ZA200110393B

ZA200110393B - Method of correlating sequence function by transfecting a nucleic acid sequence of a donor organism into a plant host in an anti-sense or positive sense orientation.

Info

Publication number: ZA200110393B
Application number: ZA200110393A
Authority: ZA
Inventors: Monto H Kumagai; Guy R Della-Cioppa; Robert L Erwin; David R Mcgee
Original assignee: Large Scale Biology Corp
Priority date: 1999-07-21
Filing date: 2001-12-19
Publication date: 2003-04-08

Description

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METHOD OF CORRELATING SEQUENCE FUNCTION BY TRANSFECTING A :

NUCLEIC ACID SEQUENCE OF A DONOR ORGANISM INTO A PLANT HOST

IN AN ANTI-SENSE OR POSITIVE SENSE ORIENTATION

This application claims priority to U.S. Application Serial Nos. 09/359,301, 09/359,305, 09/359,297, and 09/359,300, all filed on July 21, 1999.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecular biology and genetics.

Specifically, the present invention relates to a method for correlating the function of a host organism derived nucleic acid sequence by a transient expression of the nucleic acid sequence in an antisense or positive sense orientation in a plant host.

BACKGROUND OF THE INVENTION

Great interest exists in launching genome projects in human and non-human genome project. The human genome has between 2.8 million and 3.5 million base pairs, about 3 percent of which are made of genes. In June 2000, the Human Genome Project and biotech company Celera Genomics announced that a rough draft of the human genome has been completed (http://www.ncbi.nim.nih.gov). This information, however, will only represent a reference sequence of the human genome. The remaining task lies in the determination of sequence functions, which are important for the study, diagnosis, and treatment of human diseases.

The Mouse genome is also being sequenced. Genbank provides about 1.2% of the 3- billion-base mouse genome (http://www .informatics.jax.org) and a rough draft of the mouse genome is expected to be available by 2003 and a finished genome by 2005. In addition, the

Drosophila Genome Project has recently been completely (http://www _fruitfly.org).

Valuable and basic agricultural plants, including corn, soybeans and rice are also targets for genome projects because the information obtained thereby may prove very beneficial for increasing world food production and improving the quality and value of agricultural products. The United States Congress is considering launching a com genome project. By helping to unravel the genetics hidden in the corn genome, the project could aid in understanding and combating common diseases of grain crops. It could also provide a big

] boost for efforts to engineer plants to improve grain yields and resist drought, pests, salt, and other extreme environmental conditions. Such advances are critical for a world population expected to double by 2050. Currently, there are four species which provide 60% of all human food: wheat, rice, corn, and potatoes, and the strategies for increasing the productivity of these plants is dependent on rapid discovery of the presence of a trait in these plants, and the function of unknown gene sequences in these plants.

One strategy that has been proposed to assist in such efforts is to create a database of expressed sequence tags (ESTs) that can be used to identify expressed genes. Accumulation and analysis of expressed sequence tags (ESTs) have become an important component of genome research. EST data may be used to identify gene products and thereby accelerate gene cloning. Various sequence databases have been established in an effort to store and relate the tremendous amount of sequence information being generated by the ongoing sequencing efforts. Some have suggested sequencing 500,000 ESTs for corn and 100,000

ESTs each for rice, wheat, oats, barley, and sorghum. Efforts at sequencing the genomes of plant species will undoubtedly rely upon these computer databases to share the sequence data as it is generated. Arabidopsis thaliana may be an attractive target discovery of a trait and for gene function discovery because a very large set of ESTs have already been produced in this organism, and these sequences tag more than 50% of the expected

Arabidopsis genes.

Potential use of the sequence information so generated is enormous if gene function can be determined. It may become possible to engineer commercial seeds for agricultural use to convey any number of desirable traits to food and fiber crops and thereby increase agricultural production and the world food supply. Research and development of commercial seeds has so far focused primarily on traditional plant breeding, however there has been increased interest in biotechnology as it relates to plant characteristics. Knowledge of the genomes involved and the function of genes contained therein for both monocotyledonous and dicotyledonous plants is essential to realize positive effects from such technology.

The impact of genomic research in seeds is potentially far reaching. For example, gene profiling in cotton can lead to an understanding of the types of genes being expressed "primarily in fiber cells. The genes or promoters derived from these genes may be important in genetic engineering of cotton fiber for increased strength or for "built-in" fiber color. 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.

Work has been conducted in the area of developing suitable vectors for expressing foreign DNA and RNA in plant and animal hosts. Ahlquist, U.S. Patent Nos. 4,885,248 and 5,173,410 describes preliminary work done in devising transfer vectors which might be useful in transferring foreign genetic material into a plant host for the purpose of expression ) therein. Additional aspects of hybrid RNA viruses and RNA transformation vectors are described by Ahlquist ef al. in U.S. Patent Nos. 5,466,788, 5,602,242, 5,627,060 and 5,500,360. Donson er al., U.S. Patent Nos. 5,316,931, 5,589,367 and 5,866,785 demonstrate for the first time plant viral vectors suitable for the systemic expression of foreign genetic material in plants. Donson et al. describe plant viral vectors having heterologous : subgenomic promoters for the systemic expression of foreign genes. Carrington et al, U.S.

Patent 5,491,076, describe particular potyvirus vectors also useful for expressing foreign genes in plants. The expression vectors described by Carrington ef al. are characterized by utilizing the unique ability of viral polyprotein proteases to cleave heterologous proteins from viral polyproteins. These include Potyviruses such as Tobacco Etch Virus. Additional suitable vectors are described in U.S. Patent No. 5,811,653 and U.S. Patent No. 5,977,438. ‘Condreay et al., (Proc. Natl. Acad. Sci. USA 96: 127-132) disclose using baculoviruses to deliver and expresss gene efficiently in cell types of human, primate and rodent origin. Price et al, (Proc. Natl. Acad. Sci. USA 93:9465-9570 (1996)) disclose infecting insect plant and : mammalian cells with Nodaviruses.

Construction of plant RNA viruses for the introduction and expression of non-viral foreign genes in plants has also been demonstrated by Brisson et al.. Methods in Enzymology 118:659 (1986), Guzman et al., Communications in Molecular Biology: Viral Vectors, Cold

Spring Harbor Laboratory, pp. 172-189 (1988), Dawson et al., Virology 172:285-292 (1989),

Takamatsu er al., EMBO J. 6:307-311 (1987), French et al., Science 231:1294-1297 (1986), and Takamatsu er al., FEBS Letters 269:73-76 (1990). However, these viral vectors have 3 .

Amended Sheet — 2003-06-24 not been shown capable of systemic spread in the plant and expression of the non-viral foreign genes in the majority of plant cells in the whole plant. Moreover, many of these viral vectors have not proven stable for the maintenance of non-viral foreign genes.

However, the viral vectors described by Donson ez al., in U.S. Patent Nos. 5,316,931, 5,589,367, and 5,866,785, Turpen in U.S. Patent No. 5,811,653, Carrington ef al. in U.S.

Patent No. 5,491,076, and in co-pending U.S. Patent No. 5,977,438, have proven capable of infecting plant cells with foreign genetic material and systemically spreading in the plant and expressing the non-viral foreign genes contained therein in plant cells locally or systemically. Morsy et al., (Proc. Natl. Acad. Sci. USA, 95:7866-7871 *(1998)) develop a helper-dependent adenoviral vectors having up to 37Kb insert capacity } and being easily propagated.

With the recent advent of technology for cloning, genes can be selectively turned off.

One method is to create antisense RNA or DNA molecules that bind specifically with a targeted gene’s RNA message, thereby interrupting the precise molecular mechanism that expresses a gene as a protein. The antisense technology which deactivates specific genes provides a different approach from a classical genetics approach. Classical genetics usually studies the random mutations of all genes in an organism and selects the mutations responsible for specific characteristics. Antisense approach starts with a cloned gene of interest and manipulates it to elicit information about its function.

The expression of virus-derived positive sense or antisense RNA in transgenic plants provides an enhanced or reduced expression of an endogenous gene. In most cases, + introduction and subsequent expression of a transgene will increase (with a positive sense

RNA) or decrease (with an antisense RNA) the steady-state level of a specific gene product (Curr. Opin. Cell Biol. 7: 399-405 (1995)). There is also evidence that inhibition of endogenous genes occurs in transgenic plants containing sense RNA (Van der Krol er al.,

Plant Cell 2(4):291-299 (1990), Napoli er al., Plant Cell 2:279-289 (1990) and Fray er al.,

Plant Mol. Biol. 22:589-602 (1993).

Post-transcriptional gene silencing (PTGS) in transgenic plants is the manifestation of a mechanism that suppresses RNA accumulation in a sequence-specific manner. There are three models to account for the mechanism of PTGS: direct transcription of an antisense

RNA from the transgene, an antisense RNA produced in response to over expression of the 4

Amended Sheet — 2003-06-24 transgene, or an antisense RNA produced in response to the production of an aberrant sense .

RNA product of the transgene (Baulcombe, Plant Mol. Biol. 32:79-88 (1996)). The posttranscriptional gene silencing mechanism is typified by the highly specific degradation of both the transgene mRNA and the target RNA, which contains either the same or complementary nucleotide sequences. In cases that the silencing transgene is the same sense as the target endogenous gene or viral genomic RNA, it has been suggested that a plant- : encoded RNA-dependent RNA polymerase makes a complementary strand from the transgene mRNA and that the small cRNAs potentiate the degradation of the target RNA.

Antisense RNA and the hypothetical CRNAs have been proposed to act by hybridizing with the target RNA to either make the hybrid a substrate for double-stranded (ds) RNases or arrest the translation of the target RNA (Baulcombe, Plant Mol. Biol. 32: 79-88 (1996)). It is also proposed that this downregulation or “‘co-suppression” by the sense RNA might be due to the production of antisense RNA by readthrough transcription from distal promoters : located on the opposite strand of the chromosomal DNA (Grierson et al., Trends Biotechnol. 9:122-123 (1993)).

Waterhouse et al (Proc. Natl. Acad. Sci. USA. 10: 13959-64 (1998)) prepared 2 transgenic tobacco plants containing sense or antisense constructs. Pro[s] and Pro[a/s] constructs contained the PVY nuclear inclusion Pro ORF in the sense and antisense orientations, respectively. The Pro[s]-stop construct contained the PVY Pro ORF in the sense orientation but with a stop codon three codons downstream from the initiation codon.

Waterhouse ez al show when the genes of those constructs were transformed into plants, the plants exhibited immunity to the virus form which the transgene was derived. Smith ef al (Plant Cell, 6: 1441-1453, (1994)) prepared a tobacco transgenic plant containing the potato virus Y (PVY) coat protein (CP) open reading frame, which produced an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon. The expression of the untransiatable sense RNA inversely correlated with the virus resistance of the transgenic plant. Kumagai er al (Proc. Natl. Acad. Sci. USA 92:1679 (1995)) report that gene expression in transfected Nicotiana benthamiana was cytoplasmic inhibited by viral delivery of a RNA of a known sequence derived from cDNA encoding tomato (/ycopersicon esculentum) phytoene desaturase in a positive sense or an antisense orientation.

. The antisense sense and positive sense technology can be used to develop a functional genomic screening of a donor organism from Monera, Protisca, Fungi, Plantae or

Animalia. The present invention provides a method of detecting the presence of a trait in a plant host and determining the function and sequence of a nucleic acid of a donor organism by expressing the nucleic acid sequence in the plant host. GTP-binding proteins exemplify this invention. In eukaryotic cells, GTP-binding proteins function in a vanety of cellular processes, including signal transduction, cytoskeletal organization, and protein transport.

Low molecular weight (20-25 K Daltons) of GTP-binding proteins include ras and its close relatives (for example, Ran), rho and its close relatives, the rab family, and the ADP- ribosylation factor (ARF) family. The heterotrimeric and monomeric GTP-binding proteins that may be involved in secretion and intracellular transport are divided into two structural classes: the rab and the ARF families. Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. The cDNAs encoding GTP binding proteins have been isolated from a variety of plants including rice, : barley, com, tobacco, and A. thaliana. For example, Verwoert et al. (Plant Molecular Biol. 27:629-633 (1995)) report the isolation of a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family by direct genetic selection in an £. coli fabD mutant with a maize cDNA expression library. Regad et al. (FEBS 2:133-136 (1993)) isolated a cDNA clone encoding the ARF from a cDNA library of Arabidopsis thaliana cultured cells by randomly selecting and sequencing cDNA clones. Dallmann ez al. (Plant Molecular Biol. 19:847-857 (1992)) isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries using a PCR approach. Dallmann er al. prepared leaf cDNAs and use them as templates in

PCR amplifications with degenerated oligonucleotides corresponding to the highly conserved motifs, found in members of the ras superfamily, as primers. Haizel et al., (Plant

J., 11:93-103 (1997)) isolated cDNA and genomic clones encoding Ran-like small GTP binding proteins from Arabidopsis cDNA and genomic libranes using a full-length tobacco

Nt Ran cDNA as a probe. The present invention provides advantages over the above methods in identifying nucleic acid sequence encoding GTP binding proteins in that it only sequences clones that have a function and does not randomly sequence clones. The nucleic acid inserts in clones that have a function are labeled and used as probes to isolate a cDNA ) hybridizing to them.

The present invention provides a method for detecting the presence of a trait in a plant host by expressing a donor organism derived nucleic acid sequence in an antisense or positive orientation in the plant host. Once the presence of a trait is identified by phenotypic changes, the nucleic acid insert in the cDNA clone or in the vector is then sequenced. The present method provides a rapid method for determining the presence of a trait and a method for identifying a nucleic acid sequence and its function in a plant host by screening phenotypic or biochemical changes in the plant host transfected with a nucleic acid sequence of the donor organism.

SUMMARY OF THE INVENTION

The present invention essentially involves the steps of (1) introducing into a viral vector a library of host organism derived sequence inserts in a positive or antisense orientation; (2) expressing each insert in a plant host, and (3) detecting phenotypic or biochemical changes of the plant host as a result of the expression. A plant host may be a monocotyledonous or dicotyledonous plant, plant tissue or plant cell. Donor organisms include species from Monera, Protista, Fungi, Plantae, or Animalia kingdom, such as human, mouse, drosophila, etc. If the donor organism is also a plant, the donor plant and the host plant typically belong to different genus, family, order, class, subdivision, or division.

The function of sequence inserts in the library is typically unknown. The number of sequence inserts in a library is typically larger than about 10, 15, 20, 50, 100, 200, 500, 1000, 5000, or 15,000, etc. The length of each insert is typically longer than about 50, 100, 200, or 500 base pairs.

More specifically, the present invention is directed to a method of changing the phenotype or biochemistry of a plant host, a method of determining a change in phenotype or biochemistry in a plant host, and a method of determining the presence of a trait in a plant host. The method comprises the steps of expressing transiently a nucleic acid sequence of a donor organism in an antisense or positive sense orientation in a plant host, identifying changes in the plant host, and correlating the sequence expression with the phenotypic or

] biochemical changes. The nucleic acid sequence does not need to be isolated, identified, or characterized prior to transfection into the host organism.

The present invention is also directed to a method of making a functional gene profile by transiently expressing a nucleic acid sequence library in a host organism, determining the phenotypic or biochemical changes in the plant host, identifying a trait associated with the change, identifying the donor gene associated with the trait, identifying the homologous host gene, if any, and annotating the sequence with its associated phenotype or function.

The present invention is also directed to a method of determining the function of a nucleic acid sequence, including a gene, in a donor organism, by transfecting the nucleic acid sequence into a plant host in a manner so as to affect phenotypic or biochemical changes in the plant host. In one embodiment, recombinant viral nucleic acids are prepared to include the nucleic acid insert of a donor. The recombinant viral nucleic acids infect a plant host and produce antisense or positive sense RNAs in the cytoplasm which result in a reduced or enhanced expression of endogenous cellular genes in the host organism. Once the presence of a trait is identified by phenotypic or biochemical changes, the function of the nucleic acid is determined. The nucleic acid insert in a cDNA clone or in a vector is then sequenced. The nucleic acid sequence is determined by a standard sequence analysis.

One aspect of the invention is a method of identifying and determining a nucleic acid sequence in a donor organism, whose function is to silence endogenous genes in a plant host, by introducing the nucleic acid into the plant host by way of a viral nucleic acid suitable to produce expression of the nucleic acid in the transfected plant. This method utilizes the principle of post-transcription gene silencing of the endogenous host gene homologue, for example, antisense RNAs, or positive sense RNAs. Particularly, this silencing function is useful for silencing a multigene family in a donor organism. In addition, the overexpressioin of a plus sense RNA that results in overproduction of a protein may cause phenotypical or biochemical changes in a host.

Another aspect of the invention is to discover genes in a donor organism having the same function as that in a plant host. The method starts with building a cDNA library, or a genomic DNA library, or a pool of RNA of a donor organism, for example, from tissues or cells of human, mouse, or drosophila. Then, a recombinant viral nucleic acid comprising a nucleic acid insert derived from the library is prepared and is used to infect a plant host. The } infected plant host is inspected for phenotypic or biochemical changes. The recombinant viral nucleic acid that results in phenotypic or biochemical changes in the plant host is identified and the sequence of the nucleic acid insert is determined by a standard method. ) Such nucleic acid sequence in the donor organism may have substantial sequence homology as that in the plant host, e.g. the nucleic acid sequences are conserved between the donor and plant host. Once the nucleic acid is sequenced, it can be labeled and used as a probe to isolate full-length cDNAs from the donor organism. This invention provides a rapid means for elucidating the function and sequence of nucleic acids of a donor organism; such rapidly expanding information can be subsequently utilized in the field of genomics.

Another aspect of the instant invention is directed to a method of increasing yield of a grain crop. The method comprises expressing transiently a nucleic acid sequence of a donor plant in an antisense or positive sense orientation in a grain crop, wherein said expressing results in stunted growth and increased seed production of the grain crop. A preferred method comprises the steps of cloning the nucleic acid sequence into a plant viral vector and infecting the grain crop with a recombinant viral nucleic acid comprising sad nucleic acid sequence.

Another aspect of the invention is to discover genes having the same function in different plants. The method starts with a library of cDNAs, genomic DNAs, or a pool of

RNAs of a first plant. Then, a recombinant viral nucleic acid comprising a nucleic acid insert derived from the library is prepared and is used to infect a different host plant. The infected host plant is inspected for phenotypic or biochemical changes. The recombinant viral nucleic acid that results in phenotypic or biochemical changes in the host plant is identified and the sequence of the nucleic acid insert is determined by a standard method.

Such nucleic acid sequence in the first plant has substantial sequence homology as that in the host plant: the nucleic acid sequences are conserved between the two plants. This invention provides a rapid means for elucidating the function and sequence of nucleic acids of interest; such rapidly expanding information can be subsequently utilized in the field of genomics.

Another aspect of the present invention is to produce human proteins in a plant host.

After nucleic acids of similar functions from a human and a host plant are isolated and identified, the amino acid sequences derived from the DNAs are compared. The plant oo nucleic acid sequence is changed so that it encodes the same amino acid sequence as the human protein. The nucleic acid sequence can be changed according to any conventional methods, such as, site directed mutagenesis or polymerase based DNA synthesis.

Plant hosts include plants of commercial interest, such as food crops, seed crops, oil crops, omamental crops and forestry crops. For example, wheat, nice, com, potatoes, barley, tobaccos, soybean canola, maize, oilseed rape, Arabidopsis, Nicotiana can be selected as a host plant. In particular, host plants capable of being infected by a virus containing a recombinant viral nucleic acid are preferred.

A plant viral vector may comprise a native or non-native subgenomic promoter, a coat protein coding sequence, and at least one non-native nucleic acid sequence. Some viral vectors used in accordance with the present invention may be encapsidated by the coat proteins encoded by the recombinant virus. The recombinant viral nucleic acid is capable of replication in the plant host, and transcription or expression of the non-native nucleic acid in the plant host to produce a phenotypic or biochemical change. Any suitable vector : constructs useful to produce localized or systemic expression of nucleic acids in a plant host are within the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

. FIG. 1 depicts the plasmid pBS #735

FIG. 2 depicts the plasmid pBS #740.

FIG. 3 depicts the plasmid TTU51A QSEO #3. (SEQID NOs: 1 and 2)

FIG. 4 depicts the plasmid TTO1A/Ca CCS+. (SEQ ID NOs : 3 and 4)

FIG. 5S depicts the plasmid TTO1/PSY+. (SEQ ID NOs : 5 and 6)

FIG. 6 depicts the plasmid TTO1/PDS+.

FIG. 7 depicts a Monocot Viral Vector

FIG. 8 depicts the plasmid TTU51 CTP CrtB. (SEQ ID NOs : 7 and 8)

FIG. 9 depicts the plasmid pBS 740 AT #2441 (ATCC No: PTA-332).

FIG. 10 depicts the nucleotide sequence of 740 AT #2441. (SEQ ID NOs : 9 and 10)

FIG. 11 depicts the nucleotide sequence comparison of 740 AT #2441 (SEQ ID NO: 11) and

AF017991. (SEQID NO: 12)

FIG. 12 depicts the nucleotide sequence comparison of 740 AT #2441 (SEQ ID NO :13) and L16787 (SEQ ID NO :14). oo

Amended Sheet — 2003-06-24

FIG. 13 depicts the amino acid sequence comparison of 740 AT #2441 (SEQ ID NO : 15) and RAN-BI (SEQ ID NO : 16) GTP binding protein. :

FIG. 14 depicts the plasmid pBS 740 AT #120 (ATCC No: PTA-325).

FIG. 15 shows the nucleotide sequence comparison of 4. thaliana 740 AT #120 (SEQID NO: 17) and 4.thaliana est AA042085 (SEQ ID NO ; 18)

FIG. 16 shows the nucleotide sequence alignment of 740 AT #120 (SEQIDNO : 19) to rice D17760 (Oryza sativa) ADP-ribosylation factor. (SEQ D NO: 20)

FIG. 17 shows the nucleotide sequence alignment of 740 AT #120 (SEQ ID NO : 21) to human ADP-ribosylation factor P16587. (SEQ ID NO £22)

FIG. 18 shows the nucleotide sequence alignment of humanized sequence 740 AT #120 H to human ADP-ribosylation factor M33384. (SEQ ID NOs: 23, 24 and 25) ] FIG. 19 shows the plasmid KS+ Nb ARF #3 (ATCC No: PTA-324).

