N.J.) to spread the inoculm across the surface of the leaf while the leaf was supported with a plastic pot label (¾×5 2M/RL, White Thermal Pot Label, United Label). The second implemented a 3″ cotton tipped applicator (Calapro Swab, Fisher Scientific) to spread the inoculum and a gloved finger to support the leaf. Following inoculation the plants were misted with deionized water.

[0331] C. Infection.

[0332] At 13 days post inoculation (dpi), the plants were examined visually and a numerical score was assigned to each plant to indicate the extent of viral infection symptoms. 0=no infection, 1=possible infection, 2=infection symptoms limited to leaves<50-75% fully expanded, 3=typical infection, 4=atypically severe infection, often accompanied by moderate to severe wilting and/or necrosis.

[0333] XVI: Phenotypic Evaluation

[0334] At 13 dpi plants were examined and in cases where a plant's visual phenotype deviated substantially from the phenotypes of control plants, a controlled vocabulary utilizing a five-part phrase was used to describe the plants. Phrase: plant region/sub-part/modifier (optional)/symptom/severity. Plant regions: sink leaves (the upper region of the plant considered to be primarily phloem sink tissue at the time of evaluation), source leaves (expanded, fully-infected leaves considered to be phloem source tissue at the time of evaluation), bypassed leaves (leaves [three and four] that display little or no infection symptoms), inoculated leaves (leaves one and two), stem. Subparts: blade, entire, flower, foci, intervein, leaf, lower, major vein, margin, minor vein, node, petiole, shoot apex, upper, vein, viral path. Modifiers: apical, associated, banded, basal, blotchy, bright, central, crinkled, dark, epinastic, flecked, glossy, gray, hyponastic, increased, intermittent, large-spotted, light, light-colored, light-green, mottled, narrowed, orange, patchy, patterned, radial, reduced, ringspot, small-spotted, smooth, spotted, streaked, subtending, uniform, unusual, white. Symptoms: bleaching, chlorosis, color, contortion, corrugation, curling, dark green, elongation, etching, hyperbranching, mild symptoms, necrosis, patterning, recovery, stunting, texture, trichomes, wilting. Severity: 1—extremely mild/trace, 2—mild symptom (<30% of subpart affected), 3—moderate symptom (30%-70% of subpart affected), 4—severe symptom (>70% of subpart affected). Based on the symptoms a phenotypic hit value (PHV) and a herbicide hit value (HHV) were assigned to each plant phenotyped. Phenotype Hit Value: 1—no predicted value; do not request for repeat analysis, 2—of uncertain value, 3—of potential value; strong phenotype, 4—highly unusual phenotype. Herbicide Hit Value: 1—no predicted value; do not request for repeat analysis, 2—of uncertain value, 3—moderate chlorosis (especially in apical region) or necrosis, 4—Severe phytotoxicity/herbicide mode of action. Comments were added if additional information was required to complete the plant characterization. Results are presented in Table 8.

	TABLE 8
	SEQ ID NO	Library	Summary of Visual Phenotype
	SEQ ID NO:12	ABRC	Stunting
	SEQ ID NO:27	ABRC	Stunting
	SEQ ID NO:48	ABRC	Stunting
	SEQ ID NO:49	ABRC	Stunting
	SEQ ID NO:59	ABRC	Stunting
	SEQ ID NO:60	ABRC	Stunting
	SEQ ID NO:71	ARAB	Stunting
	SEQ ID NO:84	ABRC	Stunting
	SEQ ID NO:99	ABRC	Stunting
	SEQ ID NO:100	ABRC	Stunting
	SEQ ID NO:102	ABRC	Stunting
	SEQ ID NO:103	ABRC	Stunting
	SEQ ID NO:105	ABRC	Stunting
	SEQ ID NO:106	ABRC	Stunting
	SEQ ID NO:107	ABRC	Stunting
	SEQ ID NO:108	ABRC	Stunting
	SEQ ID NO:109	ABRC	Stunting
	SEQ ID NO:110	ABRC	Stunting

[0335] XVII: Metabolic Screens

[0336] A. Sample Generation.

