WO2001038513A2 - Shuffling of agrobacterium and viral genes, plasmids and genomes for improved plant transformation - Google Patents

Abstract

Methods for evolving plant vectors with improved characteristics by recursive recombination are provided. Plant vectors that are RNA or DNA polynucleotides, conjugated-DNA polynucleotides, and plasmids are provided, as are vectors that are agrobacterium strains and plant viruses. Agrobacterium vectors that have evolved such desired properties as broad host range, increased transformation efficiency, insert precision, targeted insertion, and targeting of T-DNA sequences to the chloroplast are provided. Agrobacterium strains, which are amenable to transforming a broad range of host species using simple transformation techniques such as vacuum infiltration or direct infection in planta, are provided. Plant virus vectors are provided that have evolved desired properties, including: rapid systemic spread, reduction of symptoms, and increased protein expression. Use of the evolved vectors to produce transgenic plants is provided. Methods and vectors for producing proteins in transgenic plants and for conferring pathogen-derived resistance are provided.

Description

SHUFFLING OF AGROBACTERIUM AND VIRAL GENES, PLASMIDS AND GENOMES FOR IMPROVED PLANT TRANSFORMATION COPYRIGHT NOTIFICATION PURSUANT TO 37 C.F.R. § 1.71(E)

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of United States application number 60/167,452, filed 11/23/99, and United States application number 60/202,233, filed May 5, 2000, which are incorporated in their entirety for all purposes. FIELD OF THE INVENTION

The present invention relates to vectors for use in the production of transgenic plants, and to methods of producing novel plant transformation vectors.

BACKGROUND OF THE INVENTION

Genetic modification of plant species utilizing the techniques of molecular biology has become widespread in recent years. A number of methods exist for the introduction of exogenous gene sequences into the genetic material of plants. The choice of technique is largely dependent on the application: whether stable heritable changes are sought or whether transient expression is desired, and on the plant species in question. Each technique offers a unique set of advantages and disadvantages. For example, for plant species which can be regenerated from cultured protoplasts, electroporation, the PEG method and microinjection are all suitable for introducing exogenous DNA into the protoplast nucleus. Electroporation techniques involve mixing plant protoplasts with DNA polynucleotides and subjecting the mixture to an electric pulse. The electric pulse causes the formation of holes in the protoplast cell membranes through which DNA can pass. It is a highly reproducible technique and can be adapted to both dicot and monocot species. However, it can be used only with species for which protoplast culture and regeneration protocols exist. Because it relies on the regeneration of protoplasts, production of transgenic plants is a lengthy process, often requiring several months.

The polyethylene glycol (PEG) method, while similar to electroporation in that DNA and protoplasts are mixed in vitro, relies on changes in cell membrane permeability induced by the addition of PEG to the medium. This method is simple and economical, as it requires no specialized equipment for its success. However, like electroporation, it requires that protoplast culture and regeneration techniques exist for the plant species in question.

Microinjection techniques, in which DNA or RNA polynucleotides are injected directly into the nucleus of protoplasts, are also applicable to a wide variety of species. A drawback of this technique is that it is time consuming and success is highly dependent on the skill of the individual experimentalist. Thus it is not widely applicable to the large scale generation of modified crops or other plants of commercial interest.

So-called "biolistic," or particle mediated bombardment techniques, shoot DNA-coated metal particles into plant cells or tissues at high speed. Thus, biolistic techniques are at least theoretically applicable to any tissue or species, regardless of whether culture and regeneration techniques are available. Transformation efficiency is variable and depends on selection of successful transformants.

A wide variety of dicotyledons can be readily transformed using agrobacterium mediated transformation. Agrobacterium mediated transformation relies on the ability of A. tumefaciens or A. rhizogenes to transfer DNA molecules called T-

DNA to a host plant cell. A. tumefaciens and A. rhizogenes are the causative agents of the plant neoplastic diseases crown gall and hairy root disease, respectively. Agrobacteria, which reside normally in the soil, detect soluble molecules secreted by wounded plant tissues through a specialized signal detection/transformation system. In the presence of these chemical signals, agrobacteria attach to the cell walls of wound exposed plant tissues. The agrobacteria then excise and transfer a portion of specialized DNA, designated T-DNA and delimited by "T-DNA borders," to the host plant cell nucleus where it is integrated into the chromosomal DNA. This DNA transfer system can be manipulated to transfer exogenous DNA situated between T-DNA borders to a host plant cell of choice.

However, monocots, especially those of commercially valuable species belonging to the family graminae, have proved less amenable to transformation by agrobacteria. Agrobacterial vectors which extend host range and increase transformation efficiency are of significant interest in adapting agrobacterium mediated transformation to presently recalcitrant plant species. Similarly, agrobacterium strains which are capable of introducing T-DNAs into a plants other than Arabidopsis by simple methods, such as vacuum infiltration, which do not require culture in vitro, would significantly reduce the material and labor costs associated with generating genetically modified plants. Viral vectors derived from a large selection of plant viruses have proven adaptable for use in the transformation of dicot and monocot species. The majority of plant viruses have genomes consisting of RNA polynucleotides, although a number of DNA viruses, including cauliflower mosaic virus, have been widely used. With the advent of simple in vitro transcription protocols, transformation by RNA viruses has become widespread. Typically, viral replication and expression occurs in the plant cytoplasm. Therefore virally mediated techniques are most useful where transient, rather than stable, genetic changes are the goal. For example, high levels of transient expression, suitable for the production and recovery of exogenous proteins can be achieved using plant viral vectors. Although expression of exogenous sequences via viral vectors is of significant benefit, it is not without risk. Viral vectors currently employed are capable of spreading not only within a host plant but also between plants. This makes unintentional infection of neighboring plants a potential hazard, restricting the use of many vectors to non-food crop plants such as tobacco. Thus, the development of plant viral expression vectors which safely infect valuable food and other crop species is of great interest. The present invention provides methods for developing novel and improved plant vectors for use in the production of transgenic plants. These methods are applicable to polynucleotide vectors suitable for introduction by electroporation, PEG, microinjection, particle mediated bombardment; plasmids and agrobacterium strains for use in agrobacterium mediated transformation and novel plant virus vectors. Vectors can be developed using the methods of the invention which address the issues desicribed above, as well as other disadvantages.

SUMMARY OF THE INVENTION

The present invention utilizes recursive recombination, e.g., "DNA shuffling," techniques to develop new plant vectors with improved properties. The vectors of the invention include polynucleotides such as naked RNA and DNA, conjugated DNA molecules, and plasmids, as well as plant viruses and agrobacterium strains. The methods of the invention provide for the recombination, in silico, in vitro or in vivo, of DNA fragments corresponding to plant vector components and their homologues. One or more cycle of recursive recombination is followed by screening or selection to identify recombinant vector components, or vectors, which have evolved towards a desired property. Multiple reiterations of the recombination (e.g., via shuffling) process can be employed to achieve the property desired. In some embodiments, diversity among substrate fragments is increased using error prone PCR, mutagenic amplification and/or site directed mutagenesis. Preferred embodiments include vectors which are agrobacterium, and agrobacterium derived plasmids. Other preferred embodiments are plant viruses. Methods of the invention use various screening or selection protocols to identify recombinant vectors or vector components with desired properties. Embodiments of the invention employ a variety of screening techniques, including: PCR, LCR, hybridization, proteomics and detection of reporters, (such as green fluorescent protein (GFP), β-glucuronidase (GUS), luciferase, or proteins incuded by the maize Lc gene). Screening techniques of the invention also include selection protocols including herbicide resistance and antibiotic resistance. In some cases, the selection protocols rely on detection of negatively selectable markers, such as dhll, codA, tms2 and NIA2, and their homologues.

The methods of the invention provide for the generation of Agrobacterium vectors which have evolved new and improved properties. Such properties include, but are not restricted to: insert precision, targeted insertion, broad host range, increased transformation efficiency targeting of T-DNA to the chloroplast, and improved amenability to simplified transformation techniques. These methods pertain to Agrobacterium strains, and to agrobacterium-derived plasmids, including the plasmids of binary vector systems. Embodiments of the invention provide for binary vector systems that supply necessary virulence functions on a plasmid, or alternatively, integrated into an Agrobacterium chromosome. In some embodiments, one or more agrobacterium T-DNA borders are included in the fragments to be shuffled. In preferred embodiments, the T- DNA borders are contiguous with PCR primer binding sites. In some embodiments, the fragments include agrobacterium virulence genes, e.g., virA, virB, virC, virD, virE, virG and ChvE. The fragments optionally comprise an entire agrobacterium genome. Alternative embodiments of the invention provide for Agrobacterium vectors which include A. tumefaciens or A. rhizogenes.

Preferred embodiments of the invention relate to the development of vectors which insert T-DNA sequences precisely into the host plant chromosome, or insert T-DNA sequences into a predetermined site in the host plant chromosome. In one preferred embodiment, regions of sequence similarity with a desired insertion site flank the T-DNA border or borders. In another preferred embodiment, a promoter or enhancer is included adjacent to the region of sequence similarity. The invention also provides for screening the recombinants of such embodiments in host plants or plant cells having detectable markers which are activated by insertion of the evolved vector at a predetermined site. An especially preferred embodiment relates to screening such recombinants in transgenic plants.

Alternative embodiments of the invention are directed towards evolution of agrobacterium vectors with broad host ranges, including both monocot and dicot plant species. In a preferred embodiment, virulence gene sequences are recombined, e.g., shuffled, to evolve agrobacterium vectors with a broad host range. VirA fragments are optionally recombined and the resulting recombinant sequences assessed for their ability to activate a reporter linked to a vir promoter. Embodiments include methods of screening for improved host range in intact plants and in plant cell cultures.

Methods for evolving agrobacterium vectors which target T-DNA sequences to the chloroplast are also a feature of the invention. In preferred embodiments, virD2 and/or virE2 genes are the substrates for nucleic acid diversification (e.g., DNA shuffling) procedures, optionally including one or more of a nuclear localization signal, and a chloroplast localization signal. In an especially preferred embodiment, screening for chloroplast targeting is performed by detecting a marker localized to the chloroplast.

The invention provides methods of producing plant virus vectors with novel, desired properties. The methods of the invention are equally applicable to both RNA and DNA viruses. Properties such as rapid systemic spread; reduction of viral symptoms; increased viral and exogenous gene expression; and production of proteins restricted to transgenic host plants are among those provided for by the methods of the invention.

A method for evolving a plant virus vector with rapid systemic spread is an embodiment of the invention. An embodiment provides for inoculating viruses incorporating the recombinant library generated by DNA shuffling onto leaves of an intact plant followed by recovery and analysis of viruses from leaves distal to the site of inoculation. Viral movement proteins or complete viral genomes are optionally provided as substrates for nucleic acid diversification, e.g., by DNA shuffling.

Other embodiments of the invention relate to methods of evolving viral vectors which result in a reduction in symptoms following infection. Preferred targets include viral coat proteins, viral movement proteins, viral replicases and viral recombinases, as well as entire viral genomes.

The invention includes methods for developing virus vectors which give rise to an increase in expression of either viral or exogenous DNA sequences. Optionally viral or plant promoters are provided as substrates for diversification, e.g., by DNA shuffling. For example, the promoters can be, e.g., a cauliflower mosaic virus 35S promoter. Alternatively, fragments constituting a viral genome are used as the substrate for generating recombinant libraries. A marker gene is optionally cloned between recombinant, e.g., shuffled, 5' and 3' regions of a viral genome and the resulting recombinant virus is inoculated onto the leaf of a host plant.

Methods for evolving vectors which confer pathogen-derived resistance to plant pathogens are also features of the invention, as are plant virus vectors which confer pathogen-derived resistance to plant pathogens.

A feature of the present invention is the construction of novel viral vectors for protein production in transgenic plants. Screening of such viral vectors is accomplished by expressing a recombinant virus which lacks a coat protein in a transgenic plant which supplies the missing coat protein in trans. Another aspect of the invention is a virus vector which has a polylinker in place of a coat protein. Preferred embodiments include vectors which have an exogenous polynucleotide sequence cloned into the polylinker and expressed under regulatory control of the coat protein promoter. Especially preferred embodiments include vectors which express polynucleotides that encode proteins which are biofuels, industrial enzymes, nutritional enzymes, nutraceuticals, pharmaceuticals, plantibodies, antigens, biosynthetic enzymes, or the like. A preferred embodiment is a virus which is a tobamovirus. Tobamoviruses which infect Brassicas, including Arabidopsis as well as tobamoviruses which infect legumes and other plants, including monocots are embodiments of the invention. Another feature of the invention is a method of producing proteins in transgenic plants using the recombinant plant virus lacking a coat protein of the invention. In some embodiments, the methods for evolving improved plant vectors involve diversification and screening or selection of RNA viral vectors in vivo. A plurality of RNA viral vectors containing genes of interest are introduced into a cell and the cells are grown under conditions permitting replication and recombination of the viral sequences. Optionally, the viral vectors are recovered, and the recombination is performed recursively. After recombination of the viral RNA molecules, a viral vector comprising a gene with a desired property is identified. The viral vectors are introduced into cells by inoculating the cell with infectious RNA transcripts. Alternatively, a plurality of cDNA molecules corresponding to viral transcripts are used to introduce the genes of interest into the cell. In the latter case, the plurality of cDNA molecules can be introduced by a variety of techniques including, electroporation, microinjection, biolistics, agrobacterium mediated transfromation or agroinfection. In preferred embodiments the RNA viral vectors are plant virus vectors, and the cells are plant cells. Such vectors include, tabamoviruses, potyviruses, tobraviruses, and potexviruses. In some embodiments, the plant cells are isolated cells grown in culture. In other embodiments, the plant cells are plant protoplasts, plant tissues, plant organs or intact plants.

In an embodiment, two viral vectors having complementary mutations in proteins involved in systemic infection are used to introduce nucleic acids comprising genes of interest. Upon recombination, infectivity is restored, thereby facilitating selection of recombinant genes of interest. Exemplary proteins involved in systemic infection include viral coat proteins and viral movement proteins.

Another aspect of the invention relates to libraries of recombinant sequences generated by nucleic acid diversification procedures, such as DNA shuffling. Such libraries may be vectors, vector components, vector genomes, or the like. For example, libraries composed of agrobacterial or viral sequences are included.

Agrobacterium strains and virus vectors incorporating recombinant, e.g., shuffled library sequences are also a feature of the invention. Similarly, libraries derived from transgenic plants transformed by the vectors of the invention are a feature of the invention.

Vectors produced by the methods of the invention are an aspect of the invention. Preferred embodiments include vectors which are agrobacterium plasmids or agrobacterium strains. Other embodiments of the invention are evolved plant viruses. Preferred embodiments refer to evolved plant viruses which include an exogenous DNA sequence. A preferred embodiment has a library polynucleotide inserted to give rise to a protein that is expressed as a fusion with a coat protein. Optionally, the polypeptide encoded by the inserted polynucleotide is expressed on the outside surface of a virus particle. In alternative embodiments, the exogenous polynucleotide sequence is a viral or plant promoter linked to a reporter gene, such as GFP, luciferase, β-glucuronidase, or the maize Lc regulatory gene. Other embodiments include vectors which have incorporated exogenous polynucleotide sequences which are selectable markers. Such selectable markers can be genes which confer resistance to an antibiotic or to a herbicide. Alternatively, the selectable markers can be negatively selectable markers, including homologues of the dhll, codA, tms2 and NIA2 genes. Other embodiments of the invention include evolved plant viruses which express exogenous DNA sequences under the control of a cauliflower mosaic virus 35S promoter. Alternative embodiments relate to evolved plant virus vectors which are positioned adjacent to agrobacterium borders and introduced into a plant host by Agroinfection. The invention also provides for introducing evolved viral vectors into plant protoplasts by other methods including electroporation, microinjection, biolistics and direct mechanical inoculation. Another aspect of the present invention includes plants, plant cells and plant explants transformed by the vectors of the invention to produce transgenic plants. Additionally, libraries of sequences derived from transgenic plants produced using the vectors of the invention are a feature of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 schematically illustrates a replicating Geminivirus vector system.

Figure 2 schematically illustrates recombination of Geminivirus genome to alter size selectivity of the movement proteins BR1 and BL1.

Figure 3 schematically illustrates production of a recombinant Geminivirus genome library in a common region (CR) cassette. Figure 4 schematically illustrates control (CV) and test (TV) reporter vectors for relaxing size selectivity of Geminivirus movement proteins.

Figures 5A and 5B schematically illustrate recombination of Geminivirus genomes including the common region (CR), leftward elements, including the coat protein promoter (P_CP), and Replicase (AC1) gene. The vecors illustrated in figures 5A and 5B differ by the position of a restriction enzyme recognition site (RSI). Figure 6 schematically illustrates production of a recombinant Geminivirus genome library in alternative common region cassettes lacking and possessing a visual reporter.