FIG. 20 shows the nucleotide sequence companson of A. thaliana 740 AT #120 and

N. benthamiana KS+ Nb ARF#3. (SEQ ID NO : 27) : : FIG. 21 shows a Tobacco Rattle Virus gene silencing vector. “FIG. 22 shows the plasmid pBS #740 AT #88 (SEQ ID NOs : 28 and 29) (ATCC No:

PTA-331).

FIG. 23 shows the sequence of 740 AT #88. . FIG. 24 shows the nucleotide sequence comparison of AT #88 (SEQ ID NO: 30) and Brassica rapa 135812. (SEQ ID NO: 31)!

FIG. 25 shows the nucleotide sequence companson of AT #88 (SEQ ID NO: 32) and Octopus Rhodopsin X07797. (SEQ ID NO: 33)

FIG. 26 shows the nucleotide sequence comparison of AT #88 (SEQ ID NO: 34) and Octopus Rhodopsin P31356. (SEQ ID NO: 35)

FIG. 27 shows the plasmid pBS #377 (SEQ ID NO: 36) (ATCC No: PTA-334).

FIG. 28 shows the nucleotide sequence of 740 AT #377.

FIG. 29 shows the plasmid pBS #2483 (SEQ ID NO: 37) (ATCC No: PTA-329).

FIG. 30 shows the nucleotide sequence of 740 AT #2433.

FIG. 31 shows the plasmid pBS 740 AT #909 (SEQ ID NO: 38) (ATCC No: PTA-330),

FIG. 32 shows the nucleotide sequence comparison of AT #909 and Ribosomal protein L19 (SEQ ID NO: 39)from breast cancer cell line.

FIG. 33 shows the nucleotide sequence comparison of AT #909 (SEQ ID NO: 40) and L19 P14118 60S 11

Amended Sheet — 2003-06-24

. ribosomal protein L19. (SEQ ID NO:41) . ’ EE

FIG. 34 shows the plasmid pBS AT #855 (ATCC No: PTA-326).

FIG. 35 shows the nucleotide sequence comparison of AT #855 (SEQ ID NO: 42) and HAT 7 homeobox protein ORF. (SEQ ID NO: 43)

DETAILED DESCRIPTION OF THE INVENTION :

The present invention essentially involves the steps of (1) introducing into a viral vector a library of host organism derived sequence inserts in a positive or antisense orientation; (2) expressing each insert in a plant host, and (3) detecting phenotypic or } biochemical changes of the plant host as a result of the expression. A plant host may be a monocotyledonous or dicotyledonous plant, plant tissue or plant cell. Donor organisms include species from Monera, Protista, Fungi, Plantae, or Animalia kingdom, such as human, mouse, drosophila, etc. If the donor organism is also a plant, the donor plant and the host plant typically belong to different genus, family, order, class, subdivision, or division.

More specifically, the present invention is directed to a method of changing the phenotype or biochemistry of a plant host, a method of determining a change in phenotype or biochemistry in a plant host, and a method of determining the presence of a trait in a plant host. The method comprises the steps of expressing transiently a nucleic acid sequence of a donor organism in an antisense or positive sense orientation in a plant host, identifying changes in the plant host, and correlating the sequence expression with the phenotypic or biochemical changes. The nucleic acid sequence does not need to be isolated, identified, or characterized prior to transfection into the host organism.

The present invention is also directed to a method of making a functional gene profile by transiently expressing a nucleic acid sequence library in a host organism, determining the phenotypic or biochemical changes in the plant host, identifying a trait associated with the change, identifying the donor gene associated with the trait, identifying 12 oo Amended Sheet — 2003-06-24 the homologous host gene, if any, and annotating the sequence with its associated phenotype . or function.

Another aspect of the invention is to discover genes in a donor organism having the same function as that in a plant host. The method starts with building a cDNA library, ora genomic DNA library, or a pool of RNA of a donor organism, for example, from tissues or cells of human, mouse, or drosophila. Then, a recombinant viral nucleic acid comprising a nucleic acid insert derived from the library is prepared and is used to infect a plant host. The infected plant host is inspected for phenotypic or biochemical changes. The recombinant viral nucleic acid that results in phenotypic or biochemical changes in the plant host is identified and the sequence of the nucleic acid insert is determined by a standard method.

Such nucleic acid sequence in the donor organism may have substantial sequence homology as that in the plant host, e.g. the nucleic acid sequences are conserved between the donor and

. plant host. Once the nucleic acid is sequenced, it can be labeled and used as a probe to isolate full-length cDNAs from the donor organism. This invention provides a rapid means for elucidating the function and sequence of nucleic acids of a donor organism; such rapidly expanding information can be subsequently utilized in the field of genomics.

Another aspect of the instant invention is directed to a method of increasing yield of a grain crop. The method comprises expressing transiently a nucleic acid sequence of a donor plant in an antisense or positive sense orientation in a grain crop, wherein said expressing results in stunted growth and increased seed production of the grain crop. A preferred method comprises the steps of cloning the nucleic acid sequence into a plant viral vector and infecting the grain crop with a recombinant viral nucleic acid comprising said nucleic acid sequence.

Another aspect of the present invention is directed to a method for producing human proteins in a plant host. After nucleic acids of similar functions from a human and a host plant are isolated and identified, the amino acid sequences derived from the DNAs are compared. The plant nucleic acid sequence is changed so that it encodes the same amino acid sequence as the human protein. The nucleic acid sequence can be changed according to any conventional methods, such as, site directed mutagenesis or polymerase based DNA synthesis.

L Introducing into a plant viral vector a library of sequence inserts from a donor organism.

The construction of viral expression vectors may use a variety of methods known in the art. In preferred embodiments of the instant invention, the viral vectors are derived from

RNA plant viruses. A variety of plant virus families may be used, such as Bromoviridae,

Bunyaviridae, Comoviridae, Geminiviridae, Potyvindae, and Tombusviridae, among others.

Within the plant virus families, various genera of viruses may be suitable for the instant invention, such as alfamovirus, ilarvirus, bromovirus, cucumovirus, tospovirus, carlavirus, caulimovirus, closterovirus, comovirus, nepovirus, dianthovirus, furovirus, hordeivirus, luteovirus, necrovirus, potexvirus, potyvirus, rymovirus, bymovirus, oryzavirus, sobemovirus, tobamovirus, tobravirus, carmovirus, tombusvirus, tymovirus, umbravirusa, and among others.

Within the genera of plant viruses, many species are particular preferred. They include alfalfa mosaic virus, tobacco streak virus, brome mosaic virus, broad bean mottle virus, cowpea chlorotic mottle virus, cucumber mosaic virus, tomato spotted wilt virus, carnation latent virus, caulflower mosaic virus, beet yellows virus, cowpea mosaic virus, tobacco ringspot virus, camation ringspot virus, soil-borne wheat mosaic virus, tomato golden mosaic virus, cassava latent virus, barley stripe mosaic virus, barley yellow dwarf virus, tobacco necrosis virus, tobacco etch virus, potato virus X, potato virus Y, rice necrosis virus, ryegrass mosaic virus, barley yellow mosaic virus, rice ragged stunt virus, Southern bean mosaic virus, tobacco mosaic virus, ribgrass mosaic virus, cucumber green mottle mosaic virus watermelon strain, oat mosaic virus, tobacco rattle virus, carnation mottle virus, tomato bushy stunt virus, turnip yellow mosaic virus, carrot mottle virus, among others. In addition, RNA satellite viruses, such as tobacco necrosis satellite may also be employed.

A given plant virus may contain either DNA or RNA, which may be either single- or double-stranded. One example of plant viruses containing double-stranded DNA includes, but not limited to, caulimoviruses such as cauliflower mosaic virus (CaMV). Representative plant viruses which contain single-stranded DNA are cassava latent virus, bean golden mosaic virus (BGMYV), and chloris striate mosaic virus. Rice dwarf virus and wound tumor virus are examples of double-stranded RNA plant viruses. Single-stranded RNA plant viruses include tobacco mosaic virus (TMV), tumip yellow mosaic virus (TYMV), rice necrosis virus (RNV) and brome mosaic virus (BMV). The single-stranded RNA viruses can be further divided into plus sense (or positive-stranded), minus sense (or negative- stranded), or ambisense viruses. The genomic RNA of a plus sense RNA virus is messenger sense, which makes the naked RNA infectious. Many plant viruses belong to the family of plus sense RNA viruses. They include, for example, TMV, BMV, and others. RNA plant viruses typically encode several common proteins, such as replicase/polymerase proteins essential for viral replication and mRNA synthesis, coat proteins providing protective shells for the extracellular passage, and other proteins required for the cell-to-cell movement, systemic infection and self-assembly of viruses. For general information concerning plant viruses, see Matthews, Plant Virology, 3™ Ed., Academic Press, San Diego (1991).

Selected groups of suitable plant viruses are characterized below. 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. 7 However, the invention should not be construed as limited to using these particular viruses, 2 but rather the present invention is contemplated to include all suitable viruses. Some : suitable viruses are characterized below.

TOBAMOVIRUS GROUP

The tobacco mosaic virus (TMV) is of particular interest to the instant invention because of its ability to express genes at high levels in plants. TMV is a member of the tobamovirus group. The TMV virion is a tubular filament, and comprises coat protein sub- units arranged in a single right-handed helix with the single-stranded RNA intercalated between the turns of the helix. TMV infects tobacco as well as other plants. TMV virions are 300 nm x 18 nm tubes with a 4 nm-diameter hollow canal, and consist of 2140 units of a single structural protein helically wound around a single RNA molecule. The genome is a 6395 base plus-sense RNA. The 5'-end is capped and the 3'-end contains a series of pseudoknots and a tRNA-like structure that will specifically accept histidine. The genomic

RNA functions as mRNA for the production of proteins involved in viral replication: a 126- kDa protein that initiates 68 nucleotides from the 5'-terminus, and a 183-kDa protein synthesized by readthrough of an amber termination codon approximately 10% of the time.

Only the 183-kDa and 126-kDa viral proteins are required for the TMV replication in trans.

(Ogawa et al., Virology 185:580-584 (1991)). Additional proteins are translated from subgenomic size mRNA produced during replication (Dawson, Adv. Virus Res., 38:307-342 (1990)). The 30-kDa protein is required for cell-to-cell movement; the 17.5-kDa capsid protein is the single viral structural protein. The function of the predicted 54-kDa protein is unknown.

TMV assembly apparently occurs in plant cell cytoplasm, although it has been suggested that some TMV assembly may occur in chloroplasts since transcripts of ctDNA have been detected in purified TMV virions. Initiation of TMV assembly occurs by interaction between ring-shaped aggregates (“discs”) of coat protein (each disc consisting of two layers of 17 subunits) and a unique internal nucleation site in the RNA; a hairpin region about 900 nucleotides from the 3’-end in the common strain of TMV. Any RNA, including subgenomic RNAs containing this site, may be packaged into virions. The discs apparently assume a helical form on interaction with the RNA, and assembly (elongation) then proceeds in both directions (but much more rapidly in the 3’- to 5°- direction from the nucleation site).

Another member of the Tobamoviruses, the Cucumber Green Mottle Mosaic virus watermelon strain (CGMMYV-W) is related to the cucumber virus. Nozu et al., Virology 45:577 (1971). The coat protein of CGMMYV-W interacts with RNA of both TMV and

CGMMYV to assemble viral particles in vitro. Kurisu et al., Virology 70:214 (1976).

Several strains of the tobamovirus group are divided into two subgroups, on the basis of the location of the assembly of origin. Subgroup I, which includes the vulgare, OM, and tomato strain, has an origin of assembly about 800-1000 nucleotides from the 3’-end of the

RNA genome, and outside the coat protein cistron. Lebeurier er al., Proc. Natl. Acad. Sci.

USA 74:149 (1977); and Fukuda er al., Virology 101:493 (1980). Subgroup II, which includes CGMMV-W and compea strain (Cc) has an origin of assembly about 300-500 nucleotides from the 3’-end of the RNA genome and within the coat-protein cistron. The coat protein cistron of CGMMV-W is located at nucleotides 176-661 from the 3’-end. The 3’ noncoding region is 175 nucleotides long. The origin of assembly is positioned within the coat protein cistron. Meshi er al., Virology 127:54 (1983).

. BROME MOSAIC VIRUS GROUP

Brome Mosaic virus (BMV) is a member of a group of tripartite, single-stranded,

RNA-containing plant viruses commonly referred to as the bromoviruses. Each member of the bromoviruses infects a narrow range of plants. Mechanical transmission of bromoviruses occurs readily, and some members are transmitted by beetles. In addition to

BV, other bromoviruses include broad bean mottle virus and cowpea chlorotic mottle virus.

Typically, a bromovirus virion is icosahedral, with a diameter of about 26 pm, containing a single species of coat protein. The bromovirus genome has three molecules of linear, positive-sense, single-stranded RNA, and the coat protein mRNA is also encapsidated. The RNAs each have a capped 5°-end, and a tRNA-like structure (which accepts tyrosine) at the 3’-end. Virus assembly occurs in the cytoplasm. The complete nucleotide sequence of BMV has been identified and characterized as described by Ahlquist etal., J. Mol. Biol. 153:23 (1981).

RICE NECROSIS VIRUS

Rice Necrosis virus is a member of the Potato Virus Y Group or Potyviruses. The

Rice Necrosis virion is a flexuous filament comprising one type of coat protein (molecular weight about 32,000 to about 36,000) and one molecule of linear positive-sense single- stranded RNA. The Rice Necrosis virus is transmitted by Polymyxa oraminis (a eukaryotic intracellular parasite found in plants, algae and fungi).

GEMINIVIRUSES

Geminiviruses are a group of small, single-stranded DNA-containing plant viruses with virions of unique morphology. Each virion consists of a pair of isometric particles (incomplete icosahedral), composed of a single type of protein (with a molecular weight of about 2.7-3.4X10%). Each geminivirus virion contains one molecule of circular, positive- sense, single-stranded DNA. In some geminiviruses (i.c., Cassava latent virus and bean golden mosaic virus) the genome appears to be bipartite, containing two single-stranded

DNA molecules.

POTYVIRUSES

Potyviruses are a group of plant viruses which produce polyprotein. A particularly preferred potyvirus is tobacco etch virus (TEV). TEV is a well characterized potyvirus and contains a positive-strand RNA genome of 9.5 kilobases encoding for a single, large polyprotein that is processed by three virus-specific proteinases. The nuclear inclusion protein “a” proteinase is involved in the maturation of several replication-associated proteins and capsid protein. The helper component-proteinase (HC-Pro) and 35-kDa proteinase both catalyze cleavage only at their respective C-termini. The proteolytic domain in each of these proteins is located near the C-terminus. The 35-kDa proteinase and HC-Pro derive from the

N-terminal region of the TEV polyprotein.

HORDEIVIRUS GROUP

Hordeiviruses are a group of single-stranded, positive sense RNA-containing plant viruses with three or four part genomes. Hordeiviruses have rigid, rod-shaped virions and barley stripe mosaic virus (BSMV) is the type member. BSMV infects a large number of monocot and dicot species including barley, oat, wheat, corn, rice, , spinach, and Nicotiana benthamiana. Local lesion hosts include Chenopodium amaranticolor, and Nicotiana tabacum ccv. Samsun . BSMV is not vector transmitted but is mechanically transmissable and in some hosts, such as barley, is also transmitted through pollen and seed.

Most strains of BSMV have three genomic RNAs refered to as alpha(a), beta (B), and gamma (y), At least one strain, the Argentina mild (AM) strain has a fourth geneomic

RNA that is essentially a deletion mutant of the g RNA. All genomic RNAs are capped at the 5° end and have tRNA-like structures at the 3” end. Virus replication and assembly occurs in the cytoplasm. The complete nucleotide sequence of several strains of BSMV has been identified and characterized (reviewed by Jackson, et al Annual Review of

Phytophathology 27:95-121 (1989)), and infectious cDNA clones are available (Petty, ef al.

Virology 171:342-349 (1989)).

The selection of the genetic backbone for the viral vectors of the instant invention may depend on the plant host used. The plant host may be a monocotyledonous or dicotyledonous plant, plant tissue, or plant cell. Typically, plants of commercial interest, such as food crops, seed crops, oil crops, omamental crops and forestry crops are preferred.

. For example, wheat, rice, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, lilies, grasses, orchids, irises, onions, palms, tomato, the legumes, or Arabidopsis, can be used as a plant host. Host plants may also include those readily infected by an infectious virus, such as Nicotiana, preferably, Nicotiana benthamiana, or Nicotiana clevelandii.

One feature of the present invention is the use of plant viral nucleic acids which comprise one or more non-native nucleic acid sequences capable of being transcribed in a plant host. These nucleic acid sequences may be native nucleic acid sequences that occur in a host plant. Preferably, these nucleic acid sequences are non-native nucleic acid sequences that do not normally occur in a host plant. For example, the plant viral vectors may contain sequences from more than one virus, including viruses from more than one taxonomic group. The plant viral nucleic acids may also contain sequences from non-viral sources, such as foreign genes, regulatory sequences. fragments thereof from bacteria, fungi, plants, animals or other sources. These foreign sequences may encode commercially useful proteins, polypeptides, or fusion products thereof, such as enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, antimicrobial polypeptides, and the like. Or they may 4 be sequences that regulate the transcription or translation of viral nucleic acids, package viral nucleic acid, and facilitate systemic infection in the host, among others.

In some embodiments of the instant invention, the plant viral vectors may comprise : one or more additional native or non-native subgenomic promoters which are capable of transcribing or expressing adjacent nucleic acid sequences in the plant host. These non- native subgenomic promoters are inserted into the plant viral nucleic acids without destroying the biological function of the plant viral nucleic acids using known methods in the art. For example, the CaMV promoter can be used when plant cells are to be transfected.

The subgenomic promoters are capable of functioning in the specific host plant. For example, if the host is tobacco, TMV, tomato mosaic virus, or other viruses containing subgenomic promoter may be utilized. The inserted subgenomic promoters should be compatible with the TMV nucleic acid and capable of directing transcription or expression of adjacent nucleic acid sequences in tobacco. It is specifically contemplated that two or more heterologous non-native subgenomic promoters may be used. The non-native nucleic acid sequences may be transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the products of the nucleic acids of interest.

In some embodiments of the instant invention, the recombinant plant viral nucleic acids may be further modified by conventional techniques to delete all or part of the native coat protein coding sequence or put the native coat protein coding sequence under the control of a non-native plant viral subgenomic promoter. Ifit is deleted or otherwise inactivated, a non-native coat protein coding sequence is inserted under control of one of the non-native subgenomic promoters, or optionally under control of the native coat protein gene subgenomic promoter. 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 native or non- native coat protein gene may be utilized in the recombinant plant viral nucleic acid. The non-native coat protein, as is the case for the native coat protein, may be capable of encapsidating the recombinant plant viral nucleic acid and providing for systemic spread of the recombinant plant viral nucleic acid in the host plant.

In some embodiments of the instant invention, recombinant plant viral vectors are constructed to express a fusion between a plant viral coat protein and the foreign genes or polypeptides of interest. Such a recombinant plant virus provides for high level expression of a nucleic acid of interest. The location(s) where the viral coat protein is joined to the amino acid product of the nucleic acid of interest may be referred to as the fusion joint. A given product of such a construct may have one or more fusion joints. The fusion joint may be located at the carboxyl terminus of the viral coat protein or the fusion joint may be located at the amino terminus of the coat protein portion of the construct. In instances where the nucleic acid of interest is located internal with respect to the 5° and 3’ residues of the nucleic acid sequence encoding for the viral coat protein, there are two fusion joints. That is, the nucleic acid of interest may be located 5°, 3°, upstream, downstream or within the coat protein. In some embodiments of such recombinant plant viruses, a “leaky” start or stop codon may occur at a fusion joint which sometimes does not result in translational termination.

In some embodiments of the instant invention, nucleic sequences encoding reporter protein(s) or antibiotic/herbicide resistance gene(s) may be constructed as carrier protein(s) for the polypeptides of interest, which may facilitate the detection of polypeptides of interest. For example, green fluorescent protein (GFP) may be simultaneously expressed with polypeptides of interest. In another example, a reporter gene, B-glucuronidase (GUS) may be utilized. In another example, a drug resistance marker, such as a gene whose expression results in kanamycin resistance, may be used.

Since the RNA genome is typically the infective agent, the cDNA is positioned adjacent a suitable promoter so that the RNA is produced in the production cell. The RNA is capped using conventional techniques, if the capped RNA is the infective agent. In addition, the capped RNA can be packaged in vitro with added coat protein from TMV to make assembled virions. These assembled virions can then be used to inoculate plants or plant tissues. Alternatively, an uncapped RNA may also be employed in the embodiments of the present invention. Contrary to the practiced art in scientific literature and in issued patent (Ahlquist ef al., U.S. Patent No. 5,466,788), uncapped transcripts for virus expression vectors are infective on both plants and in plant cells. Capping is not a prerequisite for establishing an infection of a virus expression vector in plants, although capping increases the efficiency of infection. In addition, nucleotides may be added between the transcription v start site of the promoter and the start of the cDNA of a viral nucleic acid to construct an : = infectious viral vector. One or more nucleotides may be added. In some embodiments of ) the present invention, the inserted nucleotide sequence may contain a G at the 5’-end.

Alternatively, the inserted nucleotide sequence may be GNN, GTN, or their multiples, oC (GNN), or (GTN),

In some embodiments of the instant invention, more than one nucleic acid is prepared for a multipartite viral vector construct. In this case, each nucleic acid may require its own origin of assembly. Each nucleic acid could be prepared to contain a subgenomic promoter and a non-native nucleic acid. Alternatively, the insertion of a non-native nucleic acid into the nucleic acid of a monopartite virus may result in the creation of two nucleic acids (i.e., the nucleic acid necessary for the creation of a bipartite viral vector). This would be advantageous when it is desirable to keep the replication and transcription or expression of the nucleic acid of interest separate from the replication and translation of some of the coding sequences of the native nucleic acid.