[0337] Individual dwarf tobacco nicotiana benthamiana, (Nb) plants were manually transfected with an unique DNA sequence at 14 or 17 days post sowing using the GENEWARETM viral vector technology (1). Plants were grown and maintained under greenhouse conditions. At 13 days after infection, an infection rating of 0, 1, 2, 3, or 4 was assigned to each plant. The infection rating documents the degree of infection based on a visual observation. A score of 0 indicates no visual infection. Scores of 1 and 2 indicate varying degrees of partial infection. A score of 4 indicates a plant with a massive overload of infection, the plant is either dead or near death. A score of 3 indicates optimum spread of systemic infection.

[0338] Samples were grouped into sets of up to 96 samples per set for inoculation, harvesting and analysis. Each sample set (SDG) included 8 negative control (reference samples), up to 80 unknown (test) samples, and 8 quality control samples.

[0339] B. Harvesting.

[0340] At 14 days after infection, infected leaf tissue, excluding stems and petioles, was harvested from plants with an infection score of 3. Infected tissue was placed in a labeled, 50-milliliter (mL), plastic centrifuge tube containing a tungsten carbide ball approximately 1 cm in diameter. The tube was immediately capped, and dipped in liquid nitrogen for approximately 20 seconds to freeze the sample as quickly as possible to minimize degradation of the sample due to biological processes triggered by the harvesting process. Harvested samples were maintained at −80 C between harvest and analysis. Each sample was assigned a unique identifier, which was used to correlate the plant tissue to the DNA sequence that the plant was transfected with. Each sample set was assigned a unique identifier, which is referred to as the harvest or meta rack ID.

[0341] C. Extraction.

[0342] Prior to analysis, the frozen sample was homogenized by placing the centrifuge tube on a mechanical shaker. The action of the tungsten carbide ball during approximately 30 seconds of vigorous shaking reduced the frozen whole leaf tissue to a finely homogenized frozen powder. Approximately 1 gram of the frozen powder was extracted with 7.5 mL of a solution of isopropanol (IPA):water 70:30 (v:v) by shaking at room temperature for 30 minutes.

[0343] D. Fractionation.

[0344] A 1200 microliter (μL) aliquot of the IPA:water extract was partitioned with 1200 μL of hexane. The hexane layer was removed to a clean glass container. This hexane extract is referred to as fraction 1 (F1). A 90 μL aliquot of the hexane extracted IPA:water extract was removed to a clean glass container. This aliquot is referred to as fraction 4 (F4). The remaining hexane extracted IPA:water extract is referred to as fraction 3 (F3). A 200 μL aliquot of the IPA:water extract was transferred to a clean glass container and referred to as fraction 2 (F2). Each fraction for each sample was assigned a unique aliquot ID (sample name).

[0345] E. Sample Preparation & Data Generation

[0346] Fraction 1:

[0347] The hexane extract was evaporated to dryness under nitrogen at room temperature. The sample containers were sealed and stored at 4 C prior to analysis, if storage was required. Immediately prior to capillary gas chromatographic analysis using flame ionization detection (GC/FID), the F1 residue was reconstituted with 120 μL of hexane containing pentacosane and hexatriacontane which were used as internal standards for the F1 analyses. The chromatographic data files generated following GC separation and flame ionization detection were named with the fraction 1 aliquot ID for each sample and stored in a folder named after the harvest rack (sample set) ID. FIG. 1 a summarizes the GC/FID parameters used to analyze fraction 1 samples.

[0348] Fraction 2:

[0349] The F2 aliquot was evaporated to dryness under nitrogen at room temperature and reconstituted in heptane containing 2 internal standards, C11:0 and C24:0. In general, fraction 2 is designed to analyze esterified fatty acids, such as phospholipids, triacylglycerides, and thioesters. In order to analyze these compounds by GC/FID, they were transmethylated to their respective methyl esters by addition of sodium methoxide in methanol and heat. Excess reagent was quenched by the addition of a small amount of water, which results in phase separation. The fatty acid methyl esters (FAMEs) were contained in the organic phase. FIG. 1b summarizes the GC/FID parameters used to analyze fraction 1 samples.