DETAILED DISCUSSION OF THE INVENTION

The present invention relates to the production of plant vectors with improved characteristics. Recursive recombination, e.g., DNA shuffling, is employed to develop plant vectors with novel and improved properties. Some embodiments of the invention are agrobacterium vectors, while other embodiments include evolved plant viruses. Preferred embodiments include agrobacterium vectors which extend the host range, and increase transformation efficiency, as well as which improve insert precision and simplify transformation techniques. Other preferred embodiments are viral vectors which have evolved properties such as rapid systemic spread, altered host range, and reduction of viral symptoms after infection. Vectors which facilitate the production of commercially valuable products in transgenic plants, while simultaneously reducing the risk of contamination of neighboring crops are provided. Vectors which confer pathogen- derived resistance to plant pathogens are provided. Another aspect of the invention relates to the production of transgenic plants using the evolved vectors and to the transgenic plants produced thereby. In one aspect, a transgenic plant expressing a viral coat protein is used to produce a vector encoded polypeptide.

DEFINITIONS Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present invention, the following terms are defined below.

The term "shuffling" is used herein to indicate recombination between non-identical sequences. For example, "DNA shuffling" involves recombination of deoxyribonucleic acid (DNA) segments. In some embodiments shuffling may include crossover via homologous recombination or via non-homologous recombination, such as via cre/lox and/or flp/frt systems. Shuffling can be carried out by employing a variety of different formats, including for example, in vitro and in vivo shuffling formats, in silico shuffling formats, shuffling formats that utilize either double-stranded or single-stranded templates, primer based shuffling formats, nucleic acid fragmentation-based shuffling formats, and oligonucleotide-mediated shuffling formats, all of which are based on recombination events between non-identical sequences and are described in more detail or referenced herein below, as well as other similar recombination-based formats. In one class of embodiments, nucleic acid shuffling involves the recursive recombination of nucleic acid sequences.

"Screening" is, in general, a two-step process in which one first determines which cells, organisms or molecules, do and do not express a detectable marker, or phenotype (or a selected level of marker or phenotype), and then physically separates the cells, organisms or molecules, having the desired property. Selection is a form of screening in which identification and physical separation are achieved simultaneously by expression of a selectable marker, which under some circumstances, allows cells expressing the marker to survive while other cells die (or vice versa). Screening reporters include luciferase, β-glucuronidase, green fluorescent protein (GFP), and the maize Lc (anthocyanin regulatory) gene. Selectable markers include antibiotic and herbicide resistance genes. A special class of selectable markers are negatively selectable markers. Cells or organisms expressing a negatively selectable marker die under appropriate selection conditions while organisms lacking or having a non- functional form of the marker survive. Examples of negatively selectable markers useful in the context of plant genetic engineering include a number of genes involved in herbicide metabolism, including: dlhl, codA, tms2 and NIA2.

The term "gene" is used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include nonexpressed polynucleotides that, for example, form recognition sequences for other proteins. Non- expressed regulatory sequences include "promoters" and "enhancers", to which regulatory proteins such as transcription factors bind, resulting in transcription of adjacent or nearby sequences. A "wild-type" gene, or DNA or RNA sequence, is a gene, or sequence, which occurs in an organism in nature.

An "exogenous" gene or "transgene" is a gene foreign (or heterologous) to the cell, or homologous to the cell, but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous genes can be expressed to yield exogenous polypeptides. A "transgenic" organism is one which has a transgene introduced into its genome. Such an organism may be either an animal or a plant. The site at which a transgene is located in the genome is referred to as its "site of insertion" or alternatively, its "insertion site." The integration of a transgene or other vector-borne sequence into a predetermined location within the host genome is referred to as "targeted insertion," while "insert precision" refers to the insertion of a single, defined copy of a T- DNA without rearrangement of the T-DNA ends.

A "vector" is a means by which an exogenous DNA is introduced into a "host cell. A vector can be a nucleic acid polynucleotide, most typically a plasmid or virus, but also including a naked RNA polynucleotide, a naked DNA polynucleotide, a poly-lysine -conjugated DNA, a pepti de-conjugated DNA, a liposome-conjugated DNA, or the like, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium. Once inside the host cell, replication and in some cases expression, are established under control of vector origin and regulatory sequences. "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including electroporation, microinjection, biolistics (or particle bombardment-mediated delivery), or agrobacterium mediated transformation. A "parental" cell, or organism, is an untransformed member of the host species giving rise to a transgenic cell, or organism.

A "host species" is the recipient of a transforming vector. A vector which is capable of transforming a wide variety of host species is said to have a "broad host range" while vectors which transform only one or a few host species is said to have a "narrow host range."

"Agrobacterium" are soil-borne plant-pathogenic bacterium, the two predominant species of which are A. tumefaciens and A. rhizogenes. Agrobacterium carry a tumor causing plasmid designated Ti, modified versions of which serve as common plant vectors. During infection, the "T region", a portion of the Ti plasmid from which "T-DNA" is derived is transferred, along with any intervening sequences, to the host plant cell. Imperfect 25 base pair repeat sequences designated the "right T-DNA border" and the "left T-DNA border" define the limits of the T-DNA, and are utilized in the transfer of the T-DNA from the agrobacterium to the plant cell.

Also located on the Ti plasmid are the "virulence" or "vir" genes of agrobacterium. The products of the vir genes contribute various functions required for transfer of the T-DNA, and include virA, virB, virC, virD, virE and the chromosomal gene, ChvE. The vir genes are regulated by a signal transduction system in which the product of the virA gene, the VirA protein, acts as sensor to detect secreted molecules from the plant. Binding of a secreted signal molecule and activation of VirA results in the induction of other vir genes via transcriptional activation. The regulatory region responsible for activation of a vir gene is referred to as a "vir promoter."

The term "Agroinfection" refers to the introduction of plant infectious agents, for example, viruses, into plants by Agrobacterium. More generally "agrobacterium mediated transformation" refers to the transfer of any DNA sequences present between T-DNA ends into a plant cell by an agrobacterium.

"Binary vector system" generally refers to a two plasmid system in which Vir function is supplied on a "helper plasmid," frequently a modified Ti plasmid, to mediate transfer of a T-DNA located on a separate plasmid. Alternatively, it is possible to supply Vir function by sequences integrated into an Agrobacterium chromosome.

"PCR," or the "polymerase chain reaction," is an in-vitro method for the amplification of specific DNA sequences. PCR utilizes multiple cycles of 1) denaturation of the target DNA sequence, 2) annealing of DNA sequences serving to prime DNA polymerization to the target DNA, and 3) polymerase mediated extension of the annealed sequence to produce multiple copies of a target DNA sequence. Single stranded oligonucleotides, typically in the range of 15-30 bases, which hybridize to portions of the target DNA, are referred to as "PCR primers." "Error prone PCR," "mutagenic amplification" and "site directed mutagenesis" are methods for introducing mutations, or alterations, in the nucleotide composition of target DNA molecules. An "inverse polymerase chain reaction" is a method of amplifying a target of an unknown sequence, for example a transgene insertion site, in which PCR primers corresponding to sequences flanking the target are provided. "LCR" or "ligase chain reaction" refers to a variation of PCR, in which T4 ligase mediates joining of fragments serving as substrates for amplification. The term "sequence similarity" means that two nucleic acid sequences are identical or share regions of sequence identity.

Viral vectors are introduced into cultured plant tissues or cells or directly onto intact plants at a "site of inoculation." "Systemic spread" refers to the replication and dissemination of the virus throughout the plant in a process which depends on the activity of viral "movement proteins." Viral "coat proteins" are used to package the replicated viral RNA or DNA genome of many plant viruses for transport within and between plant hosts. In the context of a viral vector, as elsewhere in this application, 5' refers to the left-hand or "upstream" direction of a nucleic acid molecule and 3' refers to the right- hand or "downstream" direction of a nucleic acid molecule. Hence a "5' region" of a viral nucleic acid lies upstream of a "3' region," and the direction of transcription reads from 5' to 3'. The use of nucleic acid sequences derived from a plant pathogen, most typically a virus, to confer resistance to plant pathogens in transgenic plants is referred to as "pathogen-derived resistance."

INTRODUCTION

The present invention provides a method for evolving various plant vectors, for example, agrobacterium and viruses, to acquire desired properties. Prior to the invention, construction of vectors was limited, on one hand, to the introduction of specific rational changes by site directed mutagenesis techniques, and on the other hand, to random, and often deleterious, mutagenesis techniques. The invention utilizes recursive recombination, e.g., DNA shuffling, technologies, in a process of directed evolution, to select and optimize mutations leading to desired characteristics in plant transformation vectors. These vectors are used to produce transgenic plants which are useful for a wide variety of purposes, including the production of commercially important products such as proteins, lipids, and biosynthetic polymers. Additionally, the invention relates to the production and use of evolved plant viruses which confer pathogen-derived resistance to plant pathogens.

General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics related to, e.g., the cloning and expression of agrobacterium vir genes, viral coat proteins, replicases and movement proteins, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1- 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel")). Similarly, examples of techniques sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86. 1173; Guatelli et al. d990) Proc. Natl. Acad. Sci. USA 87. 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et a/. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references therein, in which PCR amplicons of up to 40kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra.

The present invention also relates to host cells and organisms which are transformed with vectors of the invention, and the production of polypeptides of the invention, e.g. viral coat proteins and other proteins, and polypeptides encoded by exogenous DNAs, by recombinant techniques. Host cells are genetically engineered (i.e., transformed, transduced or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, an agrobacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al, Molecular Biology of Plant Tumors, (Academic Press, New York, 1982) pp. 549-560; Howell, US 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)), use of pollen as vector (WO

85/01856), or use oi Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al., Science 233, 496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci. USA 80, 4803 (1983)).

The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic plants. Plant regeneration from cultured protoplasts is described in Evans et al., "Protoplast Isolation and Culture," Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York, 1983); Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, (1983) pp. 12-29, (Birkhauser, Basal 1983); Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," Protoplasts (1983) pp. 31-41, (Birkhauser, Basel 1983); Binding, "Regeneration of Plants," Plant Protoplasts, pp. 21-73, (CRC Press, Boca Raton, 1985).

The present invention also relates to the production of transgenic organisms, which may be bacteria, yeast, fungi, or plants. A thorough discussion of techniques relevant to bacteria, unicellular eukaryotes and cell culture may be found in references enumerated above and are briefly outlined as follows. Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which may be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further, below), etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect plant cells or incorporated into Agrobacterium tumefaciens related vectors to infect plants. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, B., et al, Protein Expr. Purif 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY.

TRANSFORMING NUCLEIC ACIDS INTO PLANTS. Preferred embodiments pertain to the production of transgenic plants using the evolved vectors of the invention. Techniques for transforming plant cells with nucleic acids are generally available and can be adapted to the invention by the use of evolved plasmids, viruses, and components thereof, and by the use of agrobacterium strains comprising evolved vectors. In addition to Berger, Ausubel and Sambrook, useful general references for plant cell cloning, culture and regeneration include Jones (ed) (1995) Plant Gene Transfer and Expression Protocols— Methods in Molecular Biology, Volume 49 Humana Press Towata, NJ ("Jones"); Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY ("Payne"); and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York)

("Gamborg"). A variety of cell culture media are described in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL ("Atlas"). Additional information for plant cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma- Aldrich, Inc (St Louis, MO) (Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, MO) (Sigma-PCCS). Additional details regarding plant cell culture are found in Croy, (ed.) (1993) Plant Molecular Biology Bios Scientific Publishers, Oxford, U.K.

The nucleic acid constructs of the invention, e.g., plasmids, viruses, DNA and RNA polynucleotides, are introduced into plant cells, either in culture or in the organs of a plant by a variety of conventional techniques. To use the evolved, e.g., shuffled, sequences of the invention, recombinant DNA or RNA vectors suitable for transformation of plant cells are isolated and/or prepared. To introduce an exogenous DNA, which can itself be a recombinant, e.g., shuffled, DNA, the exogenous DNA sequence can be incorporated into an evolved vector of the invention and transformed into the plant as indicated above. Where the sequence is expressed, the sequence is optionally combined with transcriptional and translational initiation regulatory sequences which direct the transcription or translation of the sequence from the exogenous DNA in the intended tissues of the transformed plant.

The DNA constructs of the invention, for example plasmids, or naked or variously conjugated-DNA polynucleotides, (e.g., polylysine-conjugated DNA, peptide- conjugated DNA, liposome-conjugated DNA, etc.) can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant cells using ballistic methods, such as DNA particle bombardment.

Microinjection techniques for injecting e.g., cells, embryos, and protoplasts, are known in the art and well described in the scientific and patent literature. For example, a number of methods are described in Jones (ed) (1995) Plant Gene

Transfer and Expression Protocols— Methods in Molecular Biology, Volume 49 Humana Press Towata NJ, as well as in the other references noted herein and available in the literature.

For example, the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al., EMBO J. 3:2717 (1984).

Electroporation techniques are described in Fromm, et al., Proc. Nat'l. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein, et al., Nature 327:70-73 (1987). Additional details are found in Jones (1995) supra.

AGROBACTERIUM MEDIATED TRANSFORMATION In preferred embodiments, DNA constructs comprising suitable T-DNA border sequences are introduced into an Agrobacterium host vector. The virulence functions of the Agrobacterium host directs the insertion of the construct into the plant genomic DNA. For example, any one or more of the following are optionally combined with T-DNA flanking sequences: a coding sequence, a non-coding sequence, a structural gene, a disabled gene, a promoter, an enhancer and a marker and inserted into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium-mediated transformation techniques, including disarming and use of binary vectors, are also well described in the scientific literature. See, for example Horsch, et al., "A simple and general method for transferring genes into plants." Science 233:496-498 (1984), and Fraley, et al., "Expression of bacterial genes in plant cells." Proc. Nat'l. Acad. Sci. USA 80:4803 (1984) and recently reviewed in Hansen and Chilton, "Lessons in gene transfer to plants by a gifted microbe." Current Topics in Microbiology 240:22-51 (1998) and Das (1998) "DNA transfer from Agrobacterium to plant cells in crown gall tumor disease." Subcellular Biochemistry 29: Plant Microbe Interactions:343-363. These techniques are adapted in the present invention with the use of evolved vectors and vector components, and the use of such modified vectors in the production of transgenic plants.

Agrobacteria are gram-negative, soil-borne plant pathogens that cause neoplastic growth in susceptible plants. The most prevalent pathogenic strains,

Agrobacterium tumefaciens and Agrobacterium rhizogenes cause crown gall and hairy root disease, respectively. In recent years, the art of plant transgenesis has taken advantage of genetically modified agrobacterium strains to transfer exogenous DNA to host plants by means of agrobacterium mediated transformation, a process which utilizes the naturally occurring DNA transfer system of these pathogenic bacteria. The present invention specifically provides for the development of Agrobacterium plasmids and Agrobacterium strains with improved properties, for use in the production of transgenic plants.

Genes located on the Ti, or "tumor inducing," plasmid of A. tumefaciens, are involved in DNA transfer and tumor induction. The transferred DNA or T-DNA is derived from the T-region of the Ti plasmid. This region is delimited by the presence of two imperfect 25 base pair repeats designated T-DNA borders. DNA sequences, including, for example, the exogenous sequences selected for targeting to a plant genome of the invention, can be cloned between the T-DNA borders and subsequently transferred to a host plant cell. Alternatively, the exogenous sequence can be cloned adjacent to a single right T-DNA border and transferred to the host plant cell.

The virulence, or vir, gene products activate and facilitate transfer of T- DNA to a host plant cell. VirA is a transmembrane protein which acts as a sensor, detecting the presence of molecules, such as acetosyringone (3',5'-dimethoxy-4'- hydroxyacetophenone), which are secreted by wounded or metabolically active target cells. When activated by such molecules, VirA exhibits kinase activity that activates the VirG protein. In turn, VirG induces transcription of the virB, virC, VirD and virE operons. The VirB proteins are thought to form a conjugation-like pore in the bacterial surface through which the T-DNA passes upon transfer. VirC, VirD and VirE proteins are involved in the production and transfer of the T-DNA strand. In the present invention, these virulence genes, either singly or in combination are shuffled to produce virulence proteins with novel functional activities. These evolved virulence proteins are encoded by components of plasmids and the agrobacterium strains which harbor them and confer various desirable phenotypic modifications, such as increased host range, and improved T-DNA transfer efficiency under standard or simplified transformation protocols.

As the T-DNA is released from the plasmid, the VirD2 protein binds the right T-DNA border. For effective transformation, VirE2 function is transported to the host plant cell by the Agrobacterium. While it is clear that VirE2 is involved in integration of the T-DNA into the host cell chromosome, it does not appear to be essential for transfer of the T-DNA from the Agrobacterium to the host cell, and may be supplied by a second Agrobacterium or by the host cell itself (Ream "Import of Agrobacterium tumefaciens virulence proteins and transferred DNA into plant cell nuclei." Subcellular Biochemistry 29: Plant-Microbe Interactions:365-384 (1998), and references therein). Multiple molecules of VirE2, a single stranded DNA binding protein, then bind to the T- DNA strand. Both VirD2 and VirE2 possess nuclear localization signals (NLS) thought to facilitate targeting of the T-DNA to the host plant cell nucleus, where the T-DNA is integrated into the plant cell nucleus. A preferred embodiment provides for the modification or replacement of VirD2 and VirE2 NLS with sequences selected from variant, e.g., shuffled, chloroplast DNA sequences which target the T-DNA to the chloroplast.