The recombinant plant viral nucleic acid may be prepared by cloning a viral nucleic acid. If the viral nucleic acid is DNA, it can be cloned directly into a suitable vector using conventional techniques. One technique is to attach an origin of replication to the viral

DNA which is compatible with the cell to be transfected. In this manner, DNA copies of the chimeric nucleotide sequence are produced in the transfected cell. If the viral nucleic acid is )

RNA, a DNA copy of the viral nucleic acid is first prepared by well-known procedures. For example, the viral RNA is transcribed into DNA using reverse transcriptase to produce subgenomic DNA pieces, and a double-stranded DNA may be produced using DNA polymerases. The cDNA is then cloned into appropriate vectors and cloned into a cell to be transfected. In some instances, cDNA is first attached to a promoter which is compatible with the production cell. The recombinant plant viral nucleic acid can then be cloned into any suitable vector which is compatible with the production cell. Alternatively, the recombinant plant viral nucleic acid is inserted in a vector adjacent a promoter which is compatible with the production cell. In some embodiments, the cDNA ligated vector may be directly transcribed into infectious RNA in vitro and inoculated onto the plant host. The cDNA pieces are mapped and combined in proper sequence to produce a full-length DNA copy of the viral RNA genome, if necessary.

The donor organism from which a library of sequence inserts is derived mcludes

Kingdom Monera, Kingdom Protista, Kingdom Fungi, Kingdom Plantae and Kingdom

Animalia. Kingdom Monera includes subkingdom Archaebacteriobionta (archaebacternia): division Archaebacteriophyta (methane, salt and sulfolobus bacteria); subkingdom

Eubacteriobionta (true bacteria): division Eubacteriophyta; subkingdom Viroids; and subkingdom Viruses. Kingdom Protista includes subkingdom Phycobionta: division

Xanthophyta 275 (yellow-green algae), division Chrysophyta 400 (golden- brown algae), division Dinophyta (Pyrrhophyta) 1,000 (dinoflagellates), division Bacillariophyta 5,500 (diatoms), division Cryptophyta 74 (cryptophytes), division Haptophyta 250 (haptonema organisms), division Euglenophyta 550 (euglenoids), division Chlorophyta, class

Chlorophyceae 10,000 (green algae), class Charophyceae 200 (stoneworts), division

Phaeophyta 900 (brown algae), and division Rhodophyta 2,500 (red algae); subkingdom

Mastigobionta 960: division Chytridiomycota 750 (chytrids), and division Oomycota (water molds) 475; subkingdom Mvxobionta 320: division Acrasiomycota (cellular slime molds) 21, and division Myxomycota 500 (true slime molds). Kingdom Fungi includes division

Zygomycota 570 (coenocytic fungi): subdivision Zygomycotina; and division Eumycota 350 (septate fungi): subdivision Ascomycotina 56,000 (cup fungi), subdivision Basidiomycotina 23 ee

} 25,000 (club fungi), subdivision Deuteromycotina 22,000 (imperfect fungi), and subdivision

Lichenes 13,500. Kingdom Plantae includes division Bryophyta, Hepatophyta,

Anthocerophyta, Psilophyta, Lycophyta, Sphenophyta, Pterophyta, Coniferophyta,

Cycadeophyta, Ginkgophyta, Gnetophyta and Anthophyta. Kingdom Animalia includes:

Porifera (Sponges), Cnidaria (Jellyfishes), Ctenophora (Comb Jellies), Platvhelminthes (Flatworms), Nemertea (Proboscis Worms), Rotifera (Rotifers), Nematoda (Roundworms),

Mollusca (Snails, Clams, Squid & Octopus), Onychophora (Velvet Worms), Annelida (Segmented Worms), Arthropoda (Spiders & Insects), Phoronida, Bryozoa (Bryozoans),

Brachiopoda (Lamp Shells), Echinodermata (Sea Urchins & starfish), and Chordata (Vertebrata-Fish, Birds, Reptiles, Mammals). A preferred donor organism is human. Host organisms are those capable of being infected by an infectious RNA or a virus containing a recombinant viral nucleic acid. Host organisms include organisms from Monera, Protista,

Fungi and Animalia. Preferred host organisms are organisms from Fungi, such as yeast (for example, S. cerevisiae) and Anamalia, such as insects (for example, C. elegans). - To prepare a DNA insert comprising a nucleic acid sequence of a donor organism, the first step is to construct a cDNA library, a genomic DNA library, or a pool of mRNA of h the donor organism. Full-length cDNAs or genomic DNA can be obtained from public or private repositories. For example, cDNA and genomic libraries from bovine, chicken, dog, drosophila, fish, frog, human, mouse, porcine, rabbit, rat, and yeast; and retroviral libraries can be obtained from Clontech (Palo Alto, CA). Alternatively, cDNA library can be prepared from a field sample by methods known to a person of ordinary skill, for example, isolating mRNAs and transcribing mRNAs into cDNAs by reverse transcriptase (see, e.g.

Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold

Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel er al., ed. Greene Publishing and Wiley-Interscience, New York (1987)). Genomic DNAs represented in BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), or TAC (transformation-competent artificial chromosome, Lin et al., Proc. Natl. Acad. Sci.

USA, 96:6535-6540 (1999)) libraries can be obtained from public or private repositories.

Alternatively, a pool of genes, which are overexpressed in a tumor cell line compared with a normal cell line, can be prepared or obtained from public or private repositories.

Zhang et al (Science, 276: 1268-1272 (1997)) report that using a method of serial analysis of gene expression (SAGE) (Velculescu et al, Cell, 88:243 (1997)), 500 transcripts that were expressed at significantly different levels in normal and neoplastic cells were identified. The expression of DNAs that overexpresses in a tumor cell line in a host organism may cause changes in the host organism, thus a pool of such DNAs is another source for DNA inserts for this invention. The BAC/YAC/TAC DNAs, DNAs or cDNAs can be mechanically size-fractionated or digested by an enzyme to smaller fragments. The fragments are ligated to adapters with cohesive ends, and shotgun-cioned into recombinant viral nucleic acid vectors. Alternatively, the fragments can be blunt-end ligated into recombinant viral nucleic acid vectors. Recombinant viral nucleic acids containing a nucleic acid sequence derived from the cDNA library or genomic DNA library is then constructed using conventional techniques. The recombinant viral nucleic acid vectors produced comprise the nucleic acid insert derived from the donor organism. The nucleic acid sequence of the recombinant viral nucleic acid is transcribed as RNA in a host organism; the RNA is capable of regulating the expression of a phenotypic trait by a positive or anti sense mechanism. The nucleic acid sequence may also regulate the expression of more than one phenotypic trait. Nucleic acid sequences from Monera, Protista, Fungi, Plantae and Animalia may be used to assemble the

DNA libraries. This method may thus be used to discover useful dominant gene phenotypes from DNA libraries through the gene expression in a host organism.

In the case of using plant as a donor organism, the donor plant and the host plant may be genetically remote or unrelated: they may belong to different genus, family, order, class, subdivision, or division. Donor plants include plants of commercial interest, such as food crops, seed crops, oil crops, ornamental crops and forestry crops. For example, wheat, rice, corn, potatoes, barley, tobaccos, soybean canola, maize, oilseed rape, Arabidopsis, Nicotiana can be selected as a donor plant.

To prepare a DNA insert comprising a nucleic acid sequence of a donor plant, the first step is typically to construct a library of cDNAs, genomic DNAs, or a pool of RNAs of the plant of interest. Full-length cDNAs can be obtained from public or private repositories, for example, cDNA library of Arabidopsis thaliana can be obtained from the Arabidopsis

Biological Resource Center. Alternatively, cDNA library can be prepared from a field sample by methods known to a person of ordinary skill, for example, isolating mRNAs and transcribing mRNAs into cDNAs by reverse transcriptase (see, e.g., Sambrook et al,

Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor

Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed.

Greene Publishing and Wiley-Interscience, New York (1987)). Genomic DNAs represented in BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), or TAC (transformation-competent artificial chromosome, Liu et al., Proc. Natl. Acad. Sci. USA, 96:6535-6540 (1999)) libraries can be obtained from public or private repositories, for example, the Arabidopsis Biological Resource Center. The BAC/YAC/TAC DNAs or cDNAs can be mechanically size-fractionated or digested by an enzyme to smaller fragments. The fragments are ligated to adapters with cohesive ends, and shotgun-cloned into recombinant viral nucleic acid vectors. Alternatively, the fragments can be blunt-end ligated into recombinant viral nucleic acid vectors. Recombinant plant viral nucleic acids containing a nucleic acid sequence derived from the cDNA library or genomic DNA library is then constructed using conventional techniques. The recombinant viral nucleic acid vectors produced comprise the nucleic acid insert derived from the donor plant. The nucleic - acid sequence of the recombinant viral nucleic acid is transcribed as RNA in a host plant; the

RNA is capable of regulating the expression of a phenotypic trait by a positive or anti sense : mechanism. The nucleic acid sequence may also code for the expression of more than one phenotypic trait. Sequences from wheat, rice, corn, potato, barley, tobacco, soybean, canola, maize, oilseed rape, Arabidopsis, and other crop species may be used to assemble the DNA libraries. This method may thus be used to search for useful dominant gene phenotypes from DNA libraries through the gene expression.

Those skilled in the art will understand that these embodiments are representative only of many constructs suitable for the instant invention. All such constructs are contemplated and intended to be within the scope of the present invention. The invention is not intended to be limited to any particular viral constructs but specifically contemplates using all operable constructs. A person skilled in the art will be able to construct the plant viral nucleic acids based on molecular biology techniques well known in the art. Suitable techniques have been descnbed in Sambrook er al. (2nd ed.), Cold Spring Harbor

Laboratory, Cold Spring Harbor (1989); Methods in Enzymol. (Vols. 68, 100, 101, 118, and 152-155) (1979, 1983, 1986 and 1987); and DNA Cloning, D.M. Clover, Ed., IRL Press,

Oxford (1985); Walkey, Applied Plant Virology, Chapman & Hall (1991); Matthews, Plant

Virology, 3™ Ed., Academic Press, San Diego (1991); Turpen et al., J. of Virological

Methods, 42:227-240 (1993); U.S. Patent Nos. 4,885,248, 5,173,410, 5,316,931, 5,466,788, 5,491,076, 5,500,360, 5,589,367, 5,602,242, 5,627,060, 5,811,653, 5,866,785, 5,889,190, and 5,589,367, U.S. Patent No. 5,977,438. Nucleic acid manipulations and enzyme treatments are carried out in accordance with manufacturers’ recommended procedures in making such constructs.

II. Expressing members of donor organism derived sequence inserts in plant hosts . Plant hosts include plants of commercial interest, such as food crops, seed crops, oil crops, ornamental crops and forestry crops. For example, wheat, rice, corn, potatoes, barley,” tobaccos, soybean canola, maize, oilseed rape, Arabidopsis, Nicotiana can be selected as a host plant. In particular, host plants capable of being infected by a virus containing a recombinant viral nucleic acid are preferred. Preferred host plants include Nicotiana, preferably, Nicotiana benthamiana, or Nicotiana cleaviandii, :

Individual clones may be transfect into the plant host: 1) protoplasts; 2) whole ~ plants; or 3) plant tissues, such as leaves of plants (Dijkstra er al., Practical Plant Virology:

Protocols and Exercises, Springer Verlag (1998); Plant Virology Protocol: From Virus

Isolation to Transgenic Resistance in Methods in Molecular Biology, Vol. 81, Foster and

Taylor, Ed., Humana Press (1998)). In some embodiments of the instant invention, the delivery of the plant virus expression vectors into the plant may be affected by the inoculation of in vitro transcribed RNA, inoculation of virions, or internal inoculation of plant cells from nuclear cDNA, or the systemic infection resulting from any of these procedures. In all cases, the co-infection may lead to a rapid and pervasive systemic expression of the desired nucleic acid sequences in plant cells.

The host can be infected with a recombinant viral nucleic acid or a recombinant plant virus by conventional techniques. Suitable techniques include, but are not limited to, leaf abrasion, abrasion in solution, high velocity water spray, and other injury of a host as well as imbibing host seeds with water containing the recombinant viral RNA or recombinant plant virus. More specifically, suitable techniques include: (a) Hand Inoculations. Hand inoculations are performed using a neutral pH, low molanty phosphate buffer, with the addition of celite or carborundum (usually about 27

Amended Sheet — 2003-06-24

1%). One to four drops of the preparation is put onto the upper surface of a leaf and gently rubbed. (b) Mechanized Inoculations of Plant Beds. Plant bed inoculations are performed by spraying (gas-propelled) the vector solution into a tractor-driven mower while cutting the leaves. Alternatively, the plant bed is mowed and the vector solution sprayed immediately onto the cut leaves. (c) High Pressure Spray of Single Leaves. Single plant inoculations can also be performed by spraying the leaves with a narrow, directed spray (50 psi, 6-12 inches from the leaf) containing approximately 1% carborundum in the buffered vector solution. (d) Vacuum Infiltration. Inoculations may be accomplished by subjecting a host organism to a substantially vacuum pressure environment in order to facilitate infection. (e) High Speed Robotics Inoculation. Especially applicable when the organism is a plant, individual organisms may be grown in mass array such as in microtiter plates. Machinery such as robotics may then be used to transfer the nucleic acid of interest. fH) Ballistics (High Pressure Gun) Inoculation. Single plant inoculations can also be performed by particle bombardment. A ballistics particle delivery system (BioRad Laboratories, Hercules, (A) can be used to transfect plants such as N. benthamiana as described previously (Nagar ez al., Plant Cell, 7:705-719 (1995)).

An alternative method for introducing viral nucleic acids into a plant host is a technique known as agroinfection or Agrobacterium-mediated transformation (also known as Agro-infection) as described by Grimsley et al., Nature 325:177 (1987). This techmque makes use of a common feature of Agrobacterium which colonizes plants by transfernng a portion of their DNA (the T-DNA) into a host cell, where it becomes integrated into nuclear

DNA. The T-DNA is defined by border sequences which are 25 base pairs long, and any

DNA between these border sequences is transferred to the plant cells as well. The insertion of a recombinant plant viral nucleic acid between the T-DNA border sequences results in transfer of the recombinant plant viral nucleic acid to the plant cells, where the recombinant plant viral nucleic acid is replicated, and then spreads systemically through the plant. Agro- infection has been accomplished with potato spindle tuber viroid (PSTV) (Gardner et al.,

Plant Mol. Biol. 6:221 (1986); CaV (Grimsley et al., Proc. Natl. Acad. Sci. USA 83.3282 (1986); MSV (Grimsley et al., Nature 325:177 (1987)), and Lazarowitz, S., Nucl. Acids

Res. 16:229 (1988)) digitaria streak virus (Donson et al., Virology 162:248 (1988)), wheat dwarf virus (Hayes et al., J. Gen. Virol. 69:891 (1988)) and tomato golden mosaic virus (TGMV) (Elmer et al., Plant Mol. Biol. 10:225 (1988) and Gardiner et al., EMBO J. 7:899 (1988)). Therefore, agro-infection of a susceptible plant could be accomplished with a virion containing a recombinant plant viral nucleic acid based on the nucleotide sequence of any of the above viruses. Particle bombardment or electrosporation or any other methods known in the art may also be used.

In some embodiments of the instant invention, infection may also be attained by placing a selected nucleic acid sequence into an organism such as E. coli, or yeast, either integrated into the genome of such organism or not, and then applying the organism to the surface of the host organism. Such a mechanism may thereby produce secondary transfer of the selected nucleic acid sequence into a host organism. This is a particularly practical embodiment when the host organism is a plant. Likewise, infection may be attained by first packaging a selected nucleic acid sequence in a pseudovirus. Such a method is described in

WO 94/10329. Though the teachings of this reference may be specific for bacteria, those of skill in the art will readily appreciate that the same procedures could easily be adapted to other organisms.

Plant may be grown from seed in a mixture of “Peat-Lite Mix™ (Speedling, Inc. Sun

City, FI) and Nutricote™ controlled release fertilizer 14-14-14 (Chiss-Asahi Fertilizer Co.,

Tokyo, Japan). Plants may be grown in a controlled environment provided 16 hours of light and 8 hours of darkness. Sylvania “Gro-Lux/Aquarium’ wide spectrum 40 watt fluorescent grow lights. (Osram Sylvania Products, Inc. Danvers, MA) may be used. Temperatures may be kept at around 80° F during light hours and 70° F during dark hours. Humidity may be between 60 and 85%.

IOI. Detecting phenotypic or biochemical changes as a result of expression.

After a plant host is infected with individual clone of the library, one or more phenotypic or biochemical changes may be detected.

The phenotypic changes in a plant host may be determined by any known methods in the art. Phenotypic changes may include growth rate, color, or morphology changes.

Typically, these methods include visual, macroscopic or microscopic analysis. For example, growth changes, such as stunting, color changes (e.g. leaf yellowing, mottling, bleaching, chlorosis) among others are easily visualized. Examples of morphological changes include, developmental defects, wilting, necrosis, among others.

Biochemical changes can be determined by any analytical methods known in the art for detecting, quantitating, or isolating DNA, RNA, proteins, antibodies, carbohydrates, lipids, and small molecules. Selected methods may include Northern, Western blotting,

MALDI-TOF, LC/MS, GC/MS, two-dimensional IEF/SDS-PAGE, ELISA, etc. In particular, suitable methods may be performed in a high-throughput, fully automated fashion using robotics. Examples of biochemical changes may include the accumulation of substrates or products from enzymatic reactions, changes in biochemical pathways, inhibition or augmentation of endogenous gene expression in the cytoplasm of cells, changes in the RNA or protein profile. For example, the clones in the viral vector library may be functionally classified based on metabolic pathway affected or visual/selectable phenotype : produced in the organism. This process enables a rapid determination of gene function for unknown nucleic acid sequences of a donor organism as well as a host organism.

Furthermore, this process can be used to rapidly confirm function of full-length DNA’s of unknown function. Functional identification of unknown nucleic acid sequences in a library of one organism may then rapidly lead to identification of similar unknown sequences in expression libraries for other organisms based on sequence homology. Such information is useful in many aspects including in human medicine.

The biochemical or phenotypic changes in the infected host plant may be correlated to the biochemistry or phenotype of a host piant that is uninfected. Optionally, the biochemical or phenotypic changes in the infected host plant is further correlated to a host plant that is infected with a viral vector that contains a control nucleic acid of a known sequence. The control nucleic acid may have similar size but is different in sequence from the nucleic acid insert derived from the library. For example, if the nucleic acid insert derived from the library is identified as encoding a GTP binding protein in an antisense orientation, a nucleic acid derived from a gene encoding green fluorescent protein can be used as a control nucleic acid. Green fluorescent protein is known not to have the same effect as the GTP binding protein when expressed in a host plant.

In some embodiments, the phenotypic or biochemical trait may be determined by complementation analysis, that is, by observing the endogenous gene or genes whose function is replaced or augmented by introducing the nucleic acid of interest. A discussion of such phenomenon is provided by Napoli et al., The Plant Cell 2:279-289 (1990). The phenotypic or biochemical trait may also be determined by (1)analyzing the biochemical alterations in the accumulation of substrates or products from enzymatic reactions according to any means known by those skilled in the art; (2) by observing any changes in biochemical pathways which may be modified in a host organism as a result of expression of the nucleic acid; (3) by utilizing techniques known by those skilled in the art to observe inhibition of endogenous gene expression in the cytoplasm of cells as a result of expression of the nucleic acid.; (4) by utilizing techniques known by those skilled in the art to observe changes in the RNA or protein profile as a result of expression of the nucleic acid; or (5) by selection of organisms capable of growing or maintaining viability in the presence of noxious or toxic substances, such as, for example, pharmaceutical ingredients.

One useful means to determine the function of nucleic acids transfected into a host plant is to observe the effects of gene silencing. Traditionally, functional gene knockout has been achieved following inactivation due to insertion of transposable elements or random integration of T-DNA into the chromosome, followed by characterization of conditional, homozygous-recessive mutants obtained upon backcrossing. Some teachings in these regards are provided by WO 97/42210 which is herein incorporated by reference. As an alternative to traditional knockout analysis, an EST/DNA library from a donor organism, may be assembled into a viral transcription plasmid. The nucleic acid sequences in the transcription plasmid library may then be introduced into host cells as part of a functional

RNA virus which post-transcriptionally silences the homologous target gene. The

EST/DNA sequences may be introduced into a viral vector in either the plus or anti sense orientation, and the orientation can be either directed or random based on the cloning strategy. A high-throughput, automated cloning scheme based on robotics may be used to assemble and characterize the library. Alternatively, the EST/cDNA sequences can be inserted into the genomic RNA of a viral vector such that they are represented as genomic

RNA during the viral replication in host cells. The library of EST clones is then transcribed into infectious RNAs and inoculated onto a host organism susceptible to viral infection. The viral RNAs containing the EST/cDNA sequences contributed from the original library are now present in a sufficiently high concentration in the cytoplasm of host organism cells such that they cause post-transcriptional gene silencing of the endogenous gene in a host organism. Since the replication mechanism of the virus produces both sense and antisense

RNA sequences, the orientation of the EST/cDNA insert is normally irrelevant in terms of producing the desired phenotype in the host organism.

The present invention provides a method to express transiently viral-derived positive sense or antisense RNAs in transfected plants. Such method is much faster than the time required to obtain genetically engineered antisense transgenic organisms. Systemic infection and expression of viral antisense RNA occurs as short as several days post inoculation, whereas it takes several months or longer to create a single transgenic organism. The invention provides a method to identify genes invoived in the regulation of growth by inhibiting the expression of specific endogenous genes using viral vectors. This invention provides a method to characterize specific genes and biochemical pathways in donor organisms or in host plants using an RNA viral vector.

It is known that silencing of endogenous genes can be achieved with homologous sequences from the same plant family. For example, Kumagai et al., (Proc. Natl. Acad. Sci.