[0350] Fraction 3:

[0351] The F3 aliquot was evaporated to dryness under nitrogen at 40 C. In general, the metabolites in this fraction are highly polar and water-soluble. In order to analyze these compounds by GC/FID, the polar functional groups on these compounds were silylated through a 2-step derivatization process. Initially, the residue was reconstituted with 400 μL of pyridine containing hydroxylamine hydrochloride (25 mg/ml) and the internal standard, n-octyl-β-D-glucopyranoside (OXIME solution). The derivatization was completed by the addition of 400 μL of the commercially available reagent (N,O-bis[Trimethylsily] trifluoroacetamide)+1% Trimethylchlorosilane (BSTFA+1% TMCS). The chromatographic data files generated following GC separation and flame ionization detection were named with the fraction 3 aliquot ID for each sample and stored in a folder named after the harvest rack (sample set) ID. FIG. 1c summarizes the GC/FID parameters used to analyze fraction 1 samples.

[0352] Fraction 4:

[0353] The F4 aliquot was diluted with 90 μL of distilled water and 20 μL of an 0.1 N hydrochloric acid solution containing norvaline and sarcosine, which are amino acids that are used as internal standards for the amino acids analysis. Immediately prior to high performance liquid chromatographic analysis using fluorescence detection (HPLC/FLD), the amino acids in F4 are mixed in the HPLC injector at room temperature with buffered orthophtaldehyde solution, which derivatizes primary amino acids, followed by fluorenyl methyl chloroformate, which derivatizes secondary amino acids. Following HPLC separation and fluorescence detection, chromatographic data files were generated for each sample, named with a sequential number which can be tracked back to the F4 aliquot ID, and stored in a folder named after the harvest rack (sample set) ID. FIG. 1d summarizes the GC/FID parameters used to analyze fraction 1 samples.

[0354] F. Data Analysis & Hit Detection.

[0355] Two complementary methods were used to identify modifications in the metabolic profile of test samples from reference samples. These data analysis methods are called automated data analysis (ADA) and quantitative data analysis. Each fraction from each sample was analyzed by one or both of these methods to identify hits. If either method identified a fraction as a hit, the sample was called a hit for that fraction. Therefore a sample could be a hit for 1 through 4 fractions.

[0356] ADA employs a qualitative pattern recognition approach using ABNORM (U.S. Pat. No. 5,592,402), which is a proprietary software utility of the Dow Chemical Company. ADA was performed on chromatograms from all 4 fractions. The ADA process developed a statistical model from chromatograms that ideally depict unaltered (reference) metabolic profiles. This model was then used to identify test sample chromatograms that contain statistically significant differences from the normal (control) chromatograms. Updated models for each fraction were generated for each sample set. Chromatograms identified as hits by ADA, were manually reviewed and the data quality visually verified.

[0357] Quantitative data analysis is based on individual peak areas. Quantitative data analysis was applied to specific compounds of interest in fraction 2, fatty acids, and fraction 4, amino acids. The peak areas corresponding to these compounds in these fractions were generated. For fraction 2, the relative percent of the peak areas for the compounds in Table 9 were calculated for each sample. The average ({overscore (x)}) and standard deviation (STD) of the relative % of the peak areas for the individual compounds were calculated from the reference sample chromatograms analyzed within the sample set. The average and STD were used to calculate a range for each compound. Depending on the compound, this range was typically {overscore (x)}+/−3 or 5 STDs. If the relative percent of the peak area from an unknown was outside this range, the compound was considered to be significantly different from the ‘normal’ level and the sample was identified as a hit for F2. For fraction 4, the concentration, in micrograms/gram was calculated for each of the amino acids listed in Table 9, from calibration standards analyzed at the same time as the test samples. The amino acid concentrations from reference samples were used to calculate the acceptable range from the {overscore (x)} and STD for each amino acid. If the amino acid concentration for an unknown falls outside this range, the amino acid was considered to be different from normal and sample was identified as a hit for F4.\