BINARY VECTOR SYSTEMS

Because the vir gene products are soluble factors, their function is optionally supplied in trans, giving rise to the use of binary vector systems in the production of transgenic plants. In a binary vector system, vir genes are supplied on a helper plasmid, often a disarmed Ti plasmid, or, alternatively, integrated into a plant chromosome. A disarmed Ti plasmid which lacks the genes that mediate oncogenesis is much reduced in size from the native 200 kilobase pairs, thereby facilitating its manipulation in vitro. An exogenous DNA sequence, for example, a plant or bacterial structural gene, or a plant or viral promoter or enhancer, is cloned between T-DNA border repeats on a second plasmid, which typically also has a marker, e.g. an antibiotic resistance gene, to facilitate selection after introduction into Agrobacterium.

Subsequently, the exogenous DNA sequence is transferred as part of the T-DNA strand to a host plant (or plant cell or explant) where it integrates into a random site in the host plant chromosome. Preferred embodiment of the invention include evolved binary vectors and the components thereof. Individual components or combinations thereof are recombined, e.g., shuffled, in silico, in vitro or in vivo to produce novel polynucleotides with new characteristics. For example, various vir genes of a helper plasmid can be recombined (or recursively recombined) separately, together in various combinations, together as an intact helper plasmid in vitro, or together as an intact helper plasmid in the context of whole genome shuffling of bacterial genomes, and the resultant recombinant vector components and vectors screened for desirable properties.

The use of agrobacterium mediated transfer has proven a valuable technique in the production of genetically modified plant species. In addition to their utility in the transformation of plant species, Agrobacterium are readily manipulated in vitro by well established techniques of molecular biology. Such techniques are well known to those skilled in the art, and are referenced in e.g., Ausubel, Sambrook, and Berger, supra; Croy (ed) (1993) Plant Molecular Biology, Bios Scientific Publishers, Oxford, U.K., and Jones (ed) (1995) Plant Gene Transfer and Expression Protocols- Methods in Molecular Biology, Volume 49 Humana Press, Towata NJ. These are useful in the context of the present invention for the manipulation and culture of Agrobacterium cells, transformation techniques, and techniques useful for the analysis of plant cells subject to agrobacterium mediated transformation.

While dicotyledonous plants have proven most amenable to manipulation by agrobacterium mediated transformation, reports of transformation of important monocotyledonous crop plants have been forthcoming. In addition, Agrobacterium strains which are capable of transforming fungal species have also been described.

Preferred embodiments of the invention include agrobacterium strains which are capable of efficient transformation of a wide variety of fungal and plant species, (so-called broad host range) including both dicots and monocots. For example, by selecting recombinant vir genes which respond to signals from a broad range of host species, or which more effectively transduce the host plant derived signal to the activation of vir genes involved in transfer of the T-DNA, Agrobacterium strains which are capable of transforming an extended range of host plants can be developed. PLANT VIRUSES

Embodiments of the present invention also comprise vectors which are plant viruses. Plant viruses evolved to have new and desirable transformation and expression properties are preferred embodiments. Viruses are typically useful as vectors for expressing exogenous DNA sequences in a transient manner in plant hosts. In contrast to agrobacterium mediated transformation which results in the stable integration of DNA sequences in the plant genome, viral vectors are generally replicated and expressed without the need for chromosomal integration. Nonethless, in certain embodiments, stable transformation of one or more viral components can prove beneficial, e.g., to transactivate expression of an exogenous or endogenous sequence comprising appropriate cis-acting sequences.

Plant virus vectors offer a number of advantages, specifically: DNA copies of viral genomes can be readily manipulated in E.coli, and transcribed in vitro, where necessary, to produce infectious RNA copies; naked DNA, RNA, or virus particles can be easily introduced into mechanically wounded leaves of intact plants; high copy numbers of viral genomes per cell results in high expression levels of introduced genes; common laboratory plant species as well as monocot and dicot crop species are readily infected by various virus strains; infection of whole plants permits repeated tissue sampling of single library clones; recovery and purification of recombinant virus particles is simple and rapid; and because replication occurs without chromosomal insertion, expression is not subject to position effects. These many advantages are exploited by the present invention for the production of improved viral vectors.

Over six-hundred-fifty plant viruses have been identified, and are amenable directly and/or indirectly as substrates for the directed evolution processes of the invention. Plant viruses cause a range of diseases, most commonly mottled damage to leaves, so-called mosaics. Other symptoms include necrosis, deformation, outgrowths, and generalized yellowing or reddening of leaves. Plant viruses are known which infect every major food-crop, as well as most species of horticultural interest. The host range varies between viruses, with some viruses infecting a broad host range (e.g., alfalfa mosaic virus infects more than 400 species in 50 plant families) while others have a narrow host range, sometimes limited to a single species (e.g., barley yellow mosaic virus). Host range is among the many traits for which it is possible to select appropriate vectors according to the methods provided by the present invention. Approximately 75% of the known plant viruses have genomes which are single-stranded (ss) messenger sense (+) RNA polynucleotides. Major taxonomic classifications of ss-RNA(+) plant viruses include the bromovirus, capillovirus, carlavirus, carmovirus, closterovirus, comovirus, cucumovirus, fabavirus, furovirus, hordeivirus, ilarvirus, luteovirus, potexvirus, potyvirus, tobamovirus, tobravirus, tombusvirus, and many others. Other plant viruses exist which have single-stranded antisense (-) RNA (e.g., rhabdoviridae), double-stranded (ds) RNA (e.g., cryptovirus, reoviridae), or ss or ds DNA genomes (e.g., geminivirus and caulimovirus, respectively). Preferred embodiments of the invention include evolved vectors which are either RNA and DNA viruses. Examples of such embodiments include viruses selected from among: an alfamovirus, a bromovirus, a capillovirus, a carlavirus, a carmovirus, a caulimovirus, a closterovirus, a comovirus, a cryptovirus, a cucumovirus, a dianthovirus, a fabavirus, a fijivirus, a furovirus, a geminivirus, a hordeivirus, a ilarvirus, a luteovirus, a machlovirus, a maize chlorotic dwarf virus, a marafivirus, a necrovirus, a nepovirus, a parsnip yellow fleck virus, a pea enation mosaic virus, a potexvirus, a potyvirus, a reovirus, a rhabdovirus, a sobemovirus, a tenuivirus, a tobamovirus, a tobravirus, a tomato spotted wilt virus, a tombusvirus, and a tymovirus.

Plant viruses can be engineered as vectors to accomplish a variety of functions. Examples of both DNA and RNA viruses have been used as vectors for gene replacement, gene insertion, epitope presentation and complementation, (see, e.g., Scholthof, Scholthof and Jackson, (1996) "Plant virus gene vectors for transient expression of foreign proteins in plants," Annu.Rev.of Phytopathol. 34:299-323.) To evolve plant vectors which are improved for one or more properties related to the aforementioned uses, as well as for traits which are useful in the high yield production of proteins, or other products of interest, in planta, or for pathogen-derived resistance, one or more virus encoded proteins, or an entire viral genome is selected as the substrate for the diversification procedures, e.g., DNA or RNA shuffling, of the present invention.

Typically, plant viruses encode multiple proteins required for initial infection, replication and systemic spread, e.g. coat proteins, helper factors, replicases, and movement proteins. Any of these, as well as other coding and non-coding sequences, such as promoters and enhancers, can readily serve as substrates in the methods of the invention. The nucleotide sequences encoding many of these proteins are matters of public knowledge, and accessible through any of a number of databases, e.g. (Genbank: www.ncbi.nlm.nih.gov/genbank or EMBL: www.ebi.ac.uk.embl/). For example, due to the development of symptoms after viral infection by the host plant, it is sometimes difficult to recover expressed products in quantity. Reduction in the host response or alterations in the hypersensitivity reaction are of significant value in effort to increase yield from transformed plants. Sequences encoding viral proteins, in particular, coat proteins can be recombined, e.g., shuffled, separately, in combination or in the context of whole genome shuffling of plant viruses to produce the vectors of the invention which have diminished viral induced symptoms following infection.

Methods for the transformation of plants and plant cells using sequences derived from plant viruses include the direct transformation techniques described above relating to DNA molecules, see e.g., Jones, ed. (1995) Plant Gene Transfer and

Expression Protocols, Humana Press, Totowa, NJ, for a recent compilation. In addition viral sequences can be cloned adjacent T-DNA border sequences and introduced via Agrobacterium mediated transformation, or Agroinfection (see, e.g., Grimsley et al. (1986) Proc Natl Acad Sci USA 83:3282). Viral particles comprising the plant virus vectors of the invention can also be introduced by mechanical inoculation using techniques well known in the art, (see e.g., Cunningham and Porter, eds. (1997) Methods in Biotechnology, Vol.3. Recombinant Proteins from Plants: Production and Isolation of Clinically Useful Compounds, for detailed protocols). Briefly, for experimental purposes, young plant leaves are dusted with silicon carbide (carborundum), then inoculated with a solution of viral transcript, or encapsidated virus and gently rubbed. Large scale adaptations for infecting crop plants are also well known in the art, and typically involve mechanical maceration of leaves using a mower or other mechanical implement, followed by localized spraying of viral suspensions, or spraying leaves with a buffered virus/carborundum suspension at high pressure. Any of these above mentioned techniques can be adapted to the evolved vectors of the invention, and are useful for alternative applications depending on the choice of plant virus, and host species, as well as the scale of the specific transformation application. REGENERATION OF TRANSGENIC PLANTS

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124- 176, Macmillian Publishing Company, New York, (1983); and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, (1985). Regeneration can also be obtained from plant callus, explants, somatic embryos (Dandekar, et al., J. Tissue Cult. Meth. 12:145 (1989); McGranahan, et al., Plant Cell Rep. 8:512 (1990)), organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al., Ann- Rev, of Plant Phys. 38:467-486 (1987). Additional details are found in Payne (1992) and Jones (1995), both supra. These methods are adapted to the invention to produce transgenic plants using evolved vectors, including agrobacteria and viruses.

Preferred plants for the transformation and expression of the novel recombinases of this invention include agronomically and horticultural ly important species. Such species include, but are not restricted to members of the families: Graminae (including corn, rye, triticale, barley, millet, rice, wheat, oats, etc.); Leguminosae (including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea); Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower) and Rosaciae (including raspberry, apricot, almond, peach, rose, etc.), as well as nut plants (including, walnut, pecan, hazelnut, etc.), and forest trees (including Pinus, Quercus, Pseutotsuga, Sequoia, Populus,etc.)

Additionally, preferred targets for modification the evolved vectors of the invention, as well as those specified above, plants from the genera: Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena (e.g., oats), Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum (e.g., barley), Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza (e.g., rice), Panicum, Pelargonium, Pennisetum (e.g., millet), Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale (e.g., rye), Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum (e.g., wheat), Vicia, Vigna, Vitis, Zea (e.g., corn), and the Olyreae, the

Pharoideae and many others. As noted, plants in the family Graminae are a particularly preferred target plants for the methods of the invention.

Common crop plants which are targets of the present invention include corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea and nut plants (e.g., walnut, pecan, etc).

In construction of recombinant expression cassettes of the invention, which include, for example, helper plasmids comprising virulence functions, and plasmids or viruses comprising exogenous DNA sequences such as structural genes, a plant promoter fragment is optionally employed which directs expression of a nucleic acid in any or all tissues of a regenerated plant. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA oi Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers.

Any of a number of promoters which direct transcription in plant cells can be suitable. The promoter can be either constitutive or inducible. In addition to the promoters noted above, promoters of bacterial origin which operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. See, Herrara-Estrella et al. (1983), Nature. 303:209-213. Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus. See, Odell et al. (1985) Nature. 313:810-812. Other plant promoters include the ribulose-l,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer, (1988) EMBO J. 7:3315- 3327. Many other promoters are in current use and can be coupled to an exogenous DNA sequence to direct expression of the nucleic acid. If expression of a polypeptide, including various viral, bacterial and exogenous gene products, such as viral coat proteins, biosynthetic enzymes, and markers of the present invention, is desired, a polyadenylation region at the 3'-end of the coding region is typically included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from, e.g., T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions) from genes encoding expression products and transgenes of the invention will typically include a nucleic acid subsequence, a marker gene which confers a selectable, or alternatively, a screenable, phenotype on plant cells. For example, the marker may encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorosluforon, or phosphinothricin (the active ingredient in the herbicides bialaphos or Basta). See, e.g., Padgette et al. (1996) "New weed control opportunities: Development of soybeans with a Round UP Ready™ gene" In: Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis Publishers, Boca Raton ("Padgette"). For example, crop selectivity to specific herbicides can be conferred by engineering genes into crops which encode appropriate herbicide metabolizing enzymes from other organisms, such as microbes. See, Vasil (1996) "Phosphinothricin-resistant crops" In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91, CRC Lewis Publishers, Boca Raton) ("Vasil").

The invention described herein furthers the current technology by providing for improved plant transformation vectors for the introduction of exogenous DNA sequences such as the expression cassettes described above. One of skill will recognize that after the exogenous DNA sequence is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

DEVELOPMENT OF IMPROVED VECTORS

The methodology of the present invention is directed towards the development of vectors for plant transformation which have improved properties. A significant advantage of the methods of the invention is that they can be applied to the evolution of vectors which meet a wide variety of general, as well as specialized needs. The directed evolution processes described herein are limited only by the availability of screening and/or selection protocols available or developed to analyze the trait under consideration. No prior knowledge of the genes, or gene products contributing to the phenotype is essential for the successful evolution of novel plant vectors with improved characteristics, such as expanded host range, or increased transformation efficiency.

Agrobacterium vectors have traditionally proven most useful in the transformation of dicotyledonous plant species. A limited number of reports have recently emerged demonstrating agrobacterium strains which can transform monocots and other recalcitrant species. It is of significant interest to develop agrobacterium strains which are capable of transforming a broad range of monocot species, in particular food crop species, for example barley, corn, oats, rye, and wheat. Embodiments of the invention relate to evolved agrobacterium vectors with a broad host range. Another limitation of the presently available agrobacterium vectors is that they insert T-DNAs randomly in the host genome. This is further complicated by the fact that duplications, deletions and insertions frequently occur in the DNA flanking the insertion site. This results in significant variability in expression between independent transformation events, even of the same exogenous DNA sequences. This insertion site variability is not always correctable by regulatory sequences incorporated into the exogenous DNA, leading to significant unpredictability in the expression of exogenous DNAs in transgenic plants. It would be of enormous technical and commercial value to be able to select the site of transgene insertion, as well as to limit disruptions in flanking DNA during the T-DNA insertion process. Certain embodiments of the invention relate to the production of agrobacterium vectors which result in targeted insertion events and to those which have evolved the property of insert precision, that is, they insert exogenous DNA sequences into the host genome with high efficiency and result in a simple insert pattern.

Other embodiments of the invention address the need to target exogenous sequences to the chloroplast rather than the nucleus of host plant. Targeting of chloroplast derived sequences to the nucleus frequently results in unstable integration events. Such unstable integration events present numerous difficulties in the cultivation and propagation of transgenic plants, especially for commercial purposes. This problem is particularly amenable to the methods of the present invention because chloroplast targeting sequences are poorly defined and lack consistent homology consensus sequences. By evolving, e.g., by shuffling, various chloroplast DNA (cpDNA) derived sequences in concert with e.g., nuclear targeting sequences of vir genes, vectors can be evolved which target the chloroplast with increased efficiency and stability.

In addition to the traits discussed above, the methods of the invention are suitable for the development of agrobacterium strains with increased transformation efficiency as well as of strains which are amenable to simplified transformation techniques. Typically, dicotyledonous plants of such species as the experimentally popular Arabidopsis thaliana can be readily transformed by numerous agrobacterium strains through simple methods, such as vacuum infiltration of intact plants, well known in the art. However, valuable crop species belonging to monocot families, e.g., graminae, require more labor intensive and less efficient protocols, requiring plant cell culture under antibiotic conditions for successful transformation. Significant material and labor costs could be saved through the simplification of transformation protocols used for the production of transgenic crop species. The present invention addresses this need through the evolution of agrobacterium strains suitable for such protocols. Aspects of the present invention also relate to the development of improved virus vectors for the expression and recovery of non-native proteins and other products in plants. The invention provides for evolved plant viruses which exhibit, e.g., faster systemic movement, greater expression levels, increased insert size, fewer disease symptoms, reduced toxicity, higher replication levels, extended host range, and pathogen- derived resistance.

For example, high throughput screening and virus re-isolation would be faster if the time from initial infection to systemic spread was reduced. The rate of systemic spread depends largely on movement proteins and their interaction with the viral nucleic acid and the plant plasmodesmata. For example, shuffling of single virus movement proteins or family shuffling of related viral movement proteins can be used to improve the speed of systemic spread. Screening for recombinant viruses or virus components which result in rapid systemic spread can be performed by inoculating recombined, e.g., shuffled, viruses or viruses comprising recombinant, e.g., shuffled, libraries onto leaves of intact plants. At set time intervals, distal leaves can be harvested and the viruses recovered. Additional cycles of shuffling with the fastest viruses leads to a significant improvement in the rate of systemic spread. Similarly, host range can be altered by changes in viral protein-plasmodesmata interactions. Similarly, high throughput screening is facilitated by consistent high-level expression of the viral vector, and any incorporated transgene. To insure high expression of sequences derived from the introduced vector, it is desirable to improve the efficiency with which the replicase protein acts on the cis-acting origin of replication (e.g., the common region in geminiviruses). The AC2 trans-activating factor can be diversified by any of the diversification, e.g., mutation, recombination, recursive recombination, or shuffling procedures described herein, and variants which result in an increased efficiency of replication of viral derived sequences.