USA 92:1679 (1995)) report that the Nicotiana benthamiana gene for phytoene desaturase (PDS) was silenced by transfection with a viral RNA derived from a clone containing a partial tomato (Lycopersicon esculentum) cDNA encoding PDS being in an antisense orientation. Kumagai er al. demonstrate that gene encoding PDS from one plant can be silenced by transfecting a host plant with a nucleic acid of a known sequence, namely, a

PDS gene, from a donor plant of the same family. The present invention provides a method of silencing a gene in a host organism by transfecting a hon-plant host organism with a viral nucleic acid comprising a nucleic acid insert derived from a cDNA library or a genomic

DNA library or a pool of RNA from a non-plant organism. Different from Kumagai er al, the sequence of the nucleic acid insert in the present invention does not need to be identified prior to the transfection. Another feature of the present invention is that it provides a method to silence a conserved gene of a nonplant kingdom; the antisense transcript of an organism results in reducing expression of the endogenous gene of a host organism from

Monera, Protista, Fungi and Animalia. The invention is exemplified by GTP binding proteins. In eukaryotic cells, GTP-binding proteins function in a variety of cellular processes, including signal transduction, cytoskeletal organization, and protein transport.

Low molecular weight (20-25 K Daltons) of GTP-binding proteins include ras and its close relatives (for example, Ran), rho and its close relatives, the rab family, and the ADP- ribosylation factor (ARF) family. The heterotrimeric and monomeric GTP-binding proteins that may be involved in secretion and intracellular transport are divided into two structural classes: the rab and the ARF families. The ARFs from many organisms have been isolated and characterized. The ARFs share structural features with both the ras and trimeric GTP- binding protein families. The present invention demonstrates that genes of one plant, such as Nicotiana, which encode GTP binding proteins, can be silenced by transfection with infectious RNAs from a clone containing GTP binding protein open reading frame in an antisense orientation, derived from a plant of a different family, such as Arabidopsis. The present invention also demonstrates that GTP binding proteins are highly homologous in human, frog, mouse, bovine, fly and yeast, not only at the amino acid level, but also at the nucleic acid level. The present invention thus provides a method to silence a conserved gene in a host organism, by transfecting the host with infectious RNAs derived from a homologous gene of a non-plant organism.

Nucleic acid sequences that may result in changing a host phenotype include those involved in cell growth, proliferation, differentiation and development; cell communication; and the apoptotic pathway. Genes regulating growth of cells or organisms include, for example, genes encoding a GTP binding protein, a ribosomal protein L19 protein, an S18 ribosomal protein, etc. Henry et al. (Cancer Res., 53:1403-1408 (1993)) report that erb B-2 (or HER-2 or neu) gene was amplified and overexpressed in one-third of cancers of the breast, stomach, and ovary; and the mRNA encoding the ribosomal protein L19 was more abundant in breast cancer samples that express high levels of erbB-2. Lijsebettens et al. (EMBO J., 13:3378-3388 (1994)) report that in Arabidopsis, mutation at PFL caused pointed first leaves, reduced fresh weight and growth retardation. PFL codes for ribosomal protein

S18, which has a high homology with the rat S18 protein. Genes involved in development of cells or organisms include, for example, homeobox-containing genes and genes encoding

G-protein-coupled receptor proteins such as the rhodopsin family. Homeobox genes are a family of regulatory genes containing a common 183-nucleotide sequence (homeobox) and coding for specific nuclear proteins (homeoproteins) that act as transcription factors. The homeobox sequence itself encodes a 61-amino-acid domain, the homeodomain, responsible for recognition and binding of sequence-specific DNA motifs. The specificity of this binding allows homeoproteins to activate or repress the expression of batteries of down- stream target genes. Initially identified in genes controlling Drosophila development, the homeobox has subsequently been isolated in evolutionarily distant animal species, plants, and fungi. Several indications suggest the involvement of homeobox genes in the control of cell growth and, when dysregulated, in oncogenesis (Cillo et al., Exp. Cell Res., 248:1-9 (1999). Other nucleic acid sequences that may result in changes of an organism include genes encoding receptor proteins such as hormone receptors, CAMP receptors, serotonin 2 receptors, and calcitonin family of receptors; and light-regulated DNA encoding a leucine (Leu) zipper motif (Zheng er al., Plant Physiol., 116:27-35 (1998)). Deregulation or * alteration of the process of cell growth, proliferation, differentiation and development; cell communication; and the apoptotic pathways may result in cancer. Therefore, identifying the : nucleic acid sequences involved in those processes and determining their functions are beneficial to the human medicine; it also provides a tool for cancer research.

A Library of human nucleic acid sequences is cloned into vectors. The vectors are applied to the host to obtain infection. Each infected host is grown with an uninfected host and a host infected with a null vector. A null vector will show no phenotypic or biochemical change other than the effects of the virus itself. Each host is observed daily for visual differences between the infected host and its two controls. In each host displaying an observable phenotypic or biochemical change a trait is identified. The donor nucleic acid sequence is identified, the full-length gene sequence 1s obtained and the full-length gene in the host is obtained, if a gene from the host is associated with the trait. Both genes are sequenced and homology is determined. A variety of biochemical tests may also be made on the host or host tissue depending on the information that is desired. A variety of phenotypic changes or traits and biochemical tests are set forth in this document. A - : functional gene profile can be obtained by repeating the process several times. :

Large amounts of DNA sequence information are being generated in the public domain, which may be entered into a relational database. Links may be made between sequences from various species predicted to carry out similar biochemical or regulatory functions. Links may also be generated between predicted enzymatic activities and visually displayed biochemical and regulatory pathways. Likewise, links may be generated between predicted enzymatic or regulatory activity and known small molecule inhibitors, activators, substrates or substrate analogs. Phenotypic data from expression libraries expressed in transfected hosts may be automatically linked within such a relational database. Genes with™ similar predicted roles of interest in other organisms may be rapidly discovered.

The present invention is also directed to a2 method of changing the phenotype or biochemistry of a plant by expressing transiently a nucleic acid sequence from a donor plant

In an antisense orientation in a host plant, which inhibits an endogenous gene expression in the menstem of the host plant. The one or more phenotypic or biochemical changes in the host plant are detected by methods as describes previously. Transient expressing a nucleic acid sequence in a host plant can affect the gene expression in meristem. Meristems are of

Interest in plant development because plant growth is driven by the formation and activity of mernisterns throughout the entire life cycle. This invention is exemplified by a nucleic acid sequence encoding ribosomal protein S18. The activity of S18 promoter is restricted to meristems (Lijsebettesn er al., EMBO J. 13: 3378-3388). Transient expression of a nucleic acid sequence in a host plant can trigger a signal transmitting to meristems and affect the gene expression in meristem.

One problem with gene silencing in a plant host is that many plant genes exist in multigene families. Therefore, effective silencing of a gene function may be especially problematic. According to the present invention, however, nucleic acids may be inserted into the viral genome to effectively silence a particular gene function or to silence the : function of a multigene family. It is presently believed that about 20% of plant genes exist : in multigene families.

A detailed discussion of some aspects of the “gene silencing’ effect is provided in the co-pending patent application, U.S. Patent No. 5,922,602

Amended Sheet — 2003-06-24

(W095/34668 published 12/21/95) the disclosure of which is incorporated herein by reference. RNA can reduce the expression of a target gene through inhibitory RNA interactions with target mRNA that occur in the cytoplasm and/or the nucleus of a cell.

An EST/cDNA library from a plant such as Arabidopsis thaliana may be assembled into a plant viral transcription plasmid background. The cDNA sequences in the transcription plasmid library can then be introduced into plant cells as cytoplasmic RNA in order to post-transcriptionally silence the endogenous genes. The EST/cDNA sequences may be introduced into the plant viral transcription plasmid in either the plus or anti-sense orientation (or both), and the orientation can be either directed or random based on the cloning strategy. A high-throughput, automated cloning strategy using robotics can be used to assemble the library. The EST clones can be inserted behind a duplicated subgenomic promoter such that they are represented as subgenomic transcripts during viral replication in plant cells. Alternatively, the EST/cDNA sequences can be inserted into the genomic RNA of a plant viral vector such that they are represented as genomic RNA during the viral replication in plant cells. The library of EST clones is then transcribed into infectious RNAs and inoculated onto a host plant susceptible to viral infection. The viral RNAs containing the EST/cDNA sequences contributed from the original library are now present in a sufficiently high concentration in the cytoplasm of host plant cells such that they cause post- transcriptional gene silencing of the endogenous gene in a host plant. Since the replication mechanism of the virus produces both sense and antisense RNA sequences, the orientation of the EST/cDNA insert is normally irrelevant in terms of producing the desired phenotype in the host plant.

The present invention also provides a method of isolating a conserved gene such as a gene encoding a GTP binding protein, from rice, barley, com, soybean, maize, oilseed. and other plant of commercial interest, using another gene having homology with gene being isolated. Libraries containing full-length cDNAs from a donor plant such as rice, barley, corn, soybean and other important crops can be obtained from public and private sources or can be prepared from plant mRNAs. The cDNAs are inserted in viral vectors or in small subcloning vectors such as pBluescript (Strategene), pUC18, M13, or pBR322.

Transformed bacteria are then plated and individual clones selected by a standard method.

The bacteria transformants or DNAs are rearrayed at high density onto membrane filters or glass slides. Full-length cDNAs encoding GTP binding proteins can be identified by probing filters or slides with labeled nucleic acid inserts which result in changes in a host plant, or labeled probes prepared from DNAs encoding GTP binding proteins from

Arabidopsis. Useful labels include radioactive, fluorescent, or chemiluminecent molecules, enzymes, etc.

Alternatively, genomic libraries containing sequences from rice, barley, com, soybean and other important crops can be obtained from public and private sources, or be prepared from plant genomic DNAs. BAC clones containing entire plant genomes have been constructed and organized in a minimal overlapping order. Individual BACs are sheared to fragments and directly cloned into viral vectors. Clones that completely cover an entire BAC form a BAC viral vector sublibrary. Genomic clones can be identified by probing filters containing BACs with labeled nucleic acid inserts which result in changes in a host plant, or with labeled probes prepared from DNAs encoding GTP binding proteins from Arabidopsis. Useful labels include radioactive, fluorescent, or chemiluminecent molecules, enzymes, etc. BACs that hybndize to the probe are selected and their corresponding BAC viral vectors are used to produce infectious

RNAs. Plants that are transfected with the BAC sublibrary are screened for change of function, for example, change of growth rate or change of color. Once the change of function is observed, the inserts from these clones or their corresponding plasmid DNAs are characterized by dideoxy sequencing. This provides a rapid method to obtain the genomic sequence for a plant protein, for example, a GTP binding protein. Using this method, once the DNA sequence in one plant such as Arabidopsis thaliana is identified, it can be used to identify conserved sequences of similar function that exist in other plant libranes.

A functional genomics screen is set up using a tobacco mosaic virus TMV-0 coat protein capsid for infection of Nicotiana benthamiana. a plant related to the common tobacco plant. For Arabidopsis thaliana cDNA libraries are obtained from the Arabidopsis

Biological Resource Center, Bluescript® phagemid vectors are recovered by Not 1 digestion. cDNA 1s transformed into a plasmid. The plasmid is transcribed into viral vector

RNA. The inserts are in the antisense orientation as in Figure unti all of the cDNA from each cDNA library is represented on viral vectors. Each viral vector is sprayed onto the leaf of a two-week old N. benthamiana plant host with sufficient force to cause tissue injury and localized viral infection. Each infected plant is grown side by side with an uninfected plant and a plant infected with a null insert vector as controls. All plants are grown in an artificial environment having 16 hours of light and 8 hours of dark. Lumens are approximately equal on each plant. At intervals of 2 days a visual and photographic observation of phenotype is made and recorded for each infected plant and each of its controls and a comparison is made.

Data is entered into a Laboratory Information Management System database. At the end of the observation period stunted plants are grouped for analysis. The nucleic acid insert contained in the viral vector cione 740AT#120 is responsible for severe stunting of one of the plants. Clone 740AT #120 is sequenced. The homologue from the plant host 1s also sequenced. The 740AT #120 clone is found to have 80% homology to plant host nucleic acid sequence. The amino acid sequence of homology is 96%. The entire cDNA sequence of the insert is obtained by sequencing and found to code for a GTP binding protein. The host plant homologue is selected and sequenced. It also codes for a GTP binding protein.

We conclude that this GTP binding protein coding sequence is highly conserved in nature.

This information is useful in pharmaceutical development as well as in toxicology studies. ’ A complete classification scheme of gene functionality for a fully sequenced k eukaryotic organism has been established for yeast. This classification scheme may be modified for plants and divided into the appropriate categories. Such organizational structure may be utilized to rapidly identify herbicide target loci which may confer dominant : lethal phenotypes, and thereby is useful in helping to design rational herbicide programs.

The present invention is also directed to a method of increasing yield of a grain crop.

In Rice Biotechnology Quarterly 37:4 (1999) and Ashikari er al., Proc. Natl. Acad. Sci. USA 96:10284-10289 (1999), it is reported that a transgenic rice plant transformed with a rgp/ gene, which encodes a small GTP binding protein from rice, was shorter than a control plant, but it produced more seeds than the control plant. To increase the yield of a grain crop, the present method comprises expressing transiently a nucleic acid sequence of a donor plant in an antisense orientation in the grain crop, wherein said expressing results in stunted growth and increased seed production of said grain crop. A preferred method compnses the steps of cloning the nucleic acid sequence into a plant viral vector and infecting the grain crop with a recombinant viral nucleic acid comprising said nucleic acid sequence. Preferred plant viral vector is derived from a Brome Mosaic virus, a Rice Necrosis virus, or a geminivirus.

Preferred grain crops include rice, wheat, and barley. The nucleic acid expressed in the host plant, for example, comprises a GTP binding protein open reading frame having an antisense orientation. The present method provides a transiently expression of a gene to obtain a stunted plant. Because less energy is put into plant growth, more energy is available for production of seed, which results in increase yield of a grain crop. The present method has an advantage over other method using a transgenic plant, because it does not have an effect on the genome of a host plant.

In order to provide an even clearer and more consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided:

Adjacent: 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.

Anti-Sense Inhibition: 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 RNA molecules may be from either an RNA virus or mRNA from the host cells genome or from a DNA virus.

Cell Culture: A proliferating group of cells which may be in either an undifferentiated or differentiated state, growing contiguously or non-contiguously.

Chimeric Sequence or Gene: A nucleotide sequence derived from at least two heterologous parts. The sequence may comprise DNA or RNA.

Coding Sequence: A deoxyribonucleotide or nbonucleotide sequence which, when either transcribed and translated or simply translated, results in the formation of a cellular polypeptide or a ribonucleotide sequence which, when translated, results in the formation of a cellular polypeptide.

Compatible: The capability of operating with other components of a system. A vector or plant or animal 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.

Complementation Analysis: As used herein, this term 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 39 . _ a fully in its normal role. A complementary gene to the deleted or mutated gene can restore the genetic phenotype of the selected gene.

Dual Heterologous Subgenomic Promoter Expression System (DHSPES): a plus stranded RNA vector having a dual heterologous subgenomic promoter expression system to increase, decrease, or change the expression of proteins, peptides or RNAs, preferably those described in U.S. Patent Nos. 5,316,931, 5,811,653, 5,589,367, and 5,866,785, the disclosure of which is incorporated herein by reference.

Expressed sequence tags (ESTs): 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.

Expression: The term as used herein is meant to incorporate one or more of transcription, reverse transcription and translation.

A functional Gene Profile: The collection of genes of an organism which code for a : biochemical or phenotypic trait. The functional gene profile of an organism is found by screening nucleic acid sequences from a donor organism by over expression or suppression of a gene in a host organism. A functional gene profile requires a collection or library of nucleic acid sequences from a donor organism. A functional gene profile will depend on the ability of the collection or library of donor nucleic acids to cause over-expression or suppression in the host organism. Therefore, a functional gene profile will depend upon the quantity of donor genes capable of causing over-expression or suppression of host genes or of being expressed in the host organism in the absence of a homologous host gene.

Gene: A discrete nucleic acid sequence responsible for producing one or more cellular products and/or performing one or more intercellular or intracellular functions.

Gene silencing: A reduction in gene expression. A viral vector expressing gene sequences from a host may induce gene silencing of homologous gene sequences.

Homology: A degree of nucleic acid similarity in all or some portions of a gene sequence sufficient to result in gene suppression when the nucleic acid sequence 1s delivered in the antisense orentation.

Host: A cell, tissue or organism capable of replicating a nucleic acid such as a vector or viral nucleic acid and which is capable of being infected by a virus containing the viral vector or viral nucleic acid. This term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms, where appropriate. Bacteria, fungi, yeast, and animal (cell, tissues, or organisms), are examples of a host.

Infection: 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 10 integrate into a genome, chromosome or gene of a target organism.

Insert: a stretch of nucleic acid seqeunce, typically more than 20 base pairs long.

Multigene family: 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, actions, tubulins, keratins, collagens, heat shock proteins, salivary glue proteins, chorion proteins, cuticle proteins, yolk proteins, and phaseolins.

Non-Native: Any RNA or DNA sequence that does not normally occur in the cell or organism in which it is placed. Examples include recombinant viral nucleic acids and genes or ESTs contained therein. That is, an RNA or DNA sequence may be non-native with respect to a viral nucleic acid. Such an RNA or DNA sequence would not naturally occur in the viral nucleic acid. Also, an RNA or DNA sequence may be non-native with respect to a host organism. That is, such a RNA or DNA sequence would not naturaily occur in the host organism.

Nucleic acid: As used herein the term is meant to include any DNA or RNA sequence from the size of one or more nucleotides up to and including a complete gene sequence. 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.

Nucleic acid of interest: The term is intended to refer to the nucleic acid sequence whose function is to be determined. The sequence will normally be non-native to a viral vector but may be native or non-native to a host organism.

a @

Phenotypic Trait: An observable, measurable or detectable property resulting from the expression or suppression of a gene or genes.

Plant Cell: The structural and physiological unit of plants, consisting of a protoplast and the cell wall.

Plant Organ: A distinct and visibly differentiated part of a plant, such as root, stem, leaf or embryo.

Plant Tissue: Any tissue of a plant in plant 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.

Positive-sense inhibition: 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.

Promoter: The 5’-flanking, non-coding sequence substantially adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.

Protoplast: An isolated plant or bactenal cell without some or all of its cell wall.

Recombinant Viral Nucleic Acid: 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 viral nucleic acid is to be introduced.

Recombinant Virus: A virus containing the recombinant viral nucleic acid.

Subgenomic Promoter: A promoter of a subgenomic mRNA of a viral nucleic acid.

Substantial Sequence Homology: Denotes nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology are insignificant in affecting function of the gene products or an RNA coded for by such sequence.

Systemic Infection: 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.

Transient Expression: Expression of a nucleic acid sequence in a host without insertion of the nucleic acid sequence into the host genome, such as by way of a viral vector.

Transposon: 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. :

EXAMPLES

The following examples further illustrate the present invention. These examples are intended merely to be iliustrative of the present invention and are not to be construed as being hmiting.

EXAMPLE 1

Arabidopsis thaliana cDNA library construction in a dual subgenomic promoter vector. :

Arabidopsis thaliana cDNA libraries obtained from the Arabidopsis Biological

Resource Center (ABRC). The four libraries from ABRC were size-fractionated with inserts of 0.5-1 kb (CD4-13), 1-2 kb (CD4-14), 2-3 kb (CD4-15), and 3-6 kb (CD4-16). All libraries are of high quality and have been used by several dozen groups to isolate genes.

The pBluescript® phagemids from the Lambda ZAP II vector were subjected to mass excision and the libraries were recovered as plasmids according to standard procedures.

Alternatively, the cDNA inserts in the CD4-13 (Lambda ZAP II vector) were recovered by digestion with NotI. Digestion with NotI in most cases liberated the entire

Arabidopsis thaliana cDNA insert because the original library was assembled with Not] adapters. No is an 8-base cutter that infrequently cleaves plant DNA. In order to insert the

Notl fragments into a transcription plasmid, the pBS735 transcription plasmid (FIGURE 1) was digested with Pacl/Xhol and ligated to an adapter DNA sequence created from the oligonucleotides 5’-TCGAGCGGCCGCAT-3" (SEQ ID NO: 44) and 5° -GCGGCCGC-3°.

The resulting plasmid pBS740 (FIGURE 2) contains a unique Nol restriction site for bi- directional insertion of Notl fragments from the CD4-13 library. Recovered colonies were prepared from these for plasmid minipreps with a Qiagen BioRobot 9600®. The plasmid

DNA preps performed on the BioRobot 9600® were done in 96-wel] format and yield transcription quality DNA. An Arabidopsis cDNA library was transformed into the plasmid and analyzed by agarose gel electrophoresis to identify clones with inserts. Clones with inserts were transcribed in vitro and inoculated onto N. benthamiana or Arabidopsis 43

Amended Sheet — 2003-06-24 thaliana. Selected leaf disks from transfected plants were then taken for biochemical analysis.

EXAMPLE 2

Genomic DNA library construction in a recombinant viral nucleic acid vector.

Genomic DNAs represented in BAC (bactenal artificial chromosome) or YAC (yeast artificial chromosome) libraries are obtained from the Arabidopsis Biological

Resource Center (ABRC). The BAC/YAC DNAs are mechanically size-fractionated, ligated to adapters with cohesive ends, and shotgun-cloned into recombinant viral nucleic acid vectors. Alternatively, mechanically size-fractionated genomic DNAs are blunt-end ligated into a recombinant viral nucleic acid vector. Recovered colonies are prepared for plasmid minipreps with a Qiagen BioRobot 9600®. The plasmid DNA preps done on the

BioRobot 9600® are assembled in 96-well format and yield transcription quality DNA. The recombinant viral nucleic acid/Arabidopsis genomic DNA library is analyzed by agarose gel electrophoresis (template quality control step) to identify clones with inserts. Clones with inserts are then transcribed in vitro and inoculated onto N. benthamiana and/or Arabidopsis thaliana. Selected leaf disks from transfected plants are then be taken for biochemical analysis.