TABLE 9
Tobacco Metabolites Monitored in Fractions 2 and 4 by
Quantitative Analysis
	Fraction 4
Fraction 2 (Fatty Acids)	(Amino Acids)
undecanoic acid methyl ester*	C11:0	Aspartic Acid	ASP
Pentadecanoic acid methyl ester**	C15:0	Glutamic	GLU
		Acid
Pentadecanoic acid ethyl ester**	C15:0	Serine	SER
palmitic acid methyl ester	C16:0	Histidine	HIS
palmitoleic acid methyl ester	C16:1	Glycine	GLY
iso methylpentadecanoic acid methyl	C16:0:Me	Threonine	THR
ester
palmitoleic acid methyl ester	C16:2	Alanine	ALA
palmitolenic acid methyl ester	C16:3	Arginine	ARG
iso methylhexadecanoic acid methyl	C17:0Me	Tyrosine	TYR
ester
Stearic acid methyl ester	C18:0	Cystine	CY2
Oleic acid methyl ester	C18:1	Valine	VAL
Linoleic acid methyl ester	C18:2	Methionine	MET
Linolenic acid methyl ester	C18:3	Norvaline*	NVA
Arachidic acid methyl ester	C20:0	Tryptohane	TRP
Lignoceric acid methyl ester*	C24:0	Phenylalanine	PHE
		Isoleucine	ILE
		Leucine	LEU
		Lysine	LYS
		Sarcosine*	SAR
		Proline	PRO
*Internal Standard
**Surrogate Standard

[0358] Shipping Hits.

[0359] Any F1, F2, or F3 fractions identified as hits by ADA or quantitative analysis, and the most typical null for each fraction for each sample set as identified by ADA, were sent to the Function Discovery Laboratory (see Example 20) for structural characterization of the specific compounds identified. Samples were sealed, packaged on dry ice and shipped for overnight delivery.

[0360] XVIII: Identification of Metabolic Changes

[0361] This Example describes the identification of the chemical nature of genetic modifications made in tobacco plants using GENEWARE viral vector technology. The protocols involved the use of gas chromatography/mass spectrometry (GC/MS) for the analyses of three primary fractions obtained from extraction and fractionation processes.

[0362] A. Methods.

[0363] Major instruments and accessories used included Bioinformatics computer programs, mass spectral libraries, Biotech databases, Nautilus LIMS system (BLIMS; Dow), Biotech Database (eBRAD; Dow), HP Model 6890 capillary Gas Chromatograph (GC; Agilent Technologies), HP Model 5973 Mass Selective Detector (MSD; Agilent Technologies), Auto Sampler and Sample Preparation Station (Leap Technologies), Large Volume Injector system (APEX), Ultra Freezer (Revco), and model LS1006 Barcode Reader (Symbol Technologies).

[0364] Samples and corresponding References (also referred to as controls or nulls) were shipped via overnight mail. Samples were removed from the shipping container, inspected for damage, and then placed in a freezer until analysis by GC/MS.

[0365] Samples were received in vials or in titer plates with a bar-coded titer plate (TP) number, also referred to as a Rack Identification number that is used to track the sample in the BLIMS system. The barcode number is used by the FDL to extract from BLIMS pertinent information from ADA (Automated chromatographic pattern recognition Data Analysis) HIT reports and/or QUANT (a quantitative data analysis approach that makes use of individual peak areas of select peaks corresponding to specific compounds of interest in the fatty acid Fraction 2) HIT reports generated by the Metabolic Screening Laboratory. The information in these reports includes the well position of the respective HITs (Samples), the corresponding well position of the Reference, and other pertinent information, such as, aliquot identification. This information is used to generate ChemStation and Leap sequences for FDL analyses.

[0366] Samples were sequenced for analysis in the following order:

TABLE 10
Analysis Order
Solvent Blank
Instrument Performance Standard
Samples and Associated Reference
.
.
.
Performance Standard
Solvent Blank

[0367] Samples were analyzed on GC/MS systems using the following procedures. Fraction 1 samples were shipped dry and required a hexane reconstitution step. Fraction 2 and Fraction 3 samples were analyzed as received. Internal standards were added to the samples prior to analysis.

[0368] B. Fraction 1 Analysis.

[0369] The name of the GC/MS method used is BIONEUTx (where x is a revision number of the core GC/MS method). The method is retention-time locked to the retention time of pentacosane, an internal standard, using the ChemStation RT Locking algorithm.