Preferred embodiments of the invention also include plant virus vectors which confer increased expression of viral, or alternatively, exogenous proteins. In addition to strategies for recombining (e.g., shuffling) whole genomes, targeted recombination (e.g., by shuffling) of at least two different types of sequences is appropriate. Firstly, by increasing copy number of viral genomes, and/or transcripts, expression levels can be increased. For this approach, populations of DNA fragments corresponding to viral replicases are provided as substrates for recombination reactions. Alternatively, viral and/or plant promoters can be recombined, e.g., shuffled, together to produce promoter sequences which result in expression levels which are higher, or otherwise more favorable, (e.g., tissue specific, or inducible) than wild-type viral or plant promoters. Variants, e.g., shuffled variants, of viruses or viral components can be screened for quantitative differences in protein expression using reporter genes such as green fluorescent protein (GFP), β-glucuronidase (GUS), luciferase, or the maize anthocyanin regulatory gene, Lc.

Among the hundreds of known plant viruses, a large proportion are unsuitable as vectors due to the small insert size which can be stably incoporated and transmitted by the vector. For example, the geminiviruses are constrained to a genome size of approximately 2.7 Kb. Exogenous sequences, e.g., transgenes, that result in a larger genome are frequently truncated during systemic spread of the virus. This limitation is due to a lack of movement of the DNA from cell to cell and not from a failure in packaging or replication of the larger viral genome. Recently, it has been determined that both the nucleus to cyptoplasm ssDNA shuttle protein (BR1) and plasmodesmata movement proteins (BLl) have size but not sequence selectivity. By screening or selecting for variants in these proteins with relaxed size selectivity, a vector that faithfully replicates and moves systemically with large inserts can be produced.An especially preferred embodiment provides for viral vectors which express exogenous DNA sequences, only in a specific, transgenic host plant. Prior to the present invention, plant virus vectors useful for the expression of exogenous proteins in plants have been fully infectious to all plant species susceptible to the virus of the vector. Thus, the recombinant viruses are capable of escaping into the environment. The present invention provides for a viral vector that replicates and is encapsidated only in a corresponding transgenic host. A viral vector is constructed that lacks a coat protein necessary for its encapsidation, and thereby, its infectivity. The subgenomic promoter corresponding to the coat protein drives expression of an exogenous DNA sequence cloned, via a polylinker, adjacent to the promoter. The virus is then used to infect a transgenic host plant which expresses the coat protein lacking in the virus. In the transgenic host, virus particles containing the viral vector are packaged, and are capable of both short and long distance movement. However, the virus vector is not competent to form viral particles in a wild-type host plant and is not capable of escape into the environment as are autonomous viruses. One useful application is in the extension of host range of a plant virus vector. A given plant virus of the invention can be evolved by various recombination (e.g., recursive recombination or shuffling) procedures described herein, and recombinant viruses selected which have an expanded host range, without the risk of evolving new plant pathogens for those hosts. Such tailor-made viruses also have valuable applications in the screening of gene libraries inserted into such vectors in plants. Preferred embodiments relate to gene libraries which are recursively recombined, e.g., shuffled, gene libraries. The evolved vectors of this invention further provide environmentally safe viral vector systems for expression of proteins in plants, such as crop plants, in a field. The risk of escape into the environment is significantly reduced when compared to pre-existing viral expression systems. This reduces the risk associated with viral vectors that infect major crops. Currently the only commercial viral vectors for protein production are used on tobacco. The vector-transgenic plant system of the invention allows evolution of viral vectors that can infect model system plants such as Arabidopsis thaliana as well as the evolution of viral vectors for major crops such as corn and soybean without the risk of creating new plant pathogens for those crops. Alternatively, viral vectors that are capable of transactivating expression of a gene of interest can be produced. For example, the Geminivirus AC1 or "replicase" protein is capable of transactivating replication of a latent mini-replicon integrated into the genome of a plant. To limit spread of an infectious virus expressing the protein, two transgenic plant lines can be produced. The first transgenic plant expresses the replicase while the second transgenic plant possesses the integrated mini-replicon. Progeny inheriting both the replicase and the mini-replicon can express the transgene of interest at very high levels. However, the AC1 replicase is highly toxic to cell growth and differentiation as it signals the cell to enter a continuous s-phase of the cell cycle. The AC1 gene is diversified by any one or more of the recombination and/or mutagenesis methods described herein, thereby generating diverse libraries of recombinant AC1 replicase variants which are selected or screened for variants that are permissive to plant transformation while still retaining partial or full replication competence.

Another embodiment relates to the production of plant viruses that confer pathogen-derived resistance to plant viruses and other plant pathogens. Pathogen-derived resistance to plant viruses can be achieved by overexpression of coat proteins, (and occasionally other viral proteins) in transgenic plants. However, resistance is not always complete and protection is often limited to a single virus. Recombination, e.g., shuffling of viral coat proteins from a single or multiple viral strains can be used to select viral coat proteins which have, for example, increased affinity for viral RNA or DNA, or have altered assembly or encapsidation properties. Screening for recombinant coat protein genes which confer resistance to a broad range of viral pathogens can be conducted by expressing the shuffled viruses or shuffled coat proteins of the invention in transgenic plants which are then subjected to infection by wild type viruses.

DIVERSITY GENERATION

The invention provides for the evolution of plant vectors of various types that have acquired new and advantageous properties by a variety of diversification and screening or selection procedures. A variety of diversity generating protocols are available and described in the art. The procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and/or organisms with new and/or improved characteristics.

While distinctions and classifications are made in the course of the ensuing discussion for clarity, it will be appreciated that the techniques are often not mutually exclusive. Indeed, the various methods can be used singly or in combination, in parallel or in series, to access diverse sequence variants.

The result of any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids that encode proteins with or which confer desirable properties. Following diversification by one or more of the methods herein, or otherwise available to one of skill, any nucleic acids that are produced can be selected for a desired activity or property, e.g., improved or altered host range, increased efficiency orprecision of insertion, targeted insertion, increased or decreased infectivity, decreased pathogenicity, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, e.g., by any of the assays in the art as described above and in the examples below. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures for generating modified nucleic acid sequences contributing to improved plant transformation vectors are found the following publications and the references cited therein: Stemmer, et al.

(1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4:1-4; Ness et al. (1999) "DNA Shuffling of subgenomic sequences of subtilisin" Nature Biotechnology 17:893-896; Chang et al. (1999) "Evolution of a cytokine using DNA family shuffling" Nature Biotechnology 17:793-797; Minshull and Stemmer (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3:284-290; Christians et al. (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17:259-264; Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Crameri et al. (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature

Biotechnology 15:436-438; Zhang et al. (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling"

Nature Medicine 2:100-103; Crameri et al. (1996) "Improved green fluorescent protein by molecular evolution using DNA shuffling" Nature Biotechnology 14:315-319; Gates et al. (1996) "Affinity selective isolation of ligands from peptide libraries through display on a lac repressor 'headpiece dimer"' Journal of Molecular Biology 255:373-386;

Stemmer (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH Publishers, New York, pp.447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et al., (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxy- ribonucleotides" Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution." Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example, site- directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178; Dale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol. Biol. 57:369-374; Smith

(1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis" Science 229:1193-1201; Carter

(1986) "Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D.M.J. eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant Tip repressors with new DNA-binding specificities" Science 242:240-245); oligonucleotide- directed mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors" Methods in Enzymol. 100:468-500; and Zoller & Smith (1987) "Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template" Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986) "Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol. "Oligonucleotide-directed construction of mutations via gapped duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations" Nucl. Acids Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).

Additional suitable methods include point mismatch repair (Kramer et al. (1984) "Point Mismatch Repair" Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382- 403), deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res. 14: 5115), restriction-selection and restriction-selection and restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin)" Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites" Gene 34:315- 323; and Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4:450-455. "Oligonucleotide-directed double-strand break repair in plasmids oi Escherichia coli: a method for site-specific mutagenesis" Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can be found in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (February 25, 1997), "Methods for In Vitro Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (September 22, 1998) "Methods for Generating polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et al. (November 3, 1998), "DNA Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (November 10, 1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No. 5,837,458 to Minshull, et al. (November 17, 1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating Polynucleotides having Desired

Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al. "Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al. "Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et al. "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al., "Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection," WO 00/00632, "Methods for Generating Highly Diverse Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination of Polynucleotide Sequences Using Random or Defined Primers," WO 99/29902 by Arnold et al., "Method for Creating Polynucleotide and Polypeptide Sequences," WO 98/41653 by Vind, "An in Vitro Method for Construction of a DNA Library," WO 98/41622 by Borchert et al., "Method for Constructing a Library Using DNA Shuffling," and WO 98/42727 by Pati and Zarling, "Sequence Alterations using Homologous Recombination." Certain U.S. applications provide additional details regarding various diversity generating methods, including "SHUFFLING OF CODON ALTERED

GENES" by Patten et al. filed September 28, 1999, (USSN 09/407,800); "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION", by del Cardayre et al. filed July 15, 1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922); "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al, filed September 28, 1999 (USSN 09/408,392), and "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed January 18, 2000 (PCT/US00/01203); "USE OF CODON- VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., filed September 28, 1999 (USSN 09/408,393); "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES &

POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed January 18, 2000, (PCT/US00/01202) and, e.g., "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed July 18, 2000 (USSN 09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN

EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed January 18, 2000 (PCT/USOO/01138); and "SINGLE-STRANDED NUCLEIC ACID TEMPLATE- MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by Affholter, filed Sept. 6, 2000 (USSN 09/656,549). In brief, several different general classes of sequence modification methods, such as mutation, recombination, etc. are applicable to the present invention and set forth, e.g., in the references above. The following exemplify some of the different types of preferred formats for diversity generation in the context of the present invention, including, e.g., certain recombination based diversity generation formats, including a variety of DNA and RNA shuffling procedures.

Nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and or PCR reassembly of the nucleic acids. For example, sexual PCR mutagenesis can be used in which random (or pseudo random, or even non-random) fragmentation of the DNA molecule is followed by recombination, based on sequence similarity, between DNA molecules with different but related DNA sequences, in vitro, followed by fixation of the crossover by extension in a polymerase chain reaction. This process and many process variants is described in several of the references above, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Thus, any of the vectors, e.g., agrobacterium plasmids or viral vectors, or their components, described herein can be recombined in vitro according to the methods of the invention.

Similarly, nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells. Many such in vivo recombination formats are set forth in the references noted above. Such formats optionally provide direct recombination between nucleic acids of interest, or provide recombination between vectors, viruses, plasmids, etc., comprising the nucleic acids of interest, as well as other formats. Details regarding such procedures are found in the references noted above. Thus, nucleic acids comprising plant vectors, or components of such vectors can be recombined in vivo in cells to produce plant transformation vectors with improved properties as described herein. Additional details regarding one particularly favorable embodiment of in vivo recombination based on recombination of RNA viral vectors and components thereof are provided hereinbelow. Whole genome recombination methods can also be used in which whole genomes of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with desired library components (e.g., genes corresponding to the pathways of the present invention). These methods have many applications, including those in which the identity of a target gene is not known. Details on such methods are found, e.g., in WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" and in, e.g., PCT/US99/15972 by del Cardayre et al., also entitled "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination." Synthetic recombination methods can also be used, in which oligonucleotides corresponding to targets of interest are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids. Oligonucleotides can be made by standard nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic approaches. Details regarding such approaches are found in the references noted above, including, e.g., "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed September 28, 1999 (USSN 09/408,392), and "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed January 18, 2000 (PCT/USOO/01203); "USE OF CODON- VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., filed September 28, 1999 (USSN 09/408,393); "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al. , filed January 18, 2000, (PCT/USOO/01202); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer (PCT/USOO/01138), filed January 18, 2000; and, e.g., "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed July 18, 2000 (USSN 09/618,579). In silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which correspond to homologous (or even non-homologous) nucleic acids. The resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which correspond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/ gene reassembly techniques. This approach can generate random, partially random or designed variants. Many details regarding in silico recombination, including the use of genetic algorithms, genetic operators and the like in computer systems, combined with generation of corresponding nucleic acids (and/or proteins), as well as combinations of designed nucleic acids and/or proteins (e.g., based on cross-over site selection) as well as designed, pseudo-random or random recombination methods are described in "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al. , filed January 18, 2000, (PCT/USOO/01202) "METHODS OF POPULATING DATA STRUCTURES FOR USE IN

EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer (PCT/USOO/01138), filed January 18, 2000; and, e.g., "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed July 18, 2000 (USSN 09/618,579). Extensive details regarding in silico recombination methods are found in these applications. This methodology is generally applicable to the present invention in providing for recombination of plant transformation vectors and their components in silico and/ or the generation of corresponding nucleic acids or proteins.

In addition to the nucleic acid recombination methods described above, the present invention specifically provides a format for in vivo RNA recombination or "RNA shuffling" that is favorably employed in the generation of, e.g., plant virus vectors with improved properties. Nucleic acids encoding, e.g., coat proteins, movements proteins, replicases, or other components of viral vectors, or subsequences thereof, are inserted into RNA viral vectors. In the context of developing plant viral vectors and related sequences, or other sequences that have a site of action in plants, plant viruses are the vector of choice. However, it will be understood that this method is equally applicable to any type of RNA virus, and can be adapted by appropriate selection of virus and cell type to perform in vivo recombination of RNA substrates. Selection of an appropriate viral vector is within the discretion of the practitioner and can largely be determined by the cell type wherein expression is desired and/or by the mode of action or site of action of the gene of interest.

In some instances it will be desirable to insert cDNA or other DNA sequences of interest into a DNA transcription vector capable of giving rise to infectious viral RNA transcripts. The methods for so doing are well established in the art, and referenced below. For example, cDNAs, oligonucleotides, genomic fragments, or other sequences encoding, e.g., coat proteins, or subportions of coat proteins, or inactive or active gene homologs that are coat protein gene related, (or movement proteins, or replicases, or the like) can be cloned into reverse transcribed, double stranded viral cDNA molecules, which are optionally components of autonomously replicating vectors such as plasmids, episomes, T DNAs, transposons, and the like.

In either case, a population of viral vectors, each comprising a variant of the gene of interest, is introduced into plant cells or tissues such that a single plant cell or tissue receives multiple different variants of the gene of interest. If infectious transcripts are used, following inoculation, RNA transcripts are cytoplasmically replicated under the control of viral replication sequences located, typically, within the 5' terminal region of the transcript. Alternatively, after introduction, e.g., by electroporation, microinjection, or agrobacterium mediated transformation, the cDNA vector gives rise to RNA transcripts, which are then replicated in the cytoplasm of the cell by the viral RNA polymerase.

Both homologous and non-homologous recombination occur in RNA viruses, and both processes are believed to be mediated by template switching of the viral RNA-dependent RNA polymerase during replication. Specific mutations have been identified within viral RNA polymerases that affect the frequency of homologous or non- homologous RNA recombination. Accordingly, the RNA polymerase can be selected to bias the recombination process to acheive the desired outcome with respect to diversity generation. Alternatively, RNA shuffling as described herein, or other nucleic acid shuflling methods can be used to derive viral RNA polymerases with enhanced homologous and/or non-homologous RNA recombination activity. In an embodiment, viral vectors containing complementary mutations in proteins required for systemic spread of the virus are used to select variants of the vector, or of a gene of interest inserted into the vector. For example, to evolve an exogenous gene of interest, a viral vector is constructed including, in the direction of transcription: a RNA-dependent RNA polymerase (RdRp, e.g., from Potato Virus X); essential movement protein encoding sequences under regulatory control of a first subgenomic promoter; a variant of a gene of interest under regulatory control of a second subgenomic promoter; and coat protein under regulatory control of a third subgenomic promoter.

Multiple members of a population of vectors having alternative mutations in, e.g., a movement protein, a coat protein, or other viral component of interest (or in an exogenous gene of interest inserted into the vector) are introduced into, e.g., a basal leaf of an intact plant. In a preferred embodiment, the mutations are complementary such that only variants that have undergone recombination between the complementary mutations, e.g., in the viral component, or gene, of interest, will be capable of systemic infection and movement throughout the plant. Thus, sampling of distal leaves, e.g., those higher on the plant, provides a simple means of screening and selecting recombined viral vectors. In addition, this technology provides the benefit that recombination and expression are acheived in vivo in a single step. Many methods of accessing natural diversity, e.g., by hybridization of diverse nucleic acids or nucleic acid fragments to single-stranded templates, followed by polymerization and/or ligation to regenerate full-length sequences, optionally followed by degradation of the templates and recovery of the resulting modified nucleic acids can be similarly used. In one method employing a single-stranded template, the fragment population derived from the genomic library(ies) is annealed with partial, or, often approximately full length ssDNA or RNA corresponding to the opposite strand. Assembly of complex chimeric genes from this population is then mediated by nuclease- base removal of non-hybridizing fragment ends, polymerization to fill gaps between such fragments and subsequent single stranded ligation. The parental polynucleotide strand can be removed by digestion (e.g., if RNA or uracil-containing), magnetic separation under denaturing conditions (if labeled in a manner conducive to such separation) and other available separation/purification methods. Alternatively, the parental strand is optionally co-purified with the chimeric strands and removed during subsequent screening and processing steps. Additional details regarding this genral approach are found, e.g., in "SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATED

RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by Affholter, USSN 09/656,549, filed Sept. 6, 2000.