Genomic DNA from Arabidopsis typically contains a gene every 2.5 kb (kilobases) on average. Genomic DNA fragments of 0.5 to 2.5 kb obtained by random shearing of

DNA were shotgun assembled in a recombinant viral nucleic acid expression/knockout vector library. Given a genome size of Arabidopsis of approximately 120,000 kb, a random recombinant viral nucleic acid genomic DNA library would need to contain minimally 48,000 independent inserts of 2.5 kb in size to achieve 1X coverage of the Arabidopsis genome. Alternatively, a random recombinant viral nucleic acid genomic DNA library would need to contain minimally 240,000 independent inserts of 0.5 kb in size to achieve 1X coverage of the Arabidopsis genome. Assembling recombinant viral nucleic acid expression/knockout vector libraries from genomic DNA rather than cDNA has the potential to overcome known difficulties encountered when attempting to clone rare, low-abundance mRNA's in a cDNA library. A recombinant viral nucleic acid expression/knockout vector library made with genomic DNA would be especially useful as a gene silencing knockout library. In addition, the Dual Heterologous Subgenomic Promoter Expression System (DHSPES) expression/knockout vector library made with genomic DNA would be especially useful for expression of genes lacking introns. Furthermore, other plant species with moderate to small genomes (e.g. rose, approximately 80,000 kb) would be especially useful for recombinant viral nucleic acid expression/knockout vector libraries made with genomic DNA. A recombinant viral nucleic acid expression/knockout vector library can be made from existing BAC/YAC genomic DNA or from newly-prepared genomic DNAs for any plant species.

EXAMPLE 3

Genomic DNA or cDNA library construction in a DHSPES vector, and transfection of individual clones from said vector library onto T-DNA tagged or transposon tagged or mutated plants. }

Genomic DNA or cDNA library construction in a recombinant viral nucleic acid vector, and transfection of individual clones from the vector library onto T-DNA tagged or transposon tagged or mutated plants may be performed according to the procedure set forth in Examples 1 and 2. Such a protocol may be easily designed to complement mutations introduced by random insertional mutagenesis of T-DNA sequences or transposon sequences.

EXAMPLE 4

Construction of a Nicotiana benthamiana cDNA library.

Vegetative N. benthamiana plants were harvested 3.3 weeks after sowing and sliced up into three groups of tissue: leaves, stems and roots. Each group of tissue was flash frozen in liquid nitrogen and total RNA was isolated from each group separately using the following hot borate method. Frozen tissue was ground to a fine powder with a pre-chilled mortar and pestle, and then further homogenized in pre-chilled glass tissue grinder.

Immediately thereafier, 2.5 ml/g tissue hot (~82°C) XT Buffer (0.2 M borate decahydrate, mM EGTA, 1% (w/v) SDS. Adjusted pH to 9.0 with 5 N NaOH, treated with 0.1%

DEPC and autoclaved. Before use, added 1% deoxycholate (sodium salt), 10 mM dithiothreitol, 15 Nonidet P-40 (NP-40) and 2% (w/v) polyvinylpyrrolidone, MW 40,000 (PVP-40)) was added to the ground tissue. The tissue was homogenized 1-2 minutes and quickly decanted to a pre-chilled Oak Ridge centrifuge tube containing 105 pl of 20 mg/ml proteinase K in DEPC treated water. The tissue grinder was rinsed with an additional 1 ml hot XT Buffer per g tissue, which was then added to rest of the homogenate. The homogenate was incubated at 42°C at 100 rpm for 1.5 h. 2 M KCl was added to the homogenate to a final concentration of 160 mM, and the mixture was incubated on ice for 1 h to precipitate out proteins. The homogenate was centrifuged at 12,000 x g for 20 min at 4°C, and the supernatant was filtered through sterile miracloth into a clean 50 ml Oak Ridge centrifuge tube. 8 M LiCl was added to a final concentration of 2 M LiCl and incubated on ice overnight. Precipitated RNA was collected by centrifugation at 12,000 x g for 20 min at 4°C. The pellet was washed three times in 3-5 ml 4°C 2 M LiCl. Each time the pellet was resuspended with a glass rod and then spun at 12,000 x g for 20 min at 4°C. The RNA pellet was suspended in 2 ml 10 mM Tris-HCI (pH 7.5), and purified from insoluble cellular components by spinning at 12,000 x g for 20 min at 4°C. The RNA containing supernatant was transferred to a 15 ml Corex tube and precipitated overnight at -20°C with 2.5 volumes of 100 % ethanol. The RNA was pelleted by centrifugation at 9,800 x g for 30 min at 4°C.

The RNA pellet was washed in 1-2 ml cold 70°C ethanol and centrifuged at 9,800 x g for 5 min at 4°C. Residual ethanol was removed from the RNA pellet under vacuum, and the

RNA was resuspended in 200 pl DEPC treated dd-water and transferred to a 1.5 ml microfuge tube. The Corex tube was rinsed in 100 pl DEPC-treated dd-water, which was then added to the rest of the RNA. The RNA was then precipitated with 1/10 volume of 3 M sodium acetate, pH 6.0 and 2.5 volumes of cold 100% ethanol at -20°C for 1-2 h. The tube was centrifuged for 20 min at 16,000 x g, and the RNA pellet washed with cold 70% ethanol, and centrifuged for 5 min at 16,000 x g. After drying the pellet under vacuum, the

RNA was resuspended in DEPC-treated water. This is the total RNA.

Messenger RNA was purified from total RNA using an Poly(A)Pure kit (Ambion,

Austin TX), following the manufacturer's instructions. A reverse transcription reaction was used to synthesize cDNA from the mRNA template, using either the Stratagene (La Jolla,

CA) or Gibco BRL (Gaithersburg, MD) cDNA cloning kits. For the Stratagene library, the cDNAs were subcloned into bacteriophage at EcoR1/Xhol site by ligating the arms using the

Gigapack III Gold kit (Stratagene, La Jolla, CA), following the manufacturer's instructions.

For the Gibco BRL library, the cDNAs were subcloned into a tobamoviral vector that contained a fusion of TMV-U1 and TMV-US at the NotI/Xhol sites.

EXAMPLE 5

Expression of Chinese cucumber cDNA clone pQ21D in transfected plants in a positive sense confirms that it encodes a-tjchosanthin, }

We have developed a plant viral vector that directs the expression of a-trichosanthin in transfected plants. The open reading frame (ORF) for a-trichosanthin, from the genomic clone SEO, was placed under the control of the TMV coat protein subgenomic promoter.

Infectious RNA from TTUS1A QSEO #3 (FIGURE 3; nucleic acid sequence as SEQ ID

NO: 1 and amino acid sequence as (SEQ. ID NO: 2) was prepared by in vitro transcription using SP6 DNA-dependent RNA polymerase and was used to mechanically inoculate A. benthamiana. The hybrid virus spread throughout all the non-inoculated upper leaves as verified by local lesion infectivity assay, and PCR amplification. The viral symptoms consisted of plant stunting with mild chlorosis and distortion of systemic leaves. The 27- kDa a-tnchosanthin accumulated in upper leaves (14 days after inoculation) and cross- reacted with an anti-trichosanthin antibody. 47

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Plasmid Constructions. .

An 0.88-kb Xhol, Avrll fragment, containing the a-trichosanthin coding sequence, was amplified from genomic DNA isolated from Trichosanthes kirilowii Maximowicz by

PCR mutagenesis using oligonucleotides QMIX: 5°-GCC TCG AGT GCA GCA TGA TCA

GAT TCT TAG TCC TCT CTT TGC-3’ (upstream) (SEQ. ID NO: 45) and Q1266A 5° -TCC

CTA GGC TAA ATA GCA TAA CTT CCA CAT CA AAGC-3' (downstream) (SEQ ID

NO :46), The a-trichosanthin open reading frame was verified by dideoxy sequencing, and placed under the control of the TMV-UI coat protein subgenomic promoter by subcloning into TTUS1A, creating plasmid TTUS1A QSEQ #3.

In vitro Transcriptions, Inoculations, and Analysis of Transfected Plants.

N. benthaminana plants were inoculated with in vitro transcripts of Kpn I-digested

TTUS1A QSEO #3. Vinions were isolated from N. benthamiana leaves infected with

TTUS51A QSEO #3 transcripts.

Purification, Immunological Detection, and in vitro Assay of a-Trichosanthin.

Two weeks after inoculation, total soluble protein was isolated from upper, noninoculated N. benthamiana leaf tissue and assayed from cross-reactivity to a o- trichosanthin antibody. The proteins from systemically infected tissue were analyzed on a 0.1% SDS/12.5% polyacrylamide gel and transferred by electroblotting for 1] hrto a nitrocellulose membrane. The blotted membrane was incubated for 1 hr with a 2000-fold dilution of goat anti-a-trichosanthin antiserum. The enhanced chemiluminescence horseradish peroxidase-linked. rabbit anti-goat IgG assay (Cappel Laboratories) was performed according to the manufacturer's (Amersham) specifications. The blotted membrane was subjected to film exposure times of up to 10 sec. Shorter and longer chemiluminescent exposure times of the blotted membrane gave the same quantitative results. 48

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~~ - EXAMPLEG6 -

Expression of bell pepper cDNA in transfected plant in a positive sense orientation confirms : that it encodes capsanthin-capsorubin synthase.

The biosynthesis of leaf carotenoids in Nicotiana benthamiana was altered by rerouting the pathway to the synthesis of capsanthin, a non-native chromoplast-specific xanthophyll, using an RNA viral vector. A cDNA encoding capsanthin-capsorubin synthase (Ces), was placed under the transcriptional control of a tobamovirus subgenomic promoter,

Leaves from transfected plants expressing Ccs developed an orange phenotype and accumulated high levels of capsanthin. This phenomenon was associated by thylakoid membrane distortion and reduction of grana stacking. In contrast to the situation prevailing in chromoplasts, capsanthin was not esterified and its increased level was balanced by a concomitant decrease of the major leaf xanthophylls, suggesting an autoregulatory control of chloroplast carotenoid composition. Capsanthin was exclusively recruited into the trimeric and monomeric light-harvesting complexes of Photosystem II. This demonstration that higher plant antenna complexes can accommodate non-native carotenoids provides compelling evidence for functional remodeling of photosynthetic membranes by rational design of carotenoids.

Construction of the Ccs expression vector. Unique Xhol, Avrll sites were inserted into the bell pepper capsanthin-capsorubin synthase (Ccs) cDNA by polymerase chain reaction (PCR) mutagenesis using oligonucleotides: 5’-

GCCTCGAGTGCAGCATGGAAACCCTTCTAAAGCTTTTCC-3' (upstream) (SEQ ID

NO: 6), 5’-TCCCTAGGTCAAAGGCTCTCTATTGCTAGATTGCCC-3' (downstream) (SEQ ID NO: 7). The 1.6-kb Xhol, Avrll cDNA fragment was placed under the control of the TMV-U1 coat protein subgenomic promoter by subcloning into TTOIA, creating plasmid TTOIA CCS+ (FIGURE 4; nucleic acid sequence as SEQ ID NO: 3 and amino acid sequence as SEQ ID NO: 4) in the sense orientation as represented by FIGURE 4.

Carotenoid analysis. Twelve days after inoculation upper leaves from 12 plants were harvested and lyophilized. The resulting non-saponified extract was evaporated to dryness under argon and weighed to determine the total lipid content. Pigment analysis from the 49

Amended Sheet — 2003-06-24 total lipid content was performed by HPLC and also separated by thin layer chromatography on silica gel G using hexane / acetone (60:40 (V/V)). Plants transfected with TTOIA CCS+ accurnulated high levels of capsanthin (36% of total carotenoids).

EXAMPLE 7

Expression of cDNAs encoding tomato phytoene synthase and phytoene desaturase in a positive and anti sense orientation in Nicotiana benthamiana.

Isolation of tomato mosaic virus cDNA. An 861 base pair fragment (5524-6384) from the : tomato mosaic virus (fruit necrosis strain F; tom-F) containing the putative coat protein”~ ~ : subgenomic promoter, coat protein gene, and the 3'-end was isolated by PCR using primers 5-CTCGCAAAGTTTCGAACCAAATCCTC-3' (upstream) (SEQ ID NO: 49) and 5'-

CGGGGTACCTGGGCCCCAACCGGGGGTTCCGGGGG-3’ (downstream) (SEQ ID NO: 50) and subcloned into the Hincll site of pBluescript KS-. A hybrid virus consisting of

TMV-U1 and ToMV-F was constructed by swapping an 874-bp BamHI-Kpnl ToMV fragment into pPBGC152, creating plasmid TTO1. The inserted fragment was verified by dideoxynucleotide sequencing. A unique Avrll site was inserted downstream of the Xhol site in TTO1 by PCR mutagenesis, creating plasmid TTO1A, using the following oligonucleotides: 5'-TCCTCGAGCCTAGGCTCGCAAAGTTTCGAACCAAATCCTCA-3’ (upstream) (SEQ ID NO: 51), 5'- : : CGGGGTACCTGGGCCCCAACCGGGGGTTCCGGGGG-3' (downstream) (SEQ ID NO: 52).

Isolation of a cDNA encoding tomato phvtoene synthase and a partial cDNA encoding tomato phytoene desaturase. Partial cDNAs were isolated from ripening tomato fruit RNA by polymerase chain reaction (PCR) using the following oligonucleotides: PSY, 5°-

TATGTATGGTGCAGAAGAACAGAT-3" (upstream) (SEQ ID NO: 53), 5°-

AGTCGACTCTTCCTCTTCTGGCAT C-3' (downstream) (SEQ ID NO: 54); PDS, 5’-

TGCTCGAGTGTGTTCTTCAGTITTTCTGTCA-3' (SEQ ID NO: 55) (upstream), 5°-

AACTCGAGCGCTTTGATTTCTCCGAAGCTT-3' (downstream) (SEQ ID NO: 56).

Approximately 3 X 10* colonies from a Lycopersicon esculentum cDNA library were : 50

Amended Sheet — 2003-06-24 screened by colony hybridization using a 3p labeled tomato phytoene synthase PCR product. Hybridization was carried out at 42°C for 48 hours in 50% formamide, 5X SSC, 0.02 M phosphate buffer, 5X Denhart's solution, and 0.1 mg/ml sheared calf thymus DNA.

Filters were washed at 65°C in 0.1X SSC, 0.1% SDS prior to autoradiography. PCR products and the phytoene synthase cDNA clones were verified by dideoxynucleotide sequencing.

DNA sequencing and computer analysis. A PstI, BamHI fragment containing the phytoene synthase cDNA and the partial phytoene desaturase cDNA was subcloned into pBluescript®

KS+ (Stratagene, La Jolla, California). The nucleotide sequencing of KS+/PDS #38 and

KS+/ 5'3'PSY was carried out by dideoxy termination using single-stranded templates (Maniatis, Molecular Cloning, 1* Ed.) Nucleotide sequence analysis and amino acid sequence comparisons were performed using PCGENE® and DNA Inspector® [IE programs.

Construction of the tomato phytoene synthase expression vector. A Xhol fragment containing the tomato phytoene synthase cDNA was subcloned into TTO1. The vector

TTOVPSY + (FIGURE 5; nucleic acid sequence as SEQ ID NO: 18 and amino acid sequence as SEQ. ID. NO: 19) contains the phytoene synthase cDNA in the positive orientation under the control of the TMV-U1 coat protein subgenomic promoter; while, the vector TTO1/PSY - contains the phytoene synthase cDNA in the antisense orientation.

Construction of a viral vector containing a partial tomato phytoene desaturase cDNA. A

Xhol fragment containing the partial tomato phytoene desaturase cDNA was subcloned into

TTO!1. The vector TTOIA/PDS + (FIGURE 6) contains the phytoene desaturase cDNA in the positive orientation under the control of the TMV-U1 coat protein subgenomic promoter; while the vector TTOIA/PDS - contains the phytoene desaturase cDNA in the antisense orientation.

Analysis of N. benthamiana transfected by TTO1/PSY+, TTOI/PSY-, TTO1A/PDS +,

TTOL/PDS -, Infectious RNAs from TTO1/PSY+, TTO1/PSY-,TTOL/PDS +, and

TTOVPDS-, were prepared by in vitro transcription using SP6 DNA-dependent RNA polymerase as described previously (Dawson et al., Proc. Natl. Acad. Sci. USA 85:1832 (1986)) and were used to mechanically inoculate N. benthamiana. The hybrid viruses spread throughout all the non-inoculated upper leaves as verified by transmission electron microscopy, local lesion infectivity assay, and polymerase chain reaction (PCR) amplification. The viral symptoms resulting from the infection consisted of distortion of systemic leaves and plant stunting with mild chlorosis. The leaves from plants transfected with TTO1/PSY+ turned orange and accumulated high levels of phytoene while those transfected with TTO1/PDS+ and TTO1/PDS- turned white. Agarose gel electrophoresis of

PCR cDNA isolated from virion RNA and Northern blot analysis of virion RNA indicate that the vectors are maintained in an extrachromosomal state and have not undergone any detectable intramolecular rearrangements.

Purification and analysis of carotenoids from transfected plants. The carotenoids were isolated from systemically infected tissue and analyzed by HPLC chromatography.

Carotenoids were extracted in ethanol and identified by their peak retention time and absorption spectra on a 25-cm Spherisorb® ODS-15- m column using acetonitrile/methanol/2-propanol (85:10:5) as a developing solvent at a flow rate of 1 mV/min. They had identical retention time to a synthetic phytoene standard and B-carotene standards from carrot and tomato. The phytoene peak from N. benthamiana transfected with

TTO1/PSY + had an optical absorbance maxima at 276, 285, and 298 nm. Plants transfected with viral encoded phytoene synthase showed a ten-fold increase in phytoene compared to the levels in noninfected plants. The expression of sense and antisense RNA to a partial phytoene desaturase in transfected plants increased the level of phytoene and altered the biochemical pathway; it thus inhibited the synthesis of colored carotenoids and caused the systemically infected leaves to turn white. HPLC analysis of these plants revealed that they also accumulated phytoene. The white leaf phenotype was also observed in plants treated with the herbicide norflurazon which specifically inhibits phytoene desaturase.

This change in the levels of phytoene represents one of the largest increases of any carotenoid (secondary metabolite) in any genetically engineered plant. Plants transfected with viral-encoded phytoene synthase in a plus sense showed a ten-fold increase in phytoene compared to the levels in noninfected plants. In addition, the accumulation of phytoene in plants transfected with antisense phytoene desaturase suggests that viral vectors can be used ) as a potent tool to manipulate pathways in the production of secondary metabolites through cytoplasmic antisense inhibition. Leaves from systemically infected TT01 A/PDS+ plants also accumulated phytoene and developed a bleaching white phenotype; the actual mechanism of inhibition is not clear. These data are presented by Kumagai er al., Proc.

Natl. Acad. Sci. USA 92:1679-1683 (1995).

EXAMPLE 8

Expression of phytoene desaturase in transfected plants using a multipartitie viral vector -

Construction of 2 monocot viral vector. BSMV is a tripartite RNA virus that infects many agnculturally important monocot species such as oat, wheat and barley (McKinney and

Greeley, “Biological characteristics of barley stripe mosaic virus strains and their evolution”

Technical Bulletin U.S. Department of Agriculture 1324 (1965)). An expression vector derived from barley stripe mosaic virus (BSMV) was constructed by modifying a BSMV y cDNA (Gustafson et al., Virology 158(2):394-406 (1987)) (Figure 7A). In this example, we developed a monocot viral vector that directs the expression of nucleotide sequences in transfected plants. Foreign inserts can be placed under the control of the yb subgenomic promoter. The infectious BSMV y cDNA (7.42) was modified by site-directed mutagenesis.

Nucleotides 5098-5103 of v.42 were replaced with a Nhe I site. Using polymerase chain reaction (PCR) mutagenesis, a 646 bp Nhe I fragment, containing the zeomycin resistance gene as a cloning marker, was amplified from pZEr0 (Invitrogen Corporation, Carisbad, CA,

USA) using the oligonucleotides 5°

TATGCTAGCTGATTAATTAAGTCGACGAGCTGATTTAACAAATTTTAAC 3° (upstream) (SEQ ID NO: 57) and 5’

TATGCTAGCTGAGCGGCCGCGCACGTGTCAGTCCTGC

TCCTCGG 3’ (downstream) (SEQ ID NO: 58), and inserted into Nhe site of the BSMV y cDNA. This generated two plasmids, y.yb.st.P/N-zeo (positive orientation) and y.yb.st.N/P- zeo (negative orientation), with Pacl and Notl sites flanking the zeomycin resistance gene (Figure 7B). 53 .. .

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: To improve the expression of the y subgenomic RNAI, an infectious BSMV beta (b) cDNA (B42Spl) (Petty et al., Virology 179(2):712-8 (1990)) was modified by substituting the majority of the coat protein ORF by PCR mutagenesis. A 423 bp fragment was amplified from B42Spl using the oligonucleotides 5’

GGAAAGCCGGCGAACGTGGCG 3’ (upstream) (SEQ ID NO: 59) and 5°

TATATTCGAATCTAGAATCGATGCTAGCTTGCATGCTGTGAAGTGG

TAAAAGAAATGC 3' (downstream) (SEQ ID NO: 60) and cloned into the NgoMIV and

BstBI sites of creating plasmid B.APa. This construct contains only an untranslated portion of the coat protein ORF that is required for expression of the subsequent B RNA ORFs (Figure 7C). )

Construction of monocot viral vectors the contain partial maize phytoene desaturase cDNAs.

Partial cDNAs encoding phytoene desaturase (PDS) were amplified from com leaf tissue

RNA by RT-PCR using oligonucleotides pairs 175 5°

ATATTAATTAACATGGACACTGGCTGCCTGTC 3° (upstream) (SEQ. ID. NO: 61) and 180 5" TATGCGGCCGCCTACAAAGCAATCAAAATGCACTG 3' (downstream) (SEQ.

ID. NO:62) encoding PDS Met'- Leu*, pairs 177 5°

ATATTAATTAACAAGGTAGCTGCTTGGAAGGATG 3’ (upstream) (SEQ ID NO: 63) and 178 5" TATGCGGCCGCCTAGCAGGTTACTGACATGTCTGC 3’ (downstream) (SEQ. ID NO: 64) encoding PDS Lys'**-Cys*', and pairs 179 5”

ATATTAATTAACCAGTGCATTTTGATTGCTTTG 3’ (upstream) (SEQ. ID NO: 65) and 176 5° TATGCGGCCGCCTAAGATGGGACGGGAACTTCTCC 3" (downstream) (SEQ.