Internal Standard(s)
Pentacosane
Hexatriacontane
Chromatography
Column:      J & W DB-5MS
             50 M × 0.320 mm × 0.25 μm film
Mode: constant flow
             Flow: 2.0 mL/min
             Detector: MSD
             Outlet psi: vacuum
Oven:        40° C. for 2.0 min
             20° C./min to 350° C., hold 15.0 min
             Equilibration time: 1 min
Inlet:       Mode: split
             Inj Temp: 250° C.
             Split ratio: 50:1
             Gas Type: Helium
LEAP Injector:
Injector:    Inj volume: optimized to pentacosane peak intensity
             (typically 20 μL)
             Sample pumps: 2
             Wash solvent A: Hexane
             Wash solvent B: Acetone
             Preinj Solvent A washes: 2
             Preinj Solvent B washes: 2
             Postinj Solvent A washes: 2
             Postinj Solvent B washes: 2
APEX Injector
Method Name: BIONEUTx (where x is a revision number of the
             core APEX method).
Modes:       Initial: Standby (GC Split)
             Splitless: (Purge Off) 0.5 min
             GC Split: (Standby) 4 min
             ProSep Split: (Flow Select) 23 min
Temps: 50° C. for
0.0 min.
             300° C./min to 350° C., hold for 31.5 min
Mass Spectrometer
Scan: 35-800 Da at sampling rate 2 (1.96 scans/sec)
             Solvent delay: 4.0 min
Detector:    EM absolute: False
             EM offset: 0
Temps:       Transfer line: 280° C.
             Ion source: 150° C.
             MS Source: 230° C.

[0370] C. Fraction 2 Analysis:

[0371] The name of the GC/MS method used is BIOFAMEx (where x is a revision number of the core GC/MS method). The method is retention-time locked to RT of undecanoic acid, methyl ester, an internal standard, using the ChemStation RT Locking algorithm.

Internal Standard(s)
Undecanoic acid, methyl ester
Tetracosanoic acid, methyl ester
Chromatography
Column:       J & W DB-23 FAME
              60 M × 0.250 mm × 0.15 μm film
              Mode: constant flow
              Flow: 2.0 mL/min
              Detector: MSD
              Outlet psi: vacuum
Oven:         50° C. for 2.0 min
              20° C./min to 240° C., hold 10.0 min
              Equilibration time: 1 min
Inlet:        Mode: split
              Inj Temp: 240° C.
              Split ratio: 50:1
              Gas Type: Helium
LEAP Injector:
Injector:     Inj volume: optimized to undecanoic
              acid, methyl ester peak intensity
              (Typically 10 μL)
              Sample pumps: 2
              Wash solvent A: Methanol
              Wash solvent B: Methanol
              Preinj Solvent A washes: 2
              Preinj Solvent B washes: 2
              Postinj Solvent A washes: 2
              Postinj Solvent B washes: 2
APEX Injector
Method Name:  BIOFAMEx (where x is a revision
              number of the core APEX method).
Modes:        Initial: GC Split
              Splitless: 0.5 min
              GC Split: 4 min
ProSep Split: 21 min
Temps:        60° C. for 0.5 min.
              300° C./min to 250° C., hold for 20 min
              300° C./min to 260° C., hold for 5 min
Mass Spectrometer
Scan: 35-800  Da at sampling rate 2 (1.96 scans/sec)
              Solvent delay: 4.5 min
Detector:     EM absolute: False
EM offset: 0
Temps:        Transfer line: 200° C.
              Ion source: 150° C.
              MS Source: 230° C.

[0372] D. Fraction 3 Analysis.

[0373] The name of the GC/MS method used is BIOAQUAx (where x is a revision number of the core GC/MS method). Method is retention-time locked to the RT of n-Octyl-β-D-Glucopyranoside, an internal standard, using the ChemStation RT Locking algorithm.

Internal Standard(s)
n-Octyl-β-D-Glucopyranoside
Chromatography
Column:      Chrompack 7454 CP-SIL 8
             60 M × 0.320 mm × 0.25 μm film
             Mode: constant flow
             Flow: 2.0 mL/min
             Detector: MSD
             Outlet psi: vacuum
Oven:        40° C. for 2.0 min
             20° C./min to 350° C., hold 10.0 min
             Equilibration time: 1 min
Inlet: Mode: split
             Inj Temp: 250° C.
             Split ratio: 50:1
             Gas Type: Helium
LEAP Injector:
Injector:    Inj volume: Optimized to n-Octyl-β-D-
             Glucopyranoside peak intensity
(Typically 2.5 μL)
             Sample pumps: 2
             Wash solvent A: Hexane
             Wash solvent B: Acetone
             Preinj Solvent A washes: 2
             Preinj Solvent B washes: 2
             Postinj Solvent A washes: 2
             Postinj Solvent B washes: 2
APEX Injector
Method Name: BIQAQUAx (where x is a revision
             number of the core APEX method).
Modes:       Initial: GC Split
             Splitless: 0.5 min
             GC Split: 4 min
             ProSep Split: 20 min
Temps:       60° C. for 0.5 min.
             300° C./min to 350° C., hold for 21.1 min
Mass Spectrometer
Scan: 35-800 Da at sampling rate 2 (1.96 scans/sec)
             Solvent delay: 4.0 min
Detector:    EM absolute: False
EM offset: 0
Temps:       Transfer line: 280° C.
             Ion source: 150° C.
             MS Source: 230° C.