In another approach, single-stranded molecules are converted to double- stranded DNA (dsDNA) and the dsDNA molecules are bound to a solid support by ligand-mediated binding. After separation of unbound DNA, the selected DNA molecules are released from the support and introduced into a suitable host cell to generate a library enriched sequences which hybridize to the probe. A library produced in this manner provides a desirable substrate for further diversification using any of the procedures described herein. Any of the preceding general recombination formats can be practiced in a reiterative fashion (e.g., one or more cycles of mutation/recombination or other diversity generation methods, optionally followed by one or more selection methods) to generate a more diverse set of recombinant nucleic acids.

Mutagenesis employing polynucleotide chain termination methods have also been proposed (see, e.g., U.S. Patent No. 5,965,408, "Method of DNA reassembly by interrupting synthesis" to Short, and the references above), and can be applied to the present invention. In this approach, double stranded DNAs corresponding to one or more genes sharing regions of sequence similarity are combined and denatured, in the presence or absence of primers specific for the gene. The single stranded polynucleotides are then annealed and incubated in the presence of a polymerase and a chain terminating reagent (e.g., ultraviolet, gamma or X-ray irradiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated polymerization mediated by rapid thermocycling; and the like), resulting in the production of partial duplex molecules. The partial duplex molecules, e.g., containing partially extended chains, are then denatured and reannealed in subsequent rounds of replication or partial replication resulting in polynucleotides which share varying degrees of sequence similarity and which are diversified with respect to the starting population of DNA molecules. Optionally, the products, or partial pools of the products, can be amplified at one or more stages in the process. Polynucleotides produced by a chain termination method, such as described above, are suitable substrates for any other described recombination format.

Diversity also can be generated in nucleic acids or populations of nucleic acids using a recombinational procedure termed "incremental truncation for the creation of hybrid enzymes" ("ITCHY") described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17:1205. This approach can be used to generate an initial a library of variants which can optionally serve as a substrate for one or more in vitro or in vivo recombination methods. See, also, Ostermeier et al. (1999) "Combinatorial Protein Engineering by Incremental Truncation," Proc. Natl. Acad. Sci. USA. 96: 3562-67; Ostermeier et al. (1999), "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts," Biological and Medicinal Chemistry, 7: 2139-44.

Mutational methods which result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides can be favorably employed to introduce nucleotide diversity. For example, any of a variety of in vivo (e.g., exposure to chemical mutagens, passage through mutator cells lines) and/or in vitro (e.g., mutagenic PCR, site-specific mutagenesis) procedures can be used to diversify substrates corresponding to the vectors of the invention. Many mutagenesis methods are found in the above-cited references; additional details regarding mutagenesis methods can be found in following, which can also be applied to the present invention.

For example, error-prone PCR can be used to generate nucleic acid variants. Using this technique, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Examples of such techniques are found in the references above and, e.g., in Leung et al. (1989) Technique 1: 11-15 and Caldwell et al. (1992) PCR Methods Applic. 2:28-33. Similarly, assembly PCR can be used, in a process which involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions can occur in parallel in the same reaction mixture, with the products of one reaction priming the products of another reaction.

Oligonucleotide directed mutagenesis can be used to introduce site- specific mutations in a nucleic acid sequence of interest. Examples of such techniques are found in the references above and, e.g., in Reidhaar-Olson et al. (1988) Science,

241:53-57. Similarly, cassette mutagenesis can be used in a process that replaces a small region of a double stranded DNA molecule with a synthetic oligonucleotide cassette that differs from the native sequence. The oligonucleotide can contain, e.g., completely and/or partially randomized native sequence(s). Recursive ensemble mutagenesis is a process in which an algorithm for protein mutagenesis is used to produce diverse populations of phenotypically related mutants, members of which differ in amino acid sequence. This method uses a feedback mechanism to monitor successive rounds of combinatorial cassette mutagenesis. Examples of this approach are found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.

Exponential ensemble mutagenesis can be used for generating combinatorial libraries with a high percentage of unique and functional mutants. Small groups of residues in a sequence of interest are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Examples of such procedures are found in Delegrave & Youvan (1993) Biotechnology Research 11:1548- 1552.

In vivo mutagenesis can be used to generate random mutations in any cloned DNA of interest by propagating the DNA, e.g., in a strain of E. coli that carries mutations in one or more of the DNA repair pathways. These "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Such procedures are described in the references noted above. Other procedures for introducing diversity into a genome, e.g. a bacterial, fungal, animal or plant genome can be used in conjunction with the above described and/or referenced methods. For example, in addition to the methods above, techniques have been proposed which produce nucleic acid multimers suitable for transformation into a variety of species (see, e.g., Schellenberger U.S. Patent No. 5,756,316 and the references above). Transformation of a suitable host with such multimers, consisting of genes that are divergent with respect to one another, (e.g., derived from natural diversity or through application of site directed mutagenesis, error prone PCR, passage through mutagenic bacterial strains, and the like), provides a source of nucleic acid diversity for DNA diversification, e.g., by an in vivo recombination process as indicated above. Alternatively, a multiplicity of monomeric polynucleotides sharing regions of partial sequence similarity can be transformed into a host species and recombined in vivo by the host cell. Subsequent rounds of cell division can be used to generate libraries, members of which, include a single, homogenous population, or pool of monomeric polynucleotides. Alternatively, the monomeric nucleic acid can be recovered by standard techniques, e.g., PCR and/or cloning, and recombined in any of the recombination formats, including recursive recombination formats, described above.

Methods for generating multispecies expression libraries have been described (in addition to the reference noted above, see, e.g., Peterson et al. (1998) U.S. Pat. No. 5,783,431 "METHODS FOR GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS," and Thompson, et al. (1998) U.S. Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS) and their use to identify protein activities of interest has been proposed (In addition to the references noted above, see, Short (1999) U.S. Pat. No. 5,958,672 "PROTEIN ACTIVITY SCREENING OF CLONES HAVING DNA FROM UNCULTIVATED MICROORGANISMS"). Multispecies expression libraries include, in general, libraries comprising cDNA or genomic sequences from a plurality of species or strains, operably linked to appropriate regulatory sequences, in an expression cassette. The cDNA and/or genomic sequences are optionally randomly ligated to further enhance diversity. The vector can be a shuttle vector suitable for transformation and expression in more than one species of host organism, e.g., bacterial species, eukaryotic cells. In some cases, the library is biased by preselecting sequences which encode a protein of interest, or which hybridize to a nucleic acid of interest. Any such libraries can be provided as substrates for any of the methods herein described. The above descibed procedures have been largely directed to increasing nucleic acid and/ or encoded protein diversity. However, in many cases, not all of the diversity is useful, e.g., functional, and contributes merely to increasing the background of variants that must be screened or selected to identify the few favorable variants. In some applications, it is desirable to preselect or prescreen libraries (e.g., an amplified library, a genomic library, a cDNA library, a normalized library, etc.) or other substrate nucleic acids prior to diversification, e.g., by recombination-based mutagenesis procedures, or to otherwise bias the substrates towards nucleic acids that encode functional products. For example, in the case of antibody engineering, it is possible to bias the diversity generating process toward antibodies with functional antigen binding sites by taking advantage of in vivo recombination events prior to manipulation by any of the described methods. For example, recombined CDRs derived from B cell cDNA libraries can be amplified and assembled into framework regions (e.g., Jirholt et al. (1998) "Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework" Gene 215: 471) prior to diversifying according to any of the methods described herein.

Libraries can be biased towards nucleic acids which encode proteins with desirable enzyme activities. For example, after identifying a clone from a library which exhibits a specified activity, the clone can be mutagenized using any known method for introducing DNA alterations. A library comprising the mutagenized homologues is then screened for a desired activity, which can be the same as or different from the initially specified activity. An example of such a procedure is proposed in Short (1999) U.S. Patent No. 5,939,250 for "PRODUCTION OF ENZYMES HAVING DESIRED ACTIVITIES BY MUTAGENESIS." Desired activities can be identified by any method known in the art. For example, WO 99/10539 proposes that gene libraries can be screened by combining extracts from the gene library with components obtained from metabolically rich cells and identifying combinations which exhibit the desired activity. It has also been proposed (e.g., WO 98/58085) that clones with desired activities can be identified by inserting bioactive substrates into samples of the library, and detecting bioactive fluorescence corresponding to the product of a desired activity using a fluorescent analyzer, e.g., a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.

Libraries can also be biased towards nucleic acids which have specified characteristics, e.g., hybridization to a selected nucleic acid probe. For example, application WO 99/10539 proposes that polynucleotides encoding a desired activity (e.g., an enzymatic activity, for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, a hydratase, a nitrilase, a transaminase, an amidase or an acylase) can be identified from among genomic DNA sequences in the following manner. Single stranded DNA molecules from a population of genomic DNA are hybridized to a ligand-conjugated probe. The genomic DNA can be derived from either a cultivated or uncultivated microorganism, or from an environmental sample. Alternatively, the genomic DNA can be derived from a multicellular organism, or a tissue derived therefrom. Second strand synthesis can be conducted directly from the hybridization probe used in the capture, with or without prior release from the capture medium or by a wide variety of other strategies known in the art. Alternatively, the isolated single-stranded genomic DNA population can be fragmented without further cloning and used directly in, e.g., a recombination-based approach, that employs a single-stranded template, as described above.

"Non-Stochastic" methods of generating nucleic acids and polypeptides are alleged in Short "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344. These methods, including proposed non-stochastic polynucleotide reassembly and site-saturation mutagenesis methods be applied to the present invention as well. Random or semi-random mutagenesis using doped or degenerate oligonucleotides is also described in, e.g., Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10:297-300: Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208:564-86; Lim and Sauer (1991) "The role of internal packing interactions in determining the structure and stability of a protein" J. Mol. Biol. 219:359-76; Breyer and Sauer (1989) "Mutational analysis of the fine specificity of binding of monoclonal antibody 5 IF to lambda repressor" J. Biol. Chem. 264: 13355-60); and "Walk-Through Mutagenesis" (Crea, R; US Patents 5,830,650 and 5,798,208, and EP Patent 0527809 Bl.

> It will readily be appreciated that any of the above described techniques suitable for enriching a library prior to diversification can also be used to screen the products, or libraries of products, produced by the diversity generating methods.

Kits for mutagenesis, library construction and other diversity generation methods are also commercially available. For example, kits are available from, e.g., Stratagene (e.g., QuickChange site-directed mutagenesis kit; and Chameleon double- stranded, site-directed mutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkel method described above), Boehringer Mannheim Corp., Clonetech Laboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3 prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, Promega Corp., Quantum Biotechnologies, Amersham International pic (e.g., using the Eckstein method above), and Anglian Biotechnology Ltd (e.g., using the Carter/Winter method above).

The above references provide many mutational formats, including recombination, recursive recombination, recursive mutation and combinations or recombination with other forms of mutagenesis, as well as many modifications of these formats. Regardless of the diversity generation format that is used, the nucleic acids of the invention can be recombined (with each other, or with related (or even unrelated) sequences) to produce a diverse set of recombinant nucleic acids, including, e.g., sets of homologous nucleic acids, as well as corresponding polypeptides.

Following recombination and/or mutagenesis, any nucleic acids which are produced can be selected for a desired activity. In the context of the present invention, this can include testing for any property desired in a plant vector, as given in the illustrative examples above, by any of the assays in the art.

A recombinant nucleic acid produced, e.g., by recursively recombining one or more polynucleotide of the invention with one or more additional nucleic acid also forms a part of the invention. The one or more additional nucleic acid can include another polynucleotide of the invention; optionally, alternatively, or in addition, the one or more additional nucleic acid can include, e.g., a nucleic acid encoding a naturally- occurring vector or vector component, or a subsequence thereof, or any homologous sequence or subsequence thereof, (e.g., as found in Genbank or other available literature, or newly identified), or, e.g., any other homologous or non-homologous nucleic acid

(certain recombination formats noted above, notably those performed synthetically or in silico, do not require homology for recombination).

The recombining steps may be performed in vivo, in vitro, or in silico as described in more detail in the references above. Also included in the invention is a cell containing any resulting recombinant nucleic acid, nucleic acid libraries produced by recursive recombination of the nucleic acids set forth herein, and populations of cells, vectors, viruses, plasmids or the like comprising the library or comprising any recombinant nucleic acid resulting from recombination (or recursive recombination) of a nucleic acid as set forth herein with another such nucleic acid, or an additional nucleic acid. Corresponding sequence strings in a database present in a computer system or computer readable medium are a feature of the invention.

For the purposes of the present invention, the above methodologies either singly or in combination are used to evolve novel plant vectors. Such vectors can be any polynucleotide, RNA or DNA, or conjugated DNA, (e.g. poly-lysine-conjugated DNA, peptide-conjugated DNA, liposome-conjugated DNA) suitable for introduction into a host plant or host plant cell by any methodology known in the art. Such vectors can also be plasmids, derived from or incorporated into various agrobacterium strains, or indeed the Agrobacteria themselves. Similarly, the vectors can be plant viruses comprising either RNA or DNA genomes. Any of the foregoing are readily amenable to the aforementioned DNA shuffling techniques.

The methods of the invention are adapted to each application through the choice of substrates and the methods of screening or selection. For example, to evolve an Agrobacterium vector with an expanded host range, sequences, actual or virtual, corresponding to vir genes are provided as the substrates for recombination in vitro or in vivo, or in silico, respectively. The vir genes can originate as components of plasmids derived directly or indirectly from one or more agrobacterium strains, or from partial or complete Agrobacterium genomes. Additional diversity can be introduced by any one of: random mutagenesis in the generation of synthetic oligonucleotides, by error-prone PCR, mutagenic amplification, or site directed mutagenesis. After one or more cycles of recombination, e.g., shuffling, screening is performed based on the property desired. In the foregoing example, recombinant virulence genes (or plasmids, or genomic sequences) are screened or selected based on their ability to induce other virulence gene promoters, or respond to signals from a plant host. Vectors of the invention with other desirable characteristics are developed by the choice of substrate sequences, and/or screening techniques which will be apparent to one of skill in the art. In vitro techniques based on the characteristics of polynucleotides, such as PCR, LCR, nucleic acid hybridization analysis, or on the characteristics of proteins, e.g. western hybridization, proteomics, are the method of choice in some instances, e.g., evaluation of T-DNA insertion sites, as will be readily apparent to one knowledgeable in the art.

Alternatively, screening in bacteria (e.g., E.colϊ) or agrobacteria, is appropriate, for example: assays for vir gene induction used in screening for variant host range. References contained herein and previously discussed, (e.g. Sambrook, Berger, and Ausubel) provide numerous techniques relevant for the introduction of the nucleic acids of the invention, e.g., evolved viral or agrobacterial vectors or their components, into bacterial and agrobacterial vectors and strains. Bacteria, including E.coli and Agrobacteria can be evaluated for expression of recombinant genes as well as for numerous other characteristics particular to the individual circumstances.

In other cases, the screening is best performed in plant cells or intact plants. For example, evaluation of the rate of systemic spread of a virus after infection can be performed by assessing viral production in leaves distal to the site of inoculation on an intact plant. These examples are provided by way of illustration, and variants and adaptations appropriate to the specific characteristic under evaluation will be apparent to one of skill in the art.

EXAMPLES

AGROBACTERIUM VECTORS WITH EXTENDED HOST RANGE

The methods of the present invention provide for the evolution of agrobacterium vectors with a broad host range. Agrobacterium vectors currently in widespread use transform many dicot species with efficiency. However, monocot species, including most important food crop plants have been less readily amenable to agrobacterium mediated transformation. At present, transformation of many monocot species requires labor intensive plant cell culture and/or the addition of exogenous compounds to stimulate expression of vir region genes necessary for T-DNA transfer, making the procedure too inefficient for routine use. It would be extremely useful to have defined agrobacterium strains that easily and efficiently transform both dicot and monocot crop species. For example, Agrobacterium strains which can infect, in planta, leaves or cut stems of both dicot and monocot species without the addition of exogenous phytohormones, or in vitro culture, is of particular interest, and would drastically simplify the production of transgenic food crop species.