ID NO: 66) encoding PDS GIn***-Ser®”". The 0.8 Kb amplified Pac I and Not I fragments containing the partial cDNAs encoding corn PDS were placed under the control of the

BSMYV vb subgenomic promoter by subcloning into the Pacl and NotI sites y.yb.st.P/N-zeo and y. yb.st.N/P-zeo. This eliminated the Zeocin resistance gene and created plasmids with

PDS inserts in the positive orientation (y.yb.st. P/N-mPDS-N, y.yb.st. P/N-mPDS-M, and

Y.Yb.st.P/N-mPDS-C) and negative orientation (y.yb.st. /N-mPDS-N as, y.yb.st.P/N-mPDS-

M as, and v.yb.st.P/N-mPDS-C as). 54

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Analysis of barley plants transfected with y.yb.st.P/N-mPDS. Infectious BSMV RNAs from cDNA clones were prepared by in vitro transcription using T7 DNA-dependent RNA polymerase (Ambion) as described previously (Petty, et al., Virology 171(2):342-9 (1989)).

Transcripts of each of the three BSMV genomes were mixed in a 1:1:1 ratio. A 7 ul aliquot of the transcription mix was combined with 40 uL of FES and directly applied to 12 day old black hulless barley plants. The BSMV::mPDS hybrid viruses spread throughout the non- inoculated leaves. The initial viral symptoms (1-7 days post inoculation) resulting from the

PDS containing constructs displayed symptoms similar to a wild type BSMV infection. 8- days post inoculation, the BSMV-PDS plants began to exhibit streaks and patches of unusually white tissue. The affected areas lacked the necrosis or desiccation that is often associated with BSMYV induced bleaching and more like the bleached tissue found in plants treated with the chemical inhibitor of PDS, norflurazon. These white streaks were observed to some degree in all the BSMV::mPDS infected plants, although the most extensive areas of bleaching were generally found on the plants infected with BSMV containing PDS in the sense orientation.

Purification and analysis of carotenoids from transfected barley plants. The carotenoids were isolated from 50 mg of systemically infected leaf tissue 18 days post inoculation and analyzed by HPLC chromatography. Carotenoids were extracted in the dark in methanol and identified by their peak retention time and absorption spectra on a Zorbax 4.6 X 15 cm

C-18 column using acetonitrile/methanol/2-propanol (85:10:5) as a developing solvent at a flow rate of 2 ml/min. They had identical retention times to a synthetic phytoene standard and fB-carotene standards from tomato and carrot. The expression of sense and antisense

RNA to the partial maize phytoene desaturase in transfected barley inhibited the synthesis of colored carotenoids and caused the systemically infected tissue to tum white. HPLC analysis of these plants revealed that they also accumulated phytoene. The white leaf phenotype was also observed in barley plants treated with the herbicide norflurazon which specifically inhibits phytoene desaturase. Phytoene extracted from barley transfected with

BSMV-PDS was analyzed by HPLC, had a retention time similar to that of a phytoene standard, and showed a 10-60 fold increase over the levels in a BSMYV transfected control plant.

Our results that phytoene accumulated in barley plants transfected with partial antisense and positive sense phytoene desaturase suggests that plant viral vectors can be used to manipulate biosynthetic pathways in monocots through cytoplasmic inhibition of endogenous gene expression.

EXAMPLES

Expression of bacterial CrtB gene in transfected plants in a positive sense orientation confirms that jt encodes phytoene synthase.

We developed a new viral vector, TTUS1, consisting of tobacco mosaic virus strain

Ul (TMV-U1) (Goelet er al., Proc. Natl. Acad. Sci. USA 79:5818-5822 (1982)), and tobacco mild green mosaic virus (TMGMYV; US strain) (Solis er al., 177:553-8 (1990)). The open reading frame (ORF) for Erwinia herbicola phytoene synthase (CrtB) (Armstrong et al.,

Proc. Natl. Acad. Sci. USA 87:9975-9979 (1990)) was placed under the control of the tobacco mosaic virus (TMV) coat protein subgenomic promoter in the vector TTUS51. This construct also contained the gene encoding the chloroplast targeting peptide (CTP) for the small subunit of ribulose-1,5-bisphosphate carboxylase (RUBISCO) (O'Neal er al., Nucl.

Acids Res. 15:8661-8677 (1987)) and was called TTU51 CTP CrtB as represented by

FIGURE 8 (Nucleic acid sequence as SEQ. ID. NO: 30 and amino acid sequence as SEQ.

ID. NO: 31). Infectious RNA was prepared by in vitro transcription using SP6 DNA- dependent RNA polymerase (Dawson ef al, Proc. Natl. Acad. Sci. USA 83:1832-1836 (1986)); Susek et al., Cell 74:787-799 (1993)) and was used to mechanically inoculate N. benthamiana. The hybrid virus spread throughout all the non-inoculated upper leaves and was verified by local lesion infectivity assay and polymerase chain reaction (PCR) amplification. The leaves from plants transfected with TTUS1 CTP CriB developed an orange pigmentation that spread systemically during plant growth and viral replication.

Leaves from plants transfected with TTUS1 CTP CrtB had a decrease in chlorophyll content (result not shown) that exceeded the slight reduction that is usually observed during viral infection. Since previous studies have indicated that the pathways of carotenoid and chlorophyll biosynthesis are interconnected (Susck ez al., Cell 74:787-799 (1993)), we decided to compare the rate of synthesis of phytoene to chlorophyll. Two weeks post- inoculation, chloroplasts from plants infected with TTU51 CTP CrB transcripts were isolated and assayed for enzyme activity. The ratio of phytoene synthetase to chlorophyll syntheses was 0.55 in transfected plants and 0.033 in uninoculated plants (control). '

Phytoene synthase activity from plants transfected with TTUS1 CTP Cr1B was assayed using isolated chloroplasts and labeled [14C) geranylgeranyl PP. There was a large increase in phytoene and an unidentified C40 alcohol in the CrtB plants.

Phvtoene synthetase assay. .

The chloroplasts were prepared as described previously (Camara, Methods Enzymol. 214:352-365 (1993)). The phytoene synthase assays were carried out in an incubation mixture (0.5 ml final volume) buffered with Tris-HCL, pH 7.6, containing [14C]) - geranylgeranyl PP (100,000 cpm) (prepared using pepper GGPP synthase expressed in £. coli), 1 mM ATP, 5 mM MnCl, 1 mM MgCl, Triton X-100 (20 mg per mg of chloroplast protein) and chloroplast suspension equivalent to 2 mg protein. After 2 h incubation at 30°C, the reaction products were extracted with chloroform methanol (Camara, supra) and subjected to TLC onto silicagel plate developed with benzene/ethyl acetate (90/10) followed by autoradiography.

Chlorophyll} synthetase assay.

For the chlorophyll synthetase assay, the isolated chloroplasts were lysed by osmotic shock before incubation. The reaction mixture (0.2 ml, final volume) consisting of 50 mM

Tris-HCL (pH 7.6) containing [14C] geranyigeranyl PP (100,000 cpm), 5 MgCl2, | mM

ATP, and ruptured plasmid suspension equivalent to | mg protein was incubated for 1 hr at 30°C. The reaction products were analyzed as described previously.

Plasmid Constructions.

The chloroplast targeting, phytoene synthase expression vector, TTU51 CTP CrtB as represented in FIGURE 8, was constructed in several subcloning steps. First, a unique Sphl site was inserted in the start codon for the Erwinia herbicola phytoene synthase gene by polymerase chain reaction (PCR) mutagenesis (Saiki er a!., Science 230:1350-1354 (1985)) } using oligonucleotides CrtB M1S 5'-CCA AGC TTC TCG AGT GCA GCA TGC AGC --

AAC CGC CGC TGC TTG AC-3' (upstream) (SEQ ID NO: 67) and CrtB P300 5’-AAG } 57

Amended Sheet — 2003-06-24

ATC TCT CGA GCT AAA CGG GAC GCT GCC AAA GAC CGG CCG G-3 (downstream) (SEQ ID NO: 68). The CrtB PCR fragment was subcloned into pBluescript® (Stratagene) at the EcoRV site, creating plasmid pBS664. A 938 bp Sphl, Xhol CriB fragment from pBS664 was then subcloned into a vector containing the sequence encoding the N. tabacum chloroplast targeting peptide (CTP) for the small subunit of RUBISCO, creating plasmid pBS670. Next, the tobamoviral vector, TTUS1, was constructed. A 1020 base pair fragment from the tobacco mild green mosaic virus (TMGMV; U5 strain) containing the viral subgenomic promoter, coat protein gene, and the 3'-end was isolated by

PCR using TMGMYV primers 5"GGC TGT GAA ACT CGA AAA GGT TCC GG-3' (upstream) (SEQ ID NO:69) and 5-CGG GGT ACC TGG GCC GCT ACC GGC GGT oT

TAG GGG AGG-3' (downstream) (SEQ ID NO: 70), subcloned into the Hincll site of

Bluescript KS-, and verified by dideoxynucleotide sequencing. This clone contains a naturally occurring duplication of 147 base that includes the whole upstream pseudoknot domain in the 3' noncoding region. The hybrid viral cDNA consisting of TMV-U1 and

TMGMYV was constructed by swapping a 1-Kb Xhol-Kpnl TMGMYV fragment into TTO1 (Kumagai er al., Proc. Natl. Acad. Sci. USA 92:1679-1683 (1995)), creating plasmid TTUS1.

Finally, the 1.1 Kb Xhol CTP CrtB fragment from pBS670 was subcloned into the Xhol of

TTUS]1, creating plasmid TTUS1 CTP CrtB. As a CTP negative control, a 942 bp Xhol fragment containing the CrtB gene from pBS664 was subcloned into TTUS], creating plasmid TTUS51 CrtB #15.

EXAMPLE 10

Identification of nucleotide sequences involved in the regulation of plant growth bv cvioplasmic inhibition of gene expression in a positive sense orientation using viral denved

RNA, :

In this example, we show: (1) a method for producing plus sense RNA using an RNA viral vector, (2) a method to produce viral-derived sense RNA in the cytoplasm, (3) a method to enhance or suppress the expression of endogenous plant proteins in the cytoplasm by viral antisense RNA, and (4) a method to produce transfected plants containing viral plus sense RNA; such methods are much faster than the time required to obtain genetically engineered sense transgenic plants. Systemic infection and expression of viral plus sense 58

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RNA occurs as short as four days post inoculation, whereas it takes several months or longer to create a single transgenic plant. This example demonstrates that novel positive strand viral vectors, which replicate solely in the cytoplasm, can be used to identify genes involved : in the regulation of plant growth by enhancing or inhibiting the expression of specific endogenous genes. This example also enables one to characterize specific genes and biochemical pathways in transfected plants using an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of uncharacterized nucleotide sequences in transfected plants. A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. Colonies from transformed E. coli were automatically picked using a Flexys robot and transferred to a 96 well flat bottom block containing terrific broth (TB) Amp 50 ug/ml. Approximately 2000 plasmid DNAs were isolated from overnight cultures using a BioRobot and infectious RNAs from 430 independent clones were directly applied to plants. One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. One set of plants transfected with 740 AT #2441 were severely stunted. DNA sequence analysis revealed that this clone contained an Arabidopsis Ran GTP binding protein open reading frame (ORF) in a plus sense orientation. This demonstrates that an episomal RNA viral vector can be used to deliberately alter the metabolic pathway and cause plant stunting. In addition, our results show that the Arabidopsis plus sense transcript can cause phenotypic changes in N. benthamiana.

Construction of an Arabidopsis thaliana cDNA library in an RNA viral vector.

An Arabidopsis thaliana CD4-13 cDNA library was digested with Norl. DNA fragments between 500 and 1000 bp were isolated by trough elution and subcloned into the

Noil site of pBS740. E. coli C600 competent cells were transformed with the pBS740 AT library and colonies containing Arabidopsis cDNA sequences were selected on LB Amp 50 ug/ml. Recombinant C600 cells were automatically picked using a Flexys robot and then transferred to a 96 well flat bottom block containing terrific broth (TB) Amp 50 ug/ml.

Approximately 2000 plasmid DNAs were isolated from overnight cultures using a BioRobot (Qiagen) and infectious RNAs from 430 independent clones were directly applied to plants.

One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. Plants transfected with 740 AT #2441 (FIGURE 9) were severely stunted. Plasmid 740 AT #2441 contains the

TMV-U1 open reading frames (ORFs) encoding 126-, 183-, and 30-kDa proteins, the TMV-

US coat protein gene (US cp), the T7 promoter, an Arabidopsis thaliana CD4-13 Notl fragment, and part of the pUC19 plasmid. The TMV-U1 subgenomic promoter located within the minus strand of the 30-kDa ORF controls the synthesis of the CD4-13 subgenomic RNA. oT

DNA sequencing and computer analysis.

A 841 bp No!l fragment of 740 AT #2441 (FIGURE 10; nucleic acid sequence and amino acid sequence as SEO ID NOs: 9 and 10, respectively) containing the Ran GTP binding protein _ cDNA was characterized. The nucleotide sequencing of 740 AT #2441 was carried out by dideoxy termination using double stranded templates. Nucleotide sequence analysis and amino acid sequence comparisons were performed using DNA Strider, PCGENE and NCBI Blast programs. 740 AT #2441 contained an open reading frame (ORF) in the positive orientation that encodes a protein of 221 amino acids with an apparent molecular weight of 25,058 Da. The mass of the protein was calculated using the Voyager program (Perceptive Biosystems). FIGURE 11 shows the nucleotide sequence alignment of 740AT #2441 to AF017991 (SEQ. ID Nos: 11 and 12 respectively), a A. thaliana salt stress inducible small GTP binding protein Ranl. FIGURE 12 shows the nucleotide alignment of 740 AT #2441 to L16787 (SEQ. ID Nos: 13 and 14 respectively), a N. tabacum small ras-like GTP binding protein. FIGURE 13 shows the amino acid comparison of 740 AT #2441 to tobacco Ran-B1 GTP binding protein (SEQ. ID.

Nos: 15 and 16 respectively). :

The A. thaliana cDNA exhibits a high degree of homology (99% to 82%) to A4. thaliana, tomato (L. esculentum), tobacco (N. tabacum), L. japonicus and bean (V. faba) GTP binding proteins cDNAs (Table 1). The nucleotide sequence from 740 AT #2441 encodes a 60

Amended Sheet — 2003-06-24 protein that has strong similarity (100% to 95%) to A. thaliana, tomato, tobacco, and bean

GTP binding proteins (Table 2).

The #2441 DNA also exhibits a high degree of homology (67% to 83%) to human, yeast, mouse and drosophila GTP binding proteins cDNAs (Table 3). The protein also has 67%-97% identities, and 79%-98% positives to yeast, mammalian organisms such as human (Table 4)

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MALDI-TOF analysis of N. benthamiana transfected with 740 AT #2441 days after inoculation, the apical meristem, leaves, and stems from N. benthamiana transfected with 740 AT #2441. were frozen in liquid nitrogen. The soluble proteins were extracted in grinding buffer (100mM Tris, pH 7.5,2 mM EDTA, 1 mM PMSF, 10 mM BME) using a mortar and pestle. The homogenate was filtered through four layers of cheesecloth and spun at 10,000 X g for 15 min. The supernatant was decanted and spun at 100, 000 X g for 1 hr. A 500 ul aliquot of the supernant was mixed with 500 ul 20% TCA (acetone/0.07% BME) and stored at 4° C overnight. The supernant was analyzed by

MALDI-TOF. (Karas et al., Anal. Chem. 60:2299-2301 (1988)). The results showed that the soluble proteins contained a newly expressed protein of molecular weight 25,058.

Isolation of an Arabidopsis thaliana GTP binding protein genomic clone

A genomic clone encoding 4. thaliana GTP binding proteins can be isolated by probing filters containing A. thaliana BAC clones using a 12P_labelled 740 AT #2441 NotI insert.

Other members of the A. thaliana ARF multigene family have been identified using programs of the University of Wisconsin Genetic Computer Group.

EXAMPLE 11

Identification of nucleotide sequences involved in the regulation of plant growth by cytoplasmic inhibition of gene expression in an antisense ori entation using viral derived

RNA (GTP binding proteins).

In this example, we show: (1) a method for producing antisense RNA using an RNA viral vector, (2) a method to produce viral-derived antisense RNA in the cytoplasm, (3) a method to inhibit the expression of endogenous plant proteins in the cytoplasm by viral antisense RNA, and (4) a method to produce transfected plants containing viral antisense

RNA, such method is much faster than the time required to obtain genetically engineered antisense transgenic plants. Systemic infection and expression of viral antisense RNA occurs as short as several days post inoculation, whereas it takes several months or longer to create a single transgenic plant. This example demonstrates that novel positive strand viral vectors, which replicate in the cytoplasm, can be used to identify genes involved in the regulation of plant growth by inhibiting the expression of specific endogenous genes. This example enables one to characterize specific genes and biochemical pathways in transfected plants using an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of uncharacterized nucleotide sequences in transfected plants. A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. Colonies from transformed E. coli were automatically picked using a Flexys robot and transferred to a 96 well flat bottom block containing terrific broth (TB) Amp 50 ug/ml. Approximately 2000 plasmid DNAs were isolated from overnight cultures using a BioRobot and infectious RNAs from 430 independent clones were directly applied to plants. One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. One set of plants transfected with 740 AT #120 were severely stunted. DNA sequence analysis revealed that this clone contained an Arabidopsis GTP binding protein open reading frame (ORF) in the antisense orientation. This demonstrates that an episomal

RNA viral vector can be used to deliberately alter the metabolic pathway and cause plant stunting. In addition, our results suggest that the Arabidopsis antisense transcript can turn off the expression of the N. benthamiana gene.

Construction of an Arabidopsis thaliana cDNA library in an RNA viral vector.

Notl site of pBS740. E. coli C600 competent cells were transformed with the pBS740 AT library and colonies containing Arabidopsis cDNA sequences were selected on LB Amp 50 ug/ml. Recombinant C600 cells were automatically picked using a Flexys robot and then transferred to a 96 well flat bottom block containing terrific broth (TB) Amp 50 ug/ml.

Isolation of a gene encoding a GTP binding protein,

One to two weeks after inoculation, transfected Nicotiana benthamiana plants were "visually monitored for changes in growth rates, morphology, and color. Plants transfected with 740 AT #120 (FIGURE 14) were severely stunted. Plasmid 740 AT #120 contains the

TMV-Ul 126-, 183-, and 30-kDa ORFs, the TMV-US coat protein gene (US cp), the T7 promoter, an Arabidopsis thaliana CD4-13 Nol fragment, and part of the pUC19 plasmid.

The TMV-U1 subgenomic promoter located within the minus strand of the 30-kDa ORF controls the synthesis of the CD4-13 antisense subgenomic RNA.

DNA sequencing and computer analysis.

A 782 bp Nol fragment of 740 AT #120 containing the ADP-ribosylation factor (ARF) cDNA was characterized. DNA sequence of Noil fragment of 740 AT #120 (774 base pairs) is as follows: 5’- . )

CCGAAACATTCTTCGTAGTGAAGCAAAATGGGGTTGAGTTTCGCCAAGCTGTTT

AGCAGGCTTTTTGCCAAGAAGGAGATGCGAATTCTGATGGTTGGTCTTGATGCT

GCTGGTAAGACCACAATCTTGTACAAGCTCAAGCTCGGAGAGATTGTCACCACC

ATCCCTACTATTGGTTTCAATGTGGAAACTGTGGAATACAAGAACATTAGTTTCA

CCGTGTGGGATGTCGGGGGTCAGGACAAGATCCGTCCCTTGTGAGACACTACTT

CCAGAACACTCAAGGTCTAATCTTTGTTGTTGATAGCAATGACAGAGACAGAGT

TGTTGAGGCTCGAGATGAACTCCACAGGATGCTGAATGAGGACGAGCTGCGTGA

TGCTGTGTTGCTTGTGTTTGCCAACAAGCAAGATCTTCCAAATGCTATGAACGCT

GCTGAAATCACAGATAAGCTTGGCCTTCACTCCCTCCGTCAGCGTCATTGGTATA

TCCAGAGCACATGTGCCACTTCAGGTGAAGGGCTTTATGAAGGTCTGGACTGGC

TCTCCAACAACATCGCTGGCAAGGCATGATGAGGGAGAAATTGCGTTGCATCGA

GATGATTCTGTCTGCTGTGTTGGGATCTCTCTCTGTCTTGATGCAAGAGAGATTA

TAAATATTATCTGAACCTTTTTGCTTTTTITGGGTATGTGAATGTTTCTTATTGTGC

AAGTAGATGGTCTTGTACCTAAAAATTTACTAGAAGAACCCTTTTAAATAGCTTT

CGTGTATTGT-3" (SEQ. ID NO: 71).

The nucleotide sequencing of 740 AT #120 was carried out by dideoxy termination using double stranded iemplates. Nucleotide sequence analysis and amino acid sequence comparisons were performed using DNA Strider, PCGENE and NCBI Blast programs. 740

AT #120 contained an open reading frame (ORF) in the antisense orientation that encodes a protein of 181 amino acids with an apparent molecular weight of 20,579 Daltons.

SE 67 )

Amended Sheet - 2003-06-24

Sequence comparison

FIGURE 15 shows a nucleotide sequence comparison of 4. thalana 740 AT #120 and 4. thaliana est AA042085 (SEQ ID Nos: 17 and 18 respectively). The nucleotide sequence from 740 AT #120 is also compared with a rice (Oryza sativa) ADP ribosylation factor AF012896, SEQ ID NOs: 19 and 20 (FIGURE 16); which shows 82% (456/550) positives and identities.

The nucleotide sequence from 740 AT #120 exhibits a high degree of homology (81- 84% identity and positive) to nice, barley, carrot, com and 4. thaliana DNA encoding ARFs and also a high degree of homology (71-84% identity and positive) to yeast, plants, insects such as fly, amphibian such as frog, mammalian such as bovine, human, and mouse DNA encoding (Table 5).

The amino acid sequence denived from 740 AT #120 exhibits an even higher degree of homology (96-98% identity and 97-98% positive) to ARFs from rice, carrot, corn and A. thaliana and a high degree of homology (61-98% identity and 78-98% positive; even higher than nucleotide sequence homology) to ARFs from yeast, plants insects such as fly, mammalian such as bovine, human, and mouse (Table 6).