[0374] E. Performance Standard:

[0375] Two mixtures were used as instrument performance standards. One standard was run with Fraction 1 and 3 samples and the second was run with Fraction 2 samples. Below is the composition of the standards as well as approximate retention time values observed when run under the GC/MS conditions previously described. These retention time values are subject to change depending upon specific instrument and chromatographic conditions.

TABLE 11
Fraction 1 and 3 Performance Standard
Time  Compound
6.25  dimethyl malonate
7.25  dimethyl succinate
8.15  dimethyl glutarate
8.98  dimethyl adipate
11.06 dimethyl azelate
11.42 hexadecane
11.70 dimethyl sebacate
13.57 eicosane
15.36 tetracosane
16.88 octacosane
18.26 dotriacontane
19.95 hexatriacontane

[0376]

TABLE 12
Fraction 2 Performance Standard
Time  Compound
8.82  undecanoic acid, methyl ester
9.32  dodecanoic acid, methyl ester
10.24 tetradecanoic acid, methyl ester
11.07 hexadecanoic acid, methyl ester
11.84 octadecanoic acid, methyl ester
11.90 oleic acid, methyl ester
12.14 linoleic acid, methyl ester
12.39 linolenic acid, methyl ester
12.60 eicosanoic acid, methyl ester
13.42 docosanoic acid, methyl ester

[0377] F. Data Analysis.

[0378] Sample and Reference data sets were processed using the Bioinformatics computer program Maxwell. The principal elements of the program are 1) Data Reduction, 2) two-dimensional Peak Matching, 3) Quantitative Peak Differentiation (Determination of Relative Quantitative Change), 4) Peak Identification, 5) Data Sorting, and 6) Customized Reporting.

[0379] The program queries the user for the filenames of the Reference data set and Sample data set(s) to compare against the Reference. A complete listing of user inputs with example input is shown below.

TABLE 13
Bioinformatics Analysis
USER QUERY                       EXAMPLE USER INPUT
Operator Name                    M. Maxwell
Total number of data files to process 5
Which Fraction                   3
Reference (Control) File Name    AAPR0020.D
Process a specific RT Range      Y
Specific RT range                6.5-23
Internal Standard Retention Time 14.902
+/− variation in Internal Std. RT .004
Variation in peak RI, ChemStation .005
Percent variation in peak RI, Biotech .010
Database
Threshold for determining Area % change 60
Spectral Matching Value (Threshold MS- .95
XCR for peaks to be a match)
Percent to determine LOP-PM* Value 1
Percent to determine LOP-SRT** Value 3
Quality Level for Library (Library match) 80
Subtract Background              Y
Time Range for Background        21.5-22.6
SHORT SUMMARY (y/n, y = no       Y
chromatograms)
*LOP-PM - Limit of Processing for Peak Matching
**LOP-SRT - Limit of Processing for Sorting

[0380] The program integrates the Total Ion Chromatogram (TIC) of the data sets using Agilent Technologies HP ChemStation integrator parameters determined by the analyst. The corresponding raw peak areas are then normalized to the respective Internal Standard peak area. It should be noted that before the normalization is performed, the program chromatographically and spectrally identifies the Internal Standard peak. Should the identification of the Internal Standard not meet established criteria for a given Fraction, then the data set will not be further processed and it will be flagged for analyst intervention.

[0381] Peak tables from the Reference and each Sample were generated. The peak tables are comprised of retention time (RT), retention index (RI)—the retention time relative to the Internal Standard RT, raw peak areas, peak areas normalized to the Internal Standard, and other pertinent information.