The capacity to transform, or to transfer a T-DNA, is largely dependent on the ability of a given agrobacterium strain to detect and respond to phytochemical signals emitted by ihe host plant. To extend the host range of an agrobacterium strain involves tuning the receptor/signaling pathway to respond to molecules given off by the plant species to be targeted. One approach to evolving agrobacterium strains which transform monocot species is to adapt the VirA and ChvE sensor molecules to recognize the phenolic and saccharide signals of the plant. The virA gene is a plasmid-borne sequence belonging to the virulence region of the Ti plasmid in wild-type A. tumefaciens, while the chvE locus is chromosomal. Fragments representing either of the virA gene or the chvE gene can be recombined by any of the recursive recombination methodologies described above to generate novel virA or chvE sequences. Alternatively, both genes can be recombined, or recursively recombined, e.g., shuffled, simultaneously, as isolated or cloned sequences, as PCR fragments, on plasmids, or in the context of whole genome shuffling of agrobacterium strains.

Screening for virA and chvE sequences which extend host range is performed in a two step process. Firstly, recombinant VirA and/or ChvE molecules are screened for the ability to induce other genes of the virulence region. For example, a recombinant library comprising shuffled virA and or chvE sequences is transformed, e.g., by electroporation, into an agrobacterium or other bacterial strain carrying a suitably responsive reporter gene, such as a β-galactosidase (lacZ) structural gene regulated by a vir or pinF promoter. The agrobacterium are then incubated in the presence of plant extracts derived from the host species of interest and β -galactosidase activity is measured, (see, e.g., Grimsley et. al. (1989) "DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions." Mol.Gen.Genet. 217:309-316.) Secondly, agrobacterium strains which demonstrate high levels of β -galactosidase activity are evaluated for their ability to transform a broad range of host species. For example, an agrobacterium strain carrying a recombinant virA library is incubated in culture with representative plant cells, for example leaf, root, meristem, callus, protoplast or embryo, whatever is most easily regenerated for each plant species. Culture and inoculation can be performed in any high throughput format, e.g. 96 well plates. Cultures are then subjected to selection, for example for antibiotic resistance, e.g., kanamycin, imparted by integration of a T-DNA comprising the selectable marker. Those agrobacterium strains which transform a broad range of host plant species are then recovered, and if so desired, used as the starting material for additional rounds of shuffling. In a similar manner, the virG locus, responsible for the "supervir" phenotype, (Jin et. al. (1987) "Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281." J. Bacteriol. 169:4417-4425.) can be recombined. Screening and selection is performed as indicated above.

AGROBACTERIUM EXHIBITING INSERT PRECISION

For the purposes of this discussion, insert precision is defined as the insertion of a T-DNA into a host plant chromosome in a simple, defined manner without disruption of flanking sequences. It is closely related to the concept of targeted insertion of a T-DNA at a predetermined locus in the host plant chromosome. The present invention includes methods for evolving agrobacterium strains which integrate T-DNAs in a precise and/or targeted manner, as well as the agrobacterium strains which exhibit these properties. T-DNA borders are not always cut precisely in the process of their release from the agrobacterium. Furthermore, insertion into the plant chromosome is frequently flanked by deletions, duplications and insertions. The T-DNA can be concatamerized, truncated, or inserted at multiple and unlinked locations in the host genome. Agrobacterium strains influence the process of insertion as evidenced by the observation that different strains consistently result in more or less complicated insertion patterns. Typically, nopaline strains are more efficient at transformation but result in complex insertion patterns, while octopine strains are less efficient at transformation but result in simpler insertion patterns. This is of concern, because there is an inverse correlation between the number of inserts and the expression of the inserted genes. Combining the properties of high transformation efficiency and simple, single inserts would make high throughput screening more consistent and reproducible.

Insertion of a T-DNA into a plant chromosome appears to be at least partially dependent on short regions of sequence similarity between the T-DNA border and the plant chromosome, so-called "microhomology" regions. To evolve agrobacterium that insert T-DNAs simply and precisely into the host plant genome, and/or insert T-DNAs into a predetermined locus of the plant chromosome, recombination, e.g., shuffling, of at least two potential types of targets is warranted. On the one hand, the T-DNA border sequences themselves, (and in concert with adjacent regions of DNA), and on the other hand the virD and virE genes which encode proteins responsible for the excision and insertion of the T-DNA strand are selected as the substrates for recursive recombination as previously described. Sequences from both octopine and nopaline strains can be recombined and screened to identify recombinant sequences which result in targeted insertions and/or insert precision. Characterization of the insertion sites resulting from transformation by agrobacterium carrying the library of recombined sequences can be performed by southern analysis to determine copy number, and by inverse PCR to define the limits of microhomology and the nature of the flanking sequences to determine the complexity of the insertion event. Those agrobacterium which give rise to precisely integrated T-DNAs at high efficiency can be isolated, and optionally subjected to additional rounds of DNA recombination.

Agrobacterium which integrate T-DNAs to a predetermined site in the host plant genome can be developed by including regions of sequence similarity adjacent to the T-DNA borders in the fragments provided as substrates for recombination or recursive recombination (e.g., shuffling) reactions. Selection protocols based on the activation of a marker at a predetermined site in the plant genome are particularly suited to screening for targeted integration events. For example, to identify agrobacterium strains, e.g., comprising a shuffled virE library, which mediate targeted integration events, a binary vector system is employed. Shuffled virE sequences are cloned into a helper plasmid and transformed into agrobacterium carrying a plasmid comprising a T-DNA. The T-DNA is constructed to include regions of sequence similarity with a predetermined locus in the host plant genome adjacent to the T-DNA borders, or alternatively, adjacent to a single right T-DNA border. The T-DNA also includes a promoter sequence between or adjacent to the regions of sequence similarity. The agrobacterium comprising the shuffled library is then used to transform transgenic plants or plant cells which have a selectable marker, for example a gene encoding kanamycin resistance, at the aforementioned predetermined site in their genome. The resulting transformants are subjected to selection in kanamycin: only those transformants which have activated the kanamycin resistance gene by targeted integration of the promoter T-DNA will survive the selection procedure.

TARGETING OF T-DNAS TO THE CHLOROPLAST

The development of agrobacterium vectors which target T-DNAs to the chloroplast is particularly suited to the methods of the invention. The sequences responsible for guiding proteins to the chloroplast, while sharing certain features, are poorly defined, and appear to lack any clear consensus sequence, placing addition of chloroplast targeting sequences beyond simple cloning operations. Most polypeptides targeted to the chloroplast have N-terminal sequences that are rich in serine and threonine; are deficient in aspartate, glutamate, and tyrosine; and generally have a central domain rich in positively charged amino acids.

The agrobacterium VirD2 and VirE2 proteins play a role in transporting and integrating T-DNAs into the host cell nucleus. Both VirD2 and VirE2 contain nuclear localization signals (NLS) that appear to be involved in targeting the T-DNA to the plant cell nucleus. For example, the NLS of VirE2 is comprised of two amino acid sub-sequences rich in basic amino acids with the sequence NS1: KLRPEDRYVQTERYGRR; NS2: KRRYGGETEIKLKSK. While it is not certain that the VirE2 NLS are essential for transport of the T-DNA to the nucleus, they probably increase the efficiency of targeting. Recombination, e.g., shuffling, of virD and virE sequences with the 5' regions, corresponding to the N-terminal transit sequence, of genes encoding chloroplast proteins gives rise to hybrid VirD2 and VirE2 proteins capable of localizing to the chloroplast. Selection is based e.g., on detecting a T-DNA encoded marker, such as green fluorescent protein (GFP) in the chloroplast. Assessment of GFP in the chloroplast can be performed either in situ by fluorescence microscopy or by isolation of chloroplasts and fluoroscopic detection of GFP.

INCREASED INSERT CAPACITY OF VIRUS VECTORS

Another aspect of the invention relates to the evolution of improved viral vectors. For example, plant viruses known to have double stranded circular DNA forms during their life cycle such as Geminiviruses (GV) or Caulimoviruses (CaMV) have many attractive attributes as plant transformation vectors, including high level and chromosome position-independent gene expression. However, due to strict genome size limitations, both CaMV and GV vectors have achieved only limited applicability as vectors.

Geminiviruses are related to a family of viruses that includes the E. coli phages phiX174 and M13, which replicate via a rolling circle mechanism. They are the only class of plant viruses that replicate in the nucleus depending exclusively on the host encoded DNA-dependent DNA polymerases. Geminiviruses typically possess bipartite genomes, e.g., which split the genes between two DNA molecules designated A and B: subgroup III (e.g., Tomato Golden Mosaic Virus (TGMV), African Cassava Mosaic Virus (ACMV), Bean Dwarf Mosaic Virus (BDMV), Bean Golden Mosaic Virus (BGMV), etc.). In some cases, the genome is monopartite with all coding capacity residing on one DNA molecule: subgroups I and II (e.g., Maize Streak Virus (MSV), Wheat Dwarf Virus (WDV), Digitaria Streak Virus (DSV) and Tomato Leaf Curl Virus (TLCV) and tomato yellow Leaf Curl Virus (TYLCV), respectively). All Geminiviruses possess an "intergenic region" (IR) or "common region" (CR) that contains the origin of viral (+)- strand replication. In the genome of a bipartite GV, the CRs of genomes A and B are identical at the DNA sequence level and are highly conserved between closely related viruses.

The viral genomes(s) encode a Replicase protein (AC1 or AL1), a coat protein (designated CP or AV, or AR), and proteins involved in cell-to-cell movement as well as systemic spread. Typically, the Rep and coat protein are encoded by genome A and the movement functions by genome B of a bipartite genome. An exemplary experimental geminivirus system is illustrated in Figure 1. Two plasmids, designated MAXY-A and MAXY-B are constructed encoding, the replicase and a visual marker, e.g., GFP, and the BRl and BLl movement proteins, respectively.

Cell to cell movement of the bipartite GVs is governed by the BRl and BLl proteins. The BRl gene encodes a movement protein that interacts with and increases the pore size of the plasmodesmata to allow passage of unit viral genomes between cells. BRl is now known to encode a ssDNA binding protein responsible for transport of ssDNA GV genomes across the nuclear envelope membrane. BRl (or its equivalent in monopartite GVs lacking a BRl gene) is involved in systemic movement in an appropriate host plant (Timmermans et al. (1994) Ann Rev Plant Physiol Plant Mol Biol 45:79). BLl localizes to the cell periphery and interacts with BRl to shuttle the GV genome from cell to cell. The AC2 gene also plays a role in systemic movement, encoding a transcriptional activator specific for the BRl and CP promoters. Widespread use of GVs as vectors has been hampered by the observation that genomes larger that wild type become rearranged, e.g., deleted, indicating that movement is size restricted. To increase the insert size that can be stably incorporated and transmitted between cells in an infected plant, the BLl an BRl genes can be diversified, e.g., by any of the recombination, recursive recombination or shuffling procedures described herein to produce a library of movement protein variants. The components can be diversified individually, simultaneously on a single cassette or vector, or as components of intact viruses. For example, figure 2 schematically illustrates recombination of movement proteins, e.g., from 3 "parental" sequences (1, 2, and 3) to generate variant movement proteins (4). To identify variants that successfully replicate and spread a larger genome, a vector is constructed including the BLl and/or BRl variants and incorporating a visual marker, e.g., β-glucuronidase (GUS) or Green Fluorescent Protein (GFP)-GUS fusion that exceeds the size range of the wild-type GV of interest. For example, a library of recombinant movement proteins as exemplified by 4 is cut with restriction enzymes 1 and 2 (RSI and RS2) and cloned into a "common region" (CR) cassette, e.g., in a T-DNA vector suitable for agroinfection as illustrated in Figure 3. A suitable plant explant, such as leaf disks, are infected, e.g., by agro-infiltration, and arrayed for evaluation.

Alternatively, transformation is performed, e.g, in bean hypocotyledons by biolistic transformation. The arrayed explants are then co-cultivated with a replication competent/movement incompetent reporter, e.g., encoding a GUS-GFP or GFP-35S fusion protein as illustrated in figure 4, and evaluated for ability to spread a construct with a larger than wild-type insert. Expression of the GFP fusion protein is evaluated distal to the site of inoculation, e.g., using a handheld long-wave uv monitor.

RAPID SYSTEMIC SPREAD OF EVOLVED PLANT VIRUS VECTORS

One major drawback in assessing novel viral characteristics is the length of time required for the engineered virus to replicate and spread internally after inoculation. If the rate of systemic spread following initial infection were increased, high throughput screening and virus reisolation could be made significantly faster. Systemic spread depends mostly on movement proteins and their interactions with viral nucleic acids and plant plasmodesmata. Embodiments of the invention use recursive recombination, e.g., shuffling, of single virus movement proteins and/or of families of related viral movement proteins to increase the rate of systemic spread. In cases where the origin of viral assembly lies within the movement protein-coding region, oligonucleotides with the wild-type origin can be added to the recombination reaction to insure that recombinants with improved properties will be able to form intact viral particles. The resulting libraries of recombinant viral movement proteins are cloned into a viral vector and inoculated onto leaves of intact plants. At set time intervals, distal * leaves are harvested and virus recovered. Those library members which yield high titers at earlier time points than a wild-type reference virus are subjected to additional cycles of shuffling. Similarly, host range can be expanded by manipulating the viral protein- plasmodesmata interaction, and assessing the ability of recombinant viruses to infect host plant species of interest. REDUCTION OF VIRAL SYMPTOMS FOLLOWING INFECTION

In some host-virus reactions, viral symptoms are severe, making it difficult to assess infected leaves for biochemical properties or to purify protein from infected leaves. In most cases, viral coat proteins are the most important determinants of the host response, including the hypersensitivity reaction. Viral replicases and movement proteins may also play significant roles. An embodiment of the present invention relates to the evolution of plant viruses that result in diminished viral symptoms. Genes encoding coat protein, replicases, and movement proteins are all suitable substrates for the development of plant viruses which evoke reduced symptoms. Such sequences can be recombined, e.g., shuffled, individually, in combinations of families of related genes, simultaneously including members of multiple gene families, or together in the context of whole genome shuffling.

The critical determinant in evolving plant virus vectors which do not elicit significant detrimental symptoms or hypersensitive responses is the manner in which the recombinant library is screened. To evolve plant viruses which provoke diminished plant responses, screening is based on an observed reduction in symptoms. Typically, a hypersensitive response (HR) results in necrotic lesions near the infection site. Lesions are characterized by an accumulation of cytotoxic phytoalexins, alterations in the cell walls, the accumulation of pathogenesis-related (PR) proteins and rapid cell death, and as such can be evaluated both visually and biochemically. Investigations into individual host-pathogen interactions have led to the cloning of individual genes involved in the HR, (see, for example Karrer et. al., (1998) "Cloning of tobacco genes that elicit the hypersensitive response." Plant Molecular Biology 36: 681-190.) However, such investigations are time consuming and laborious. In contrast, recursive recombination of viral genes, and genomes rapidly results in the rapid isolation of viruses which fail to elicit the HR response, as well as those which exhibit reduced symptoms in general. Alternatively, toxicity due to a viral component can prevent high level expression of a virus or virus-derived gene of interest. For example, high levels of the AC1 (AL1) replicase of Geminiviruses induces efficient replication and, thus, expression of a gene of interest incorporated by a Geminivirus (e.g., African cassava mosaic virus, ACMV) vector. However, the AC1 replicase is highly toxic to the cell, inducing a continuous S-phase of the cell cycle.

Replication of a geminivirus genome requires three basic components: a Rep protein (Rep) encoded by AC1 (AL1), a cognate CR, and host DNA synthesis machinery. Although Rep is the only viral encoded protein required for DNA replication, another small protein encoded by the viral AC3 gene greatly enhances replication (Sunter et al. (1990) Virology 179:69). Amplification of double stranded viral DNA as well as rolling circle production of viral (+) ssDNA is initiated by Rep protein. This protein binds to a specific recognition sequence in the CR, creates a ssDNA nick then covalently links a conserved tyrosine residue within itself to a specific base within the nearby conserved loop structure on the (+) DNA strand. This results in the initiation of (+) strand DNA replication by the host cell DNA synthesis machinery. In addition to this function, the Rep proteins have a putative retinoblastoma susceptibility protein (Rb) binding domain as has been demonstrated for the related tumorigenic SV40 T-antigen. In mammailian cells, Rb inhibits the activity of a family of transcription factors that induce expression of genes required for DNA synthesis. The T-antigen of SV40, and other related proteins, send quiescent cells into S-phase thru binding and deactivating Rb and related proteins. In addition, the Rep proteins may also directly activate transcription via interaction with plant proliferating cell nuclear antigen (PCNA). Rep proteins of the geminiviurses WDV and TGMV have been shown to bind to Rb proteins. Furthermore, the WDV Rep protein contains the conserved Rb binding motif LXCXE found in SV40 T-antigen (and related proteins). In addition, over-expression of the maize homologue of Rb inhibits WDV replication in plant cells. The tumorigenic capacity of SV40 T-antigen can be completely uncoupled from its Rep function provided the host DNA replication machinery is active (Cooper et al. (1997) Proc Natl Acad Sci USA 94:6450). The mutant T-antigen is more active in promoting viral replication since binding to Rb as well as p53 compete for its function as a "replicase" protein.