The high homology of DNAs encoding GTP binding proteins from yeast, plants, insects, human, mice, and amphibians indicates that DNAs from one donor organism can be transfected into another host organism and silence the endogenous gene of the host organism 68 “7 1ded Sheet — 2003-06-24

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The protein encoded by 740 AT #120, 120P, contained three conserved domains: the phosphate binding loop motif, GLDAAGKT, (consensus GXXXXGKS/T ); the G’ motif,

DVGGQ, (consensus DXXGQ), a sequence which interacts with the gamma-phosphate of

GTP; and the G motif NKQD, (consesus NKXD), which is specific for guanidinyl binding.

The 120P contains a putative glycine-myristoylation site at position 2, a potential N- Eg glycosylation site (NXS) at position 60, and several putative serine/threonine phosphorylations sites.

Humanizing DNA

The nucleotide sequence from 740 AT #120 is also compared with a human ADP ribosylation factor (ARF3) M33384, which shows a strong similarity (76% identity at the nucleotide level and 87% identity at the amino acid level). The amino acid sequence alignment of 740 AT #120 to human ADP-ribosylation factor (ARF3) P16587 is compared in FIGURE 17 (SEQ. ID. Nos: 55-57), which shows 87% identity and 90% positive. : The high homology of the nucleic acid and amino acid sequence between the two makes humanizing 740 #AT120 practical. A humanized sequence, 740 AT#120 H nucleic acid sequence is prepared by changing the 740 AT#120 nucleic acid sequence so that it encodes the same amino acid sequence as the human M33384 encodes. The nucleic acid is changed by a standard method such as site directed mutagenisis or DNA synthesis. FIGURE 18 (SEQ. ID. Nos: 58 and 59 for nucleotide sequences and SEQ. ID. NO: 60 for amino acid sequence) shows the sequence alignment of 740 AT #120H to human ARF3 M33384.

Isolation of an Arabidopsis thaliana ARF genomic clone

A genomic clone encoding A. thaliana ARF can be isolated by probing filters containing A. thaliana BAC clones using a 2P labeled 740 AT #120 NotI insert. Other members of the 4. thaliana ARF multigene family have been identified using programs of the University of Wisconsin Genetic Computer Group. The BAC clone T0813 located on chromosome II has a high degree of homology to 740 AT #120 (78% to 86% identity at the - nucleotide level).

Amended Sheet — 2003-06-24

Isolation and characterization of a cDNA encoding Nicotiana benthamiana ARF,

A 488 bp cDNA from N. benthamiana stem cDNA library was isolated by polymerase chain reaction (PCR) using the following oligonucleotides: ATARFK1S, 5'

AAG AAG GAG ATG CGA ATT CTG ATG GT 3' (upstream) (SEQ ID NO:72),

ATARFN176, 5' ATG TTG TTG GAG AGC CAG TCC AGA CC 3' (downstream) (SEQ ID

NO:73). The vent polymerase in the reaction was inactivated using phenol/chloroform, and the PCR product was directly cloned into the Hincll site in Bluescript KS+ (Strategene).

The plasmid map of KS+ Nb ARF #3, which contains the N. benthamiaca ARF ORF in pBluescript KS+ is shown in FIGURE 19. The nucleotide sequence of N. benthamiana KS+

Nb ARF#3, which contains partial ADP-ribosylation factor ORF, was determined by ~~ dideoxynucleotide sequencing. The nucleotide sequence from KS+ Nb ARF#3 had a strong similarity to other plant ADP-ribosylation factor sequences (82 to 87% identities at the nucleotide level). The nucleotide sequence comparison of N. benthamiana KS+ Nb ARF#3 and A. thaliana 740 AT #120 shows a high homology between them (FIGURE 20, SEQ. ID.

Nos: 63 and 64 respectively). The nucleotide sequence of KS+ NbARF #3 exhibits a high degree of homology (77-87% identities and positives) to plant, yeast and mammalian DNA encoding ARFs (Table 7). Again, the high homology of DNAs encoding GTP binding proteins from yeast, plants, human, bovine and mice indicates that DNAs from one donor organism can be transfected into another host organism and effectively silence the endogenous gene of the host organism. 72

Amended Sheet — 2003-06-24

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A full-length cDNA encoding ARF is isolated by screening the N. benthamiana cDNA library by colony hybridization using a 3?P-labeled N. benthamiana KS+/Nb ARF #3 probe. Hybridization is carried out at 42°C for 48 hours in 50% formamide, 5X SSC, 0.02

M phosphate buffer, 5X Denhart's solution, and 0.1 mg/ml sheared calf thymus DNA.

Filters are washed at 65°C in 0.1X SSC, 0.1% SDS prior to autoradiography.

Rapid isolation of cDNAs encoding ARF, GTP binding proteins from rice, barley, corn, soybean, and other plants

Libraries containing full-length cDNAs from rice, barley, corn, soybean and other important crops are obtained from public and private sources or can be prepared from plant mRNAs. The cDNAs are inserted in viral vectors or in small subcloning vectors such as pBluescript (Strategene), pUC18, M13. or pBR322. Transformed bacteria (E. coli) are then plated on large petri plates or bioassay plates containing the appropriate media and antibiotic. Individual clones are selected using a robotic colony picker and arrayed into 96 well microtiter plates. The cultures are incubated at 37°C until the transformed cells reach log phase. Aliquots are removed to prepare glycerol stocks for long term storage at -80°C.

The remainder of the culture is used to inoculate an additional 96 well microtiter plate containing selective media and grown overnight. DNAs are isolated from the cultures and stored at -20°C. Using a robotic unit such as the Qbot (Genetix), the E. coli transformants or

DNAs are rearrayed at high density on nylon filters or glass slides. Full-length cDNAs encoding ARFs from rice, barley, corn, soybean and other important crops are isolated by screening the various filters of slides by hybridization using a 3?P-labeled or fluorescent NV. benthamiana KS+/Nb ARF #3 probe. or Arabidopsis 740 AT #120 NotI insert.

Rapid isolation of genomic clones encoding ARF, GTP binding proteins from nice. barley corn, soybean, and other plants

Genomic libraries containing sequences from rice, barley, com, soybean and other important crops are obtained from public and private sources, or are prepared from plant genomic DNAs. BAC clones containing entire plant genomes have been constructed and organized in minimal overlapping order. Individual BAC: are sheared to 500-1000 bp fragments and directly cloned into viral vectors. Approximate 200-500 clones that completely cover an entire BAC will form a BAC viral vector sublibrary. These libraries can be stored as bacterial glycerol stocks at -80C and as DNA at -20C. Genomic clones are identified by first probing filters of BACs with a 32P.1abeled or fluorescent N. benthamiana

KS+/Nb ARF #3 probe, or *’P-labeled Arabidopsis 740 AT #120 Not insert. BACs that hybridize to the probe are selected and their corresponding BAC viral vector sublibrary is used to produce infectious RNA. Plants that are transfected with the BAC sublibrary are screened for loss of function (for example, stunted plants). The inserts from these clones or their corresponding plasmid DNAs are characterized by dideoxy sequencing. This provides a rapid method to obtain the genomic sequence for the plant ARFs or GTP binding proteins.

Rapid isolation of cDNAs encoding human ADP-ribosylation factor

Libraries containing full-length human cDNAs from organisms such as brain, liver, breast, lung, etc. are obtained from public and private sources or prepared from human mRNAs. The cDNAs are inserted in viral vectors or in small subcloning vectors such as pBluescript (Strategene), pUC18, M13, or pBR322. Transformed bacteria (E. coli) are then plated on large petri plates or bioassay plates containing the appropriate media and antibiotic. Individual clones are selected using a robotic colony picker and arrayed into 96 well microtiter plates. The cultures are incubated at 37°C until the transformed cells reach log phase. Aliquots are removed to prepare glycerol stocks for long term storage at -80°C.

DNAs are rearrayed at high density on nylon or nitrocellulose filters or glass slides. Full- length cDNAs encoding ARFs from human brain, liver, breast, lung, etc. are isolated by screening the various filters or slides by hybridization with a 32P-labeled or fluorescent N. benthamiana KS+/Nb ARF #3 probe or Arabidopsis 740 AT #120 NotI insert.

Construction of a viral vector containing human cDNAs,

An ARFS5 clone containing nucleic acid inserts from a human brain cDNA library (Bobak et al., Proc. Natl. Acad. Su. USA 86:6101-6105 (1989)) was isolated by polymerase chain reaction (PCR) using the following oligonucleotides: HARFMIA, 5' TAC CTA GGG

WO (1/07600 PCT/US00/20261

CAA TAT CTT TGG AAA CCT TCT CAA G 3' (upstream) (SEQ ID NO:74),

HARFKI181X, 5 CGC TCGAGT CACTICTIGTITTIGAGC TGA TTG GCC AG 3 (downstream) (SEQ ID NO:75). The vent polymerase in the reaction was inactivated using phenol/chloroform. The PCR products are directly cloned into the Xhol, Avrll site TTO1A.

EXAMPLE 12

Silencing of phvioene desaturase in nicotiana benthamiana using a tobravirus vector.

Tobacco rattle tobravirus (TRV) is a bipartite positive-sense, single-stranded RNA - virus. TRV is able to infect a wide range of plant hosts, including Arabidopsis thaliana (unpublished data), Nicotiana species, Brassica campestris, Capsicum annuum,

Chenopodium amaranticolor, Glycine max, Lycopersicon esculentum, Narcissus pseudonarcissus, Petunia X hybrida, Pisum sativum, Solanum tuberosum, Spinacia oleracea, Vicia faba, : (http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/72010004.htm#SymptHost). TRV RNA-1 encodes proteins involved in viral replication (Replicase, 134/194 kDa) and movement (Movement Protein (mp) 29 kDa), as well as Cysteine Rich Protein ((CRP) 16 kDa) (Figure 21.A). An improved mutant of TRV RNA-1, pLSB-1, was isolated from an N. benthamiana plant that had been inoculated with a passaged sap extract of PpK20 (MacFarlane and

Popovich. Efficient expression of foreign proteins in roots from tobravirus veciors.

Virology, 267, 29-35 (2000)) from another N. benthamiana plant. Plants inoculated with pLSB-1 RNA-1 exhibit gene silencing more extensively compared to those inoculated with

PpK20 RNA-1. Virions were purified from the leaf tissue by a PEG precipitation method (Gooding GV Jr, Hebert TT (1967) A simple technigue for purification of tobacco mosaic virus in large quantities. Phytopathology 57(11):1285), RNA was isolated using the RNeasy

Mini Kit (Qiagen®), then cDNA was made using the cDNA Synthesis System (Gibco

BRL®) using the oligonucleotide 5'-

TTAATTAAGCATGCGGATCCCGTACGGGCGTAATAACGCTTACGTAGGCGAGGG

GTTTTAC-3’ (SEQ ID NO: 76). The full length TRV RNA-1 was PCR amplified using the oligonucleotides 5°- 76

Amended Sheet - 2003-06-24

ATGAAGAGCATGCTAATACGACTCACTATAGATAAAACATTITCAATCCTTTGAA

CGC-3' (upstream) (SEQ ID NO:77) and 5°- :

TTCATCTGGATCCCGGGCGTAATAACGCTTACGTAGGCG-3' (downstream) (SEQ ID NO:78) and cloned into pUC18 at the Sph I/Bam Hl sites. This TRV RNA-1 construct, pLSB-1, was verified by dideoxynucleotide sequencing and found to have 29 point mutations compared with the published sequence for PpK20 RNA-1 (Visser,P.B. and Bol,J.F. (1999).

ACCESSION AF166084). All of these point mutations are in the replicase gene, and many code for amino acid substitutions. The sequence of the mutant TRV RNA-1 viral sequence contained within pLSB-1 is as follows. 5'-

ATAAAACATTTCAATCCTTTGAACGCGGTAGAACGTGCTAATTGGATTTTGGTG ~

AGAACGCGGTAGAACGTACTTATCACCTACAGTTTTATTTTGTTTITCTITTITGGT

TTAATCTATCCAGCTTAGTACCGAGTGGGGGAAAGTGACTGGTGTGCCTAAAAC

CTTTTCTTTGATACTTTGTAAAAATACATACAGATACAATGGCGAACGGTAACTT

CAAGTTGTCTCAATTGCTCAATGTGGACGAGATGTCTGCTGAGCAGAGGAGTCA

TTTCTTTGACTTGATGCTGACTAAACCTGATTGTGAGATCGGGCAAATGATGCAA

AGAGTTGTTGTTGATAAAGTCGATGACATGATTAGAGAAAGAAAGACTAAAGAT

CCAGTGATTGTTCATGAAGTTCTTTCTCAGAAGGAACAGAACAAGTTGATGGAA

ATTTATCCTGAATTCAATATCGTGTTTAAAGACGACAAAAACATGGTTCATGGG

TTTGCGGCTGCTGAGCGAAAACTACAAGCTTTATTGCTTTTAGATAGAGTTCCTG

CTCTGCAAGAGGTGGATGACATCGGTGGTCAATGGTCGTTTTGGGTAACTAGAG

GTGAGAAAAGGATTCATTCCTGTTGTCCAAATCTAGATATTICGGGATGATCAGA

GAGAAATTTCTCGACAGATATTTCTTACTGCTATTGGTGATCAAGCTAGAAGTG

GTAAGAGACAGATGTCGGAGAATGAGCTGTGGATGTATGACCAATTITCGTGAAA

ATATTGCTGCGCCTAACGCGGTTAGGTGCAATAATACATATCAGGGTTGTACAT

GTAGGGGTTTTTCTGATGGTAAGAAGAAAGGCGCGCAGTATGCGATAGCTCTTC

ACAGCCTGTATGACTTCAAGTTGAAAGACTTGATGGCTACTATGGTTGAGAAGA

AAACTAAAGTGGTTCATGCTGCTATGCTTTTTGCTCCTGAAAGTATGTTAGTGGA

CGAAGGTCCATTACCTTCTGTTGACGGTTACTACATGAAGAAGAACGGGAAGAT

CTATTTCGGTTTTGAGAAAGATCCTTCCTTTTCTTACATTCATGACTGGGAAGAG

TACAAGAAGTATCTACTGGGGAAGCCAGTGAGTTACCAAGGGGATGTGTICTAC

TTCGAACCGTGGCAGGTGAGAGGAGACACAATGCTTTTTTCGATCTACAGGATA

77

Amended Sheet — 2003-06-24

GCTGGAGTTCCGAGGAGGTCTCTATCATCGCAAGAGTACT ACCGAAGAATATAT

ATCAGTAGATGGGAAAACATGGTTGTTGTCCCAATTTTCGATCT GGTCGAATCA

ACGCGAGAGTTGGTCAAGAAAGAC CTGTTTGTAGAGAAACAATTCATGGACAA

GTGTTTGGATTACATAGCTAGGTTATCTGACC AGCAGCTGACCATAAGCAATGT

TAAATCATACTTGAGTTCAAATAATTGGGTCTTATTCATAAAC GGGGCGGCCGT

GAAGAACAAGCAAAGTGTAGATTCTCGAGATTTACAGTTGTTGGCTCAAACTTT

GCTAGTGAAGGAACAAGTGGCGAGACCT GTCATGAGGGAGTTGCGTGAAGCAA

TTCTGACTGAGACGAAACCTATC ACGTCATTGACTGATGTGCTGGGTTTAATATC

AAGAAAACTGTGGAAGCAGTTTGCTAACAAGATCGCAGTCGGCGGATTCGTTGG

CATGGTTGGTACTCTAATTGGATTCTATC CAAAGAAGGTACTAACCTGGGCGAA

GGACACACCAAATGGTCCAGAACTATGTTACGAGAACTCGCACAAAACCAAGG

TGATAGTATTTCTGAGTGTTGTGTATGC CATTGGAGGAATCACGCTTATGCGTCG

AGACATCCGAGATGGACTGGTGAAAAAACT ATGTGATATGTTTGATATCAAACG

GGGGGCCCATGTCTTAGACGTTGAGAATCCGTGCCGCT ATTATGAAATCAACGA

TTTCTTTAGCAGTCTGTATTCGGCATCTGAGTCCGGTGAGAC CGTTTTACCAGAT

TTATCCGAGGTAAAAGCCAAGTCTGATAAGCT ATTGCAGCAGAAGAAAGAAAT

CGCTGACGAGTTTCTAAGTGCAAAATTCTCTAACTATTCT GGCAGTTCGGTGAGA

ACTTCTCCACCATCGGTGGTCGGTTCATCTCGAAGCGGACT GGGTCTGTTGTTGG

AAGACAGTAACGTGCTGACCCAAGCT AGAGTTGGAGTTTCAAGAAAGGTAGAC

GATGAGGAGATCATGGAGCAGTTTCTGAGTGGTCTTATTGACACTGAAGCAGAA

ATTGACGAGGTTGTTTCAGCCTTTTCAGCTGAATGTGAAAGAGGGGAAACAAGC

GGTACAAAGGTGTTGTGTAAACCTTTAACGCCAC CAGGATTTGAGAACGTGTTG

CCAGCTGTCAAACCTTTGGTCAGCAAAGGAAAAACGGTCAAACGTGTCGATTAC

TTCCAAGTGATGGGAGGTGAGAGATTACCAAAAAGGCCGGTTGTCAGTGGAGA

CGATTCTGTGGACGCT AGAAGAGAGTTTCTGTACTACTTAGATGCGGAGAGAGT

CGCTCAAAATGATGAAATTATGTCTCTGTATCGTGACTATTCGAGAGGAGTTATT

CGAACTGGAGGTCAGAATTACCCGCACGGACT GGGAGTGTGGGATGTGGAGAT

GAAGAACT GGTGCATACGTCCAGTGGTCACTGAACATGCTTATGTGTTCCAACC

AGACAAACGTATGGATGATTGGTCGGGATACTTAGAAGTGGCTGTTTGGGAAC G

AGGTATGTTGGTCAACGACTTC GCGGTCGAAAGGATGAGTGATTATGTCATAGT

TTGCGATCAGACGTATCTTTGCAATAACAGGTTGATCTTGGACAATTTAAGTGCC

CTGGATCTAGGACCAGTTAACTGTTCTTTTGAATTAGTTGACGGTGTACCTGGTT

GTGGTAAGTCGACAATGATTGTCAACTCAGCTAATCCTTGTGTCGATGTGGTTCT

CTCTACTGGGAGAGCAGCAACCGACGACTTGATCGAGAGATTCGCGAGCAAAG

GTTTTCCATGCAAATTGAAAAGGAGAGTGAAGACGGTTGATTCTTTTTTGATGC

ATTGTGTCGATGGTTCTTTAACCGGAGACGTGTTGCATTTCGACGAAGCTCTCAT

GGCCCATGCTGGTATGGTGTACTTTTGCGCTCAGATAGCTGGTGCTAAACGATGT

ATCTGTCAAGGAGATCAGAATCAAATTTCTTTCAAGCCTAGGGTATCTCAAGTT

GATTTGAGGTTTTCTAGTCTGGTCGGAAAGTTTGACATTGTTACAGAAAAAAGA

GAAACTTACAGAAGTCCAGCAGATGTGGCTGCCGTATTGAACAAGTACTATACT

GGAGATGTCAGAACACATAACGCGACTGCTAATTCGATGACGGTGAGGAAGAT

TGTGTCTAAAGAACAGGTTTCTTTGAAGCCCGGTGCTCAGTACATAACTTTCCTT

: CAGTCTGAGAAGAAGGAGTTGGTAAATTTGTTGGCATTGAGGAAAGTGGCAGCT

AAAGTGAGTACAGTACACGAGTCGCAAGGAGAGACATTCAAAGATGTAGTCCT

AGTCAGGACGAAACCTACGGATGACTCAATCGCTAGAGGTCGGGAGTACTTAAT

CGTGGCGTTGTCGCGTCACACACAATCACTTGTGTATGAAACTGTGAAAGAGGA

CGATGTAAGCAAAGAGATCAGGGAAAGTGCCGCGCTTACGAAGGCGGCTTTGG

: CAAGATTTTTTGTTACTGAGACCGTCTTATGACGGTTTCGGTCTAGGTTTGATGT

CTTTAGACATCATGAAGGGCCTTGCGCCGTTCCAGATTCAGGTACGATTACGGA

CTTGGAGATGTGGTACGACGCTTTGTTTCCGGGAAATTCGTTAAGAGACTCAAG

CCTAGACGGGTATTTGGTGGCAACGACTGATTGCAATTTGCGATTAGACAATGT

TACGATCAAAAGTGGAAACTGGAAAGACAAGTTTGCTGAAAAAGAAACGTTTC

TGAAACCGGTTATTCGTACTGCTATGCCTGACAAAAGGAAGACTACTCAGTTGG

AGAGTTTGTTAGCATTGCAGAAAAGGAACCAAGCGGCACCCGATCTACAAGAA

AATGTGCACGCGACAGTTCTAATCGAAGAGACGATGAAGAAGCTGAAATCTGTT

GTCTACGATGTGGGAAAAATTCGGGCTGATCCTATTGTCAATAGAGCTCAAATG

GAGAGATGGTGGAGAAATCAAAGCACAGCGGTACAGGCTAAGGTAGTAGCAGA

TGTGAGAGAGTTACATGAAATAGACTATTCGTCTTACATGTATATGATCAAATCT

GACGTGAAACCTAAGACTGATTTAACACCGCAATTTGAATACTCAGCTCTACAG

ACTGTTGTGTATCACGAGAAGTTGATCAACTCGTTGTTCGGTCCAATTTTCAAAG

AAATTAATGAACGCAAGTTGGATGCTATGCAACCACATTTTGTGTTCAACACGA

GAATGACATCGAGTGATTTAAACGATCGAGTGAAGTTCTTAAATACGGAAGCGG

CTTACGACTTTGTTGAGATAGACATGTCTAAATTCGACAAGTCGGCAAATCGCTT

CCATTTACAACTGCAGCTGGAGATTTACAGGTTATTTGGGCTGGATGAGTGGGC

GGCCTTCCTTTGGGAGGTGTCGCACACTCAAACTACTGTGAGAGATATTCAAAA

TGGTATGATGGCGCATATTTGGTACCAACAAAAGAGTGGAGATGCTGATACTTA

TAATGCAAATTCAGATAGAACACTGTGTGCGCTCTTGTCTGAATTACCATTGGA

GAAAGCAGTCATGGTTACATATGGAGGAGATGACTCACTGATTGCGTTTCCTAG

AGGAACGCAGTTTGTTGATCCGTGTCCAAAGTTGGCTACTAAGTGGAATTTCGA

GTGCAAGATTTTTAAGTACGATGTCCCAATGTTTTGTGGGAAGTTCTTIGCTTAAG

ACGTCATCGTGTTACGAGTTCGTGCCAGATCCGGTAAAAGTTCTGACGAAGTTG

GGGAAAAAGAGTATAAAGGATGTGCAACATTTGGCCGAGATCTACATCTCGCTG

AATGATTCCAATAGAGCTCTTGGGAACTACATGGTGGTATCCAAACTGTCCGAG

TCTGTTTCAGACCGGTATTTGTACAAAGGTGATTCTGTTCATGCGCTTTGTGCGC

TATGGAAGCATATTAAGAGTTTTACAGCTCTGTGTACATTATTCCGAGACGAAA

ACGATAAGGAATTGAACCCGGCTAAGGTTGATTGGAAGAAGGCACAGAGAGCT

GTGTCAAACTTTTACGACTGGTAATATGGAAGACAAGTCATTGGTCACCTTGAA

GAAGAAGACTTTCGAAGTCTCAAAATTCTCAAATCTAGGGGCCATTGAATTGTT

TGTGGACGGTAGGAGGAAGAGACCGAAGTATTTTCACAGAAGAAGAGAAACTG

TCCTAAATCATGTTGGTGGGAAGAAGAGTGAACACAAGTTAGACGTTITTITGACC

AAAGGGATTACAAAATGATTAAATCTTACGCGTTTCTAAAGATAGTAGGTGTAC

AACTAGTTGTAACATCACATCTACCTGCAGATACGCCTGGGTTCATTCAAATCG

ATCTGTTGGATTCGAGACTTACTGAGAAAAGAAAGAGAGGAAAGACTATTCAG

AGATTCAAAGCTCGAGCTTGCGATAACTGTTCAGTTGCGCAGTACAAGGTTGAA

TACAGTATTTCCACACAGGAGAACGTACTTGATGTCTGGAAGGTGGGTTGTATT

TCTGAGGGCGTTCCGGTCTGTGACGGTACATACCCTTTCAGTATCGAAGTGTCGC

TAATATGGGTTGCTACTGATTCGACTAGGCGCCTCAATGTGGAAGAACTGAACA

GTTCGGATTACATTGAAGGCGATTTTACCGATCAAGAGGTTTTCGGTGAGTTCAT

GTCTTTGAAACAAGTGGAGATGAAGACGATTGAGGCGAAGTACGATGGTCCTTA

CAGACCAGCTACTACTAGACCTAAGTCATTATTGTCAAGTGAAGATGTTAAGAG

AGCGTCTAATAAGAAAAACTCGTCTTAATGCATAAAGAAATTTATTGTCAATAT

GACGTGTGTACTCAAGGGTTGTGTGAATGAAGTCACTGTTCTTGGTCACGAGAC

GTGTAGTATCGGTCATGCTAACAAATTGCGAAAGCAAGTTGCTGACATGGTTGG

Co TGTCACACGTAGGTGTGCGGAAAATAATTIGTGGATGGTTTGTGTGTGTTGTTATC

AATGATTTTACTTTTGATGTGTATAATTGTTGTGGCCGTAGTCACCTTGAAAAGT :