[0382] The first of two filtering criteria, established by the analyst was then invoked and must be met before a peak is further processed. The criterion is based upon a peak's normalized area. All normalized peaks having values below the Limit of Processing for Peak Matching (LOP-PM), were considered to be “background”. These “peaks” were not carried forth for any type of mathematical calculation or spectral comparison.

[0383] In the initial peak-matching step, the Sample peak table was compared to the Reference peak table and peaks between the two were paired based upon their respective RI values matching one another (within a given variable window). The next step in the peak matching routine utilized mass spectral data. Sample and Reference peaks that have been chromatographically matched were then compared spectrally. The spectral matching was performed using a mass spectral cross-correlation algorithm within the Agilent Technologies HP ChemStation software. The cross-correlation algorithm generates an equivalence value based upon spectral “fit” that was used to determine whether the chromatographically matched peaks are spectrally similar or not. This equivalence value is referred to as the MS-XCR value and must meet or exceed a predetermined value for a pair of peaks to be “MATCHED,” which means they appear to be the same compound in both the Reference and the Sample. The MS-XCR value can also be used to judge peak purity. This two-dimensional peak matching process was repeated until all potential peak matches were processed. At the end of the process, peaks are categorized into two categories, MATCHED and UNMATCHED.

[0384] A second filtering criterion was next invoked, again based upon the normalized area of the MATCHED or UNMATCHED peak. For a peak to be reported and further processed, its normalized area must meet or exceed the predetermined Limit of Processing for Sorting (LOP-SRT).

[0385] Peaks that are UNMATCHED are immediately flagged as different. UNMATCHED peaks are of two types. There are those that are reported in the Reference but appear to be absent in the Sample (based upon criteria for quantitation and reporting). These peaks were designated in the Analyst Report with a percent change of “−100 percent” and the description “UNMATCHED IN SAMPLE.” The second types of peaks are those that were not reported in the Reference (again, based upon criteria for quantitation and reporting) but were reported in the Sample, thus appearing to be “new” peaks. These peaks were designated in the Analyst Report with a percent change of “100 percent” and the description “NEW PEAK UNMATCHED IN NULL.”

[0386] MATCHED peaks were processed further for relative quantitative differentiation. This quantitative differentiation is expressed as a percent change of the Sample peak area relative to the area of the Reference peak. A predetermined threshold for change must be observed for the change to be determined biochemical and statistically significant. The change threshold is based upon previously observed biological and analytical variability factors. Only changes above the threshold for change were reported.

[0387] Peaks were then processed through the peak identification process as follows. The mass spectra of the peaks were first searched against mass spectral plant metabolite libraries. The equivalence value assigned to the library match was used as an indication of a proper identification.

[0388] To provide additional confirmation to the identity of a peak, or to suggest other possibilities, library hits were searched further against a Biotechnology database. The Biotechnology database is based on the Access database program from Accelrys (formerly Synopsis) and utilizes Accord for Access (also available from Accelrys) to incorporate chemical structures into the database.

[0389] The Chemical Abstract Services (CAS) number of the compound from the library was searched against those contained in the database. If a match was found, the CAS number in the database was then correlated to the data acquisition method for that record. If the method was matched, the program then compared the retention index (RI), in the Peak Table, of the component against the value contained in the database for that given method. Should the RI's match (within a given window of variability) then the peak identity was given a high degree of certainty. Components in the Sample that are not identified by this process were assigned a unique identifier based upon Fraction Number and RI (example: F1-U0.555). The unique identifier was used to track unknown components. The program then sorts the data and generates an Analyst Report.

[0390] An Analyst Report is an interim report consisting of PBM algorithm match quality value (equivalence value), RT, Normalized Peak Area, RI (Sample), RI (database) Peak Identification status [peak identity of high certainty (peaks were identified by the program based on the pre-established criteria) or criteria not met (program did not positively identify the component)], Component Name, CAS Number, Mass Spectral Library (containing spectrum most closely matched to that of the component), Unknown ID (unique identifier used to track unidentified components), MS-XCR value, Relative % Change, Notes (MATCHED UNMATCHED), and other miscellaneous information. The Analyst Report was reviewed manually by the analyst who determined what further analysis was necessary. The analyst also generated a modified report, for further processing by the program, by editing the Analyst Report accordingly.