Thus, to decrease toxicity and permit increased expression of Rep proteins in transgenic plants, variants of the AC1 gene that exhibit replication competence, but lack the activities, e.g., Rb, PCNA binding, that result in induction of S-phase are selected following diversification of the AC1 gene by any one or more of the recombination or recursive recombination, e.g., shuffling, procedures described herein. For example, Figures 5A and 5B illustrate two alternative constructs for recombining parental nucleic acids encoding GV Rep proteins (1, 2, and 3) to generate a library of nucleic acids variants (4) encoding Rep proteins. Figures 5A and 5B differ by the placement of restriction enzyme recognition sites useful for facilitating manipulation of the substrate and recombinant sequences. Figure 6 illustrates alternative embodiments of vectors favorable for evolution of Rep proteins with reduced toxicity. Preceding or following recombination, the AC1 (optionally including the AC2 and AC3 genes) are inserted into common region (CR) cassettes with or without a reporter, (e.g., GFP). Such variants can be selected by phenotypic evaluation of transformed plant cells or explants as described above, or by assessing DNA content of the transformed cells by any of the methods known in the art. If necessary to insure high-level replication of a such a GV vector, induction of the DNA synthesis machinery can be regulated by alternative methods such as spraying the plant with cytokinins, etc.

PLANT VIRUS VECTORS WITH ENHANCED EXPRESSION LEVELS

High expression levels of vector derived sequences are valuable in a number of commercially relevant applications. Embodiments of the invention provide for evolved viruses which express high levels of a viral protein, or alternatively engender high levels of expression of an exogenous protein inserted into the vector. Viruses offer several advantages over stable transformation procedures for expressing proteins in plants. Proteins encoded by chimeric viruses can be produced in 1-2 weeks rather than the 3-12 month time frame required for production using stable transformed plants. The expression levels of proteins encoded by viruses can exceed the levels obtained by stable transgenes. In addition viruses, are also amenable to high throughput systems that are useful for screening libraries of diverse genes such as those produced by DNA shuffling and other directed evolution technologies.

Because of the organization and limited size of viral genomes, wild-type viral or closely related promoters are typically used to drive expression. Promoters which lead to high levels of constitutive expression or to other expression patterns (e.g. tissue specific, or inducible) of interest can be evolved using the methods of the invention. Promoter sequences from various plant viruses as well as plant promoter sequences are recombined to generate a library of recombinant promoters. The recombinant promoters are cloned into a viral vector adjacent to a reporter gene, such as GFP, β -glucuronidase, or the maize anthocyanin regulatory gene, Lc, such that expression of the reporter is dependent on the recombinant promoter. Quantitative monitoring of the reporter gene is used to evaluate promoter strength, selectivity, and inducibility, (and any other desired property). Promoters identified in the screening can then be used to drive expression of exogenous genes other than the reporter gene, or cloned to replace wild-type, or other sub-optimal promoters of other vectors. In one preferred embodiment, whole genome shuffling is used to recombine a virus, or a group of closely related viruses. The entire genome (less than 10 kb) is treated to induce fragmentation (for example, by DNAse treatment or by sonication) and then recombined as described above. A marker gene is then cloned between the 5' and 3' shuffled regions. The recombinant viruses can be transcribed in vitro and the transcripts used to inoculate leaves of a host plant via manual inoculation procedures as previously described. Assessment of the marker gene, by either qualitative or quantitative methods allows concurrent screening of promoter strength, replication level, and systemic spread. Concurrent screening of multiple parameters offers the unique benefit of simultaneously optimizing the variables which act in concert to determine protein production. The viral expression system is particularly suited for promoter shuffling and evaluation because there is no affect on expression due to chromosomal position effects. Thus, comparisons between transformants will provide a more accurate reflection of variation in promoter activity.

The invention also provides for plant virus vectors for the production of proteins in planta. Such vectors are useful for the expression and screening of recombinant, including, e.g., shuffled, libraries as well as for large scale production of shuffled and other proteins in plants. Some viruses such as the potato virus X (PVX) and tobacco mosaic virus (TMV) can accept large genes and still package virions effectively. Other viruses can be employed by replacing genes non-essential for replication with exogenous sequences, incli'ding library sequences.

The present invention offers a number of advantages for the screening and evaluation of recombinant, e.g., shuffled, libraries. For example, the evolved plant viruses of the invention can be introduced by electroporation, or other equivalent methods, into plant protoplasts where they can replicate autonomously and accumulate to high levels. Chromosomal insertion is unnecessary and evolved protein function can be assayed within hours of introduction.

The choice of vector can be made to optimize production of protein. For example the cauliflower mosaic virus (CaMV) 35S promoter is a well characterized constitutive promoter, which in some cases can yield as much as 2% of total soluble protein recovered from a plant cell. The CaMV 35S promoter or the evolved promoters of the invention can be used to generate high levels of protein expression. Production of some proteins (for example, sparingly soluble proteins) is simplified by cloning the exogenous gene, or gene library (including a shuffled library) as a fusion with the viral coat protein. Fusion proteins can then be simply and effectively recovered from virus particle preparations. Such coat protein fusions are particularly useful in the production of phage display vectors. Antigenic epitopes are fused to the coat protein such that they are displayed on the outside of the virus particle. This application is especially useful for vaccine production.

PROTEIN PRODUCTION IN TRANSGENIC PLANTS

A potential drawback of using viral vectors to produce proteins in plants is the possibility that symptomatic infection can result, and that this infection could spread from the engineered plants to nearby crop plants. A preferred embodiment of the invention provides for viral vectors which are capable of propagation only within a transgenic host, reducing the risk of escape into the environment. This technology allows the use of DNA shuffling to evolve wider host ranges for virus vectors without the risk of evolving new plant pathogens for those hosts.

The present invention supplies a viral coat protein in trans by expression of the coat protein in a transgenic plant. The corresponding viral promoter drives expression of an exogenous sequence to be expressed for protein production. For purposes of illustration, development of a tobacco mosaic virus (TMV) vector which is operable in transgenic cruciferae is described. However, the methods can be applied equally to viruses which infect other plant species, and to transgenic plants of such species which serve as hosts. At least 4 strains of TMV are known to infect cruciferous crops and are able to replicate in Arabidopsis. The sequences of these strains are available in Genbank, and accordingly, polynucleotides corresponding to their sequences can be isolated and cloned by techniques well known in the art. Similarly, DNA sequences of strains which infect soybean are available in Genbank and can be used to develop viral vectors which infect soybeans and other legumes. Several strains that infect odontoglossum and ribgrass are known, and can be used to evolve vectors for monocot crops such as com, rice and wheat.

Several publications suggest that coat proteins and movement proteins are the major determinants of host range, (see, e.g., Spitsin et al. (1999) Proc. Nat'l. Acad. Sci. USA 96:2549-2553; Hilf and Dawson (1993) Virology 193:106-114.) Cloning sites are introduced into an infectious TMV clone, allowing the introduction of heterologous movement proteins and coat proteins into the vector backbone. This provides the basis of evolving tobamovirus vectors for any host. Libraries of oligonucleotides of crucifer infecting tobamovirus movement and coat proteins are constructed and cloned into the TMV backbone. Arabidopsis plants are infected with the library and variants capable of replication and long distance movement are harvested from the distal leaves. Several passages of infection and recovery can be used to select for virus fitness, and several independent isolates developed and sequenced.

After developing a virus capable of forming high titers on Arabidopsis, the coat protein is excised and cloned into a vector for the production of transgenic plants. The recombinant, e.g., coat protein gene, under control of a constitutive promoter, is then introduced (e.g., by agrobacterium mediated transformation using standard vacuum infiltration techniques) into Arabidopsis plants (or plant cells). Transgenic plants are then propagated by techniques well established in the art, and described above.

The site formerly occupied by the excised coat protein, and retaining the coat protein promoter then provides the cloning site for exogenous sequences to be expressed in the transgenic plants. For example, sequences corresponding to a visual marker, such as green fluorescent protein (GFP), can be cloned adjacent to the coat protein promoter and the virus vector used to infect the transgenic Arabidopsis. Visual assessment under appropriate light conditions enables selection of recombinant GFP with improved or increased fluorescent properties. Similarly, the evolved vector can be used for screening and production of protein pharmaceuticals, nutraceuticals, biosynthetic enzymes, and biodegradable polymers and polymer components among others.

Further cycles of recombination and selection are then used to evolve vectors which infect crop plants of interest. The present invention provides for the production of protein and other biosynthetic products of interest in plant species which have previously posed significant risks of infection. The virus vector of the invention is unable to spread systemically and incapable of infecting wild-type plants in experimental or field settings. Only when used to infect transgenic plants can the virus vector replicate, be encapsidated and spread systemically, as is required for efficient protein production. In addition to the crucifer vector described above, preferred embodiments relate to virus vectors and to methods for their development, which are suitable for protein production in legumes, such as soybeans, and graminae, such as corn. Proteins to be expressed include such valuable products as biofuels; biodegradable polymers (including plastics) and their components; industrial enzymes; enzymes for improved animal and human nutrition; nutraceuticals; protein pharmaceuticals; antigens; plantibodies; and biosynthetic enzymes (e.g., of lipid, or carbohydrate synthetic pathways). The invention also provides for transgenic plants used for the expression of such evolved viruses.

PATHOGEN-DERIVED RESISTANCE TO PLANT PATHOGENS

Plant viruses can cause considerable damage to commercially valuable crops. The present invention provides for evolved plant viruses and viral genes that confer pathogen-derived resistance to plant viruses, as well as for a method of evolving viruses or viral genes to confer pathogen-derived resistance to plant viruses. Pathogen- derived resistance refers to the observation that overexpression of a pathogen derived component, in many cases a viral coat protein, can protect a transgenic plant which expresses it from infection by that virus. However, resistance is not always complete and protection is most frequently limited to a single virus from which the coat protein originated.

It is likely that resistance occurs because the high concentration of coat proteins prevents the origin of replication from being exposed long enough for the virus to replicate in the plant cell cytoplasm. However, proof of this mechanism is not required for the effective application of the methods or for the deployment of the evolved viruses of the invention. Recursive recomibnation, e.g., DNA or RNA shuffling, is applied to a single viral coat protein, or to families of several coat proteins, and assayed for increased affinity for RNA or DNA, or for processivity in assembly. For example, recombinant viral proteins can be recovered and pooled for evaluation by binding to RNA or DNA- conjugated columns, on matrices such as agarose or sepharose. Washing conditions are determined empirically for each input coat protein to select only recombinant coat proteins which bind the viral nucleic acid with greater affinity. Specificity for the viral origin of replication can be assessed by using synthetic oligonucleotides corresponding to the viral origin as the column supported substrate. By evaluating binding to oligonucleotides corresponding to the origin of replication of various viruses, the spectrum of viruses to which resistance is provided can be expanded. Details pertaining to employment of the above techniques, are available in the previously cited references (e.g., Ausubel, Berger, and Sambrook) and are known to those of skill in the art. Similarly, other well known techniques for the binding of proteins to nucleic acids can be used to evaluate evolved coat proteins, and are available to persons skilled in the art. After pre-screening based on molecular characteristics, the ability to confer resistance in planta is assayed. Recombinant, e.g., shuffled, coat protein encoding sequences with favorable binding (or other characteristics) identified in the previous screening step, are introduced into transgenic plants by any of the techniques routinely used to generate stable transformants, e.g., microinjection, electroporation, biolistics, Agrobacterium mediated transformation. Stable integrants are selected and regenerated to constitute whole plants which express the recombinant coat protein. Such transgenic plants are subjected to infection by wild-type virus. For example, to evolve viruses which protect important crucifer crops, (e.g., cabbage, cauliflower, broccoli, mustards and rapes, including forage and oilseed rape), the model plant Arabidopsis thaliana is used. Many viruses that infect Arabidopsis also infect other crucifers, and their sequences and corresponding polynucleotides are readily available, see e.g., Aguilar et al. (1996) "Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana. Plant Mol. Bio. 30:191-197. After one or more rounds of diversification and screening in Arabidopsis has yielded recombinant viral sequences that confer resistance, candidate clones are tested in specific crop species to confirm protection. While the foregoing invention has been described in some detail for puposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, vectors with various desirable properties, not explicitly described, which are useful for specific applications can readily be developed by the methods of the present invention. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of evolving a plant vector, the method comprising:

(a) providing a population of nucleic acid fragments comprising at least one component of a plant vector;

(b) recursively recombining the population of nucleic acid fragments to generate a library of recombinant polynucleotides;

(c) optionally repeating the recombination process of steps (a) and (b) one or more times;

(d) screening the library of recombinant polynucleotides to identify at least one recombinant polynucleotide, wherein the recombinant polynucleotide is a component of an evolved vector which has acquired or evolved a desired property; (e) repeating steps (a) through (d) until the evolved plant vector has acquired the desired property.

2. The method of claim 1, wherein the nucleic acid fragments provided in step (a) are produced by one or more of: error prone PCR, mutagenic amplification, and site directed mutagenesis.

3. The method of claim 1 , comprising providing a population of DNA fragments.

4. The method of claim 1, comprising providing a population of RNA fragments.

5. A library of recombinant polynucleotides produced by the method of claim 1 or 2.

6. The library of claim 5, wherein the library is introduced into an Agrobacterium.

7. The library of claim 5, wherein the library is introduced into a plant virus.

8. An evolved vector produced by the method of claim 1 or 2.

9. A transgenic plant or a transgenic plant cell or a transgenic plant explant made by transforming a parental plant cell with the evolved vector of claim 1 or

2.

10. The transgenic plant cell or transgenic plant explant of claim 9, wherein the plant cell or plant explant is a stem, a leaf, a root, a meristem, a callus, or an embryo.

11. A library of recombinant polynucleotides, wherein the recombinant polynucleotides comprise at least one sequence derived from a genome of the transgenic plant, or the transgenic plant cell, or the transgenic plant explant of claim 9.

12. The method of claim 1, wherein the vector comprises one or more of: a plasmid, a virus, a bacterium, an agrobacterium, a RNA polynucleotide, a naked DNA polynucleotide, a poly-lysine-conjugated DNA, a pepti de-conjugated DNA and a liposome-conjugated DNA.

13. The method of claim 12, wherein the vector is an agrobacterium vector, the population of nucleic acid fragments provided in step (a) comprising at least one agrobacterium gene, wherein the recombinant polynucleotide identified in step (d) is a component of an evolved agrobacterium strain which has acquired or evolved a desired property, step (e) comprising repeating steps (a) through (d) until the evolved agrobacterium strain has acquired the desired property.

14. The method of claim 13, wherein the evolved agrobacterium vector comprises a binary vector system having a helper plasmid, which helper plasmid comprises vtr function.

15. The method of claim 13, wherein a DNA sequence comprising vir function is integrated into a chromosome of an agrobacterium.

16. The method of claim 13, wherein the evolved agrobacterium strain is A. tumefaciens.

17. The method of claim 13, wherein the evolved agrobacterium strain is A. rhizogenes.

18. The method of claim 13, wherein the nucleic acid fragments provided in step (a) include at least one agrobacterium right T-DNA border.

19. The method of claim 18, wherein the nucleic acid fragments provided in step (a) include at least one agrobacterium right T-DNA border and at least one agrobacterium left T-DNA border.

20. The method of claim 19, wherein the at least one right agrobacterium

T-DNA border and the at least one left agrobacterium T-DNA border provided in step (a), each further comprise a PCR primer binding site.

21. The method of claim 13, wherein the fragments provided in step (a) comprise at least one of: a virA gene, a virB gene, a virC gene, a virD gene, a virE gene, a virG gene or a chvE gene.

22. The method of claim 13, wherein the nucleic acid fragments provided in step (a) comprise at least one agrobacterium genome.

23. The method of claim 13, wherein the desired property comprises one or more of: insert precision, targeted insertion, improved host range, transformation efficiency, in planta transformation of leaves, in planta transformation of cut stems, in planta transformation in the absence of exogenous phytohormones, transformation without in vitro culture, and chloroplast targeting.

24. The method of claim 23, wherein the desired property is insert precision.

25. The method of claim 23, wherein the nucleic acid fragments provided in step (a) include at least one agrobacterium right T-DNA border.

26. The method of claim 25, wherein the screening of step (d) is performed by one or more of polymerase chain reaction, hybridization, ligase chain reaction, and proteomics.

27. The method of claim 26, wherein the screening of step (d) is performed by an inverse polymerase chain reaction of at least one junction between an integrated T- DNA and its site of insertion.

28. The method of claim 23, wherein the desired property is targeted insertion.

29. The method of claim 28, wherein the nucleic acid fragments provided in step (a) include at least one Agrobacterium right T-DNA border.

30. The method of claim 29, wherein the screening of step (d) is performed by one or more of polymerase chain reaction, hybridization, ligase chain reaction and proteomic s.

31. The method of claim 30, wherein the screening of step (d) is performed by an inverse polymerase chain reaction of at least one junction between an integrated T- DNA and its site of insertion.

32. The method of claim 31, wherein the nucleic acid fragments provided in step (a) further comprise a region of sequence similarity with a desired insertion site, which region of sequence similarity is attached adjacent and contiguous to the at least one agrobacterium T-DNA border.

33. The method of claim 32, wherein a promoter or an enhancer is included in the nucleic acid fragments provided in step (a), which promoter or enhancer is located adjacent to the region of sequence similarity.