GTCGTAAACGTGTTGAAACAAGAAATCGAGAAATTTGGAAACAAATTCGACGA

AATCAAGCTGAAAACATGTCTGCGACAGCTAAAAAGTCTCATAATTCGAAGACC

TCTAAGAAGAAATTCAAAGAGGACAGAGAATTTGGGACACCAAAAAGATTTTT

AAGAGATGATGTTCCTTTCGGGATTGATCGTTTIGTTTIGCTTTTTGATTITTATTTTA

TATTGTTATCTGTTTCTGTGTATAGACTGTTTGAGATTGGCGCTTGGCCGACTCA

TTGTCTTACCATAGGGGAACGGACTTTGTTTGTGTTGTTATTTTATTTGTATTTITA

TTAAAATTCTCAATGATCTGAAAAGGCCTCGAGGCTAAGAGATTATTGGGGGGT

GAGTAAGTACTTTTAAAGTGATGATGGTTACAAAGGCAAAAGGGGTAAAACCC +

CTCGCCTACGTAAGCGTTATTACGCCC-3' (SEQ ID NO:79).

RNA-2 encodes the capsid protein and two non-structural proteins, 2b and 2¢ (Figure 21.A.) A TRV RNA-2 construct expressing GFP was denived from a full-length clone of RNA2 of TRV isolate PpK20 (Mueller et al 1997. Journal of General Virology, 78, 2085-2088 (1997), MacFarlane and Popovich. Efficient expression of foreign proteins in roots from tobravirus vectors. Virology, 267, 29-35 (2000)). This TRV-GFP construct has the 2c gene of TRV RNA-2 replaced with the pea early browning virus (PEBV) coat protein promoter linked to GFP (MacFarlane and Popovich, 2000). This TRV-GFP construct was further modified by replacing the GFP gene with Pst 1 and Not I cloning sites to produce the plasmid pK20-2b-P/N. The phytoene desaturase (PDS) gene from N. benthamiana was PCR amplified from the plasmid pWPF187 using the following oligonucleotides 5’-

TGGTTCTGCAGTTATG (SEQ ID NO:80)

CATGCCCCAAATTGGACTTG-3' (upstream) (SEQ ID NO: 81) and 5’-TTTTCCTTTT )

GCGGCCGCTAAACTACGCTTGCTTCTG-3’ (downstream) (SEQ ID NO: 82). This PCR product was then subcloned into pK20-2b-P/N in the positive orientation. The resulting construct, TRV-PDS (Figure 21.B.), was linearized with Sma I and transcribed using T7

RNA polymerase (Ambion mMessage mMachine). Transcript RNA2 was mixed with transcripts from a full-length clone of TRV RNA-1 (pLSB-1).

TRV-PDS was inoculated onto N. benthamiana. After 6-7 days, chlorotic areas began to develop in the upper emerging leaves. After 8-10 days, these chlorotic areas developed into white areas. Samples from TRV-PDS infected plants were analyzed using 81

Amended Sheet — 2003-06-24 oo HPLC. HPLC analysis revealed a dramatically elevated level of phytoerie in TRV-PDS infected plants when compared to an uninoculated control.

EXAMPLE 13

Identification of nucleotide sequences involved in the regulation of plant development by cytoplasmic inhibition of gene expression in an anti sense orientation using viral derived

RNA (G-protein coupled receptor).

This example again demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a signal transduction pathway in plants. In addition, our results suggest that the Arabidopsis antisense transcript can turn off the expression of the N. ) _ benthamiana gene.

A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. Colonies from transformed E. coli were automatically picked using a Flexys robot and transferred to a 96 well flat bottom block containing terrific broth (TB) Amp 50 ug/ml. Approximately 2000 plasmid DNAs were isolated from overnight cultures using a BioRobot and infectious RNAs from 430 independent clones were directly applied to plants. One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. One set of plants transfected with 740 AT #88 (FIGURE 22) developed a white phenotype on the infected leaf issue. DNA sequence analysis revealed that this clone contained an Arabidopsis G-protein coupled receptor open reading frame (ORF) in the antisense orientation.

DNA sequencing and computer analysis.

A 758 bp Not! fragment of 740 AT #88 containing the G-protein coupled receptor cDNA was characterized. The nucleotide sequencing of 740 AT #88 was carried out by dideoxy termination using double stranded templates. Nucleotide sequence analysis and amino acid sequence comparisons were performed using DNA Strider, PCGENE and NCBI

Blast programs. FIGURE 23 shows the partial nucleotide sequence (SEQ ID NO:28) and amino acid sequence (SEQ ID NO:29) of 740 AT #88 insert. The nucleotide sequence from 740 AT #88 was compared with Brassica rapa cDNA L35812 (FIGURE 24, SEQ. ID. Nos: 82 : Amended Sheet — 2003-06-24

71 and 72), 91% identities and positives; and the octopus rhodopsin cDNA X07797 (FIGURE 25, SEQ ID NOs: 73 and 74), 68% identities and positives. The homology of :

DNAs encoding rhodopsin from plant and animal rhodopsin indicates that genes from one kingdom can inhibit the expression of gene of another kingdom. The amino acid sequence derived from 740 AT #88 was compared with octopus rhodopsin P31356 (FIGURE 26,

SEQ. ID. Nos: 75-77), 65% identities and positives. Table 8 shows the amino acid sequence comparison of 740 AT #88 with D. discoideum and Octopus rhodopsin: 58 — 62% identities and 63 — 65% positives.

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EXAMPLE 14

Identification of nucleotide sequences containing an Arabidopsis S18 ribosomal protein open reading frame.

One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. one set of plants transfected with 740 AT #377 (FIGURE 27) were severely stunted. DNA sequence analysis (FIGURE 28, SEQ ID NO: 78) revealed that this clone contained an

Identification of L 19 ribosomal protein gene involved in the regulation of plant growth by cytoplasmic inhibition of gene expression using viral derived RNA.

One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. One set of plants transfected with 740 AT #2483 (FIGURE 29) were severely stunted. DNA sequence analysis (FIGURE 30, SEQ ID NO:37) revealed that this clone contained an

Arabidopsis L19 ribosomal protein open reading frame (ORF) in the antisense onentation. The 740 AT #2483 nucleotide sequence exhibited a high degree of homology (77-78% idenuties and positives) to plant, L19 nbosomal proteins genes (Table 9). In addition, The 740 AT #2483 nucleotide sequence exhibited a high degree of homology (71 - 79% identities and positives) to yeast, insect and human L19 nbosomal proteins genes (Table 9). The 740 AT #2483 amino acid sequence comparison with human, insect and yeast nbosomal protein L19 shows 38 - 88% identities and 61 — 88% positives (Table 10). The high homology of DNAs encoding nbosomal L19 protein from human, plant, yeast and insect indicates that genes from one organism can inhibit the gene expression of an organism from another kingdom. 85

Amended Sheet —- 2003-06-24

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DNA sequencing and computer analysis.

The bp NotI fragment of 740 AT #909 containing the nbosomal protein L19 cDNA was characterized. The nucleotide sequencing of 740 AT #909 (FIGURE 31) was carried out by dideoxy termination using double stranded templates. Nucleotide sequence analysis and amino acid sequence comparisons were performed using DNA

Strider, PCGENE and NCBI Blast programs. FIGURE 32 shows nucleotide alignment of 740 AT #909 to human S5 6985 ribosomal protein L19 cDNA (SEQ ID NOs: 80 and 81 respectively). FIGURE 33 (SEQ ID NOs: 40 and 41) shows the amino acid sequence alignment of 740 AT #909 to human P14118 60S ribosomal protein L19. Table 11 ~~ shows the 740 AT #909 nucleotide sequence comparison to plant, drosophila, yeast, and human: 63-79% identities and positives. Table 12 shows the 740 AT #909 amino acid comparison to plant, human, mouse, yeast, and insect L19 ribosomal protein: 65-88% identities and 80-92% positives. 88

Amended Sheet — 2003-06-24

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. EXAMPLE 16 i

Construction of a cvtoplasmic inhibition vector in a positive sense containing A. thaliana HAT? homeobox-leucine zipper nucleotide sequence.

An Arabidopsis thaliana CD4-13 cDNA library was digested with NotI. DNA + fragments between 500 and 1000 bp were isolated by trough elution and subcloned into the

Notl site of pBS740. £. coli C600 competent cells were transformed with the pBS740 AT library and colonies containing Arabidopsis cDNA sequences were selected on LB Amp 50 pg/ml. : [solatjon of a gene encoding HAT? homeobox-leucine zipper. }

One to two weeks after inoculation, transfected Nicotiana benthamiana plants were visually monitored for changes in growth rates, morphology, and color. Plants transfected with 740 AT #855 (FIGURE 34) were moderately stunted. Plasmid 740 AT #855 contains the TMV-U1 126-, 193-, and 30-kDa ORFs, the TMV-US coat protein gene (US cp), the T7 promoter, an Arabidopsis thaliana CD4-13 Notl fragment, and part of the pUC19 plasmid.

The TMV-U1 subgenomic promoter located within the minus strand of the 30-kDa ORF controls the synthesis of the CD4-13 subgenomic RNA.

DNA sequencing and computer analysis.

The Notl fragment of 740 AT #855 was characterized: nucleotide sequence analysis and amino acid sequence comparisons were performed using DNA Strider, PCGENE and

NCBI Blast programs 740 AT #855 contained A. thaliana HAT 7 homeobox-luecine zipper cDNA sequence. The nucleotide sequence alignment of 740 AT #855 and Arabidopsis thaliana HATT homeobox protein ORF (U09340) was compared. FIGURE 35 (SEQ. ID. ’ Nos: 42 and 43) shows the nucleotide sequences of 740 #855 and A.thaliana HAT? homeobox protein ORF, and the amino acid sequence of A. thaliana HAT7 homeobox protein ORFs. The result show that 740 AT #855 contains a 3’ — untranslated region (UTR) of the 4.thaliana HAT7 homeobox protein ORF in a positive orientation, thus inhibited the expression of HAT 7 homeobox protein in the transfected N. benthamiana. Table 13 shows the 740 AT #855 nucleotide sequence comparison with A. thaliana, rat and human: 65-98% identities and positives 90

Amended Sheet — 2003-06-24

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EXAMPLE 17

Identification of human nucleotide sequences involved in the regulation of plant growth by cytoplasmic inhibition of gene expression using viral derived RNA containing human nucleotide sequences.

A human brain cDNA library are obtained from public and private sources or prepared from human mRNAs. The cDNAs are inserted in viral rectors or in small subcloning vectors and the cDNA inserts are isolated from the cloning vectors with appropriate enzymes, modified, and Not! linkers are attached to the cDNA blunt ends. The human cDNA inserts are subcloned into the Nol site of pBS740. E. coli C600 competent cells are transformed with the pBS740 sublibrary and colonies containing human cDNA sequences are selected on LB Amp 50 ug/ml. DNAs containing the viral human brain cDNA library are purified from the transformed colonies and used to make infectious RNAs that are directly applied to plants. One to three weeks post transfection, the plants developing severe stunting phenotypes are identified and their corresponding viral vector inserts are characterized by nucleic acid sequencing. organism by inhibition of an endogenous gene expression using viral derived RNA containing human nucleotide sequences,

A human brain cDNA library are obtained from public and private sources or prepared from human mRNAs. The cDNAs are inserted in viral vectors or in small subcloning vectors and the cDNA inserts are isolated from the cloning vectors with appropriate enzymes, modified, and Nol linkers are attached to the cDNA blunt ends. The human cDNA inserts are subcloned into the Nol site of pFastBacl. The human cDNA insert is removed from the shuttle plasmid pFastBac-HcDNA containing the human cDNA insert to pFastBacMam] as an EcoRI-Xbal fragment to construct pFastBacMam1-HcDNA according to Condreay ef al., (Proc. Natl. Acad. Sci.USA, 96: 127-132 (1999).

Recombinant virus is generated using the Bac-t0-Bac system (Life Technologies). Virus is further amplified by propagation in Spodoptera frugiperda cells. Phenotypic changes such as doubling rate, shape, size, kinase activity, cytokine release, response to excipients (e.g. toxic compounds, pathogens, etc.), division of cell culture, serum-free growth, activation of gene, and expression of receptor are detected microscopically, macroscopically, or by a biochemical method. Cells with phenotypic or biochemical changes are detected and the nucleic acid insert in the cDNA clone or in the vector that results in changes is then sequenced.

Humanizing plant homologue for expression of plant denved human protein

In order to obtain the corresponding plant cDNAs, the human clones responsible for causing changes in the transfected plant phenotype (for example, stunting) are used as probes. Full-length plant cDNAs are isolated by hybridizing filters or slides containing N. benthamiana cDNAs with **P-labelled or fluorescent human cDNA insert probes. The positive plant clones are characterized by nucleic acid sequencing and compared with their human homologs. Plant codons that encode for different amino acids are changed by site directed mutagenesis to codons that encode for the same amino acids as their human homologs. The resulting “humanized” plant cDNAs encode an identical protein as the human clone. The “humanized” plant clones are used to produce human proteins in either transfected or transgenic plants by standard techniques. Because the “humanized” cDNA is from a plant origin, it is optimal for expression in plants.

EXAMPLE 18

Gene silencing/co-suppression of genes induced by delivering an RNA capable of base pairing with itself to form double stranded regions.

Gene silencing has been used to down regulate gene expression in transgenic plants.

Recent experimental evidence suggests that double stranded RNA may be an effective stimulator of gene silencing/co-suppression phenomenon in transgenic plant. For example,

Waterhouse et al. (Proc. Natl. Acad. Sci. USA 95:13959-13964 (1998), incorporated herein by reference) described that virus resistance and gene silencing in plants could be induced by simultaneous expression of sense and antisense RNA. Gene silencing/co-suppression of plant genes may be induced by delivering an RNA capable of base pairing with itself to form double stranded regions.

Coe

This example shows: (1) a novel method for generating an RNA virus vector capable oo of producing an RNA capable of forming double stranded regions, and (2) a process to silence plant genes by using such a viral vector.

Step 1: Construction of a DNA sequence which after it is transcribed would generate an RNA molecule capable of base pairing with itself. Two identical, or nearly identical, ds

DNA sequences are ligated together in an inverted onentation to each other (i.e., in either a head to tail or tail to head orientation) with or without a linking nucleotide sequence between the homologous sequences. The resulting DNA sequence is then be cloned into a cDNA copy of a plant viral vector genome.

Step 2: Cloning, screening, transcription of clones of interest using known methods ~ in the art.

Step 3: Infect plant cells with transcripts from clones.

As virus expresses foreign gene sequence, RNA from foreign gene forms base pair upon itself, forming double-stranded RNA regions. This approach is used with any plant or non-plant gene and used to silence plant gene homologous to assist in identification of the function of a particular gene sequence.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.

All publications, patents, patent applications, and web sites are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, patent application, or web site was specifically and individually indicated to be incorporated by reference in its entirety.

Units which are used in this specification and which are not in accordance with the metric system may be converted to the metric system with the aid of the following table: 1 psi =6.895 x 10° Pa 1 inch =254x 107m 1°C = (°F -32) 5/9 94

Amended Sheet — 2003-06-24

Claims

1. A method for correlating a nucleic acid sequence of a donor organism with its function comprising the steps of: (a) introducing into a plant viral vector a library of a donor organism derived cDNA, genomic DNA or RNA inserts in a positive sense orientation; (b) expressing each insert of said library in a host plant; (c) detecting a phenotypic or biochemical change of said plant host resulting from the expression of an insert of said library; (d) determining the sequence of said insert; and (e) correlating the sequence of said insert with said phenotypic or biochemical change.

2. A method for correlating sequence with function comprising the steps of: (a) introducing into a plant viral vector a library of a donor organism derived cDNA, genomic DNA or RNA inserts in an anti-sense orientation; (b) expressing each insert of said library in a host plant; (c) detecting a phenotypic or biochemical change of said plant host resulting from the expression of an insert of said library; (d) determining the sequence of said insert; and (e) correlating the sequence of said insert with said phenotypic or biochemical change.

3. The method according claim 1 or 2 wherein said donor organism is selected from the group consisting of Monera, Protoctista, Fungi, and Animalia.

4. The method according to claim 1 or 2 wherein said donor organism is human.

5. The method according to claim 1 or 2 wherein said donor organism is mouse.

6. The method according to claim 1 or 2 wherein said donor organism is drosophila.

7. The method according to any one of claims 4-6 wherein said library is derived from tumor cells.

8 The method according to any one of claims 4-6 wherein said library is derived from ESTs.

9. The method according to claim 1 or 2 wherein said donor organism is a plant and said donor plant and said host plant belong to different family, order, class, subdivision, or division.

10. The method according to claim 1 or 2, wherein said donor plant is selected from the group consisting of food crops, seed crops, oil crops, omamental crops and forestry.

11. The method according to claim 1 or 2, wherein said host plant is selected from the group consisting of food crops, seed crops, oil crops, ornamental crops and forestry.

12. The method according to claim 1 or 2, wherein said host plant is Nicotiana.

13. The method according to claim 12, wherein said host plant is Nicotiana benthamina or Nicotiana cleaviandii.

14. The method according to claim 1 or 2, wherein said host plant is a monocot.

15. The method according to claim 1 or 2, wherein said plant viral vector is derived from a single strand, plus sense RNA virus.

16. The method according to claim 15, wherein said plant viral vector derived from the group consisting of a potyvirus, a tobamovirus, a bromovirus, a geminivirus, a hordivirus and a tobravirus.

17. The method according to claim 15, wherein said single strand, plus sense RNA virus is a multipartite virus.

18. The method of claim 1 or 2, wherein said plant viral vector comprises a native or non-native subgenomic promoter.

19. The method according to claim 1 or 2, wherein said insert encodes a protein selected from the group consisting of ribosomal proteins, GTP binding proteins, tumor suppressor, and G-protein coupled receptors.

20. The method according to claim 1 or 2, wherein said phenotypic change comprises growth rate, morphology or color changes.

21. The method of claim 1 wherein the plant viral vector containing said insert causes cytoplasmic inhibition of a gene expression.

22. The method of claim 1 wherein the plant viral vector containing said insert causes overexpression of a polypeptide product.

23. The method according to claim 1 or 2 further comprising the step of annotating each insert sequence with its associated phenotypic or biochemical change.

24. The method according to claim 1 or 2 where said method further comprising the steps of; (1) determining the nucleic acid sequence homology between human and plant sequences (2) altering the nucleic sequence of the plant sequence such that the altered plant sequence encodes same amino acid sequence as the human sequence.

25. The method accoding to claim 1 or 2 wherein said method further comprisng the steps of utilizing the function of sequence in step (d) in increasing the yield of a grain crop.

26. The method according to claim 16 wherein said plant viral vector is derived from a Tobacco Rattle Virus.

27. The method of claim 15 wherein said plant viral vector is derived from a barley stripe mosaic virus

28. A nucleic acid prodcut is comprising the sequence of step (d) of claim 1 or 2.

29. A polypeptide product encoding the sequence of step (d) of claim 1 or 2.