[0391] For Fractions 2 and 3, derivatization procedures were performed prior to analysis to make the certain components more amenable to gas chromatography. Thus, the compound names in the modified analyst report (MAR) were those of the derivatives. To accurately reflect the true components of these fractions, the MAR was further processed using information contained in an additional database. This database cross-references the observed derivatized compound to that of the original, underivatized “parent” compound by way of their respective CAS numbers and replaces derivatives with parent names and information for the final report. In addition, any unidentified components were assigned a “999999-99-9” CAS number.

[0392] The Modified Analyst Report also contains a HIT Score of 0, 1, or 2. The value is assigned by the analyst to the data set of the Sample aliquot based on the following criteria:

[0393] 0 No FDL data on Sample

[0394] 1 FDL data collected; Sample not FDL HIT

[0395] 2 FDL data collected; Sample is FDL HIT

[0396] An FDL HIT is defined as a reportable percent change (modification) observed in a Sample relative to Reference in a component of biochemical significance.

[0397] An electronic copy of the final report is entered into the Nautilus LIMS system (BLIMS) and subsequently into eBRAD (Biotech database). The program also generated a hardcopy of the pinpointed TIC and the respective mass spectrum of each component that was reported to have changed.

[0398] “NQ” and “NEW” are two terms used in the final report. Both terms refer to UNMATCHED peaks whose percent changes cannot be reported in a numerically quantitative fashion. These terms are defined as follows:

[0399] “NQ” is used in the case where there was a peak reported in the Reference for which there was no match in the Sample (either because there was no peak in the Sample or, if there was, the area of the peak did not satisfy the Limit of Processing for Peak Matching). The percent change designation of “−100%” used in the Analyst report is replaced with “NQ”.

[0400] “NEW” is used in those situations where a peak was reported in the Sample but for which there was no corresponding match in the Reference (either because there was no peak in the Reference or, if there was, the area of the peak did not satisfy the Limit of Processing for Peak Matching). For these situations, the percent change designation of “100%” used in the Analyst Report is replaced with “NEW”. The designation of “NEW” in the final report to a component that is present in the Sample but not in the Reference was necessary to eliminate any ambiguity with the appearance of “100%” for MATCHED peaks. A “100%” designation in the final report exclusively refers to a component with modification that doubled in the Sample relative to the Reference.

[0401] G. Results.

[0402] The results of the metabolic screening revealed that transfection with 55 of the inserts resulted in measurable metabolic changes.

Claims

1. A method of creating a transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype comprising: expressing in the plant the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122:

2. A method of creating a transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype comprising the steps of: (a) providing a viral inoculum capable of infecting a plant comprising the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122; (b) applying said viral inoculum to a plant; whereby the plant is infected and the DNA or the mRNA is expressed in the plant.

3. The method of claims 1 or 2 wherein the plant is turfgrass.

4. The method of claims 1 or 2 wherein the plant is fir tree.

5. A transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype made by the method comprising: expressing in the plant the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122.

6. The transfected or transgenic plant of claim 5 wherein the plant is turfgrass.

7. The transfected or transgenic plant of claim 5 wherein the plant is fir tree.

8. A transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype made by the method comprising the steps of: (a) providing a viral inoculum capable of infecting a plant comprising the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122; (b) applying said viral inoculum to a plant; whereby the plant is infected and the DNA or the mRNA is expressed in the plant.

9. The transfected or transgenic plant of claim 8 wherein the plant is turfgrass.

10. The transfected or transgenic plant of claim 8 wherein the plant is fir tree.

11. A method of producing multiple crops of the plant of claims 5-10 comprising the steps of: (a) planting a reproductive unit of the plant; (b) growing the planted reproductive unit under natural light conditions; (c) harvesting the plant; and (d) repeating steps (a) through (c) at least once in the year.

12. A method of manufacturing a biopharmaceutical comprising: (a) providing a plant that expresses a biopharmaceutical in the plant; (b) providing a viral inoculum capable of infecting a plant comprising the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group of SEQ. ID NO: 1-122; (c) applying said viral inoculum to the plant; whereby the plant is infected, exhibits a dwarf phenotype, and expresses the biopharmaceutical.