34. The method of claim 33, wherein the screening of step (d) further comprises: transforming the library of recombinant polynucleotides into a host plant or a host plant cell, which host plant or host plant cell has a detectable marker at a desired insertion site, whereby insertion of a library member at the desired insertion site results in activation of the detectable marker.

35. The method of claim 34, wherein the host plant or host plant cell in step (d) is a transgenic plant or cultured transgenic plant explant having a marker transgene at the desired insertion site.

36. The method of claim 35, wherein the marker transgene of step (d) is green fluorescent protein (GFP), β-glucuronidase, luciferase or a maize Lc (anthocyanin regulatory) gene.

37. The method of claim 35, wherein the marker transgene of step (d) is a selectable marker.

38. The method of claim 37, wherein the marker transgene of step (d) confers resistance to an antibiotic or a herbicide.

39. The method of claim 37, wherein the marker transgene of step (d) is a negatively selectable marker.

40. The method of claim 39, wherein the marker transgene is a homologue of a dhll gene, a codA gene, a tms2 gene, or a N7A2 gene.

41. The method of claim 40, wherein the marker transgene is a nonfunctional homologue of a dhll gene, a codA gene, a tms2 gene, or a NIA2 gene.

42. The method of claim 23, wherein the desired property is the ability to transform a broad range of host plant species.

43. The method of claim 42, wherein the nucleic acid fragments provided in step (a) comprise at least one gene selected from among: virA, virB, virC, virD, virE, virG and chvE.

44. The method of claim 43, wherein the nucleic acid fragments provided in step (a) comprise at least one virA gene, and wherein the screening of step (d) is performed by detecting expression of a marker operably linked to a vir promoter.

45. The method of claim 44, wherein the vir promoter is bound by a VirG protein, which VirG protein is activated by a recombinant VirA protein.

46. The method of claim 42, wherein the screening of step (d) is performed in an intact plant or in a plant culture of one or more of a stem, a leaf, a root, a protoplast, a callus, a meristem, and an embryo.

47. The method of claim 42, wherein the host plant species comprises a dicot or a monocot.

48. The method of claim 23, wherein the desired property is the ability of the Agrobacterium to target a T-DΝA to the chloroplast.

49. The method of claim 48, wherein the nucleic acid fragments provided in step (a) comprise at least one of a virD2 DNA sequence and a virE2 DNA sequence.

50. The method of claim 48, wherein the nucleic acid fragments provided in step (a) comprise at least one of a chloroplast localization signal and a nuclear localization signal.

51. The method of claim 48, wherein the screening of step (d) is performed by screening for localization to a chloroplast.

52. A library of recombinant polynucleotides produced by the method of claim 13.

53. The library of claim 52, wherein the library is introduced into an Agrobacterium.

54. An evolved agrobacterium strain produced by the method of claim 13.

55. A transgenic plant or a transgenic plant cell or a transgenic plant explant made by transforming a parental plant cell with the evolved agrobacterium strain of claim 54.

56. The transgenic plant cell or transgenic plant explant of claim 55, wherein the plant cell or plant explant is a stem, a leaf, a root, a meristem, a callus, or an embryo.

57. A library of recombinant polynucleotides, wherein the recombinant polynucleotides comprise at least one sequence derived from a genome of the transgenic plant, or the transgenic plant cell, or the transgenic plant explant of claim 55.

58. The method of claim 12, wherein the vector comprises a virus, the population of nucleic acid fragments provided in (a) comprising at least one component of a plant virus, wherein the recombinant polynucleotide identified in the screening of step (d) is a component of an evolved plant virus, which evolved plant virus has acquired or evolved a desired property, step (e) comprising repeating steps (a) through (d) until the evolved plant virus has acquired the desired property.

59. The method of claim 58, wherein the plant virus is selected from the group: a RNA virus, a DNA virus, an alfamovirus, a bromovirus, a capillovirus, a carlavirus, a carmovirus, a caulimovirus, a closterovirus, a comovirus, a cryptovirus, a cucumovirus, a dianthovirus, a fabavirus, a fijivirus, a furovirus, a geminivirus, a hordeivirus, a ilarvirus, a luteovirus, a machlovirus, a maize chlorotic dwarf virus, a marafivirus, a necrovirus, a nepovirus, a parsnip yellow fleck virus, a pea enation mosaic virus, a potexvirus, a potyvirus, a reovirus, a rhabdovirus, a sobemovirus, a tenuivirus, a tobamovirus, a tobravirus, a tomato spotted wilt virus, a tombusvirus, and a tymovirus

60. The method of claim 58, wherein the desired property is systemic spread of an evolved virus incorporating an insert, which insert is larger than an insert spread by a wild-type virus.

61. The method of claim 60, wherein the nucleic acid fragments provided in step (a) encode at least one movement protein.

62. The method of claim 61, wherein the nucleic acid fragments provided in step (a) comprise at least one viral genome.

63. The method of 58, wherein the desired property is rapid systemic spread of an evolved plant virus^' following infection.

64. The method of claim 60, wherein the library of recombinant polynucleotides is cloned into a virus vector, which recombinant virus vector is inoculated onto at least one leaf of an intact plant followed by harvest and recovery of a plurality of virus particles from leaves distal to the at least one leaf whereon lies the site of inoculation

65. The method of claim 60, wherein the nucleic acid fragments provided in step (a) encode at least one movement protein.

66. The method of claim 60, wherein the nucleic acid fragments provided in step (a) comprise at least one viral genome.

67. The method of claim 58, wherein the desired property is reduction of viral symptoms after infection.

68. The method of claim 67, wherein the nucleic acid fragments provided in step (a) comprise at least one viral protein.

69. The method of claim 68, wherein the nucleic acid fragments provided in step (a) encode at least one of a viral coat protein, a viral replicase, a viral recombinase and a movement protein.

70. The method of claim 67, wherein the nucleic acid fragments provided in step (a) comprise at least one viral genome.

71. The method of claim 58, wherein the desired property is reduced toxicity following introduction of the virus.

72. The method of claim 71, wherein the nucleic acid fragments provided in (a) encode at least one viral protein.

73. The method of claim 72, wherein the nucleic acid fragments encode at least one of a viral coat protein, a viral replicase a viral recombinase and a viral movement protein.

74. The method of claim 71, wherein the nucleic acid fragments provided in (a) comprise at least one viral genome.

75. The method of claim 58, wherein the desired property is a higher level of viral gene expression as compared to the level of expression generated by a wild-type virus.

76. The method of claim 75, wherein the nucleic acid fragments provided in step (a) comprise at least one viral or plant promoter.

77. The method of claim 76, wherein the nucleic acid fragments provided in step (a) comprise a cauliflower mosaic virus 35S promoter.

78. The method of claim 77, wherein the nucleic acid fragments provided in step (a) comprise at least one viral genome.

79. The method of claim 58, wherein the desired property is a higher constitutive level of exogenous gene expression than is expressed under control of a wild- type promoter provided in step (a).

80. The method of claim 79, wherein the nucleic acid fragments provided in step (a) comprise at least one viral or plant promoter.

81. The method of claim 80, wherein the nucleic acid fragments provided in step (a) comprise a cauliflower mosaic virus 35S promoter.

82. The method of claim 79, wherein the nucleic acid fragments provided in step (a) comprise at least one viral genome.

83. The method of claim 82, wherein the screening of step (d) comprises the steps of cloning a marker gene between a 5' shuffled region and a 3' shuffled region of the at least one viral genome to generate a recombinant virus, and inoculating the recombinant virus onto a leaf of a host plant.

84. The method of claim 58, wherein the screening step of (d) is performed in a transgenic plant constitutively expressing a viral coat protein, and wherein the evolved vector comprises a polycloning site, which polycloning site replaces a viral coat protein structural gene and is contiguous with a corresponding viral coat protein promoter, such that an exogenous DNA inserted into the polycloning site is expressed under control of the viral coat protein promoter.

85. A library of recombinant polynucleotides produced by the method of claim 58.

86. The library of claim 85, wherein the library is introduced into a plant virus.

87. An evolved plant virus produced by the method of claim 58.

88. The evolved plant virus of claim 87, wherein a polynucleotide is inserted into the evolved plant virus, which polynucleotide comprises a product of a gene library.

89. The evolved plant virus of claim 88, wherein the gene library is produced by recursive recombination.

90. The evolved plant virus of claim 89, wherein the gene library is produced by DNA or RNA shuffling.

91. The evolved plant virus of claim 89, wherein the polynucleotide is inserted into a viral coat protein gene resulting in translation of a fusion protein, which fusion protein comprises a polypeptide encoded by the polynucleotide fused to a viral coat protein.

92. The evolved plant virus of claim 91, wherein the polypeptide encoded by the polynucleotide is displayed on an outside surface of a virus particle.

93. The evolved plant virus of claim 89, wherein the polynucleotide inserted into the evolved plant virus is a plant or viral promoter, which promoter is operably linked to a reporter gene.

94. The evolved plant virus of claim 93, wherein the reporter gene is green fluorescent protein (GFP), luciferase, β-glucuronidase or a maize Lc (anthocyanin regulatory) gene.

95. The evolved plant virus of claim 93, wherein the reporter gene of step (d) is a selectable marker.

96. The evolved plant virus 95, wherein the reporter gene of step (d) confers resistance to an antibiotic or a herbicide.

97. The evolved plant virus of claim 95, wherein the reporter gene of step

(d) is a negatively selectable marker.

98. The evolved plant virus of claim 97, wherein the reporter gene is a homologue of a dhll gene, a codA gene, a tms2 gene^ or a NIA2 gene.

99. The evolved plant virus of claim 98, wherein the reporter gene is a non-functional homologue of a dhll gene, a codA gene, a tms2 gene, or a A2 gene.

100. The evolved plant virus of claim 89, wherein the polynucleotide is inserted contiguous to a cauliflower mosaic virus 35S promoter resulting in expression of a protein encoded by the polynucleotide under the control of the cauliflower mosaic virus 35S promoter.

101. The evolved plant virus of claim 89, wherein a viral genome of the evolved plant virus is positioned adjacent to at least a right agrobacterium T-DNA border.

102. A method of using the evolved plant virus of claim 101, comprising introducing the evolved plant virus into a host plant cell by Agroinfection.

103. A method of using the evolved plant virus of claim 89. Wherein the vector is introduced into plant protoplasts by one or more of electroporation, microinjection, biolistics, or direct mechanical inoculation.

104. A transgenic plant or a transgenic plant cell or a transgenic plant explant made by transforming a host plant or host plant cell or host plant explant with the evolved plant virus of claim 87.

105. The transgenic plant cell or transgenic plant explant of claim 104, wherein the plant cell or plant explant is a stem, a leaf, a root, a meristem, a callus, or an embryo.

106. A library of recombinant polynucleotides, wherein the polynucleotides comprise at least one sequence derived from a genome of the transgenic plant, or the transgenic plant cell, or the transgenic plant explant of claim 104.

107. A recombinant plant virus, which recombinant plant virus comprises a polycloning site, which polycloning site replaces a viral coat protein structural gene and is contiguous with a corresponding viral coat protein promoter, such that an exogenous DNA inserted into the polycloning site is expressed under control of the viral coat protein promoter.

108. The recombinant plant virus of claim 107, wherein the viral vector is a tobamovirus.

109. The recombinant plant virus of claim 108, wherein the desired property is the ability to infect a transgenic Brassica.

110. The recombinant plant virus of claim 109, wherein the Brassica is Arabidopsis thaliana.

111. The recombinant plant virus of claim 107, wherein the desired property is the ability to infect a transgenic plant, which transgenic plant is selected from the family Leguminosae.

112. The recombinant plant virus of claim 107, wherein the desired property is the ability to infect a transgenic plant, which transgenic plant is a monocot.

113. The recombinant plant virus of claim 112, wherein the transgenic plant is a crop plant selected from among the genera: Eleusine, Lollium, Bambusa,

Dactylis, Sorghum, Pennisetum, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Coix.

114. The recombinant plant virus of claim 107, further comprising an exogenous DNA, which exogenous DNA is inserted into the polycloning site.

115. The recombinant plant virus of claim 114 wherein the exogenous

DNA encodes a polypeptide.

116. The recombinant plant virus of claim 115, wherein the polypeptide is a protein, which protein is a biofuel, an industrial enzyme, a nutritional enzyme, a nutraceutical, a protein pharmaceutical, a plantibody, an antigen, or a biosynthetic enzyme.

117. The recombinant plant virus of claim 116, wherein the protein is a biosynthetic enzyme, which biosynthetic enzyme comprises a component of a biochemical pathway producing at least one of an oil, a starch, a biodegradable plastic, a component of a biodegradable plastic, a biopolymer, and a component of a biopolymer.

118. A method of producing a polypeptide or a biosynthetic product, the method comprising: transforming a transgenic plant, or plant cell or plant explant, which transgenic plant or plant cell or plant explant expresses a viral coat protein, with the recombinant plant virus of claim 107.

119. A method of evolving a plant virus or viral component to confer pathogen-derived resistance to plant viruses, the method comprising: (a) providing a population of plant virus nucleic acid fragments;

(b) recursively recombining the population of nucleic acid fragments to generate a library of recombinant polynucleotides;

(c) optionally repeating the recombination process of steps (a) and (b) one or more times;

(d) screening the library to identify at least one recombinant polynucleotide, wherein the at least one recombinant polynucleotide comprises one or more evolved viral components, which one or more evolved viral components have acquired or evolved the property of conferring resistance to infection by at least a second plant virus when the one or more evolved viral components are stably introduced and expressed in a host plant, wherein the at least second plant virus is the same or different from the evolved plant virus or the one or more evolved viral components;

(e) repeating steps (a) through (d) until the one or more evolved viral components have acquired the ability to confer pathogen-derived resistance to plant viruses.

120. An evolved plant virus or viral component produced by the method of claim 119.

121. A methqd for producing a gene with a desired property, the method comprising:

(a) introducing a plurality of RNA viral vectors comprising one or more gene of interest into at least one cell;

(b) growing the cell under conditions permitting cytoplasmic recombination between the plurality of RNA viral vectors, thereby producing a library of recombinant RNA viral vectors;

(c) optionally recovering at least one recombinant viral vector and repeating steps (a) and (b);

(d) identifying at least one RNA viral vector comprising a gene with a desired property.

122. The method of claim 121, comprising introducing the plurality of

RNA viral vectors by inoculating at least one cell with infectious viral transcripts.

123. The method of claim 121, comprising introducing the plurality of RNA viral vectors by introducing a plurality of cDNA molecules corresponding to viral transcripts.

124. The method of claim 123, wherein viral transcripts comprising the plurality of cDNA molecules are produced in the cytoplasm of the at least one cell.

125. The method of claim 123, wherein the plurality of cDNA molecules are introduced by electroporation, microinjection, biolistics, agrobacterium mediated transformation or agroinfection.

126. The method of claim 121, wherein the RNA viral vector comprises a plant viral vector.

127. The method of claim 126, wherein the RNA viral vector is selected from among a tobamovirus, a potyvirus, a tobravirus and a potexvirus.

128. The method of claim 126, wherein the RNA viral vector comprises a Tobacco Mosaic Virus (TMV), a TMV homolog or an engineered viral vector derived from a TMV or TMV homolog.

129. The method of claim 121, wherein the gene of interest comprises a protein coding sequence.

130. The method of claim 121, wherein the at least one cell comprises a plant cell.

131. The method of claim 130, wherein the plant cell comprises an isolated plant cell, a protoplast, a plant explant, a plant tissue or an intact plant.

132. The method of claim 130, comprising growing the plant cell in suspension culture.

133. The method of claim 130, comprising growing at least one intact plant comprising the plant cell.

134. The method of claim 121, wherein the cytoplasmic recombination is mediated by template switching of an RNA polymerase expressed by the at least one cell.

135. The method of claim 134, wherein the RNA polymerase is a plant viral RNA polymerase.

136. The method of claim 134, wherein the RNA polymerase is a mutant or engineered viral RNA polymerase that enhances the frequency of homologous or non-homologous RNA recombination relative to a wild-type plant viral RNA polymerase.

137. The method of claim 136, wherein the mutant or engineered viral RNA polymerase is produced by a directed evolution process.

138. The method of claim 137, wherein the directed evolution process comprises DNA or RNA shuffling.

139. The method of claim 121, comprising recovering at least one recombinant viral vector by isolating RNA from the at least one cell.

140. The method of claim 121, comprising identifying the at least one

RNA viral vector comprising a gene with a desired property by selection or screening.

141. The method of claim 121, comprising introducing at least a first RNA viral vector incapable of systemic infection in a plant and a second RNA viral vector incapable of systemic infection in a plant, which first and second viral vectors have complementary mutations in genes essential for systemic infection, and identifying at least one recombinant RNA viral vector by selecting or screening for RNA viral vectors capable of systemic infection.

142. The method of claim 141, wherein the genes having complementary mutations comprise one or more of a gene encoding a viral movement protein or a gene encoding a viral coat protein.

143. The method of claim 141, wherein selecting or screening is performed by sampling a plant cell or tissue remote from the site of introduction.